synthetic studies of some biologically important molecules

Synthetic Studies of Some Biologically Important M olecules

A Thesis presented in partial fulfillm ent o f the degree of Doctor o f Philosophy from the University of London

b y

Peter George Robins

UCL Chemistry Department, 20 Gordon Street,

London.

October 1994

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A b s t r a c t

A b s t r a c t

Potass ium channels play a crucial part in ce llu la r hom eostasis and

disruptions in their function are implicated in a variety of disease states

including diabetes and cardiac arrhythmias. Consequently , a wide variety

of potassium channel modulators are in therapeutic use but few of them

appear to act as antagonists. However, apamin, from bee venom has been

shown to be an extremely potent blocker of the small conductance,

calcium-activated potassium channel (SK) and the main body of this thesis

is concerned with ex tend ing previous w ork on the s truc tu re -ac tiv i ty

rela tionships of this compound. Two salient, pos itively-charged nitrogen

atoms on ad jacent arginine residues in the pep tide , arranged at an

appropriate spacing seem to be the seat of activity and the assumption that

this is the pharmacophore provides the basis of the work. Thus a series of

bis-quaternary ammonium salts were synthesised by the substitu tion of

various amines onto one of a number of rigid, aromatic frameworks which

were chosen because they arranged the cationic centres approximately 11

Â apart, the posited crucial distance, as well as providing a hydrophobic

b r id g e . 2 ,6- and 2 ,7 -d im e th y l a n th ra c e n e , 3 ,6- and 1,6 -d im e th y l -

ph en an th ren e and t r a n s - 2 , 4 ' - and t r a n s 4 , 4 ' - d im e th y I s t i lb e n e w ere

synthesised according to established procedures. All compounds, including

p -x y len e and 2 ,6 -d im ethy l n a p h th a le n e , w ere b i s -b ro m in a te d in the

benzylic position with N-bromosuccinimide or bromine. The first three

fram ew orks in the list above were then substi tued with p iperid ine ,

pyrrolidine and morpholine and quaternised with iodom ethane to give 9

compounds of medium to negligible activity in blocking the SK current in

ra t s y m p a th e t ic n e u ro n e s . S u b s t i tu t io n o f a m ix tu re of 2 ,5 -

dim ethylpyrrolid ines followed by iodom éthylation produced a series of

compounds with good activity whilst the synthesis of a series of compounds

co n ta in in g cf j -2 ,6 -d im e th y lp cou ld not be com ple ted . 1,4-

diazabicyclo[2 .2 .2]octane (DABCO) and N-substitu ted derivatives, together

with quinuclidine were also used as substituents, but did not improve on

previous results. An attempt to make tricationic derivatives of benzene

could not be completed in the time available. The results of the assay and

A b s t r a c t

in fe ren c e s are p resen ted toge the r with spec tra l da ta for a ll new

c o m p o u n d s .

The second part of the thesis deals with a series of attempts to

generate the lithium dianion of pyruvic acid and effect its addition to

various electrophiles. No evidence was found for the presence of the ion so

a ttem pts were made to stabilise it by the use o f esters and hindered

th ioes te rs o f the acid which included ter t-buty l and 1, 1-d ie thy lpropy l

th iopyruvate and methyl and ethyl pyruvate. A num ber o f 0 - tr ia lky ls i ly l

enol ethers w a 5 also synthesised and their reaction^ with electrophiles in

the presence of Lewis acids were investigated. No conditions were found

under which reaction was observed to occur so the Diels-Alder and inverse-

dem and D ie ls -A lde r reac tions o f the silyl enol e thers were brief ly

i n v e s t ig a t e d .

A c k n o w l e d g e m e n t

A c k n o w l e d g e m e n t

I would like to thank my supervisor, P. J. Garratt, for the support he

has provided over the years of my attendance at UCL and for his help

during an often trying time.

I would also like to thank the various members of my group who

have provided the usual supply of chemicals, amity and conflict that have

made this course so varied. These include Stefan, Rob, Ashley, Sundram and

Najeeb. Many other people, too numerous to mention, were also on hand to

provide respite, but especially Aneela, Dan, Bruce, Duncan, Tara and Dave.

Thanks to Stefan for various nmr spectra, Karen for the nOe spectrum, and

Keith Wibley for drawing the M E? and energy-minimised diagrams.

F u n d in g for this course was p rov ided by the S c ience and

Engineering Research Council and I would like to extend my thanks to

them, and those responsible for securing my grant.

To Graham: thank you for supporting me over the years

The errors of a wise man make your rule

Rather than the perfections of a fool.

- William Blake

This thesis is dedicated to my father.

C o n t e n t s

Contents

A b s t r a c t .............................................................................................................................................. 2

A c k n o w l e d g e m e n t ..........................................................................................................................4

Dedication.............................................................................................................................................. 5

Table of Contents............................................................................................................................... 6

G l o s s a r y ................................................................................................................................................9

1. In t ro d u c t io n ............................................................................................................................... 11

1.1. The Basis of the Cellular Potassium Current............................................12

1.1.1. The Action Potential............................................................................. 12

1.1.2. Discovery of the Role of K+................................................................14

1.1.3. Classification of Potassium Channels........................................... 15

1.2. Potassium Channel diversity............................................................................. 17

1.3. Potassium Channel Structure............................................................................ 20

1.3.1. The Selectivity Filter............................................................................20

1.3.2. Use of Channel Antagonists..............................................................21

1.3.3 Detailed Channel Structure D educed From SAR- and 24

Point Mutation-Studies

1.4. Electrophysiological Characterisation........................................................... 28

1.5. Specific Potassium Channel Modulators......................................................31

1.5.1. Animal Toxins..........................................................................................31

1.5.2. Apamin-A specific Blocker of SK ...................................................32

1.5.3. Characterisation of Apamin Binding Sites ..................................33

1.6 The Structure of Apamin............................................................. 33

1.6.1. Primary Sequence.................................................................................. 33

1.6.2. Structure/Activity Relationships.................................................... 34

1.6.3. Configuration of Apamin................................................................... 38

1.6.4. Molecules Containing Bis-Quaternary N itrogen ..................... 42

Atoms as Mimics of Apamin

1.7. Therapeutic Applications of Potassium Channel A ntagonists 44

1.7.1. Mechanisms of Interaction With the C hannel..................... 44

1.7.2. Clinical Applications.............................................................................46

1.8. References.................................................................................................................. 48

2. Results and Discussion............................................................................................................ 52

2.1 Routes to Dimethylated Aromatic Hydrocarbons.................................... 53

C o n t e n t s

2.1.1. D im ethylphenanthrenes.......................................................................54

2.1.2. Dimethylanthracenes.............................................................................57

2.2. Benzylic Functionalisation of Aromatic H ydrocarbons ..................... 63

2.3. Synthesis of D im ethyl-phenanthrenes and -an th racenes.................. 67

2.3.1. Phenanthrenes.................................................................................. 67

2.3.2. Anthracenes.............................................................................................. 71

2.4. Other Hydrocarbons................................................................................................ 75

2.5. Formation of Amines..............................................................................................76

2.5.1. Derivatives of Piperidine, Pyf/olidine and M orpholine..76

2.5.2. Quaternisation of Amines................................................................... 80

2.5.3. Derivatives of 2,5-Dimethylpyrrolidine..................................... 82

2.5.4. Derivatives of DABCO..........................................................................84

2.5.5. Derivatives of Quinuclidine.............................................................. 88

2.5.6. Attempted Synthesis of Derivatives of c is -2 ,6 - ....................... 89

Dimethylpiperidine

2.5.7. Other Compounds...................................................................................90

2.6. References................................................................................................................... 92

3. Pharmacological Evaluation...................................................................................................95

Glossary.................................................................................................................................96

3.1. Introduction................................................................................................................ 97

3.2. Preparation of Rat Sympathetic Ganglia...................................................100

3.3. Derivatives of Piperidine, Pyrrolidine and M orpholine .................103

3.4. Derivatives of 2,5-Dimethylpyrrolidine...................................................... 104

3.5. A Comparison of Electrostatic Potentials................................................... 107

3.6. 2 ,6-B is[(cis-2 ,6-dim ethyl)piperidom ethyl]- ............................................... 114

Dimethiodide

3.7. Derivatives of DABCO and Quinuclidine.....................................................115

3.8. The Effect of N-N Distance.............................................................................. 119

3.9. Conclusion..................................................................................................................122

3.10. References............................................................................................................... 122

4. Experimental..............................................................................................................................124

4.1. Apparatus and Reagents......................................................................................125

4.2. Experimental.............................................................................................................126

4.3. References................................................................................................................. 181

5. N u c le o p h i le Addition of Pyruvic Acid Synthons.................................................182

5.1.Introduction................................................................................................................. 183

C o n t e n t s

5.1.1. Carboxylic Acid Dianions...............................................................183

5.1.2. Pyruvic Acid......................................................................................... 187

5.1.3. Aims of the Project...........................................................................190

5.2. Results and Discussion.......................................................................................193

5.3. Experimental...........................................................................................................210

5.3.1. Apparatus and Reagents.................................................................. 210

5.3.2. Experiments........................................................................................... 211

5.4. References................................................................................................................218

Appendix........................................................................................................................................... 221

Corrigenda and Addendum....................................................................................................... 225

G l o s s a r y

Glossary

KC - potassium channel;

ICF - intracellular fluid;

ECF - extracellular fluid;

ATP - adenosine triphosphate;

gx ’ conductance of x in a given medium;

AP - action potential;

C a j 2 + - intracellular Ca^+;

Nai'*’ - intracellular Na"*";

A T Pi - intracellular ATP;

I k - current associated with a given potassium channel K;

K m - muscarine-sensitive potassium channel;

K r / K i - inward rectifying potassium channel;

K v - outward (delayed) rectifying potassium channel;

K a - potassium channel carrying the transient A-current;

K a T P / ^ g - adenosine triphosphate-sensitive potassium channel;

BK - large conductance, Ca^"*"-activated potassium channel;

SK - small conductance, Ca^'*’-activated potassium channel;

TEA - tetraethylammonium ion;

QA - quaternary ammonium salt;

K d (v ) - voltage-dependent affinity of a putative blocker for a given

channel recep tor;

5 - (i) parts per million of applied field, relative to tetramethylsilane;

(ii) electrical distance;

z - ionic charge;

SAR - s tructure/activity relationship;

Sn - membrane-spanning segment of a potassium channel;

H5 - extracellular connecting region between S5 and S6 of most potassium

channels;

Shaker, RCK - two closely related families of genes from d r o s o p h i l a

melanogaster which encode a series of A- and delayed

rectifier-type channels;

L D 50 - median lethal dose; the dose required to effec t 50% mortality in vivo'.

G l o s s a r y

A23187 - a Ca^"^ ionophore used to stimulate response of Ca^'^-dependent

p ro c e s s e s ;

nOe - nuclear Overhauser effect;

HPLC - high-pressure liquid chromatography;

DABCO - l,4-diazabicyclo[2.2.2]octane;

HIV - human immunodeficiency virus;

FAB - fast atom bombardment mass spectrometry;

Cq - quaternary carbon atom;

Ct - tertiary carbon atom;

Cs - secondary carbon atom;

Gly, G - glycine;

Tyr, Y - tyrosine;

Asp, D - aspartic acid;

Thr, T - threonine;

Met, M - methionine;

Val, V - valine;

His, H - histidine;

Cys, C - cysteine;

Asn, N - asparagine;

Pro, P - proline;

Glu, E - glutamic acid;

Leu, L - leucine;

Arg, R - arginine;

S - serine;

F - phenylalanine;

K - lysine;

I - isoleucine;

A - alanine;

W - tryptophan;

Har - homoarginine;

Orn - ornithine;

T h r-11 - eleventh amino acid residue from the C-terminus.

10

Chapter 1

In trod u ction

I n t r o d u c t i o n

I n t r od u c t i on

1.1 T he Basis O f T he C e l lu la r P o ta s s iu m C u r r e n t

1.1.1 T he A c t io n P o te n t i a l

All living cells exhibit a resting membrane potential, but a characteristic

of excitable cells (that is, muscle cells and neurons ) is that they are able to

a l te r th a t po ten t ia l by va r ia t ions in the io n -p e rm e a b i l i ty o f the

m e m b r a n e ^ . In a normal cell, this potential difference at rest is 50-100 mV

with the interior of the cell negative and is due to the uneven distribution

of ions between the intracellular fluid (ICF) and the extracellu lar fluid

(ECF). This difference is maintained by a slow but continuous process of

active transport whereby enzymes powered by the metabolism of adenosine

triphosphate (ATP-ases) pump Na+ ions from the cell and K+ ions into the

cell resulting in concentrations of K+ in the ECF and ICF of 4 and 150 mM

respectively. The diffusion rate for this process is small, typically 1 p m o 1

m ’ ^ s ’ ^, but very much more rapid ion flow occurs when the stimulus

applied to an excitable cell (the release of acetylcholine at a synapse or

motor end-plate is the usual means by which this occurs) is sufficiently

strong to generate the so-called action potential (AP) (figure 1 .1 ) . The

stim ulus reduces the negative resting m em brane po ten t ia l to a less

negative value, a process called depolarisation. When a critical voltage, the

threshold potential, is reached activation of the voltage dependent Na+

channels occurs, causing a sudden, large increase in the Na+ conductance (gN a) of the cell membrane, and a fast Na+ influx. During this phase of the

AP, the negative state inside the cell is not only reversed but the membrane

potential even reaches positive values (overshoot). Prior to this, however, gNa begins to decrease (this “inactivation” occurs after approximately 0.1

ms) accompanied by a slow rise in K"*" conductance (g ^ ) . This permits K+

efflux and leads to the re-estab lishm ent o f the negative potentia l orrepolarisation. For a few milliseconds before g ^ returns to its resting value,

the m em brane po ten tia l may be even m ore n ega tive than at rest

(hyperpo larisa tion) . Besides m uscle contraction and the p ropagation of

nerve impulses, the AP is associated with a number of phenomena such as

12


— - 90 mV

1. Resting state ^ + 20 mV

A

2. Depolarization

jovershoo t

3. Repolarization

Threshold 4 ' / \/ \

Hyperpolarization

A. Depolarization and repolarization

Restingmembranepotential

Time

Figure 1.1: The Action Potential

13


an increase in the intracellular concentration of ATP or excretion from

endocrine cells, and these form the basis of any clinical exploitation.

1.1.2 Discovery Of The Role Of K+

The nature of the action potential has been known for a number of years.

The rôle of Na+ was established first since it was known that the external

c o n c e n tra t io n of the ca tion was m uch g rea te r than the in te rn a l

concen tra tion , rendering a ltera tions in experim ental cond it ions a more

fac ile p rocess . A series o f experim ents on a num ber of c e llu la r

preparations such as frog muscle^ and squid giant axon^ revealed that if

this concentration fell below a certain level, or was replaced by a Na**‘- f r e e

medium, then progressive depression of the amplitude of the AP, leading

ultimately to the inexcitability of the cell was observed. This was easily

m easured using voltage-c lam p techniques. It was apprec ia ted , as the

importance of Na"*" in the AP was established, that if the early phase of the

AP was due to the influx of N a + , then some degree of rectification was

necessary in order to explain the falling phase of the AP and also to restore

the potential of the cell membrane to a resting value. This was rather more

d iff icu lt to a ttr ibute experim entally to K"*" since the flow of ions was

outward and the later part of the current during constant depolarisation

was found to be broadly the same, regardless of the composition of the

external medium^.

H od g k in and H ux ley m oun ted an e x te n s iv e s tudy o f the

electrophysiology of the delayed rectifier channel in the giant axon of the

squid, Lol igo fo rbes i , first establishing the relationship between current

and voltage, then dissecting the contributions due to Na"^ and Under

vo ltage-c lam p cond it ions , when the res t ing m em brane po ten t ia l was

subjected to a sudden displacement, then held constant for 10-50 ms, the

transient inward current due to the influx of Na"^ was observed to pass over

into a long-lasting outward current, the magnitude of which depended on

the s treng th of the depo la r isa tion and the tem pera tu re . Though the

ou tw ard curren ts reached in a vo ltage-c lam p experim en t were often

considerably greater than those observed in the AP, this was thought to be

because the duration of the AP was not sufficient to allow the outward

current to reach its maximum value, and it seemed that this current

14


associated with prolonged depolarisation was the same current responsible

for the falling phase of the AP. There was strong evidence that the latter

(and therefore the former) was caused by K+ leaving the axon^, and,

together with the results of tracer experiments^, the rôle of K" in the AP

was thus established.

1 .1 .3 C la s s i f ic a t io n O f P o ta s s iu m C h a n n e l s

The rem arkable feature o f all ion channels is the ir se lec tiv ity to a

particular ion and in the case of potassium channels, the heteromorphism

that can lead to the presence of several different types within the same

c e l l ^ . There are at least 30 distinguishable types o f potassium channel

( K C ) ^ , differing in the means through which they are regulated or “gated”

and this forms the basis for an ad hoc classification. Though designation of

a particular channel depends on the criteria used for electrophysiological

c h a r a c t e r i s a t i o n ^ , three basic types of gating have been identif ied ,

a c c o r d in g to w h ic h the f o l l o w in g c h a n n e l ty p e s m ay be

d isc r im in a te d ^ ® ’ 1, 12.

(i) V o l ta g e - s e n s i t iv e c h a n n e ls , a c t iv a te d by c h a n g e s in

membrane potential throughout the AP;

(ii) Ion-activated channels which are gated by changes in the

i n t r a c e l lu la r ion c o n c e n t r a t io n s . C a i^ + is capable of

activating and Nai+ of blocking some classes of K"*" channels;

( ii i) K+ c h a n n e ls g a ted by n e û ro t r a n s m i t te r s such as

a c e ty lc h o l in e .

The best characterised of these channels are given in table 1 . 1

along with conductance measurements and those agents which are known

to block individual channels. Conductance can also be used as a distinct

basis for classification but varies according to tissue type and the method

by which it is obtained. For these purposes, conductances are given in

picoSiemens (pS) as determined by the patch-clamp technique (see section

1 .4 ) where a channel with a conductance of 10 pS will carry 1 pA of

current when the potential is 100 mV away from equilibrium^^.

It is now well established that potassium channels are also present in

most non-excitab le cells. For instance, the rhythm ic e lec tr ica l activ ity

evoked by glucose is closely involved in the regulation of insulin secretion

15

T y p e Single C h a n n e l C o n d u c t a n c e Blocking Agents

V o l t a g e - S e n s i t i v e C h a n n e l s

Oulward Rectifier (Ik v )Inward Rectifier (Ir / Ik i ) Transient Outward A-Current

5-60 ps TEA, aminopyridines, Cs^, Ba^+, Zn^+, stryehninc, quinine5-30 ps TEA, Cs+ (potent), Rb+, Na+, Li+, Sr^+, formaldehyde20 ps Aminopyridines (potent), TEA (weak), dendrotoxins, quinidine

L i g a n d - A c t i v a t e d C h a n n e l s

On

High Conductance Ca^^-Activated(BK) Intermediate Conductanee Ca^+ Activated (IK) Low Conductance Ca^^-Aetivatcd (SK) ATP-Sensitive ChanneI(KATP/Kc)5-HT Inactivated Channel (Is)Na+-Activated K+ Channel (K^a)

100-250 ps TEA (potent), charybdotoxin, quinine, Ba^ +18-50 ps Quinine, eharybdotoxin, Cs+, dicarboeyanine dyes10-14 ps Apamin, neuromuscular blockers20-60 ps Tolbutamide, glibenclamide, TEA (weak), lignocaine, tetroeaine 55 ps Cs+ (partial), Ba^+, TEA (weak), 4-aminopyridine (weak)220 ps TEA, 4-aminopyridine, tetrodotoxin (reduces Na^ entry)

oa .

R e c e p t o r - C o u p l e d C h a n n e l s

Atrial (AChm) Channel 25-50 ps Cs+, Ba^+, 4-aminopyridine

Tabic 1.1; Classification Of Potassium Channels (adapted from ref. I I )


and depends on variations in the K"*" permeability of the p-cells in the

p a n c r e a s ( s e e section 1 .7 .2 ) . The rates of K"*" flux in non-excitable cells

are clearly independent of the action potential and may be modulated by

hormonal effects (such as those induced by insulin or epinephrine) or by

sympathetic stimulation, but the specific physiological function of some of

the channels is still not clear. However, there are no broad differences

between the electronic properties of channels found in non-excitable cells

and their excitable counterparts. Recombinant DNA techniques have been

important in establishing the molecular basis of KC diversity, but also the

structural homology seen in an enormously diverse range of species^

M ost work has involved the so-called “Shaker” gene o f the fru it-f ly

Drosophi la melanogaster which, upon cloning and expression in X e n o p u s

oocytes yields a family of voltage-gated KCs with distinct biophysical and

pharm acological properties but sharing a core of transm em brane domains

flanked by variable N- and C-terminal r e g i o n s ^ W i t h the addition of the

genes Shal, Shab and Shaw, which, together with Shaker comprise an

extended gene family encoding A- and delayed rectif ier-type channels^ ^

(see next section), four subfamilies have been defined. The fact that shared

amino acid identity is greater between proteins encoded by the individual

d rosoph ila genes and those encoded by m am m alian hom ologues than

between the drosophila genes of the four subfamilies suggests that these

arose be fore the d ivergence o f vertebra te and in v er teb ra te species.

Further, the discovery of genes homologous to Shaker in plant tissue lends

weight to the idea that voltage-gated channels at least, ubiquitous in

eukaryotic cells, have evolved as structural variations of channels encoded

by a gene which dates from the Precambrian era, before the divergence of

P la n ta e and A n im a l ia .

1.2 P o tass iu m Channel D ive r s i ty

KCs thus play a crucial rôle in de te rm in ing the res t ing m em brane

potential, time course amplitude and polarity of electrical changes in most

types of cells. Their exceptional diversity makes it instructive to detail,

beyond the information given in Table 1 .1 , the range of channel types and

the importance of the current they carry.

17


(i) M-current,

The M -current is a small, sub-threshold, voltage-dependent, outward

K + current. It determines the general level of excitability since it becomes

activated in the region between the resting and threshold potentials and

can therefore limit repetitive activity and firing frequency. It has been

i d e n t i f i e d ^ ^ in a num ber of different ganglia and neurons^® and is

inhibited by m uscarinic ACh-receptor agonists, including muscarine itse lf

(hence M -cu rren t) .

(ii) Inward (Anomalous) Rectifier, Ir / I k i

Unusually, opening of this channel permits an inward K+ current^ ^.

The m echanism of action of the channel is thought to be a voltage- dependen t steric b lockade by M gj^ + ; h y p e rp o la r isa t io n at po ten t ia ls

negative to E r causes Kg"*" to enter the cell, leading to displacement of the

ion and an increase in gR . At depolarisation, M g^+ blocks the channel

again. Assigning a function to the channel is difficult but it could play a

rôle in countering local elevations in intercellular K"*" concentra tion .

(iii) Delayed (Outward) Rectifier, I ^ y

As discussed, the delayed rectifier channel was the first kind of

potassium channel to be characterised. It carries the curren t which is

mainly responsible for the repolarisation phase of the AP. It is activated

there fo re by d epo la r is ing voltages and occurs , on average , severa l

milliseconds after depolarisation, hence the name. They are also ubiquitous

in non-exc itab le cells^® but the function of the current in these cases is

u n c l e a r .

(iv) Transient K"** or A-current, I ^

Along with Ij^ and I r , the A-current contributes significantly to the

resting m em brane potential in vertebrate and invertebrate neurons. It is

active in the sub-th resho ld region of the m em brane po ten tia l , being

activated by depolarisation after a period of hyperpolarisation. It therefore

plays a role in de term ining the firing frequency and is observed to

prolong the inter-spike interval during bursts in various neurons^^.

(v) ATP-sensitive K+ Channels, Kyi^Tp/^G

18


These channels are sensitive to the concentration of ATPj and occur

in the heart, pancreatic (3-cells and skeletal muscle; they are associated

with specific physio log ica l functions in each case. The ac tivation of ca rd iac has been suggested as a possible source o f therapeutic

in tervention for ischaemic hypoxia; the subsequent loss of K+ would

coun terac t the increased excitability of the heart and shorten the time

during which it is susceptible to arrhythmias of ischaemic origin.Harrison and Ashcroft identified a channel, K q , at pancreatic p - c e l l s

which appeared to be m odulated by glucose concen tra tion^^ . It was later

suggested that glucose, whose m etabolism increases in trace l lu la r ATP concentration, leads to closure of K ^ t p » which was therefore identical to

Kg -

(vi) Stretch-Activated K"*" Channels

Ion channels sensitive to the tension of the cell m em brane are

known in a number of tissues but only a few are -selective. The channels

show sim ilar properties to the intermediate conductance C a ^ " ^ -a c t iv a te d

channels and it is generally assumed that they play a crucial rôle in

volume regulation and cell death^'^’^^.

(vii) Ca2+-activated K+ Channels

Given their rôle in the generation of the AP, it is not surprising that

these channels are widespread in excitable cells. Two types may be clearly

d istinguished , v/z., voltage-dependent channels of large conductance (BK),

with conductances of the order of 100-250 pS, and channels o f small

conductance (10-14 pS) with little or no voltage-dependence (SK). Channels

with intermediate conductances are also widespread, but are less defined in

their function. BK is the best characterised of the three since its large

conductance yields a high signal/noise ratio during record ing and these

channels have been iden tif ied by the patch-clam p techn ique in nearly

every cell type^^ . The SK channel is much less extensive and its small

conductance means that it is often overlooked unless suppression of BK

with tetraethylammonium ion (TEA, a general KC blocker discussed in the

ne x t s e c t io n ) is c a r r ie d o u t ^ ^ . It is re sp o n s ib le fo r a slow afte rhyperpo larisa tion curren t (AHP) so is often ca lled K y ^ y p ; AHP is

19


known to trigger repetitive firing in a num ber of cells such as rat

sym pathe tic n e u ro n s and cultured cells from rat skeletal muscle^^. This

current is selectively blocked by apamin.

1.3. P o t a s s iu m Channe l S t r u c tu r e

1.3.1 The Se lect iv ity F i l ter

Potassium channels, like all ion channels, are water-filled pores but are

a lso enzym es w hich ca ta ly se the norm ally im p o ss ib le t ra n sp o rt of

potassium ions across the lipid bilayer. Potassium movem ent through the

channel is rapid (conductances correspond to a rate of 10^ - 1 0 s"^) and the

activation energy is low so is, physically speaking, s im ilar to aqueous

d i f f u s io n ^ ^ . However, any model of channel architecture must account for

the observed limiting of K+ diffusion at high concentrations and the fact

that flux can be competitively inhibited by other cations, much as enzymes

are inh ib ited by substrate analogues. After in tensive study, however, a

model of gross channel architecture has emerged.

The most in tr igu ing aspect of potassium channels is their high

selectivity in allowing only K'*' to pass, particularly in view of the smaller

radius of the sodium cation (95 pm against 133 pm for K"^). This “selectivity

filter” occurs at the narrowest part of the channel and its mode of action is

electrostatic in basis^.

Ions in aqueous solution interact strongly with the water around

them; a positively charged ion attracts the negative end of the water dipole,

i . e. the oxygen atom, and though the water m olecules are in therm al

ag ita tion , they m ain ta in their hydrogen-bonded s truc tu re as m uch as

possible and point the oxygen, on average, towards the ion. This interaction

lowers the energy of the ion significantly; alkali metal cations prefer this

environm ent to à vacuum by approximately 70-130 kcal mol" ^ , an energy

comparable to that of a covalent bond. Measurement of flux rates indicate

that K+ experiences energy changes of, at most, a few kcal mol" and the

channel must therefore compensate for the lost energy of hydration almost

completely by electrostatic and chemical interactions. The work required to

move the ion to the interior of the channel is thus the difference between

two energy terms and these depend inversely on the size of the ion.

20


E isenm ann and Horn^^'^ explained the equilibrium binding selectivity of

cation-selective glass electrodes as a model for the channel binding site by

calculating the interaction of a cation of radius r with an anionic site of

radius rA and deduced that the energy of interaction is proportional to

( r + r A ) '^ - For a large anionic site (a low field-strength site) with rA >» r, the

importance of ion radius is minimised and the barrier to entry dominated

by hydration energies. Since this is less for larger ions, this provides the

necessary selectivity of K+ over Na+.

S im ilarly Bezanilla and Armstrong^ suggested a bracelet of oxygen

atoms ringing the narrowest part of the channel, which would substitute

for the water molecules and select for the ion with the closest fit. This is

s im ilar to E isenm ann’s low field-strength model since it m inimises the

effect o f ion radius by delocalising negative charge.

The high selectivity of potassium channels is not however consistent

with the high transport rates that are observed. A crude calculation based

on the bulk resistivity of a salt solution bathing the channel^ reveals that,

if a pore 6 A in diameter and 50 A long were filled with 120 mM KCl, its

calculated resistance would be 18 GO (corresponding to a conductance of 55

pS) or 20 GG (g = 50 pS) if access resistance due to diffusion up to the mouth

of the pore is taken into account. Since the resis tance of some real

channels is as low as 4 G Q , these channels must only have a short, narrow

region with large antechambers at one or both ends that m inimise the

contribu tion of diffusion. This allows one to construct a p icture of a

“typica l” potassium channel (figure 1 . 2 ).

1.3.2 Use O f C h a n n e l A n ta g o n is t s

C ruc ia l to the unders tand ing o f channel a rch i tec tu re has been the

availability of blocking agents. These provide a ready means of disturbing

channel function in ways which can be controlled and, more importantly,

quantified. A problem has traditionally hampered elec trophysio logica l and

b iophysica l charac te r isa tion of potassium channels how ever, vi z . , those

m odu la to rs ava ilab le were typ ica lly unselec tive and often lacking in

potency. Of those that have been used, by far the most useful have been the

a m i n o p y r i d i n e s , 4 - a m i n o p y r i d in e an d 3 ,4 - d i am i n o p y r i d i n e , and

quaternary ammonium ions (QA), derivatives of TEA (figure 1 .3 ) .

21


The binding affinity of many potassium channel blockers is voltage-

mmrnm

m m ## # # #

F igure 1.2: Diagram of general potassium channel structure inferred

from conductance measurements and showing tunnel (T),

mouths (M) and antechambers (A).

N.

NH21

Et —

Et

N — Et

Et

3

Figure 1.3; Classical potassium channel blockers: 4-aminopyridine 1,

3, 4-diaminopyridine 2 and tetraethylammonium ion (TEA) 3.

d e p e n d en t^ ^ . For instance, TEA itself blocks the squid axon delayed rectifier

channel when perfused inside the cell and it is found that its affinity is

higher if the inner electrical potential is made more positive^^. Woodhull

devised a model which quantified this voltage dependence and related it to


the position of the site of the blocking a c t i o n ^ I n t u i t i v e l y , we may

imagine that a more positive potential inside pushes the ion outward (i.e.

into the channel). The blocking ion will partition according to a Boltzmann

distribution so that the voltage-dependent affinity, K j(V ), is given by:

Kd(V) = Kd(0)e(zôFV/RT) eq. 1.1

where Kd(0) is the affinity with no applied voltage, z is the valence of the

ion, F, R and T have the standard meaning (RT/F = 25 mV at 25 °C) and 5 is the

fraction of the field traversed by the blocker in moving to the blocking

s i te 3 5 ,3 6 this last quantity, sometimes called the electrical distance, we

have a way of assessing the relative penetration of various blockers into

the channel and thus a means to infer the size and shape of its entrance.

A set of experiments by Miller^ supports the notion that the blocking

site is within the membrane field and relates the electrical distance, S, to

the physica l distance. W orking with the delayed rec tif ie r channel from

rabbit sarcoplasm ic reticulum, he used a series o f bis-quaternary amines

(two trim ethylam ino groups linked by an alkyl chain of varying length)

and noted that for short chain lengths, the ions behaved as simple divalent

c o m p o u n d s ( z 6 = 1.3) with twice the effective valence of trimethyl

amm onium ion (TMA, for which z5 = 0.65). As the length was increased,

however, the effective valence gradually decreased to that of TMA; whilst it

may have been expected that the aggregate effective valence would be

given by ô = 6 % + 6 2 , it seems that one end was entering as far into the

channel as possible whilst the other, repelled by the positive charge of the

first, stayed as far out as possible. A chain length of 4-5 carbon atoms was

found to be just enough to get the second end out of the field (Ô2 = 0, z5 = 6 1 ).

Thus 65% of the field falls within a distance of 6Â. Given that the

m em brane is some 50 Â thick, this implies the fo llow ing s tructura l

f e a t u r e s :

(i) an antechamber so large that little potential drops across it and

whose resistance is negligible compared to the narrowest part of the

c h a n n e l ;

(ii) a mouth which the QA enters and blocks;

(iii) a tunnel which only the smallest ions can enter.

This accords well with the model predicted on the basis o f conductance

23


m e a s u r e m e n t s .

Besides voltage, there is one other, perhaps more important factor,

which affects the affinity of quaternary am m onium derivatives for the

c h a n n e l , v i z . , the length of the hydrocarbon side chains that are

subs ti tu ted for the ethyl groups of From TEA to

trie thylnonylam m onium ion (TEnonA) affinity increases 600 cal mol"^ for

each added carbon atom, suggesting that the leng thened side-chain is

binding to a hydrophobic moiety adjacent to the channel m outh^^. Though

it now seems probable that the walls of the channel are lined by a - h e l i c e s

that have hydrophilic or charged sidegroups, it is possible that the long

arm o f T E nonA ex tends beyond the h y d ro p h i l ic l in in g in to the

hydrophobic material behind, thus contributing to the b inding energy^

This e ffec t can also be produced by adding a benzene ring whilst

hyd roph il ic groups, like hydroxy l, reduce the b ind ing a ff in ity . This

feature, a QA site that binds a wide range of differently sized ions with the

same 5 and that prefers hydrophobic groupings, is seen in a wide range of

c h a n n e l s .

1.3.3 Deta i l ed Channel Structure Deduced From SAR And Point

M u t a t i o n S t u d i e s

Studies of the action of TEA on voltage-gated channels have revealed that

the in ternal and external binding sites for TEA are distinct; whilst, as

discussed, in ternal binding is dependent on m em brane potentia l, external

binding is insensitive to such changes. Mutational analysis o f the channels

encoded by the Shaker gene from drosophila has given im portant clues

about the nature of the TEA binding site and thus about the channel pore. It

is important to see this in terms of the generally accepted model of KC

s tru c tu re , how ever . The express ion of genes f rom severa l species ,

including mouse, rat, human and d r o s o p h i l a itself has produced a series of

po tass ium channel-form ing proteins with very sim ilar prim ary sequences.

A lthough there is little crystallographic information available on intrinsic

m em brane proteins it is generally believed that hydrophobic stretches of

n in e te en or m ore am ino acids in any sequence , d e te rm in ed from

h y d ro p a th y p lo ts , are m em brane-spanning'^^*'^^. This has consequences

24


for channel structure since these regions are seen in all known sequences.

Voltage-gated KCs belong to a superfamily of voltage-gated and secondary

m e s s e n g e r -g a te d c h a n n e l s ^ ^ including the voltage-dependent Na+ and

C a 2 + channels and C a- + -activated KCs which, because of sequence

sim ilarities, are thought to contain either one or four copies of an

underly ing structural motif"^^. This consists of six m em brane-spanning

segments (S1-S6), five of which are hydrophobic, the remaining segment

(S4) being positively charged and amphipathic (Figure 1 .4 ) .

ex trace llu lar

T h e p r o p o s e d m e m b r a n e - s p a n n i ng or i en ta t i on o f one protein subuni t o f a vo l tage- gated KC: four units probably ase mbl e to make a funct ional c h a n n e l . C - t e r m i n i o f t e n c o n t a i n a p h o s p h o r y l a t i o n s i t e ( P) and the s e q u e n c e b e t we e n s e g me n t s SI and 52 is f r eq ue nt ly N - g l y c o s y l a t e d , ind icated in this diagram by smal l branches .

in tracellu lar

Figure 1.4: Diagram of a voltage-gated potassium channel deduced

from hydropathy plots and site-directed mutagenesis studies (from ref. 45)

There is also an extended hydrophobic loop (H5) connecting segments S5

and S6 which is tucked into the lipid bilayer from the extracellular side.

The Shaker-like KCs are proposed to consist of four subunits arranged into

an outer cylinder of sixteen a -helices (formed from segments S1-S3 and

S5), surrounding an inner cylinder of eight a -helices (S6 and 54)^^. The

results of site-directed mutagenesis studies have provided insight into

which of these domains are involved in gating, selectivity and inactivation.

For instance, the positively charged 54 is most likely to be the voltage

25


sensor of voltage-gated KC’s, moving towards the extracellular surface by a

helical screw mechanism (figure 1 .5 ) during the transition o f the channel

from a closed to an activated state^^.

0. ' ' © '

At the resting membrane potential, all positively-charged residues are paired with fixed negative charges on other transmembrane segments of the channel and the transmembrane segment is held in that position by the negative internalmembrane potential. Depolarisation reduces the force holding the positive charges in their inward position. The S4helix is then proposed to undergo a screw-like motion through a rotation of approximately 60® and an outward displacement of approximately 5Â. This movement leaves an unpaired negative charge on the inward surface of themembrane and reveals an unpaired positive charge on the outward surfaceto give net charge transfer of +1.

F ig u re 1.5: The helical screw mechanism of the putative voltage

sensor of voltage-gated potassium channels (from ref. 47).

S45, the intracellular loop connecting S4 and S5, is also thought to form an

am phipathic helix that takes part in the regulation o f the gating of the

channel; its positive charges point towards the centre of the pore, forming

an e lec tros ta t ic barrier to the passage of cations whilst m ovem ent away

from the pore would cause opening. This latter m ovem ent implies a

rotation of the helix which would move its positive charges away from the

pore and b r ing in a negative charge which could take part in the

formation o f a negatively charged ring at the intracellular to the pore.

The H5 loop is strongly implicated in pore formation"^^. It is highly

conserved am ong known KC proteins and invariably consists o f nineteen

amino acids with a Gly-Tyr-Gly-Asp sequence motif towards the C-terminus

and a Thr-Met-Thr-Thr-Val motif in the middle (figure 1 .6 ) .

26


X

ou tsideKj

w)

T h e d i ag r a m s h o w s , in s ing l e letter code, the S 5 - S 6 l inker region o f v o l t a g e - g a t e d Sh ak er channe l s wi th the c o n s e r v e d H5 r e g i on i nser ted into the m e mb r a n e from the o u t s i d e . T h e p r e s u m e d pore f ormi ng res idue , t yros i ne ( Y) can be seen whi l s t those res idues with d i a g o n a l h a t c h i n g s e e m to be respons i bl e for b i ndi ng T E A from the intracel lular s ide.

Figure 1.6: The amino acid sequence of the H5 loop

The pore is assumed to be formed from four (3-hairpin structures of

H5, one from each of the subunits. The narrowest part of the pore, the part

that separates intracellular and extracellular space, may be formed by four

tyrosine residues (again, one from each subunit) since tyrosine occurs in

the H5 region of every known KC subunit sequence. Variations in the H5

region are m ajor determ inants of KC pharm aco logy , a ltering the

sensitivity of a given channel toward either internal or external blockers.

Experiments have also indicated that the H5 region is the location of

the binding site for both internally- and externally- applied TEA. The

series of channels encoded by the Shaker-related RCK family of genes

showed distinct affinities for external TEA and characterisation of the

proteins revealed substantial differences at residue 19 of the otherwise

h ig h ly - c o n s e r v e d H5 r e g i o n ^ B . S ite -d irec ted m utagenes is to give

positively-charged residues at position 1 also caused a significant, but

much less substantial alteration to the sensitivity to external TEA. This

suggests that a negatively-charged residue 1 attracts TEA, directing it to

residue 19 which then takes over with a van-der-Waals type interaction.

The most useful information regarding the internal blocking site

has come from chimeric KCs in which the H5 region of one is replaced by

a n o t h e r ^ ^ . The fact that the S5-S6 linker region carries properties such as

27


TEA sensitivity and single channel conductance over from its parent also

supports the idea that H5 is the pore-forming region. Substitution of Thr-11

specifically alters block of Shaker channels by internal TEA as well as the

se lec tiv ity properties of the pore and this residue has therefore been

suggested as participating in the form ation o f the selectiv ity f il te r of

Shaker K C ’s as well as providing a binding site for internal TEA.

U nfo r tu n a te ly , s i te -d irec ted m utagenes is s tud ies have not been

carried out on SK, the small conductance, C a^"^-ac tiva ted KC b locked

specifically by apamin. The problem arises out of this specificity, however,

since no channels have yet been cloned which have been found to be

blocked by the peptide. The value of mutational analysis may be seen from

such work carried out on voltage-gated channels encoded by Shaker and

RCK using dendrotoxin and charybdotoxin where, despite the difficulties

involved in elucidating the structure of the peptides themselves because of

their size, much is now known about the receptor site (the majority of

which seems to be located on the H5 region for both toxins, with some

degree of overlap of the two)^®.

The paradigm of KC topology illustrated here by the Shaker A-

channels has however been found to hold for almost all the KC proteins

expressed so far, though the m olecular features of many do, admittedly,

remain to be elucidated. Recently, however. Ho et. al. expressed an ATP-

regulated KC from rat kidney whose structure represents a major departure

from this model^^. Analysis of the protein’s primary structure allowed the

prediction of a structural model (figure 1 .7 ) which con ta ined only two

m e m b ra n e -sp a n n in g segm en ts bu t w hich c o n se rv e s an am ino ac id

segment analogous to H5 of voltage-gated KCs, providing further evidence

that this region forms the ion-permeation pathway.

1 .4 . E l e c t r o p h y s i o l o g i c a l C h a r a c t e r i s a t i o n

S AR studies o f KC modulators have been important in establishing the

common features and, presumably, ancestry of potassium channels but this

lack o f specif ic ity has f rus tra ted attem pts to ch a ra c te r ise ind iv idual

channels . One o f the most im portant advances has been the increasing

soph is t ica tion of e lec trophysio log ica l techniques. These even allow the

properties of a single channel to be investigated in the absence

28


] P O 4 l o o p

C O O H

Figure 1.7: Diagram of an ATP-regulated potassium channel from rat

kidney (from ref. 15).

of noise from other ion channels which, under standard voltage-clamp

conditions, may obscure in terpreta tion of the resu lts . In the past,

macroscopic currents due to potassium ion flux have been characterised,

but the problem now arises of dissecting them and ascertaining the

contributions due to the various classes of single channels. The observed

variability of single channel properties with respect to conductance, ion

29


se lec tiv ity , open probability and k inetics , as well as dependence on

a g o n is t s , a n ta g o n is ts , s e c o n d a ry m e s s e n g e rs , c y to p la s m ic c a lc iu m

concentration and even mechanical stress, has increased enormously as a

result o f research into this problem, but has created an incongruity: far

more ion channel types have been identified than there are known ion

currents. Thus any macroscopic current must consist of contributions from

several populations of channels.

Two methods, more than any others, have been instrum ental in

these advances. In one of a num ber of patch-clamp techniques developed

by N eher and S a k m a n n ^ a micropipette is attached to a patch of cell

membrane then disturbances to potassium channel function are caused and

the ion flux (current) is m onito red with tim e v ia changes in the

com position o f the saline solution contained within the p ipette (figure

1 .8 ) . Single channels can be detected electrically because they have a very

high turnover rate ( 10^ ions s" giving rise to a current of approximately

20 pA). This is now the most common method for measurem ent of ion

c h a n n e l c u rre n t .

bath solution

ATPpipette solution

no transmitter)

c -A MP

PROTEI Si KINASE

cytoplasm

receptor

transmitter receptor complex

channel

Diagram illustrating the suitability of patch-clamp current recording to investigate channel regulation by transmitters acting via secondary messengers. By applying the transmitter to the cell membrane outside the pipette opening, the modulation of membrane channels of the patch in the pipette tip can be examined. In this example, The hypothetical pathway of KC regulation by serotonin in A p l y s i a sensory neurones is illustrated.

Figure 1.8: P a tc h -c la m p re c o rd in g

30


A lternative ly , ion-conducting molecules can be studied in model

p h o s p h o l i p i d m e m b r a n e s ^ ^ T h e s e are typ ica lly art if ic ia l p lanar

bilayers formed across a 150 pM diameter hole in a Teflon f ilm ^^’^^. Using

this method, the voltage and the peptide or protein concentration can be

adjusted to allow the measurement of single ion channels. The large size

and complexity of channel proteins, which has made elucidation of detailed

channel structure so difficult, makes this technique particularly attractive.

A “minimalist” approach to the design of protein analogues may be posited:

first, the structural basis for a given function is predicted then a simplified

pep tide or pro te in sequence is designed which con ta ins the putative

functional features whilst d iscard ing , or rep lac ing with sequences of

m inim al com plex ity , those considered to be sup e rf lu o u s^ ^ . This latter

technique has been particularly useful in establishing a great deal of the

secondary structure of channel proteins.

1.5 Spec i f i c Po tass ium C h anne l M o d u la to r s

1.5.1 An im al Toxins

Ultimately, the lack of specificity of the QA ions and the ubiquity of the

binding site meant that the results of studies which used them had to be

in terpreted cautiously. M oreover, both QA ions and 4-am inopyridine are

known to block receptors for a wide variety of neurotransmitters including

muscarinic-, Ü2 -, a l - , a 2-, 5 H T i a - and 5HT2 -recep to rs^^ . Other compounds,

such as quinine, local anaesthetics and some ions, like Cs2+ and Zn^+ also

effectively block all, or at least some, types o f potassium channel, but they

also have additional, often more specific, effects on other classes of ion

c h a n n e l .

A number of animal toxins have been discovered to act as potent,

specific antagonists of KCs however. For instance, dendrotoxin, comprising

about 2.5% of the total venom protein o f the Eastern green mamba,

D e n d ro a p s is a u g u s tic e p s , is a peptide of 7077 Da which blocks transient

outward KCs^^. Similarly, charybdotoxin, a minor constituent of the venom

of the Mideastern scorpion, L e iu ru s q u in q u e s tr ia tu s var. hebraeus with a

mass of 4353 Da is selective for large conductance, Ca^"^-activated KCs^^.

31


1.5.2 A p am in , A Specific B locke r O f SK

A pam in, in com parison with o ther p ro te inous venom cons ti tuen ts , is

relatively small with a mass of only 2039 Da. It is a specific blocker for a

class of small conductance, Ca^ + -activa ted KCs and has an extensively

studied pharmacology. It is isolated from the venom of the Honey bee. A p is

m e lli fe r a , which, besides apamin, contains several toxic components, such

as h istam ine and phospholipase A, and o ther peptide toxins, such as

m elittin and m ast-cell degranulating peptide, all o f which have been

p u r i f ie d to h o m o g e n e i ty by H a b e rm a n n and c o -w o rk e r s ^ ® . Its

exceptionally small size (see section 1 .6 . 1 ) means that it is the only peptide

known to cross the blood-brain barrier.

Although the neurotoxicity of apamin is high (LD50 = 4 mg/kg upon

in travenous adm in is tra t ion in m ouse)^^ , the first indication of its site of

action came from studies on non-neuronal cell cultures of the guinea-pig.

A pam in was found to be se lec tive in in h ib i t in g the A T P -induced

hyperpolarisation of in testinal smooth muscle ce lls^^ as well as K" loss

from hepatocytes^^ . The K+ loss observed with the Ca^+ ionophore A23187

was also inhibited by apamin, leading Banks and co-workers to suggest that

it was acting on a Ca^ + -d e p e n d e n t K C ^^ . The fact that apamin did not

however inhibit a Ca^''"-dependent potassium current in erythrocytes gave

early evidence of the m ultifarious nature of this fam ily o f channels,

iK(Ca). This finding was followed by the observation that charybdotoxin

s p e c if ic a l ly b locked a Ca^ -a c t iv a te d KC c h a ra c te r i s e d by large64conductance (100-250ps) as previously described . However, the target for

apamin in cultured rat skeletal muscle was postulated as a C a ^ '* '-ac t iv a ted ,27QA-insensitive channel of much smaller conductance (10-14ps) . Further,

this channel showed only a weak voltage dependence and much greater

sensitivity to C a ^ ^ concentration at negative membrane potentials than the

charybdotox in-sensitive channel. That the d ifferen t Ca^ " ' '-dependent K"*"

currents are due to different channels is now well-established and it is

accepted that apamin is highly selective for ju s t one of them: the Ca^'*'-

ac tiva ted , Q A -res is tan t , nearly v o l ta g e - in d e p en d e n t ch an n e l o f sm all

conductance. These characteristics make it ideally suited to generate the

long-lasting after-hyperpolarisation that is seen in the many cells which5 8are known to contain this channel

32


1.5.3 C harac ter i sa t ion Of A p am in B in d in g Sites

A pam in may be radiolabelled with iodine at its h istid ine residue^

w hils t re ta in ing its b io logica l activity . This has a llow ed rad iograph ic

localisation and quantification of binding sites for ^ ^ I ] - m o n o i o d o a p a m i n

in the central nervous system of a number of species with an affinity that

accords well with its neurotoxicity. The binding to peripheral organs was

observed to be much less, though there were exceptions. Cook and Haylett

were the first to observe the binding of ^ ^ I] -m o n o io d o ap a m in to in tac t

ce lls unde r p h ys io log ica l cond it ions us ing an assay o f g u in ea -p ig

h e p a to c y te s ^ G . The dissociation constant, K j , for the iodinated derivative

(350 pM) compared well with that for native apamin (K j = 376 pM for the

same assay). It is known that a-adrenoreceptor agonists cause a loss of

from hepa tocy tes and the same workers dem onstra ted that increas ing

co n cen tra t io n s o f apam in p roduced a p rog ress ive dep ress io n o f the

dose/response curve to (-)-phenylephrine, measured as a function of K"*"

loss from a suspension of the cells. These two results strongly suggested a

binding o f labelled apamin that was directly related to the presence of

a p a m in -s e n s i t iv e , Ca^ +-activated KCs and that this was consistent with

simple competition at a single class of binding site. The binding of

m onoiodoapamin to rat hepatocytes and human erythrocytes was observed

to be much less and could not be reliably quantified, suggesting the lack of

apam in-sensitive Ca^+-activated KCs in these cell types.

1.6. The Structure O f Ap amin

1.6.1 P r im a r y S e q u e n c e

Given its potency, that the structure of apamin has been the source of a

great deal of research is not surprising. It was established by Habermann^ ^

to be a peptide containing eighteen residues with two disulphide bridges

whilst Callewaert and co-workers^^ deduced its amino acid sequence as that

shown in figure 1 .9 .

33


I ICys-Asn-Çys-Lys-AIa-Pro-GIu-Thr-Ala-Leu-Cys-AIa-Arg-Arg-<Zys-Gln-Gln-His-NH 2

1 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8

4*F igu re 1.9: A p a m i n

The position of the disulphide bridge was unequivocally established

by the same w o r k e r s ^ T r y p s i c hydrolysis of native apamin gave only one

peptide and arginine. Since N-term inal analysis revea led only alanine,

then cleavage must have occurred between positions 4 and 5, 13 and 14, and

14 and 15, i.e. the bridges could not have been between residues 1 and 3 and

residues 11 and 15. Of the two remaining choices, the bridge was found to be

as shown in figure 1 .9 . A complete cycle of Edman degradation removed

cyste ine-1 and thus destroyed one bridge. Trypsic hydrolysis of the product

gave arginine and a single peptide, a result which could only have arisen

if a disulphide bridge was linking cysteine-3 and cyste ine-15.

1 .6 .2 S t r u c t u r e / A c t i v i t y R e l a t i o n s h i p S t u d i e s

A number of teams have investigated the SAR of apamin using a number of

m odified peptides and synthetic analogues. V incen t and co-w orkers^ ®

undertook a series of specific chemical modifications o f the peptide and

found that most did not have a great effect on peptide activity, determined

by the median lethal dose (LD5 0 ) of subcutaneous in jec tion in mouse.

Transformation of the e-amino group of lysine-4 5 into hom oarginine

34


NHH (CH2)4NH2 NH2

(i) y + CH3 0 — < ( +^ y HSO4- ^ ^ 2

H (C H 2>4N H ^ y NH2+ CH3OH

< Vly s in e

5H Œ 2S-

( ii) XHjN y

o o H CH2S

MeCNH ' y + CH3COOH

c y s te in e

7O

O

H (C H 2)4N H 2 o o

/ y • - J - . x

ly s in e 5

H (CH2)4NHCCH3 + CH3COOH

< V9 n

( i i i ) H (CH2)2C 02H h y ELN=C=N(CH2)3NMe2 H (CH2)2CNHCH2C02Et

. A . A ,Vglutamic acid

1 0

(iv)

H2N COOEt

O

+ (CH3CH20C)20

h is t id in e

NH-, O

H (CH2)3NH ( +

1 3

OEt

+ MeCH20C02H

OH

H (CH2)3NH (

y . /arg in in e

1 4

OH

1 5

Figu r e 1.10

(H ar) using m ethy lisourea hydrogen su lpha te (f igu re 1 .1 0 ( 1 ) ) had no

discernible effect, whilst variously acétylation of lysine-4 and of the a -

am in o g ro u p o f c y s te in e -1 7 w ith a c e t ic a n h y d r id e ( f ig u re

1 .1 0 ( i i ) ) p r o d u c e d a reduction in activity o f only 2.5 tim es. Similarly,

modification of the carbonyl of glutamine-7 10 by formation of an amide

bond with glycine ethyl ester (figure 1 .1 0 (1 1 !) ) produced only a 50%

decrease in activity whilst carbéthoxylation of h is t id ine-18 12 with diethyl

pyrocarbonate (figure 1 . 10(1 v))produced no detectable loss o f activity at

35


all. These effects were synergistic, however; acétylation of cysteine-1 and

lysine-4 together with carbéthoxylation of histidine-18 produced a peptide

devoid of neurotoxicity. Reduction of the disulphide bridges with NaBH^.

fo llow ed by a lkylation of the resulting thiol with iodoacetam ide also

e lim inated toxicity , confirming an earlier result by Habermann^®. Most

interesting, however, was that modification of arginine-13 and arginine-14

with cyc lohexaned ione (figure l . l O ( v ) ) or trypsic cleavage, resulting in

the removal of arginine-14 and the formation of a two-chain peptide (see

Callewaert, above) also destroyed the toxicity, giving the first indication of

the singular importance of this part of the sequence.

These observations were expanded by G ran ier et a lP Using a

method that they had established in the solid-phase synthesis of apamin, a

num ber of synthetic analogues, differing in the arginine region by one,

two or three residues from apamin itself, were synthesised. Replacement of

either o f the arginine residues in positions 13 and 14 with lysine resulted

in values for the LD50 (in the same assay as the previous workers) very

close to that of apamin. Replacement of both, however, led to a peptide with

an activity that was very faint but which could be enhanced by reaction

with 0 -methylisourea sulphate, to give homoarginine residues in positions

4, 13 and 14 and a relative activity 0.15 times that of apamin, indicating the

favourable effect of the guanidinium structure on activity. The com par ison

with [Har-4]-apamin, prepared by Vincent and which shows no loss of

activity, suggests that positive charge in positions 13 and 14 is not of

unique importance and that the length of the arm bearing the functional

group is crucial. The synthesis of [O rnith ine-13, O rn i th in e -14]-apamin

(figure 1 . 1 1 ) and its complete lack of neurotoxicity would seem to support

th is^ ^ .

H CH2-CH2-CH2-NH2

''''COOH

16F ig u re 1.11: O r n i t h i n e

G a n e l l in and co -w o rk e rs^ synthesised a series of com pounds

containing a bis-guanidinium grouping and a number of small peptides with

ad jacent a rg in ine or arg in ine-lysine residues. O f the la t te r group of

36


compounds, all were found to be almost completely lacking in activity,

su g g e s t in g that this d ip ep tid e s tru c tu re does not convey enough

information for effective recognition by the receptor site. The possibility

of steric hindrance from derivatising groups was discounted by the lack of

activ ity o f the conform ationa lly f lex ib le 1, 10-b isguan idodecane (figure

1 . 1 2 ), synthesised because the decane chain was considered as equivalent

to the eleven bonds separating the guanidine groups in the peptide.

NH

NH

F igu r e 1.12

It is possible that the inactivity of the compounds was due to the inability

of the groups to achieve an appropriate spatial separation, but it seems

more likely that the guanidinium groups are a necessary, but not sufficient

contribution to the toxicity of the peptide.

L abbé-Ju lié et also found it difficult to correlate the observed

high affinity of apamin (K j = 10 pM) solely with the position of two

guanidinium groups in positions 13 and 14 and undertook a study to

determine which other of apam in’s structural features were responsible, at

least in part, for its action. For this, they were helped by two very sensitive

assays which were able to determine activities below the threshold of

detectability of previous studies, v iz ., inhibition of I ] - m o n o i o d o a p a m i n

b ind ing to rat brain synaptic m em branes and in trace reb ro ven tr icu la r

injection in mice.

Firstly, it was known by this time from proton NMR studies that Leu-

10, the only hydrophobic residue in apamin, was in proxim ity to the

arg in ine s ide -cha ins^^ , but replacement of this residue by alanine led only

to slight loss of potency, implying no close interaction with the receptor.

However, a more profound effect was noted by the sequential shortening of

the C-terminus. Loss of His-18 led to a peptide with 16 % of the toxic effect

and 1 % of the binding activity of apamin. Since these values were lower

than those obtained for a peptide in which His-18 had been chem ically

m o d i f i e d ^ ® ’^ ^ , it seem ed that there was a po ten t ia l ly s ign if ican t

37


involvement of the histidine residue.

Loss of GIn-17 and GIn-16 led to a greatly decreased activity but since

both gave similar values (0.35 and 0.43% toxicity respectively and 0.01%

binding efficiency), G in-17 was implicated as an important element in the

interaction of apamin with its receptor, probably through the amide side

chain. D espite the drastic loss o f activ ity how ever, bo th behaved as

com plete apamin agonists, with a dose-response curve paralle l to that

ob ta ined fo r na tive apamin in the b ind ing assay. G ln-17 could not

therefore be an essential residue for the specific activity o f apamin.

The m odification o f Arg-13 and Arg-14 using cyc lohexaned ione

produced a peptide with a previously undetectable activity (see above) but

measurable in the new assays. As was the case for the glycine-deficient

p e p tid e s , th is ana logue was ab le to sp e c i f ic a l ly in h ib i t ^ I ] -

m ono iodoapam in binding and exh ib ited the charac te r is t ic sym ptom s of

apamin poisoning in mice (0.002 and 0.04% relative activity compared to

apamin). Though the rôle o f the positive charges o f the arginine side-

chains was well established, being considered as part o f a well-defined

conformation which fitted a specific pocket on the receptor with negative

charges at the bottom, this was the first time that other forces of attraction

around Arg-13 and Arg-14 were shown to be involved in the specific

activity of apamin. It appears that the side-chains o f the arginine residues

are involved in hydrophobic bonding to the receptor, com plem enting the

ionic a ttrac tion .

1 .6 .3 C o n f i g u r a t i o n

N aturally enough, in the light of these observations, the secondary and

tertiary structure of apamin was of great interest. Early studies on the

c ircular dichroism spectrum of apamin by M iroshnikov et a l J ^ indicated

that a large part of the molecule existed as an a -he lix , and also showed that

the c o n f i g u r a t i o n was not greatly influenced by m odification of the side-

c h a in s . T h e s e w o rk e rs d e m o n s t ra te d th a t r e l a t iv e c o n s ta n c y o f

c o n f i g u r a t i o n observed over a wide pH range and in the presence of

organic solvents and 6M guanidinium HCl, indicating a stability thought to

be due to the established presence of the d isulphide links. Subsequent

studies concentrated on locating the a -he lix . Bystrov et a l J ^ undertook an

38


extensive NMR study of the peptide in D%0 solution and, stressing the

helical nature of fhe c o n f ig u r a t io n ,p r o p o s e d a tentative structure based on

the m easu red shifts, sp in-spin couplings and to rs ional angles, (figure

1 .1 3 ) .

Thi

Alq

NHAla

F ig u re 1.13; Proposed Structure of Apamin (from ref. 75)

H igh-resolution NMR studies revealed that alm ost all o f the amide NH

protons o f the molecule resolved into individual signals. By observing the

exchange rates with D 2 O, it was found that the individual amide groups

possessed m arkedly d ifferen t ha lf- lives , a fac t a t t r ibu ted to hydrogen

bonding and/or screening of these groups from the solvent molecules. This

latter explanation was later refuted by Englander and K allenbach^^ who

suggested that apam in’s small size would almost certainly perm it solvent

accessibility and therefore slow amide exchange was most likely due solely

to hydrogen bonding and not to screening o f the am ide group by the

peptide architecture. The rigidity of the molecule was therefore due to a

combination of disulphide linkage and strong hydrogen bonding.

H id e r and R agnarsson^® predicted that it was not possible to

construct the disulphide linkages if the entire 4-17 peptide segment was in

39


an a -h e l ic a l configuration. However, limiting it to segments 8-17 or 4-12

pe rm itted c ross l ink ing . The fo rm er was p refe rred , b e in g s trong ly in

accord with the NMR study o f Bystrov et and allowed for the

formation of two p-turns in the N-terminal octapeptide which would orient

the half-cysteines into the correct position for linkage.

Subsequently , Bystrov et a l j ^ refined their prediction to include an

a -h e l ix in the region o f peptides 6-13 and suggested the most plausible

system of in tram olecular hydrogen bonding, to include the a - h e l ix and

three p-turns in the C-terminal residue, based on the m easurements of the

exchange rates of the NH groups of Ala-5, Cys-15 and Gln-16.(figure 1 ,14)

-H* • 0

H -N

H -N

H -N

F ig u re 1.14: Diagram of in tram olecular H-bonds in apamin

This allowed construction of a spatial structure which also took account of

the coupling constants of the side-chains and their p red ic ted geometry,

(figure 1 .1 5 )

This did not fu lly reso lve the crucial ques tion o f the precise

conformation of the guanidinium groups of the two arginine residues. In

40


CH3NH-

:n h

F i g u r e 1 .15

an extensive 2D NMR study, Pease and Wemmer^^ posited a structure that

was genera lly consistent with previous models (figure 1 .1 6 ) but in which

the s ide -cha ins were shown to have far fewer constra in ts and exhibit

grea ter variability in their movements than did the backbone that held

t h e m .

TeA9

L i e

E7

P6R13

A12

AS

N 2R14'

0 1 7

K40 1 6

C 3

H 1 8

F ig u re 1.16: Ribbon Diagram of Apamin (from ref. 81).

41


In particular, the lack of nOe signals involving the side-chain proton of

the a rg in in e res idues made it im poss ib le to d e te rm in e the exac t

conform ation of the side-chains and suggested that both were mobile in

solution. It would seem that, providing the residues are positioned in a well-

defined way, then the inherent flexibility of the side-chains allows the

correc t pos ition ing of the guanidinium groups in the recep to r pocket,

corroborating the SAR studies of Granier et al

1 .6 .4 M o le c u le s C o n ta in in g B i s - q u a t e r n a r y N i t r o g e n A to m s As

M im ics O f A p a m in

In 1985, Cook and Haylett^^ correlated the ability of several compounds to

inhibit ^Î]-m onoiodoapam in binding with their ability to inhibit Ca^ + -

m ediated K+ efflux from guinea-pig hepatocytes. Tetraethylam m onium ion

and q u in in e were e ffec tive only in h igh c o n c e n t ra t io n w h ils t 9-

am inoacrid ine , quinacrine and chloroquine were slightly m ore effective.

By far the most active compounds, besides apam in i tse lf , were the

n e u ro m u sc u la r b lock ing agents tubocura rine 1 8 , pancuron ium 21 and

a tracu r ium 2 0 , the structures of which are given in fig 1.17 along with

the inhibition constants against [^^Î]-monoiodo-apamin b ind ing for these

and re la ted compounds. As previously d iscussed, the neu ro tox ic ity of

apamin is crucially dependent on the two adjacent, posit ive ly charged

amino acid residues in positions 13 and 14 and it was therefore postulated

that the ability of these compounds to behave in such a way may depend on

the distance between the two charged nitrogen atoms, present in them all

and which were thought to mimic the active peptide m oiety. Certainly,

there was a high correlation between their affinity for apam in binding

sites and their ability to inhibit K+ efflux from hepatocytes and, with the

compelling evidence that these binding sites corresponded to KCs, that the

compounds were acting as competitive antagonists at the apamin binding

site in the SK channel seemed an obvious conclusion. The subsequent

discovery that dequalinium inhibited [^^Î]-m ono iodoapam in b ind ing even

m ore e ffec tiv e ly supported this and led to the sy n th es is o f 3,6-

b is (p ip e r id o m eth y l)p h en an th ren e d im eth iod ide 23 . This was selected

42


O M e“l

OH

MeO OH Me

2+ Me Me

N-(CH2) io -N ^ y - N H z

1 8

Tubocurarine Chloride; K , = 9.2±0.4

“ O

1 9

Dequalinium Iodide; K j = 1.1 ±0.1

CH2CH2C0(CH2)50CCH2CH2>^N. ^

Me Me

OMeOMe OMe

2Ct'2 0

Atracurium Chloride; K j = 4.3±0.2 MeCOO

2+

MeMe

Me

H

2+

2Br'

Pancuronium Bromide (R=Me), Vecuronium Bromide (R=H) 2 1 2 2

Ki = 3.2+0.2 Kj = 3.6±0.5

Me Me

2+

2r2 3

3,6-Bis(piperidomethyl)phenanthrene Dimethiodide; K , = 9.9±1.3

F igure 1.17: Potent, selective blockers of SK (all values in | iM )

43


because its rigid framework was proposed to space the two quaternary

n itrogen atom s at an appropriate distance with the m ethy lene bridges

affo rd ing a s light degree of freedom that could enhance any supposed

in te rac t io n with the receptor, whilst the p ip e r id in e ring was chosen

because , with the exception of dequalin ium , it is a s truc tura l feature,

w hether substitu ted or unsubstitu ted , of all the neu rom uscu la r b lockers

illustrated. Though it was 3-fold less effective at inh ib iting radiolabelled

apam in b ind ing than pancuronium , it appeared that, d esp ite the large

difference in the nature of the backbone, the interaction was of the same

nature, and provided a starting point for SAR studies of this class of

c o m p o u n d .

1.7. Therapeut ic Appli ca t ions of Potassium C h a n n e l A n ta g o n i s t s .

1.7.1 M ech an ism s O f Interac t io n With The Channel

V oltage-gated KCs exist in several conform ational sta tes which may be

regarded as closed resting, closed activated, open and inactivated. At normal

resting potential, most KCs are closed. Upon a shift o f m embrane potential

to more positive values, a voltage-dependent conform ational change takes

place, but the subsequent opening and closing of the activated channel is

voltage independent. These transitions may be sum m arised in the state

diagram in figure 1 .18 . Two modes of interaction are well understood®^: N-

type and C-type inactivation, referring to which terminus o f the protein is

C : closed resting state; II € * , C **, activated closedIt \ states; O , open state; I,

^ P * _^ P** ^ P ^ , inactivated state. C-type^ u ^ icN and N-type ( I n )

inatpena'n. | | ^ inac t iva t io n may occurV Tl / y independently or s imul

taneously ( I c n )-

F ig u re 1.18: Transition state diagram of a voltage-gated potassium

c h a n n e l

44


involved. The N-terminal sequence is thought to behave like a ball that

blocks the pore by swinging in and out of a cytoplasm ic receptor site

be tw een segm ents S4 and S5, w hilst C -type in ac t iv a t io n is due to

c o n fo rm at io n a l changes in the reg ion tow ards the C -term inus which

involves a constriction of the channel’s extracellu lar mouth. Deletions or

a lte ra tions in the carboxy-term inal cytoplasm ic dom ain do not markedly

affect this process, so it is unlikely to involve the terminus itself in a ball

and chain mechanism like that suggested for N -type inactivation. Both

term ini are believed to be in tracellu lar which has im portant consequences

for any attempted pharmacological modification since the large majority of

the protein is located on the inner side of the cell, with the exception of a

few res idues betw een a lte rna te pairs of m em brane -spann ing segments.

Therefore, any putative modulators which cannot penetrate the membrane

can only bind to a small area of the protein. The fact that a large part of

this area is the H5 segment which, as a lready d iscussed , varies little

between different channel proteins is one of the reasons why the variety

o f KC blockers is much less than the number of channels themselves, and

also accounts for the ubiquity of the QA binding site.

H - drugs

îpriiîîiin i/;/iLAJiiiimivi

m f \ Y J

k m _A_ j

blockade of open closed

ion channels

ch an g eof

channel gating

Possible modes of interaction of drugs with voltage- or Ca^+- activated KCs: drugs may directly block open or closed channels (left). Alternatively, a change in channel gating can occur, resulting in an increase or decrease of channel open time or the probability of channel opening; this may result from drug binding to one or more allosteric sites at subunits of the channel protein or from alterations in the lipid environment of the channel (right).

F i g u r e 1 .19

45


We may posit four ways in which drugs may interact with KCs^^:

(i) by binding directly to the open channel, thereby blocking the

current, but also preventing closure of the channel;

(ii) by binding to the closed channel, preventing opening;

(iii) by binding to an allosteric site on the protein and thereby

changing the channel’s gating properties;

(iv) by altering the lipid environment of the channel.

These are summarised in figure 1 .19 .

1 .7 .2 C l in ic a l A p p l i c a t i o n s

Several marketed drugs appear to function as selective KC b l o c k e r s ^ F o r

instance, as a result of the SAR of TEA derivatives, clofilium 24 (figure

1 . 2 0 ) was found to be an effective treatment for disrhythmias of the heart.

E t

C l— < ^ ( C H 2 ) 4 N ( C H 2 ) 6 C H 3

2 4

F i g u r e 1 .20

It was the first specifically designed class III antiarrhythmic and works by

b lock ing card iac delayed rec tif ie r channels , thus leng then ing the AP

duration of cardiac cells in a selective manner.

Am ong the most im portant of the known KC blockers are the

antid iabetic sulphonylureas g lyburide 25 , g l ip iz id e 26, and tolbutamide 2 7

(figure 1 . 2 1 )

\ / '-”"N(CH2)2— \ S—N -C —N\ = / H \ _ / ^ H H

OCH3 2 5

glyburide

46


/ ~ \ y / — \ V 9

2 6

g lip iz id e

— \ ° °H3C—^ a— S -N -C — N— (CH 2)30 H 3^ ^ H H

2 7to lbutam ide

F ig u r e 1.21

It is known that increases in intracellular glucose in pancreatic p - c e l l s

leads to a corresponding increase in cytoplasmic ATP concentration. This

causes the closure of Ka t p » resulting in membrane depolarisation and the

opening of vo ltage-dependen t Ca^+ channels. The resultant increase in

C a j 2 + c o n c en tra t io n causes ex o c y to t ic se c re t io n o f in su l in . The

su lphony lu reas apparen tly function by b lock ing the pancrea tic ATP-

dependent KC.

The historical lack of potent antagonists has meant, however, that

the field of phanvitfcological modulation of KCs compared, say, to that for

sodium channels, is still in relative infancy. Current industrial research

centres around the su lphonylureas as an tid iabetics , and derivatives of

sotalol, a mixed class I l/class III antiarrhythm ic whose class III effect

seems to reside in the D-isomer. It seems likely, however, that as the

architec ture o f potassium channels becomes clearer, drug design should

become more sophisticated and produce a range of modulators that reflects

the diversity and pharmacological importance of the channels.

47


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(61

(62

(63

(64

(65

(66(67

(68

(69

(70

(71

(72

(73

(74

(75

(76

(77

(78

(79

Habermann, E. Science 1972 , 7 77, 314.

Habermann, E. Naunym Schm iedeberg 's Arch. Pharm acol. 1 9 7 7 , 3 0 0 ,

189.

Banks, B.; Brown, C.; Burgess, G. M.; Claret, M.; Cocks, T. M.; Jenkinson,

D. H. Nature 1979, 282, 415.

Burgess, G. M.; Clavet, M.; Jenkinson, D. H. J. Physiol. 1 9 8 1 ,5 7 7, 67.

Miller, C.; Moczydlowski, E.; Latorre, R.; Phillips, M. Nature 1985, 313,

316.

Hugues, M.; Duval, D.; Kitabgi, P.; Lazdunski, M.; Vincent, J. P. J. Biol.

Chem. 1982, 257, 2762.

Cook, N. S.; Haylett, D. G. J. Physiol. 1985, 358, 373.

Habermann, E.; Reiz, K. G. Biochem. Z. 1965, 343 , 192.

Shipolini, R.; Bradbury, A. P.; Callewaert, G. L.; Vemon, C. A. Chem.

Commun. 1967, 679.

Callewaert, G. L.; Shipolini, R.; Vemon, C. A. FEBS Lett. 1 9 6 8 ,7 , 111.

Vincent, J. P.; Schweitz, H.; Lazdunski, M. Biochem istry 1975, 14,

2521.

Granier, C.; Pedroso Muller, E.; Van Rietschoten, J. Eur. J. Biochem.

1978, 82, 293.

Cosand, W. C.; Merrifield, R. B. Proc. Natl. Acad. Sci. USA 1977, 74,

2771.

Demonchaux, P.; Ganellin, C. R.; Dunn, P. M.; Haylett, D. G.; Jenkinsef^

D. H. Eur. J. Med. Chem. 1991, 26, 915.

Labbé-Jullié, C.; Granier, C.; Albericio, P.; Defendini, M.-L.; Ceard, B.;

Rochat, H.; Van Rietschoten, J. Eur. J. Biochem. 1991, 196, 639.

Bystrov, V. P.; Okhanov, V. V.; Miroshnikov, A. P.; Ovchinnikov, Y. A.

FEBS.Lett. 1980 ,779 , 113.

Habermann, E.; Pisher, K. A dvances In C ytopharm acology, Raven

Press: NY, 1979.

Miroshnikov, A. L; Elyakova, E. G.; Kudelin, A. B.; Senyavina, A. B.

Bioor g. Khim. 1978, 4, 1022.

Bystrov, V. P.; Arseniev, A. S.; Gavrilov, Y. D. J. Magn. Res. 1978, 30,

151.

Englander, J. W.; Kallenbach, N. R. Q. Rev. Biophys. 1984, 16, 521.

50


(8 0 ) Hider,

(8 1 ) Pease,

(8 2 ) Hoshi,

51

Chapter 2

Results and Discussion

Re s u l t s a n d D i s c u s s i o n

2. Results And Discussion

T he m o d e s t s u c c e s s o f 3 , 6 - b i s ( p i p e r i d o m e t h y l ) p h e n a n t h r e n e

dimethiodide as an apamin antagonist led us to wonder how the structure of

the compound could be altered to improve its binding affinity. The two most

obvious ways in which modifications could be achieved were by changing:

(i) the substitu ted amine; (ii) the spacing fram ew ork . The envisaged

synthesis (scheme 2 . 1 ) of analogues of 23 made the former a simple task

since this merely required the use of different amines in the S n 2 reaction

in the penultimate stage of the synthesis whilst the la tte r required the

availability of a number of dimethylated aromatic hydrocarbons.

PN-Br

bMe ■Me BrH jC-f-V

2 8 2 9

RlNHzC-\ CHjI +

-j|— CH^NR; - R 2N(CH3)H2C t - J —CH2(CH3)NR2

^ 21'

3 0 3 1

S c h e m e 2.1

These are, generally speaking , quite rare and w here com m erc ia lly

ava ilab le are often p roh ib itive ly expensive , so it was necessa ry to

synthesise the majority of those that were needed. The synthetic routes to

such compounds are broadly the same as those used to obtain the parent

hydrocarbons, but with the additional problem of reg iospecific ity of the

m ethyl groups.

2.1 R o u te s To D im e th y la te d A r o m a t i c H y d r o c a r b o n s

The means by which these are obtained fall usually into one of two

categories: cyclodehydration of ortho-substi tu ted deriva tives (3 2 , scheme

2 .2 . 1 ) or photolytically-induced cyclisation of suitably substitu ted acyclic

precursors (35 , scheme 2 .2 .2 ) .

53

4 - R

Re su l t s a n d D i s c u s s i o n

Rt—

X = COOH, CHO 3 2

R

R

S c h e m e 2 .2 .1

hv/I]

S c h e m e 2 .2 .2

2 .1 .1 D i m e t h y l p h e n a n t h r e n e s

Early investiga tions into the synthesis of phenanthrenes were prom pted

by the need to identify, by comparison with authentic samples, certain

hydrocarbons obtained by the dehydrogenation of bile acid; these were

predicted, on the basis of of absorption spectra and X-ray crystallographic

m e a s u re m e n ts , to be d e r iv a t iv e s o f p h e n a n th r e n e ^ . B a rd h am and

S e n g u p t a ^ d e ve loped a syn thes is based on e th y lc y c lo h e x a n o n e -2 -

carboxylate 37 (scheme 2 .3 ) .

C0 2 Et

O

EtOzC

(i) K (s)/C6Hô

^ (ii)37

Br

(i) 10% KOH/A

(ii) HVH2O

(iii) H 2O38

Na/Et 2O

= / H2O

P20s/A/6mm Hg = \ Se, 300-320'C

41

Scheme 2.3

42

The potassium enolate of this was reacted with p-phenylethyl bromide, then

hydrolysed to the cyclohexanone 3 $ . Reduction with sodium followed by

cyclisation with phosphorus pentoxide and dehydrogenation with selenium

gave phenan threne 42 in unstated yield. Using appropria te ly substituted

54

R e su l t s a n d D i s c u s s i o n

m a te r i a l s , they w ere a lso ab le to s y n th e s i s e 1,4- and 1,7-

d im e thy lphenan th rene . By using a s im ila r but in d ep en d en tly derived

route , H aw orth and co-w orkers^ extended this to include all the 9,10-

unsubstituted analogues with the exception of the 3,5- and 4,5-derivatives,

but observed a troublesome shift of the methyl group between positions 4

and 1 during the dehydrogenation of some of the compounds.

3 ,6 - D im e th y lp h e n a n th r e n e 4 4 was synthesised by Sengupta and

C h a t t e r j e e ^ via the ca ta ly t ic d e h y d ro g e n a tio n o f 7 -m e th y l - l ,2 ,3 ,4-

t e t r a h y d r o n a p h th a l e n e - 2 - s p i r o ( 3 ’- m e th y lc y c lo p e n ta n e ) 4 3 (scheme 2 .4 ) .

Me.Me

43

Pt/C(cat.)

300-310 'C 36%

S c h e m e 2 .4

2,6- and 2,7-Dimethylanthracene were also formed as side products but in

yields too low to make separation viable. Later, Sengupta and co-workers^

synthesised the same phenanthrene by an alternative route (scheme 2 .5 ) .

Me Me0

Xylene.0

OMe Me

46 4745

Me

10% Pd/C

300-3 20“C

Me44

(i) KOH

(ii) sodalime

Me

S c h e m e 2 .5

4 ,5 -d im e th y lp h e n a n th re n e 54 is of interest since twisting of the sp^

framework can be achieved by alkyl substitution at these positions and 4,5-

d isubstitu ted phenanthrenes are the smallest polycyclic m olecules with an

enforced helical structure; if the barrier to racém isation is sufficiently

high, the compounds may be resolved. The strain on the framework is one

55

Re s ul t s a n d D i s c us s io n

of the reasons why this phenanthrene was the last of its family to be

s y n t h e s i s e d ^ ; Newman and W hitehouse ’s syn thes is s ta r ted with the

o z o n o ly s is o f py rene 49 then p roceeded as show n to g ive the

phenan th rene 54 in reasonable yield (Scheme 2 .6 ) .

L iA lW u / = ( > =

EtOH,

H+(cat.) 76% C2H5O

P, HI

165°CH3C Œ 3

CgHg, EhO ^90% HOH2C CH2OH 90%

5 2

S c h e m e 2.6

4,5-Dimethylphenanthrene was also prepared in low yield by Frim and co-

w o r k e r s ^ ’ using a method which is now the most com m only used to

prepare phenanthrenes , i .e ., formation of a stilbene by the Wittig reaction

of an aldehyde and the ylide derived from the phosphonium salt of a benzyl

halide, followed by photocyclisation (scheme 2 .7 ) .

PPh r Br

(i)n-BuLi, THF

( T o i 2h. Me

10%

C-C(3Hi2» I2» h v /==■

20-50%

56 Me

Scheme 2.7

Me Me

Couture and co-w orkers^ used a directly analogous method to prepare 3,6-

and 1,8-dim ethylphenanthrene, the latter in relatively m odest yield (45%)

which they attributed to the presence of the two m ethyl groups in the

ortho pos it ions in the parent s tilbene. This m ethod has also been

e x te n s iv e ly used to p repare 3 ,6 -d im ethy l p h e n a n th re n e in y ie ld s o f

between 46 and 819&10.11 and its use is now preferred since it is quite

general and cyclisation is successful with stilbenes bearing halo, methoxy,

trifluoromethyl, phenyl and carboxy substituents (though not nitro, acetyl

56

R e s u l t s a n d D i s c u s s i o n

or d i m e t h y l a m i n o ) ^ 2 The general procedure for the photoirradiation step

was e s ta b l ish e d by M allo ry who found that the most sa tisfac tory

conditions were using 0.01 moles of the stilbene dissolved in 1 litre of

cyclohexane under an atmosphere of air and with iodine as oxidant.

2 . 1 . 2 D i m e t h y l a n t h r a c e n e s

D im e th y la n th ra c e n e s w ere h is to r ic a l ly sy n th e s ise d by F r ie d e l -C ra f ts

reactions of toluene with aluminium chloride and various alkyl or benzyl

halides but most of these syntheses resulted in isom eric m i x t u r e s ^ T h e

first workers to devise a specific synthesis o f dimethylanthracenes by

the use of a Friedel-Crafts approach were Morgan and Coulson^^. Their

route to 2,6- and 2,7-dim ethylanthracene (scheme 2 .8 ) was established in

order to obtain reference samples of the two h y d r o c a r b o n s after studies

on the constituents of low-boiling tar and also to settle the confusion

surrounding the structures of products of earlier syntheses.

Me57(a) R=3-Me 57(b) R=4-Me

Me

AICI3 .CS2

82%Rir

58Me 'Me

59(a) R = 4-Me 59(b) R = 5-Me

6-12 hrs r4 -

60(a) R=6-Me 60(b) R=7-Me

Scheme 2.8

Thus, 2 ,4 ,4 '- and 2 ,5 ,4 '- tr im ethylbenzophenone 59 were obtained by

the reaction of p-to luoyl ch lo ride and the app rop ria te xylene. These

benzophenones were reported to cyclise with loss of w ater by prolonged

dry reflux to give the respective d imethylanthracene. The smoothness of

the reac tion was la ter con tes ted by Pepper and co -w orkers^ ^ , who

demonstrated that a large part of the crystalline precipitate observed upon

cooling of the reaction m ixture was in fact d im ethy lan th rone , though

reduc tion o f this com pound was easily effected . In fact, substitu ted

57

R e s u l t s a n d D i s c u s s i o n

an th racenes have often been synthesised by the red u c t io n o f the

co rre sp o n d in g an th rone or an th raqu inone but aga in , ea rly syn theses

were p lagued by ambiguity. In an early m ethod, for instance. Seer^ 4 obtained anthraquinones by the Friedel-C rafts condensa tion o f m -toluoyl

chloride 61 (scheme 2 .9 ) but he was not able to unequivocally assign the

structure o f the products until Morgan and Coulson characterised the two

anthraquinones formed by oxidation o f their anthracenes.

H3C COCl

61

AICI3. 130-140X

62

Scheme 2.9

M eth o d s fo r the red u c t io n o f the a p p ro p r ia te a n th ro n e or

anthraquinone include the classical Clemmensen m ethod for the reduction

of aromatic k e t o n e s ( s c h e m e 2 . 1 0 ).

63

Z n, H 2 O, HCl

A, 48hrs

Scheme 2.10

H . H

H H

This system is weak enough to give only the 9,10-dihydroanthracene 64 but

a modification by M artin^^, using an alkaline two-phase system, proceeds

to completion and is useful for compounds not appreciably soluble in the

acidic mixture above or molten at the reflux temperature (scheme 2 . 1 1 ).

65

Zn, N aO H , P hM e

A, 12hrs, 93%

Scheme 2.11

66

58

R e s u l t s a n d D i s c u s s io n

A m ong o th e r rea g e n ts tha t have been u sed is a lu m in iu m

tr is (cyc lohexy l) oxide which G aylord and S tepân^^ used to specifically

reduce a num ber of m ethylated anthraquinones, including both 2 ,6- and

2 ,7 -d im e th y lan th raq u in o n e (schem e 2 ,1 2 ) .

Me MeAl, HgCl 2, CeHiiOH

CCI 4(cat), A, 2h, 69%

60(b)

Scheme 2.12

A stra ightforw ard and apparently general m ethod was established

by C risw ell and Klandermann^®, the later part o f which simply required

re f lu x o f the in te rm e d ia te an th ro n e w ith so d iu m b o ro h y d r id e in

isopropanol (scheme 2 ,1 3 ) .

H H

H+, H2O, A

^ 6 CH3OH. NaBH4 Me

A, 88%

Me MePrOH, NaBH4

A, 24-36hrs

Scheme 2.13

a GH

HO H

60(b)

Klemm, Kohlik and Desai^^ obtained disubstituted anthraquinones by

the D ie ls -A lder reaction o f 4 -m ethy lbenzoqu inone 71 and isoprene 7 2 ,

followed by base-assisted dehydrogenation of the adduct (scheme 2 .1 4 ) .

(i) Eton, 75*C Me

-

72

(ii) 5% KOH, EtOH O 2, 46 hrs

Scheme 2.14

“rMe

59


This gave an isomeric mixture of 2,6- and 2,7-dim ethylanthraquinone from

which the isom ers were separa ted by f rac tiona l c ry s ta l l is a t io n from

ethanol. Reduction with zinc dust in ethanoic acid gave the respective

anthracenes, but the overall yield for the synthesis was very low.

1,5- and 1 ,8-D im ethylan thracenes 76 and have been formed by

the cyclodehydration of the appropriate o-benzoylbenzoic acid by Cristol

and Caspar^^ (scheme 2.15).

Me OH 73

C.H 2SO4, 95'C 3-4 hrs, 73%

O Me

Zn, C.NH3 50'C, 16 hrs

72%Me

Zn, C.NH3 50‘C, 2hrs

65% Me

Me 76 78

Scheme 2.15

The acid catalysed cyclisation of either of the benzoic acid derivatives led to

a good yield (the same, regardless of which starting material) of a

60


Me O Me OMe O

82 O Me81 O Me

Me OH Me CO

Me OMe OH

87 O83 O Me7 9 0 M e

Me O MeMe

O

Figure 2.1: The Hayashi Rearrangement

m ixture o f the two anthraquinones due to a H ayashi rearrangem ent^

(figure 2 .1 )

NH2 NH,NBS, DMF

91-95%

BrCHO

8 8 8 9

(i) NaNO 2 . H+

(ii) C H 2 =N 0 H

69%9 0

89%

HO H

Br Mb Mb

N aB H 4 (i) CuCNCF3 COOH

94%

(i) CuCN NMS, A, 4 hrs

9 2 (ii) NH 3 , H2 O 9 3 69%

CH O

— ' o c o “. . i ' c ô a “(i) NaOH. EtOH, A 0-20“C THF 69%(ii)H 2Û,H+ ^ 6 3

82%6 0 ( b )

S c h e m e 2 .1 6

61

R e su l t s a n d D i s c u s s io n

The two com pounds were separated by f rac tional c rys ta ll isa t ion from

ethanol prior to reduction with zinc dust in concentrated aqueous ammonia

s o lu t io n .

M ost recently , Lai and Peck^^ devised a syn thesis of 2 ,7 -d im ethy l

an th racene w hich is po ten t ia l ly genera l (schem e 2 . 1 6 ) , Again, the

a n th ra ce n e is ob ta ined from the an th rone , i t s e l f ob ta in ed by the

cyclodehydration of the carboxylic acid 94 . This is conceptually similar to

Cristal and Caspar’s method (see scheme 2 .1 5 , above) but represents an

improvement since that of the earlier workers relied on the availability of

a selectively substituted phthalic anhydride. The new method involves the

reaction of the Grignard reagent derived from p-chlorotoluene and 3-

bromotoluAlckhyJe 90 (easily obtained as shown) followed by dehydration and

conversion to, first, the nitrile 93 then the acid 9 4 . Yields in the synthesis

are good to excellent and though long, it could prove to have considerable

p o t e n t i a l .

A num ber of compounds are known to give d im ethylan thracenes

when pyrolysed under a variety of conditions. For instance, Errede and

C a s s i d y r e p o r t e d that 2 ,6-d im ethylan thracene 6 0 ( b ) was formed in

good y ield from the low -pressu re , fas t-f low py ro ly s is o f p - to ly l-p -

xy ly lm ethane 95 (scheme 2 .1 7 ) .

Me

Me95

Me 970°C,0.03s

lmmHg,55%

S c h e m e 2 .17

60(b)

Trahanovsky and Suker^^ observed that dimers ofo-quinodim ethane 9 6

gave su b s ti tu ted an th racenes under s im ila r cond it ions , bu t with an

interesting alteration of regiochem istry (scheme 2 .1 8 ) .

920“C

60(b)

S c h e m e 2 .1 8

62

R e s u l t s a n d D is c u s s io n

The conversion was found to be highly specific and indicated that the

dimer was not reverting to the monomeric state. S im ilarly , l ,2 ,4 ,5 -d i(3 -

m e th y lb e n z o )c y c lo h e p ta n e 97 was found to give 1, 8-dimethylanthracene

7 8 upon pyrolysis, but with a retention of re la tive s tereochem istry

(scheme 2 .1 9 ) .

2 7

Me Me

97

730’C

78

S c h e m e 2 .19

2.2 B e n z y l ic F u n c t i o n a l i s a t i o n O f A r o m a t i c H y d r o c a r b o n s

O ne of the easiest ways to functionalise benzylic carbon atoms is by

b ro m in a t io n . This has usua lly been ach ieved by rea c t io n o f the

hydrocarbon in one of two ways and there are many examples of both

th roughou t the literature . The first is the reaction with brom ine in

te t r a c h lo ro m e th a n e so lu t ion under h igh in te n s i ty i l lu m in a t io n . This

method has been used to prepare a number of derivatives such as 9,10-

b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e ^ ^ 9 9 and 9 ,1 0 - b i s ( b r o m o m e th y l ) -

anthracene 1 0 1 ^ ^ ’^® (scheme 2 . 2 0 ).

Br 2, CCI4

hv(200W sunlamp) 77%

Br 2 > CCI 4 , A benzoyl peroxide

99

9h, 500W tungsten lamp 85% 101

Scheme 2.20

63


Other compounds have been prepared in this way and it is a useful method

w here m u lt ib ro m i na tion is r e q u i re d or fo r c o m p o u n d s such as

t e t r a m e t h y l f l u o r a n t h e n e ^ 1 1 0 2 , where failure is observed through other

methods (scheme 2 . 2 1 ).

Complex mixtureNBS, e c u initiator ^

Me Me

MeMe

Br], CCI4 500W lamp

56%

103

Br

Scheme 2.21

The form ation of 9 ,10 -b is (b rom om ethy l)an th racene has also been

effected without the aid of ultraviolet i llum ination by H a u p t m a n n ^ and

Berner et a l^^ , who found that gentle heating of the reaction mixture was

merely required to give the p roduct (in unspecif ied yield) but this is

apparently unusual. In any case, the reaction proceeds via a free-radical

chain mechanism and the second major class of reactions differs in the

source and the means by which the radicals are genera ted , though the

mechanism of reaction is the same. N-Bromosuccinimide is now known to

generate a low, steady-state concentration of bromine which is available to

react as described. In the absence of an external energy source, a radical

initiator such as benzoyl peroxide is used and the whole carried out again

in a non-in teracting solvent such as tetrachlorom ethane. These conditions

have been used to prepare b is(b rom om ethy l)phenan threnes by a num ber

of workers.

___ NBS, CCI4 A, 4hrs

\ / \ — / AIBN, benzoyl peroxideMe 570/0 Br 104

Scheme 2.22

64


For instance, Staab and co-workers were the first group to synthesise 3,6-

b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e ^ '^ ’^^ 1 0 4 (scheme 2 .2 2 ) and in doing so,

established a method that was used, with small m odifications, by several

s u b s e q u e n t g roups in the sy n th es is of, am ong o th e r c om pounds ,

c y c l o p h a n e s ^ ^ , h e l ic e n e s ^ ^ , ch ira l p h e n a n th re n e d e r iv a t iv e s ^ ^ and

c o r o n e n e ^ ^ . Staab and co-workers carried out the reaction using a 1:1

mixture of benzoyl peroxide and aza-bis(isobutyronitrile), while the latter

in it ia to r alone was used to prepare 2, 7 -b is (b rom om ethy l)phenan th rene

1 0 6 from its bis-iodinated derivative by Jenny and co-workers'^® (schem e

2 .2 3 ) .

1 0 5

N B S , e c u , A

l e h r s , 5 5 % , A IB N

I Br1 0 6

= ' Br

Scheme 2.23

HOOC OOŒI

107 108NaBH4 , diglyme, BFgiEl 2 0 ,50°C,

3hrs, 17%

LiAlH 4 ,THF, Soxhlet, 24hrs

HD CH

109

MeaSiBr, CH C l 3 , 50“C, 8 hrs, 58%

110(b)

Scheme 2.24

Staab and co-workers'* I»'* also synthesised 2 ,7 -b is (b rom om ethy l)-

a n th r a c e n e 1 1 0 ( b ) bu t by a ra the r d i ffe ren t m ethod , p roceed ing

a l te rn a t iv e ly v i a the methyl ester of the carboxylic acid 1 0 8 or the

65


an lhroned icarboxylic acid 107 through the b is(hydroxym ethy l) compound

109 (scheme 2 .2 4 ) .

O th e r ro u te s to b is (b ro m o m e th y l ) d e r iv a t iv e s o f a ro m a tic

h y d r o c a r b o n s in c lu d e tha t o f G o ld e n ^ ^ who o b ta in e d 9 ,1 0 -b is -

( b ro m o m e th y l) a n th ra c e n e 101 by the reaction of 9 ,10-bis(chlorom ethyl)-

a n th ra c e n e 1 1 1 with sodium bromide in refluxing acetone to give the

product in good yield (scheme 2 .2 5 ) .

NaBr, Ac ] 0

A, 82%

S c h e m e 2 .25

i n 10 1

The bis(hydroxymethyl) derivative has also been used to produce a variety

o f dibrom inated aromatic compounds (scheme 2 .2 6 ) .

CH2OH CH2OH CH2Br CH2BrC 3 H5 N, PBrj

THF, 2hrs, rt

32% HBr, AcOH

A, 20min, 94%

1 1 3

1 1 4 ^HO

i i )

HOOK1 1 5

HOH2 Ç ÇH2 OH

( iv )

1 1 7

C 5 H 5 N, PBrg, CgHg

50°C, 2hrs, 85%

Br Br1 1 6

BrH2C CH2Br

C5 H5 N. PBrj.CgHg

60°C, 2hrs, 87%

References: (i) 44, 45, 46 (ii) 47; (iii) 48; (iv) 4 9

S c h e m e 2 . 2 6

1 1 8

66


2.3 S y n t h e s i s O f Dim e t h y l - p h e n a n t h r e n e s and - a n t h r a c e n e s

Our initia l targets were 3 ,6-dim ethylphenanthrene, the spacing group in

our lead m olecule , and 2,6- and 2 ,7 -d im ethylan thracene . As discussed,

e s tab lished syntheses ex isted for all three com pounds and these were

followed in the first instance.

2 . 3 . 1 P h e n a n t h r e n e s

M allo ry 's m ethod^^ for the preparation of 3 ,6-dim ethylphenanthrene from

the photolytically induced cyclisation of r r a n j - 4 ,4 ' - d im e th y l s t i l b e n e was

used initially. Plentiful supplies of the stilbene were available but, in any

case, it is easily prepared from p-xylene and p-tolualdehyde in good yield.

The yields for the cyclisation, typically carried out on 1.0 g of starting

m aterial in 1 litre of cyclohexane, were satisfactory at around 35% after

recrysta llisa tion from ethanol, but much less than the reported yield of

8 1 % ^ . A modification to the earlier procedure was recently reported by Liu

and Katz^®, developed as part of a general route to [5]-helicenes^ ^ . They

found that cyclisation of molecules contain ing benzylic e ther functions

was spoilt by elimination of the ethers to give alkenes because of the HI

genera ted . Addition of p ropylene oxide to consum e the nascent acid

p r e v e n te d e l im in a t io n w ith the o n ly r e q u i r e m e n t b e in g th a t

stoichiometric amounts of iodine needed to be used since air, in the absence

of the sequestered iodine, was an ineffective oxidant. Interestingly, yields

were found to be improved even for m olecules w ithout benzylic ether

functions and, using this method, a crude yield of 95% was claimed for the

cyclisation of t rans -4 , 4 '-dimethylstilbene. No significant increase in yield

was observed, however, using this methodology when attempts were made

by us to repeat it, possibly due to the presence of small amounts of dissolved

oxygen , and since M allo ry 's m ethod had the advan tage of re la tive

insensitivity to changes in reaction conditions, its use was preferred.

The H nm r spectrum of 3 ,6 -d im ethy lphenan th rene (figure 2 .2 )

illustrates many of the features characteristic of the spectra of polycyclic

arom atic com pounds and their m ethylated derivatives. All the arom atic

protons are well resolved, even at 60 MHz, and a number of broadenings

and splittings are observed.

67


H9. HIO

HI. H8H4, H5

CDCI3

H2, H7

Q

Figure 2.2

The spectra of such compounds are a mixture o f ortho, meta and para

coupling and long range coupling transmitted through the ring. Alkylated

derivatives also present the possibility of benzylic coupling which, because

o f the much larger differences in the chemical shifts o f the protons

involved, tends to be better documented and more obvious than coupling

betw een ring pro tons. How ever, the fou r-bond coup ling observed in

protons 2 and 7 of 3 ,6-dimethylphenanthrene is probably due to coupling

with protons 4 and 5 respectively which, though themselves singlets, are

observed to be broadened. This is presumably due to a combination of

unreso lved meta, para, benzylic and possibly long-range coupling. Any

multi-bond coupling in protons 9 and 10 is not observed, even at 400 MHz,

and the signal appears as a singlet.

The mass spectrum of this compound is also typical of compounds of

this type and polycyclic aromatics in general. The m olecular ion peak at

m/z 206 forms the baseline and strong peaks are observed at m/z 191 and

176 corresponding to loss of the methyl groups. Peaks at m/z 165 and 150

due to loss of ethylene from each of these ions are also evident as well as

minor peaks resulting from cleavage and further loss of e thene from these

io n s .

The e lec tron ic spectra of po lynuclear a rom atic com pounds are

usually complicated and for this reason they are useful as fingerprints for

the identification of unknown compounds of this type, such as are often

68


observed in the degradation of natural products. Furtherm ore, substituents,

regardless of their nature or position, have only a small effect on the

lineshape and position of the absorption maxima rela tive to the parent

hydrocarbon. This is i llustrated in figure 2 .3 w here three com pounds,

d i f fe r in g in the pos it ion and e lec tronega tiv ity o f th e ir subs ti tuen ts

produce spectra that are unm istakably phenan threno id in charac te r and

with only minor differences in the wavelength of m aximum absorption.

+ 1 . 00A

0 . 20 0 ( A / [I I V . )

+ 0 . 0 0 A

2 0 0 . 0 1 0 0 . 0 ( M M / D I V . >

N + O

V . V V A'%V',

V y y . y 10 0 . 0 ( NM/ DI V . )+ 2 .

+ ü .

600*!!0

NM ■5 0 0 . 0

Me

>Me

HM

F i g u r e 2 .3

69


The nex t step in our syn thes is invo lved fu n c t io n a l is a t io n o f the

phenanthrene. Of those methods with the hydrocarbon as substrate , that

using N -brom osucc in im ide seem ed the m ost a t trac tive because of the

apparent ease of reaction, good yields and the fact that, through its use, a

precedent existed for the formation of at least one of the target compounds.

A m odif ica tion of S taab 's m e t h o d ^ w a s em ployed in which catalytic

benzoyl peroxide was used as the sole radical initiator and this gave post

crystallisation yields typically around 75%, something of an improvem ent

on published yields of 5 6 ^ ^ ^ and 57%^"^’^^ . 3 ,6 - B i s ( b r o m o m e th y l ) -

phenanthrene was obtained as a white powder and gave spectra that were

entirely consistent with its structure. The H nmr spectrum has the same

profile as that of the parent phenanthrene except that there is a downfield

shift of 2 ppm for the alkyl protons and 0.5 ppm for the ortho ring protons

at positions 2 and 7 and 4 and 5. The mass spectrum gives the correct

pattern of isotope peaks corresponding to the molecular ion at m/z 364 and

for [M - Br]+ at m/z 283 and 285 with the predicted fragmentation pattern at

m/z 204 and below. The electronic spectrum also has the lineshape typical

o f phenanthrenes and the wavelength of maximum absorp tion appears at

258.5 nm compared to 253.5 nm in the unbrominated compound.

1,6 -D im e th y lp h e n a n th re n e 1 2 3 may be unequivocally synthesised

by the photocyclisation of r r a n 5 -2 ,4 ’-d im ethy ls t i lbene 1 2 2 using M allo ry’s

m e th o d ^ ^ .

Me Me Me

1 1 9 A, 2 hrs 1 2 0

PPh 3+B f

12 1

(i) Li, MeOH

( i i ) Me

Me h v , l 2, C6H .2

12-24 hrs

1 2 2 1 2 3

= benzoyl peroxide

S c h e m e 2.27

70


The latter compound was prepared as shown in scheme 2 .2 7 using the same

m eth o d o lo g y as that used to prepare the 4 ,4 ’-isom er: p -xy lene was

b ro m in a te d using N -b ro m o su cc in im id e (50% ) or b ro m in e under the

i llum ination of a sodium lamp (11%), then the triphenylphosphonium salt

form ed. Form ation of the stilbene proved to be a d isappoin ting ly low-

yielding reaction for reasons that are not clear but are presumably steric

in origin. Yields were insensitive to changes in reaction conditions such as

d ifferent bases (either Li/MeOH or Na/MeOH) or protracted reaction time

and reflux. Precipitation of the product was also difficult since this rarely

occurred spontaneously to any great extent, so the crude reaction mixtures

w ere co o le d to -78 °C. This p rov ided a m ix tu re o f s ti lbene and

t r ip h e n y lp h o s p h in e ox ide w hich was e as i ly s e p a ra te d by c o lu m n

chrom atography to give the product as white crystals in a yield of only

16%. The final stage o f the synthesis gave, a fte r chrom atography in

hexane, a 36% yield of the phenanthrene as shiny colourless plates; this

yield was not incidentally improved by the use of Liu and K atz’s procedure

in v o lv in g s to ic h io m e tr ic am ounts I2 and p ropylene oxide. The mass

spectrum is almost exactly as that for 3,6-isomer and the UV spectrum has

already been given in this chapter (see figure 2.3)

2 .3 .2 A n t h r a c e n e s

O f the many routes, established and putative, that exist to 2,6- and 2,7-

d im e th y la n th ra c e n e , M organ and C oulson 's m ethod^^ seemed the most

prom ising since, despite the relative crudeness of the reaction conditions,

the route was very short and the starting m aterials cheap and readily

available . M oreover, in contrast to other, earlier m ethods, this could be

used to synthesise both isomers in a manner that was both regiospecific

and po ten t ia l ly h igh-y ie ld ing . The in itia l step (see schem e 2 .8 ) , the

F r ie d e l -C ra f ts acy la tion o f xy lene w ith p - to luoy l c h lo r id e p roceeded

sm oothly to give 2,4,4 '-trim ethylbenzophenone as a pale yellow oil and

2 ,5 ,4 '- tr im ethy lbenzophenone as a low-melting yellow solid in yields of

64% and 77% respectively. The cyclisations proved to be much less facile

than the original paper suggests, however. The authors were alert to the

dange rs in h eren t in a techn ique as fo rc ing as dry ref lux o f the

benzophenones in air (when we carried out the experiment, a Woods metal

71


bath was used and temperatures as high as 360 °C were observed) and their

suggestion that the product be removed at in tervals from the cooled

reaction mixture proved both advantageous to the overall yield and simple

since the highly crystalline product was easily removed by filtration and

subsequent washing with E t2 0 to remove the more soluble benzophenone.

Extensive decomposition was observed after 12-15 hours' reflux and the

reaction was generally discontinued after three or four cycles of heating,

cooling, filtration and evaporation, but even at this stage, the yield was

much low er than reported. The brown solid obtained was purif ied by

vacuum sublimation giving bright yellow plates that mass spectral analysis

revealed to be a mixture of the anthracene 60 (m/z 206) and a compound

with a molecular mass of 223, presumably the anthrone 6 3 . The H nmr

spectrum showed 60 and 63 to be present in the ratio of approximately 3:7

respectively in a yield that, over many experiments, never exceeded 17%

(based on converted benzophenone). No mention was made of this in

Morgan and Coulson's original paper but it was subsequently repeated by

P e p p e r , H o w e ll and R ob inson^ who s u b je c te d 2 ,4 ,4 ' - t r im e th y l -

benzophenone to the reaction conditions and obtained the two compounds

in unspec if ied proportion . The anthracene was separab le by repeated

fractional crystallisation from ethanol, but the low overall yield made this

unattractive and wasteful. As discussed in section 2 .1 .2 , there are many

methods for the reduction of aromatic ketones, the most attractive of which

was that of Criswell and Klandermann^®, which simply required reflux of

the in term edia te anthrone with sodium borohydride in isopropanol. This

was there fo re carried out on the sublim ed, but u n separa ted reaction

mixtures of both compounds and gave, in each case, a quantitative crude

yield o f the anthracene. The two were purified by recrysta llisa tion from

ethano l and subsequen t colum n chrom atography using hexane as the

eluant g iv ing white, h ighly crysta lline products w ith the in tense blue

fluorescence characteristic o f anthracenes. The H nmr spectrum of each

compound is relatively simple because of the high degree o f symmetry of

the m olecules and again, all the signals are well resolved. Thus, the

spec trum of 2 ,6 -d im e th y lan th racen e 6 0 ( a ) appears as a pair of singlets

and a doublet o f doublets, the higher field pair of which, corresponding to

protons 3 and 7, are observed to be further split into doublets due to

coupling with the methyl groups. Like 3 ,6 -d im ethylphenanthrene 4 4 , the

72

R esu lts a n d D isc u s s io n

Other pair o f ortho protons in pos it ions 1 and 5, do not appear as a doublet ,

but are ob se r ve d to be broadened. Simi larly, the s ignal due to protons 1 and

8 o f 2 , 7 - d i m e t h y l a n t h r a c e n e 6 0 ( b ) is a broad s i n g l e t w i t h the ortho

protons 3 and 6 appearing as a doublet o f doublet s at 57 . 2 7 . Protons 9 and 10

h a v e b e e n a s s i g n e d p r e v i o u s l y in the l i t erature u s i n g n O e e x p e r i m e n t s .

T h e m a s s sp e c tr a o f both c o m p o u n d s are very s i m i l a r w i t h a s trong

m o le c u l a r ion peak and peaks at 191 ( [M - C H 3 ] + ), 178 and 89, whi l s t the

e l e c t r o n i c s p e c t r a e x h i b i t m a x i m u m a b s o r p t i o n at 2 5 5 . 5 nm for both

c o m p o u n d s .

B r o m i n a t i o n o f the two anthracenes was e f f e c t e d o n c e aga in by the

N - b r o m o s u c c i n i m i d e / b e n z o y l p e r o x i d e s y s t e m d e v i s e d by Staab ^^ and was

sa t i s f ac to ry , th ou gh the react ion was much l e s s s m o o th than that o b se r v e d

for the p h e n a n t h r e n e . A s d i s c u s s e d , 2 , 7 - b i s ( b r o m o m e t h y l ) a n t h r a c e n e has

b e e n m a d e b e f o r e by Staab^ ( s e e s c h e m e 2 . 2 4 , s e c t io n 2 . 2 ) but the

s y n t h e s i s w a s not ap p l i ca b le in this ca se s i n ce the b ro m in at ed c o m p o u n d

w a s o b t a i n e d from the d ie s t er and d i c a r b o x y l i c ac i d o f the anth rac ene ,

rather than the h yd ro carb on i t se l f . This was unf ortunate in v i e w o f the

g o o d y i e l d but S t a a b ’s a l t e r n a t iv e m e t h o d w a s s a t i s f a c t o r y und er the

c i r c u m s t a n c e s . Th e two c o m p o u n d s were thus obta i ned , af ter cry s t a l l i s a t i on

f rom a var iety o f so l ve nts , as h i g h- m el t i n g y e l l o w n e e d le s in y i e l d s o f abou!-

$0 % . O n c e aga in , l i tt le ch a n g e is obser ved in the H nmr spectra o f the

c o m p o u n d s c o m p a r e d to the ir p recurso rs , apart f r o m a d o w n f i e l d sh i f t

more n ot ic ab l e in those protons ortho to the subs t ituted m et hy l group. The

m a s s s p e c t r a g i v e s a t i s f a c t o r y i s o t o p e a b u n d a n c e p e a k s for the t w o

b r o m i n e a t o m s a n d are s i m i l a r to t h a t o b s e r v e d f o r 3 , 6 -

b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e . F i n a l l y , the U V s p e c t r a g a v e m a x i m u m

absorpt ion for the 2 , 6 - i s o m e r as 265 .5 nm and 2 62 .5 nm for the 2 , 7 - i s o m e r

w i t h the c h a r a c t e r i s t i c l i n e s h a p e .

T h e sy m m e tr ic a l 1,5- and 1, 8 - d im et hy la n t hr ac en es w e r e a l s o se le c te d

as ta rget s for the in it ial s ta ge o f the s y n t h e s e s s i n c e th e y p r o v id e d an

appropr ia te sp a c i n g for the two methy l groups (e s t im ated to be 10.0 Â and

7 .3 Â r e s p e c t i v e l y co m p a r e d to 9 .5 Â for 3 , 6 - d i m e t h y l p h e n a n t h r e n e ) and

w e r e c o n v e n i e n t l y prepared in Cri s tol and C a s p a r ’s d i v e r g e n t syn th es i s^ “

d e s c r i b e d in s c h e m e 2 .1 5 . T h e H a y a s h i r e ar r a ng e m e nt in the c y c l i s a t i o n

73

R e su l ts a n d D is c u s s io n

m eant tha t both an th raqu inones could be p rep a re d from a s ingle

precursor, 6 -m ethy l-2 -(2 -m ethy lbenzoyl)benzo ic acid and this i tse lf was

prepared in an apparently straightforward three-step procedure based on

work with benzoylbenzoic acid esters by Newman and co-workers^

( s c h e m e 2.28).

C.H2SO4

1 2 4

Me

E^O, A, 4 hrs

O1 2 6

•5°C

Me o Me ^ M g B rV . C6H6. X

E^O, A

12 7 7 3 0 Me

S c h e m e 2.28

When attempts were made by us to repeat the synthesis, the initial Diels-

A lder reaction o f 2 -m ethylfuran 1 2 4 with maleic anhydride gave the

adduc t 6 -m e th y l-2 ,4 -d io x o -3 ,1 0 -d io x a t r ic y c lo [5 ,2 , l ,0 ^ ’^ ]dec-7-ene 126 as a

low-melting, pale pink powder and was routinely accomplished in yields of

70% or grea ter, but the subsequent dehydra tion step p roved to be

c o n s id e ra b ly m ore p ro b le m a t ic . The au tho rs e v id e n t ly e x p e r ie n c e d

difficulty also and report that among the usual reagents found unsuitable

for this step were hydrogen ch loride gas, phospho rus pen tox ide in

benzene, sodium sulphate, acetyl chloride and acetic anhydride whilst the

only reagent found suitable was 90% sulphuric acid. We found that the

temperature of the reaction and thus the degree of agitation of the mixture

was critical and established a slightly modified procedure in which rather

than a ttem pt isolation of the product by fil tra tion o f the neutra lised

reaction mixture (this often did not occur, even despite strong basification)

the product was extracted into chloroform and evaporated. This gave the

product as a dark oil or an unstable brown solid which could be crystallised

from propan-2-ol to give the anhydride as large, white needles, but yields

were never impressive, rarely exceeding 10% from the adduct.

74


Formation of the acid 73 was accomplished by the addition of the

Grignard reagent derived from o-bromotoluene to an ethereal solution of

the phthalic anhydride giving the benzoic acid as a pale yellow powder in

yields of the order of those reported. Cristol and C aspar’s method for the

cyc lodehydration o f this compound involved heating it in concentra ted

sulphuric acid for at 95 °C for four hours, somewhat stringent conditions

that m erely resulted in the form ation of an in trac tab le oil when we

rep ea ted the experim en t, desp ite the a u th o rs ’ c la im s tha t the two

anthraquinones were stable to the reaction conditions. With the failure of

this reaction, however, it was decided to discontinue attempts to make the

anthracenes since the modest yield at all but the first stage of the synthesis

and the d if f icu l ty in rep l ica t in g e x p e r im e n ta l p ro c e d u re m ade it

uneconomical in view of the limited time available.

2.4 O th e r H y d r o c a r b o n s

r e a c " .

t i m e /

h o u r s

y i e l d /

%

^ J h - h /

Hz

" Jr - h /

Hz

5c H2 ^ m a x /

n m

12 8

2 68 8.2 - 4.51 322.0

1 2 9 V 2 13 8.2 -

4 .51 -H l

4.61-H14 313.5

1 3 01 36 8.3 1.7 4.64 227.0

' O X . .1 3 1

2 51 - - 4.48 218.0

y ^ ^

B r - ^ ^ 13 23 41

9.8

(H 7) -

4 .79-HI"

5 .0 2 -H l ' 256.5

Values fo r are given fo r protons ortho to the methylene group and four-bond coupling o f theseprotons to the ring protons where applicable. For prOton assignments, see experimental section.

Table 2.1

75

R e su l t s a n d D is c u s s io n

O f the remaining four dimethylated hydrocarbons which were used,

two had already been synthesised “en route” to the two phenanthrenes,v/z.

t r a n s - 4 ,4 ' - and rraAZ5-2,4’-d im ethylstiIbene and the rem ain ing pair, 2,6-

d im ethylnaphthalene and p-xylene were com m ercially available. All were

brom inated, with varying degrees of success, using S taab ’s system^'^ and

the yields and reaction time, together with selected spectral data are given

in table 2.1 above.

2,5 Formation O f Amines

2.5 .1 D e r iv a t iv es O f P ip e r id in e , P y r r o l id in e and M o r p h o l in e

As ind ica ted , the next stage in the synthesis o f the b is-quaternary

an a lo g u es of 3 ,6 -b is (p ipe r idom ethy l)phenan th rene d im e th io d id e involved

the reaction of the b is(bromom ethyl) hydrocarbons with various amines.

F o llow ing the earlier synthesis of the p iper ido -substi tu ted phenanthrene

we attempted in the first instance to make a series based on substitution

onto the same skeleton of a number of cyclic amines chosen because of the

h igh crysta llin ity that their semirigidity would hopefully confer on the

product and also because of their structural similarity to portions of the

neurom uscular blockers described in Chapter 1. The conditions employed

in itia lly involved stirring the dibromide with sto ich iom etric amounts of

amine in tetrahydrofuran, but after reaction under these conditions for a

day, the reaction was observed to have turned a dark-brown colour and

work-up produced an oil in the complicated H nmr spectrum of which,

none of the desired amine could be detected. Though it is undesirable to use

an excess of amine in reactions of this kind because, of the risk of competing

side-reactions, an attempt was made using the dibromide and a large excess

of base to determine its effect on the reaction, but once again, only an

in tractable brown oil was produced; this, and the earlier result were the

same regardless of the amine used. However, upon a change of solvent to

ethanol and a reduction in reaction time, work-up again produced a dark-

brown oil. Reverse phase HPLC revealed how ever that this consisted

primarily of a single compound. Moreover, the H nmr spectrum indicated

that this was the bis-aminated phenanthrene. Since the oil would not yield

76


to c ry s ta ll isa t ion or tr i tu ra tion with a num ber o f so lvents includ ing

ethanol, petroleum spirit and chloroform and would not run on silica gel,

the problem remained of isolating the compound in crystalline form. This

was eventually solved by establishment of the procedure detailed in the

ex p e r im en ta l section: a fte r reac tion , the e thano l was rem oved then

chloroform or ether added to the crude reaction mixture. This was extracted

with 10% HCl(aq) to remove the basic product and any salt that may have

form ed and these extracts made alkaline with aqueous amm onia. The

precip ita te thus formed was exhaustively extracted into ether, dried and

evaporated to give a clear, usually slightly coloured oil and this was then

seeded or triturated to give the product as an off-white or yellow powder.

This procedure produced most of the compounds u ltim ately synthesised

with occasional modifications that are detailed as they were found to be

n e c e s s a r y .

u c ;1 3 3 1 3 4 1 3 5

F i g u r e 2.4

The f irs t three compounds to be thus form ed were 3 ,6 -b is (pyrro lido-

m e th y l ) - , 3 , 6 - b i s ( p ip e r id o m e th y l ) - and 3 ,6 - b i s ( m o r p h o l i n o m e t h y l ) -

p h e n a n th re n e (133, 134, 135, figure 2 .4 ) in yields which were initially

found to be modest but improved with repetition. The three compounds also

gave spectral data completely in accord with the given structures and show

no signs of either monosubstitution or the presence of basic side products.

Thus, the H nmr spectra of the three are midway in character between the

d ib rom ide and the parent phenan threne and 4 -bond coup ling to the

b&Y pro(;b'AS is observed in the ortho protons of positions 2 and 7 in all.

The mass spectra gave molecular ion peaks and the expected fragmentation

pattern corresponding to the loss of the two substituents. Two points are

worthy of note. Firstly, the molecular ion of compound 135 appears at m/z

77


377 corresponding to [M + H]+. This feature was found to be common to all

the m o rpho lino -subs ti tu ted com pounds subsequen tly syn thes ised and is

rem arkab le not only because of the unpred ic tab le occurrence o f this

phenom enon with the other analogues, but also because a com parison of

the pK a values o f the con juga te acids of the am ines revea ls the

m orpho lin ium ion to be considerably m ore acidic than that fo r the

remaining pair of compounds (Table 2 .2 ) .

pKa

m o r p h o l in i u m ion 8.330

p i p e r i d in i u m ion 11.123

p y r r o l i d i n i u m ion 11.270

From “T he C R C H andbook O f C hem istry A n d P hysics”, 5 9 th ed ., 1 9 7 8

T a b l e 2 .2

Secondly, besides the peak one would expect from the sequential loss of the

substituents at m/z 204, there is a peak of almost equal intensity two units

h ig h e r , c o r re s p o n d in g to [phenanthrene]"*". This is perhaps due to a

parallel decomposition pathway in which a hydrogen atom is transferred

from the protonated amine onto the adjacent carbon atom, prior to the loss

of the amine itself. The electronic spectra of the three compounds again

support the structure and each exhibit essentially the same wavelength of

maximum absorption (255.0 nm for compounds 134 and 1 3 5 and 254.0 nm

for compound 1 3 3 ) .

The piperido-, pyrrolido- and m orpholino-methyl derivatives o f the

two anthracenes were the next targets and the synthesis of most of these

was accomplished with little trouble in yields that were good to very good

u s in g the s tanda rd m eth o d o lo g y . D uring the p re p a ra t io n o f 2 ,7-

b is (p i p e r i do m e th y l) a n th r a c e n e 1 4 4 however, fine, w hite needles were

observed to precipitate from the reaction mixture. These were acid-soluble,

so no fu ther work-up was perform ed on the crysta ls w hich spectra l

analysis revealed to be the desired diamine. Otherwise, the compounds were

obtained as off-white powders with spectral data in accord with the given

78


s t r u c tu r e s . In p a r t ic u la r , the H nmr sp ec tra o f a ll bu t 2 ,6-

b i s ( p y r r o l id o m e th y l ) a n t h r a c e n e 1 4 0 exhibit benzylic coupling. All six

compounds gave mass spectra where the molecular ion appears at [M -f- H]+

bu t the f rag m en ta tio n pa tte rns are o therw ise as ex p ec ted and the

e le c tro n ic sp e c tra all e xh ib i t the h igh ly c h a ra c te r is t ic an th ra ce n o id

lineshape (figure 2 .5 ) .

5 0 A

)

A0M M

1 0 0 . 0 ( N M D I U . > 0 0L* 0 0 . 0

F igu re 2.5: Electronic Spectrum Of Compound 154 , Show ing A nthracene

L i n e s h a p e

The wavelengths of maximum absorption are given in table 2 . 3

along with other, selected spectral data.

79

Results and D iscussion

^max nm ^Jh-h/H z "Jh-h/H z 5cH2

OXCClO 258.0 8.7 1.4 3.64

138 (139)(260.0) (8.7) - (4.79)

CTCCLO 258.0 8.6 - 3.84

140 (141)(258.0) (9.2) (1.3) 4.77

oCJXCCLnC? 258.0 8.7 1.2 3.67

142 (143)(260.5) (8.7) - (4.89)

a ^ x x r o 261.5 9.1 1.3 3.71

144 (145)(260.0) (8.8) - (4.93)

a x O T o 262.0 8.5 1.2 3.87

146 (147)(260.5) (8.8) - (4.93)

oOf'XxXr'O 256.0 8.7 1.5 3.71

148 (149)(259.0) (8.3) - (4.89)

r f h 2 5 4 .0 8.1 1.5 3.88

(255.0) (8.1) - (4.35)

\ _ / ^ 134 (23)

2 5 5 .0 7.9 - 3.77

(255.0) (8.0) - (4.85)

0 0 ^ 135 (137)

255.5 9.1 1.4 3.76

(255.5) (8.1) - (4.46)

Figures in brackets refer to di-N-methylated analogue, iso la ted as diiodide.

Table 2.3

2.5.2 Q uatern i sa t ion O f A m ines

I o d o m é t h y l a t i o n o f th e se c o m p o u n d s w a s n e c e s s a r y in or de r to rend er the

n i t r o g e n s q u a t er na r y l i k e t h o s e o f the n e u r o m u s c u l a r b l o c k e r s w e w e r e

a i m i n g to m i m i c and a l so to c o n f e r s o lu b i l i t y in the a s s a y by w h i c h they

w e r e b e i n g tes ted . C lea r l y , wi th a react ion as e s t a b l i s h e d as q ua te r n i sa t io n

80


of a nitrogen atom, many methods and reagents exist but the method of

S ten lake et al using iodom ethane or m ethyl m ethane-su lphona te in

ace ton itr ile was selected because of the superf ic ia l s im ilarity of the

com pounds that were used in this reference ( them selves neurom uscular

blocking agents) and the similarities of scale; we intended to carry out

reactions on 10-30 mg of material and the paper detailed precautions that

were necessary as a result. Thus, our compounds were stirred in 1-2 ml of

acetonitrile for a period of time that varied between 24 and 48 hours with a

la rge ex cess o f io d o m e th a n e . The q u a te rn is e d co m p o u n d u sua lly

precipitated from this mixture, often after only 10 minutes, but in any case,

the product was isolated by pouring into dry ether which gave the products

as flocculent white or pale-yellow precipitates which were then isolated by

filtration in yields that were as high as 91% but more often around 50%.

These h igh -m elting salts were com plete ly unde tec tab le using standard

e lec tron im pact mass spectrom etry techniques but y ie lded spectra that

exh ib i ted certa in charac te r is t ic features under fast atom bom bardm ent

conditions. Thus, the molecular ion was observed as [M + I]+ where M refers

to the respective dication (under this nom enclature, the indicated charge

refers to the overall charge of the ion and not to a charge in excess of that

already on M) , then a small or non-existent peak due to M+ and peaks due

to loss of a methyl group, loss of the amine and loss of the two together. The

unsubstituted hydrocarbon framework was also seen as strong peak at m/z

204 and many of the compounds exhibited a prominent peak in this region,

corresponding to No conditions were found under which the diiodide

could be observed or the molecular ion made more prominent, but the

sp e c tra w ere n o n e th e less su f f ic ie n t ly c h a ra c te r i s t ic . The e le c tro n ic

spectra are, for the most part, virtually ind istinguishable from those of

their unquatern ised precursors, retaining the lineshapes and exhibiting a

shift of at most 2 nm. The nmr spectra, on the other hand, illustrate the

effect that increasing the degree of substitution on the nitrogen atom (and

thereby restric ting inversion processes) has on the ring protons of the

amine; the rigid tetrahedral structure means that the equatorial and axial

protons of the heterocyclic ring are no longer equivalent and the signals

due to the |3-protons are now resolved into distinct multiplets for all except

the m eth iodide salts of 2 ,7 -b is (py rro lidom ethy l)an th racene 1 4 7 and 2,6-

b i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e 1 4 3 . For both, there is evidence of

81


d iffe ren tia t io n which none the less rem ains incom ple te , so the values

quoted for these two compounds in the experim ental section are for a

single multiplet due to all eight protons. Which of the signals was due to

which proton was solved by an nOe experiment: irradiation of the protons

of the methyl groups of compound 141 at 62.98 produced an enhancem ent

of the multiplet at 53.68 which, it is therefore assumed, belongs to the four

pyrrolidinyLprotons syn to the methyl group.

Otherwise, the nmr spectra of the compounds are similar to those of

their unquaternised precursors, though interestingly, most of them fail to

exhibit secondary splitting of the signal due to the ortho protons of the

aromatic ring. One can understand this in terms of the altered electronic

environment in the vicinity of the charged nitrogen atom, but the problem

is compounded by the fact that the extent of the splitting is often so small

that it is simply not fully resolved

2 .5 .3 D e r iv a t iv e s O f 2 , 5 - D i m e t h y l p y r r o l i d i n e

Thus synthesised, this first batch of compounds was submitted for testing

but, as d iscussed in the fo llowing chapter, the results represented no

improvement on any of our lead compounds. Given the structural similarity

of the three amines, substitution of an amine with different steric, rather

than electronic properties seemed the most likely way of influencing these

values w hilst s tay ing w ith in our o rig ina l b r ie f . The serend ip itous

d iscovery of 2 ,5-dim ethylpyrrolid ine and its subsequent substitution onto

the various hydrocarbons led us to just such a compound. Our sample, a

commercially available mixture, consisted of 64% of the cis-isomer and 36%

of the trans-isomer as determined by nmr analysis and comparison with a

pure sample of the cis compound. In the first instance, this amine was

reacted with 2 ,7 -b is(b rom om ethy l)an th racene but this was in itia lly not

without problems. Isolating the compound from the dark oil that was

routinely produced was found to be difficult and the product was eventually

obtained by precipita tion with the careful addition of water and the

removal and washing (with cold ethanol) of the product. Luckily, there

appears to have been a preference for the dibromide to react with the cis-

isomer of the amine since the H nmr spectrum of the product reveals the

product of substitution by this isomer to be six times more abundant than

82

Results and D iscussion

for the trans-isomer. Thus, the syn and anti protons of the pyrrolidine ring

appear as a pair of complex multiplets, the a-p ro tons as a broadened quartet

and the methyl groups as a dominating doublet with a smaller set of peaks

to the side of these corresponding to the less abundant isomer.

The quaternisation of this diamine illustrated a problem that was to

occur with a number of later compounds when it was found that after

pouring of the reaction mixture into ether in the usual way, a precipitate

was obtained but which, upon filtration, collapsed to an oil or a hard, glassy

solid. The problem seemed to be solved by allowing them to dry in a vacuum

so that if, after pouring into ether, the precipita tes were settled, either

through gravity or centrifugation, and the solvent decanted, they could be

obtained by evaporation of the remaining solvent in vacuo. O bv iously ,

because the solvent was largely ether, this had to be carried out with great

care, but the procedure was succesful for all those com pounds which

ex h ib i te d this problem . 2 ,7-Bis [ (2 ,5 -d im e th y l )p y r ro l id o m e th y l ] a n th racene

d im e th iod ide 1 5 3 was thus obtained as a yellow powder whose spectra

exhibited all the characteristics of its previous homologues.

Upon pharmacological testing, this compound was found to be some

twenty times more active than the lead phenanthrene derivative so we set

about the synthesis of a number of derivatives using this amine. The full

series of analogues made in the light of this observation is given in table

2 .4 . These were obtained generally without incident but a num ber of

amines could only be obtained as oils. It was found, after b r ie f

experimentation that they could be quaternised directly with methyl iodide

to give, in all cases, the salt as an off-white or pale-brown solid though the

problem of oiling-out discussed above was also observed for some products.

Prior to this however, when attempts to make the bis-substituted benzene,

1,4 -b is [ (2 ,5 -d im e th y l )p y r ro l id o m e th y l ]b e n z e n e 1 5 8 produced only a dark-

b row n oil, an a l te rn a t iv e p ro ce d u re was d e v ise d in w hich the

dibromoxylene was added to a stoichiometric solution of the pyrrolidine in

sodium methoxide and methanol. No improvement was noted, however and

the com pound, which gave satisfactory data was reacted as described.

S im i la r ly , t ra n s -4 ,4 '- b i s [ ( 2 ,5 -d im e th y l ) p y r r o l id o m e th y l ] s t i lb e n e 1 6 0 was

obtained as a brown oil by the dropwise addition of a solution of the lithium

salt of the pyrrolidine to the d ibrom ide but in satisfactory yield. All

compounds provided consistent spectral data and the pharm acological data

83


are discussed in the next chapter.

Yield/% ÔCH2 ^max

Me

150 (151)

4 8

(50)

8.1, 9.1

(9.8, 7.6)

4.05, 4.11

(4.80, 5.15)

2 5 8 . 5

( 2 5 6 . 5 )

Me Metnoccrü152 (153)

92( 12)

8 . 2

(8.6)

1.1 3 . 9 5

( 4 . 88 )

2 6 2 . 5

( 2 6 2 . 0 )

“trpco!Me

154 (155)

5 0

( 92)

8. 7

(8. 8)

1.6

( 1 . 3)

3 . 91

( 4 . 76)

2 5 8 .

( 2 6 0 . 0 )

Me Me

Me

156 (157)

17

(64)

8. 8

(8.2)

0 . 8 3 . 8 7

( 4 . 72 )

2 3 6 . 0

( 23 0 . 5 )

Me

158 (159)

59

(48)

3 . 7 6

( 4 . 58)

2 2 4 . 0

( 22 2 . 0 )

160 (161)

5 0

(38)

8 . 0

(7. 8)

3 . 7 8

( 4 . 53 )

3 1 4 . 0

( 31 6 . 5 )

Me

162 (163)

41

( 39)

7.5

(7.6) -

3.95, 4.03

(4.84, 5.04)

3 0 7 . 5

( 30 7 . 5 )

Figures in brackets refer to di-N-methylated analogue, isolated as diiodide.

T a b le 2.4

2.5.4 D eriva t ives O f D A BC O

It was of interest to us to alter the electronic properties of the compounds

in order to assess the effect on their binding properties and an obvious way

84


in which to do this was to in troduce more charge in the term inal

substituents; this seemed sensible in view of the high basicity of the

g u a n id in iu m groups of arginine (pKa for arginine is estimated to be about

12, rendering it h ighly charged at physio log ica l p H ^ ^ ) . F u r th e rm o re ,

dequalin ium , one of our lead compounds, not only contains a pair of

aromatic ring systems, but also has a pair of terminal amine groups which,

though not strictly basic, are likely to greatly alter the electronic profile of

the molecule. The amine we selected was l ,4 -d iaza -[2 .2 .2 ]-b icyc looc tane

(DABCO, figure 2.6) for several reasons.

F igure 2.6: DABCO

F irs t ly , the n i trogen a tom , exposed becau se o f the “ fo ld e d -b a c k ”

substituents , is rendered much more reactive than the equivalen t open-

chain or secondary amine and the consequent expected facility of the

substitu tion reaction was borne out in practice. A lso, the restric tions

placed on the molecule as a result of the rigid structure mean that the lone

pairs on the nitrogen atoms extend in opposite d irections to one another

and the electronic environment at one is unusually insensitive to reaction

at the other; this is further enhanced by the n itrogen atoms being

constrained to adopt a near-tetrahedral structure, m inim ising the effect of

substitution on overall molecular structure. The use of a tertiary amine also

obviates the need for a subsequent quaternisation reaction, an im portant

consideration in view of the small scale at which these syntheses were

carried out. There is also a precedent for the use o f the dibasic DABCO

molecule in the formation of pharmacologically active agents.

O+ ATA + / ---- \ V n R2

V N — ( CH 2 ) n — N— 'R2N-

1 6 5

F i g u r e 2 .7

85


Diederich and co-workers^^ prepared a series of tetracationic bis-DABCO

derivatives 1 6 5 of the form shown in figure 2.7 as carriers for nucleotide

5 ’-tr iphosphates - potential chain-term inating inh ib itors o f HIV reverse

transcriptase but hampered by poor cellular uptake. The DABCO derivatives

formed 1:1 complexes with the nucleotides and were found to exhibit the

correct l ipophilic ity to partition into the organic phase during transport

e x p e r i m e n t s

The conditions under which our syntheses were carried out were the

same as those for previous quatern isa tion reactions. Thus, when the

respective dibromide was stirred with an excess of DABCO (this was possible

becouse there was no risk of side-reactions, given the quaternary nature of

the product) then almost instantly, a precipitate was observed to form. The

reactions were nonetheless treated as previous substitu tions and heated

under gentle reflux for four hours in order to e lim inate the risk of

monosubstitution. It was not found necessary to pour the compounds into

ether since precipitates were observed in all cases after the given reaction

time and these were removed by filtration (with no attendant problems)

and washed with ether to give the products as white or off-white powders

in good to excellent yields (table 2 .5 ) .

Yield/% ^Jh-h/ H z "Jh-h/ H z 5cH2 ^max/ nm

S X X t S

1 6 6

92 - - 4 . 5 9 2 1 7 . 5

nS ^ ^ C O ^ nS "

1 6 7

69 8.4 - 4 . 7 8 2 3 1 . 0

1 6 8

96 8.8 1.3 4 . 7 5 2 6 2 . 0

1 6 9

4 1 8.9 - 4 . 8 6 25 9 . 5

1 7 0

58 - - 4 . 5 4 3 1 6 . 5

All compounds isolated as dibromide

T a b l e 2.5

86


The 1 H nmr spectra of the com pounds are re la tive ly sim ple since

quaternisation of the nitrogen atom does not give rise to diastereotopic

protons at the ot-carbon atom. Thus, the ring protons of the DABCO moiety

appear as a large pair of triplets, broadened due to their proximity to the

quaternised and the uncharged nitrogen atoms. Benzylic coupling is only

obse rved fo r the 2 ,6 -d im e thy lan th racene d e r iv a tiv e 1 6 8 but the nmr

spectra are otherwise as expected for the individual compounds. The mass

spectra are much the same in character as those for the methiodide salts of

the amines so that peaks are seen for [M + Br]'*’ in the appropriate ratio, a

negligible or absent molecular ion peak and peaks corresponding to the

sequential loss o f the two DABCO units. [DABCO]+ itself appears as a strong

peak at m/z 112 in all spectra and once again, [M^ + ] is observed for a

number of the compounds. Wavelengths of maximum absorption are given

in table 2 .5 .

The synthesis of the compounds 171 and 1 7 2 in figure 2 .8 gave a

pair of compounds which resem bled the nucleotide carriers of Diederich

and c o -w o rk e rs ^ ^ (figure 2 .7 ) . Carrying four charges, these m olecules

were therefore superficially similar t o the DABCO derivatives which, it is

a s s u m e d , will be protonated in vivo , but the substitu tion alters the

lipophilicity and steric profile around these extra charges.

Me +

17 2

F i g u r e 2.8

These com pounds were read i ly p repared by s t i r r in g l ,4 - b i s - ( l ,4 -

d iaza [2 .2 .2 ]b icyclooctano-m ethy l)benzene dibrom ide with iodom ethane and

4 - (b ro m o m e th y l ) to lu e n e (a-b rom o-p-xy lene) respectively for 24 hours in

acetonitrile. The starting material was observed to disappear into solution a

short while after the addition of the halide, followed by the precipitation of

87


the product. Isolation was as before, by pouring into ether and filtration'

giving the two compounds as white powders in very good yields. The high

degree o f symm etry of the m olecules and the ir m oie ties resu lts in

com paritively simple nmr spectra, but neither provides a particularly neat

mass spectrum, even under FAB conditions. Identification is made possible,

however, by the presence of fragmentation products

2.5.5 D er ivat ives O f Q u in u c l id in e

The extent to which the basic nitrogen atom within these compounds was

responsible for changes in binding efficiency could be assessed by the

synthesis of a set of homologues in which this atom was not present. The

appropria te base, l -a z a - [2 .2 .2]-b icyclooctane , or qu inuc l id ine , was thus

substituted for DABCO in the previous reaction and generated a new family

of compounds, illustrated with yields and appropriate spectral data in table

2 .6 . The synthesis of the compounds was less straightforward than before

because of problems during filtration and yields were generally lower, but

the spectral details were obviously very similar to those of the DABCO

d e r iv a t iv e s .

Yield/% ^Jh-h/H z ^Jh-h/H z SCH2 ^max

1 7 3

100 - - 4.48 217.0

0 ^1 0 0 :0 17 4

25 8.4 1.1 4.62 230.0

0-XCCX;gi17 5

7 8.9 1.1 4.71 262.5

17 6

10 8.8 - 4.89 267.5

1 7 7 +

64 8.8 1.3 4 .?4 255.0

T a b le 2.6

88


2.5.6 A t te m p te d S y n th e s i s O f D e r iv a t iv e s O f c / s - 2 ,6 - D im e th y I p ip e r id in e

2 ,6 -D im ethylp iperid ine may be purchased as its configura tiona lly

pure c i5-form and its substitution onto the hydrocarbon frameworks would

allow us to assess the effect that expansion of the heterocyclic ring would

have on the re la tive potency of the 2 ,5 -d im e th y lp y rro l id o -su b s t i tu ted

compounds. The extra steric strain associated with the a -b ran ch in g on the

larger ring meant that when the reactions were carried out, a h igher

boiling solvent was used, in this case, propan-2-ol. Previously, a number of

u nsuccess fu l a ttempts had been made to subs ti tu te qu inald ine and 4-

aminoquinaldine (the latter, when substituted at positions 1 and 10 of a

decyl chain, giving dequalinium, one of the lead compounds) onto the bis-

bromomethyl compounds in a number of solvents of decreasing volatility,

starting at ethanol and moving onto butanone (methyl ethyl ketone) and 2 ,

6-dimethylheptan-4-one (diisobutylketone) with a boiling point of 169 °C.

At this tem pera tu re , ex tensive decom position o f the d ib rom ides was

observed to occur, and with no attendant reaction, it was clear that

offsetting steric strain by raising the temperature of the reaction was of

only limited use. However, despite the lack of reaction in this case, propan-

2-ol was established as a good compromise since, on the basis of the limited

extent to which decolourisation occurred after reflux, decom position was

kept to a minimum. Upon reaction of s to ich iom etr ic amounts of the

p iperid ine with 2, 7 -b is(b rom om ethy l)an th racene , how ever, a p rec ip ita te

was observed but which analysis revealed to be the salt of the starting

amine. The formation of side products such as this had only been observed

to o ccu r once be fo re w hen, du r ing an a ttem p t to rem ake 2,7-

b is (m o rp h o l in o m e th y l )a n th ra c e n e 1 4 8 , the only product obtained was the

m orpholine salt (presumably the hydrobrom ide). Several fu rther attempts

at substitu ting 2 ,6-dimethy Ipiperidine produced the same result, but the

reaction was found to be successful in producing the derivative 2 ,6-bis[(cis-

2 ,6 - d im e th y l ) p i p e r i d o m e th y l ] n a p h t h a l e n e 1 7 8 when the solvent was

changed to butanone.

89


Me Me

F ig u r e 2.9

Me Me

W e w e r e u n a b le to r e p r o d u c e th i s r e s u l t w i th 1 ,4 -

b is (b ro m o m e th y l )b e n z e n e , f ir s t in b u tan o n e , then , fo r c o m p a r iso n ,

ethanol, so it was decided to alter the reaction conditions in an attempt to

synthesise other members of this series. Thus, a solution of the dibromide

in ethanol was stirred with 0.1 M silver nitrate solution and the piperidine

added upon formation of a precipitate of silver bromide. Once again, only

the salt o f the amine was formed, appearing as a highly crystalline residue

in the evaporated mixture, by a mechanism that is unclear. Lack of time,

how ever, did not allow us to investigate further and the naphthalene

derivative remained the only compound of this type that we were able to

synthesise. It was quaternised as before with iodomethane in acetonitrile

and ap a r t from the problem of o iling-ou t, p reven ted as p rev ious ly

described, proceeded smoothly to give the dimethylated compound 179 . The

two com pounds once again illustrate the effect that quaternisation of an

endocyclic n itrogen atom has on the H nmr signals o f the protons

adjacent to it since 1 7 9 displays separate signals co rresponding to the

equatorial and axial protons at positions 3 and 4 of the piperidine ring.

Irradiation of the N-methyl group at 52.85 produces an enhancem ent of the

signal at 51.68 which therefore belongs to the axial proton at this carbon

a tom .

2 .5 .7 O t h e r C o m p o u n d s

The effec t o f increased charge over the whole molecule had previously

been addressed via the synthesis of the DABCO and N-substituted DABCO

derivatives described earlier. By combining this with multiple substitution,

both o f the central hydrocarbon and of the substituents them selves we

conceived the idea of a dendritic “net” that would hopefully provide a

balance o f hydrophilicity and lipophilicity with a degree of flexibility that

would m aximise interaction with the receptor. The concept of this type of

90


molecule, referred to as arboranes or dendrimers if polynuclear^ '^, is not

new and synthetic routes to the molecules proceed, not surprisingly, by a

series o f sequential substitutions of a central template. The first targets

(which at this stage, remained unbranched) are shown in figure 2 . 1 0 .

+

180 R = methyl181 R = p-xylyl

6 B r '

R

F ig u r e 2.10

The proven facility of the reaction of DABCO with benzylic halogens meant

that the both the molecules shown could potentially be synthesised in two

steps from 1, 3, 5-tris(bromomethyl)benzene ( a , a ' , a " - t r i b r o m o m e s i t y l e n e ) .

The synthesis of this compound seemed simple given the ease with which

p-xylene had been dibrominated, but our first attempt, in which mesitylene

was reacted with three equivalents of N -brom osuccin im ide in refluxing

te trach lo rom ethane produced a c lear oil which c rys ta ll ised but which

contained several similar products. These were not separable in this state

by rec ry s ta l l i sa t io n or th in - la y e r c h rom a tog raphy us ing any so lven t

system that we were able to find. The alternative method, using liquid

bromine under h igh-in tensity illum ination was prom ising because it had

been used to m ultiply brom inate a num ber of m olecules in precedent

reactions in good yields as d iscussed earl ie r in this chapter. Using

mesitylene, however, the same mixture of products as before was observed,

regardless of the conditions that were employed in the reaction. This is odd

because s to ich io m e tr ic am ounts o f b rom ine would be abso rbed to

colourlessness during the reaction, but whether it was added over five

minutes or two hours a messy H nmr spectrum was still produced with

m any p eak s e x tra n e o u s to the two th a t w ere e x p e c te d . The

tris(b rom om ethy l) com pound has been made before^^ and the method is

tacit acknow ledgem ent o f the failure of the two concerted techniques

91


discussed above (scheme 2 .2 9 ) . It begins with the tricarboxylic acid 1 8 2

which is esterified then reduced with lithium alum inium hydride to the

triol 1 8 4 Reaction with phosphorus tribromide gives the tribromide 185 in

45% overall yield

COOH C02MeMe0 H/H2S04(cat) UAIH4/THF

HOOC COOH1 8 2

Me02C^ ^ C02Me 1 8 3

OH

HO. OH

1 8 4

Br

Hr. Br

18 5

PBr 3/Et 2O

S c h e m e 2 .29

It seemed that the best way of controlling this unpredictability but

still using the attractive NBS reaction was by a process of sequential

bromination where, it was reasoned, the addition of only one equivalent of

bromine atoms each time would would limit the number o f possible side-

products and make purification simpler. This was in fact mostly borne out

in practice so that reaction of mesitylene with one equivalent of NBS

produced , a f te r d is t i l la t ion , 5 -b rom om ethy l-m -xy lene 1 8 6 as colourless

l iqu id in good y ield . F u r th e r reac tion gave the b is (b ro m o m e th y l)

deriva tive 187 as white crystals, but much less smoothly and only after

chrom atography in a chloroform /hexane eluant. The final step, however

p roved as frus tra t ing ly im precise as be fore and the syn thes is had,

unfortunately to be abandoned.

2 .6 R e f e r e n c e s

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( 2 ) Bardham, J. C.; Sengupta, S. C. J. Chem. Soc. 1932, 2520.

(3 ) Haworth, R. D.; Mavin, C. R.; Sheldrick, G. J. Chem. Soc. 1934 (I), 454.

(4 ) Sengupta, S. C.; Chatterjee, D. N. J. Ind. Chem. Soc. 1953, iO, 27.

(5 ) Sengupta, S. C.; Sachchidananda, A. B.; Mitra, A. J. Ind. Chem. Soc.

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1960, 37, 597.

6) Newman, M. S.; Whitehouse, H. S. JACS 1949, 71, 3664.

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10) Davy, J. R.; Jessup, P. J.; Reiss, J. A. J. Chem. Ed. 1975, 52, 747.

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13) Wood, C. S.; Mallory. F. B. J. Org. Chem. 1964, 29, 3373.

14) Seer, C. Monatsh. 1 9 1 1 ,5 2 , 143.

15) Morgan, G. T.; Coulson, E. A. J. Chem. Soc. 1929, 2203.

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625.

22 ) Cristol, S. J.; Caspar, M. L. J. Org. Chem. 1968, 55, 2020.

23) Newman, M. S.; Ihrman, K. G. JACS 1958, 80, 3652.

24 ) Lai, Y.-H.; Peck, T.-G. Aust. J. Chem. 1 9 9 2 ,4 5 , 2067.

25 ) Errede, L. A.; Cassidy, J. P. JACS 1960, 82, 3653.

26) Trahanovsky, W. S.; Surber, B. W. JACS 1985, 107, 4995.

27 ) Tsuge, A.; Nago, H.; Mataka, S.; Tashiro, M. J. Chem.Soc. Perkin Trans.

1 1992, 1179.

28) Cheng, S. K. T.; Wong, H. N. C. Synth. Commun. 1990, 20, 3053.

29) Millar, I. T.; Wilson, K. V. J. Chem. Soc. 1964, 2121.

30) De Briijn, P. Compt. Rend. 1950, 257, 295.

31 ) Li, Y. Ph. D. Thesis, Chinese University of Hong Kong, 1993.

32) Hauptmann, S. Chem. Ber. 1960, 95, 2604.

33) Berner, E.; Gramstad, T.; Vister, T. Acta Chem. Scand. 1953, 7, 1255.

34) Staab, H. A.; Meissner, Ü. E.; Meissner, B. Chem. Ber. 1976, 109, 3875.

35) Meissner, U.; Meissner, B.; Staab, H. A. Angew. Chem. Int. Ed. Engl.

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1973, 12, 916.

(3 6 ) Leach, D. N.; Reiss, J. A. Aust. J. Chem. 1 9 7 9 ,5 2 , 361,

(3 7 ) Moradpour, A.; Kagan, H.; Baes, M.; Morren, G.; Martin, R. H.

Terahedron 1 9 7 5 , 5 7 , 2139.

(3 8 ) Laarhoven, W. H.; Peters, W. H. M.; Tinnemans, A. H. A. Terahedron

1978, 34, 769.

(3 9 ) Craig, J. T.; Halton, B.; Lo, S.-F. Aust. J. Chem. 1 9 7 5 ,2 5 , 913.

(4 0 ) Baumgartner, P.; Paioni, R.; Jenny, W. Helv. Chim. Acta 1 9 7 1 , 5 4 , 266.

(4 1 ) Staab, H. A.; Sauer, M. Liebigs Ann. Chem. 1984, 742.

(4 2 ) Sauer, M.; Staab, H. A. Liebigs Ann. Chem. 1984, 615.

(4 3 ) Golden, J. H. J. Chem. Soc. 1961, 3W *

(4 4 ) Akiyama, S.; Misumi, S.; Nakagawa, M. Bull. Chem. Soc. Jpn. 1960, 55,

1293.

(4 5 ) Akiyama, S.; Misumi, S.; Nakagawa, M. Bull. Chem. Soc. Jpn. 1962, 55,

1826.

(4 6 ) Akiyama, S.; Nakagawa, M. Bull. Chem. Soc. Jpn. 1971, 44 , 3158.

(4 7 ) Du Vernet, R, B.; Wennerstrdm, O.; Lawson, J.; Otsubo, T.; Bockelheide,

V. JACS 1978, 700, 2457.

(4 8 ) Badger, G. M.; Campbell, J. E.; Cook, J. W.; Raphael, R. A.; Scott, A. I. J.

Chem. Soc. 1950, 2326.

(49 ) Bergmann, E. D.; Ikan, R. J. Organomet. Chem. 1958, 25 , 907.

(5 0 ) Liu, L.; Yang, B.; Katz, T. J.; Poindexter, M. K. J. Org-

1991, 56, 3769.

(5 1 ) Liu, L.; Katz, T. J. le t. Lett. 19 9 1 ,5 2 , 6831.

(52 ) Newman, M. S.; Me Cleary, C. D. JACS 1941 ,65 , 1537.

(5 3 ) Newman, M. S.; Lord, B. T. JACS 1944, 66, 733.

(5 4 ) Stenlake, J. B.; Waigh, R. D.; Dewar, G. H.; Hughes, R.; Chappie, D. J.;

Coker, G. C. Eur. J. Med. Chem.-Chimica Therapeutica 1981, 16, 515.

(5 5 ) Creighton, T. E. Proteins: Structures and M olecular Properties',

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(5 6 ) Li, T.; Krasne, S. J.; Persson, B.; Kaback, H. R.; Diederich, F. J.

Organomet. Chem. 1993, 58, 380.

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630.

94

Chapter 3

Pharmacological Evaluation

P h a r m a c o l o g i c a l E v a l u a t i o n

G l o s s a r y

Besides the terms given in the glossary on page 9 of this thesis, the

following abbreviations are also used in this chapter:

K j . the inhibition constant, a measure of the ability of a drug to inhibit the

response at a receptor. For practical purposes, Kj and the IC50 , an

empirical measurement of the dose of drug required to effect 50% of

the observed maximal response, are the same.

Hepes - (N -[2 -hyd roxye thy l]p ipe raz ine -N '-[2 -e thanesu lphon ic acid]), used

as a buffer in tissue culture media;

HESS - Hank's Balanced Salt Solution;

U - a measure of enzyme activity: one unit of collagenase hydrolyses 1 pM

of furylacryloyl Leu-Gly-Pro-Ala (FALGPA) per minute at 25 °C at pH 7.5

in the presence of Ca^+ ions;

MEP - molecular electrostatic potential;

V(r) - electrostatic potential at point r.

96

P h arm aco log ica l E valuation


3.1 I n t r o d u c t i o n

JL he sm all-conductance, ca lc ium -activa ted po tass ium channel, SK, has

been found to be widely distributed in excitable cells. Its low conductance

and h igh channel dens ity in m ost m em brane p a tch es , r e su l t in g in

complicated kinetics, has meant that neither the sequence of ion selectivity

nor single channel kinetics are known in detail. However, the current for

which it is responsible, a late after-hyperpolarisation, is readily detectable,

since suppress ion of the large-conductance, ca lc ium -ac t iva ted po tass ium

channel, BK (which is usually present alongside SK), is readily achieved by

the use o f TEA in m illimolar quantities, at which concentra tion SK is

u n a f f e c t e d ^ . The channel has been thus identified in bullfrog sympathetic

g a n g l i o n ^ , cultured cells from rat skeletal muscle^ and m em brane patches

of p r im ary ra t m uscle cu l tu re s^ . The discovery o f apam in and its

apparen tly unique suppression of the AHP current has led to further

d iscoveries . For instance, nanom olar concentrations o f the pep tide can

block this current in both the spinal motorneurones^ and neurones of the

m otor cortex^ of the cat and inhibit the neurotensin induced relaxation of

gu inea-p ig co lon^ .

The presence of apamin binding sites, p resum ed to be apam in-

sensitive potassium channels, in smooth muscle seems to be dependent on

the degree of innervation of the tissue. For instance^, rat m yotubes, known

to contain these channels, when co-cultured with neurones from the rat

spinal cord were found to exhibit action potentials no longer followed by

the AHP. Similarly , an apamin-sensitive AHP was observed in rat leg

muscles two days after transection of the sciatic n« rve^ . It seems that

apamin-sensitive KCs are fully expressed in denervated m ammalian muscle

cells and completely absent in innervated ones, but which factor controls

the expression of this type of channel is not known.

Until the small conductance Ca^'*'-activated KC can be expressed and

th e r e f o r e s e q u e n c e d th ro u g h r e c o m b in a n t D N A t e c h n i q u e s , a ll

inform ation about the channel has of necessity been estab lished through

97

P h arm aco log ica l Evaluation

kinetic measurements or competitive binding studies. Clearly, apamin has

been crucial in this regard and, perhaps more importantly, has provided a

starting point for SAR studies. The first compounds to be studied on this

basis are detailed in Table 3.1 and their structures given in figure 3 .1 . The

firs t colum n of the table lists the d issoc ia tion constan t, Ki of the

recep to r /[^ ^ ^ I] -m ono iodoapam in complex in the presence of the inhibitor,

and the second, the IC50 (that is, the concentration required to effect 50%

inhibition) for angiotensin-evoked K+ loss from guinea-pig hepatocytes

K i (pM ) IC 50 (pM )

Q u in in e 510+78 150

Q u in id in e 340+46 240

Q u in a c r in e 77+11 73

C h lo r o q u in e 140+14 200

P r i m a q u i n e 890+99 970

9 - A m in o a c r id in e 70±10 120

S t r y c h n i n e 180+18 190

A t r a c u r iu m 4.5+0.2 3.0

T u b o c u r a r i n e 7.5±0.7 3.0

P a n c u r o n i u m 6 .8+0 .9 3.5

G a l la m in e 14±2 12

D é c a m é th o n iu m 620±80 450

H e x a m é th o n iu m 760+90 2000

D ib u c a in e 810±100 470

(TEA)+ 5800+1300 7900

B a2+ 14000 _

A p a m in 0.376+0.083x10-3 1.0x10-3

Adapted from ref. 9

T a b le 3.1

Evidently , the greatest degree of block was observed with the three

neu rom uscu lar blockers, atracurium , tubocurarine and pancuronium .

98


= CHHQ

MeO

quinine(quinidine = dextro isomer)

Me

OMe

MeO

Me

chloroquine

quinacrine

NHz

prim aquine

Os try ch n in e

Q — ( C H 2 ) 2 N E t 3

q(CH2)2NEt3

9-am inoacr id ine

Me3N-(CH2)n"NMe3 ^

NEti

O(CH2)2NEt3

gailamine

hexaméthonium (n = 6)décaméthonium (n = 10) d ib u ca in e

COOMeMe

Me 'N +MeMe

MeCOO

pancuronium

Me OMeMeOH

N +

MeMeO OHtubocurarine

O O

. (CH2)2C0(CH2)50C(CH2)2\'N

atracuriumMeO OMe

F ig u r e 3.1

As p re v io u s ly d i scussed, this abil i ty was though t to c o r re l a t e wi th the

a r r a n g e m e n t o f the two p o s i t i v e ly c h a r g e d n i t r o g e n a t o m s , s p a c e d

99


approximately 11Â apart in each molecule and was presumed distinct from

their neurom uscular blocking effects. This suggestion was corroborated by

la te r w o rk ^ . D e q u a l in iu m and 3 ,6 - b i s ( p ip e r id o m e t h y l ) p h e n a n th r e n e

d im e th io d id e 2 3 , ne ither of which have an appreciab le neurom uscular

b locking capability, were tested in the same assay described above and

found, particu larly in the form er case, to have a sign ificant b locking

effect (Table 3 .2 ) .

Ki/pM ICso/liM

V e c u r o n iu m 3.6+0.5 4.9+0.3

D e q u a l in iu m 1.1+0.1 1.9+0.1

2 3 9.9+1.3 8.7+1.1

from re fs 9, 10

T a b le 3.2

3.2 P r e p a r a t i o n o f R a t S y m p a th e t i c G a n g l ia

T he choice of compounds such as 3 ,6 -b is (p iper idom ethy l)phenan th rene

d im e th iod ide 23 as suitable for development thus seem ed reasonable and

the consequent syntheses of analogues of this compound are detailed in the

previous chapter. This chapter deals with the results of the assay by which

they were tested for their ability to block SK. Pharm acological data was

collected by Mr P. M. Dunn of the pharmacology department, UCL. This

assay is based on the fact that the AP in rat sym pathetic neurones is

fo llow ed by an apamin-sensitive, SK -m ediated AH P (/. e. the membrane

potential becomes more negative than in the resting ce ll)^^ . This is studied

in single neurones either by using an in tracellu lar m icroelectrode or by

whole-cell patch clamping. Thus seventeen day old Sprague Dawley rats

were k illed by inhalation of a r ising concen tra tion o f nitrous oxide.

S u p e r io r c e rv ica l g an g lia w ere rem o v ed and p la c e d in ic e -c o ld

unsupplemented L-15 medium. The pooled ganglia from 2 to 4 animals were

desheathed and 3 or 4 deep cuts were made in each ganglion using fine

iridectomy scissors. The ganglia were transferred to 4ml Hanks Ca^'*'- and

M g ^ ^ - f re e saline buffered with 10 mM Hepes (pH 7.4) (HBSS), containing

6mg/ml BSA and 372 U/ml collagenase and incubated for 35 minutes at 37

100

P harm aco log ica l E valuation

°C. The ganglia were then incubated for a further 15 minutes in 4 ml HBSS

containing Im g/m l trypsin. The enzyme solution was next rem oved and

enzyme activity stopped by adding 1ml growth medium to the ganglia. The

ganglia were then dissociated by gently passing them 5-10 times through a

fire polished Pasteur pipette. Undissociated pieces of tissue were allowed to

settle out and the supernatan t (conta in ing the ce ll suspension) was

removed, made up to 5 ml and centrifuged at 800 g for 5 minutes. The pellet

was resuspended in 1-2 ml growth medium and d ispensed into prepared

culture dishes containing 1 ml growth medium.

35 mm plastic culture dishes were treated with laminin (10 pg /m l in

HBSS) for 90 minutes then rinsed with HBSS. A glass ring (diameter 13 mm,

height 2 mm) was placed in the centre of each disk (to retain the neurones

in the center of the dish) and 1 ml growth medium was added.

Cells were grown in L-15 medium supplemented with 10% foetal calf

serum, 0.2 mM glutamine, 0.6% (w/v) D-glucose, 0.19% (w/v) N aH C O ],

penicill in (100 U/ml), streptomycin (100 pg /m l) and nerve growth factor

(0.05 pg/ml). Cells were maintained at 37 °C in a humidified atmosphere of

95% O 2 , 5% C O 2 for up to 10 days b e fo re be ing taken for

e le c tro p h y s io log ica l record ing .

Electrical recording was carried out by placing the dishes on the

stage o f an inverted microscope and perfused at a rate of 7 ml/minute with

Krebs solution containing (mM): NaCl, 118; KCl, 4.8; CaCl2 , 4.5; NaHCO], 25;

K H 2P O 4 , 2.28; MgS0 4 , 1.19; glucose, 11; equilibrated with 95% O2, 5% CO2.

Drugs were were applied by perfusing the bath at the required final

c o n c e n t r a t io n . I n t r a c e l lu l a r r e c o r d in g s w e re m a d e w ith g la s s

microelectrodes drawn from 1mm diameter glass tubing, filled with IM KCl

(res is tance 80-120 M Q ) and connected to a bridge balance amplifier to

permit sim ultaneous current in jection and potential recording.

Action potentials were evoked by in jection of 30 ms pulses of

depolarising current at a frequency of 0.2 Hz. The signals were digitised at

1 KHz and averages of 3 or 4 action potentials were obtained before, during

and a f te r drug app lication . The time cou rse o f the d ru g -se n s i t iv e

component of the AHP was determined by subtracting the record obtained

in the presence of the drug from the average of the pre- and post-drug

control records. The reduction in the amplitude of the AHP at the time of

m axim um d iffe rence was exp ressed as a pe rcen tage o f the con tro l

101

P h a rm aco log ica l Evaluation

am plitude at that time point. W here appropriate , this was repeated at

several concentrations and the Hill equation was fitted to the data using an

ite rative least-squares curve-fitt ing routine, providing an estimate of the

IC 50 value.

Obviously, because this assay is different from that originally used to

test the neuromuscular blockers as well as our lead compound, the results

obtained through testing of the new analogues are not strictly comparable,

but the new assay has the great advantage that, unlike the guinea-pig

hepatocyte assay which consists of a suspension o f cells in a physiological

solution, this uses single cells which are thus freely accessible to the drug,

c rea ting no d iffus ion delays. M oreover, several com pounds , including

apamin itself, have been retested on the new assay, providing a basis for

comparison. These values are given in Table 3 .3 .

Ki/|iM IC sq/^M

D e q u a l in iu m 1.1±0.05^^

T u b o c u r a r i n e 11.6±3.3l2 20

A p a m in 3x10-313 3x10-3

T a b le 3.3

A num ber of the compounds were also tested on a separate assay as the

result o f a random screening programme. This consists of a section of

rabbit in tes tine , the jejunum , which contracts spontaneously and whose

a c t iv i ty is in h ib i te d by A T P, b ra d y k in in , n e u ro te n s in and a -

adrenoreceptor agonists. Since these are known to act by opening K(Ca)

channels, the degree of inhibition of contraction by any putative drug is a

measure of its ability to block these channels. Of the three compounds

tested, only one, / r a « j - 4 , 4 ' - b i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o m e t h y 1]s t i l b e n e

dimethiodide, showed reasonable activity, but not of the order to make more

extensive investigation worthwhile. Apamin exhibits an IC50 of 0.92± 0.19

nm in this assay and the results for the three compounds are given in the

appropria te sections below.

102


3 .3 . D e r iv a t iv e s o f P i p e r i d in e , P y r r o l i d i n e a n d M o r p h o l i n e

T T he first group of compounds that were submitted for testing are detailed

in Table 3.4 along with values obtained in the bioassay. The upper figure

refers to the % inhibition of K+ loss at 10 pM whilst the lower is the IC5 0 ,

es tim ated by in terpo la tion of the above figures. The la tter value is

o rd inarily obtained by testing over a range o f concentra tions but the

estimated values reveal these to be too low to make more than one run of

the technically demanding assay viable.

VTOCCr-/

O -C H .

1 4 5

32.1+4.3

20.25pM*

1 3 9

20.4±8.8

37 .OpM*

2 3

8.0+4.0

60.OpM *

C '^ H i

1 4 7

32.9+14.9

20.0pM*

1 4 1

25.1+2.2

29.OpM*

1 3 6

23.8+8.6

30.5pM *

1 4 9

22.0+2.4

33.0pM*

1 4 3

26.7+11.2

27.0pM *

1 3 7

25.7+11.3

28.0pM *

' / C 5 0 value, est im a ted by interpolation

Table 3.4: % Inhibtion at 10 pM of AHP in rat sympathetic neurones for

derivatives of piperidine, pyrrolidine and m orpholine

Figure 3 .2 gives a dose-response curve of the two compounds and 136

against dequalin ium and i llustrates the generally poor activ ity of the

compounds compared to the latter. The most significant detail of table 3 .4

h o w e v e r , is the im p o ten c y o f 3 ,6 - b i s ( p ip e r id o m e th y l )p h e n a n th r e n e

d imethiodide, 23 . This was our lead compound and showed an activity in the

e a rl ie r assay based on gu inea-p ig hepa tocy tes w hich com pared with

dequalinium (see figure 1 .1 7 , Chapter 1). Its drastic loss of activity in this

assay, particularly in light of later results with s im ilar compounds, is

103

Ph arm aco log ica l Evaluation

difficult to ascribe, but in the context of the rather poor activities of all the

compounds listed, is not in itself significant. There are no clear trends in

the table (the estimated values of the IC50 would render these of only

questionable value anyway), but it is interesting to note the slight decrease

in the value for the IC50 in going from the anthracenes (average N-N

distance 12.0 Â) to the phenanthrene (N-N distance 9.5 Â) which accords

with the prem ise o f our project. The figures are o therwise som ew hat

d i s a p p o i n t i n g .

3 .4 . D e r i v a t i v e s o f 2 , 5 - D i m e t h y l p y r r o I i d i n e

T he next group of com pounds to be ana lysed were the (2,5-

d im ethyl)pyrro lidom ethyl analogues of the previously form ed compounds.

As discussed in Chapter 2 , the discovery of this amine was a fortunate

coincidence, but was a nonetheless obvious way of a ltering the steric

p roperties of the com pounds around the n itrogen atom s, the pu tative

pharmacophore. The first of these to be tested, the 2,7-dimethylanthracene

derivative 153 showed a dramatic increase in activity, approaching that of

d equa lin ium (F igure 3 .3 ) . Incidentally , this diagram gives an IC 50 for

dequalinium of 0.75 mM, which was obtained on a different batch of cells

and has been found to be within the experimental error of this assay. This

im provem ent was also observed in the 2 ,6 -d im ethy lan th racene analogue

15 5 . The values obtained in the assay for these two compounds and for the

other derivatives made in the light of these observations are given in Table

3 .5 . The comparable activity of the dimethylanthracene analogues is again

seen and the exponential increase in IC50 going from anthracene through

naphthalene to benzene is again in accord with our initial hypothesis that

activity is in someway dependent on transmolecular distance. The reason

for the marked overall increase in this series of analogues is less obvious

however, but is apparently steric, originating in the a -m e th y l groups of

the p y r ro l id in e r ing ; 2 ,7 -b is (p y r ro l id o m e th y l)a n th ra c e n e d im e th io d id e ,

which merely lacks these methyl groups, is some eighteen times less active.

104


N-N dist/Â % in h ib " (lOpM) IC5o/|iMMe Me Me Me

C Q O O r j Ô11.9 - 1.1

Me fvie Me

Me 155

12.2 42.8±7.8t 3.2*

Me Me Me 10.0 34.4±3.6t 5.8*

Me Me 159

7.9 32.0 33.0*

Me Me

Me Me 1 6 1

14.2 [45.0] -

Me Me

M e ^ ^ A ^ ^ ^ y U e

^ 163

11.0 51.0 9.2*

^ M e ^ ^ __

Me 151

10.8 40.0 13.5*

Me Me

Me M e 179a

10.0 54.9 8.0*

*estim ated va lu e

t at 3,uM

[ ] va lu e obtained on rabbit jejenu m

Table 3.5: % Inhibition of AHP in rat sympathetic neurones for

derivatives of 2 ,5-dim ethyl pyrrolid ine and 2 ,6-dime thy Ipiperidine^

105

P h a rm a co lo g ica l Evaluation

.o■EJC

X

Me Me100

902 1 *

80

70

60

50Me Me

40I 143 (26pM )

136 (30jiM )

30

20

10

0 L_ 0.01 0.1 101

[ B l o c k e r ] ( u M )

F ig u re 3.2: Dose-response curves of compounds 136 and 143 against

d e q u a l i n i u m

jCC

X

100 MeMe

+ N—(CH2)io —X90153 ( M u M )

2 1 *80

70

60

50

40 Me Me

30Me Me

20

0.01 0.1 1 10

[ B l o c k e r ] ( u M )

F ig u re 3.3: Dose-response curve of compound 153 compared to

d e q u a l i n i u m

106


A ssum ing that dequalin ium contorts i t s e l f into a conform ation

w here the term inal qu inald in ium groups are sp a t ia l ly a rranged at a

distance comparable to that in our compounds and that there is negligible

e lec tros ta t ic transmission along the decyl chain, then we may make a

d irec t com parison between the end groups o f the two molecules. The

similarity of the two moieties rests on the substitution around the nitrogen

a tom excep t that one o f the a - m e t h y l g r o u p s in the b is (2 ,5 -

d im ethyO pyrro lido derivative is extended to form part of the ring in

quinald ine (Figure 3 .4 ) .

M e

— N. +

M e

M e

F igure 3.4

O f course , the com parison is not complete s ince the trigonal p lanar

n itrogen atoms of dequalinium mean that the free m ethyl group is co-

p lanar with the quinaldine ring, but the apparent spatial overlap of the

two, together with the fact that the salient guanidinium groups in apamin

also con ta in tetrahedral nitrogen atoms m akes this a likely point of

i n t e r a c t i o n .

3.5 A Com parison of the E lec tros ta t ic P o te n t ia l s

Steric accessibility is always the most crucial aspect of b inding, but most

drugs exert their function via in te rm olecu la r in te ra c t io n s which are

e lec trosta tic in origin. Therefore, for compounds of approx im ate ly equal

steric volume, the molecular electrostatic potential (MEP) can be used to

assess the likelihood of binding to a receptor with affimity for a ligand

whose MEP profile is known^^. It is calculated as the interaction between a

107


positive unit charge and the molecule at different points around the latter,

using equation 3 .1 :

V(r) = (Z Z a /IR a - rl) - (J p ( r ’)dr7lr’ - rl) eq. 3.1

where V(r) is the electrostatic potential at point r, ( r’) the electron density

function at r ’ and Z& the charge of the atom A found at Ra- By varying the

distance between the unit charge and the molecule, a 3D map is constructed

which serves as an electrostatic “fingerprint” of a compound. The MEPs at

longer d is tances (>3 Â) have been suggested to reflect a “distance

pharm acophore” , essential for aligning the ligand prior to contact, whilst

the M EP at the van der W aal’s contact distance (1.75 Â) constitutes a

“con tac t p h a rm aco p h o re” , requ ired for op tim al b i n d i n g ^ F i g u r e s 3 . 5

gives the 3D MEP diagrams for our most active compounds, viz., 2,6- 155 and

2 ,7 - b is [ (2 ,5 -d im e th y l)p y r ro l id o m e th y l]an th rac e n e d im e th io d id e 153 whilst

figure 3 .6 gives an equivalent diagram of dequalinium . These diagrams,

which were generated on a Silicon Graphics display using the SYBYL

package from TRIPOS associates are based on certain assumptions. Firstly,

the two anthracenes are shown with the methyl groups of the pyrrolidyl

ring cis with respect to each other since this was revealed to be by far the

most abundant isom er after substitution of the d im ethylpyrro lid ine onto

the anthracene nucleus, as previously discussed. Secondly, the two cyclic

methyl groups are themselves anti to the methyl group attached to the

nitrogen atom since the approach of the methyl group from the syn side

would be h ighly d isfavoured . The activity of the two com pounds is

therefore assumed to reside in this configuration, despite the fact that its

purity is not complete (figure 3 .7 ) .

Me/,,

F i g u r e 3.7

The illustated conformation of the two molecules are global energy minima

based on an iterative energy minimisation programme on the COSMIC

108


F ig u r e 3.5: MEP Diagrams for 2,7- (top) and 2,6-bis[(2,5-

d im e th y O p y r r o l i d o m e t h y l ] a n th r a c e n e D im e th io d id e .

109

Pharm acological Evaluation

Figure 3.6: MEP Diagram of Dequalinium

110


Structure drawing p a c k a g e ^ ^ ^ ^ and these are given in figures 3 .8 and 3 .9 .

As may be expected, these conformations place the ring substituents on

opposite sides with respect to the aromatic ring plane, and the diagrams

give views of the molecules through and perpendicular to the ring plane.

Thirdly, given the large number of degrees of freedom of the decyl

chain of dequalinium, generation of an energy minimised conformation is

not m eaningful. Therefore, an arbitrary conform ation which places the

pair of endocyclic, quaternary nitrogen atoms some 13.5 Â apart has been

used as the basis of the MEP diagram. It is also assumed that dequalinium

remains unprotonated at physiological pH.

Inspection of the diagrams reveals im portant d ifferences between

the essentially indistinguishable hourglass shaped MEP diagrams of the two

anthracenes and the much less uniform diagram of dequalin ium . The

isopotentia l shells of the diagrams are co lour coded according to the

following scheme: red, 20.0; orange, 15.0; yellow 10.0; green, 5.0 kcal mol"^,

c o r r e s p o n d in g to p e rp e n d ic u la r d is ta n c e s from the m o le c u le of

approximately 1, 2, 4 and 7 Â. The transections of the isopotential contour

maps of the two anthracenes are roughly 3 Â above the aromatic ring

plane whilst that of dequalinium is the same distance above the axis of the

decyl chain, which, in this diagram, is linear. The diagrams reveal the

d isrup tion to the e lec trosta tic p rofile that has occurred around the

terminal moieties as a result of the aromatic ring and the para amino

group. Given that these views represent distance pharm acophores for the

compounds, it seems reasonable to conclude, on a purely qualitatitive basis,

that their initial contact with the receptor will be of a d ifferent type

because of the extensive delocalisation in the quinaldine ring system of

dequalinium . W hether the nature of the binding is u ltim ately different

once the ligands have docked is not of course possible to predict but since

MEPs are a representation of what the receptor actually "sees", the

poss ib i li ty exists that this is the case. This hypo thes is could not

u n fo r tu n a te ly be c o n f i rm e d by the te s t in g o f 2 ,6 - and 2 ,7-

b is (q u in a ld in o m e th y l)an th ra c e n e s ince , as d isc u sse d in the p rev ious

chapter, these compounds could not be synthesised. Their synthesis would

have allowed an assessment of the degree to which areas of the molecules

111


F ig u r e 3.8: Energy-Minimised Conform at ion of 2 ,7-Bis[(2,5-

d im ethy Opy rro l idom ethyl ] an th ra cene D im e th io d id e 1 5 3

112


Figure 3.9: En er gy -M in im ised Con fo rm at ion of 2 ,6-B is[ (2 ,5

d im e th y l ) p y r ro l id o m e th y l ] a n th r a cen e D im e t h i o d id e 1 5 5

113


remote to the putative pharmacophores were contributing to secondary

binding, but in the absence of this information, it is assumed that the major

determinant o f binding ability o f the 2,5-dim ethyl pyrrolido derivatives is

steric, originating in the area around the charged centres. If, as the weight

of evidence suggests, it is ch iefly the interaction o f two nitrogen atoms

with a receptor site in the base o f well-defined pocket which determines

the degree o f block of apamin and analogous compounds, then it is easy to

understand that the substitution pattern around these atoms w ou ld be

c r u c ia l .

3 .6 . 2 , 6 - B i s [ ( c i 5 - 2 , 6 - d i m e t h y l ) p i p e r i d o n i e t h y 1]

n a p h t h a l e n e D i m e t h i o d i d e

A n obvious modification to the compounds given in Table 3 .5 would be

expansion o f the heterocyclic ring v i a the use o f 2,6-dimethylpiperidine.

T his co m p o u n d has the ad vantage that it m ay be p u rch ased

configurationally pure, thus preventing the formation o f isom ers upon

quaternisation o f the nitrogen atom, a process d iscussed in Chapter 2 .

H ow ever , 2 ,6 - b i s [ ( c i j - 2 ,6 - d im e t h y l )p ip e r id o m e th y l ] -n a p h th a le n e 1 7 9 was

the only member o f this series synthesised due to a persistent problem

whereby the salt o f the amine was formed exclusively in the reaction. The

comparable activity o f this compound to its pyrrolidomethyl analogue 1 5 7

(see table 3 . 5 ) suggests that our assumption that the activity o f the latter

compound rests in the most abundant isomer and not in one o f the minor

isomers, is correct. However, the increased steric volume o f the substituted

piperidine, together with the fact that the a -m e th y l groups are more

proximate to the nitrogen atom mean that the approach o f the cationic

centres to the receptor is occluded and this presumably explains the small

drop in the value o f the IC5 0 . Though one ob v iou sly cannot draw firm

conclusions from a single compound, this result is enough to suggest that

the change in the values o f the IC5 0 of the other compounds in this series,

had they yielded to synthesis, would not have been dramatic.

114

P harmaco log ica l Evaluat ion

3.7 Derivatives of DABCO and Q uin uc l id ine

O ubsVitotion of DABCO onto the hydrocarbons gave a series of compounds

with markedly different electronic and steric profiles from those previous.

Most significantly, there are now two further basic nitrogen atoms in the

molecules, which we considered significant given the high basicity of the

guanidiniorn moiety of arginine (pKa is estimated to be about 12) and the

high charge it consequently carries at physiological pH. As discussed in

Chap ter 1, there is presumably an effect due to this contributing to the

action of apamin at its binding site since there is no pharmacological effect

observed when the two arginine residues o f the peptide are substituted

with ornithine (in which the guanidine group is replaced by the much less

basic amino group).

% inhibition IC50 (pM)

at lOpM

gross' 0 532Br' 166

y

grcoug?2 B f 167

11.4

[ 12]*

47

* Value obtained on rabbit jejenum

T ab le 3.6: % Inhibition of AHP in rat sympathetic neurones

by derivatives of DABCO

Assuming, therefore, that the second set of nitrogen atoms of the DABCO

moieties are also protonated in vivo then the use of this amine provides a

way of introducing this increased charge, despite the fact that it is not

delocalised as in arginine. Five compounds were synthesised in this series,

but the results with the first two compounds tested indicated that there was

little value in further application of the d ifficult assay (table 3 .6 ) ; the

small reduction in the IC50 between the benzene and the naphthalene

derivatives suggested that the values for the anthracenes would not be

significantly higher, and given their low values anyway, it was apparent

115

Pharmaco log ica l Evaluat ion

that a modified approach was needed. Therefore, two sets of compounds

were syn thes ised in which the res idual bas ic ity o f the b is-D A B C O

compounds was removed by: (i) replacement of the second nitrogen atom

with carbon to give derivatives of quinuclidine, lacking the excess charge

of the previous analogues; (ii) substitution at the nitrogen atom to give a

set o f com pounds which re ta ined this charge but su rrounded by a

lipophilic environm ent. The first series, form ed by the substitu tion of

quinuclidine instead of DABCO onto the bis-bromomethyl compounds, gave

a set o f compounds that are in all respects but one the same as the bis-

DABCO derivatives above. This assumes that, in the former series, the second

nitrogen atom makes a discrete contribution to the electronic profile of the

m olecule since there is unlikely to be significant transm ission along the

aliphatic bridges which connect it to the rest o f the molecule (and as

previously discussed, the disposition of the nitrogen atom places its lone

pair away from the m olecule , and quatern isa tion does little to affect

m o lecu la r s truc ture). Use o f qu inuclid ine therefo re p rov ides a d irec t

measure of the contribution of this atom to the binding efficiency of the

bis-substituted DABCO derivatives. The second set of analogues are a pair of

compounds formed by substitution of a hydrocarbon onto the bis-DABCO

deriva tive of p-xylene to give an extended te traca tion ic species with

several degrees of freedom and alternating areas of hydrophilic ity and

lipophilic ity . The high overall charge of the m olecule would seem to

m it iga te ag a in s t op tim al b ind ing e ff ic iency s ince the re is a lso a

hydrophobic contribution to the binding of apamin. It has further been

suggested that the inability of certain peptide analogues to mimic the

blocking action of the dicationic neurom uscular blocking agents initially

cited by Cook and Haylett was because they were too polar in comparison to

the latter which have a large, hydrophobic spacer between the two ionic

c e n t r e s ^ . It was hoped, however, that a ba lance betw een the two

properties would be achieved in our compounds, as had been done in the

structurally sim ilar nucleotide carriers of Diederich^^. The values obtained

for the IC50 in the rat ganglion assay for both types of compound are given

in table 3 .7 . The importance of the steric contribution to to binding is

further underlined by the near-complete inactivity of the bis-DABCO and

N-substituted bis-DABCO derivatives given in tables 3 .6 and 3 .7 . Since the

second nitrogen atom of the DABCO moiety will be protonated also at

116

Pha rm aco log ica l Evaluat ion

physiological pH (pKa for DABCO = 8.8 and 3.0), all these compounds are

te tracation ic and therefore have electrostatic profiles which have certain

elements in common with dequalinium.

% inhib ition

at lOpM

IC50 (pM )

2 B f 1731 1 .0 ± 8 4 7

2 B f 1742 1 .9 ± 2 3 3

2 B f 1 7 55 3 .0 ± 6 8 .8

- -

” 3*= 2 B f 1 7 11 5 ± 1 1 4 0 .5

2 B f , 2 r

1 7 2

2 3 ± 1 0

[ 6 *]

3 0 .5

Values for % inhibition are given as the average of 3 - 5 experim ents

* % inhibition o f ATP responses in rabbit jejunum at lOpM

Table 3.7

Unlike the previous compounds however, they all lack branching at the 2-

positions o f the rings and are considerably more ste r ica lly congested

around the putative pharm acophore. Given that the 3D geom etry of a

structure is only a necessary, but not sufficient, cause of activity (since

e lectronic and hydrophobic forces are, as d iscussed, also required for

117

Phar maco log ica l Evaluat ion

response), this would seem to suggest that the nature of the binding of

dequa lin ium and our pa ir o f active m olecu les is, at least part ia l ly ,

electrostatically distinct. W hether these results further suggest that these

bindings are qualitatively different or indeed, occurs at distinct areas of

the receptor, is too early to say, however.

Though becom ing a l i t tle rem oved from our in it ia l rem it, the

com pounds 180 and 181 (figure 3 .1 0 ) would have provided an interesting

extension of the concept that lay behind the the synthesis o f the previous

compounds. Again, the overall charge of the molecule is high, certainly

rendering the molecule too polar to cross the b lood-b ra in ba rr ier for

instance, but it was reasoned that the presence o f the cationic centres over

a relatively wide area coupled with an enhanced flexibility would provide

an opportunity for any two of them to position them selves over the

receptor site in the optimum position for block.

Me

_ Me

3 B r , 31 *

Me 180

M e

6Br*

181

Me"

Figure 3.10

Unfortunately, synthesis of the compounds was not achieved in the time

available, but compounds of the type shown in figure 3 .1 0 are the subject

of continued investigation by other workers.

118


3.8 T he E f fe c t o f N i t r o g e n - N i t r o g e n D is ta n c e

Our original hypothesis, based on the observation by Cook and Haylett, was

that, in the absence of m odula ting fac tors , then the t ran sm o lecu la r

distance between the two nitrogen atoms in our compounds was of singular

im portance. Leaving aside the e lectronic and steric con tr ibu tions which

are inferred as equally important, it is helpful to assess the extent to which

the h y p o th es is holds. V alues for these in te rc a t io n ic d is tan ces are

essentia lly unaffected by the nature o f the subs ti tuen t and though the

early m easurem ents which suggested the su itab il i ty o f our com pounds

were from Dreiding models, the values quoted in table 3 .8 were obtained on

the CO SM IC structure drawing package. It is assum ed that d iffe ren t

substituents on the hydrocarbon fram ework will have only a neglig ible

effect on the transm olecular distance and so the m easurem ents , which

were taken as the average of the ten lowest energy conformations of the

b is[(2 ,5 -d im ethy l)py rro lidom ethy l] analogues, are quo ted as genera l for

each hydrocarbon spacer.

Average N-N distance/Â

3,6-dimethylphenanthrene 9.5

1,6-dimethyIphenanthrene 10.8

fraAij-4,4'-dimethylstilbene 142

rranj-2,4’-dimethylstiIbene 11.0

2,6-dimethylnaphthalene 10.0

2,7-dimethylanthracene 11.9

p-xylene 7.9

2,6-dimethylanthracene 12.2

Table 3.8

U nfortuna te ly , the limited set o f data means that not all c lasses o f

compound can be assessed for this correlation but the results for two sets of

com pounds are given below. The first, the de riva tives of pyrro lid ine ,

provide an excellent corroboration of the hypothesis, despite the limited

activity of the compounds (figure 3 .1 1 )

119

Pha rm aco log ica l Evaluat ion

B is (p y rro l id o m eth y l) d e r iv a t iv e s

y = - 21.341 + 4.8384X - 0 .22283x^2 R^2 = 1 .0005.0

■ Column 24.9 -

oU

4 . 8 -

4 . 7 -

4 . 6 -

4.510 1 1 12 1 39

N-N distance

F i g u r e 3 .11

These da ta p red ic t tha t op tim um b in d in g o f b i s (p y r ro l id o m e th y l )

substituted compounds will occur when the distance betw een the nitrogen

atoms is approxim ate ly 10.8 Â. S im ilarly , a p lo t o f the ca lcu la ted

transmolecular distances versus -log IC50 for our three b is(p iperidom ethy l)

substitu ted compounds (figure 3 .1 2 ) provides a figure of the same order,

approximately 11 Â. These diagrams provide reasonable evidence of the

veracity of the hypothesis in some cases, but the poor activity of the six

compounds above underlines the fact that it cannot be used to predict

absolute or even relative activity; obviously, two cationic nitrogen atoms

are not in themselves a sufficient constituent of the pharmacophore.

120

oU

y =

Phar maco log ica l Evaluat ion

B is (p ip e r id o m eth y l) d e r iv a t iv e s 41.725 + 8.5448% - 0.39049x^2 R ^2 = 1.000

5.0 ■ Colum n 2

4.9

4.8

4.7

4.6

4.5

4.4

4.3

4.29 1 0 1 1 1 2 13

y =

N -N distance

F igu r e 3.12

B is (2 ,5 -D im eth y lp y rro l id o m e th y l) D e r iv a t iv e s

71.330 + 23.094X - 2.3196x''2 + 7.7457e-2x^3 R' 2 = 0.7206

■ Column 2

5

48 97 10 1 1 1 2 1 3

olo

00o

N -N distance

F i g u r e 3 .1 3

121


In the case of our most active set of compounds, the derivatives of 2,5-

dimethylpyrrolidine, the contribution of these atoms to the activity and its

correlation with their relative disposition becomes even more tenuous and

a s im ple second order polynom ial function cannot be constructed that

gives an optimum value for the transmolecular distance (figure 3.13 g i v e s

the third order function which exhibits a m inim um at an N-N distance

corresponding to 10.6 Â). The efficacy of these compounds in blocking SK

is c learly m itiga ted by factors o ther than the d is tance betw een the

nitrogen atoms which is in accord with the generally accepted view of the

nature of the binding of apamin to its receptor^

3.9 . C o n c l u s i o n

L is quite clear from this limited data set that a pair of cationic nitrogen

atoms is a useful starting point for construction of compounds with SK

agonistic properties. However, the only lim ited success with a set of

compounds synthesised using this as the sole criterion implies that the

steric contribution to the binding of apamin mimics is greater than this

simple theory would suggest. The fact that dequalin ium , apparently the

most potent non-peptide blocker of the small conductance, C a^"^ -ac tiva ted

KC so far discovered^ ^ , and 2 ,7 - b i s [ ( 2 ,5 - d im e th y l ) p y r r o l id o m e th y l ) ]

an th racene d im eth iod ide 1 5 3 , which has an activity approaching that of

dequalinium, share a degree of steric crowding around the supposed active

site and that, in the absence of these substituents, the activity of the latter

is decimated, gives an indication of a new site of synthetic intervention.

3 .1 0 R e f e r e n c e s

(1 ) Romey, G.; Lazdunski, M. Biochem. Biophys. Res. Commun. 1 9 8 4 ,7 7 5 ,

669.

(2 ) Pennefather, P.; Lancaster, B.; Adams, P. R.; Nicoll, R. A. Proc. Natl.

Acad. Sci. USA 1984, 52, 3040.

(3 ) Blatz, A. L.; Magleby, K. L. Nature 1 9 8 6 ,5 2 5 , 718.

122


(4 ) Zhang, L.; Krnjevic, K. Neurosci. Lett. 1987 , 74, 58.

(5 ) Szente, M. B.; Baranyi, A.; Woody, C. D. Brain Res. 1 9 8 8 ,4 6 7 , 64.

( 6 ) Hugues, M.; Duval, D.; Schmid, H.; Kitabgi, P.; Lazdunski, M.; Vincent,

J. P. Life Sci. 1982, 31, 437.

(7 ) Schmid-Antomarchi, H.; Renaud, J.-F.; Romey, G.; Hugues, M.; Schmid,

A.; Lazdunski, M. Proc. Natl. Acad. Sci. USA 1982, 82, 2188.

( 8 ) Castle, N. Ph. D. Thesis, UCL, 1987.

(9 ) Cook, N. S.; Haylett, D. 0 . Br. J. Pharmacol. 1983, 546P.

(1 0 ) Cook, N. S.; Haylett, D. G. J. Physiol. 1 9 8 5 ,5 5 5 , 373.

(1 1 ) Dunn, P. M. Eur. J. Pharmacol. 1994, 252 , 189.

(1 2 ) Kawai, T.; Watanabe, M. Br. J. Pharmacol. 1986 87, 225 .

(1 3 ) Dunn, P. M., Personal Communication.

(1 4 ) Hogberg, T. H.; Norinder, U. In A Textbook o f Drug Design and

D eve lo p m en t', P. Krogsgaard-Larsen and H. Bundgaard, Ed.; Harwood:

Chur, 1991.

(1 5 ) Vintner, J. G.; Davis, A.; Saunders, M. R. J. Comp. Aid. Mol. Design

1987, 7, 31.

(1 6 ) Morley, S. D.; Abraham, R. J.; Haworth, I. S.; Jackson, D. E.; Saunders,

M. R.; Vintner, J. G. J. Comp. Aid. Mol. Design 1991, 5, 475.

(1 7 ) Demonchaux, P.; Ganellin, C. R.; Dunn, P. M.; Haylett, D. G.; Jenkinson,

D. H. Eur. J. Med. Chem. 1991, 26, 915.

(1 8 ) Li, T.; Krasne, S. J.; Persson, B.; Kaback, H. R.; Diederich, F. J. Org.

Chem. 1 9 9 3 ,5 5 , 380.

123

Chapter 4

Exper im en ta l

E x p e r i m e n t a l

4. Experimental

4.1 A p p a r a t u s a n d R e a g e n t s

Chemical reagents were purchased from Aldrich Chemical Co., Lancaster,

Fisons and BDH. Solvents were purified by standard m ethodologies. All

experim ents using water sensitive reagents were carried out under an

a tm osphere of dry nitrogen.

M icroanalysis samples were prepared by d ry ing in vacuo at room

temperature over silica gel. The analyses were carried out by Jill Maxwell

or Alan Stones in the Microanalytical Section of the Chemistry Department,

U niversity College London, with a Perk in -E lm er 2400 CHN Elem ental

A n a ly s e r .

Melting points were determined on a Reichert melting point apparatus.

Both melting and boiling points are uncorrected.

U ltrav io le t (UV) spectra were recorded on a Shim adzu UV 160A

u ltrav io let spectrometer using quartz cells of 1 cm pathlength and in

either absolute ethanol or deionised water as indicated.

Proton nuclear magnetic resonance H nmr) spectra were recorded on

a Varian VXR-400 (400 MHz) or VXR-200 (200 MHz) spectrometer. Chemical

shifts (6 ) and coupling constants (J) are reported in ppm and Hz,

respectively. The spectra were recorded in deuterochloroform (CDCI3 ) or

d im e th y lsu lp h o x id e -d 6 (d^-DMSO) solution. Residual protic solvent ie. CHCI3

( 5 h = 7.26 ppm) or CD3 S O C D 2 H (§ h = 2.52 ppm) was used as internal

reference. The following abbreviations are used in signal assignments: s

(singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad).

Carbon nuclear magnetic resonance (^ ^C -nm r) spectra were recorded at

100 MHz on a Varian VXR-400 spectrometer. Signals are reported as 5 values,

using the resonances of CDCI3 (5 c 77.0 ppm, t) or (CD3 )2 SO (5 c 39.7 ppm,

hep tup le t) as reference. W here ind ica ted , the signal m u ltip lic i ty was

determined by an APT pulse sequence.

Mass spectra were recorded on a VG7070H mass spectrom eter with

Finnigan Incos II data system at University College London, or on a VG ZAB-

2F (EIMS) or VG12-250 (Cl) mass spectrometer at the London School of

Pharm acy . A ccurate Mass de te rm ina tions were made on a V acuum

125


Generators VG ZAB SE mass spectrometer at the London School of Pharmacy.

For analytical thin layer chromatography (TLC) Merck Kieselgel 60 F 2 5 4

plates were used. Compounds were visualised by ultra-violet light or by

heat development using a p-anisaldehyde-based (350 ml 95 % EtOH, 12 ml

conc. H 2 S O 4 , 8 ml p -an isa ldehyde , 6 ml g lacial acetic acid) s ta in ing

preparation . Column chrom atography was perform ed using M erck flash

silica gel 60 (200-400 mesh) or Sorbsil C60-A (40-60 pm) flash silica.

Apart from the numbering used for assignm ent o f nmr signals the

nam ing and num bering of com pounds th ro u g h o u t the e x p e r im e n ta l

section adheres to Chemical Abstract nomenclature.

4.2 E x p e r i m e n t a l

1. P r e p a r a t i o n of 3 , 6 - D i m e t h y l p h e n a n t h r e n e 44

CH

7’r a / i 5'-4 ,4 '-dimethylstilbene (1.00 g, 4.80 mmol) and iodine (0.10 g,

0.39 mmol) were dissolved in warm cyclohexane (1 litre) then this was

allowed to cool and the turbid solution filtered. The mixture was irradiated

under UV light for 24-48 hours, washed with N a 2 S 2 0 3(aq) and the solvent

removed in vacuo giving a pale brown solid which was chrom atographed

(hexane 67-70 °C) to give the phenanthrene as white flakes (0.20 g, 0.97

mmol, 20%), m. p. 143-145 °C (lit. 146 ° C \ 142 °C^, 142-143 °C^).

126


2. P r e p a r a t i o n o f 2 , 4 , 4 ' - t r i m e t h y l b e n z o p h e n o n e 59 (b )

CHCH

This was prepared according to the literature method"^ giving, after

distillation, a very pale-yellow oil in 64% yield, b.p. 138-140 °C/0.07 mm Hg

(lit. 169 °C/4 mm Hg).

3. P r e p a r a t i o n O f 2 , 7 - d im e th y l a n t h r a c e n e 60(b)

Dry 2 ,4 ,4 '-trimethylbenzophenone (10.00 g, 44.59 mmol) was heated

to gentle reflux using a W o o d ’s M etal bath and m aintained at this

temperature for 4-6 hours. The precipitate which formed upon cooling of

the ketone was filtered off and washed lightly with E t2 0 to give a brown

solid, then the filtrate was evaporated and returned to reflux. This was

repeated four or five times. The combined precipitates were then vacuum

sublimed at approximately 100 °C to give yellow plates and needles that

consisted of a mixture of the anthracene and the anthrone (1.57 g). This

was reduced directly by adding the unseparated mixture (0.50 g) to sodium

borohydride (0.50 g, 13.21 mmol) in ‘PrOH, then refluxing for 24-48 hours.

The reaction mixture was poured into a slurry of ice (200 ml) made acidic

by the addition of a few drops of conc. HCl, then the yellow precipitate (0.46

g) was filtered off, dried and column chromatographed (hexane) to give the

highly crystalline anthracene (0.089 g, 0.43 mmol, 1% ) m.p. 230-235 °C (lit.

241 °C" , 240-243 °C^, 238-239 °C^).

127


4. P r e p a r a t i o n o f 2 ,5 ,4 - T r i m e t h y I b e n z o p h e n o n e 5 9 (a )

CH CH

This was prepared according to the literature method^ producing the

title benzophenone as a yellow oil in 11% yield. This crysta llised on

standing and was used without further purification m.p. 4 9 -51°C (lit. 54 °C).

5. P r e p a r a t i o n o f 2, 6 - D i m e t h y l a n t h r a c e n e 6 0 (a )

This was prepared from 2 ,5 ,4 '-trim ethylbenzophenone (18.00 g, 87.3

mmol) using the same method as described in experiment 3, giving a crude

yield of 4.10 g. This an thracene/anthrone m ixture (0.23 g) was then

suspended in *PrOH (25 ml) with sodium borohydride (0.23 g, 6.21 mmol) and

heated to reflux for 24 hours. The product was collected as before, and

recrystallised from hexane (67-70 °C), giving the product as pale-yellow

flakes (0.1085 g, 0.53 mmol, 1%), m. p. 228-230 °C (lit. 230-235 250 °C"^).

6 . P r e p a r a t i o n o f 3 , 6 - B i s ( b r o m o m e t h y l ) p h e n a n t h r e n e 104

Br Br

3,6-Dimethylphenanthrene (1.20 g, 5.81 mmol) was heated to reflux

128


in e c u (15 ml) with N-bromosuccinimide (2.03 g, 11.40 mmol) and benzoyl

peroxide (36 mg) for 6 hours. Filtration, followed by removal of the solvent

in vacuo produced a pale-yellow powder which was recrysta llised from

ethanol to give the dibromide as an off-white powder (1.73 g, 4.75 mmol,

82%), m. p. 160-162 °C (lit. 173-175 °C^).

7. P r e p a r a t i o n of 2 , 7 - B i s ( b r o m o m e t h y l ) a n t h r a c e n e 110 (b)

2,7-D im ethy lan th racene (0.30 g, 1.45 mmol), N -b rom osucc in im ide

(0.50 g, 2.80 mmol) and benzoyl peroxide (10 mg) were heated to reflux in

C C I 4 for 3 hours. The solvent was removed in vacuo and the yellow solid

obtained was recrystallised from EtOH, giving the dibromide as a yellow

powder (0.1778 g, 5.24 mmol, 34%), m. pt. 190-198 °C (lit. 202-204 °C^).

8 . P r e p a r a t i o n of 2 , 6 < B i s ( b r o m o m e t h y l ) a n t h r a c e n e 1 1 0 ( a )

2 ,6 -D im ethylan thracene (0.10 g, 0.48 mmol), N -b rom osucc in im ide

(0.17 g, 0.95 mmol) and benzoyl peroxide (5 mg) were heated to reflux in

C C I 4 (20 ml) for two hours. The solution was filtered, evaporated in vacuo

and recrystallised from 'PrOH, giving the dibromide as a yellow powder

(49.7 mg, 0.14 mmol, 28%), m.p. 186-188 °C.

nmr: 4.70 (s, 2H, H D ; 7.49 (d, 2H, ^ j ^ . h = 7.7Hz, H3,7);

7.97 (s, 2H, Hl,5); 7.98 (d, 2H, ^ J ^ .h = 8.0Hz, H4,8);

8.37 (s, 2H, H9,10)

^^C nmr 35.7; 125.6; 126.8; 127.5; 127.9; 130.4; 131.1; 134.8

129


Mass Spectrum (m/z): 366 [(C i6H i 2* 'B r 2)+, 11%]; 364

[ (C i6H i 2*‘B r79Br)+, 22%]; 362 [ (C if iH i2’ ®Br2>+,

11%]; 285 [(C i6H i 2^^Br)+, 100%]; 283

[(C if iH i2™Br)+, 99%]; 204 [ ( C ie H n ) * , 27%]; 203

[ (C l6H i i )+ , 33%]; 202 [(C ieH io )* , 43%]

UV (EtOH): "max = 265.5nm; 8 = 67760

Accurate Mass: Expected for C i ^ H i i B r ] , 362.9302

Found 362.9305

9. P r e p a r a t i o n of 3 , 6 - B i s ( p i p e r i d o m e t h y l ) p h e n a n t h r e n e 134

3,6-Bis(brom om ethyl)phenanthrene (0.10 g, 0.27 mmol) was heated

to reflux in ethanol (5 ml) then piperidine (0.05 g, 0.06 ml, 0.60 mmol),

made up to 1 ml in ethanol, was added dropwise. After reflux for four hours,

the mixture was diluted with Et20 (20 ml) then washed with 10% H C l(aq)

(2x20 ml). The aqueous layer was removed and the product precipitated by

the careful addition of NH](aq). The product was extracted with Et2 0 (3x2 0

ml), d r ied (M gS 0 4 ) then evaporated to give an orange oil which was

triturated with E t2 0 to give a pale orange powder. After washing with cold

petroleum spirit (b.p. 80-100 °C), the title amine was obtained as a white

powder (26.4 mg, 0.07 mmol, 26%), m.p. 108-110 °C.

200MHz iR nmr (CDCI3): 1.45 (m, 4H, H51; 1.63 (m, 8H, H4'); 2.47 (m, 8H,

H3');3.74 (s, 4H, H r ) ; 7.59 (dd, 2H, = 8.1Hz,

4Jh -h = 1.5Hz, H2,7); 7.68 (s, 2H, H9,10); 7.82 (d, 2H,

^JH-H = 8.1 Hz, H l ,8); 8.60 (d, 2H, ^Jh-H = 0.64Hz,

130


nmr (CDCI3):

H4,5)

24.4; 25.9; 54.6; 64.3; 123.2; 126.3; 128.0; 128.3; 130.0;

131.3; 136.4

Mass Spectrum (m/z): 372 [M'*', 12%]; 288 [(M - Cs H iqN)'^, 27%]; 206

[(C i 6 H i 4)+, 100%); 204 [(C i6H i2)+ , 75%]

UV (EtOH): Xmax = 255.0nm; 8 = 103480

Accurate Mass: Expected for C26H 32N 2, 372.2565

Found, 372.2561

10. Preparat ion of 3,6- Bis( morph ol in ome thy I) p h e n a n t h r e n e 135

M orpholine (0.05 g, 0.05 ml, 0.60 mmol) was treated with 3,6-

b is (b rom om ethy l)phenan th rene (0.10 g, 0 ..27 m m ol) as d esc r ibed in

experim ent 9 , giving a clear oil This was triturated with E t2 0 , giving the

title amine as white crystals (20.0 mg, 0.05 mmol, 20%) m. p. 105-109 °C.

200M Hz iH nmr (CDCI3); 2.53 (m, 8H, H41; 3.7Zf-(m, 8H, H31; 3.77 (s, 4H,

H D ; 7.61 (dd, 2H, ^ J ^ .h = S.2Hz, ^Jh-H = 14Hz,

H2,7); 7.70 (s, 2H, H9,10); 7.84 (d, 2H, = 8.2Hz,

H l ,8); 8.60 (s, 2H, H4,5)

nmr (CDCI3 ): 53.8; 64.0; 67.0; 123.1; 126.4; 127.9; 128.6; 130.0;

131.4; 136.1

Mass Spectrum (m/z): 377 [(M H)+, 100%]; 292 [(M - C4H gN0 )+, 15%];

131


UV (EtOH):

Accurate Mass

206 [(Ci 6 H i 4)+, 10%]; 204 [(C i6H i2)+ , 4%]

^max - 255.Onm; £ = 81410

Expected for C24H 29N 2O 2, 377.2229

Found, 377.2224

11. P r e p a r a t i o n of 3 , 6 - B i s ( p y r r o l i d o m e t h y l ) p h e n a n t h r e n e 133

Pyrrolid ine (0.04 g, 0.05 ml, 0.60 mmol) was treated with 3,6-

b is (b ro m o m eth y l)p h en an th ren e (0 .10 g, 0.27 m m ol) as d esc r ib ed in

experim ent 9. After work-up and trituration with E t2 0 , the title amine was

obtained as pale brown crystals (23.70 mg, 0.07 mmol, 25%) m. p. 95-98 °C.

200 MHz iH nmr (CDCI3): 1.81 (m, 8H, H41; 2.59 (m, 8H, H3'); 3.88 (s, 4H,

m y , 7.60 (dd, 2H, 3Jh_h = 8.1Hz, ^ J ^ .h = 1.5Hz,

H2,7); 7.67 (s, 2H, H9,10); 7.83 (d, 2H, =

8.0Hz,Hl,8); 8.63 (s, 2H,H4,5)

13c nmr (CDCI3): 23.4; 54.2; 61.1; 123.1; 126.4; 127.8; 128.6; 130.1;

131.4; 136.7

Mass Spectrum (m/z): 34.1k[ M+, 10%]; 2^6- [(M - 9 N)+, 27%]; 206

[(C i6H i4)+ , 100%]; 204[(Ci6H 12)+, 74%]

UV (EtOH): ^max = 254.0nm; e = 109110


Found, 345.2330

132


12 . P r e p a r a t i o n o f 6 - M e t h y I - 2 , 4 - d i o x o - 3 , 1 0 - d i o x a t r i c y c I o

[5 .2 .1 .0 i*5]dec-7-ene 126

Me

This was prepared according to the literature method^®, giving off-

white crystals in 77% yield, m.p. 58-59 °C (lit. 59-63 °C).

13. P r e p a r a t i o n o f 3 - M e t h y l p h t h a l i c a n h y d r i d e 127

Me

C o m p o u n d 126 (50.00 g, 0.28 mol) was crushed to a fine powder and

added portionwise to vigorously stirred sulphuric acid (500 ml) held at -2

°C. The temperature of the reaction was raised to 10°C, then the mixture was

poured onto ice (1.5 kg). The yellow precipitate which formed was collected

and washed with ice water, then dissolved in chloroform (100 ml). The

aqueous layer was discarded and the organic layer dried (M gS0 4 ) and

evapora ted in vacuo to give a sticky brown solid which was recrystallised

twice from ^PrOH to give large white needles (6.05 g, 0.04 mol, 14%), m.p.

115-118 °C (lit. 10 117-119 °C).

133


14. P r e p a r a t i o n of 2 - ( 2 - M e t h y l b e n z o y l ) - 6 - m e t h y l b e n z o i c ac id 73

Me OH

This was prepared according to the literature method^®, except 3-

methylphthalic anhydride (5.00 g, 0.031 mol) was added to a solution of o-

tolyl magnesium bromide (c. 1 M in Et2 0 ). This produced an orange powder

which was dissolved in Et20 (50 ml) and washed with 10% HCl(aq) (50 ml).

The organic layer was then dried (M gS0 4 ) and the solvent removed in

v a c u o to give a pale yellow powder (3.68 g, 48%), m. p. 113-115 °C (lit.^®

116-118 °C)

15. Preparation of 3,6 - B i s ( p i p e r i d o m e t h y l ) p h e n a n t h r e n e dimethiodide 23

N++ N-

MeMe 6 '

3 ,6 -B is(p ip e r id o m eth y l)p h en an th ren e (15.0 mg, 0 .04 mmol) was

stirred under N2(g) with iodomethane (0.12 g, 0.05 ml, 0.87 mmol) in freshly

distilled, dry acetonitrile (1 ml) for 24 hours. After pouring into Et2 0 (400

ml), the pale yellow precipitate was collected by filtration and dried (20.0

mg, 0.03 mmol, 76%), m.p. >230°C.

nmr (CDCI3): 1.68 (m, 4H, H51; 1.92 (m, 8H, H41; 2.95 (s. 6H,

H61; 3.43 (m, 8H, H3'); 4.85 (s, 2H, H T); 7.34 (d, 2H,

3Jh-H = 8.0Hz, H2,7); 7.54 (s, 2H, H9,10), 7.68 (d, 2H,

3 Jh -h = 8.1Hz, H l ,8); 8.55 (s, 2H, H4,5)

134


13c nmr (CDCI3): 19.0; 20.8; 42.8; 60.3; 63.6; 125.6; 128.2; 128.4; 129.6;

129.9; 131.1; 132.4

Mass spectrum (m/z): 529 [(M + I)+, 6%]; 303 [(M - C5H 10N - CH])""", 16%];

204 [(C i6H i 2)+, 100%]; 201 [M2+, 35%]

UV (EtOH):

Analysis (%):

^max = 255.0nm; e = 82150

Calculated for C28H 38N 2I2. C: 51.23; H: 5.83; N: 4.27

Found, C: 50.65; H: 6.14; N: 3.99

16. P r e p a r a t i o n o f 3 , 6 - B i s ( p y r r o l i d o m e t h y l ) p h e n a n t h r e n e

d i m e t h i o d i d e 136

+ .N'

Me

3,6 -B is(py rro l idom ethy l)phenan th rene (20.0 mg, 0 .06 m m ol) was stirred under N 2 (g ) with iodomethane (0.16 g, 0.07 ml, 1.16 mmol) in

acetonitrile (1 ml) for 48 hours. The white precipitate which formed in this

time was removed by filtration, washed with Et2 0 and dried in vacuo, giving

the title salt as a white powder (18.2 mg, 0.06 mmol, 50%) m. p. >230 °C.

nmr (d^-DMSO):

13c nmr (d^-DMSO):

1.68 (m, 8H, m y , 2.52 (s, 6H, H5'); 2.99 (m, 4H,

H3aO; 3.23 (m, 4H, H3sO; 4.35 (s, 4H, HU); 7.38 (d,

2H, 3 J h .h = 8.1Hz, H2,7); 7.55 (s, 2H, H9,10); 7.69

(d, 2H, 3 J h .h = 8.1Hz, H l ,8); 8.63 (s, 2H, H4,5)

20.9; 47.6; 63.0; 65.8; 127.6; 127.7; 127.9; 129.3; 129.5;

130.7; 132.5

135


Mass Spectrum (m/z): 501 [(M + I)+, 12%]; 359 ((M - CH3)+, 2%]; 289 [(M

C4H iiN )+ , 11%]; 274 [(M - C4H 11N - CH3)+, 3%];

204 [(Ci6H|2)+.15%]; 187 [M2+, 7%]

UV (H2O):

Analysis, %:

^max = 255.0nm; £ = 64620

Calculated for C26H 34N 2I2. C: 49.70; H:5.45; N: 4.46

Found, C: 49.33; H: 5.74; N: 4.19

17. P r e p a r a t i o n of 3, 6 - B i s ( m o r p h o l i n o m e t h y l ) p h e n a n t h r e n e


+ N-

5 Me Me

3,6-B is(m orpholinom ethyl)phenanthrene (15.0 mg, 0.04 mmol) was

quaternised as described in experiment 15 using iodomethane (0.16 g, 0.07

ml, 1.16 mmol). The reaction mixture was then poured into dry Et2 0 (100 ml)

and the resultant precipitate was removed by filtration to give the title salt

as a white powder (20.0 mg, 0.03 mmol, 76%) m. p. >230 °C.

I r nmr (dg-DMSO): 2.00 (m, 8H, H41; 2.67 (s, 6H, H5'); 3.51 (m, 8H, H31;

4.46 (s, 4H); 7.37(d, 2H, = 8.1Hz, H2,7); 7.56

(s,2H, H9,10); 7.69 (d, 2H, = 8.1Hz, H l ,8); 8.57

(m, 2H, H4,5)

13c nmr (d6-D M S0): 45.3; 58.9; 60.0; 125.8; 128.0; 128.5; 129.3; 129.4;

131.3; 132.6

Mass Spectrum (m/z): 533 [(M + I)+, 12%]; 405 [(M - H)"*", 2%]; 391

[(M - CH3)+, 11%]; 305 [(M - C 4 H n N 0 ) + , 10%];

288[(M - C4H 11NO - CH3)+, 5%]; 204 [(C i6H i 2)+,

136


UV (H2O):

Analysis (%):

48%]; 202 [M^+, 7%]

Imax = 255.5nm; E = 68940

Cklculatedfor C26H 34N2O 2I2, C: 47.29; H: 5.19; N:4.24

Found: C: 46.57; H: 5.29; N: 3.88

18. P r e p a r a t i o n o f 2 , 6 -B is ( p ipe rid om e t h y l ) a n t h r a c e n e 138

2,6-Bis(bromom ethyl)anthracene (0.10 g, 0.27 mmol) was heated to

reflux in ethanol (5 ml) then piperidine (0.05 g, 0.06 ml, 0.60 mmol) in

ethanol (1 ml) was added dropwise. Reflux was continued for four hours

then the mixture was worked up as described in experiment 9 to give a pale

yellow powder (54.1 mg, 0.15 mmol, 54%), m.p. 184-188 °C.

1h nmr (CDCI3):

nmr (CDCI3):

1.43 (m, 4H, H5'); 1.59 (m, 4H, H4'); 2.42 (m, 4H,

H31; 3.64 (s,4H, H I ') ; 7.46 (dd, 2H, ^j ^ . h = 8.7Hz,

4Jh -h = 1.4Hz, H3,7); 7.83 (s, 2H, Hl,5); 7.92 (d, 2H,

3Jh.h = 8.7Hz, H4,8); 8.32 (s,2H, H9,10)

24.4; 25.9; 54.6; 64.1; 125.6; 127.3; 127.4; 127.9; 131.2;

131.4; 135.3

Mass Spectrum (m/z): 373 [(M + H)+, 100%]; 290 [(M - C5HioN)+, 8%]; 206

[ (C i 6H i 4)+, 5%]

UV (EtOH): ^max “ 258.Onm, £ — 110640


Found, 373.2640

137


19. P r e p a r a t i o n o f 2 , 6 - B i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e 142

2,6-Bis(bromom ethyl)anthracene (0.10 g, 0.27 mmol) was treated as

experim ent 17 with morpholine (0.05 g, 0.05 ml, 0.60 mmol). Work up as

before gave a yellow oil which was triturated with Et2 0 to give pale yellow

crystals (55.7 mg, 0.15 mmol, 55%), m.p. 204-206 °C.

nmr (CDCI3): 2.51 (br s, 8H, H4'); 3.67 (s,4H, H I ') ; 3.73 (m,8H,

H3'); 7.48 (dd, 2H, 3j h _h = 8.7Hz, = 12Hz,

H3,7); 7.84 (s,2H, Hl,5); 7.94 (d, 2H, 3J h .h = 8.7Hz,

H4,8); 8.33 (s,2H, H9,10)

13c nmr (CDCI3): 53.7; 63.7; 63.8; 125.6; 125.7; 127.1; 127.6; 128.2;

131.3; 131.4

Mass Spectrum (m/z): 377 [(M + H)+, 100%]; 292 [(M - C4H 8NO + 2H)+,

13%]; 206 [(Ci 6H i 4)+, 7%]

UV (EtOH): Xmax - 258.0; £ = 25000

Accurate Mass: Expected for C24H 28N 2O 2» 376.2150

Found 376.2155

20 . P r e p a r a t i o n o f 2 , 6 - B i s ( p y r r o l i d o m e t h y l ) a n t h r a c e n e 140

138


2,6-Bis(bromom ethyl)anthracene (0.10 g, 0.27 mmol) was treated as

described in experiment 17 with pyrrolidine (0.04 g, 0.05 ml, 0.60 mmol).

Work up as before gave the title amine as an off-white powder (47.8 mg,

0.14 mmol, 51%), m.p. >230 °C (dec.).

nmr (CDCI3); 1.83 (br s, 8H. H4'); 2.64 (br s, 8H, H31; 3.84 (s,4H,

H I ') ; 7.50 (d, 2H, 3 J h .h = 8.6Hz. H3,7); 7.89 (s, 2H,

H I,5); 7.94 (d, 2H, = 8.6Hz, H4,8); 8.34 (s, 2H,

H9,10)

nmr (CDCI3): 23.4; 54.2; 60.8; 125.8; 127.1; 127.3; 128.3; 131.2;

131.5; 135.2

Mass Spectrum (m/z): 345 [(M + H)+, 100%]; 276 [(M - C4H 8N + 2H)+, 5%];

206 [(Ci6Hi4)+. 6%]

UV (EtOH): Xmax - 258.0nm; £ = 141840


Found 345.2334

21. P r e p a r a t i o n of 2,6- B is ( p ip e r id o m e th y 1)a n t h r a c e n e d im e th io d id e 139

Me

2,6-B is(p iperidom ethyl)an thracene (40.0 mg, 0.11 mol) was stirred

under N 2(g) with iodomethane (0.32 g, 0.14 ml, 2.32 mmol) in acetonitrile (1

ml) for four days. The mixture was then poured into dry E t2 0 and the

resultant precipitate was filtered and dried, giving the title salt as a white

powder (33.4mg, 0.05 mmol, 47%), m.p. >230 °C.

139


nmr (dg-DMSO):

nmr (d^-DMSO):

1.53 (br m, 2H, H5a'); 1.63 (hr m, 2H, H5s'); 1.92

(br m, 8H, H4'); 3.02 (s, 6H, H6 '); 3.39 (m. 8H, H3');

4.79 (s,4H, H r ) ; 7.65 (d, 2H, = 8.8Hz, H3,7);

8.27 (d. 2H, 3Jh _h = 8.7Hz, H4,8); 8.34 (s, 2H, Hl,5);

8.78 (s, 2H, H9,10)

19.4; 20.9; 43.1; 60.0; 63.4; 125.7; 127.3; 128.9; 129.3;

131.2; 131.3; 134.1

Mass Spectrum (m/z): 529 [(M + I)+, 10%]; 401 [(M - H)+, 2%]; 387 [(M

C H 3)+, 3%]; 303 [(M - C e H n N ) ^ 22%]; 288 [(M -

C6H 13N - CH3)+, 6%]; 204 [ (C iô H n )^ , 100%]

UV (H2O):

Analysis (%):

Xmax = 260.0nm; 6 = 230790

Calculated for C28H 38N 2I2, C: 51.23; H: 5.83; N: 4.27

Found, C: 49.86; H: 5.97; N: 4.15

2 2 . P r e p a r a t i o n o f 2 , 6 - B l s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e


Me

2 ,6 -B is(m orpho linom ethy l)an th racene (40.0 mg, 0.11 m m ol) was

treated with iodomethane (0.32 g, 0.14 ml, 2.32 mmol) as experiment 20. This

produced the title salt as a pale yellow powder (34.0 mg, 0.05 mmol, 48%),

m.p. >230 °C.

nmr (d^-DMSO): 2.49 (m, 8H, H41; 3.16 (s, 6H, H51; 4.01 (m, 8H,

H31; 4.89 (s, 4H, HU); 7.67 (d, 2H, 3Jh_h = 8.7Hz,

140


nmr (d^-DMSO):

H3,7); 8.28 (d, 2H. 3Jh_h = 8.8Hz, H4,8); 8.37 (s, 2H,

H I ,5); 8.78 (s, 2H, H9,10)

45.2; 58.8; 59.9; 67.9; 125.2; 127.4; 129.0; 129.3; 131.2;

131.3; 134.3

Mass Spectrum (m/z): 533 [(M + I)+, 17%]; 405 [(M)+; 1%]; 391 [(M -

C H 3)+; 12%); 305 [(M - C5H n N O ) + , 37%]; 288 [(M

C 5H 11NO - CH3>+ 5%]; 204 [(C i6H i 2)+, 95%]; 202 [

M2+, 11%]

UV (H2O):

Analysis (%):

^max — 260.5nm; 6 = 240500

Calculated for C26H34N2O2I2. C: 47.29; H: 5.19; N:4.24

Found: C: 46.38; H: 5.33; N: 3.92

23 P r e p a r a t i o n of 2, 6- B i s ( p y r r o l i d o m e t h y l ) a n t h r a c e n e d im e th io d id e 141

Me

2 ,6 -B is (p y r ro l id o m e th y l )a n th ra c e n e (40 .0 mg, 0 .12 m m ol) was

treated with iodomethane (0.32 g, 0.14 ml, 2.32 mmol) as described in

experiment 20. This produced the title salt as a pale yellow powder (23.8 mg,

0.04 mmol, 32%), m.p. >230 °C.

nmr (dg-DMSO): 2.18 (m, 8H, H40; 2.98 (s, 6H, H5'); 3.47 (m, 4H,

H 3 a l; 3.68 (m, 4H, H3s'); 4.77 (s, 4H, HU); 7.68 (dd,

2H, 3j h _h = 9.2Hz; = 13Hz, H3,7); 8.27 (d, 2H,

3Jh -h = 8.7Hz, H4,8); 8.37 (s, 2H, Hl,5); 8.77 (s, 2H,

H9,10)

141


nmr (d^-DMSO): 20.9 (C4?); 47.5; 63.0; 65.4; 127.0; 127.3; 128.8; 129.1;

131.26; 131.33; 133.4

Mass Spectrum (m/z): 501 [(M + I)+, 28%]; 359 [(M - CH3>+, 12%]; 289 [(M

- C5H i i N)+, 34%]; 274 [(M - C 5H nN -C H 3)+ 26%];

204 [(Ci 6H i 2)+, 100%]; 202 [M2+, 10%]

UV (H2O):

Analysis, %:

^max - 258.0nm; e = 192510


Found, C: 48.92; H: 5.55; N: 4.39

24 . P r e p a r a t i o n o f 2 , 7 - B i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e 148

2 ,7-B is(b rom om ethy l)an th racene (0.10 g, 0.27 m m ol) was reacted

with morpholine (0.05 g, 0.05 ml, 0.60 mmol) using the method described for

2 ,6 -b is (b ro m o m e th y l)a n th ra ce n e in ex p e r im en t 18 . This gave an orange

gum which was triturated with E t2 0 to give the title amine as a yellow

powder (34.8 mg, 0.09 mmol, 34%), m.p. 177-180 °C.

nmr (CDCI3):

13c nmr (CDCI3):

2.54 (m, 8H, H4'); 3.71 (s, 4H, H I ') ; 3.76 (m, 8H,

H3'); 7.50 (dd, 2H, = 8.7Hz, ^j ^ . h = 1.5Hz,

H3,6); 7.87 (s, 2H, Hl,8); 7.96 (d, 2H, 3Jh _h = 8.7Hz,

H4,5); 8.34 (s, IH, H9); 8.38 (s, IH, HIO)

53.7; 63.7; 66.9; 125.6; 125.8; 127.0; 127.7; 128.3;

131.1; 131.6; 135.0;

Mass Spectrum (m/z) 377 [(M + H)+, 100%; 292 [(M - C 4H gN 0)+ , 6%]; 206

[(Ci 6Hi4)+, 2%]

142


UV (EtOH): Xmax = 256.Onm; £ = 154730

Accurate Mass: Expected for C24H 29N 2O 2, 377.2229

Found, 377.2233

25 . P r e p a r a t i o n o f 2 , 7 - B i s ( p y r r o l i d o m e t h y l ) a n t h r a c e n e 146

2 ,7-B is(b rom om ethy l)an th racene (0.10 g, 0.27 m mol) was trea ted

with pyrrolidine (0.04 g, 0.05 ml, 0.60 mmol) in ethanol (15 ml) using the

m ethod described for 2 ,6-b is(brom om ethyl)an thracene in experim en t 19 .

Work up as before produced a yellow oil which was triturated with E t2 0 to

give the title amine as a pale yellow powder (58.9 mg, 0.17 mmol, 63%), m.p.

182-184 °C.

iH nmr (CDCI3): 1.83 (m, 8H, H4'); 2.61 (m, 8H, H3'); 3.87 (s, 4H,

HU); 7.56 (dd, 2H, % _ H = 8.6Hz, 4Jh_h = 1.2Hz,

H3,6); 7.94 (dd, 2H, = 8.5Hz, ^ J ^ .h = 1.2Hz,

H4,5); 8.35 (s, 3H, HI, H8, H9); 8.38 (s, IH, HIO)

13c nmr (CDCI3): 23.5; 54.2; 61.1; 126.6; 127.2; 128.8; 128.8; 130.6;

130.9; 131.5; 138.1

Mass Spectrum (m/z): 345 [(M + H)+, 20%]; 284 [(M - C4HgN)+, 13%]; 205

[(C i6H i3 )+ , 7%]

UV (EtOH): ^max - 262.0nm; £ = 81470

Accurate Mass: Expected for C24H29N 2, 345.2343

Found, 345.2331

143


26 . P r e p a r a t i o n o f 2 , 7 - B i s ( p l p e r i d o m e t h y l ) a n t h r a c e n e 14 tf.

2 ,7 -B is(b rom om ethy l)an th racene (0.10 g, 0 .27 m m ol) was reacted

with piperidine (0.05 g, 0.06 ml, 0.60 mmol) using the method described for

2 ,6 -b is (b ro m o m e th y l)a n th ra ce n e in expe rim en t 1 7 . Upon cooling of the

reac tio n m ix tu re , f ine w hite needles o f an ac id so lu b le m ate r ia l

p rec ip ita ted and these were filtered off, washed with cold e thanol and

dried, giving the title amine, (54.2 mg). Partial evaporation of the filtrate

provided a further crop of material (6.9 mg, 0.16 mmol, 61% total yield) m.p.

166-168 °C.

nmr (CDCI3): 1.46 (m, 4H, H5'); 162 (m, 8H, H4'); 3.71 (s, 4H,

HU); 2.45 (m, 8H, H 307.56 (dd, 2H, ^ j ^ . h = 9.1Hz,

% - H = 1.3Hz, H3,6); 7.93 (d, 2H, ^ j ^ . h = 8.6Hz,

H4,5); 8.33 (s, 3H, HI, H8, H9); 8.37 (s, IH, HIO)

nmr (CDCI3): 24.4; 26.0; 54.6; 64.2; 121.5; 126.5; 126.7; 127.3; 128.6;

130.5; 131.5; 137.9

Mass Spectrum (m/z) 345 [(M + H)+, 19%]; 284 [(M - C4HgN)+, 14%]; 205

[(Ci 6H i 3)+. 8%]

UV (EtOH): ^max - 261.5nm; £ = 135850

Accurate Mass: Expected for C26H 33N2 , 373.2641

Found 373.2644

27. P r e p a r a t i o n of 2 ,7 - B i s ( m o r p h o l i n o m e t h y l ) a n t h r a c e n e d im e th io d id e 149

144


Me

21'

2 ,7 -B is(m orpho linom ethy l)an th racene (35.2 mg, 0 .09 m m ol) was

stirred with iodomethane (0.12 g, 0.06 ml, 0.88 mmol) in acetonitrile (2 ml)

for 24 hours. After pouring the reaction m ixture into dry E t2 0 , the

resultant precipitate was removed by filtration and dried, to give the title

salt as a white powder (36.2 mg, 0.05 mmol, 59%) m.p. >230 ®C.

nmr (d^-DMSO): 2.49 (m, 8H, H4'); 3.15 (s, 6H, H5'); 4.01 (m, 8H,

H30; 4.89 (s, 4H, HK); 7.68 (d, 2H, ^ J ^ .h = 8.3Hz,

H3,6); 8.26 (d, 2H, = 8.1Hz, H4,5); 8.39 (s, 2H,

H l ,8); 8.76 (s, IH, H9); 8.79 (s, IH, HIO)

nmr (dg-DMSO): 43.6; 58.8; 59.9; 67.6; 124.9; 128.90; 128.94; 129.0;

130.6; 130.9; 131.8; 134.4

Mass Spectrum (m/z): 407 [(M + H)+, 2%]; 391 [M - CH3)+, 22%]; 305 [(M -

C 5 H iiN 0 )+ , 10%]; 288 [(M - C5H 11NO - CH3)+, 4%];

204 [(C i6H i 2)- . 23%]

UV (H2O):

Analysis (%):

%max = 259.0nm; £ = 123190

Calculated for C26H34N 2O 2I2» C: 47.29; H: 5.19; N:4.24

Found; C: 46.67; H: 5.29; N: 4.05

145


28. P r e p a r a t i o n of 2 ,7 -B i s ( p y r r o l i d o m e t h y 1)a n t h r a c e n e d im e t h i o d i d e 147

, Me Me

21'

2,7-Bis(pyrrolidomethyl)anthracene (40.0 mg, 0.12 mmol) was stirred

under N 2(g) in acetonitrile (1 ml) with iodomethane (0.32 g, 0.14 ml, 2.32

mmol) for 48 hours. The mixture was then poured into Et2 0 (100 ml) and the

resultant precipitate filtered and dried, giving the title salt as a white

powder, (11.9mg, 0.02 mmol, 16%) m.p. >230°C.

H nmr (dg-DMSO): 2.19 (m, 8H, H4'); 3.00 (s, 6H, H5'); 3.50 (m, 4H,

H3a'); 3.69 (m, 4H, H 3 s l ; 4.90 (s, 4H, HI ); 7.78 (dd,

2H, 3Jh_h = 8.8Hz, 4Jh_h = 1.3Hz, H3,6); 8.35 (d, 2H,

3 Jh -h = 8.8Hz, H4,5); 8.68 (s, 3H,H3,8,9); 8.91 (s,

IH, HIO)

nmr (d^-DMSO): 20.8; 47.3; 63.0; 65.3; 122.8; 127.7; 129.30; 129.33;

129.7; 130.0; 132.1; 132.3

Mass Spectrum (m/z): 359 [(M - CH3)+, 1%]; 289 [(M - C4H 8N - % ) + , 2%];

206 [(C i6H i 4)+, 6%]; 85 [(C4H 8N + CH3)+, 100%]; 70

[(C4H 8N)+, 25%]

UV (H2O):

Analysis, %:

^max = 260.5nm; £ = 134680


Found, C: 48.34; H: 5.54; N: 4.17

146


29. P r e p a r a t i o n of 2 , 7 - B i s ( p i p e r i d o m e t h y l ) a n t h r a c e n e d i m e t h i o d i d e 145

Me

21"

2,7-Bis(piperidomethyl)anthracene (40.0 mg, 0.11 mmol) was stirred

with iodomethane (0.16 g, 0.07 ml, 1.16 mmol) in acetonitrile (1 ml) for 48

hours then poured into dry Et2 0 (100 ml). The resultant precipitate was

then filtered and dried, giving the title salt as an off-white powder (64.0

mg, 0.10 mmol, 91%) m. p. >230°C.

nmr (dg-DMSO): 1.75 (m, 4H, H50; 1.94 (m, 8H, H4'); 3.05; (s, 6H,

H60; 3.48 (m, 8H, H3'); 4.93 (s, 4H, HI ); 7.76 (d,

2H, = 8.8Hz, H3,6); 8.34 (d, 2H, ^ J ^ .h = 8.9Hz,

H4,5); 8.67 (s, 3H, Hl,8,9); 8.92 (s, IH, HIO)

nmr (d^-DMSO): 19.4; 20.8; 28.4; 46.2; 60.0; 122.8; 127.6; 128.0; 129.6;

129.76; 129.80; 132.3; 132.9

Mass Spectrum (m/z): 387 [(M - CH3)+, 10%]; 303 [(M - C5H 10N - CH3)+,

5%]; 206 [(C :6H i 2)+, 45%]; 201 [M2+, 5%] 99

[(C5H 10N + CH3)+, 98%]

UV (H2O):

Analysis (%):

A«max “ 260.Onm; 6 — 121400

Calculated for C28H 38N 2I2» C: 51.23; H: 5.83; N: 4.27

Found, C: 50.87; H: 6.01; N: 4.12

147


30. P r e p a r a t i o n o f 2 , 7 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o m e t h y l ] a n t h r a c e n e 152

Me

Me Me'

2 ,7-B is(brom om ethyl)an thracene (0.10 g, 0.27 mmol) was heated to

reflux in EtOH (10 ml) then 2,5-dimethylpyrrolidine (0.06 g, 0.07 ml, 0.60

mmol) was added. Reflux was continued for one hour then the solvent

vo lum e was halved and w ater carefu lly added to the m ixture until

precipitation just began to occur. After cooling and standing overnight, a

pale yellow precipitate formed which was removed by filtration. This solid

was then dissolved in 10%HCl(aq) and precipitated by the careful addition

of aqueous amm onia solution. Extraction of this precipita te with E t2 0

fo llowed by drying (M gS0 4 ) and vacuum removal of the solvent gave the

title amine as a yellow powder (62.4 mg, 0.16 mmol, 58%), m.p. 142-147 °C.

200 MHz 1 h nmr (CDCI3): 1.05 (d, l OHz, H5c'); 1.08 (d, 6 H, ^ j ^ . h =

1.0 Hz, H5tO; 1.42 (m, 4H, H4'); 1.82 (m, 4H, H41;

2.70 (m, 4H, H3 ); 3.95 (s, 4H, H r ) ; 7..53 (dd, 2H,

3 Jh -H = 8.2Hz, 4 jh _ h = l .lH z, H3,6); 7.90 (d, 2H,

3JH_H = 8.6Hz, H4,5); 8.34 (s, 3H, Hl,8,9); 8.36 (s,

IH, HIO)

13c nmr (CDCI3): 20.8; 31.4; 56.5; 60.5; 126.2; 126.46; 126.50; 127.8;

128.1; 128.2; 130.5; 130.4

Mass Spectrum (m/z): 401 [(M + H)+, 22%]; 304 [(M - C6H 12N + 2H)+,

16%]; 205 [(C i6H i 3)+. 12%]

UV (EtOH) ^max - 262.5nm; £ = 81260

Accurate Mass: C 28H 36N 2 requires 400.2879

Found 400.2873

148


31. P r e p a r a t i o n o f 2 , 7 - B l s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o m e t h y l ] a n t h r a c e n e

d im e th iodide 153

Me5 MeMe

Me Me

21*

2 ,7 -8 is [ (2 ,5 -d im e th y l)p y rro l id o m e th y l] an th racene (35 .0 mg, 0 .09

mmol) was stirred with iodomethane (0.25 g, 0.11 ml, 1.77 mmol) in

acetonitrile (1 ml) for 24 hours. After pouring into E t2 0 (400 ml), the

resultant precipitate was allowed to settle, then the ether was decanted and

the m oist solid carefully dried in vacuo to give the title salt as yellow

powder (7.4mg, 0.01 mmol, 12%), m.p. >230 °C.

nmr (d^-DMSO):

13c nmr (d6-D M S0):

1.43 (d, 12H, 3Jh . h = 6.5Hz, H5'); 168 (m, 4H, H4');

2.08 (m, 4H, H4'); 2.79 (s, 6H, H6'); 3.63 (m, 4H,

H31; 4.88 (s, 4H, H I ') ; 7.68 (d, 2H, ^j ^ . h = 8.6Hz,

H3,6); 8.34 (d, 2H, = 8.7Hz, H4,5); 8.62 (s, 3H,

Hl,8,9); 8.92 (s, IH, HIO)

13.4; 26.5; 36.3; 60.7; 67.6; 125.5; 128.1; 129.3; 129.6;

130.0; 130.7; 132.3; 132.5

Mass Spectrum (m/z): 557 [(M + I)+, 4%]; 401 [(M - CH3)+, 5%]; 317 [(M

C 6H 12N - CH3)+, 15%; 215 [M2+, 5%] ; 204

[(Ci6Hi2)+. 100%]

UV (H2O):

Analysis (%):

Im ax = 262.0nm; e = 135320

Calculated for C28H 42N 2I2. C: 52.64; H; 6.19; N:4.09

Found C: 51.02; H: 6.31; N: 3.87

149


32. P r e p a r a t i o n of 2 , 6 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o n i e t h y l ] a n t h r a c e n e 154

5 'Me

Me

Me

Me

2,6-Bis(bromom ethyi)anthracene (0.10 g, 0.27 mmol) was treated as

described in experiment 30 . Reflux was maintained for two hours then the

reaction was allowed to cool overnight, producing a precipita te of pale

yellow plates. These were filtered off, washed with cold ethanol and dried,

giving the title amine (53.8 mg, 0.14 mmol, 50%), m. p. 142-146 °C.

nmr (CDCI3): 1.07 (d, 3Jh _h = 2.3Hz, HSc'); 110 (d, 6H, HSt'); 1.43

(hr s, 4H, H40; 1.87 (m, 4H, H4'); 2.70 (m, 4H, H3');

3.91 (s, 4H, H r ) ; 7.45 (dd, 2H, 3Jh _h = 8.7Hz, 4Jh . h =

1.6Hz, H3,7); 7.85 (s, 2H, Hl,5); 7.91 (d, 2H, =

8.5Hz, H4,8); 8.34 (s, 2H, H9,10)

nmr (CDCI3): 20.4; 31.0; 55.8; 60.4; 125.7; 127.5; 127.75; 127.82;

131.2; 131.4; 135.0

Mass Spectrum (m/z); 401 [(M + H)+, 100%]; 304 [(M - C6H 12N + 2H)+,

7%]; 206 [(Ci 6 H i 4)+. 4%]

UV (EtOH): Xmax = 258.0nm; E = 175300

Accurate Mass: C 28H 36N 2 requires 400.2879

Found 400.2876

150


33. P r e p a r a t i o n of 2 , 6 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l l d o m e t h y l ] a n t h r a c e n e

d im eth io d id e 155

5 MeMe

Me

N +

Me MeMe21*

2 ,6 -B is [ (2 ,5 -d im e th y l )p y r ro l id o m e th y l ]a n th ra c e n e (0 .03 g, 0 .075

mmol) was treated as described in experiment 31 . The reaction mixture was

then poured into dry Et2 0 and the resultant precipitate filtered, giving the

title salt as a white powder (47.3 mg, 0.07 mmol, 92%), m. p. >230 °C.

nmr (d^-DMSO):

nmr (d^-DMSO):

1.43 (d, 12H, = 6.5Hz, H5'); 1.68 (m, 4H, H4');

2.07 (m, 4H, H4'); 2.78 (s, 6H, H6'); 3.65 (m, 4H,

m y , 4.76 (s, 4H, H I ') ; 7.58 (dd, 2H, = 8.8Hz,

4Jh -h = 1.3Hz, H3,7); 8.26 (d, 2H, = 8.9Hz,

H4,8); 8.31 (s, 2H, Hl,5); 8.80 (s, 2H, H9,10)

13.5; 26.4; 36.3; 60.8; 67.4; 125.7; 127.27; 127.29;

129.1; 131.1; 131.2; 133.7

Mass Spectrum (m/z): 557 [(M + I)+, 28%]; 429 [(M - H)+, 5%]; 401 [(M

CH3)+, 3%]; 317 [(M - C 6H i2N )+ , 35%]; 302 [(M -

C6H 12N - CH3)+, 8%], 215 [M2+, 15%]

204 [(C i 6 H i 2)+, 100%]

UV (H2O):

Analysis (%):

A-max ~ 260.Onm, £ — 239340

Calculated for C30H 42N 2I2, C: 52.64; H: 6.19; N:4.09

Found, C: 51.76; H: 6.37; N: 3.87

151


34 . P r e p a r a t i o n of 2 , 6 - B l s ( b r o m o m e t h y l ) n a p h t h a l e n e 130

2,6-Dimethylnaphthalene (0.30 g, 1.92 mmol) was heated to reflux in

C C I4 (20 ml) with benzoyl peroxide (15.7 mg) and N-bromosuccinimide (0.68

g, 3.81 mmol) for one hour. Filtration of the reaction mixture followed by

evaporation gave a light-sensitive, yellow solid which was recrysta llised

from ethanol to give the title dibromide as white crystals (0.22 g, 0.61 mmol,

32%), m.p. 101-103 °C.

nmr (CDCI3): 4.64 (s, 4H, H I'); 7.50 (dd, 2H, = 8.3Hz,

H = 1.7Hz, H3,7); 7.79 (d, 2H, ^ J^ .h = 8.5Hz, H4,8);

7.80 (s, 2H, Hl,5)

13c nmr (CDCI3): 33.8 ( C r ); 127.4 (Ct); 127.7 (Q ); 128.8 (Q);

132.8 (Cq); 135.9 (Cq)

Mass Spectrum (m/z): 316 [(C i2H i o * ‘B r2)+, 19%]; 314

[ (C i2H i o ’ ^B r8 'B r)+ , 40%]; 312 [ (C i2H io ' '^ B r 2)=

20%]; 235 [(C12H io**Br)+, 99%]; 233

[(Ci2Hio'79Br)+, 99%]; 154 [ (C ,2H io )+ . 100%]

UV (EtOH): ^max = 227.Onm; £ = 79490

Accurate Mass: Expected for C i 2H io B r2 311 .9158

Found 311.9149

152


35. P r e p a r a t i o n of 2 , 6 - B i s [ ( 2 , 5 - d i m e t h y l ) p y r r o l i d o n i e t h y l ] n a p h t h a l e n e 156

5 M eM e

M eM e

2,6-B is(brom om ethyl)naphthalene (0.10 g, 0.32 mmol) was refluxed

in ethanol (10 ml) with 2, 5-dimethylpyrrolidine (0.06 g, 0.08 ml, 0.65 mmol)

for two hours. After work-up as described in experiment 9 , a sticky yellow

oil was obtained which resisted trituration with a number of solvents and

so was isolated by prec ip ita tion from ethanol solution by the careful

addition of water. This gave a turbid solution which crystallised overnight

to give white flakes of the title amine which were filtered off and dried

(18.6 mg, 0.05 mmol, 17%), m.p. 50-52 °C.

nmr (CDCI3): 1.07 (d, 12H, 3Jh _h = 5.4Hz, H5'); 1.38 (m, 4M, H41;

1.77 (m, 4H, H4'); 2.59 (m, 4H, H3'); 3.87 (s, 4H,

H r ) ; 7.43 (dd, 2H, = 8.8Hz, ^ J ^ .h = 0.8Hz,

H3,7); 7.68 (s, 2H, Hl,5); 7.72 (d, 2H, 3J h _h = 8.3Hz,

H4,8)

nmr (CDCI3): 20.7 ( C4-' , m), 30.0 ( C 5 ' , m); 31.2

(C5‘); 59.8 ( C D ; 127.3 (Q); 127.5 (Q); 128.0 (Q);

132.4 (Cq); 135.2 (Cq)

Mass Spectrum (m/z): 351 [(M + H)+, 100%]; 252 [(M - C6H i 2N)+, 5%]; 154

[ (C i2H io)+ , 3%]

UV (EtOH): ^max = 236.0nm; £ = 65150


Found 350. 2716

153


36 . P r e p a r a t i o n of 2 ,6 - B i s [ ( 2 ,5 - d i m e t h y l ) p y r r o l id o m e th y l ] n a p h t h a i e n e

d im e th io d id e 157

MeM e

M eM e M e

2 ,6 -B is [ (2 ,5 -d im e th y l)p y rro l id o m e th y l ]n a p h th a le n e (12 .0 mg, 0.03

mmol) was stirred in acetonitrile (1 ml) with iodomethane (60.0 mg, 0.03 ml,

0.44 mmol). A white precipitate formed which gave a f locculent material

after pouring into dry E t2 0 (200 ml). This was filtered off and dried, giving

the title salt as a white powder (14.0 mg, 0.02 mmol, 64%), m. p. >230 °C.

nmr (dg-DMSO): 1.42 (d, 12H, 3Jh _h = 6.3Hz, H5'); 1.66 (m, 4H, H41;

2.04 (m, 4H, H41; 2.75 (s, 6H, H6'); 3.54 (m, 4H,

H3'); 4.72 (s, 4H, HK); 7.64 (d, 2H, ^j ^ . h = 8.3Hz,

H3,7); 8.14 (d, 2H, = 8.0Hz, H4,8); 8.15 (s, 2H,

H l ,5 )

nmr (d^-DMSO): 13.4 ; 26.4 ; 36.3 ; 60.4 ; 67.1 ; 126.9; 129.2; 129.9;

132.68; 132.74

Mass Spectrum (m/z): 267 [(M - C6H 12N - CH3)+, 49%]; 252 [(M - C g H ^ N

2CH3)+, 1%]; 190 [M2+, 24%]; 98 [(C6Hi2N)+, 65%]

UV (H2O):

Analysis (%):

^max = 230.5nm; £ = 192220

Calculated for C26H40N 2I2, C: 49.22; H;6.36; N:4.42

Found, C: 48.87; H: 6.15; N; 4.11

37. P r e p a r a t i o n of T r a w s - 4 , 4 ' - b i s ( b r o m o m e t h y l ) s t i l b e n e 1 2 8

154


7 raAi5-4 ,4 '-dimethylstilbene (0.50 g, 1.37 mmol) was heated to reflux

in CCI4 (25 ml) with benzoyl peroxide (25 mg) and N-bromosuccinimide

(0.47 g, 2.65 mmol) for 2 hours. Filtration of the reaction mixture followed

by evaporation in vacuo and recrystallisation from EtOH produced the title

dibromide as a pale yellow powder (0.60 g, 0.93 mmol, 68%), m.p. 155-158 °C.

nmr (CDCI3): 4.51 (s, 4H, HI); 7.09 (s, 2H, H6); 7.38 (d, 4H, ^ j ^ . h

= 8.2Hz, H3 or 4); 7.48 (d, 4H, = 8.3Hz, H3 or 4)

13c nmr (CDCI3):

Mass Spectrum (m/z):

33.4 (Cl); 126.9 (Ct); 128.6 (Ct); 129.5 (Ct);

137.2 (Cq); 137.3 (Cq)

368 [(C i6H i48lB r2)+ , 2%]; 366

[ (C i6 H i4 ’ 5Br8lBr)+ , 4%] ; 364 [ ( C i e H u ’ ^Braj-^,

2%]; 287 [(C i6H i48 'B r)+ , 65%]; 285

[(C i6Hi479Br)+, 59%]; 206 [(C :6H i4)+ , 100%]

UV (EtOH): ^max = 322.Onm; £ = 36820

Accurate Mass: Expected for 364.9462

Found, 364.9469

38. Preparation of T r a / i s - 2 , 4 ' - b i s ( b r o m o m e t h y l ) s t i l b e n e 1 2 9

155


7’r a « 5-2 ,4 '-dimethylstilbene (0.50 g, 2.40 mmol) was refluxed in CCI4

(20 ml) with benzoyl peroxide (20.0 mg) and N-bromosuccinimide (0.85 g,

4.78 mmol) for two hours. Reduction of the crude, filtered mixture gave a

yellow oil which was flash chrom atographed (CH2 C I 2 ) to give a sticky

yellow powder. This was triturated with E t2 0 , giving the title dibromide as a

white powder (0.11 g, 0.31 mmol, 13%), m. p. 90-95 °C.

1 h nmr (CDCI3); 4.51 (s, 2H, HI); 4.61 (s, 2H, H14); 7.06 (d, IH, ^ . h

= 16.2 Hz, H6); 7.24-7.53 (m, 7H); 7.62 (d, IH, ^ j ^ . h

= 7.5 Hz); 7.69 (m, IH)

13c nmr (CDCI3): 32.0; 33.4; 125.7; 126.4; 127.2; 128.1; 128.8; 129.3;

129.5; 130.4; 134.9; 136.7; 137.4; 137.5

Mass Spectrum (m/z): 368 [(C i6H i48lBr2)+ , 7%]; 366

( ( C i 6H i 4’ ’ B r8 'B r )+ , 17%] ; 364 [(C i6H i 4’ ^ B r2)+,

7%]; 287 [(C i6H i 4^ 'B r)+ , 100%]; 285

[(C i6H i479Br)+ , 100%]; 206 [(C i6H l4)+ , 100%]; 205

[ ( C i e H n r , 100%]

UV (EtOH): -max = 313.5 nm; £ = 31250

Accurate Mass: Expected for i 4^ ^B r2, 364.9462

Found, 364.9460

39. Preparat ion of 1,4-Bis [ ( 2 ,5 - d i m e t h y l ) p y r r o l i d o m e t h y l ]b e n z e n e 158

5'MeMe

MeMe

156


2,5-Dimethylpyrrolidine (0.50 ml, 0.41 g, 4.08 mmol) was dissolved in

a so lu t ion of sodium (0.09 g) in m ethano l (15 ml) then 1,4-

bis(bromomethyl)benzene (0.34 g, 2.03 mmol) was added slowly and with

vigorous stirring. The mixture was then ref luxed for two hours and

standard work-up gave the amine as a brown oil (0.27 g, 2.41 mmol, 59%).

nmr (CDCI3): 1.02 (d, 12H, = 6.0Hz, H5tO; 1.09 (d, 12H,

H = 6.1Hz, H 5c l; 1.40 (m, 4H, H4'); 1.79 (m, 4H,

H4'); 2.59 (br q, 4H, ^ J ^ . h = 5.5Hz, H 31 3.76 (s, 4H,

H I ') ; 7.24 (s, 4H, H2,3,5,6)

13c nmr (CDCI3): 20.3 (C5’); 31.1 (C4’); 54.5 (C3’); 59.3 ( C l ’;

127.4 (C l ,4); 148.4 (C2,3,5,6)

Mass Spectrum (m/z): 301 [(M + H)+, 32%]; 202 [(M - C6H i 2N)-i-,

7%]; 104 [(CgH8)+, 6%]; 98 [(C6Hi2N)+, 10%]

UV (EtOH): ^max = 224.0 nm; e = 28520

Accurate Mass: Expected for C 10H 16N 2, 164.1313

Found: 164.1311

40. Preparat ion of l , 4 -B i s - [ ( 2 ,5 - d im e t h y l ) p y r r o l id o m e t h y l ] b e n z e n e

dimethiodide 159

5 'M e M eM e

M eM e

M e2 1 "

1,4 -B is[(2 ,5 -d im ethy l)py rro lidom ethy l]benzene (0.17 g, 0.55 mmol)

was dissolved in acetonitrile (1 ml) and stirred with iodomethane (0.50 g,

0.22 ml, 3.52 mmol) for 24 hours. During this time, a white precipitate formed and this was removed by filtration, washed with E t2 0 and dried,

157


giving the title salt as a creamy-white powder (0.15 g, 0.26 mmol, 48%), m.p.

>260 °C (dec.).

nmr (d^-DMSO):

13c nmr (d^-DMSO):

1.36 (d, 6H, 3J h _h = 6.5Hz, H5"); 166 (m, 4H, H4');

2.08 (m, 4H, H4'); 2.71 (s, 6H, H60; 3.56 (m, 4H,

my, 4.58 (s, 4H, H r ) ; 7.59 (s, 4H, H2,3,5,6)

13.4 (C 50; 26.4 (C 40; 36.5 (C6'); 59.7 (C3'); 67.2

(C r ) ; 129.7 (C l,4); 133.2 (C2,3,5,6 )

Mass Spectrum (m/z): 315 [(M - CH3)+, 3%]; 202 [(M - C6H 12N - 2CHs)+,

49%]; 187 [(C i3H i9N )+ , 12%]; 104 [(CgH8)+, 64%];

98 [(C6Hi2N)+, 80%]

UV (H2O):

Analysis (%):

^max “ 222.Onm; 6 — 42540

Calculated for C22H 38N 2I2, C: 45.22; H: 6.55; N:4.80

Found, C: 44.14; H: 6.43; N: 4.33

41. Preparation of rra / i5- 4 ,4 "-bis[(2 ,5 - d im e t h y l ) p y r r o l i d o m e t h y l ]

s t i lb e n e 160

3 M e

M eM e

M e

2,5-Dimethylpyrrolidine (0.07 g, 0.07 ml, 0.57 mmol) was added to

stirred, dry THF (10 ml) at 0 °C under N 2(g) then n-BuLi (0.36 ml of a 1.6 M

solution in hexanes, 0.57 mmol) was added dropwise. After 10 minutes, the

solution was removed with a syringe and added dropwise to a stirred

suspension of f r o n j -4 ,4 '-b is(brom om ethyl)s ti lbene (0.10 g, 0.27 mmol) in

dry THF (5 ml). The mixture was stirred at 0 °C for 30 minutes then standard

158


work-up gave the title amine as a brown oil (54.3 mg, 0.29 mmol, 50%).

nmr (CDCI3): 1.11 (d, 6H, 3Jh_h = 5.6Hz, H3); 1.39 (m, 4H, HI);

1.78 (m, 4H, HI); 2.60 (m, 4H, H2); 3.78 (s, 4H,

H5); 7.06 (s, 2H, HIO); 7.27 (d, 4H, ^ J ^ .h = 8.0Hz, H7

or 8); 7.43 (d, 4H, 3Jh_h = 8.2Hz, H7 or 8)

nmr (CDCI3): 20.3 (Cl or C3); 31.21 (Cl or C3); 54.4; 59.5; 126.0;

128.0; 129.7; 130.2; 136.0

Mass Spectrum (m/z): 403 [(M + H)+, 11%]; 304 [(M - C6H12N)+, 23%]; 206

[(Ci6Hi4)+, 100%]; 98 [(C6H i 2N)+, 23%]

UV (EtOH):

Accurate Mass:

^max = 314.0nm; e = 57820

Expected for C28H 39N 2, 403. 3113

Found, 403.3108

42. P r e p a r a t i o n of 7 r a w s - 4 ,4 ' - b i s [ ( 2 ,5 - d im e th y l ) p y r r o l id o m e th y l ] s t i l b e n e

d im e th io d id e 161

5 Me

M eM e

2 1 '

M e M e

r r a n 5-4 ,4 '-b is [ (2 ,5 -d im ethy l)py rro lidom ethy l] s ti lbene (54.3 mg.

0.13 mmol) was added to acetonitrile (3 ml) and stirred with iodomethane(0.20 ml, 0.46 g, 3.21 mmol) for 24 hours. After pouring into E t iO (100 ml),

the mixture was centrifuged (x3000, 5minutes) then the E t2 0 decanted. The

moist solid was carefully dried in vacuo giving the title salt as yellow

powder (21.5 mg, 0.05 mmol, 38%), m. p. >230 °C.

159


nmr (d^-DMSO): 1.37 (d, I2H, 3Jh _h = 6.4Hz, H3); 1.65 (m, 4H, HI);

2.08 (m, 4H, HI); 2.69 (s, 6H, H5); 3.49 (m, 4H,

H2); 4.53 (s, 4H, H6); 7.42 (s, 2H, H l l ) ; 7.45 (d, 4H,

3Jh -H = 7.8Hz, H8 or 9); 7.73 (d, 4H, = 7.7Hz,

H8 or 9)

nmr (d^-DMSO): 13.4; 26.4; 36.2; 60.3; 67.0; 127.1; 127.2; 129.2; 133.1

138.6

Mass Spectrum (m/z); 402 [(M - 2CH3)+, 2%]; 304 [(M - C6H 12N - 2CH3)+,

2%]; 206 [(C i6H i4)+ , 91%]; 201 [M2+, 1%]

UV (H2O):

Analysis (%):

^max = 316.5 nm; e = 31490

Calculated for C30H44N 2I2, C: 52.49, H: 6.46; N: 4.08

Found: C: 51.03; H: 6.23; N: 3.96

43. Preparation of 7>a/î5:-2 ,4 ' -bis[(2 ,5 - d i m e t h y l )pyrro l idom eth yI]

s t i lbe ne 162

Me

3 Me

MeMe

14

7 r a n j - 2 ,4 ’-bis(bromomethyl)stilbene (0.08 g, 0.22 mol) was refluxed

in EtOH (10ml) with 2,5-dimethylpyrrolidine (0.04 g, 0.05 ml 0.44 mmol) for

two hours. Standard work-up gave the title amine as a pale yellow oil

(35.7mg, 0.09 mmol, 41%).

^H nmr (CDCI3): 0.89 (d, 12H, 3JH-H = 5.4Hz, H3); 1.14 (m, 4H, HI);

1.80 (m, 4H, HI); 2.68 (m, 4H, H2); 3.95 (s, 2H, H5);

4.03 (s, 2H, H18); 7.06 (d, IH, ^j ^ . h = 16.0Hz); 7.35

160


(m, 7H); 7.54 (d, IH, 3JH-H = 7.5Hz); 7.56 (m, IH)

Mass Spectrum (m/z): 403 [(M + H)+, 8%]; 305 [(M + H - CôH nN)"^, 35%];

207 [(C i6H i 5)+, 57%]; 91 [(C?H7)+, 100%]

UV (EtOH):

Accurate Mass:

Xmax = 307.5; e = 28500

Expected for C28H 39N 2, 403. 3113

Found, 403.3112

44. Preparation of r r a n 5- 2 ,4 ' -bis[(2 ,5 -d im e th y I ) p y r r o l i d o m e th y l ] s t i lb e n e

dimethiodide 163

2 i M e

20

M eM e

1421'

7 raA îj-2 ,4 '-b is [(2 ,5 -d im ethy l)py rro lidom ethy l]s t i lbene (13.0 mg, 0.03

mmol) was dissolved in acetonitrile (1 ml) and stirred with iodomethane

(0.34 g, 0.15 ml, 2.41 mmol) for 24 hours. The mixture was then poured into

E t 2 0 (100 ml) and centrifuged (x3500, 4 minutes). Decanting of the solvent

followed by careful drying in vacuo of the moist solid produced the title

salt as a pale brown powder (8.7 mg, 0.01 mmol, 39%), m. p. >230 °C.

^H nmr (d^-DMSO): 1.17 (d, 6H, 3Jh_h = 6.4Hz); 1.28 (d, 6H, 3Jh_h =

6.5Hz); 1.58 (m, 4H, HI); 1.99 (m, 4H, HI); 2.60 (s,

3H, H4 or 19); 2.61 (s, 3H, H4 or 19); 3.52 (m, 4H,

H2,20); 4.84 (s, 2H, H5); 5.04 (s, 2H, H18); 7.43 (d,

IH, 3Jh_h = 16.6Hz); 7.63 (m, 7H); 7.86 (d, IH,

H = 7.6Hz); 7.89 (m, IH)

Mass Spectrum (m/z): 560 [(M + H + I)+, 6%]; 320 [(M - C6H 12N - CH3 +

161


UV (EtOH):

Analysis (%):

H)+, 75%]; 207 [(C i6H i5)+ , 68%]; 98 [ (C ^H iiN )^ ,

100%]

^max = 307.5 nm; £ = 19220

Calculated for C30H44N 2I2» C: 52.49, H: 6.46; N: 4.08

Found: C: 51.67; H: 6.66; N: 3.96

45. Preparat ion of l , l ’- (P ‘ X y ly l )b i s ( l -aza -4 -azon iab ic yc lo [2 .2 .2 ]oc tan e )

dibromide 166

2Br'

l,4-Bis(bromomethyl)benzene (100.0 mg, 0.38 mmol) was dissolved in

acetonitrile (10 ml) then DABCO (90.0 mg, 0.80 mmol) was added and the

reaction stirred for five minutes. The precipitate which formed was filtered

and washed with E t2 0 giving the title salt as a white powder (131.3 mg, 0.035

mmol, 92%), m.p. >320 °C.

nmr (d^-DMSO):

nmr (d^-DMSO):

3.20 (br t, 12H, ^j ^ . h = 7.3Hz, H4'); 3.50 (br t, 12H,

3Jh -h = 7.3Hz, H31; 4.59 (s, 4H, HU); 7.67 (s, 4H,

H2,3,5,6)

46.8 (C4-); 54.7 (C3'); 70.1 (CU); 131.2 (C l,4); 136.4

(C2,3 5,6)

Mass Spectrum (m/z): 409 [(M + 8lBr)+, 41%]; 407 [(M + 79sr)+ , 42%]; 328

[M+, 0.1%]; 327 [(M - H)+, 5%]; 216 [(M -

C6H i2N 2)+ , 27%]; 215 [(M - C6H 12N 2 - H)+, 31%];

164 [M2+, 8%] 112 [(C6H i 2N 2)+, 100%]; 104

[(CgH8)+, 7%]

162


UV (H2O):

Analysis (%):

^max = 217.5 nm; £ = 8170

Calculated for € 20X32^ 4 6 :2, C: 49.19, H: 6.61; N: 11.47

Found: C: 49.03; H: 6.68; N: 11.38

46. Preparat ion of l , l ’* ( 2 ,6 -d i m e t h y l n a p h t h a l e n y l ) b i s ( l -a z a - 4 -

azoniabicyc lo [2 .2 .2 ]octane) dibromide 167

2 B r '

2,6-Bis(bromom ethyl)naphthalene (100.0 mg, 0.32 mmol) was heated

to reflux for four hours with DABCO (70.0 mg, 0.64 mmol) in acetonitrile (10

ml). Filtration of the precipitate which formed followed by washing with

E t 2 0 gave the title salt as a white powder (119.0 mg, 0.54 mmol, 69%), m.p.

>320 °C.

nmr (d^-DMSO):

13c nmr (d6-D M S0):


UV (H2O):

Analysis (%):

3.05 (br t, 12H, = 6.9Hz, H4'); 3.42 (br t, 12H,

3Jh -h = 6.8Hz, H3'); 4.78 (s, 4H, HU), 7.71 (d, 2H,

3Jh-H = 8.4Hz, H3,7); 8.13 (d, 2H, ^ J ^ .h = 8.4Hz,

H4,8); 8.20 (s, 2H, Hl,5)

44.6 (C40; 51.6 (C3'); 6 6 .2 ( C U ) ; 126.4; 129.0; 130.5;

132.8; 133.3

459 [(M + 8 lBr)+, 3%]; 457 [(M + 79Br)+, 3%]; 266

[(M - C6H i 2N 2)+, 4%]; 265 [(M - C6H 12N 2 - CH3)+,

1%]; 189 [M2+, 0.15%]; 154 [(C i2 H io)+ , 100%]; 112

[(C6H i 2N 2)+]

Xmax = 231.0 nm; £ = 55570

Cklculatedfor C24H32N 4Br2, C: 53.74, H: 6.01; N: 10.45

163


Found: C: 53.54; H: 6.12; N: 10.32

47. P r e p a r a t i o n o f 1 , 1 -^ ' 'û ws-4 ,4 ' -d im ethyI s t i l b e n y 1) b is ( l - a z a - 4 -

azon i a b i c y c lo [2 .2 .2]oc tan e) d ib r o m id e 170

2Br'

j -4 ,4 '-b is (b ro m o m eth y I)s t i lb en e (100.0 mg, 0.27 mmol) was

treated with DABCO (60.0 mg, 0.55 mmol) as described in experiment 4 6 ,

giving the title salt as a pale yellow powder (74.4 mg, 0.16 mmol, 58%), m. p.

>320 °C.

nmr (d^-DM SO):

nmr (d^-DMSG):

UV (HiO):

Analysis (%):

3.03 (br t, 12H, ^ J ^ . h = 7.1Hz, H40; 3.33 (br t, 12H

3Jh -h = 7.6Hz, H31; 4.54 (s, 4H, HU); 7.45 (s, 2H,

H7); 7.54 (d, 4H, = 8.1Hz, H2 or 3); 7.77 (d, 4H,

= 8.1Hz, H2 or 3)

44.7 (C41; 51.6 (C31; 66.1 (CU); 126.6; 127.0;

129.1; 133.6; 138.5

Mass Spectrum (m/z): 318 [(M - C ôH nN )'' ' , 46%]; 221 [M^+, 13%]; 206

[ (C i 6 H i 4)+, 100%]; 112 [ (C eH n N z )^ , 72%]

X-max — 316.5nm; £ — 20180

Calculated for C2gH3gN4Br2, C: 56.96, H: 6.49; N: 9.49

Found: C: 55.09; H: 6.61; N: 7.96

164


48. P re p a r a t i o n o f l , l "“( 2 , 6 - d i n i e t h y l a n t h r a c e n y l ) b i s ( l - a z a - 4 -

a z o n i a b i c y c l o [ 2 .2 . 2 ] o c t a n e ) d ib r o m id e 168

2Br"

2,6-Bis(bromom ethyl)anthracene (100.0 mg, 0.27 mmol) was treated

with DABCO (60.0 mg, 0.55 mmol) as described in experiment 46 giving the

title salt as a white powder (124.0 mg, 0.26 mmol, 96%), m.p. >320 °C (dec.).

nmr (d^-DMSO):

nmr (d^-DMSO):

3.04 (br t, 12H, = 7.2Hz, H4'); 3.43 (br t, 12H,

3Jh -h = 7.2Hz, H30; 4.75 (s, 4H, H I ') ; 7.63 (dd, 2H,

3Jh -H = 8.8Hz, 4Jh . h = 13Hz, H3,7); 8.29 (d, 2H,

H = 8.7Hz, H4,8); 8.33 (s, 2H, H I,5), 8.77 (s, 2H,

H9,10)

44.7 (C41; 51.7 (C3'); 66.5 (CT); 125.2; 127.2; 129.0;

129.3; 131.2; 131.3; 134.2

Mass Spectrum (m/z): 509 [(M + 8lBr)+, 30%]; 507 [(M + 79b f )+, 30%]; 427

[(M - H)+, 7%]; 316 [(M - C6Hi2N)+, 60%]; 214

[(M2+, 25%]; 204 [(Ci6Hi2)+, 100%]; 112

[(C6Hi2N2)+, 41%]

UV (HiO):

Analysis (%):

^max = 262.0 nm; e = 57400

Calculated for C2gH36N 4B r2, C:57.14; H:6.17; N: 9.52

Found, C: 56.59; H: 5.88; N: 8.73

49. P r e p a r a t i o n of 1 ,1 - (p -x y ly l ) b i s ( 4 - m e t h y l - 1,4-d iazon iab ic y d o

[2 .2 .2]octane) diiodide d ib ro m id e 171

165


Me

2 B f 21-

C o m p o u n d 166 (50.0 mg, 0.13 mmol) was added to acetonitrile (5 ml)

with iodomethane (0.40 ml, 0.91 g, 6.43 mmol) and stirred for 24 hours at

room temperature. Filtration of the precipitate which formed followed by

washing with E t2 0 gave the title salt as a pale yellow powder (67.8 mg, 0.13

mmol, 100%) m.p. >320 °C.

1H nmr (d^-DMSO): 3.27 (s, 6H, H5'); 3.94 (m, 24H, H3',4 '); 4.95 (s, 4H,

H D ; 7.73 (s, 4H, H2,3,5,6)

13c nmr (de-DMSO): 50.2 ; 51.8 ; 52.5 ; 65.5 ; 128.8; 133.9

Mass Spectrum (m/z): 358 [M+, 0.2%]; 231 [(M - C6H 12N 2 - CHg)'"', 94%];

142 [(C6H 12N2 + CH3)+, 71%]; 112 [(C6Hi2N2)+,

31%]; 104 [(CgH8)+, 78%]

UV (H2O):

Analysis (%):

X.max “ 223.0 nm; £ — 37400

Qlculatedfor C22H3gN4Br2l2, C: 34.22; H: 4.96; N: 7.26

Found: C: 33.80; H: 4.79; N: 6.86

50 . P r e p a r a t i o n o f l , l - ( p - x y l y l ) b : s ( 4 - p - x y l y l - l , 4 -

d i a z o n i a b i c y c lo [ 2 .2 .2 ] o c t a n e ) t e t r a b r o m i d e 172

4Br"

166


C o m p o u n d 166 (50.0 mg, 0.13 mmol) was stirred at room temperature

with 4-bromomethyltoluene (50.0 mg, 0.27 mmol) in acetonitrile (5 ml) for 24 hours. Filtration of the precipitate followed by washing with E t2 0 gave

the title salt as a white powder (61.4 mg, 0.08 mmol, 63%), m.p. >280 °C (dec.).

nmr (d^-DMSO): 3.10 (s, 6H); 3.40 (m, 24H); 4.62 (s, 4H); 4.93 (s, 4H);

7.65 (m, 12H)

Mass Spectrum (m/z): 409 [(M - ZCgHg + S 'B r)+ . 30%]; 407 [(M - ZCgHg +

7^Br)+. 31%]; 217 [(M - IC gH g - C6H 12N 2 + H)+

68%]; 112 [(C6Hi2N2)+, 100%]; 105 [(CgH9)+, 45%];

104 [(C8Hg)+, 5%]

UV (H2O):

Analysis (%):

Xmax ~ 218.0 nmj £ — 28220

Calculated for C36H 5oN4Br4, C: 50.37: H: 5.87;N: 6.53

Found: C: 47.12; H: 6.61; N: 6.14

51. Preparation of l , l ' - ( 2 , 7 - d i m e t h y l a n t h r a c e n y l ) b i s ( l - a z a - 4 -

azoniabicyclo [2 .2 .2 ]octane) dibromide 169

2Br

2,7-Bis(bromomethyl)anthracene (0.10 g, 0.27 mmol) was stirred for

two hours with DABCO (60.0 mg, 0.05 mmol) in acetonitrile (10 ml). The

reaction mixture was poured into Et2 0 (200 ml) producing a flocculent

precipitate. A fter settling, the solvent was decanted and the moist solid

carefully dried in vacuo, giving the title salt as a yellow powder (65.0 mg,

0.11 mmol, 41%), m.p. >320 °C.

nmr (d^-DMSO): 3.02 (m, 12H, H4'); 3.41 (m, 12H, H3'); 4.86 (s, 4H,

HI'); 7.72 (d, 2H, 3jH_H = 8.9Hz, H3,6); 8.34 (d, 2H,

167


nmr (d^-DMSO):

3Jh -H = 8.7Hz, H4,5); 8.62 (s, 3H, H I ,8,9); 8.90 (s, IH,

HID)

44.8; 51.7; 66.3; 122.8; 127.6; 129.7; 129.8; 130.0;

132.4; 133.0; 137.2

Mass Spectrum (m/z): 509 [(M + 8lBr)+, 2%]; 507 [(M + '79Br)+, 2%]; 428

[M+, 1%]; 316 [(M - C 6H i 2N2)+, 4%]; 204

[(C i 6 H i 2)+, 9%]; 112 [(C6Hi2N2)+, 64%]

UV (H2O):

Analysis (%):

%max = 259.5 nm; e = 98400

Calculated for C2sH 36N 4Br2, C:57.14; H:6.17; N: 9.52

Found, C: 55.59; H: 5.93; N: 8.62

52. P r e p a r a t i o n of 2 ,6 - B ls - [ ( c i s - 2 ,6 - d im e th y l )p ip e r id o m e th y 1]

n a p h t h a l e n e 178

6'Me

Me

Me

Me

2,6-Bis(bromomethyl)naphthalene (100.0 mg, 0.32 mmol) and c is -2 ,6 -

dimethylpiperidine (70.0 mg, 0.08 ml, 0.64 mmol) were refluxed in butanone

(10 ml) for two hours. This produced a precipitate which was filtered off

and recrysta llised from isopropanol giving the amine as white needles

(26.2 mg, 0.07 mmol, 22%), m.p. 64-67 °C

^H nmr (CDCI3): 1.14 (d, 12H, = 5.9Hz, H6'); 1.33 (m, 4H, H5');

1.60 (m, 8H, H4'); 2.54 (m, 4H, H31; 3.95 (s, 4H,

H D ; 7.45 (dd, 2H, ^ J ^ . h = 8.4Hz, = 1.2Hz,

H3,7); 7.72 (d, 2H, ^j ^ . h = 8.4Hz, H4,8); 7.80 (s, 2H,

H l ,5 )

168


13c nmr (CDCI3): 22.1; 24.2; 34.5; 53.6; 57.3; 126.2; 126.7; 127.2; 127.6;

132.3

Mass Spectrum (m/z): 379 [(M + H)+, 100%]; 266 [(M - C 7H i4N )+ , 78%];

154 [(C i 2 H io )+, 69%]; 112 [(CvH m N )^ 36%]

UV (EtOH): ^max = 234nm; e = 46750

Accurate Mass: Expected for C26H 38N 2 378.3035

Found 378.3035

53. P repara t ion of 2 ,6 -Bi^^is -2 ,6 -d imeth y l )p ip e r id o m e th y 1]

n a p h t h a l e n e dimethiodide 179

6' M eM e

M e

M eM e

21" M e

Compound 179 (10.0 mg, 0.03 mmol) was dissolved in dry acetonitrile

(1 ml) with iodomethane (0.16 g, 0.07 ml, 1.16 mmol) and stirred for 24

hours. After pouring into E t2 0 (200 ml), the compound was isolated by

decanting the solvent and placing the near-dry solid under low vacuum,

giving the title salt as an off-white powder (14.2 mg, 0.02 mmol, 80%), m.p.

>230 °C.

nmr (d^-DM SO): 1.10 (m, 2H, H5'e); 144 (m, 2H, H5'a); 1.54 (d, 12H,

^Jh -H = 6.2Hz, H60; 1.62 (m, 4H, H4"g); 168 (m, 4H,

H4'a); 2.85 (s, 6H, H7'); 3.19 (m, 4H, H3'); 4.78 (s,

4H, HU); 7.58 (d, 2H, 3Jh _h = 8.5Hz, H3,7); 8.10 (s,

2H, Hl,5); 8.10 (d, 2H, = 8.4Hz, H4,8)

Mass spectrum (m/z): 535[(M 4- I)+, 2%]; 393 [(M - CH3)+, 1%]; 281 [(M -

C H 3 - C 7H i 4N)+, 29%]; 204 [M^+, 11%]; 154

169

UV (EtOH):

Analysis (%):


[ (C i 2 H io)+, 100%]

^max = 231.0 nm; e = 192320


Found, C: 48.82; H: 6.41; N: 3.87

54. Preparat ion of l , l ' - ( p - x y ly l ) b i s ( l - a z o n ia b ic y c lo [ 2 .2 .2 ] o c t a n e )

dibromide 173

2 B r ‘

l,4-Bis(brom omethyl)benzene (80.0 mg, 0.30 mmol) was stirred with

quinuclid ine (70.0 mg, 0.65 mmol) in acetonitrile (10 ml) for 24 hours. The

precipita te which formed was filtered off, washed with E t2 Û and dried,

giving the title salt as a white powder (146.9 mg, 0.30 mmol, 100%),

m.p. >230 °C.

nmr (d^-DMSO):

nmr (d^-DMSO):


UV (H2O):

Analysis (%):

1.85 (m, 12H, H4'); 2.04 (br t, 2H, ^j ^ . h = 3.0Hz,

H5'); 3.45 (t, 12H, ^ J ^ .h = 7.6Hz, H3'); 4.48 (s,

4 H ,H D ; 7.61 (s, 4H, H2,3,5,6)

19.5; 23.3; 53.7; 65.5; 129.5; 133.4

407 [(M + 8lBr)+, 48%]; 405 [(M + 79Br)+, 51%]; 325

[(M - H)+, 1%]; 215 [(M - C7H i 3N)+, 18%]; 104

[(CgH8)+, 27%]

A-max “ 217.0 nm; £ = 16980

Calculated for C22H 34N 2B r2, C: 54.33; H: 7.05; N: 5.76

Found, C: 54.17; H: 7.09; N: 5.71

170


55. P r e p a r a t i o n o f 1 , 1 - ( 2 , 6 - d i m e t h y l n a p h t h a l e n y l ) b : s ( l

a z o n ia b i c y c l o [ 2 .2 . 2 ] o c t a n e ) d ib r o m i d e 174

2 B r ’

2,6-Bis-(bromomethyl)naphthalene (50.0 mg, 0.16 mmol) was reacted

with qu inuc l id ine (40.0 mg, 0.32 mmol) as described in experiment 5 3 ,

giving the title salt as white powder (21.5 mg, 0.04 mmol, 25%), m.p. >230 °C.

nmr (dg-DMSO):

nmr (d^-DMSO):

1.85, (br s, 12H, H4'); 2.04 (m, 2H, H5'); 3.47 (br t,

12H, 3Jh_h = 7.8Hz, H3'); 4.62 (s, 4H, HT); 7.69 (dd,

2H, = 8.4Hz, 4Jh_h = l . lH z, H3,7); 8.11 (d, 2H,

3Jh . h = 8.3Hz, H4,8); 8.17 (s, 2H, Hl,5)

19.5 (CSy, 23.4 (C4-); 53.8 (C31; 66.1 (C l '); 126.8;

128.9; 130.4; 132.7; 133.0

Mass Spectrum (m/z): 457 [(M + 8lBr)+, 78%]; 485 [(M + 79Br)+, 75%]; 375

[(M + H)+, 4%]; 265 [(M - C v H ib N )^ , 100%];

188 [M2+, 19%]; 155 [(C i2H u ) + , 11%]; 112

[(C7H 13N + H)+, 40%]; 111 [(C7H i 3N)+, 36%]

UV (H2O):

Analysis (%):

^max = 230.0 nm; £ = 88350

Calculated for C26H 36N 2B r2, C: 58.22; H: 6.77; N: 5.22

Found, C: 58.87; H: 6.91; N: 5.03

171

E x p e r i m e n t a!

56. P r e p a r a t io n of l , l ' - ( 2 , 7 - d i m e t h y l a n t h r a c e n y I ) b i s ( l -

a z o n i a b i c y c l o [ 2 .2 . 2 ] o c t a n e ) d ib r o m i d e 176

2 B r

2,7-B is(brom om ethyl)an thracene (20.0 mg, 0.05 mmol) was stirred

with quinuclidine (13.3 mg, 0.12 mmol) in acetonitrile (5 ml) for 24 hours.

The mixture was then poured into E t2 0 (200 ml) producing a flocculent

yellow precipitate which was allowed to settle then the solvent decanted

with a pipette. The moist solid was carefully dried in vacuo giving the title

salt as a yellow powder (3.2 mg, 0.01 mmol, 10%), m.p. >230 °C (dec.).

nmr (d^-DMSO): 1.86 (m, 12H, H4'); 2.04 (m, 2H, H5'); 3.48 (br t, 12H,

3Jh-H = 7.6Hz, H3'); 4.89 (s, 4H, H I ') ; 7.70 (d, 2H,

3Jh-H = 8.8Hz, H3,6); 8.32 (brs,2H, H4,5); 8.62 (s, 3H,

H1,8,H9); 8.90 (s, IH, HIO)

Mass Spectrum (m/z): 507 ((M + 5%]; 505 [(M + '^9Br)+, 5%]; 213

[(M - C7H i 3N)+, 12%]; 206 [ (C ie H u ) ^ , 100%]; 204

[ ( C |6H i 2)+. 89%]

U V ( H 2O ): ^max = 267.5 nm; e = 70710

57. Preparation of l , l ’- ( 2 , 6 - d i m e t h y l a n t h r a c e n y l ) b i s ( l -

azoniabicyc lo[2 .2 .2 ]octane) dibromide 175

2 B r ’

172


2,6-B is(brom om ethyl)an thracene (70.0 mg, 0.19 mmol) was stirred

with quinuclidine (40.0 mg, 0.40 mmol) in acetonitrile (10 ml) then worked-

up as described in experiment 55 to give the title salt as a yellow powder

(8.0 mg, 0.01 mmol, 7%), m.p. >230°C.

nmr (d^-DM SO): 1.86 (m, 12H, H41; 2.05 (m, 2H, H51; 3.51 (m, 12H,

H3 ); 4.72 (s, 4H, H I ') ; 7.71 (dd, = 8.9Hz,

= l .lH z, H3,7); 8.26 (d, 3J h . h = 8.9Hz, H4,8); 8.38 (s,

IH, Hl,5); 8.75 (s, IH, H9,10)

Mass Spectrum (m/z): 507 [(M + 8>Br)+, 11%]; 505 [(M + 13%]; 315

[(M - C7H i 3N)+, 27%]; 213 [M2+, 7%]; 204

[ (C i6H i 2)+. 29%]

UV (H2O);

Analysis (%):

^max - 262.5 nm; e = 15470

Calculated for C3oH3gN2B r2, C: 61.44; H: 6.53; N: 4.78

Found, C: 59.89; H: 6.23; N: 4.52

58. Preparation of l ,6 -b l s [ ( 2 ,5 - d im e th y l ) p y r r o l id o m e t h y I ]

p h e n a n t h r e n e 150

Me

S' Me 2 7 Me

Me

1,6 -Bis(bromomethyl)phenanthrene (50.0 mg, 0.14 mmol) was heated

to reflux for three hours in EtOH (2 ml) with 2,5-dimethylpyrrolidine (0.035

ml, 0 .028 g, 0.29 mmol). After cooling overn ight, the solution was

evaporated and E t2 0 (20 ml) added. This was extracted with 10% HCl(aq) (20

173


ml) then the aqueous layer was made basic by the addition of NH3(aq) and

extracted with E t2 0 (3x20 ml). Drying (M gS0 4 ) and evaporation of the

organic extracts gave the title amine as a yellow oil (27.0 mg, 0.07 mmol,

489%).

1h nmr (CDCI3): 0.90 (d, 12H, 3 J h .h = 5.5Hz, H5'); 1.17 (m, 4H, H41;

1.85 (m, 4H, H40; 2.74 (m, 4H, H3'); 4.05 (m, 2H,

H r ' ) ; 4.11 (s, 2H, H D ; 7.56 (d, IH, 3Jh _h = 7.6Hz,

H9 or HIO); 7.57 (d, IH, = 8.1Hz, H2); 7.68 (d,

IH, 3Jh _h = 6.8Hz, H9 or HIO); 7.74 (d, IH, ^Jh . r =

9.1Hz, H7); 7.82 (d, IH, ^j ^ . h = 8.1Hz, HI); 8.28 (br

d, IH, 3Jh . h = 8.8Hz, H6); 8.59 (s, IH, H4); 8.62 (d,

IH, ^Jh -H = 8.6Hz, H5)

Mass Spectrum (m/z): 400 [M+, 3%]; 302 [(M - C6Hi2N)+, 91%]; 206

[(C i6H i4)+ , 100%]; 205 [(C i6Hi3)+, 100%]; 204

[ (C i 6 H i 2)+. 75%]

UV (EtOH): Imax = 258.5 nm; e = 13100


Found, 400.2882

59. Preparation of l , 6 - B i s [ ( 2 ,5 - d i m e t h y l ) p y r r o I id o m e t h y l ] p h e n a n t h r e n e

dimeth iodide 151

MeMe

5' Me 2 7 Me

+ N-

Me2 F

Me

174


1,6 -B is [ (2 ,5 -d im e thy l)py rro l idom ethy l]phenan th rene (12.2 mg, 0.03

mmol) was stirred with iodomethane (0.12 g, 0.05 ml, 0.87 mmol) in

acetonitrile (1 ml) for 72 hours. After pouring into ether (200 ml), the

precipitate was centrifuged (x2000, 5 minutes) then the solvent decanted.

The moist solid was dried in a low vacuum, giving the title salt as an off-

white powder (10.3 mg, 0.02 mmol, 50%), m.p. >320 °C

nmr (d^-DMSO): 1.16 (d, 6H, 3 Jh .h = 5.9Hz); 1.43 (d, 6H, 3Jh_h = 5.6

Hz); 1.67 (m, 4H, H4'); 2.08 (m, 4H, H4'); 2.76 (s, 3H,

H6 ' or 6 "), 2.79 (s, 3H, H6 or 6 "); 3.67 (m, 2H,

H31, 3.95 (m, 2H, H3"); 4.80 (s, 2H, H I ') ; 5.15 (s,2H, H I" ) ; 7.71 (dd, IH, 3 % .^ = 7.8Hz, 4 % . ^ = l.OHz,

H9); 7.79 (d, IH, = 7.6Hz, H7); 7.91 (dd, IH,

^Jr-H = 7.6Hz, '^Jh-H = 0.8Hz, H6); 8.11 (d, IH, ^Jh-H

= 9.0Hz, HI); 8.20 (d, IH, 3Jh_h = 7.8Hz, HIO); 8.53

(dd, IH, 3Jh_h = 9.8Hz, ^ j ^ . h = 0.7Hz, H2); 9.04 (m,

IH, H4); 9.08 (d, IH, = 7.8Hz, H5)

Mass Spectrum (m/z): 415 [(M - CH])"*", 46%]; 318 [(M - CH3 - CgHioN)^,

52%]; 205 [(C i6H i 3)+, 45%]; 215 [M2+, 24%]; 204

[ (C i6H i 2)+, 60%]

UV (H2O):

Analysis (%):

X-max = 256.5 nm; 8 = 44900


Found, C: 51.79;H: 6.30;N: 3.87

60. Preparation of 1 , 1 - ( 3 , 6 - d i m e t h y l p h e n a n t h r e n y l ) b i s ( l , 4 -

diazoniab icyclo [2 .2 .2 ]octane) dibromide 1?^

2Br*

175


3,6-Bis(bromomethyl)phenanthrene (50.0 mg, 0.14 mmol) was stirred

in acetonitrile (5 ml). Addition of quinuclidine (0.16 g, 0.14 mol) caused the

dibromide to dissolve. After 10 minutes, a fine white precipitate was formed

and stirring was continued for further 24 hours. The reaction mixture was

poured into Et2 0 (100 ml) then centrifuged (x2000, 5 minutes), decanted and

evaporated to dryness in a low vacuum, producing the title salt as creamy-

white powder (52.8 mg, 0.09 mmol, 64%), m.p. >320 °C.

nmr (d^-DMSO): 1.86 (m, 12H, H4'); 2.06 (m, 2H, H5'); 3.57 (br t,

12H, 3 J h .h = 7.6Hz, H3'); 4.74 (s, 4H, H I ') ; 7.80 (d,

2H, 3 J h .h = 8.1Hz, H2, 7); 8.02 (s, 2H, H9, 10); 8.13

(d, 2H, 3Jh_h = 8.2Hz, HI, 8); 9.28 (s, 2H, H4, 5)

nmr (d^-DMSO): 19.5; 23.4; 54.0; 66.5; 126.5; 127.8; 128.7; 129.1; 129.5;

131.1; 132.4

Mass Spectrum (m/z): 507 [(M 4- 8 lBr)+, 95%]; 505 [M -k 79Br)+, 94%]; 425

[(M - H)+, 4%]; 315 [(M - C yH isN )^ , 3%]; 213 [M2+.

14%]; 204 [(C i6H i 2)+, 100%]; 112 [(CyHisN )^, 33%]

UV (HiO): Xrnax = 255.0 nm; e = 77080

Analysis (%): Calculated for C3oH3gN2Br2, C: 61.44; H: 6.53; N: 4.78

Found, C: 60.61; H: 6.60; N: 4.71

61. Preparation of T r ip h e n y l - p - x y l y l p h o s p h o n i u m bromide 121

PPh 3+ Br

This was prepared according to the literature method, giving the

title salt as a fine white powder in 70% yield, m.p. 265-270 °C (lit.^^ 276-277

°C).

176


62. P r e p a r a t i o n O f - 2 , 4 ' - d i m e t h y l s t i l b e n e 1 2 2

Me14 Me

Sodium (1.38g, O.Oômol) was dissolved in methanol (150 ml) and then

triphenyl-p-xylyl phosphonium bromide (13.43 g, 30.0 mmol) was added

with stirring followed by o-tolualdehyde (3.60 g, 3.45 ml, 30.0 mmol).

Stirring was continued for three hours, then the crude product was isolated

by crystallisation at -78 °C (4.24 g). This was dissolved in hot cyclohexane

and filtered, then column chromatographed (CHClgiC^H 12, 0:1 rising to 1:10)

giving the title alkene as white crystals (1.39 g, 4.80 mmol, 16%), m.p. 44-47

°C.

nmr (CDCI3): 2.37 (s, 3H, HI); 2.43 (s, 3H, H14); 6.98 (d, IH,

= 16Hz, H6); 7.18 (d, IH, = 7.9Hz); 7.20 (m,

3H); 7.29 (d, IH, 3 J h .h = 8.0Hz); 7.43 (d, IH, =

16.0Hz, H7); 7.51 (d, IH, ^j ^ . h = 7.3Hz)

nmr (CDCI3): l9.9(tj);2J-3(Cs);l7b.2;125.5; 126.2; 126.4; 127.3; 129.4;

129.9; 130.3 (all Q ) ;

134.9; 135.7; 136.5; 137.5 (all Cq)

Mass Spectrum (m/z): 208 [M+, 100%]; 193 [(M - CH3)+, 52%]; 178 [(M -

2C H 3)+ , 52%]; 165 [(Ci3H9)+, 10%]; 115 [(CgH?)^,

32%]; 91 [(C7H7)+, 15%]

UV (EtOH): ^max - 302.0 nm, £ = 24370

Accurate Mass: Expected for C 16H 16, 208.1252

Found 208.1252

177


63. P re p a r a t i o n of 1 , 6 - d i m e t h y l p h e n a n t h r e n e 123

Me

Me

7'ra/i5-2,4"-dimethylstilbene (0.13 g, 0.62 mmol) was d issolved in

cyclohexane (600 ml) with iodine (20.0 mg, 0.08 mmol) and irradiated for 6 hours. The reaction mixture was then washed with N a 2 S 2 0 g ( a q ) (200 ml),

d r ied (M g S 0 4 ) and evaporated in vacuo to give an orange oil. This was

column chromatographed (hexane, 67-70 °C) to give white crystals of the

phenanthrene (46.5 mg, 0.22 mmol, 36%), m.p. 85-87 °C (lit. 82.5-83.5 °C^,

87-88

1h nmr (CDCI3):

nmr (CDCI3);

2.65 (s, 3H, H I" ) , 2.77 (s, 3H, HT); 7.46 (m, 2H, H7

and H9 orlO); 7.55 (d, IH, ^Jr -H = 8 3Hz, H9 or 10);

7.79 (d, IH, 3Jh _h = 7.8Hz, H2); 7.82 (d, IH, 3J h _h =

7.9Hz, H8); 7.91 (d, IH, % . H = 9.0Hz, H3); 8.52 (s,

IH, H5); 8.60 (d, IH, = 8.4Hz, H4)

20.0; 22.2; 120.8; 121.8; 122.6; 125.8; 126.5; 127.6;

128.1; 128.3; 129.6; 130.0; 130.7; 130.9; 134.7; 136.2

Mass Spectrum (m/z): 206 [M+, 100%]; 191 [(M - % ) + , 65%]; 178 [(M

C 2H4)+, 14%]; 165 [(M - CH3 - C2H2)+, 10%]

UV (EtOH): A.max = 256.5 nm; 8 = 39640

Accurate Mass: Expected for C 16H 14, 206.1096

Found 206.1091

64. P r e p a r a t i o n o f l , 6 - b i s ( b r o m o m e t h y l ) p h e n a n t h r e n e 132

178


Br

Br

1,6 -Dimethylphenanthrene (0.18 g, 0.87 mmol) was refluxed in CCI4

(10 ml) with N-bromosuccinimide (0.30 g, 1.70 mmol) and benzoyl peroxide

(10.3 mg) for three hours. After cooling and filtration, the solution was

evaporated to give a yellow solid which was recrystallised from EtOH to give

the dibromide as a cream coloured powder (130.8 mg, 0.36 mmol, 41%), m.p.

104-110 °C.

nmr (CDCI3): 4.79 (s, 2H, m ' y , 5.02 (s, 2H, H I ') ; 7.64 (d, IH,

H = 8.4Hz, H9 or HIO); 7.67 (m, 2H, H7 and H9 or

HIO); 7.90 (d, IH, ^ J ^ .h = 9.8Hz, H2); 7.93 (d, IH,

3Jh.H = 8.1Hz, H8); 8.12 (d, IH, ^ J ^ .h = 9.1Hz, H3);

8.70 (s, IH, H5); 8.73 (d, IH, ^ j ^ . h = 8.1Hz, H4)

13c nmr (CDCI3): 31.9; 34.2; 122.8; 123.4; 124.1; 126.4; 127.3; 127.8;

128.7; 129.4; 130.1; 130.5; 130.8; 131.6; 134.0; 136.2

Mass spectrum (m/z): 366 [ (Q 6 H i 2 8l B r 2r , 7%]; 364 [ ( C i e H n ’ ^B rS lB r) '

15%]; 362 [(C i6H i 2’ ’ B r2)+, 7%]; 285

[ (C i6H i 2* 'B r)+ , 100%]; 283 [(C i6H i 2"'’ Br)+,

100%]; 204 [(C i6H i 2)+, 35%]

UV (EtOH); A.max - 256.5nm; 6 = 36040

Accurate Mass: Expected for C i 6H i 2B r2, 362.9302

Found 362.9299

65 . P r e p a r a t i o n o f l , 3 - D i m e t h y l - 5 - ( b r o m o m e t h y l ) b e n z e n e 18 6

179


M e Me

Mesitylene (1.90 g, 2.20 ml, 0.015 mol) was heated to reflux for four

hours with N-bromosuccinimide (5.34 g, 0.03 mol) and benzoyl peroxide (15

mg) in CCI4 (50 ml). The newly-formed succinimide was then removed by

f itra tion and the filtrate evaporated in vacuo. The crude product was

vacuum distilled (Kugelrohr) to give the title bromide as a colourless oil

(0.48 g, 2.30 mmol, 15%).

66 . P r e p a r a t i o n o f l - M e t h y I - 3 , 5 - b i s ( b r o m o m e t h y l ) b e n z e n e 187

Me

This was prepared acording to experiment 66 from 1,3-dim ethyl-5-

(brom om ethyl)benzene (1.00 g, 5.02 mmol), N -brom osucc in im ide (1.80 g,

10.10 mmol) and benzoyl peroxide (10 mg). The crude product, a sem i

crysta lline yellow oil, was column chromatographed ( 10:1 hexane:CH 2C l 2)

to give a fraction with Rf 0.49 which was evaporated to give white crystals

of the title dibromide (0.12 g, 0.45 mmol, 9%), m.p. 38-42 ®C (lit.^^ 41.5-42.5

®C).

180


5.3 R e f e r e n c e s

( 1 ) Laarhoven, W. H.; Peters, W. H. M.; Tinnemans, A. H. A. Tetrahedron

1978, 34, 769.

( 2 ) Haworth, R. D.; Mavin, C. R.; Sheldrick, G. J. Chem. Sac. 1934 (I), 454.

( 3 ) Newman, M. S.; Lilje, K. C. J. Org. Chem. 1 9 7 9 ,4 4 , 4944.

( 4 ) Morgan, 0 . T.; Coulson, E. A. J. Chem. Sac. 1929, 2203.

( 5 ) Pepper, J. M.; Howell, M.; Robinson, B. P. Can. J. Chem. 1 9 6 4 ,4 2 , 1242.

( 6 ) Lai, Y.-H.; Peck, T.-G. Aust. J. Chem. 19 9 1 ,4 5 , 2067.

( 7 ) Klemm, I. H.; Kohlik, A. J.; Desai, K. B. J. Org. Chem. 1 9 6 3 ,2 5 , 625.

( 8 ) Du Vernet, R. B.; Wennerstrom, O.; Lawson, J.; Otsubo, T.; Bockelheide,

V. JACS. 1978, 100, 2457.

( 9 ) Staab, H. A.; Sauer, M. Liebigs Ann. Chem. 1984, 742.

(1 0 ) Cristol, S. J.; Caspar, M. L. J. Organomet. Chem. 1968, 33, 2020.

(1 1 ) Davy, J. R.; Jessup, P. J.; Reiss, J. A. J. Chem. Ed. 1975, 52, 747.

(1 2 ) Jacobs, W. A. J. Org. Chem. 1951, 16, 1593.

(13) Vogtle, P.; Zuber, M.; Lichtenthaler, R. G.; Chem. Ber. 1973, 106, 717.

181

Chapter 5

Nucleophilic Addition o f Pyruvic A cid Synthons

N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S y n th o n s

5. Nucleophilic Addition O f Pyruvic Acid Synthons

5.1 I n t r o d u c t i o n

The chemistry of carbonyl compounds with activated hydrogen atoms is an

e n o rm o u s ly d ive rse and syn th e t ic a l ly im p o r ta n t a sp ec t o f o rgan ic

chemistry. The phenomenon of a-hydrogen acidity in such compounds is

due to the keto-enol tau tom erism they undergo and the subsequen t

resonance stabilisation of the resulting depro tonated anion 1 8 9 (figure

5 .1 ) .

O

R ' Rl H H

18 8

OR,

H

O'

18 9

R Ri

H

F i g u r e 5.1

Historical attempts at generating the a-an ions of carboxylic acids as shown

were largely unsuccessful since decomposition usually occurs under the

cond it ions needed for their form ation. However, recent advances have

made this a facile process for many acids and this introduction provides a

brief overview of the history of such chemistry, together with a review of

the enolate chemistry of synthons of pyruvic acid, a compound which has

so far eluded such advances.

5.1.1 C a rb o x y l ic A cid D ia n io n s

The modern chemistry of carboxylic acid dianions can be traced to an

experim ent in 1938 by Morton, Fulwell and Palmer^ which inferred the

form ation of the a -a n io n of phenylaceta te and hexanoate anions using

p heny lsod ium as base. Thus, reactions with carbon d iox ide produced

p h e n y lm a lo n ic - 1 9 1 and butylm alonic-acids in yields of 60 and 17%

respective ly (scheme 5 .1 ) .

183

N u c le o p h i l ic A d d i t i o n o f P y r u v ic A c i d S y n th o n s

19 0 19 1

(i) CO2

(ii)

H COOH

19 2

S c h e m e 5.1

The pheny lace ta te dianion 1 9 1 was subsequently form ed, in 1956, by

H auser and Chambers^ in a system that consisted of sodium or potassium

amide in liquid ammonia (scheme 5 .2 ) .

H H

2KNH2, NH3(1)

19 0 19 1

S c h e m e 5.2

H rOOHC6H5CH2C 1

Later still in 1958, DePree and Closson^ formed the dianion of acetic

acid 195 using sodium amide at approximately 200 °C (scheme 5 .3 ) .

O

M e ^ O - N a ^ +200 °C

1 9 4

O' Na+

H2C ^ O' Na+

19 5

S c h e m e 5.3

Under these rather forcing conditions, sodium amide acts as both base and

solvent, and the workers claim to have isolated the salt as a dry-air stable

solid which reacted with standard reagents such as benzyl chloride or

carbon dioxide.

H ow ever, the inherent d isadvantages o f such system s, v iz . , the

ex p e r im en ta l d iff icu lty or the in stab il i ty o f the d ian ions under the

p reva i l ing conditions m eant that the syn the tic op p o r tu n it ie s of such

potentially useful chemistry was not explored for a num ber o f years. In

1967 how ever , Creger^ published a paper detailing his use of lithium

diisopropylam ide (LDA) in tetrahydrofuran (THF)Zhexane solution to form

184

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S y n th o n s

the a -a n io n s of sec-butanoic acid 197 and 2-methylbutanoic acid and their

subsequent alkylkation in high yield (scheme 5 .4 ) .

Y RM e /' COOH -------------------^ M e'/^C O O ' Na+ — M e”/ COOHMe Me (ii) H Me

1 9 6 1 9 7 41-89% 1 9 8

S c h e m e 5.4

Though his method was limited in scope (yields of alkylated products from

other straight chain or a -b ranched carboxylic acids were found to be low

in the region of 30-60%), his demonstration of the use of LDA changed the

course o f such reactions and soon, a whole chem istry was constructed

around the use of similar strong, organic-solvent soluble bases. The success

of such bases (typically hindered lithium amides which are easily produced

by the reaction of bu ty ll i th ium with the appropria te am ine in THF

solution) can be considered as arising from three factors:

(i) they are soluble in aprotic solvents, with which no complexation

takes place, and this aids the observed, almost quantitative, metallation of

the a - c e n t r e ^ ;

(ii) the steric hindrance of the groups on the amide reduces the

nucleophilicity of the base, as well as the amine subsequently formed, and

reduces com petitive s ide-reactions^;

( i i i) the low tem pera tu res needed fo r the r e a c t io n p reven t

d eco m p o s i t io n (and, as later d iscussed , reduce the r isk of C la isen

c o n d e n s a t io n s ) .

A significant improvement to Creger’s system was made by Pfeffer

and Silbert who, noting that formation of the lithiated dianion of straight-

chain carboxylic acids produced a cloudy, he terogeneous solution, added

the highly polar aprotic solvent hexamethylphosphoram ide (HMPA) in the

belief that this would facilitate dispersion of the dianion. This produced a

clear solution and immediately raised yields to 90% or grea ter^’ The rôle

that the co-solvent plays can be inferred from the observation that best

results are obtained when it and the dianion are in a 1:1 molar ratio; it is

know n that o rg an o l i th iu m com pounds form h igh m o le c u la r w eight

a g g r e g a te s ^ and that for lithium dianions in THF, polymers may be formed

185

N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S yn th o ns

where n = 65 - 250 (accounting for their colloidal state)^. HMPA would thus

seem responsible for disrupting the aggregated species and forming what

has been d escr ibed as “ an assoc ia tion be tw een one m olecu le of

hexam ethylphosphoram ide and the counter-ion in a solvent-separated ion-

pa ir^® ” . It is interesting to note however, the deleterious effect that HMPA

has on reactions of dianions of a-branched acids. These are soluble in THF

and addition of the co-solvent produces a sharp decrease in yield as the

length of the side-chain is increased, accompanied by an increase in the

formation of the alkene derived from the alkyl halide. HMPA is somehow

thought to amplify the steric effect of branching but the method of its

agency is unclear.

Concurrent with the use of lithium amides was a second method

which, though not reaching the popularity of the first, has nonetheless

been found to be useful in a number of cases. It was devised by Normant

and Angelo who used as their base aromatic radical anions formed by

d is s o lv in g sod ium in n ap h th a len e or p h e n a n th r e n e ^ ^ . Yields of a -

a lkylated products were quite low for many aliphatic acids but satisfactory

for aromatic acids, such as phenylacetic acid (scheme 5 .5 ) .

^ " Y ^ C O O H (ii) ‘PrBr k j

1 9 0 1 9 9

S c h e m e 5.5

Later, Angelo showed that a similar system could be extended to

include reactions with carbonyl compounds to produce (3-hydroxy acids

(scheme 5 .6 ) .

O (i) L i , lQ lJ , THF, 50-60 °C----------------------/---\ D H

M e - ^ O H (ii) 0 = 0 \ ---0

2 0 0 38% 2 0 1 COOH

S c h e m e 5 .6

186


He found tha t the l i th iu m /n a p h th a le n e sy s tem was p re fe ra b le to

l i th iu m /p h e n a n th re n e or sod ium /naph tha lene sy s tem used b e fo re and

allowed him a greater degree of success with purely aliphatic acids.

5 .1 .2 P y r u v ic A c id

P y ruv ic ac id or 2 -oxo -p ropano ic acid is a c o m m on m o lecu le in

p h y s io lo g ica l processes where, in its enol form , ( f ig u re 5 . 2 ) it is

considered to be a key intermediate in enzymatic reactions catalysed by

py ru v a te k inase , phosphoeno lpyruva te c a rb o x y lase , p y ru v a te phospha te

dikinase, malic enzyme and oxalacetate decarboxylase^^.

0,0H

MeO

2 0 2

OH.OH

H jCO

2 0 3

F i g u r e 5.2

Though the enol form has been generated in solution and is reasonably

stable ( t i /2 = 7 minutes at 20 °C in D2 O s o l u t i o n ^ t h e acid has proved

uny ie ld ing to direct a lkylation using both c lass ica l and more modern

methods. The reasons for this are unclear. Tapia et aZ."^suggest that this may

be due to the insolubility of its dianion in organic solvents, but given the

rem arkab le efficacy of HMPA in solvating a - l i t h i a t e d s t r a ig h t - c h a in

a c id s^ , this seems puzzling. As a consequence of these apparent limitations,

a num ber of synthetic equivalents of pyruvic acid have been prepared.

These vary in their degree of complexity, but all can be regarded as

m olecules of pyruvic acid with their chemistry m odified so that, after

reaction and suitable deprotection, three carbon hom ologation is observed.

Most recently, Tapia et al.^^ showed that conversion of the a - k e t o

function of the acid to its d imethylhydrazone derivative 2 0 4 fac ili ta tes

formation of the dianion 2 0 5 (scheme 5 .7 ) .

187

N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th o n s

O

MeOH M6 2NNH2

O

2 0 2

E^O

N “ U+OH 2MeLi, THE, HMPA, 0 °C

O' LtMe

2 0 4 2 0 5

S c h e m e 5.7

This dianion is a strong nucleophile , reacting with a variety of alkyl

halides as well as aldehydes and ketones in reasonable yield. An added

advantage is that deprotection proceeds spontaneously in the acidic w ork

up (scheme 5 .8 ) .

Me2NN ~ L i +

o2 0 5

OH+

40%

OH

2 0 6

Q d

2 0 7

S c h e m e 5.8

Similar work has been carried out by W illiams and Benbow ^^, who

used tert-butyl esters of pyruvate oxime ethers 2 0 8 to effect C-alkylation

(scheme 5 .9 ) .

M eO . ^O . MeO^ , 0 .N

MeO^Bu

02 0 8

(i) 3LDA, THF, -78 °C

(ii) RBr

5 1 -7 7 %

RN

0 ‘Bu

O2 0 9

S c h e m e 5.9

Three interesting points emerge from this work:

(i) three equivalents of base were needed (use of two equivalents

yielded only unreacted starting material);

(ii) use of the parent oxime gave complete decomposition under a

variety of conditions;

188

N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S y n th o n s

(iii) excess alkylating agent was needed and only benzylic or allylic

h a lid es reac ted su ff ic ien tly rap id ly to c om pe te w ith d eco m p o s i t io n

p a th w a y s .

The problem in (i) was overcome by the addition of two equivalents

of lithium bromide prior to reaction, followed by 1.5 equivalents of base.

This led to good yields, so the workers assumed that the oxime ethers must

have coordinated to two equivalents of lithium base in unreactive clusters

prior to deprotonation.

Classically, oxalacetic acid has been used as a pyruvate equivalent.

C o r n f o r t h , F i r th and G o t t s c h a lk ^ ^ a t te m p te d sy n th e s is o f N-

a c e ty ln e u ra m in ic acid (N A N A , 212.) by the aldol condensation of N-

acetylhexosamine and pyruvic acid, but observed no reaction. By replacing

pyruvic acid with oxaloacetic acid 211 however, in the expectation that the

more reactive methylene group would help drive the reaction, it was found

to be successful, though it proceeded in very low yield. The superfluous

carboxyl group was lost in the pH-regulated medium they used (scheme

5 .9 ) .

CHOH-

HO-H-H-

■NHAc ■H OH OH

O

OH "O2CO2'

CH2OH

210 211

(i) p H 9 - * i l , 2 3 °C, 48hr

(ii) pH 6.8, 0“C

-CO2

S c h e m e 5.9

HH*^ H

HO H H

ÇO2'= 0

HOH NHAc HOH OH

CH2OH

212

A more elaborate approach was developed by Schmidt and B e tz^^’^^.

The first step in their synthesis of the related system 3-deoxy-D-manno-2-

octulosonic acid (KDO) was the reaction of their pyruvate synthon, 2-

benzyloxy-3-(phenylth io )acry lic acid N -m ethylam ide 2 1 3 with 2 ,3 :4 ,5 -d i-0 -

isop ropy lidene-D -arab inose (schem e 5 .1 0 ) .

189


HNMe

2 1 3

LDA, THF, HMPA, -80 °C

CHO

Ov MeMe O

O Me

M e. P"«

Q, MeMe O

O Me2 1 4

S c h e m e 5 .10

They chose such functionalisa tion fo llow ing investiga tion of a - a l k o x y

s u b s t i tu te d a c ry la te s and fou n d tha t a p - t h i o s u b s t i t u e n t and

m onosubs t i tu ted amide group supported p -carbon lithiation, as well as

in c re a s in g n u c leoph il ic r ea c t iv i ty and d ia s te re o s e le c t iv i ty . Q uench ing

r e v e a le d tha t , in the p re s e n c e o f tw o e q u iv a le n ts o f l i th iu m

diisopropylamide, formation of the dianion was practically quantitative. A

further advantage was the ease of deprotection: heating in high-boiling

petrol followed by treatment with Raney nickel produced good yields at

each stage.

5.1.3 A im s O f T he P ro je c t

3 -D eo x y -D -m an n o -2 -o c tu lo so n ic acid (KDO, see p rev ious section) is

currently the subject of much research interest since it is found in the

lipopolysaccharides (LPS) of all gram -negative bacteria which have been

s t u d i e d ^ ^ . The KDO residues are situated at the reducing ends of the

polysaccharide domains, linking them by ketoside bonds to the fatty-acid

subs ti tu ted 2-am ino-2-deoxy-D -g lucosy l d isaccharides know n as lipid A.

Figure 5 .3 is a block diagram indicating the location of KDO in the LPS

from Salmonella. The incorporation of KDO appears to be an vital step in

LPS biosynthesis and indeed in the growth o f gram negative bacteria,

accounting for the enormous in terest in the b iochem istry and synthetic

carbohydrate chemistry of KDO and derivatives. KDO itself is thought to be

form ed in vivo from D -arabinose-5-phosphate and phosphoenol pyruvate

p r e c u r s o r s .

190


£iN^£IN p:

NH-P~

(121NH- (U)

P~

- N H

0 i i d e chain Outer core Di h e p lose (KDO)j region reg ion

L i p i d A

■(Hi-■(12)-

- d i ) -i l SJ-

3 - OH' t e t r o d e c a n o ic dodecanoie te t r a d ec o n o c he»adecano<

k acid re i idv ts

B lock diagram representing the con stituents o f the gram -negative bacterial lipopolysaccharide. The inside o f the cell is at the right and the suroundings o f the cell at the left o f the drawing. The diheptose-K DO region is som etim es referred to as the "inner core". P' represents phosphate groups (from ref. 19).

F i g u r e 5.3

S yn the tic approaches to KDO have the re fo re ty p ic a l ly invo lved the

biom im etic addition of some pyruvate equ ivalen t to p ro tec ted arabinose

d e r iv a t iv e s .

COOHOHHO

OHA c

HOOH

N A N A 2 1 2 -

COOHOHHO

OH

HOOH

KDN 2 15

F i g u r e 5 .4

C rich ^ ® has postulated a route to the related sytems N-acetylneuraminic

acid (NANA, 2 1 2 see scheme 5 .9 and figure 5 .4 ) and 3-deoxy-D -m anno-2-

191


nonulosonic acid (KDN, 2 1 5 ) (figure 5 .4) which includes a similar addition

of some pyruvate equivalent as the initial step (scheme 5 .1 1 ) .

Me

Me O

[H]

COOH

f" CH2CCOOR" /

Me

CHO

MeMe Me

2 1 6 2 17 2 18

S c h e m e 5.11

Our brief was therefore to investigate conditions under which pyruvic acid

or synthons thereof could be used to facilitate this initial reaction.

192

N u c le o p h i l i c A d d i t i o n o f P y ru v ic A c i d S yn th o n s

5.2 R e s u l ts A nd D isc u ss io n

Given the apparent absence in the l i te ra tu re re la t ing to the d irec t

alkylation of pyruvic acid, this seemed to be a good starting point for

investigations into the pyruvic acid system. Though the failure of standard

m ethodolog ies is im plic it in certain papers^ in itia l attem pts centred

around a mixed THF/hexane/HM PA solvent and lithium diisopropylamide as

base (scheme 5 .1 2 ) .

OMe OH

OJ L OH (i) LDA. THF, HMPA

» N2(g), -78 °CO (ii) Mel L O

2 0 2 2 1 9

S c h e m e 5.12

Several attempts were made, using iodomethane as the alkylating agent, but

all resulted in a complex mixture of products. The H nmr spectra of the

c rude p ro d u c ts were not p a r t ic u la r ly h e lp fu l in " id e n t i fy in g the

com ponents ; com plete decom position of the subs tra te had apparen tly

occurred since there were no signals relating either to pyruvic acid [5 2 .5 2

(s, 3H); 58.31 (s, IH)] or the supposed product, 2-oxo-butanoic acid [51 .04

(t, 2H); 52.84 (q, 3H) and a broad downfield singlet]. Inspection of the

aqueous layer by saturation with sodium chloride, fo llowed by extraction

with E t2 0 revealed only the presence of HMPA. A sim ilar experiment,

conduc ted w ithou t the use o f the co -so lven t p ro d u ced a s im ila r ly

unremarkable result, though peaks due to pyruvic acid are present among

the num erous peaks presum ably due to decom position in the H nm r

spectrum of the product.

We checked that this failure was not due to procedural error by

repeating the reaction using standard procedure on a known substra te^.

Thus, alkylation using 1-bromobutane, of octanoic acid 2 2 0 at 0 °C gave 2-

butyloctanoic acid 221 in 42% yield (scheme 5 .1 3 ) .

193


(i) LDA. THF ,HMPA, N2(g),0 °C

(ii) CH3(CH2)3Br

2 2 0 2 2 1

S c h e m e 5 .13

HMe

All analy tica l details support the s tructure and though the y ield was

rela tively low, it clearly demonstrated the correctness o f the method. A

repeat run was therefore carried out on pyruvic acid under precisely the

same conditions, but the result was the same as for previous reactions of

this type. The mass spectrum is dominated at high m/z by HMPA but at

low er values, strong alkyl signals are present, perhaps ind ica ting the

presence of octane, form ed in a s ide-reaction by the attack of excess

b u ty ll i th ium on brom obutane.

A tten tion was then turned to rela ted pyruvic acid system s. The

m odulating efect that estérification with bulky alkyl groups has on the

reactivity of acids in the alkylation reaction (as well as their propensity to

undergo self-condensation) is well noted 15,22 this, together with the

fac t that Ley and co-w orkers have pub lished w ork on the term ina l

acylation of P-ketothioesters such as 2 2 2 23 ,24,25 (scheme 5 .1 4 ) led us to

believe that similar a-ke to th ioeste r systems may prove susceptible to anion

f o r m a t i o n .

(i) NaH, DME, 0 °CO O (ii) nBuLi. DME .0 °C H O H O O

(ii)

2 2 2 (iii) If 2 2 383%

S c h e m e 5 .14

L e y ’s route was unfortunately not applicable to com pounds of this type

since his procedure involved the attack on diketene o f sodium thio-tert-

butoxide 2 2 5 as shown in scheme 5 .15 .

194

N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c id S y n th o n s

MeNaH, THF, 0 “C

M e ' / ^ S HMe

2 2 4

Me

M e ^ '/^ S 'N a '"Me

2 2 5

H2C

O

O O

63% M e " " ^ ^ ^ ^ ^ S ‘Bu

2 2 3

S c h e m e 5 .15

Therefore, a classical estérification route was chosen, i. e., the addition of

the acid chloride to the thiol, the standard method for preparing thio-

c a rb o x y l ic e s t e r s ^ P y r u v o y l chloride 2 2 8 is a reasonably stable liquid

which resists synthesis from the acid using standard reagents such as

phosphorus trichloride, phosgene, thionyl chloride or oxalyl chloride

The acid chloride has been synthesised from trimethylsilyl pyruvate^ but

the m ost successful synthesis in the literature is due to Ottenheijm and

DeM an who suggested oc,a-dichloromethyl methyl ether 2 2 6 as a suitable

chlorine source (scheme 5 .1 6 ) .

^OH A,30minM e' V + CI2CHOCH3 --------------

O2 0 2

HCl2 2 6

Me

O

O

2 2 8

OO. ,OMe

Me ) <q CI h

2 2 7

OCl + ¥

H OMe

2 2 9

S c h e m e 5 .16

The c h lo r in a te d e ther is o f ten avo ided on accoun t o f its acu te

carcinogenicity, but, it was claimed, was able to give yields of up to 51% in

this reaction. We found that some modification was neeeded to reach these

yields, for instance, p ro longing the reaction time and conducting the

experim ent under a nitrogen atmosphere. Also, the sole H nmr signal,

supposedly at 52.59 in CDCI3 was found actually to be 52.48, but the synthesis

195

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S yn th o n s

was nonetheless successful and a typical run appears in the experimental

section of this chapter.

Despite the noted lability of the acid chloride, the reaction with 1,1-

d im e th y le th a n e th io l 2 3 0 proved to be much less facile than anticipated.

Fractional distillation of the thiol was found to be necessary because it

consisted of several compounds, exhibiting several spots on a TLC plate

(this is a noted problem with tertiary thiols and in the absence of base,

yields for the reaction were tiny. Triethylamine was eventually found to be

the most successful base and the novel thioester 2 3 1 was obtained as a

yellow oil in reasonable yield (scheme 5 .1 7 ) .

EfaN, EtzO, A

S c h e m e 5.17

A series of a ttempted alkylations were then carried out on the

thioester under a variety of conditions. LDA was the first base chosen with

the entire reaction carried out at -78 °C. Once again, only the decomposition

products were evident in the crude reaction mixture, the only signals in

the nmr spectrum, apart from those due to HMPA, appearing in the alkyl

region. This is perhaps not surprising, since a study on the synthetic

u til ity o f a series of th ioes te r enolate anions inc lud ing ter t-bu ty l

thioacetate carried out by Edwin Wilson and Hess^®, revealed no reaction of

the m agnesium or lith ium enolates with iodom ethane or m ethyl vinyl

ketone at 0 °C, though the Claisen condensation product was observed in

both cases (scheme 5 .1 8 ) .

9 ( i ) ‘PrMgBr, EfeO, THF, -20— 0 °C/100min O O MeMe

Me (ii) îT, H2OMe

Me2 3 2 2 2 2

S c h e m e 5 .1 8

196

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S y n th o n s

No explanation for this preference for self-condensation at the expense of

alkylation was provided so we sought an alternative base.

The proven efficacy of lithium isopropylcyclohexylamide (LICAD) in

generating the a -an ions of a wide variety of esters was demonstrated by

Rathke and Lindert ^, so it was tried next using standard conditions on the

same thioester. This method produced a small quantity of yellow solid which

is almost certainly not the desired product, since the carbonyl stretch in

the infra-red spectrum is negligibly weak and in the wrong region besides.

Its id en t i ty rem ains unc lear. The mass sp ec tru m has a com p lex

f ragm en ta tion pattern (a pers is ten t problem with these system s, with

neither El or FAB methods giving satisfactory results, even with otherwise

fully characterised compounds), so is not useful in helping to identify the

product of this reaction.

A repeat run using the same base produced a further quantity of

crystalline material though with a markedly different melting point (>200

°C against 126-130 °C, the decomposition point of the material yielded in the

previous experiment). The H nmr spectrum is not helpful in identifying

this material but reveals that the solid is contaminated with the thioester

w h ich w h e th e r re g e n e ra te d or u n re a c te d , im p l ie s th a t c o m p le te

decomposition of the substrate has at least been prevented. The infrared

spectrum confirms this presence with a strong signal at 1718 cm" ^

However, neither of the two samples yielded to recrystallisation and

remain unidentified. Use of the base was nonetheless continued for the

reasons given above and the reaction of the thioester with an aldehyde was

investigated. This was chemistry more relevant to the aim of the project

since the first step of the Crich synthesis involved, as d iscussed, the

addition of some pyruvate dianion equivalent to a sugar derivative The

aldehyde chosen in this case was benzaldehyde in order to add some detail

to the previously empty lowfield part of the spectrum (scheme 5 .1 9 ) .

O

Me

(i) LICAD, THF, HMPA

Me ^2(g). -78 °C

O Me Me (ii) PhCHO

2 3 1

S c h e m e 5 .1 9

H OH ?

O M e Me

2 3 3

197


The result of this experiment, which, like all those previously, was

carried out at -78 °C, was a mixture of unidentified products, none of which

appeared to be the desired aldol. The 200 MHz H nmr spectrum includes a

profusion of weak signals between 50 and 55 whose origin is unclear whilst

the absence of the normally strong tert-butyl peak around 51.4 is striking

and perhaps indicates that some kind of hydrolysis may have occurred.

D e u te ra t io n and c a rb o n y la t io n are s ta n d a rd te c h n iq u e s fo r

determining the existence and degree of formation of the a - a n i o n s ^ and

water may be used to the same end to reform the substrate in situations

where extensive decomposition is observed to occur. The fact that simple

hydrolysis of the reaction mixture in the experiment above produced only

negligible amounts of starting material meant that we embarked upon the

s y n th e s is o f a new, m ore h indered th io e s e te r . 1 ,1 -D ie th y lp ro p y l

th iopyruvate ( tert-heptyl thiopyruvate) was chosen for the same reasons

as before and its synthesis followed a similar route with the exception of

the fact that the appropriate thiol 2 3 6 was com m ercially unavailab le and

had to be synthesised from the alcohol 2 3 5 . The alcohol i tse lf was

synthesised from 234 by addition to ethyl magnesium bromide in good yield

(>90%) whilst the thiol was prepared in approxim ately 50% yield in a

s tandard substitution reaction using a method after Barton and Crich^

(scheme 5 .2 0 ) .

Me

O (i) Et MgBr, Et^O, A (i) HiS, CH iCh

(ii) P f, H iO (ii)93% 51%

2 3 4

OM eC (0)C 0C k Et,N ^N 2 (g ),E t2 0 , A, 1 h ^

52% Me

2 3 7

S c h e m e 5 .20

198


The novel thioester 237 was obtained as a sweet-smelling oil in yields of up

to 55% and gave consistent spectral data. It is pertinent at this point to talk

of the nature of the carbonyl stretch in these compounds . In any a , p -

unsa tu ra ted system, two conform ations are possib le and two peaks are

therefore seen in the in fra -red corresponding to the two conform ers

where the unsaturated systems are disposed s-cis or s-trans to one another.

The lower of the two is always assumed to be due to the s-trans form since

delocalisation is expected to be greater. Spectroscopic and crystallographic

data of a -d ik e to systems however, indicates that the s-trans form of the

com pounds is the stable conformer^ Hence, we should only expect one

band in the infra-red. There is also little difference observed between the

IR and R am an ac tive f r e q u e n c ie s ^ ^ , in d ic a t in g l i t t l e m e c h a n ic a l

interaction, (presumably as a result of a fairly weak central C-C bond and a

low degree of conjugation). In pyruvic acid, the two carbonyl groups

absorb at 1745 c rn 'l and appear as one peak and all the systems synthesised

in this project exhibit single absorptions in the carbonyl region of the

i n f r a r e d .

R eac tion o f the th ioes te r with b rom obu tane us ing L ICA D in

T H F /H M P A at -78 ®C p roduced a very e n co u rag in g resu lt . A fte r

chromatography of the mixture, a clear oil was obtained in an apparently

good yield of 47%. The 400 MHz ^H nmr spectrum of the compound seems to

support fo rm ation of the product, 1 ,1-diethylpropyl 2 -oxo-th iohep tanoa te

2 3 8 (scheme 5 .2 1 ) and the IR spectrum shows a strong carbonyl stretch

(similar to the thioester at 1718 cm '^ ) .

OOÏ (i) LICAD, THF, HMPA

Me N2(g). -78 °C Me" ^ ^ ^ Me

Me (") CH3(CH2)3Br ^ ^Me

2 3 7 2 3 8

S c h e m e 5.21

The C nmr spectrum shows only a slight sign o f the two low-field

carbonyl peaks one would expect but the spectrum of the parent thioester

shows these to be very weak. The mass spectrum gives c lear alkyl

f ragm en ta tion from butyl dow nw ards, as well as as the te r t-hep ty l

199


fragm ent, a fragm ent due to ester cleavage [(C2 H 5 ) 3 0 8 ■*■] and a weak

molecular ion peak at m/z 258. It was found that formation of the presumed

product was arrested if the temperature of the reaction was allowed to rise

to 0 °C prior to the addition of the euectrophile and we had therefore

established, we hoped, a working procedure for the reaction. However, we

were not able to capitalise on this success when the reaction of the

th ioester with benzaldehyde under the same conditions as before produced,

after chrom atography, an oil whose H nm r spectrum was com plete ly

devoid of aromatic signals and whose IR spectrum showed no aromatic

stretches and only a weak carbonyl stretch at 1715 cm-1.

The loss of all signals in the nmr spectrum apart from those in the

alkyl region in this reaction seemed to indicate that alkane formation was

becom ing a prevalent side reaction. It was decided therefore to use 1-

bromodecane as the quenching agent for two reasons:

(i) the formation of any alkane would hopefully lead to material of

h igh enough m o lecu la r w eigh t to g ive a c lea r and c h a ra c te r is t ic

fragm entation pattern in the mass spectrum;

(ii) the higher molecular weight would also give more material by

w e i g h t .

In the subsequent reaction, carried out entirely at -78 ®C, none of

the desired product was apparently formed. The sample displays a triplet at

52.30 in the nmr spectrum, perhaps due to a pair of methylene protons a

to the diketo system (the singlet in the parent thioester occurs at 52.33 and

the a -p ro to n s of bromobutane as a triplet at 53 .40) but once again, no

signals due to the carbonyl groups or the carbon atoms to which they are

attached are present in the infra-red or nmr spectrum.

It was unclear exactly what had been occurring in the preceding

reactions and it was difficult to make sense of the conflicting data. It

seemed fairly reasonable however, to assume that, had the reactions been

working at all, they were doing so in yields that were undetectable and this

led us to change the base system under investigation. The system of Tapia

et al. uses methyllithium as base and the entire reaction is carried out at

0 There is tacit acknowledgm ent of the failure o f this system to

genera te the pyruvate dianion within the paper and the use of the

d im ethy lhyd razone presum ably arose from this observa tion . It becam e

clear however, when we tried to use the system on our thioester that the

200

N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c id S yn th o n s

gain in stability of the dianion caused by functionalisation of the a - k e t o

group and which allowed such relatively mild conditions to be employed,

was not m atched by estérification; tert-hepty l th iopyruva te was reacted

under these conditions and quenched with a tw o-fold excess of various

e le c tro p h i le s inc lud ing b rom odecane , b en z a ld e h y d e and b ro m o b u tan e ,

p ro d u c in g in each case only the r e g e n e ra te d th io e s te r and the

electrophile . Quenching with water produced, after work-up, only an oil

with an IR spectrum identical to the thioester. M odification of the a - k e t o

function, by far the most common m ethod of genera ting pyruvic acid

synthons, would thus seem to be a more effective remedy than the remote

functionalisa tion we had been employing up to this point, though the

single success with tert-heptyl th iopyruvate suggested that perhaps the

two strategies could be made to work in concert.

Enolates have for some time been trapped as their trialkylsilyl enol

ethers, in the expectation that the ether function would be readily cleaved

by a num ber of organometallic reagents to generate a h ighly reactive

enola te , and are easily prepared by the reaction o f the ketone with

trialkylsilyl chlorides in the presence of any of a num ber of bases^^ . A

large, stereospecific chemistry now exists for reaction of these compounds

with electrophiles and there seemed no obvious reason why pyruvic acid or

its derivatives would not be amenable to at least formation of the silyl enol

e th e r , e s p e c ia l ly in view of the s ta b i l i ty o f the d e p ro to n a te d

d im ethy lhydrazone of Tapia et a l^^ . The b is-tr im ethy ls i ly l derivative of

pyruvic acid has been synthesised and was the source of some interesting

kinetic data which deduced that ketonisation of the c leaved ether was

s u f f i c i e n t ly s lo w ^ ^ (see section 5 . 1 .2 ) to suggest that reaction of the

nascent enolate was a real possibility. For our first attempt at synthesis of

the t r im e thy ls i ly l derivative of te r t-hep ty l th iopyruva te , we retu rned to

our original system and attempted generation of the lithium enolate at -78

°C using lithium diisopropylamide in THF, fo llowed by quenching with

ch lo ro tr im e thy ls i lane (TM S-Cl). The fact that this produced only the

th ioeste r was further evidence that the lithium enolate, were it being

generated at all was insufficiently stable under the reaction conditions to

react. A m ore conventional system was then em ployed. Follow ing the

method of House et. al. triethylamine (EtgN), then TMS-Cl were added to a

201

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S yn th o ns

solution of the thioester in dimethylformamide. After two hours’ stirring,

extraction with hexane gave a low yield of the silylated enol 2 3 9 (scheme

5 .2 2 ) , easily detectable in the nmr spectrum of the unpurified reaction

mixture because of the pair of doublets around 55.0 due to the newly-formed

pair of vinylic protons (see Appendix, p. 222).

O

Me

O

2 3 7

S ^ ^ E t EtgN, M c 3 SiCl, DM F

E t E t N i(g ) , 2 hours

50%

0SiMc3

o E t E t

2 3 9

S c h e m e 5.22

This material proved to be surprisingly resistant to hydrolysis, unaffected

by D2O after three hours at room temperature or by stirring in water at pH4

for two hours, and this allowed us to employ an aqueous work-up which had

the twin advantage of removing any residual solvent and dissolving the

amine salt which was always formed. Thus, by extracting the mixture with

hexane, then washing with H2O and drying briefly over MgSO^, yields of up

to 54% were possible. The pure material was however unstable over days in

air, so was stored at -20 °C in hexane solution and used in this form. For the

reaction of this compound with electrophiles, we intended to follow the

methods of House et. and Rasmussen^^ which involve metathesis of

the silyl enol ether with methyllithium followed by reaction in situ with an

alkyl or benzyl halide. The reaction was m odified to account for the

p robab le decom position of the anion under the ref lux ing cond it ions

employed by House, and was carried out at -40 °C. A quenching reaction was

the first to be tried (scheme 5 . 2 3 ) which regenerated the th ioester as

expected, but in less than quantitative yield due to decomposition.

OLiE t MeLi, DME

-40°C, H (

40 mins

2 4 02 3 9

HiO, A

30 m ins

34%

H 3C

O Et Et

2 3 7

S c h e m e 5 .23

202

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S y n th o n s

This became the overriding pathway when repeated with benzaldehyde and

no product could be detected in the crude reaction m ixture, despite the

disappearance of all nmr signals relating to the silyl enol ether. The

aqueous layer was examined also in the unlikely event that the target aldol

was w a te r - so lu b le , but the p ro d u c t appeared to c o n s is t only o f

benzaldehyde and regenerated thioester.

M u k a iy a m a and c o - w o r k e r s ^ ^ d isc o v e re d tha t t i tan iu m

te t r a c h lo r id e (T iC U ) could be used to promote the reaction of ketone

trim ethylsily l enol ethers with ketones or aldehydes w ithout the problem

of self-condensation that limits the usefulness o f o ther aldol syntheses.

They proposed that the reaction proceeded via the form ation of the

titanium enol ether 2 4 2 (scheme 5 .2 4 ) which added to the electrophile to

give the chelate 2 4 3 .

OSiMea

2 4 1

OTiCl]

R i ' ^ C R j Rs

2 4 2

+ MegSiCl

OI I

R4 CR5

Cb

RiR2 R3

2 4 3

R4

R5

HiO OH

Ri

2 4 4

+ TiCyOH

Sc h e m e 5 .24

Hydrolysis yielded the desired product. Other Lewis acids were later found

capable of promoting the reaction^ \ but we decided to use M ukaiyam a’s

original system and test its suitability for our th ioester derivative. O-

" f r im e th y l s i l y l - t e r t - h e p ty l th io p y r u v a te 2 3 9 was thus reac ted with

benzaldehyde in the presence of TiCU at -78 °C to give a yellow oil that nmr

analysis revealed to consist once again of unreacted benzaldehyde and

regenerated thioester, the latter recovered by chrom atography in almost

q uan ti ta t ive y ield. No change was obse rved upon e lev a tio n o f the

tem pera tu re of reaction though increas ing am ounts o f d ecom pos it ion

became apparent above 40 °C. We checked for any procedural errors by

203

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c id S yn th o n s

s y n th e s i s in g 1- t r im e th y Is i iy I c y c lo h e x e n e f ro m c y c lo h e x a n o n e and

reacting it with benzaldehyde to give the aldol shown in scheme 5 .2 5 , a

reaction carried out in our source paper'^®.

0SiMc3 ÇHO

2 4 5

TiCU, CH2CI2

N 2(g), -78 °C, I hr 33%

S c h e m e 5.25

9 h o H

2 4 6

We obtained the product as yellow crystals (the ratio of the threo isomer to

the erythro isom er was not investigated) though in considerably lower

yield than claimed in the reference. This did however suggest that the

thioester derivative was not suitable for reaction under these conditons.

Further investiga tion of this reac tion was carr ied out on the

trim ethy ls i ly l enol derivatives of methyl and ethyl pyruvate . The two

esters are commercially available and their use at this stage was expedient

given the tim e-consum ing synthesis of the th ioester (the use of which

could be continued at a later stage). The two silyl enol ethers (248 and 2 4 9

for the derivatives of m ethyl and ethyl pyruvate respec tive ly ) were

syn thes ised w ithou t inc iden t as schem e 5 . 2 4 by the reaction of the

respective ester with TMS-Cl and EtgN in DMF at room temperature and

obtained as clear oils in reasonable yield. The reaction of 0-tr im ethylsily l

enol(ethyl pyruvate) 2 4 9 with benzaldehyde at a number of temperatures

using M ukaiyam a's system did not produce markedly different results from

those described above and once again, the tendency to decomposition was

considerably increased above 40 °C.

It was at this point that we discovered a pair of papers by Sugimura

and co-workers which detailed the use of the trimethylsilyl enol ethers of

both these pyruvic acid acid esters as well as tert-butyl pyruvate, in a Lewis

acid-m ediated aldol reaction. Sugimura^^'"^^ was also aware of the potential

for the synthesis of the y - h y d r o x y - a - k e t o function which occurs in KDO

and used his reaction to synthesise a number of 1,4-lactone derivatives of

KDO. His conditions were essentially the same as those of Mukaiyama that

we had adopted though interestingly, of all the Lewis acids used in the

204

N u c le o p h i l i c A d d i t i o n o f P y ru v ic A c i d S yn th o n s

r e a c t io n , T iC l4 was found to be the least effective in promoting the

reaction, B F 3 :E t2 0 the best (scheme 5 .26).

OMe OSiMej Lewis acid, CH 2C I2 9

2 4 7 2 4 9 250 Yield: 10% (TiCU)86% (BI^:0Et2)

S c h e m e 5.26

All reactions were carried out on acetals to prevent the further substitution

which was found to be a problem with unprotected keto functions (scheme

5 .2 7 ) .

O SiM e,

BF,:E^O, CH,Cb

O N 2 (g), -78— 8 °C2 5 1 2 4 9

OH O

252 - 10%

C02Et

253 4 3

S c h e m e 5.27

S u g im u ra ’s m ethod for m aking the silyl enol ethers was also slightly

different; 4-(dimethylam ino)pyridine (DMAP) was found to be an efficient

cata lyst for the formation of the ether in benzene solution and was a

modification of an earlier procedure developed by Sekine^^ who used the

conditions to form bis-( tr im ethy ls ily l)eno l ethyl pyruvate in 80% yield

(com pared to 51%, obtained by Peliska and O'Leary in the absence of a

c a ta ly s t ^ ^ ) . However, we observed no significant increase in the yield of

the ethers com pared to our earlier attempts, also in the absence of a

catalyst, and yields were stable at around 40%. This figure was unaffected if

Sug im ura ’s careful anhydrous and anaerobic work-up was replaced by a

cycle o f rapid washing and drying. For our first attempts at reaction of the

ethers under the new conditions, an aldehyde, benzaldehyde, was used in

place o f the ketals used in the paper since at this stage, m ultip le

205

N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S yn th o n s

substitutions were not of primary concern to us but the only products of

the reaction , using boron tri fluoride diethyl etherate as promoter, were

u n r e a c te d a ld e h y d e and r e g e n e ra te d e s te r . W hen re p e a te d w ith

benzaldehyde dimethyl acetal 2 5 4 however, the presence of a small amount

o f the c in n am ald éh y d e d e r iv a tiv e 2 6 3 was the first indication of a

successfu l reac tion since the product was p resum ably form ed by the

e l im in a t io n o f m e th a n o l f ro m the a lk o x y k e to e s te r d u r in g

chrom atography of the crude reaction mixture (scheme 5 .2 8 ) .

9^® OSiMes OMe O

° N2(g), -78 °C °2 5 4 2 4 9

H O

H+

2 6 3

S c h e m e 5 .28

Though the rem aining m aterial was the expected m ixture of ester and

aldehyde (regenerated from the acetal under the same conditions that had

p ro d u ce d e lim in a tio n o f the p roduct) this resu l t was encou rag ing .

However, elevation of the temperature or an increased reaction time failed

to p rov ide fu rthe r ev idence of product fo rm ation , desp ite S ug im ura

claiming a yield of 71% for this reaction. In common with him however, we

observed that total consumption of the acid accompanied all attempts at its

reaction and assumed therefore that though cleavage o f the e ther was

occurring as predicted, decomposition pathways were com peting with the

reaction pathway and the reaction was therefore extrem ely sensitive to

a lterations in conditions.

W ith one exception (propanal dimethyl acetal), all of Sug im ura’s

electrophiles were aromatic, so we briefly investigated the reaction of some

aliphatic aldehydes with the silyl enol ethers to assess whether this would

affect the course of the reaction. The use of acetaldehyde at -78 °C on O-

trim ethylsily l-enol(ethylpyruvate) gave only a small amount of brown oil

(scheme 5 .2 9 ) .

206

N u c le o p h i l i c A d d i t i o n o f P y r u v ic A c i d S yn th ons

0 0S iMe3 HO H O1 + 1 O (i) BI^iEtzO, CH2CI2, -78— 0 °C II ^

M e " ^ H 'E t " 'E tO (ii) Pf, H2O ^

2 64- 2 4 9 2 6 5

S c h e m e 5 .29

This exhibited a broad peak in the infrared at 3431 cm"^ but no evidence of

a successful reaction was otherwise obtained. Similarly, the use of hexanal

produced only a complex mixture of products which included unreacted

aldehyde, despite it being used in stoichiometric quantities.

In o n e f in a l a t t e m p t , 0 - t r i m e t h y l s i l y l e n o l ( t e r t - h e p t y l

thiopyruvate) was reacted with benzaldehyde but merely gave a yellow oil

tha t c o n s is ted m ain ly of the regenera ted th io es te r and w hich was

recovered in 36% yield after chromatography in chloroform. Any further

attempts at nucleophilic addition of these compounds was abandoned at this

p o in t .

T hough our attempts at u til is ing these esters and th ioesters o f

pyruvic acid as synthons for the nucleophilic addition of the acid itse lf

were abandoned, we decided to make one final investiga tion into the

chemistry of the silyl enol ethers. Inspection of the compounds reveals a

double bond substi tu ted by two e lec tronega tive g roups, c rea ting , we

imagined, a potentia lly strong dienophile and so the reactiv ity of O-

t r i m e t h y l s i ly l - e n o l ( e th y lp y r u v a te ) 2 4 9 in the D iels-A lder reaction with

cyclopentadiene was investigated (scheme 5 .3 0 )

OMejSi Œ 2CI2, A

ÇH2

2 66 2 4 9

S c h e m e 5.30

OEt

In an tic ipa tion o f a fac ile reaction, we in it ia l ly ref luxed the

subs tra te with freshly cracked cyc lopen tad iene only b r ie f ly but this

procedure merely produced an intractable brown oil. By carrying out the

207

N u c le o p h i l i c A d d i t io n o f P y ru v ic A c i d S yn th o n s

reaction at a lower temperature (-78 °C, rising to 0 °C) no reaction was

observed at all, so the apparent instability of the silyl enol ether under the

reoction conditions was not easily offset by making the conditions more

m ild .

1 ,3-D iphenylisobenzofuran is an excellent diene in the D iels-Alder

r e a c t io n '^ ^ since there is a strong driving force from the aromatisation of

the cyclohexadiene ring, and it therefore seemed a useful alternative to

cyclopen tad iene (scheme 5 .3 1 ) .

2 5 6

OEtaSiO

OEt

CH-

2 5 7

C H iC k , A Ph

OEtPh

2 5 8

S c h e m e 5.31

Upon reflux of 0-tr ie thylsily l-enol(ethyl pyruvate) 2 5 7 in CH2 C I 2 for 1-2

hours, the fluorescent colour of the diene was observed to discharge and

work-up provided a yellow solid that was recrystallised from iPrOH to give

white crystals with a melting point some twenty degrees higher than the

diene. The H nmr spectrum of this compound gave no indication of the

presence of the triethylsilyl function, however. The low melting points of

the two isomers of l ,3 -d ihydro - l ,3 -d ipheny lisobenzofu ran (86-87 ®C and

99.5-100.5 °C for the cis and trans compounds respectively"^^) rule out the

possibility that reduction of the furan had occurred and the absence of a

peak at m/z 272 in the mass spectrum confirm s this (Mr for 1,3-

diphenylisobenzofuran is 270.31). The mass spectrum does display a peak at

m/z 363 and peaks corresponding to the loss of one and two phenyl groups

from this ion at 286 and 209 respectively, but it is not clear to what species

these figures refer since no pattern of f ragm enta tion of the desired

product seems to account for them.

As a final attempt we decided to use hexachlorocyclopentadiene in an

inverse dem and D iels-A lder reaction. Such reactions typ ica lly require

208


som ew hat forcing conditions such as reaction in a Carius tube at high

temperature, but are well established as a way of forcing the reaction of

e le c t ro n - r ic h a lkenes and in the m anufac tu re o f h igh ly c h lo r in a te d

p e s t i c id e s ^ ^ . The efficacy of the reaction was once again established with a

trial run of a method drawn from the literature*^^ so that 1-brom opropene

2 6 0 , after sealing and heating in a Carius tube at 170 °C for 19 hours with

h e x a c h lo r o c y c lo p e n ta d ie n e 2 5 9 , gave the adduct 2 6 1 shown in scheme

5 .3 1 in reasonable yield.

Cl Cl

+ Carius Tube, 170°C , 19hrs Cl^^ < ‘ *Br

H s C ^ H 25%

2 6 0

S c h e m e 5.31

Cl Cl

2 6 1

H o w ev er , the reac tion o f s to ich io m e tr ic am oun ts o f O - t r ie th y ls i ly l

enol(ethyl pyruvate) with the diene in a sealed tube at 150°C for four days

produced only an oily black material with no trace of the silyl enol ether

visible in its nmr spectrum. A repeat run at a higher temperature and for a

shorter time (180 °C for 24 hours) did however provide a solid material.

Breakage of the tube was accompanied by the release of an acidic-smelling

gas and the product was obtained as dry, shiny black flakes. The mass

spectrum of this material, however showed the only volatile constituent to

be h e x a c h lo ro c y c lo p e n ta d ie n e , the rem a in d e r p re su m a b ly be ing h igh

m olecu la r w eight tars form ed from the decom position o f the c learly

unstable silyl enol ether. With this failure, no further reactions o f the

compounds were investigated

209


5.3 E x p e r i m e n t a l

5.3.1 A p p a r a tu s and R e a g e n t s

All details for this section are as for chapter 4 except:

(i) In fra-red (IR) spectra were recorded on a P erk in -E lm er PE-983

sp e c tro m e te r using a liqu id film and d ich lo rom e thane so lvent. M ain

absorption bands are reported with wavenum ber n in cm" and intensity

(s, strong; m, medium; w, weak; br, broad);

(ii) Purif ica tion of te trahydrofuran for use in a lky la tion experim ents

was by reflux o v e r sodium wire with benzophenone as indicator.

The general procedure for the alkylation reactions described in the

p re v io u s s e c t io n was as fo llo w s : d i is o p ro p y la m in e or iso p ro p y l-

cyclohexylamine was dissolved in dry THF (to approximately 0.5 M) under

nitrogen gas in a two-necked flask connected to a nitrogen bubbler. The

tem pera ture was lowered to -78 °C and a sto ichiom etric amount of n-

butyllithium was added dropwise via the use of a syringe and septum, then

the temperature raised to 0 °C for fifteen minutes. After lowering to -78 °C,

the carbonyl compound was added and allowed to react for fifteen to thirty

minutes at this temperature or at 0 °C. The electrophile (approximately IM

in hexamethylphosphoramide) was next added and allowed to react at -78 °C

or 0 °C for a period of time between thirty minutes and twelve hours.

S tandard work-up refers to quenching with aqueous am m onium chloride,

fo llow ed by ex traction with d iethyl ether. The o rganic ex tracts were

washed with water and saturated sodium chloride solution, then dried and

evaporated in vacuo. All unsuccessful experiments have been indicated in

the previous section by placing the product in square brackets.

210


5 .3 .2 E x p e r i m e n t s

1. P r e p a r a t io n of 1 ,1 -D im e th y le th y l t h io p y r u v a te 231

OMe Me

l* l-D im e th y le th an e th io l (13.5 ml, 10.83 g, 0.12 mol) and dry

triethylamine (3.70 ml, 2.66 g, 0.03 mol) were added to dry ether (15 ml)

under an atmosphere of nitrogen then heated to reflux. Pyruvoyl chloride

(3.70 ml, 0.03 mol), made up to 10ml in dry ether was then added dropwise,

producing a vigorous reaction and a white precipitate. A further portion of

dry e ther (30 ml) was added to facilitate stirring, then reflux was

c o n t in u e d fo r 30 m in u te s . The p r e c ip i t a t e d t r i e th y l a m m o n iu m

hydrochloride was dissolved in the minimum amount of sodium chloride

solution then the organic layer was dried (MgS0 4 ), evaporated in vacuo and

distilled through a Vigreux column, giving the thioester as a yellow oil

(1.32 g, 8.23 mmol, 33%), b.p. 50-70 °C/17 mm Hg.

nmr (CDCI3): 1.51 (s, 9H); 2.38 (s, 3H)

13c nmr (CDCI3); 22.6; 28.4; 62.4; 189.8; 193.3

Mass Spectrum:

IR ( c m ' l ) :

160 [M+, 1%]; 57 [(CMe3)+, 64%]

1014.2 (s); 1093.7 (m); 1164.1 (m); 1210.0 (m);

1363.0 (m); 1448.7 (w); 1659.8 (s); 1717.9 (s); 1766.9

(m); 3994.2 (br)

A n a ly s is : Calculated for C7H 12O 2S, C:52.46; H: 7.56

Found, C: 52.16, H: 7.89

211


2. P r e p a r a t io n of 3 -E t h y l - p e n t a n - 3 -ol 235

MeMeOH

Me

This was prepared according to the l i te ra tu re m ethod^ then

distilled, giving the title alcohol as a clear oil (23,72 g, 0.20 mol, 93%), b.p.

140-144 °C (lit. 140-142 °C).

3. P r e p a r a t i o n of 3 - E t h y l - p e n t a n - 3 - t h i o l 236

Me MeSH

Me

This was prepared according to the l i te ra tu re m ethod^ ^ , then

distilled, giving the title thiol as a yellow oil which was used immediately

without further purification (11.75 g, 0.09 mol, 51%).

4. P r e p a r a t i o n of 1 , 1 - D ie t h y lp r o p y l t h i o p y r u v a t e 237

Me Me

Me

3-Ethyl-pentan-3-thiol (4.00 g, 0.03 mol) and triethylamine (3.10 g,

4.27 ml, 0.03 mol) were added to dry ether (15 ml), followed by pyruvoyl

chloride (3.30 g, 0.03 mol) in dry ether (5 ml). The mixture was refluxed for

1 hour then worked-up by the addition of aqueous ammonium chloride (15

ml). The organic layer was washed with water (15 ml) then dried (MgS0 4 )

and evaporated in vacuo to give a dark oil which was distilled (Kugelrohr),

giving the title thioester as a yellow oil (3.27 g, 16.16 mmol, 52%), b.p. 110-

115 °C/30 mm Hg.

212

N u c le o p h i l i c A d d i t io n o f P y r u v ic A c i d S yn th o n s

I r nmr (CDCI3):

nmr (CDCI3):


IR (cm‘ ^):

A n a ly s is :

0.85 (t, 9H, ^ Jh -H = 7.4Hz); 1.82 (q, 6H, ^JR-H =

7.7Rz); 2.33 (s, 3R)

8.2; 23.7; 27.2; 61.3; 190.9; 194.6

202 [M+, 5%]; 99 [(CEt3)+, 67%]; 29 [ (C iR s)^ , 100%]

1011.1 (m); 1130.5 (m); 1350.8 (m); 1378.3 (m);

1454.8 (m); 1655.6 (vs); 1717 .9 (vs); 1756.3 (s);

2874.0 (s); 2935.2 (s)

Calculated for C 10R I 8O 2S, C: 59.37; R: 8.97

Found, C: 59.26; R: 8.91.

5. P r e p a r a t i o n o f 1 ,1 - D i e t h y l p r o p y I 2 - o x o - t h i o l h e p t a n o a t e 238

Me Me

Me

Isopropylcyclohexylamine (0.06 g, 0.08 ml, 0.50 mmol) was added to

dry TRF (1.5 ml) and cooled to -78 °C then n-butyllithium (0.2 ml of a 2.5 M

solution in hexane, 0.50 mmol) was added. The temperature was raised to 0

°C for 15 m inutes then low ered again to -78 °C. 1 ,1 -D ie thy lpropy l

thiopyruvate (0.10 g, 0.49 mmol) in dry TRF (0.5 ml) was added at this

tem perature and stirred for 30 minutes. RM PA (0.59 ml) was then added,

followed 30 minutes later by bromobutane (0.14 g, 0.10 ml, 0.10 mmol) and

after warming to 0 °C and standard work-up, a yellow oil was obtained. This

was co lum n chrom atographed (E t2 0 :petroleum spirit, b.p. 40-60 °C, 0:1

rising to 1:0) to give a clear oil which spectral data indicated to be the title

thioester (0.06 g, 0.23 mmol, 47%).

213


nmr (CDCI3): 0.87 (t, 9H, ^Jh -H = 5.4Hz); 0.89 (t, 3H, ^JR-H =

7.5Hz); 1.43 (m, 12H), 2.30 (t, 2H, ^ J r -R = 7.3Rz)

nmr (CDCI3): 1.0; 8.1; 13.8; 22.4; 26.2; 28.1; 31.5; 53.1; 191.5;

197.2

Mass Spectrum (m/z): 258 [M+, 1%]; 131 [(Et3CS)+, 28%]; 9 9 [ (E t3 0 + , 5%];

57 [(C4R9)+, 100%]; 43 [(C3R 7)+, 76%]; 29 [(C2R5)+,

64%]

IR ( c m ' l ) : 1014.2 (m); 1090.7 (m); 1259.0 (w); 1378.3 (m);

1451.7 (w); 1598.6 (m); 1653.7 (w); 1717.9 (s);

2867.9 (w); 2922.9 (m); 2959 7 (s)

A n a ly s is : Calculated for C 14R 24O 2S, C: 65.58; R: 9.43

Found, C: 65.12; R: 9.12

6. Preparat ion o f 2 -b u ty lo c ta n o ic ac id 221

.COOKMe

Me

This was prepared according to the literature method^, followed by column

c h ro m a to g ra p h y (p e n ta n e :E t2 0 , 1:0 rising to 1:1), giving the title acid as a

clear oil (1.50 g, 7.50 mmol, 42%).

7. Preparation of P yruvoy l C h lo r ide 228

Me

214


This was prepared according to the literature m ethod^ ^ , giving the acid

chloride as a pale green oil (2.36 g, 0.02 mol, 24%), b.p. 24-37 °C/100-105 mm

Hg (lit. 53 °C/126 mm Hg).

8. P r e p a r a t i o n of O - T r i m e t h y l s i l y l -

e n o l ( l , l - d i e t h y I p r o p y l t h i o p y r u v a t e ) 2 3 9

Me

To a solution of ^heptyl thiopyruvate (0.40 g, 2.00 mmol) in dry DMF under

n itrogen was added trie thylam ine (0.63 g, 0 .36 ml, 6 .20 mmol) and

ch lo ro trim ethy ls i lane (0.67 g, 0.79 ml, 6.20 mmol), p roducing a white

precipitate. The mixture was stirred for two hours then dry hexane (10 ml)

was added and stirred and the hexane (upper) layer syringed out. The

m ate r ia l was s tored under n itrogen in hexane so lu t io n at reduced

temperature (0.30g, l.SOmmol, 54%).

^H nmr (CDCI3):

13c nmr (CDCI3):

0.24 (s, 9H); 0.84 (t, 9H, = 7.2Hz); 1.78 (q, 6H,

^JH-H = 7.4Hz); 4.50 (d, IH, 3j h _h = 1.6Hz); 5.32 (d, IH, 3Jh _h = 1.5Hz)

5.8; 7.9; 21.2; 58.8; 101.4; 140.3; 159.7

IR (cm’ l ) : 1075.4 (s); 1252.9 (s); 1399.7 (s); 1497.6 (s);

1611.7 (m); 1682.3 (vs); 1724.9 (vs); 1941.3 (w);

2763.8 (m); 3369.7 (w)

215

N u c le o p h i l ic A d d i t io n o f P y ru v ic A c i d S y n th o n s

9 . P r e p a r a t i o n o f 0 - T r i n i e t h y s i I y l - e n o l ( m e t h y l p y r u v a t e ) 2 4 8

Me

This was prepared according to the literature method'^^^, giving the ether as

a colourless oil (0.87 g, 4.99 mmol, 28%), b.p. 50-55 °C/10 mm Hg (lit. 75-76.5

°C/41 mm Hg).

10. P r e p a r a t i o n of 0 - T r i m e t h y s i l y l - e n o l ( e t h y I p y r u v a t e ) 249

Me

This was prepared according to the literature method'^ giving the ether as

a colourless oil (2.57 g, 13.65 mmol, 43%), b.p. 62-67 °C/15 mm Hg (lit. 69-70

°C/18mm Hg).

11. P r e p a r a t i o n of 0 - T r i e t h y s i l y I - e n o l ( e t h y l p y r u v a t e ) 257

Me

Ethyl pyruvate (5.0 g, 4.72 ml, 0.043 mol) was added to benzene with 4-

dim ethylam inopyrid ine (10 mg) and ch lorotriethylsilane (6.78 g, 7.55 ml,

0.045 mol) and heated to reflux. Triethylamine (4.53 g, 6.27 ml, 0.045 mol)

was then added, causing the immediate formation of a white precipitate.

Heat was maintained for three hours then the mixture was washed with

w ater (5 ml), dried (M g S 0 4 ) and distilled, giving the title ether as a

colourless oil (1.95 g, 0.01 mol, 17%) b.p. 150-155 °C/22 mm Hg.

216

N u c le o p h i l i c A d d i t io n o f P y ru v ic A c id S yn th ons

1 h nmr (CDCI3): 0.67 (q, 6H, ^ J r - H = 8.0Hz); 0.95 (t, 9H, ^ J r - H =

7.9Hz); 1.26 (t, 3R, ^Jh -R = 7.1Hz); 4.17 (q, 2H, 3JH-

H = 7.1Hz); 4.81 (d, IH, ^J j j . r = l.lHz); 5.44 (d, IH,

^JH-H = l .lH z)

13c nmr (CDCI3): 4.7; 6.5; 14.1; 61.0; 103.3; 147.5; 164.4

Mass Spectrum (m/z): 230 [M+, 1%] ; 115 [(SiEt3)+, 76%]; 29 [(C2H 5)+, 100%]

IR (cm-1): 861.2 (m); 1001.9 (m); 1237.6 (m); 1320.2 (s); 1620.0

(s); 1684.3 (s); 1727.1 (s); 3449.2 (br)

A n a ly s is : Calculated for C n H 2 2 0 3 S i , C: 57.34; H: 9.63

Found: C: 56.99; H: 9.52

12. P r e p a r a t io n o f ( 2 -oxo )c ycIoh e xy l pheny l m eth a n o l 246

This was prepared according to the literature m e t h o d ^ g i v i n g a yellow

solid which was recrystallised from EtOAc to give the title aldol as yellow

crystals (0.80 g, 3.92 mmol, 33%), m.p. 103-105 °C (lit. 103.5-104.5 °C for

erythro, 75 °C for threo isom er^^).

13. P r e p a r a t i o n o f 5 - B r o m o - l , 2 , 3 , 4 , 7 , 7 - h e x a c h I o r o - 6 -

m e t h y l n o r b o r n - 2 - e n e 2 6 1

Cl p

217

N u c le o p h i l ic A d d i t io n o f P y r u v ic A c i d S yn th o n s

This was prepared according to the literature method^ ^ from a mixture of

cis- and trans- 1-bromopropene (2.50 g, 1.77 ml, 17.69 mmol), giving the

title compound as a waxy white solid (1.39 g, 4.46 mmol, 25%), m.p. 187-188

°C (lit. 193-195 °C for 5-endo, 6-endo isomer^ ^).

14. At tem pted Preparation of the A d d u c t o f O -T rim ethy l s i ly l -

e n o l ( e t h y l p y r u v a t e ) a n d H e x a c h l o r o c y c l o p e n t a d i e n e

0 -T r im e th y ls i ly l -en o l(e th y l py ruvate ) (1 .78 g, 9 .45 m m ol) and

hexachlorocyclopentadiene (2.58 g, 9.45 mmol) were sealed in a Carius tube

and heated to 180 °C for 24 hours. Breakage of the tube was accompanied by

the release of an acidic-smelling gas. The tube was then rinsed in CH 2 C I 2

and evaporated to give a shiny black powder which consisted of unreacted

hexach lorocyc lopen tad iene and decom position p roducts .

5.4 R e f e r e n c e s

( 1 ) Morton, A. A.; Ful^well Jnr., F.; Palmer, L. JACS 1 9 3 8 ,6 0 , 1426.

(2 ) Hauser, C. R.; Chambers, W. J. JACS 1956, 75, 4942.

(3 ) De Free, D. O.; Closson, R. D. JACS 1958, SO, 2311.

(4 ) Creger, P. L. JACS. 1967, 89, 2500.

(5 ) Pfeffer, P. E.; Silbert, L. S. 36. 1971, 5290 , .

( 6 ) Petragnani, N.; Yonashiro, M. Synthesis. 19 8 2 , 521.

(7 ) Pfeffer, P. E.; Silbert, L. S. J. Org. Chem. 1970, 55, 262.

(8 ) Pfeffer, P. E.; Silbert, L. S.;Chirinko Jr., J. M. J. Org. Chem. 1 9 7 2 ,5 7 ,

451.

(9 ) Malian, J. M.; Bebb, R. L. Chem. Rev. 1969, 69, 693.

(1 0 ) Chan, L. L.; Smid, J. JACS 1968, 90, 4654.

(1 1 ) Normant, H.; Angelo, B. Bull. Chim. Soc. Fr. 1962, 810.

(1 2 ) Angelo, B. Bull. Chim. Soc. Fr. 1970, 1848.

(1 3 ) Peliska, J. A.; O'Leary, M. H. JACS 1 9 9 1 ,1 1 3 , 1841.

(1 4 ) Tapia, I.; Alcazar, V.; Moran, J. R.; Caballero, C.; Grande, M. Chem. Lett.

1990 , 697.

(1 5 ) Williams, D. R.; Benbow, J. W. Tet. Lett. 1 9 9 0 ,5 7 , 5881.

(1 6 ) Comforth, J. W.; Firth, M. E.; Gottschalk, A. Biochem. J. 1 9 5 7 ,6 5 , 57.

218

N u c le o p h i l i c A d d i t io n o f P y ru v ic A c i d S y n th o n s

(1 7 ) Schmidt, R. R.; Betz, R. Angew. Chem. Int. Ed. Engl. 1 9 8 4 ,2 5 , 430.

(1 8 ) Esswein, A. A.; Betz, R.; Schmidt, R. R, Helv. Chim. Acfa.1989, 72, 213.

(1 9 ) Unger, F. M. Adv. Carbohydr. Chem. Biochem. 1 9 8 1 ,5 5 , 323.

(2 0 ) Crich, D. SERC Earmarked Application. 1990.

(2 1 ) "Aldrich Library O f NMR Spectra", Edition II, Vol I . Aldrich

Chemical Co., 1983.

(2 2 ) Hauser, C. R.; Puterbaugh, W. H. JACS 1953, 75, 1068.

(2 3 ) Booth, P. M.; Fox, C. M. J.; Ley, S. V. J. Chem. Soc. Perkin Trans. I. 1987,

121 .

(2 4 ) Clarke, T.; Ley, S. V. J. Chem. Soc. Perkin Trans. I. 1987, 131.

(2 5 ) Ley, S. V.; Woodward, P. R. Tet. Lett. 1987, 28, 345.

(2 6 ) Voss, J. Synthesis o f Thioesters and Thiolactones. Vol. 6 of

Com prehensive O rganic Synthesis. 8 vols. Oxford: Pergamon, 1991.

(2 7 ) Ottenheijm, H. C. J.; De Man, J. M. M. Synthesis. 1975, 163.

(2 8 ) Hausler, J.; Schmidt, U. Chem. Ber. 1974, 107, 145.

(2 9 ) Spencer, M. D.; Ph. D., UCL, 1990.

(3 0 ) Edwin Wilson Jr., G.; Hess, A. J. Org. Chem. 1 9 8 0 ,4 5 , 2766.

(3 1 ) Rathke, M. W.; Lindert, A. JACS 1971, 95, 3318.

(3 2 ) Barton, D. H. R.; Crich, D. J. Chem. Soc. Perkin Trans. I. 1986, 1603.

(3 3 ) Bellamy, L. J. The Infrared Spectra O f Complex M olecules. Vol. 1 of 2

vols. NY: Chapman & Hall, 1975. .

(3 4 ) Rasmussen, R. J.; Tunnicliff, D. D.; Robert Brittain, R. JACS. 1949, 71,

1068.

(3 5 ) Stork, 0 .; Hudrlik, P. F. JACS 1 9 6 8 ,9 0 , 4462.

(3 6 ) House, H. 0 .; Czuba, L. J.; Gall, M.; Olmstead, H. D. J. Org. Chem. 1969 ,54 ,

2324.

(3 7 ) House, H. O.; Gall, M.; Olmstead, H. D. J. Org. Chem. 1 9 7 1 ,5 6 , 2361.

(3 8 ) Rasmussen, J. K. Synthesis. 1977 , 92.

(3 9 ) Mukaiyama, T.; Narasaka, K.; Banno, K. Chem. Lett. 1973, 1011.

(4 0 ) Mukaiyama, T.; Narasaka, K.; Banno, K. JACS 1974, 96, 7503.

(4 1 ) Mukaiyama, T.; Murakami, M. Synthesis. 1987 , 1043.

(4 2 ) Sugimura, H. Noguchi Kenkyusho Jiho. 1989 , 52 , 33.

(4 3 ) Sugimura, H.; Shigekawa, Y.; Uematsu, M. Syn. Lett. 1991, 153.

(4 4 ) Sekine, M.; Futatsugi, T.; Yamada, K.; Hata, T. J. Chem. Soc. Perkin

Trans. I. 1982, 2509.

219

N u c le o p h i l ic A d d i t io n o f P y r u v ic A c i d S y n th o n s

(4 5 ) Greenhouse, R.; Borden, W. T.; Hirotsu, K.; Clardy, J. JACS 1977, 99,

1664.

(4 6 ) Smith, J. G.; McCall, R. B. J. Org. Chem. 1 9 8 0 ,4 5 , 3982.

(4 7 ) Boger, D. L.; Patel, M. Prog. Heterocycl. Chem. 1989, 7, 30.

(4 8 ) Alexander, R.; Davies, D. I. J. Chem. Soc. Perkin Trans. 1. 1973, 83.

220

A p p e n d i x

K>K)to

5

:k"3atv.

1. nmr Spectrum of O-Trimctliylsilyl cnol( I , I -diethylpropyl thiopyruvate) 23 9

K)toOJ

T— 1— I— I— I— I— I— I— r —r

r

T

2. h l mm specl tum ol O-'Irimelhylsi lyl cnoKclhyl pyiiiv:ile) 2 4 V

K>K)

L- V iU l -J L _

r

f

j

T I I j r 1—I—:—r "1 j : i I i i ! i I i J i i ~

? I r'pM

3. 'I I nmr spectrum of 0-Trielhylsilyl enol(elhyl pyruvate) 2 5 7

C or r i gen da

Throughout this thesis:

(i) “pyrrolido” and “pyrrolidyl” should be read as pyrrolidino and

p y r ro l id in y l , resp e c t iv e ly ;

(ii) “piperido” should be read as piperidino;

(iii) “chloroform ” should be read as trichloromethane;

(iv) “ 1, 4-diaza[2.2.2]bicyclooctane” and “ l-aza[2 .2 .2]b icyclooctane”

should be read as 1, 4-diazabicyclo[2.2,2]octane and 1-

azab icyc lo [2 .2 .2 ]oc tane , respec tive ly ;

(v) “/A C 5 ” should be read as J. Am. Chem. Soc.

A d d e n d u m

The express ions “NHg (aq .)” or “aqueous am m onia” refer to an

aqueous ammonia solution at a concentration of approximately 2M.

225

synthetic studies of some biologically important molecules

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