chapter 1 synthesis c s and g i activity o p conidine a...

CHAPTER 1

SYNTHESIS, COMPUTATIONAL STUDY AND GLYCOSIDASE INHIBITORY

ACTIVITY OF POLYHYDROXYLATED CONIDINE ALKALOIDS - A BICYCLIC

IMINOSUGAR

Chapter I: Conidine Iminosugars

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CHAPTER 1

Synthesis, computational study and glycosidase inhibitory activity of polyhydroxylated conidine alkaloids - a bicyclic iminosugar

Section A: Introduction and Literature Survey for Conidine and Related

Iminosugars

1.1 INTRODUCTION TO IMINOSUGARS/AZASUGARS

There is growing interest amongst scientific community in a heterogeneous group of

hydrophilic plant alkaloids owing to their potential biological activities and their possible

ecological and taxonomic significance.1 Amongst these compounds, the

polyhydroxylated derivatives of the monocyclic piperidine, pyrrolidine and bicyclic

indolizidine, pyrrolizidine, quinolizidine and nortropane alkaloids have attracted a great

attention in last couple of years.2 Although these alkaloids have generally been grouped

together, their relationships are largely conceptual based on the fact that the hydroxyl

groups are held in a fixed stereochemistry by the heterocyclic ring system in a way

which mimics the stereochemical positioning of the hydroxyl groups in carbohydrates.

Members of these groups of alkaloids have been given various generic names such as

iminosugars,3 polyhydroxy alkaloids,1a azasugars,4 aminosugars5 or sugar-shaped

alkaloids6 so as to indicate structural resemblance to sugars.

Thus, iminosugars are polyhydroxylated alkaloids in which the endocyclic

oxygen atom of the sugar ring is replaced with the basic nitrogen.7 Mere this atomic

substitution confers these compounds with the ability to mimic sugars and inhibit various

carbohydrate processing enzymes; most significantly the glycosidases (glycoside

hydrolases) which are intimately involved in a huge array of biological functions.

Compounds which inhibit these enzymes consistently possess much potential as

medicinal agents for the treatment of a variety of diseases. As such, iminosugars

undoubtedly form the most attractive class of carbohydrate mimics reported so far. The


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densely-packed functionality and stereochemical information present in iminosugars

make them challenging targets for asymmetric synthesis, whereas carbohydrates are

apparently very attractive as chiral-pool starting materials for this purpose. Indeed, the

majority of the most successful syntheses of iminosugars use the latter approach. The

origin of their therapeutic use goes back to ancient times and traditional Chinese

phytomedicines. In Occident (the Western World), Haarlem oil – the first medication

produced on an industrial scale in the 17th century, was recommended for the treatment

of diabetes and for whitening the skin. One of the major constituents of Haarlem oil was

an extract from leaves of Morus alba – the white mulberry, an extremely rich source of

iminosugars.8 The scientific history of iminosugars began in the early 1960’s with the

almost simultaneous reports of the synthesis of sugar derivatives containing a nitrogen

atom in the ring by the groups of Paulsen,9 Jones10 and Hanessian.11 During that time the

replacement of an endocyclic oxygen atom in sugars by heteroatoms (N, S, P)12 to form

‘heteroses’13 was purely an academic exercise. In 1967, Paulsen published the first

synthesis of 1-deoxynojirimycin (DNJ)14 (Figure 1).

OHOHO R

OH

OH

HNHO

HOOH

OH

RR = OH NojirimycinR = H 1-Deoxynojirimycin (DNJ)

R = OH GlucoseR = NH2 Glcosamine

NHOHO

OH

OHNHO

HOOH

OH OH

n-BDNJ (ZavescaTM)in the treatment of Gauchers disease

Miglitol (GlycetTM)oral anti-diabetic drug

Figure 1: Some representative iminosugars as a natural products and ‘drugs’.

In the same year, Inouye et al.15 isolated nojirimycin from bacteria (Streptomyces) and

identified its antibiotic properties. The first rebirth of iminosugars came from the

isolation of DNJ from natural sources and the finding of its biological activity as a -

glucosidase inhibitor by Bayer chemists in 1976. Diversity of enzymes inhibited by

iminosugars promises a new generation of medicines in a wide range of diseases such as

diabetes, viral infections, lysosomal storage disorders or tumor metastasis.16 Various


4

structures are currently involved in clinical trials and the first successes are being

recorded. Recently, two iminosugar-based drugs have been approved: GlycetTM

(commonly known as Miglitol) (Figure 1) in 1996 for the treatment of complications

associated with type II diabetes, and ZavescaTM in 2003 as the first oral treatment for

Gaucher disease, a severe lysosomal storage disorder.

Since the late 1990s, the rate of discoveries has increased dramatically. In order

to find a potent analogue, original structures of iminosugar such as higher ring

homologues (seven- or eight-membered iminoalditols), conformationally constrained or

extended side chain analogues have been designed and synthesized. For example, a

constrained iminosugar consisting of β-lactam fused with polyhydroxylated piperidine

ring as the potential -galactosidase inhibitor17 and very recently after publishing our

work, the azetidine iminosugars reported by G.W.J. Fleet et al.18

1.1.1 CLASSIFICATION OF IMINOSUGARS:

In general, iminosugars are broadly classified as (a) monocyclic and (b) bicyclic

polyhydroxylated alkaloids. In that naturally occurring are classified into five structural

classes: polyhydroxylated piperidines, pyrrolidines are monocyclic while; indolizidines,

pyrrolizidines and nortropanes2 are bicyclic iminosugars. Many novel synthetic

iminosugars are also known and doubt many more are waiting for discovery. A account

of these iminosugars is given herewith.

1.1.1.1 MONOCYCLIC IMINOSUGARS

(a) Piperidines

The six-membered polyhydroxylated nitrogen atom containing ring compounds are

considered as polyhydroxylated piperidines. The first naturally occurring iminosugar to

be discovered was the piperidine iminosugar which was originally isolated as an

antibiotic from Streptomyces sp.19 but, following structural characterization,20 it was


5

given the trivial name nojirimycin (NJ) 1 after its isolation from Streptomyces

nojiriensis.21 In addition to its antimicrobial activity, nojirimycin was found to be a

potent inhibitor of - and β-glucosidases,22 as might be expected from its structural

imitation of glucose. Some of the other piperidine iminosugars later discovered, either

isolated from nature or synthesized in laboratory, are fagomine 2 ((2R,3R,4R)-2-

(hydroxymethyl)piperidine-3,4-diol) isolated from Fagopyrum esculentum

(Polygonaceae)23 followed by moranoline ((2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-

3,4,5-triol) from a species of Morus (Moraceae)24 commonly known as 1-

deoxynojirimycin 3 (DNJ), isofagomine 425 a glycogen phosphorylase inhibitor, and

miglitol 5.

HN R1

R2HOOH

HO

1 R1 = R2 = OH (NJ)2 R1 = R2= H3 R1 = H, R2 = OH (DNJ)

HN

OHOH

HO

4 isofagomine

N

OHHOOH

HO

5 miglitol

OH

Figure 2: Representative examples of piperidine iminosugars.

(b) Pyrrolidines

The existence of sugars in five membered furanose form tempted scientists to look for

the five-membered polyhydroxylated nitrogen heterocycles – commonly known as

pyrrolidine alkaloids. The tetrahydroxy pyrrolidine (2R,3R,4R,5R)-2,5-

bis(hydroxymethyl)pyrrolidine-3,4-diol (DMDP) 6 was isolated from species of the

legume genus Lonchocarpus.26


6

HN

OHHO

HO OH

HN

OHHO

HO

HN

HO

HO

6 (DMDP) 7 (DAB1) 8 (CYB3)

Figure 3: Representative examples of pyrrolidine iminosugars.

Some of other examples of pyrrolidine iminosugars are the 1-deoxy derivative of D-

arabinosimine DAB-l 7,1b isolated from the legume Angylocalyx boutiqueanus.27 The

1,2-dideoxy derivative was obtained from Castanospermum australe27 and subsequently

became known as CYB-3 8.1b

(c) Azepanes and Azocanes

Amongst monocyclic iminosugars the seven-membered iminocyclitols, commonly

known as azaseptanoses or polyhydroxy azepanes 9-1314,28 or ring homologues of

piperidine iminosugars, were synthesized and found to be promising glycosidase and

glycosyltransferase inhibitors. The hydrophilicity and conformational flexibility of

polyhydroxyazepanes made them as potentially useful minor groove binding ligands

(MGBLs).

HN

OHHO

OH

HN

OHHO

OHHO

R1

NHOHO

OH

OHH

NHO

OH

H OH

OH

OH

9 10 R1 = H11 R1 = CH2OH

12 13

HO

Figure 4: Representative azepane and azocane iminosugars.

Recently synthesized (2004)29 other higher ring analogues of piperidine iminosugars are

the eight-membered iminoalditols commonly known as polyhydroxylated azocanes

12/13 whose biological activity is yet unknown.


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1.1.1.2 BICYCLIC IMINOSUGARS

(a) Indolizidines

Indolizidine alkaloids are the six-membered ring fused with the five-membered ring with

nitrogen atom at the ring fusion 14-17. The first of the polyhydroxylated bicyclic

alkaloids discovered was swainsonine 14, a trihydroxyindolizidine alkaloid, isolated

from the legume Swainsona canescens.30

N

OHHO

OH

HON

OHOH

OH N

OH

OHN

OAc

H2N

14 15 16 17

HH H H N

H OH

OH

HOH2COH

I

Figure 5: Representative indolizidine iminosugars.

Subsequently seeds of another Australian legume, Castanospermum australe were found

to have another polyhydroxyindolizidine, castanospermine 15.31 Some of other

representatives of naturally occurring polyhydroxy indolizidine alkaloids are

lentiginosine 1632 and slaframine 17.33 Many more analogues castanospermine,

swainsonine and lentiginosine are either isolated or synthesized in the laboratory and

investigated for glycosidase inhibitory activity.

(b) Pyrrolizidines

Polyhydroxylated pyrrolizidine alkaloids are the bicyclic five-five fused ring structures

with the nitrogen atom at the ring junction. The first two polyhydroxypyrrolizidines

discovered are australine 18 and alexine 19 which were isolated at about the same time

from Castanospermum australe and Alexa leiopetala, respectively.34


8

N

OH

OH

18 -H (Australine)19 -H ( Alexine)

20 21

HO H

OH

N

OH

OH

HO H

OH

HO N

OH

OH

H

OH

Figure 6: Representative pyrrolizidine iminosugars.

The most recent polyhydroxypyrrolizidine is casuarine 20 which was isolated from

Casuarina equisetifolia (casuarinaceae). A library of hyacinthacine(s) 21, yet another

naturally occurring polyhydroxypyrrolizidine alkaloid, is also known in the literature.35

(c) Quinolizidines

Polyhydroxylated quinolizidine alkaloids (six-membered ring fused to six-membered

ring with the nitrogen atom at the ring fusion) are isosteric homologues of indolizidines

such as, tetrahydroxyquinolizidine 2236 and trihydroxy quinolizidine alkaloids 23.37

N

OHHO

OH

HO

22 23

N

HOOH

HO

H H

Figure 7: Representative quinolizidine iminosugars.

(d) Perhydroaza-azulenes

The synthetic 1-azabicyclo-[5,3,0]-decane compound is known as polyhydroxylated

perhydroaza-azulenes. These are fused five-seven bicyclic ring systems with nitrogen

atom at the ring junction. This includes trihydroxy-4-azabicyclo-[5.3.0]-decane 2438 and

pentahydroxy-4-azabicyclo-[5.3.0]-decane 25.39


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NOH

NOH

OH

HO

OHHO

HO

24 25

H OH H

Figure 8: Representative perhydroaza-azulene iminosugars.

(e) Nortropanes

The nortropanes are members of the most recently documented group of [5.2.1] bridged

iminosugars with the nitrogen atom at the bridge head and named as the calystegines An

– Cn where A, B and C stands for three, four and five hydroxyl groups in the compound

and the subscript indicates their order in chromatography. These are tri-, tetra- and

pentahydroxy nortropane alkaloids which were isolated from roots and root exudates of

Calystegia sepium (Convolvulaceae).40 The nomenclature used for calystegines is

derived from their chromatographic behavior during the original isolation from

Calystegia sp. The tri- and tetra-hydroxy calystegines A – C were initially separated as

two spots, A and B, after paper electrophoresis, and each of these spots were then

resolved into their isomeric components by liquid chromatography, to give calystegines

A1, A2, A3, A4 and B1, B2.41 Of these, only the structures of calystegines A3, B1 and B2

have been elucidated.41 Calystegines have been differentiated from other tropane

alkaloids by the lack of an N-methyl group and the presence of a hydroxyl group at the

bridge head of the bicyclic ring.


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NHOH

HOHO

NHOH

HONH

OHHO

NHOH

HOHO

NHOHOH

HONH

OHHO

HO

NHOH

HOHO

NHOHOH

HONH

OHHO

HO

HO OH

HO HO

HO

HOOH OH OH

HO

calystegine A3 calystegine A5 calystegine A6

calystegine B2 calystegine B3 calystegine B4

calystegine C1 calystegine C2 calystegine B1

Figure 9: Representative nortropane iminosugars (calystegines).

(f) Bicyclic diiminosugars

There are also entities incorporating extra nitrogen in more than one position, including

the one in the ring. For example, the six- and five-membered ring fused skeleton having

one nitrogen atom at the ring junction and another at the anomeric position with hydroxy

substituents in either of the rings, are reported in literature and generally known as

bicyclic diiminosugars. Kifunensine 26 – a potent immunomodulator and inhibitor of the

glycoprotein processing mannosidase-I, was isolated from the Actinomycete

Kitasatosporia kifunense 9482.42 Another well known example of naturally occurring

bicyclic di-iminosugar is the nagstatin 27 which is a potent N-acetyl-β-D-

glucosaminidase inhibitor.43

26 27

NOH

HO

OH

NHAcN

COOHNHO

OHOH

HO

N

O

O

H

Miscellaneous examples of constrained azasugars are nortropanes 28a-c,44 aziridines

29,45 30,46 3147 and their analogues.


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28a R1 = R2 = H28b R1 = OH, R2 = H28c R1 = H, R2 = OH

HN

HO

OHHO R1

R2 NH

OHHO OH

HO NHO

OHHO

NHHO

HO OH

29 30 31

1.1.2 BIOLOGICAL ACTIVITY AND THERAPEUTIC APPLICATIONS OF

IMINOSUGARS

To date, many reviews on the biological activity of iminosugars focused mainly on their

ability to inhibit glycosidases.1,2 In addition, a number of other biological activities of

iminosugars are also known.

1.1.2.1 Antidiabetic activity

Diabetes mellitus, generally referred to as diabetes, is a medical situation associated with

abnormally high level of glucose (or sugar) in the blood (termed as hyperglycemia).48

Diabetes Mellitus is a chronic disorder. In 2006, according to the World Health

Organization (WHO), almost 171 million people worldwide suffer from diabetes.

Modern scientific evidence reveals that much of the morbidity and deaths of diabetes can

be eliminated by insistent treatment with diet, exercise, and new pharmacological

approaches to attain better control of blood glucose level. Furthermore, the opportunity

of avoiding the commencement of diabetes using dietary supplements and/or herbal

medicines has attracted increasing attention. There are two main types of diabetes

mellitus known as Type 1 and Type 2. In addition to the use of various approaches to

cure diabetes, the use of -glucosidase inhibitor is one of the alternative therapeutic

approaches. In the 1970s, it was recognized that inhibition of all or some of the intestinal

disaccharidases and pancreatic -amylase by inhibitors could regulate the assimilation of

carbohydrate and these inhibitors could be used therapeutically in the oral treatment of


12

the non-insulin-dependent diabetes mellitus (NIDDM), commonly called as Type 2

diabetes. Even though the -glucosidase inhibitory activity of nojirimycin 1 and DNJ 3

in vitro was excellent, their efficacy in vivo was only moderate.49 Therefore, a large

number of DNJ derivatives were designed and synthesized in the hope of increasing the

in vivo activity and this led to the discovery of miglitol 5 which was chosen as the most

favorable inhibitor out of a large number of in vitro active agents. In Type 2 diabetes,

hepatic glucose production is augmented.50 A potential way to suppress hepatic glucose

production and lower blood glucose in type 2 diabetes patients may be effected by

inhibition of hepatic glycogen phosphorylase. In enzyme assays, 1,4-dideoxy-1,4-imino-

D-arabinitol (DAB1) 7, was found to be a potent inhibitor of hepatic glycogen

phosphorylase.51 DAB1 further inhibited hepatic glycogen breakdown in vivo and

displayed an accompanying antihyperglycemic effect, which was most pronounced in

obese mice.52 Recently the synthetic piperidine iminosugar isofagomine 4 has been

reported to inhibit effectively the hepatic glycogen phosphorylase with an IC50 value of

0.7 µM, and to prevent basal and glucagon stimulated glycogen degradation in cultured

hepatocytes with IC50 values of 2-3 µM.53

1.1.2.2 Anti-viral activity

A number of iminosugars have anti-viral activities, in particular against Human

Immunodeficiency Virus (HIV) – the virus responsible for Acquired Immune Deficiency

Syndrome (AIDS).54 Swainsonine 14, Castanospermine 15 and DNJ 3 have been found

as effective antiviral agents.55

1.1.2.3 Lysosomal diseases

Domestic animals in Australia that consume species of Swainsona which contains the

alkaloid swainsonine suffer from a neurophysiological disorder called 'peastruck'.56 As

swainsonine is ambiphilic i.e. both water and lipid soluble, it is capable of crossing


13

plasma membranes and accumulates within the lysosomes. Therefore, if animals were

feeding on plants containing swainsonine for a short period of time, they could consume

sufficient amount to cause neurophysiological disorders. In 1990, two other iminosugars

that inhibit glycosidases, namely lentiginosine 16 and 2-epi-lentiginosine have been

isolated from species of Astragalus and could contribute to the toxicity of the plants.32

1.1.2.4 Anti-cancer properties

There is evidence that oligosaccharides on the surface of tumor cells play an important

role in malignant phenotype and tumor growth. Iminosugars have been tested for their

capability to inhibit glycosylation or processing of aspargine linked oligosaccharides that

could inhibit tumor growth and metastasis. To date, swainsonine 14 and castanospermine

15 has attracted the most attention as they inhibit tumor growth and stimulate the

immune response.57 Swainsonine also enhances bone marrow cellularity, stimulates

lymphocyte proliferation and caused a decrease in metastatic foci in the lungs.58 It was

also found to improve the activities of the mouse immune system in vitro59 monitored the

antibody response of sheep red blood cells (SRBC) in immunodeficient mice when

treated with an immunosuppressive factor such as sarcoma tumors.

1.1.2.5 Nematicidal activity

Plant parasitic nematodes cause widespread crop damage around 20% of the world’s

total crop production and are responsible for severe financial loss. The pyrrolizidine

alkaloid DMDP 6 was found to possess a range of activities against several species of

parasitic plant nematodes. Birch et al.60 showed that DMDP can be used as a foliar spray,

soil drench or seed coating. To date, the mechanism underlying the nematicidal activity

is speculative. DMDP may be acting directly on the nematodes by inhibiting enzymes or

indirectly by activating or enhancing the resistance system in the plant roots.


14

1.1.2.6 Plant growth regulatory activity

Castanospermine 15 has been shown to be a potent plant growth regulator, inhibiting

root elongation in dicotyledons by 50 % at a concentration of 300 ppb. Swainsonine did

not inhibit elongation in either root type at any concentration used.61 It is not known if

these compounds occur naturally in root exudates of iminosugar producing plants; if they

do, these results indicate that some iminosugars might have allelopathic properties.

1.1.2.7 Immunomodulatory activity

Immunomodulation is a process which changes the immune system of an organism in

both ways i.e. immunostimulation and immunosuppression. Immunomodulator is a

substance which regulates the immune system to give optimum response. The

immunosuppressive activity of indolizidine alkaloids such as swainsonine 14,

castanospermine 15, and kifunensine 26 has been reported.62 Recently, synthetic

analogues of nojirimycin – a piperidine iminosugar were also found to act as potential

immunosuppressive agents.63 The indolizidine alkaloids I reported from our group

shown in Figure 5 are found to be promising immuno-potentiating property at µM

levels.81l

1.1.2.8 Gaucher disease

Gaucher disease is a lysosomal storage disorder (LSD) and is relatively rare hereditary

disorder as it is caused by deficiency in lysosomal acid β-glucosidase (GlcCerase) – the

enzyme responsible for the catabolism of glucosylceramide. Defects in the performance

of this enzyme lead to the accumulation of undegraded glucosylceramide (GlcCer) in

macrophages and to severe symptoms. Iminosugar such as N-butyl-1-deoxynojirimycin

(Zavesca®) inhibits the biogenesis of GlcCer avoiding the accumulation of

glucosylceramide (GlcCer).64


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1.1.2.9 Iminosugars as glycosidase inhibitors

As previously mentioned, iminosugars are potent inhibitors of very important class of

enzymes – glycosidases which are widespread in biological systems.65 These enzymes

catalyze the hydrolysis of glycosidic bond in carbohydrates and glycoconjugates. The

overall effect is the release of low-molecular weight monosaccharides and

oligosaccharides. Due to importance of glycosidases in many biochemical systems, it is

obvious that compounds that inhibit them exhibit biological activity. Thus, glycosidase

inhibitors may cause detrimental effects but they can also be used as valuable tools while

investigating the physiological role of glycosidase enzymes. The enzymatic activity of

iminosugars has been ascribed to their structural resemblance to simple sugars. The

scope and selectivity of the inhibition was considered to be dependent on the position,

number and stereochemistry of the hydroxyl groups on the molecule. However,

experimental data have shown that the chirality of the hydroxyl groups on the

iminosugars was not sufficient to predict their ability to inhibit enzymes. Enzyme

inhibition can be extremely variable and influenced by the source of the enzyme as well

as experimental conditions, such as pH.66 The overall glycosidases process involves

cleavage of the glycosidic bond linking anomeric carbon of the sugar with an oligo- or

polysaccharide or a nucleoside diphosphate group. The released glycosyl group is further

transferred to water (by glycosidases) or to some other nucleophilic acceptor (by

transferases).

1.1.3 GLYCOSIDASE MECHANISM

The glycosidase mechanism was proposed by Koshland67 in 1953 and was subsequently

developed by many workers. The presently accepted form of this mechanism is

demonstrated using the example of D-galactose as one of the saccharide unit as shown in

Scheme 1. -Glycosidase mechanism is generally believed to occur through an E2 type


16

elimination process, in which a positively charged aglycon (the leaving group) and the

lone pair of the ring oxygen are placed antiperiplanar, cooperatively assisting the

glycosidic bond cleavage reaction via intermediate I.68 In the case of the β-glycosidase

reaction, if the enzyme proceeds via an E2 type mechanism, similar to that of the -

glycosidases, the protonated substrate has to go through a highly strained intermediate II

that may not favor further reaction. Therefore, it is considered that in the case of a β-

glycosidase reaction, the positively charged aglycon departs via an E1 like mechanism

involving the glycosyl cation III which is further stabilized by the lone pair of electrons

on the ring oxygen to give IV. Although, the final reaction intermediate in both the

reaction mechanisms is the same flattened, half chair oxocarbonium ion IV, the first

intermediate in the case of β-glycosidase reaction differs with respect to the position of

charge development. Glycosidases are also classified on the basis of the stereochemical

outcome of the newly formed anomeric bond namely into - and β-glycosidases. The

enzymatic cleavage of the glycosidic bond liberates a sugar hemiacetal with either the

same configuration as the substrate (retention) or less commonly, the opposite

configuration (inversion) and based on this criterion glycosidases are classified as

retaining or inverting glycosidase.


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A. -Glycosidase reaction I

E2 like elimin.

Monosaccharide

O

OOHHO

OH OH

R

O

HOOHHO

OH OH

R

Protonationby enzyme

OO

OHHO

OH OH

R

OH

HOOH

O

O

R

OH

Protonationby enzyme

O

OHHO

OH OH

O

OHHO

OH OH

+ H2O

O

OHHO

OH OH

OH

E1 like elimination

E1 like elimin.

B. -Glycosidase reaction

OO

OHO

OH OH

RR =

polysaccharides

II

III

IV

VFirst intermediate

First intermediate

Second intermediate

Scheme 1: Glycosidase reaction mechanism involved in - and β- glycosidases.

The chemical entity that is capable of mimicking either the charge or shape (or both) of

the substrate or that of any of the transition states, can act as a reversible inhibitor of that

particular glycosidase. These entities are termed as glycosidase inhibitors. The biological

activity of azasugars – the glycomimetics arises from the conformational resemblance to

natural sugar. The protonation of the ring nitrogen at physiological pH, mimics the

developing charge of an intermediate oxocarbenium ion during glycosidic bond

cleavage.

HN

OHN

OHHHPhysiological

pHHO HOO

OH

HO

Scheme 2: Conformational resemblance of protonated iminosugars with oxocarbenium intermediate.


18

Thus, the presence of iminosugar moiety inhibits the process of glycosyl bond cleavage

and this intrinsic capability of azasugars makes them to be identified as prospective

therapeutic agents for the treatment of various human disorders.

The current chapter deals with the synthesis and conformational study of conidine

iminosugars and testing of their glycosidase inhibitory activity. Their detailed

introduction and literature methods are described herein.

1.2 CONIDINE IMINOSUGARS/ AZETIDINE IMINOSUGARS:

In general, the six/four ring fused compounds with nitrogen atom at the ring fusion of

type 32 are called as conidine69 alkaloids and its 25 oligomer is known to be heparin

antagonist.70 Although, a few examples of alkyl substituted conidines are known;71 there

was not a single report on conidine iminosugars before this work has been published.72

Only related example describing β-lactam iminosugar hybrid 33 which is a competitive

potent galactosidase inhibitor is reported by Pandey et al.17. Later on G.W.J. Fleet et al.18

have synthesized polyhydroxylated conidine and azetidine iminosugars and studied their

glycosidase inhibitory activity. G. I. Georg et al.73 have reported the synthesis of eight

and four membered iminosugar analogues as inhibitors of testicular ceramide-specific

glucosyltransferase, testicular β-glucosidase 2, and other glycosidases.

NN NO

H

OH

OHOH

H OH

OH

OH

3332

-H = 34a-H = 34b

1 2

34

56

7

8

Figure 10: Conidine iminosugars.

This chapter of the thesis deals with the synthesis of six-four fused ‘Conidine’ bicyclic

iminosugars 34a and 34b using chiron approach, their conformational study using 1H

NMR information and Density Functional Theory (DFT) study and evaluation of their


19

glycosidase inhibition against various enzymes. Therefore, it is more appropriate to

confine our discussion related to polyhydroxylated conidine or analogous azetidine

iminosugars. A brief account of these iminosugars in particular synthetic account and

biological activities of this class of iminosugars is given below.

1.3.1 REPORTED METHODS FOR THE SYNTHESIS OF

POLYHYDROXYLATED CONIDINE AND RELATED AZETIDINE

IMINOSUGAR ANALOGUES

1.3.1.1 Method due to Hall and Inch74

Hall and Inch reported the azetidine derivative of monosaccharide for the first time

(Scheme 3). They observed an unexpected formation of the fused galactose azetidines 37

during their attempt to synthesize the 4,6-bis-amino galactose derivatives 36 from the

corresponding glucose 4,6-di-tosylates 35 by treatment with ethanolic methylamine.

They have not extended the work on the azetidine compound 37.

O

OROR

ROTsO

OTs

Scheme 3: Reagents and conditions: (a) MeNH2, EtOH, 120 oC

O

OROR

RO

NMe

O

OROR

ROMeHN

NHMe

+

R = Me, Bn R = Me, 30%R = Bn, 4%

R = Me, 30%R = Bn, 63%

35 36 37

a

1.3.1.2 Method due to Pandey et al.17

A β-lactam azasugar hybrid (polyhydroxylated carbacephem) 33 as a competitive potent

galactosidase inhibitor was synthesized by Pandey and co-workers. The key intermediate

43, obtained by convergent synthesis from L-(+)-tartaric acid and 3-aminopropanol using

the chemical transformations is shown in scheme 4.


20

L-(+)-Tartaric acid

3-Aminopropanol

O

O OH

N

O

Boc

Ref. 73O

O O

H

H2N

TMS

OH

O

OHN

TMS

OH

O

O N O

TMS

a

b, c d, e

f

g

Scheme 4: Reagents and conditions: (a) IBX, EtOAc, reflux, 9 h, 85%; (b) (Boc)2O, TEA, 18 h; (c) CH3CH(OEt)2,PPTS, benzene, reflux, 24 h, 77% over 2 steps; (d) s-BuLi, TMEDA, 78 oC, 3 h, then TMSCl, 78 oC to rt, 3 h; (e)2 N HCl, dioxane, 80 oC, 45 min, 88% over 2 steps; (f) NaBH(OAc)3, 1,2-dichloroethane, 12 h, then 2 N NaOH, 2 h, 71%; (g) (CH2O)n, benzene, Dean–Stark, 4 h, 95%; (h) h, 450 W, lamp, CH3CN:i-PrOH (3:1), 4 h, 60%; (i) OsO4,K3Fe(CN)6, K2CO3, py, t-BuOH/H2O (1:1), rt, 16 h, 90%; (j) NaIO4, silica gel, 15 min; (k) NaBH4, MeOH, rt, 4 h,82% over 2 steps; (l) BnBr, NaH, THF, reflux, 12 h, 86%; (m) 1 N HCl, MeOH, rt, 4 h; (n) BnBr, NaH, TBAI, THF,reflux, 24 h, 78% over 2 steps; (o) 6 N HCl, dioxane–MeOH, reflux, 48 h; (p) (Boc)2O, TEA, rt, DCM, 8 h; (q) PDC,DMF, rt, 8 h, 57% over 3 steps; (r) TFA, DCM, 0 oC, 3 h; (s) 2-chloro-1-methylpyridinium iodide, TEA, CH3CN, 60oC – rt, 32 h, 53% over 2 steps; (t) H2, Pd/C, 60 psi, MeOH, 6 h, 95%.

38 39

40 41

42

43

O

O N O

TMS

N O

HO

O N O

HO

O

HOHO

N O

HHO

O

O

N O

HBnO

O

ON O

HBnO

BnO

BnON OH

HBnO

BnO

BnO

O

BocN

HBnO

BnO

BnO O

N

HHO

HO

HO O

Similarly, D-(+)-Tartaric acidN

HHO

HO

HO O

h i j, k

l

m, no, p, qr, s

t

43 44 45 46

47484950

33

51, ent-33

The intermediate 43 was cyclized employing a protocol already reported from their

group75 by irradiating a dilute solution of 43 (3 mmol) and 1,4-dicyanonaphthalene (0.4

mmol) in a mixture of acetonitrile:iso-propanol (3:1, 250 mL) in a Pyrex vessel using a

450 W Hanovia medium pressure lamp, to give 44 as a single diastereomer in a 60%

yield. The dihydroxylation of 44 using osmium tetraoxide produced 45 in a 90% yield.


21

Single crystal X-ray diffraction analysis confirmed the stereochemistry of newly

generated stereocentre in 45. The diol, upon sodium metaperiodate oxidation afforded

the corresponding ketone, which was immediately subjected to sodium borohydride

reduction to afford 46 in a 82% yield as the only diastereomer. The stereochemistry of 46

was also confirmed from 1D as well as 2D 1H NMR spectroscopy of the corresponding

benzylated derivative 47.

A selective deprotection of the acetonide moiety of 47 and benzyl protection of

the resultant diol gave the corresponding tribenzylated molecule 48. Subsequently, the

1,3-oxazine ring moiety in 48 was opened by refluxing with 6 N HCl in dioxane–

methanol for 48 h. The resultant secondary amine was re-protected as its N-Boc

derivative prior to PDC oxidation to the corresponding acid 49. The deprotection of the

N-Boc moiety of 49 by stirring with TFA in DCM at 0 oC for 3 h followed by the

treatment with 2-chloro-1-methylpyridinium iodide (Mukaiyama’s reagent) in the

presence of excess triethylamine afforded β-lactam 50 in a 53% yield. The removal of

the O-benzyl groups by hydrogenation at 60 psi afforded β-lactam-azasugar hybrid

molecule 33 in a 95% yield.

In order to correlate the enzyme specific inhibition property of 33, Pandey et al.

also synthesized its (L-galacto configured) enantiomer 51 (ent-33) using similar synthetic

path starting from D-(−)-tartaric acid. Since, it is observed by the same group76 that 1-N-

iminosugar 53 showed better inhibitory activity for the β-glucosidase (Ki = 30 µM) than

52 (Ki = 90 µM), it would be interesting to evaluate the enzyme inhibition activity of 54

as well. In this context, they synthesized compound 54 following an analogous route to

that described for 33, starting from alcohol 55 (Scheme 5).


22

OH

O

ON

O

O

O

TMS

N

O

O O

H

Scheme 5: Reagents and Conditions: (a) IBX, EtOAc, reflux, 9 h; (b) 42, NaBH(OAc)3, 1,2-dichloroethane, 12 h, then 2 N NaOH, 2 h; (c) (CH2O)n, benzene, Dean–Stark, 4 h, 75% over 3 steps; (d) h, 450 W lamp, CH3CN:i-PrOH (3:1), 4 h, 60%.

a, b, c d Scheme 5

52 53 54

55

NHHO

OHOH

HO

NHHO

HON

HO

HO

H

O

56

54

57

The inhibitory activities of 33, 51 and 54 were assessed against β-galactosidase

(Aspergillus oryzae), -galactosidase (coffee beans), β-glucosidase/β-mannosidase

(almonds), -glucosidase (yeast) and -mannosidase (jack beans). The D-galacto-

configured β-lactam 33 exhibited competitive and specific inhibition only against β-

galactosidase. It inhibited -galactosidase very poorly and showed no inhibition against

-/β-glucosidase and -/β-mannosidase. This enzyme specific inhibition of 33 is in good

agreement with its D-galacto-configured structure. Furthermore, compounds 51 and 54

have shown no inhibition against any of these enzymes under study.

1.3.1.3 Method due to Soengas et al.77

In 2011, Soengas and co-workers have synthesized 3,3-dimethyl azetidine tri-acetates

from various polyhydroxylated β-lactams. Reduction of β-lactams 58 and 59 with lithium

aluminium hydride gave azetidines 60 and 61 which on subsequent hydrolysis of

acetonide and silyl deprotection using aqueous trifluoroacetic acid, followed by per-

acetylation afforded the azetidine tri-acetates 62 and 63 (Scheme 6).


23

NBn O

OO

TBDPSON

Bn

OO

TBDPSO

Scheme 6: Reagents and conditions: (a) LiAlH4, THF, ; (b) i. TFA/H2O; ii. Ac2O, pyr., DMAP

NBn

OAcAcO

AcO

58 (6R)59 (6S)

60 (6R)61 (6S)

62 (6R)63 (6S)

a b

1.3.1.4 Method due to Fleet et al.18a

G. W. J. Fleet and co-workers utilized a highly efficient and flexible method for the key

azetidine ring formation which is demonstrated by the cyclizations of 3,5-di-O-triflates

of pentoses and hexoses, and of a 2,4-di-O-triflate of glucose, with various primary

amines. They have generalized this methodology to synthesize the two enantiomeric

pairs of azetidine iminosugars 69a/69b and ent-69a/ent-69b. The use of D- and L-sugars,

to reach the common intermediate 64, by simple chemical conversions is shown in

Scheme 7.

O

O

O

TfO

TfOO

O

O

HO

HOO

O

O

NR

PentosesNH

OHOH

OH

HexosesO

O

O

HO

O

O

O

O

O

HO

HOHO

NH

OHOH

OH NH

OHOH

OH NH

OHOH

OHNH

OHOH

OH

a b c

d

e

Scheme 7: Reagents and conditions: (a) Tf2O, Pyr., CH2Cl2 -30 oC, 1-2 h, 88-95%; (b) R-NH2, MeCN, or R-NH2,DIPEA, MeCN, rt, 5-24 h, 54-97%; (c) i. 1:1, TFA/H2O, rt, 15-48 h, ii. NaBH4, H2O, rt, 2-24 h, 17-98% over 2 steps, iii.Pd/C, NH4HCO2, MeOH, reflux, 20-30 min, 82-99%; (d) H2SO4. (1%, aq), MeOH, rt, 1.5 h or 80% AcOH, H2O, rt, 18 h, 81-85%; (e) i. NaIO4, H2O, rt, 4 h, ii. NaBH4, EtOH, rt, 1 h, 96-97% over 2 steps.

R = PhCH2R = MeR = Me(CH2)3R = Me(CH2)8R = p-OMe-C6H4-CH2

64

65

66

67 68 69

69a 70a, ent-69a 69b 70b, ent-69b

The intermediate 64 obtained from hexoses was converted to respective ditriflate

derivatives 67 that on treatment with amine eventually get converted to azetidine ring

fused furanose compounds 68 is the key step in their synthesis. 1,2-Acetonide


24

functionality in 68 was cleaved in 1:1 TFA/H2O or 80% AcOH and resultant hemiacetal

was reduced using NaBH4 to corresponding primary alcohol. The protecting group on

nitrogen was then removed under hydrogenolysis conditions to afford azetidine

iminosugars 69a/69b and 70a/70b (ent-69a/ent-69b) (Scheme 7). These iminosugars

were tested for glycosidase inhibitory activity, and L-xylo- and L-arabino azetidine

iminosugars show particularly significant and specific inhibition of nonmammalian -

glucosidases.

1.3.1.5 Method due to Fleet et al.18b

In this sequel publication, G. W. J. Fleet and coworkers extended the above methodology

from -furanosides to various β-pyranosides. The required ditriflate 73D was accessed

from the diacetone D-allose 71D using three steps. Thus compound 71D was converted

into monosilylated diol 72D (selective removal of 5,6-acetonide followed by

monosilylation of resultant primary alcohol) which on reaction with triflic anhydride in

pyridine gave compound 73D. The SN2 displacement of ditriflate 73D with various

primary amines afforded the azetidine ring skeleton in good yields. Removal of 1,2-

acetonide functionality using trifluoroacetic acid in water, reduction of resultant

hemiacetal with sodium borohydride in water at room temperature, per-acetylation of

intermediate tetra-ol in acetic anhydride and pyridine gave compound 75D for isolation

and characterization purpose and finally global deprotection gave azetidine iminosugars

76D, 79D and 80D. Similar reactions were performed with enantiomeric compound 71L

to afford the corresponding azetidine iminosugars 76L, 79L and 80L (Scheme 8).


25

O

O

O

O

O

HO

O

O

O

TBDMSO

HO

HO

aO

O

O

TBDMSO

TfO

TfO

b c

d

O

O

O

NR

TBDMSO

e

fO

O

O

NBn

TBDMSOg

BnN

OAc

CH2OAc

OAcAcOH2C

HN

OH

CH2OH

OHHOH2C N

OH

CH2OH

OHHOH2C

R

Scheme 8: Reagents and Conditions: (a) i. CH3COOH/H2O (7:3), rt, 18 h, 81-85%, ii. tBuMe2SiCl, Imidazole, DMF, 20 to 10 oC, 1.5 h, 94-79%; (b) Tf2O, pyr., CH2Cl2, 30 to 10 oC, 2h, 98-96%; (c) MeNH2, DIPEA, MeCN, 40 oC, 15h, 90-65% for Me and BuNH2, DIPEA, MeCN, 40 oC, 16 h, 88-84% for Bu; (d) TFA/H2O (9:1), rt, 70-72 h; then NaBH4, H2O, rt, 18-24 h, 47-92% for Me over 2 steps and 88-98% for Bu over 2 steps; (e) BnNH2, DIPEA, MeCN, 50 oC, 25 h,93-84%; (f) TFA/H2O (9:1), rt, 3 h; then NaBH4, H2O, rt, 18 h; then Ac2O, pyr., rt, 23 h, 68% over 3 steps; (g) i.NaOMe, MeOH, rt, 21 h, 98-88%, ii. HCOONH4, 10% Pd/C, anhydrous MeOH, reflux, 30 min, 100-71%.

77D, R = Me78D, R = n-Bu

79D, R = Me80D, R = n-Bu

75D76D 74D

73D72D71D

O

O

O

O

O

HO

HN

OH

CH2OH

OHHOH2C

N

OH

CH2OH

OHHOH2C

R

79L, R = Me80L, R = n-Bu76L71L

and

They have also exploited this strategy in the synthesis of similar azetidine iminosugars

using 1,3-di-triflate displacement in pyranose form of sugars. Thus, benzylation of

compound 81L followed by its conversion to pyranose form by 1,2-acetonide opening

and subsequent acetylation afforded tetra-acetate 83L. Treatment of 83L with hydrogen

bromide in acetic acid, followed by treatment of the resulting crude bromide with

methanol in the presence of silver carbonate, afforded the fully protected β-glucoside

84L. Deprotection of all acetates followed by selective protection of primary alcohol

with trityl chloride in pyridine in the presence of DMAP afforded the diol 85L which

subsequently treated with triflic anhydride and pyridine to give ditriflate. Further the

smooth addition of N-benzyl amine led to the bicyclic azetidine compound 86L. Acid

hydrolysis of compound 86L afforded both trityl and anomeric deprotected compound.

Sodium borohydride reduction of resulting hemiacetal led to the triol which was further

converted into corresponding triacetate 87L. Removal of acetates with sodium

methoxide in methanol and deprotection of N- and O-benzyl groups with ammonium


26

formate, Pd/C in anhydrous methanol gave the azetidine iminosugar 88L in 8% overall

yield from compound 81L. Similar reaction sequence was performed with diacetone D-

glucose 81D to give enantiomeric azetidine iminosugar 89D (ent-88L) in 22% overall

yield (Scheme 9).

O

O

O

O

O

HO

O

O

O

O

O

BnO

b

OAcO

OBnAcO OAc

CH2OAc

c

OMeO

OBnAcO OAc

CH2OAc

d

OMeO

OBnHO OH

CH2OTr

eO

TrOH2C

MeO OBn

BnN

NBn

OBn

CH2OAcAcO

AcOH2Cg

NH

OH

CH2OHHO

HOH2C NH

OH

CH2OHHO

HOH2C

Scheme 9: Reagents and Conditions: (a) BnBr, NaH, DMF, rt, 23 h, 88-94%; (b) i. TFA/H2O (1:1), rt, 24 h, 81-86%, ii. Ac2O, pyr., rt, 15 h, 75-85%; (c) i. HBr, AcOH, CH2Cl2, 5 oC, 20 h, ii. AgCO3, MeOH, rt, 6 h, 53-62% over 2 steps; (d)i. NaOMe, MeOH, 40 oC, 43 h, ii. TrCl, DMAP, pyr., rt, 16 h, 57-90% over 2 steps; (e) i. Tf2O, pyr., CH2Cl2, 10 oC, 2h, 83-90%, ii. BnNH2, MeCN, 70 oC, 2h, 86-80%; (f) i. 1,4-dioxane/2M HCl (1:1), 50 oC, 22 h, ii. NaBH4, MeOH, rt, 2.5h, iii. Ac2O, pyr., rt, 16 h, 70-89% over 3 steps; (g) i. NaOMe, MeOH, 45 oC, 23 h, ii. HCCNH4, 10% Pd/C, AnhydrousMeOH, reflux, 30 min, 100-91% over 2 steps.

a

f

O

O

O

O

O

HO

81L 82L 83L 84L

85L86L87L

81D88L 89D, ent-88L

1.3.1.6 Method due to Fleet et al.18c

In one more subsequent publication by Fleet and coworkers, they turned their attention to

the hydroxyconidine iminosugars – more resembles with our work. They have compared

the final hydroxyconidine compound 90 as the constrained analogue of swainsonine, 14

and azetidine iminosugar 92 as the constrained analogue of DIM [1,4-dideoxy-1,4-

imino-D-mannitol], 91 (Figure 11). The sterically constrained and functionalized

conidine 90 was remarkably stable to acid, contrary to the ready acid catalyzed

polymerization of conidine itself.78


27

N

OH

OH

HO

NOH

HO OH

NH

HO OH

OH

CH2OHNH

OH

CH2OH

OHHO

14 90 91 92

Figure 11: Swainsonine analogues.

The synthesis starts with D-altrose 95 which was formed in a batch reactor from D-

fructose 93 by epimerization of C3 by D-tagatose-3-epimerase (DTE) to D-psicose 94

which was equilibrated in situ by D-arabinose isomerase (DAI) (Scheme 11) without the

need for the isolation of intermediates.

HOH2CCHO

OH

OHOH

OHHOH2C

CH2OH

O

OHOH

OHHOH2C

CH2OH

O

OHOH

OH

DTE DAI

93 94 95Scheme 10: Formation of D-Altrose 95 from D-Fructose 93.

Protection of D-altrose 95 with acetone in the presence of sulfuric acid and anhydrous

copper (II) sulfate gave an inseparable mixture of the pyranose 96 and furanose 97

diacetonides in 85% yield in a ratio of 2:3 (Scheme 11). Partial hydrolysis of the mixture

of 96 and 97 with aqueous acetic acid resulted in selective hydrolysis of the 5,6-

acetonide of the furanose isomer 98 which allowed isolation of the required

monoacetonide 98 (50%) together with pure pyranose diacetonide 96 (31%). Treatment

of 98 with TBDMS chloride in DMF in the presence of imidazole afforded selective

protection of the primary alcohol to give the silyl ether 99 (75%). Esterification of the

two remaining hydroxyl groups in 99 with triflic anhydride in dichloromethane in the

presence of pyridine formed the ditriflate 100 (97%). Reaction of 100 with benzyl amine

in acetonitrile in the presence of diisopropylethylamine (DIPEA) gave the key azetidine

101 (Scheme 11).


28

HOH2CCHO

OH

OHOH

OH

Scheme 11: Reagents and Conditions: (a) Me2CO, CuSO4, concd. H2SO4, rt, 48 h, 85%; (b) MeCOOH:H2O, 4:1, rt,7 h, 50% 98 + 31% 96; (c) TBDMSCl, Imidazole, DMF, 30 to 20 oC, 2.5 h, 75%; (d) Tf2O, pyr., CH2Cl2, 30 to 20 oC, 50 min, 97%; (e) BnNH2, DIPEA, MeCN, 60 oC, 36 h, 21%.

O

O

O

OO

CH2OH

O

O

O

OO

HO

O

O

O

HOHO

HO

+

O

O

O

HOTBDMSO

HO

O

O

O

TfOTBDMSO

TfO

O

O

O

NTBDMSO

Bn

a b

c

de

95 97 98

99100101

96

Hydrolysis of 101 with aqueous trifluoroacetic acid removed both the silyl ether and

acetonide protecting groups to give 102 which, on reduction by sodium borohydride in

water, gave the N-benzylazetidine 103 (80% yield from 101) (Scheme 12). Transfer

hydrogenation of 103 by ammonium formate in the presence of palladium on carbon

gave the azetidine analogue 104 of DIM. In another sequence, reaction of 102 with the

stabilized ylide, Bu3P=CHCO2Me, in 1,4-dioxane afforded the Wittig product 105 (48%

from 101). Transfer hydrogenation of 105 caused debenzylation and reduction of the

C=C and gave a mixture of products from which the bicyclic lactam 106 was isolated in

34% yield. All the hydroxyl groups in 105 were protected as the corresponding trisilyl

ether 107 by treatment with TBDMS triflate in DMF in the presence of 2,6-lutidine

(75%). Transfer hydrogenation of 107 in methanol gave a mixture of an ester together

with lactam 108; further heating of the reaction mixture in acetonitrile allowed the

isolation of pure fully protected lactam 108 albeit only in low yield (30%). Reduction of

lactam 108 with borane in THF gave the borane adduct 109 in which four substituents on

the azetidine ring are cis. Treatment of 109 with acidic ion-exchange resin in methanol

cleaved the borane complex and removed the silyl ethers to allow the isolation of the free

trihydroxyconidine 110. Inhibition of the various glycosidases by the azetidines 103,

104, 106, and 110 was studied. The monocyclic azetidine analogue of DIM 104 showed


29

no inhibition of any glycosidase; its N-benzyl derivative 103 was a specific weak

inhibitor of almond β-glucosidase [IC50 545 µM]. Both the conidine analogue of

swainsonine 110 and the corresponding lactam 106 were weak inhibitors of β-

galactosidase [IC50 492 and 341 µM, respectively]. Azetidine swainsonine 110 also

showed weak inhibition of -mannosidase [IC50 640 µM].

O

O

O

NTBDMSO

Bn NBn

OH

CHO

OHHONBn

OH

CH2OH

OHHOa

NH

OH

CH2OH

OHHO

NBn

OHOHHO

CO2Me

N

OHHO

O

OHNBn

OTBDMSOTBDMSTBDMSO

CO2Me

N

OTBDMSTBDMSO

O

OTBDMS

N

OTBDMSTBDMSO OTBDMS

N

OHHO OH

b c

d

e f

g

hi

101 102 103 104

105106

107

108109110

Scheme 12: Reagents and Conditions: (a) CF3COOH, H2O, 9:1, rt, 3.5 h; (b) NaBH4, H2O, rt, 15 h, 80% from 101; (c) HCO2NH4, 10% Pd/C, MeOH, reflux, 0.5 h, 95%; (d) Bu3P=CHCO2Me, 1,4-dioxane, rt, 4 h, 48% from 101; (e)HCO2NH4, 10% Pd/C, MeOH, reflux, 68 h, 34%; (f) TBDMSOTf, 2,6-lutidine, DMF, rt, 23 h, 75%; (g) HCO2NH4, 10%Pd/C, MeOH, reflux, 20 h, 30%; (h) THF:BH3, THF, reflux, 26 h, 30%; (i) Dowex H+ resin, MeOH, rt, 7 d, 92%.

H3B

1.3.1.7 Method due to Tiwari et al.79

Recently, Tiwari and co-workers have synthesized the iminosugar 34b (identical to one

that we synthesized earlier) using the key step of [NMM]+[HSO4]− promoted conjugate

addition and Mitsunobu reaction (Scheme 13) and termed compound 34b as 1-deoxy-

norcastanospermine. The Michael addition of benzyl amine to ,β-unsaturated ester 111

using ionic liquid [NMM]+[HSO4]− showed the reversed diastereoselelctivity (D-gluco

being major diastereomer) than that observed under conditions employed by our

group.80b The reduction of sugar β-amino ester 112 with LiAlH4 in anhydrous THF

resulted in corresponding β-amino alcohol 113 in 96% yield. Further Mitsunobu reaction


30

conditions were optimized to reach the conclusion of using 1.0 equiv. of β-amino alcohol

113, 1.2 equiv. Ph3P, 0.2 equiv. DIAD and 1.5 equiv. PhI(OAc)2 afforded the azetidine

114 in 72% yield. In the next steps, hydrolysis of 1,2-acetonide using TFA/H2O and

reductive aminocyclization under hydrogenation conditions afforded the titled compound

34b.

O

BnO

OEt

O

O

OO

BnO

OEt

O

O

OHN

Ph

O

BnO

OEt

O

O

OHN

Ph

Minor Major

+D-Glucose5 Steps a

b

O

BnO

HO

O

OHN

Ph

O

BnO O

ON

Ph

H

S c or de, fN

H

HO OHOH

1-deoxy-norcastanospermine

Scheme 13: Reagents and conditions: (a) benzyl amine, EtOH, [NMM]+[HSO4]-, rt, 15 min, 95% (ratio 80 : 20); (b)LiAlH4, THF, rt, 3 h, 96%; (c) PPh3, DIAD, PhI(OAc)2, THF, rt, 10 h, 72%; (d) PPh3 and DIAD (each 1.5 equiv.), THF, rt, 12 h, 70% (e) TFA–H2O (3 : 2), rt, 12 h, 90%; (f) 10%Pd/C, H2, 80 psi, MeOH, 12 h, 60%.

111 112a 112b

11311434b


31

CHAPTER 1


Section B: Synthesis of conidine iminosugars

1.2.1 Introduction

In Chapter 1, Section A, we have given an account of different types of iminosugars, its

biological importance and recently known synthetic methods for the preparation of

conidine and azetidine iminosugars. In the continuation of our interest in the area of

iminosugars,81 we now report the synthesis of conidine iminosugars, their

conformational analysis, glycosidase inhibitory activity that is substantiated by

molecular docking studies.

1.2.2 Present work

1.2.2.1 Retrosynthetic analysis

As shown in Scheme 14, we visualized the conidine iminosugars 34a and 34b from the

sugar derived β-lactams 115a and 115b in three steps viz. reduction of β-lactam to

azetidine, 1,2-acetonide deprotection and reductive aminocyclization. The β-lactams 115

could be derived from β-amino esters 112a and 112b in two steps viz. hydrolysis of ester

to acid and acid-amine coupling. The β-amino esters 112a and 112b were in turn derived

from intermolecular Michael addition of N-benzyl amine to a common -β-unsaturated

ester intermediate 111 which was earlier reported from our group using D-glucose.80


32

NOH

OHHOH

NOH

OHHOH

O

O

O

BnO

NBn

O

O

O

BnO

NBnO

O

O

O

O

BnO

EtO2CNHBn

O

O

O

BnO

EtO2CNHBn

O

O

O

BnO

EtO2C

D-Glucose

34a

34b

115a

115b

112a

112b

111

Scheme 14: Retrosynthetic analysis of compounds 34a and 34b.

1.2.2.2 Synthesis of D-glucose derived β-amino esters (112a and 112b)

The synthesis of ,β-unsaturated ester was achieved from D-glucose (Scheme 15). Thus,

D-glucose was treated with acetone in the presence of catalytic amount of H2SO4 to get

diacetone D-glucose 116 as a white solid in good yield; mp = 109-110 oC, [lit82 mp =

110-111 oC]. The C-3 hydroxyl group in 116 was protected as benzyl ether using benzyl

bromide and sodium hydroxide to give 3-O-benzyl protected diacetone D-glucose 117.83

Selective deprotection of 5,6-O-isopropylidene functionality using 10% H2SO4 (to get

diol 118) and oxidative cleavage of corresponding diol using NaIO4 afforded 1,2-O-

isopropylidene--D-xylo-pentodialdose 11984 in overall 68% yield from D-glucose. The

Wittig reaction of 119 with Ph3P=CHCOOEt in dichloromethane at reflux afforded β-

unsaturated ester 111a and 111b as a geometric mixture (E:Z = 4:1) in 86% yield

(Scheme 15). Although the difference in Rf values of the geometric isomers 111a and

111b was small, a careful separation by flash chromatography afforded E and Z isomers

in pure form. The spectral and analytical data as well as specific rotation values were

found to be in agreement with the data reported by us.80 In the next step, we studied the

intermolecular conjugate addition reaction of benzyl amine to ,β-unsaturated ester 111.

Thus, the intermolecular Michael addition of N-benzyl amine to ,β-unsaturated ester


33

111 under neat conditions was found to be stereoselective to obtain 112a and 112b (L-

ido and D-gluco configured) in the diastereomeric ratio of 70:30 respectively in 85%

yield (Scheme 15).

D-GlucoseO

O

O

BnO

HO

HO

+

Major Minor70 : 30112a 112b

O

O

O

RO

O

Oa

b 116, R = H117, R = Bn

cO

O

O

BnO

H

O

d

O

O

O

BnO

EtO2C

H

H

111a = E111b = Z 4:1

e

fEtO

O NHBnO

O

O

BnO

EtO

O NHBnO

O

O

BnO

Scheme 15: Reagents and conditions: (a) Me2CO, anhydrous CuSO4, H2SO4, rt, 30 h, 68%; (b) NaOH, BnBr, TBAI, 125 oC, 2 h, 93%; (c) 10% H2SO4, MeOH-H2O, 5 h, 88%; (d) NaIO4, Me2CO-H2O, 0 oC, 1.5 h, 68%; (e)Ph3P=CHCOOEt, CH2Cl2, reflux, 30 h, 86%; (f) BnNH2, rt, 12 h, (dr 70/30 = 112a/112b), 90%.

118 119

1.2.2.2.1 Assignment of the relative stereochemistry at C5 of the β-amino esters

112a and 112b

The assignment of stereochemistry at newly generated stereocentre C-5 was achieved in

the following way. The reduction of the ester functionality in 112a and 112b, separately,

with LiAlH4 in THF afforded the β-amino alcohols 120a and 120b in 87% and 84%

yield, respectively (Scheme 16).

D-Glucose

O

O

O

BnO

EtO2C

111

O

O

O

BnO

EtO2C

112a R1 = NHBn; R2 = H112b R1 = H; R2 = NHBn

R2R1O

O

O

RO

R2R1

HO

120a R = Bn; R1 = NHBn; R2 = H120b R = Bn; R1 = H; R2 = NHBn

Scheme 16: Reagents and Conditions: (a) BnNH2, rt, 12 h, (dr 70/30 = 112a/112b) 90%; (b) LiAlH4, THF, rt, 2 h, 84% for 120a and 87% for 120b;

a b

The comparative 1H NMR data of the C5-epimeric pair 120a and 120b turned out to be

informative. It is known that for a given C5-epimeric pair, derived from D-gluco

furanose, the J4,5 in the L-ido isomer (threo-relationship) is consistently larger than that


34

of the corresponding D-gluco isomer (erythro-relationship).85 The higher value of J4,5

observed in the diastereomer 120a (9.5 Hz), as compared to 120b (6.9 Hz) indicated the

L-ido configuration for 120a and the D-gluco configuration for 120b. This assignment

was further supported by a comparison of the chemical shifts of H3 in both the isomers.

The chemical shift of H3 is reported to be diagnostic such that, in the L-ido-isomer, it is

significantly upfield (δ ~3.6) as compared to that in the D-gluco (δ ~4.0).82 In 120a, the

H3 appeared upfield at δ 3.86 as compared to 120b at 4.03 δ further supporting the D-

gluco- and L-ido- configuration at C5 to 120b and 120a, respectively. This fixed the

configurations at C5 in 112a and 112b as 5S and 5R, respectively.

1.2.2.2.2 Explanation for observed stereoselectivity in the Michael addition

The observed stereoselectivity in the conjugate addition to 111a and 111b was

rationalized by us in terms of Felkin-Anh like transition states.86 As shown in Figure 12,

four transition states I, II, III and IV were anticipated for 111a and 111b. In

conformations I and II, the more electronegative C-O group of the furanose ring,

whereas in III and IV the largest C3-benzyloxy substituent were placed at right angles to

the C=C bond. It is well-known in case of the Felkin-Anh like model that the nucleophile

attacks from the face opposite to the group perpendicular to the C=C. In the case of 111a

and 111b, the re face attack in conformer I (to give D-gluco-isomer) and si face attack in

conformer II (to give L-ido-isomer) is hindered by the C3-benzyloxy group. Therefore,

we considered conformers III and IV. It is apparent that the conformer III has the

preference over IV due to the favorable alkene-arene π-stacking effect87 in which the si

face attack of the N-benzyl amine, by chelation with furanose ring oxygen, explains the

diastereoselective formation of L-ido-isomer.


35

H

EtO2C H

OH

O

OOBnH

H

CO2EtH

OH

O

OOBnH

Re attack

Si attack

O O

O

OBnH

H H

HEtO2CO O

O

OBnH

H

H

EtO2C HRe attack

Si attack

I II

IVIII

112b(D-gluco)112a (L-ido)

Unfavourable due to steric hindrance of C3 -OBn

Figure 12: Felkin-Anh like transition states for 111a and 111b

1.2.2.3 SYNTHESIS OF CONIDINE IMINOSUGARS (34a and 34b)

Having β-aminoesters 112a and 112b in hand with good amount, we thought of

exploiting both of these diastereomers for the synthesis of targeted conidine iminosugars.

In the first attempt, we thought of utilizing the major isomer 112a to get target molecule.

As shown in scheme 17, the L-ido configurated β-aminoester 112a was treated

with lithium hydroxide in methanol-water that afforded a white solid in 98% yield. The

IR spectrum of crude product showed band at 3450-2800 cm-1 indicative of presence of

the carboxylic acid and amine groups.

O

O

O

BnO

NHBn

121a

aO

O

O

BnO

NHBn

112a

EtO

O

HO

O

Scheme 17: Reagents and conditions: (a) LiOH.H2O, MeOH-H2O, 0 oC to 25 oC, 2 h, 98%.


36

The absence of signals at 1745 cm-1 due to the ester carbonyl indicated the hydrolysis of

the ester group.

Figure 13: 1H NMR (300 MHz, CDCl3) Spectrum of compound 121a

Figure 14: 13C NMR (75 MHz, CDCl3) Spectrum of compound 121a


37

The 1H (Figure 13) and 13C NMR (Figure 14) spectrum of the product showed the

absence of signals due to the -CH2CH3 group of ethyl ester group. An appearance of two

exchangeable protons due to carboxylic acid and secondary amine group at δ 7.40-8.20

indicated the hydrolysis of ester to acid. The compound was analyzed for the molecular

formula C24H29NO6. Based on the spectral and analytical data, structure of the compound

was assigned as 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5-(N-benzylamino)-β-L-

ido-heptofuranuronic acid (121a)88.

Having β-amino acid in good amount in hand, we thought of synthesizing the

sugar derived β-lactam. A vast number of methods for the preparation of β-lactams are

known in the literature.89 The major approaches for the synthesis of azetidin-2-one (β-

lactam) ring system are summarized in Figure 15.

NO

R1

R2 R4R3

R5

R1

C

R2

O

R4R3

NR5

R4R3

NR5

R1R2

R5 NO

ClO

H

+ Et3N +

+

R3 R4R1,2

+

N2R1,2

NOR5

R3,4

R2R1

ORLiO R5 N

R4R3

+OH

O

HNR5

R1 R2 R3R4

R NNTsNa

R4

NR5

R3

+NR5

R2

R1 R4R3

+ CO

+CO

Figure 15: Different approaches for the synthesis of β-lactam

However, we have chosen an approach of intramolecular acid-amine coupling reaction to

reach the target. The advantages of this route are, (i) this route is intramolecular and


38

devoid of problem of regioselectivity, (ii) most of other approaches utilize the metal

activation which is not economic, and (iii) the precursor 112 was well established in our

laboratory from cheap and commercially available D-glucose.

While proceeding through the literature in search of suitable reagent for acid-

amine coupling, we scanned a number of peptide coupling reagents such as DCC,

HOBT, HATU, etc. The mixed anhydride method using iso-butylchloroformate and

triethylamine was giving mixture of compounds along with the desired product.

Alternatively, we turned our attention to the Mukaiyama reagent which is the pyridinium

salt containing a good leaving group at 2-position of the pyridine ring. The plausible

mechanism of the Mukaiyama reagent in β-lactam formation is shown in Scheme 18.

The mechanism includes the activation of an acid group by pyridinium salt to give

intermediate I. In the next step, the nucleophilic attack of secondary amine at the

carbonyl group with elimination of N-methyl-2-pyridone led to β-lactam formation. The

product is formed in excellent yield without any side reaction. The by-product 1-methyl-

2-pyridone is easy to remove facilitating efficient purification of the β-lactam.

NMe

ClI

NMe

ClI

NEt3-amino acid

Et3N

NMeIO

O R

HNBnH O

O R

HNBn

Cl

MeO

O R

HNBnN

I

NBn

R

O

N OMe

+

I

Scheme 18: Plausible mechanism of Mukaiyama reagent for β-lactam formation

Thus, in the subsequent step, treatment of 121a with 2-chloro-1-methylpyridinium iodide

(Mukaiyama reagent)90 and triethylamine in dichloromethane afforded a white solid in

89% yield (Scheme 19). The IR spectrum of the product showed the characteristic band


39

at 1748 cm-1 that was assigned to the strained 4-membered lactam (β-lactam). The

absence of IR absorption bands at 3450-2800 cm-1 due to COOH and NH indicated the

formation of β-lactam. The 1H NMR (Figure 16) spectrum showed two methylene

protons, to the lactam carbonyl, at δ 2.37 (dd, J = 14.6, 2.5 Hz) and δ 2.83 (dd, J =

14.6, 5.2 Hz). A signal at δ 3.79 integrating for one proton showed ddd (J = 8.5, 5.2, 2.5

Hz) that was assigned to the H-5. The downfield shift of the signals (δ 2-3) due to two H-

6 protons indicated the formation of strained β-lactam ring.

O

O

O

BnO

NHBn

121a

aHO

OO

O

O

BnO

NBnO

115aScheme 19: Reagents and conditions: (a) 2-Chloro-1-methylpyridinium iodide, Et3N, Dry CH2Cl2, 25 oC , 2 h, 89%.


The 13C NMR (Figure 17) showed β-lactam carbonyl at δ 165.9. The signal due to the C-

6 appeared at δ 39.0 which was at δ 31.3 in the starting compound indicated the


40

formation of β-lactam. The compound was analyzed for the molecular formula

C24H27NO5. Based on the spectral and analytical data, structure of the compound was

assigned as 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5,7-(N-benzylimino)-β-L-ido-

sept-1,4-furan-7-ulose (115a).


Having sugar appended β-lactam 115a in hand, we have attempted the reduction of β-

lactam to get azetidine ring system.

A rapid and efficient reduction of N-substituted azetidin-2-ones to N-substituted

azetidines is generally carried out with diborane in tetrahydrofuran, LiAlH4, and Raney

nickel. In view of this, β-lactam 115a was reacted with LiAlH4 (1 equiv.) in THF. The

reaction at 0 C was sluggish, however at room temperature, β-lactam ring opened

product namely 3-aminopropanol derivative is obtained in 20% yield as evident from the

spectral data. We also attempted electrophilic reducing agents such as DIBAL-H and

borane. Under variety of reaction conditions of solvent and temperature with different


41

ratios of reagents, we could not isolate expected β-lactam reduced product. The

attempted reduction of 115a to 122a with 9-BBN (9-Borabicyclo-(3.3.1)-nonane),

diborane under variety of reaction conditions gave no conversion, and starting material is

recovered at the end of the reaction. On the contrary, DIBAL-H at −78 C led to complex

reaction mixture. Based on above observation, we turned our attention towards the use of

chloroalanes as reducing agent in this reaction. It was thought that the use of three

equivalents of LiAlH4 and AlCl3 in each gave very small amount of required compound

and highly polar major product which may arise from the 1,2-acetonide opening under

strong Lewis acid (AlCl3) conditions. The 1.5 equivalents of LiAlH4 and 0.5 equivalents

of AlCl3 forms chloroalane at lower temperature which is the active species in the

reaction. Our attempts in the direction of reduction of β-lactam 115a to the

corresponding azetidine 122a are summarized in Table 1.

Table 1: Reduction of β-Lactam 115 to azetidine 122

Sr.

No.

Reaction Conditions Remarks

Reagent Solvent Temperature Yield

1 9-BBN THF −20 C - Starting recovered

2 rt - Starting recovered

3 reflux - Starting recovered

4 BH3.THF THF −20 C - Starting recovered

5 rt - Starting recovered

6 reflux - Starting recovered

7 DIBAL-H THF −78 C - Complex mixture

8 LiAlH4 THF 0 C to rt - Lactam opening

product

9 LiAlH4 : AlCl3 (3.0 equiv. each)

THF 0 C to rt 20% Low yield

10 LiAlH4 : AlCl3 (1.5 : 0.5 equiv.)

THF −20 C to 0 C 83-85% Product with good

yield


42

Alternatively, we thought of chloroalane as a choice of reducing agent. Ojima and co-

workers were the first to discover the potency and selectivity of chloroalanes (AlH2Cl or

AlHCl2) in reductions of β-lactams.91 They applied this reagent to a large array of

substrates showing the generality and effectiveness of the process.92 Alcaide and co-

workers93 applied the chloroalane reduction method to a series of monocyclic and

polycyclic β-lactams. It is noteworthy that these reductions are highly chemoselective (β-

lactam is reduced to azetidine selectively) in the presence of conjugated double bonds,

isolated double and triple bonds and other functionalities prone to reduction. Carreira and

co-workers94 observed that if the electron-rich C-4 substituent present in the β-lactam,

the newly formed azetidine rearranges to the ring expansion product, in the presence of

Lewis acids during the reduction with alanes. The chloroalanes are formed in situ from

lithium aluminum hydride (3 equiv.) and aluminum chloride (1 equiv.) at 0 C. When the

ratio of LiAlH4 to AlCl3 is 3:l, new reducing species AlH2Cl and/or AlHC12 is produced.

Cole and co-workers95 have studied the formation of chloroalanes by trapping

them with their reaction with -diimine. The plausible mechanism of chloroalanes92a for

the reduction of β-lactam to azetidine is shown in Scheme 20.

NO Bn

R

H Al NO Bn

R

Al NO Bn

R

Al NO Bn

R

Al

NBn

R

OAl+H AlN

Bn

R

AlOAlOAl Al

NBn

R

Scheme 20: Plausible mechanism for reduction of β-lactam to azetidine using chloroalane


43

Thus, chloroalane was prepared in situ from LiAlH4:AlCl3 (1.5 equiv. : 0.5 equiv.) in

anhydrous THF at −20 C to 0 C and to this compound, 115a in THF was added at −20

C. The reaction after workup gave a thick oil 83% yield (Scheme 21). The IR spectrum

showed absence of lactam carbonyl stretching at 1748 cm-1 indicating the reduction of β-

lactam. In the 1H NMR spectrum (Figure 18), appearance of one more methylene group

protons - to N-benzyl indicates the controlled reduction. Absence of any exchangeable

proton suggests that no ring opened product (β-amino alcohol) was obtained. Three

multiplets in the region of δ 1.80 to 3.25 integrating for four protons and one multiplet at

δ 3.60 for one proton suggested the presence azetidine ring.

aO

O

O

BnO

NBnO

115a

O

O

O

BnO

NBn

122aScheme 21: Reagents and conditions: (a) LiAlH4/AlCl3, anhyd. THF, 20 oC to 0 oC, 10 min, 83%.



44

In the 13C NMR (Figure 19), absence of carbonyl carbon at 165.9 and presence of one

extra CH2 confirmed the formation of azetidine ring. Furthermore, the compound was

analyzed for the molecular formula C24H29NO4. Based on the 1H and 13C NMR

spectroscopic and analytical data, structure of the compound was assigned as 1,2-O-

isopropylidine-3-O-benzyl-5,6,7-trideoxy-5,7-(N-benzylimino)-β-L-ido-sept-1,4-

furanose (122a).


Targeting towards the conidine iminosugars, the azetidine sugar derivative 122a was

treated with TFA:water (3:2) at 0-30 C that afforded hemiacetal (as evident from the 1H

NMR of the crude product). The hemiacetal mixture was purified by chromatography

and then subjected to hydrogenation using H2, 10% Pd/C for 5 days that afforded thick

oil which on silica gel column chromatography gives a white moisture sensitive

compound in 78% yield (Scheme 22). The IR spectrum showed the broad signal at 3200-

3600 cm-1 indicating presence of hydroxyl groups. The 1H NMR (Figure 20) of this

compound in D2O showed the absence of methyl groups of acetonide. Absence of

aromatic protons suggested the removal of N- and O-benzyl groups. Downfield shift of

methylene protons of azetidine ring indicated the robustness of azetidine under


45

hydrogenation condition. The overall integration of 1H NMR spectrum was found to be

corresponding to 9 protons instead of expected 10 protons. It was thought that H-3

proton being attached to the oxygenated carbon might have shifted to downfield and

probably got merged in HDO signal. To confirm this feature, the 1H NMR was recorded

in methanol-d4. The NMR spectrum showed the shift of all signals. The 1H NMR

spectrum recorded in methanol-d4 (Figure 21) shows all 10 protons expected for

compound 34a. The signals are well resolved in this spectrum and shifted to upfield by ~

δ 0.3.

O

O

O

BnO

NBn

122a

aN

H OHOH

OH

34a

Scheme 22: Reagents and conditions: (a) (i) TFA/H2O (3/2), 0 oC to 25 oC, 4 h; (ii) H2, 10% Pd/C, 80 psi, 25 oC, 5 days, 78% over 2 steps.

Figure 20: 1H NMR (300 MHz, D2O) Spectrum of compound 34a


46

In the 13C NMR (Figure 22) spectrum, absence of two signals due to methyl groups

indicated the removal of 1,2-acetonide group. Absence of aromatic and benzylic carbons

suggests the complete deprotection of the N- and O-benzyl groups. Presence of

additional CH2 attached to nitrogen (at δ 53.7) indicates the reductive aminocyclization.

The compound was analyzed for the molecular formula C7H13NO3. Based on the IR

spectrum, 1H and 13C NMR spectroscopic and analytical data, structure of the compound

was assigned as (3S,4R,5R,6S)-3,4,5-trihydroxyconidine (34a ).

The characteristic azetidine ring protons were also assigned indicating that the

azetidine ring is stable towards hydrogenation although some reports89a showed the

cleavage of azetidine ring which otherwise may give eight membered iminosugar.

Figure 21: 1H NMR (300 MHz, Methanol-d4) spectrum of compound 34a


47

Figure 22: 13C NMR (75 MHz, D2O) Spectrum of compound 34a

After successful synthesis of conidine iminosugar 34a, we turned our attention for the

synthesis of conidine iminosugar 34b starting from the minor sugar β-amino ester

intermediate 112b. Thus, hydrolysis of 112b with LiOH.H2O in methanol-water led to a

white solid in 96% yield (Scheme 23). As evident from the IR spectrum, 1H (Figure 23),

13C (Figure 24) NMR and analytical data, the structure was assigned as 1,2-O-

isopropylidine-3-O-benzyl-5,6-dideoxy-5-(N-benzylamino)--D-gluco-heptofuranuronic

acid (121b).

O

O

O

BnO

NHBn

121b

aO

O

O

BnO

NHBn

112b

EtO

O

HO

O

Scheme 23: Reagents and conditions: (a) LiOH.H2O, MeOH-H2O, 0 oC to 25 oC, 2 h, 96%.

In the next step, the intramolecular coupling of acid and amine groups of compound

121b using Mukaiyama reagent afforded a white solid in 91% yield (Scheme 24).

O

O

O

BnO

NHBn

121b

aHO

OO

O

O

BnO

NBnO

115bScheme 24: Reagents and conditions: (a) 2-Chloro-1-methylpyridinium iodide, Et3N, Dry CH2Cl2, 25 oC , 2 h, 91%.


48

Figure 23: 1H NMR (300 MHz, CDCl3) spectrum of compound 121b

Figure 24: 13C NMR (75 MHz, CDCl3) spectrum of compound 121b


49

Figure 25: 1H NMR (300 MHz, CDCl3) spectrum of compound 115b

Figure 26: 13C NMR (75 MHz, CDCl3) Spectrum of compound 115b


50

As evident from the IR spectrum, 1H (Figure 25), 13C (Figure 26) NMR and analytical

data, the structure was assigned as 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5,7-(N-

benzylimino)--D-gluco-sept-1,4-furan-7-ulose (115b).

In the subsequent step, the reduction of lactam compound 115b using chloroalane

reagent generated in situ from LiAlH4 and AlCl3 afforded a white solid in 85% yield

(Scheme 25). As apparent from the IR spectrum, the 1H (Figure 27), 13C (Figure 28)

NMR and analytical data, the structure was assigned as 1,2-O-isopropylidine-3-O-

benzyl-5,6,7-trideoxy-5,7-(N-benzylimino)--D-gluco-sept-1,4-furanose (122b).

aO

O

O

BnO

NBnO

115b

O

O

O

BnO

NBn

122bScheme 25: Reagents and conditions: (a) LiAlH4/AlCl3, anhyd. THF, 20 oC to 0 oC, 10 min, 85%.

Figure 27: 1H NMR (300 MHz, CDCl3) Spectrum of compound 122b


51

Figure 28: 13C NMR (75 MHz, CDCl3) Spectrum of compound 122b

In the penultimate step, sugar azetidine compound 122b was treated with TFA:water

(3:2) to give the anomeric mixture of hemiacetal which was characterized by 1H NMR of

crude product. Silica gel chromatography of crude hemiacetal afforded thick colourless

oil which was treated in the final step with Pd/C and H2 at 80 psi for 5 days to give

compound as a white solid after purification in 71% yield (Scheme 26). The IR spectrum

showed the absorption at 3200-3600 cm-1 indicated the presence of hydroxyl groups. As

apparent from the 1H (Figure 29), 13C (Figure 30) and analytical data, the structure was

assigned as (3S,4R,5R,6R)-3,4,5-trihydroxyconidine (34b ).

O

O

O

BnO

NBn

122a

aN

H OHOH

OH

34a

Scheme 26: Reagents and conditions: (a) (i) TFA/H2O (3/2), 0 oC to 25 oC, 4 h; (ii) H2, 10% Pd/C, 80 psi, 25 oC, 5 days, 71% over 2 steps.


52

After successful synthesis of conidine iminosugars 34a/b, we studied their

conformational aspects and glycosidase inhibitory activity, which follows in next

sections.

Figure 29: 1H NMR (75 MHz, D2O) Spectrum of compound 34b

Figure 30: 13C NMR (75 MHz, D2O) Spectrum of compound 34b


53

1.2.3 EXPERIMENTAL SECTION

Expt. No. 1.2.3.1 Preparation of 1,2:5,6-di-O-isopropylidene--D-gluco-furanose

(116)

O

O

O

HO

HO

OD-Glucose

Acetone, Conc. H2SO4 anhy.CuSO4, 30 °C, 30 h, 68%

116

To a stirred solution of anhydrous copper sulfate (100 g, w/w) and D-glucose (100 g,

w/w) in dry acetone was added conc. sulfuric acid (5.0 mL) at 0 °C. The reaction mixture

was stirred at room temperature for 30 h. Saturated solution of potassium carbonate was

added slowly. Acetone was evaporated under reduced pressure; the residue was extracted

with chloroform (3 X 150 mL). The organic layer was dried and concentrated to afford

white solid, which was recrystallized from chloroform-hexane to give 116 in 65 g, 68 %

yield. mp 108-110 °C. [lit82. mp 110-111 °C].

Expt. No. 1.2.3.2 Preparation of 1,2:5,6-di-O-isopropylidene-3-O-benzyl--D-gluco-

furanose (117).

O

O

O

HO

O

OO

O

O

BnO

O

ONaOH, BnBr, TBAI

125 oC, 2 h, 93%116 117

Diacetone D-glucose 116 (20.0 g, 76.9 mmol), benzyl bromide (15.8 g, 92.3 mmol) and

NaOH (9.3 g, 230.7 mmol) was heated at 125 °C for 2 h. Reaction mixture was cooled

to room temperature; water (40 mL) was added and extracted with chloroform (3 X 60

mL). The combined organic layer was washed with water followed by brine, dried

(Na2SO4) and concentrated to afford a thick liquid that on purification by column


54

chromatography (hexane/ethyl acetate = 9.5/0.5) gave pure 117 (26.0 g, 93 %) as a thick

liquid. [] 25D = −20.2 (c 0.4, CHCl3) [lit.83 []D = −20.8 (c 0.1, MeOH)].

Expt. No. 1.2.3.3 Preparation of 1,2-O-isopropylidene-3-O-benzyl--D-gluco-

furanose (118).

O

O

O

BnO

O

O

117

O

O

O

BnO

HO

HO

118

10% H2SO4, MeOH-H2O

0 oC to rt, 5 h, 88%

The 3-O-Benzyl protected diacetone D-glucose 117 (26 g, 74.3 mmol) was dissolved in

methanol (130 mL) and water (45 mL) and 10% H2SO4 (21.5 mL) and stirred at room

temperature. After 5 hours, saturated potassium carbonate was added to neutralize the

reaction mixture to pH = 7-8. Methanol was evaporated and the residue extracted with

chloroform (30 mL X 4), organic layer dried (Na2SO4) and evaporated to give a thick

liquid, which after column purification (hexane/ethyl acetate = 8.5/1.5) gave 118 (20 g,

88%). [] 25D = −34.8 (c 0.4, CHCl3) [lit.96 [] 25

D = −35 (c 0.6, CHCl3)].

Expt. No. 1.2.3.4 Preparation of 1,2-O-ispropylidene-3-O-benzyl--D-xylo-

pentodialdo-1,4-furanose (119).

O

O

O

BnO

HO

HOO

O

O

BnO

H

O

118 119

NaIO4, Me2CO-H2O,

0 oC to rt, 1.5 h, 68%

A solution of compound 118 (23.00 g, 74.20 mmol) in acetone (130 mL) and water (20

mL) was cooled to 0 °C. Sodium metaperiodate (2.85 g, 13.30 mmol) was added in

portions to the cooled solution and stirred for 1.5 hours. Ethylene glycol (2 mL) was

added to the reaction mixture and extracted with chloroform (3 X 40 mL). The

chloroform layer dried and evaporated to afford a thick liquid which on column


55

purification (hexane/ethyl acetate = 8.5/1.5) afforded the pentodialdose 119 (18.50 g,

92%). [] 25D = −90.1 (c 1.0, CHCl3) [lit.84 [] 25

D = −86.5 (c 2.7, CHCl3)].

Expt. No. 1.2.3.5 Preparation of triphenylethoxycarbonylmethylene phosphorane.

Ph3P + BrCH2COOEtToulene

. BrNaOHPh3PCH2COOEt Ph3P=CHCOOEt

To a solution of triphenylphosphine (30.0 g, 114 mmol) in dry toluene (58.0 mL) was

added ethyl bromoacetate (13.5 mL, 121 mmol) and the reaction was left overnight at

room temperature. The phosphonium salt thus obtained was filtered, washed with dry

toluene and dried. A solution of this salt in water (200 mL) and toluene (75 mL),

containing pinch of phenolphthalein was neutralized with aqueous sodium hydroxide till

the pink colour persists. The combined toluene layer was dried and concentrated to about

one fourth of its volume. Addition of hexane resulted in the separation of the crystalline

solid which was crystallized from toluene to get phosphorane (36.0 g, 90%). mp = 125

C (lit.97 mp 125-127 C).

Expt. No. 1.2.3.6 Preparation of (E+Z) ethyl 1,2-O-isopropylidine-3-O-benzyl 5,6-

dideoxy--D-xylo-5-eno-heptofuranuronoate (111a and 111b).

O

O

O

BnO

H

O O

O

O

BnO

EtO2C

H

H

111a = E isomer111b = Z isomer

119

Ph3P=CHCOOEt, CH2Cl2,

reflux, 12 h, 90%

To a solution of aldehyde 119 (1.8 g, 6.40 mmol) in dry dichloromethane (20 mL) was

added triphenylethoxycarbonylmethylene phosphorane (2.9 g, 8.33 mmol). The reaction

mixture was refluxed for 12 h and concentrated on rotary evaporator to give a thick

liquid. Column chromatography on silica gel (pet ether/ ethyl acetate = 95/5) gave a


56

mixture of (E+Z) ,β-unsaturated ester 111 as an oil (1.99 g, 90%) in the ratio of E/Z =

27/73 (ratio determined from 1H NMR).

Expt. No. 1.2.3.7 Preparation of ethyl 1,2-O-isopropylidene-3-O-benzyl-5-(N-

benzylamino)-5,6-dideoxy--D-gluco-heptofuranuronate (112a) and ethyl 1,2-O-

isopropylidene-3-O-benzyl-5-(N-benzylamino)-5,6-dideoxy-β-L-ido-

heptofuranuronate (112b).

O

O

O

BnO

EtO2C

H

H

111a = E111b = Z

+

112a 112b

EtO

O NHBnO

O

O

BnO

EtO

O NHBnO

O

O

BnO

BnNH2, rt

12 h, 90%

A solution of 111a,b (1.00 g, 2,87 mmol) and N-benzyl amine (0.76 g, 7.18 mmol) was

stirred at room temperature under N2. After 12 h, the reaction mixture was directly

loaded on the flash chromatography column. Elution first with pet ether/ ethyl acetate =

98/2 afforded β-amino ester 112a (0.35 g, 27%) as a thick liquid and further elution with

pet ether/ ethyl acetate = 95/5 gave 112b (0.82 g, 63%) as a pale yellow solid. Both

isomers are characterized fully as they are already reported from our group.80b

Expt. No. 1.2.3.8 Preparation of 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5-(N-

benzylamino)-β-L-ido-heptofuranuronic acid (121a).

O

O

O

BnO

HOOCNHBn

O

O

O

BnO

EtOOCNHBn

LiOH.H2O, MeOH-H2O,

0 oC to 25 oC, 2 h, 98%;

112a 121a

To an ice cooled solution of β-L-ido-β-amino ester 112a (0.200 g, 0.44 mmol) in

methanol/water (5 mL, 4/1) was added lithium hydroxide monohydrate (0.112 g, 2.66

mmol) at 0 oC. The mixture was brought to 25 oC, stirred for 2.5 h and neutralized to pH

7 by addition of 0.5M H3PO4. Methanol was evaporated and residue was extracted with


57

chloroform (3 x 7 mL), combined chloroform layer was washed with water, dried with

anhydrous Na2SO4 and solvent removed under reduced pressure to give pale yellow

solid which on purification by column chromatography (chloroform/methanol = 9.5/0.5)

afforded β-L-ido acid 121a (0.184 g, 98%) as a white solid.

mp: 165-166 oC;

Rf 0.30 (chloroform/methanol = 9/1);

[]D32 −32.17 (c 1.08, CHCl3);

IR (neat) 3445 (COOH), 1611 (NH), 2928, 1084 cm1;

1H NMR (300 MHz, CDCl3): δ 1.32 (s, 3H, CH3), 1.48 (s, 3H, CH3), 2.20 (dd, J = 17.1,

5.1 Hz, 1H, H-6a), 2.58 (dd, J = 17.1, 4.95 Hz, 1H, H-6b), 3.30-3.56 (m, 1H, H-5), 3.88

(d, J = 12.7Hz, 1H, N-CH2Ph), 3.98 (d, J = 3.0 Hz, 1H, H-3), 4.02 (d, J = 12.7 Hz, 1H,

N-CH2Ph), 4.25 (dd, J = 9.5, 3.0 Hz, 1H, H-4), 4.48 (d, J = 11.7 Hz, 1H, O-CH2Ph), 4.67

(d, J = 3.6 Hz, 1H, H-2), 4.69 (d, J = 11.7 Hz, 1H, O-CH2Ph), 5.94 (d, J = 3.6 Hz, 1H, H-

1), 7.23-7.45 (m, 10H, Ar-H) 7.40-8.20 (br, 2H, Exchangeable with D2O, NH and

COOH); 13C NMR (75 MHz, CDCl3): δ 26.1 (CH3), 26.7 (CH3), 31.3 (C-6), 50.2 (C-5), 53.3 (N-

CH2Ph), 72.0 (O-CH2Ph), 80.3, 81.1, 81.7 (C-2/C-3/C-4), 101.8 (C-1), 112.2 (OCO),

128.2, 128.4, 128.7, 128.9, 135.9, 136.5 (Ar-C), 172.6 (COOH).

Expt. No. 1.2.3.9 Preparation of 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5-(N-

benzylamino)--D-gluco-heptofuranuronic acid (121b).

O

O

O

BnO

HOOCNHBn

O

O

O

BnO

EtOOCNHBn

LiOH.H2O, MeOH-H2O,

0 oC to 25 oC, 2 h, 96%;

112b 121b


58

Following the same procedure, -D-gluco-β-amino ester 112b (0.250 g, 0.55 mmol) was

hydrolyzed with LiOH.H2O (0.140 g, 3.34 mmol) in methanol/water (5 mL, 4/1) for 3 h

to give the -D-gluco acid 121b (0.225 g, 96%) after silica gel column purification

(chloroform/methanol = 9.5/0.5) as a white solid.

mp: 162-163 oC;


[]D32 −5.75 (c 1.02, CHCl3);

IR (neat) 3438 (COOH), 1625 (NH), 2938, 1062 cm1;

1H NMR (300 MHz, CDCl3) δ1.34 (s, 3H, CH3), 1.49 (s, 3H, CH3), 2.17 (dd, J = 15.3,

11.0 Hz, 1H, H-6a), 2.48 (dd, J = 15.3, 4.0 Hz, 1H, H-6b), 3.55 (dt, J = 11.0, 4.0 Hz, 1H,

H-5), 3.82 (d, J = 12.3 Hz, 1H, -NCH2Ph), 4.09 (d, J = 3.3 Hz, 1H, H-3), 4.16 (d, J =

12.3 Hz, 1H, -NCH2Ph), 4.18 (dd, J = 4.7, 3.3 Hz, 1H, H-4), 4.46 (d, J = 11.4 Hz, 1H, -

OCH2Ph), 4.64 (d, J = 3.9 Hz, 1H, H-2), 4.68 (d, J = 11.4 Hz, 1H, -OCH2Ph), 6.05 (d, J

= 3.9 Hz, 1H, H-1), 6.20-6-90 (br, 2H, Exchangeable with D2O, NH and COOH), 7.15-

7.38 (m, 10H, Ar-H);

13C NMR (75 MHz, CDCl3) δ26.1 (CH3), 26.7 (CH3), 33.2 (C-6), 51.4 (C-5), 53.6 (-

NCH2Ph), 71.9 (OCH2Ph), 79.3, 81.2, 82.1 (C-2/C-3/C-4), 104.5 (C-1), 112.1 (OCO),

128.2, 128.3, 128.5, 128.8, 128.9, 135.9, 135.9 (Ar-C), 173.2 (COOH).

Expt. No. 1.2.3.10 Preparation of 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5,7-

(N-benzylimino)-β-L-ido-sept-1,4-furan-7-ulose (115a).

O

O

O

BnO

HOOCNHBn

121a

O

O

O

BnO

NBnO

115a

2-Chloro-1-methylpyridinium iodide,

Et3N, Dry CH2Cl2, 25 oC , 2h, 89%


59

To a suspension of 2-chloro-1-methylpyridinium iodide (0.329 g, 1.29 mmol) and Et3N

(0.359 mL, 2.58 mmol) in dry dichloromethane (6 mL) at 25 oC was added a solution of

β-L-ido acid 121a (0.500 g, 1.17 mmol) in dry dichloromethane (2 mL) drop wise. The

solution was stirred at 25 oC for 2 h and water (2 mL) was added. Reaction mixture was

extracted with ethyl acetate (3 x 10 mL), organic layer dried on anhydrous Na2SO4 and

solvent removed under reduced pressure to give a thick liquid which on column

purification (n-hexane/ethyl acetate = 8.5/1.5) afforded β-L-ido-β-lactam 115a as white

crystalline solid (0.430 g, 89%).

mp: 129-131 oC;

Rf 0.45 (n-hexane/ethyl acetate = 7/3);

[]D32 −39.86 (c 2.61, CHCl3);

IR (neat) 1748 (C=O), 1065, 1380 cm1;


2.5 Hz, 1H, H-6a), 2.83 (dd, J = 14.6, 5.2 Hz, 1H, H-6b), 3.79 (ddd, J = 8.5, 5.2, 2.5 Hz,

1H, H-5), 3.87 (d, J = 3.6 Hz, 1H, H-3), 4.11 (dd, J = 8.5, 3.6 Hz, 1H, H-4), 4.22 (d, J =

14.7 Hz, 1H, N-CH2Ph). 4.35 (d, J = 12.3 Hz, 1H, O-CH2Ph), 4.58 (d, J = 3.6 Hz, 1H, H-

2), 4.64 (d, J = 14.7 Hz, 1H, N-CH2Ph), 4.67 (d, J = 12.3 Hz, 1H, O-CH2Ph), 5.98 (d, J =

3.6 Hz, 1H, H-1), 7.18-7.40 (m, 10H, Ar-H);

13C NMR (75 MHz, CDCl3) δ26.2 (CH3), 26.7 (CH3), 39.0 (C-6), 45.6 (N-CH2Ph), 49.7

(C-5), 71.7 (O-CH2Ph), 81.6, 81.8,83.7 (C-2/C-3/C-4), 105.7 (C-1), 111.8 (OCO), 127.3,

128.1, 128.3, 128.5, 128.5, 128.6, 136.5, 136.6 (Ar-C), 165.9 (CO).


60

Expt. No. 1.2.3.11 Preparation of 1,2-O-isopropylidine-3-O-benzyl-5,6-dideoxy-5,7-

(N-benzylimino)--D-gluco-sept-1,4-furan-7-ulose (115b).

O

O

O

BnO

HOOCNHBn

121b

O

O

O

BnO

NBnO

115b

2-Chloro-1-methylpyridinium iodide,

Et3N, Dry CH2Cl2, 25 oC , 2h, 91%

As above, the reaction of -D-gluco acid 121b (0.700 g, 1.64 mmol) with 2-chloro-1-

methylpyridinium iodide (0.461 g, 1.80 mmol) and Et3N (0.503 g, 3.61 mmol) in dry

dichloromethane (10 mL) at 25 oC for 3 h gave a thick liquid which on silica gel column

purification (n-hexane/ethyl acetate = 8.7/1.3) afforded -D-gluco-β-lactam 115b (0.610

g, 91%) as white solid.

mp: 126-127 oC;


[]D32 −38.38 (c 2.58, CHCl3);

IR (neat) 1744 (C=O), 1080, 1385 cm1;


4.9 Hz, 1H, H-6a), 3.15 (dd, J = 14.7, 2.4 Hz, 1H, H-6b), 3.66 (d, J = 3.6 Hz, 1H, H-3),

3.83 (dt, J = 4.9, 2.4 Hz, 1H, H-5), 4.18 (dd, J = 4.9, 3.6 Hz, 1H, H-4), 4.28 (d, J = 15.6

Hz, 1H, -NCH2Ph). 4.30 (d, J = 12.0 Hz, 1H, -OCH2Ph), 4.45 (d, J = 15.6 Hz, 1H, -

NCH2Ph), 4.57 (d, J = 3.9 Hz, 1H, H-2), 4.59 (d, J = 12.0 Hz, 1H, -OCH2Ph), 5.89 (d, J

= 3.9 Hz, 1H, H-1), 7.18-7.40 (m, 10H, Ar-H);

13C NMR (75 MHz, CDCl3) δ26.2 (CH3), 26.7 (CH3), 40.2 (C-6), 44.8 (N-CH2Ph), 50.0

(C-5), 71.4 (O-CH2Ph), 78.6, 81.8, 82.3 (C-2/C-3/C-4), 104.7 (C-1), 111.7 (OCO),

127.2, 127.5, 127.6, 127.9, 128.4, 128.6, 136.1, 137.0 (Ar-C), 168.0 (CO).


61

Expt. No. 1.2.3.12 Preparation of 1,2-O-isopropylidine-3-O-benzyl-5,6,7-trideoxy-

5,7-(N-benzylimino)-β-L-ido-sept-1,4-furanose (122a).

O

O

O

BnO

NBnO

115a

O

O

O

BnO

NBn

122a

LiAlH4/AlCl3, anhyd. THF,

20 oC to 0 oC, 10 min, 83%

To a cooled (ice-salt mixture) suspension of LiAlH4 (0.014 g, 0.36 mmol) in anhydrous

THF (2 mL) was added a solution of AlCl3 (0.016 g, 0.12 mmol) in anhydrous THF (2

mL) and stirred at −20 oC for 30 mins. Then a solution of β-L-ido-β-lactam 115a (0.100

g, 0.24 mmol) in anhydrous THF (3 mL) was added drop wise at −20 oC for 5 mins and

further stirred for 5 mins at 0 oC. Reaction was quenched with saturated aqueous Na2SO4

at 0 oC slowly. The reaction mixture was filtered through celite, solvent removed under

reduced pressure to give a colorless oil which was chromatographed (n-hexane/ethyl

acetate = 8.8/1.2) to give β-L-ido-azetidine 122a (0.082 g, 83%) as white solid.

mp: 94-96 oC;


[]D32 −131.68 (c 2.14, CHCl3);

IR (neat) 1060, 1375 cm1;

1H NMR (300 MHz, CDCl3) δ 1.33 (s, 3H, CH3), 1.53 (s, 3H, CH3), 1.82 – 1.94 (m, 2H,

H-6a and H-6b), 2.76-2.88 (m, 1H, H-7a), 3.13- 3.24 (m, 1H, H-7b), 3.37 (d, J = 12.6

Hz, 1H, -NCH2Ph), 3.60 (m, 1H, H-5), 3.89 (d, J = 3.3 Hz, 1H, H-3), 4.09 (d, J = 12.6

Hz, 1H, -NCH2Ph), 4.38 (dd, J = 4.9, 3.3 Hz, 1H, H-4), 4.42 (d, J = 12.0 Hz, 1H, -


= 3.7 Hz, 1H, H-1), 7.18 – 7.40 (m, 10H, Ar-H);


62

13C NMR (75 MHz, CDCl3) δC-6), 26.4 (CH3), 26.8 (CH3), 51.2 (C-7), 62.4 (C-5),

64.3 (-NCH2Ph), 71.8 (-OCH2Ph), 81.6, 81.8, 84.8 (C-2/C-3/C-4), 105.4 (C-1), 111.5

(OCO), 126.8, 127.7, 127.8, 128.1, 128.3, 128.5, 128.9, 137.1, 137.6 (Ar-C).

Expt. No. 1.2.3.13 Preparation of 1,2-O-isopropylidine-3-O-benzyl-5,6,7-trideoxy-

5,7-(N-benzylimino)--D-gluco-sept-1,4-furanose (122b).

O

O

O

BnO

NBnO

115b

O

O

O

BnO

NBn

122b

LiAlH4/AlCl3, anhyd. THF,

20 oC to 0 oC, 10 min, 85%

The reduction of -D-gluco-β-lactam 115bg, 1.22 mmolwith LiAlH4 (0.070 g,

1.83 mmol)and AlCl3 (0.082 g, 0.61 mmol) in anhydrous THF (10 mL) at −20 oC to 0

oC in 10 min gave the colorless oil which on column purification (n-hexane/ethyl acetate

= 9/1) afforded -D-gluco-azetidine 122b (0.410 g, 85%) as a white solid.

mp: 101-103 oC;


[]D32 −24.43 (c 2.12, CHCl3);

IR (neat) 1070, 1355 cm1;

1H NMR (300 MHz, CDCl3) δ 1.31 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.08-2.16 (m, 1H,

H-6a), 2.24-2.41 (m, 1H, H-6a), 2.84-2.88 (m, 1H, H-7a), 3.35 (td, J = 9.0, 2.4 Hz, 1H,

H-7b), 3.50 (d, J = 12.9 Hz, 1H, -NCH2Ph), 3.56-3.70 (m, 2H, -NCH2Ph, H-5), 3.81 (d, J

= 3.0 Hz, 1H, H-3), 4.06 (dd, J = 6.0, 3.0 Hz, 1H, H-4), 4.38 (d, J = 12.0 Hz, 1H, -


= 3.9 Hz, 1H, H-1), 7.18 – 7.40 (m, 10H, Ar-H);


63

13C NMR (75 MHz, CDCl3) δC-6), 26.4 (CH3), 26.7 (CH3), 52.4 (C-7), 62.7 (C-5),

63.4 (-NCH2Ph), 71.2 (-OCH2Ph), 81.8, 82.0, 82.6 (C-2/C-3/C-4), 104.8 (C-1), 111.5

(OCO), 126.9, 127.4, 127.8, 128.0, 128.2, 128.4, 128.7, 137.4, 138.4 (Ar-C).

Expt. No. 1.2.3.14 Preparation of (3S,4R,5R,6S)-3,4,5-trihydroxy-conidine (34a ).

O

O

O

BnO

NBn

122a

(i) TFA/H2O (3/2), 0 oC to 25 oC, 4 h

(ii) H2, 10% Pd/C, 80 psi, 25 oC, 5 days,78% over 2 steps

N

H OH

OH

OH

34a

A cooled (0 oC) solution of 122a (0.41 g, 1.04 mmol) in TFA-H2O (5 mL, 3:2) was

stirred for 1 h, brought to 25 °C, and stirred for additional 2 h. TFA was evaporated at

high vacuum to yield the crude hemiacetal (0.32 g). A solution of the above product in

dry methanol (8 mL) was hydrogenated in the presence of 10% Pd/C (0.07 g) at 80 psi

for 5 days. The catalyst was filtered and washed with methanol, and the filtrate was

concentrated to afford a thick liquid. Purification by column chromatography on silica

gel chloroform/methanol (5.5/4.5) afforded 34a (0.13 g, 78%) as a moisture sensitive

colorless solid.

Rf 0.30 (methanol);

[]D32 −.43 (c 1.12, MeOH);

IR (neat) 3600-3200 (broad, OH) cm1;

1H NMR (300 MHz, D2O) δ 2.44-2.58 (m, 1H, H-7a), 2.78-2.96 (m, 1H, H-7b), 3.34 (dd,

J = 12.6, 11.0 Hz, 1H, H-2a), 3.63 (dd, J = 12.6, 5.0 Hz, 1H, H-2b), 3.67-3.76 (m, 1H, H-

6), 3.82 (dt, J = 10.0, 4.7 Hz, 1H, H-8a), 3.90 (t, J = 6.9 Hz, 1H, H-4), 3.95 (dd, J = 6.9

Hz, 1H, H-5), 4.45 (dt, J = 10.0, 8.8 Hz, 1H, H-8b), 4.88-5.05 (m, merged with HDO

signal, 1H, H-3);


64

13C NMR (75 MHz, D2O) δ 22.2 (C-7), 49.3 (C-8), 53.7 (C-6), 62.9 (C-2), 68.3 (C-3),

71.5 (C-5), 73.7 (C-4).

Expt. No. 1.2.3.15 Preparation of (3S,4R,5R,6R)-3,4,5-trihydroxy-conidine (34b ).

O

O

O

BnO

NBn

122b

(i) TFA/H2O (3/2), 0 oC to 25 oC, 4 h

(ii) H2, 10% Pd/C, 80 psi, 25 oC, 5 days,71% over 2 steps

N

H OH

OH

OH

34b

Following a similar manner, compound 122b (0.35 g, 0.89 mmol) was treated with TFA-

H2O (5 mL, 3:2), and the resultant hemiacetal was hydrogenated over 10% Pd/C (0.05 g)

for 5 days to furnish 34b (0.1 g, 71%) as a moisture sensitive colorless solid, after

isolation and purification by column chromatography on silica gel (chloroform/methanol

= 7.5/2.5).


[]D32 +44.21 (c 1.12, MeOH);

IR (neat) 3600-3200 (broad, OH) cm1;

1H NMR (300 MHz, D2O) δ 2.34-2.50 (m, 1H, H-7a), 2.74-2.90 (m, 1H, H-7b), 3.25 (dd,

J = 13.5, 6.0 Hz, 1H, H-2a), 3.43 (dd, J = 13.5, 3.3 Hz, 1H, H-2b), 3.56 (dd, J = 8.2, 4.8

Hz, 1H, H-4), 4.06-4.14 (m, 1H, H-3), 4.14-4.27 (m, 3H, H-5, H-8a, H-8b), 4.27-4.36

(m, 1H, H-6);

13C NMR (75 MHz, D2O) δ 18.8 (C-7), 50.2 (C-8), 55.0 (C-6), 61.3 (C-2), 67.2 (C-3),

68.3 (C-5), 73.4 (C-4).


65

CHAPTER 1


Section C: Conformational study of conidine iminosugars

1.3.1 Conformations of 34a and 34b using 1H NMR information

In bicyclic iminosugars, the binding ability and their function as glycosidase inhibitor

depend on their conformation. For example, indolizidine iminosugar (six-five ring fused

system) namely 1-deoxy-castanospermine 123 exists in 8C5 conformation I (Figure 31a)

and exhibits the potent enzyme inhibition toward the - and β-glucosidase and -

mannosidase whereas; 1-deoxy-8a-epi-castanospermine exists in 5C8 conformation II

and shows selective inhibition of - and β-galactosidase and β-glucosidase.98

H

HH

HH

OH

HO N

HHO

I 8C5

OH

H

HOH

H

HNH

OH

H

II 5C8

1

2

3 4 5

6

78

8aN

OH

OHOH

H123

Figure 31a: Conformations of 1-deoxy-castanospermine 123

In case of conidine iminosugars 34a and 34b (six-four ring fused system), the 1H NMR

spectra were found to be dramatically different and knowing that the four membered

fused azetidine ring system will change the conformation of six membered piperidine

ring, it is expected that both target molecules could exist in different conformations. In

order to assign the conformations, we studied the 1H NMR spectra of 34a and 34b and

the chemical shifts as well as the coupling constants were obtained by the decoupling

experiments and are given in Table 2.


66

Table 2: 1H NMR Spectra of 34a and 34b

H-2a H-2e H-3 H-4 H-5

34a 3.34 (dd)

J2a,2e = 12.6 Hz

J2a,3a = 11.0 Hz

3.63 (dd)

J2a,2e = 12.6 Hz

J2e,3a = 5.0 Hz

4.88-5.05 (m) 3.90 (t)

J4,3 = J4,5 =

6.9 Hz

3.95 (dd)

J5,4 = 6.9 Hz

J5,6 = 6.0 Hz

34b 3.25 (dd)

J2a,2e =13.5 Hz

J2a,3a = 6.0 Hz

3.43 (dd)

J2a,2e =13.5 Hz

J2e,3a = 3.3 Hz

4.06-4.14 (m) 3.56 (dd)

J4,5 = 8.2 Hz

J4,3 = 4.8 Hz

4.14-4.27 (m)

H-6 H-7a H-7e H-8a H-8e

34a 3.67-3.76 (m) 2.44-2.58 (m) 2.78-2.96 (m) 3.82 (dt)

J8a,8e =J8a,7a =

10.0 Hz

J8a,7e = 4.7 Hz

4.45 (dt)

J8a,8e =J8a,7e =

10.0 Hz

J8e,7a = 8.8 Hz

34b 4.27-4.36 (m) 2.34-2.50 (m) 2.74-2.90 (m) 4.14-4.27 (m) 4.14-4.27 (m)

For compound 34a, we assumed three conformations A, A1 and A2 (Figure 31b). In the

1H NMR spectrum of 34a, the H-2a proton appeared as a doublet of doublet with

geminal and vicinal coupling constants of 12.6 Hz and 11.0 Hz, respectively. The large

vicinal coupling constant J2a,3a = 11.0 Hz requires the axial-axial relative orientation of

the H-2 and H-3 and therefore the H-3 was assigned the axial orientation. This

established the possibility of conformation A and ruled out conformation A1. In the

precursor 122a, the relative stereochemistry of H-2/H-3 and H-3/H-4 is trans and

assuming that the same stereochemistry is retained in compound 34a, the H-3/H-4 and

H-4/H-5 should have axial-axial orientation. In accordance with this, the H-4 appeared as

a triplet (J4a,3a = J4a,5a = 6.9 Hz). The relatively small coupling constant for axially

oriented H-3/H-4 (J3a,4a = 6.9 Hz) required little distortion from the usual chair

conformation of piperidine ring. Thus, dihedral angle of ~160 between the H-3 and H-4

protons (rather than 180o in axial-axial) accounts for coupling constant of 6.9 Hz. In


67

agreement with this, the axially oriented H-5 showed doublet of doublet with J4a,5a = 6.9

Hz and J5a,6e= 6.0 Hz wherein, relatively large coupling constant for axial-equatorial

protons indicates the dihedral angle between H-5a and H-6e ~10o. This ruled out the

conformation A1. To account these features, the half chair conformation A2 (as shown

below) was assigned to 34a wherein, the fused four membered strained azetidine ring

forced the piperidine ring to adopt distorted half chair conformation.

A1 2C5

H

H

HH H

OH

HO N

HHO

A 5C2

N

HOH

H

HHO

HO

H

H HN

OH

OH

OHH

1 2

3

456

7

8

34a

OH

H

H HOH

H

HNH

OHA2

Figure 31b: Conformations of compounds 34a.

In case of 34b, three conformers B, B1 and B2 (Figure 31c) were considered. In

the 1H NMR of 34b, one of the H-2 appeared as doublet of doublet with geminal

coupling constants of 13.5 Hz (J2a,2e) and vicinal coupling constant of 6.0 Hz (J2a,3)

while, other H-2 appeared as doublet of doublet with coupling constants of 13.5 Hz and

3.3 Hz. The small vicinal coupling constant of J2e,3e = 3.3 Hz ruled out the 5C2

conformation B as it requires large coupling constant between the axially oriented H-

2/H-3 protons. To decide the conformations between B1 and B2, we considered the H-4

proton. In conformation B1, the H-4 is equatorial and is expected to show doublet of

doublet with small coupling constants. However, the H-4 showed doublet of doublet with

J4,5 = 8.2 Hz and J4,3 = 4.8 Hz. The appearance of H-4 with large coupling constant of J4,5

= 8.2 Hz ruled out the 2C5 conformation B1. To account this aspect, we assumed the

most probable boat conformation B2 wherein the quasi-axially oriented H-4 has large

coupling constant of 8.2 Hz with the quasi-axial H-5 and small coupling constant of 4.8

Hz with the quasi-equatorial H-3. In accordance with this, the H-5 showed doublet of


68

doublet with large coupling constants of 8.2 Hz with H-4 and 9.3 Hz with H-6 suggesting

that the ring fusion proton (H-6) is quasi-axial. Thus, the boat conformation B2 (as

shown below) was assigned to compound 34b wherein the two fused rings are the

farthest away from each other and –OH groups are positioned on either at quasi-

equatorial or quasi-axial.

OH

HH

HOH

H

HNH

OHB1 2C5

H

HH

HH

OH

HO N

HHO

B 5C2

N

H H

OH

H

OH

H

H

HOH

B2

N

HOH

OH

OH

34b

1

234

56

78

Figure 31c: Conformations of compounds 34b.

1.3.2 Conformations of 34a and 34b using Density Functional Theory (DFT)

Calculations

Thus, based on the 1H NMR studies, conidine iminosugar 34a and 34b were found to

exist in half chair (A2) and boat or twist boat (B2) conformations respectively. This

observation was substantiated by employing density functional theory (DFT). Thus,

different conformers of 34a and 34b were generated by varying the C4-C5-C6-N1 and

C2-N1-C6-C5 dihedral angles from –80° to +80° within the framework of HF/3-21G(d)

theory. The corresponding energy profiles for 34a and 34b are displayed in Figures 32

and 33 respectively.

d(C4-C5-C6-N1) scan d(C2-N1–C6 –C5) scan

34a_A1 34a_A2


69

34a_B1 34a_B2

34a_C1 34a_C2

34a_D1 34a_D2

Figure 32: Relative energy profiles for the conformers of 34a with different hydrogen bonding patterns as

a function of dihedral angle. The minimum energy conformer among all the profiles is considered as a

reference.

d(C4-C5-C6-N1) scan d(C2-N1–C6 –C5) scan

34b_A1 34b_A2


70

34b_B1 34b_B2

34b_C1 34b_C2

34b_D1 34b_D2

Figure 33: Relative energy profiles for the conformers of 34b with different hydrogen bonding pattern as a

function of dihedral angle. The minimum energy conformer among all the profiles is considered as a

reference.

As revealed from these profiles, ring conformers with chair (C), half chair (HC), twisted

chair (TC) and twisted boat (TB) were included. Minima (24 for 34a and 20 for 34b) on

the potential energy surface using the dihedral angle as reaction coordinate, were

optimized at the B3LYP/6-31G(d,p) level of theory. Stationary point geometries thus

obtained were confirmed to be local minima from the vibrational frequencies, all of

which turn out to be real. Relative stabilization energies (ΔERel) were calculated by


71

subtracting the energy of lowest energy conformer from those of rest of the conformers

(Table 3).

Table 3: B3LYP/6-31G(d,p) calculated relative stabilization of energies of 34a and 34b conformers (local

minima on scan).

34a ΔERel 34b ΔERel ΔE*

A103 1.5 A127 10.4 18.1

A112 2.0 A131 7.3 15.0

A119 1.8 A140 1.2 9.0

A135 18.3 A206 0.0 7.7

A204 1.5 A220 7.3 15.0

A211 9.2 B126 7.7 15.5

A230 9.3 B139 29.4 37.1

B103 0.0 B206 2.5 10.3

B112 6.4 B229 10.7 18.4

B205 0.0 C113 2.9 10.6

B212 6.3 C128 7.3 15.0

B232 9.3 C140 1.2 9.0

C104 0.4 C206 0.0 7.7

C111 1.8 C220 7.3 15.0

C133 18.3 D113 4.9 12.6

C138 18.3 D128 10.5 18.2

C208 0.4 D140 1.2 9.0

C215 1.8 D207 8.1 15.8

C232 18.5 D220 10.5 18.2

D104 2.7 D238 21.2 28.9

D111 4.4 * The relative energies of

conformers of 34b are

calculated with reference to

minimum energy conformers

of 34a.

D208 2.7

D214 4.4

D232 10.5

It has been observed that the some of the local minima (conformers) located on the

different dihedral scan possess the same geometry as well as the same energy.


72

Some of these geometries from the scan of both the C4-C5-C6-N1 and C2-N1-

C6-C5 dihedral angles exhibiting different hydrogen bonding patterns finally converged

to identical conformers. Out of several local minima possessing similar six membered

ring conformations as well as the orientation of -OH groups, one conformer was chosen

for subsequent calculations. The gas phase structures were reoptimized employing

SCRF-PCM model using water as solvent. The ΔERel and Boltzmann contribution (BC)

values in gas phase as well as in water are given in Table 4 specifying the ring

conformation.

Table 4: B3LYP/6-31G(d,p) stabilization energies (ΔERel, in kJ mol-1) and Boltzmann contribution (BC

in %) of the minima along the dihedral scan.

34a 34b

Gas phase SCRF-

PCM

Ring

Config

Gas phase SCRF-PCM

Ring

Config

ΔERel BC ΔERel BC ΔERel BC ΔERel BC

A103 1.5 13.8 11.4 0.4 6C3 A127 10.4 0.6 8.4 1.6 4HC

A112 2.0 11.3 0.0 36.5 NC4 A131 7.3 2.0 9.2 1.2 3TB6

A119 1.8 12.3 0.9 25.4 NC4 A140 1.2 23.6 11.9 0.4 NTB5

A135 18.3 0.0 26.2 0.0 NTB4 A206 0.0 38.3 0.0 48.2 3TB5

A211 9.2 0.6 4.6 5.7 NC4 B126 7.7 1.7 7.4 2.4 NTB4

B103 0.0 25.3 11.4 0.4 6C3 B139 29.4 0.0 29.3 0.0 NTB4

B112 6.4 1.9 5.0 4.9 NC4 B206 2.5 14.0 2.3 19.1 2TB5

C104 0.4 21.6 12.3 0.3 NC4 B229 10.7 0.5 6.0 4.3 2TB5

C232 16.6 0.1 13.1 0.2 3HC C113 2.9 11.9 9.4 1.1 3TC6

D111 4.4 4.3 1.1 23.4 NC4 D113 4.9 5.3 12.3 0.3 2TC5

D208 2.7 8.5 7.9 1.5 NC4 D128 10.5 0.6 10.3 0.8 NTB4

D232 10.5 0.4 8.5 1.2 2TB5 D207 8.1 1.5 2.3 19.1 2TB5

D238 21.2 0.0 8.6 1.5 2TB5

Only one conformer has been reported out of all the conformers having same ΔERel

values and the geometry.


73

Conformer

name

Scan

Point

No.

D1 Ring conformer

Conformer

name

Scan

Point

No.

D1 Ring

conformer

34a_A101 01 -80.0

34a_A125 25 16.0

34a_A205 05 -64.0

34a_A130 30 36.0

34a_A110 10 -44.0

34a_A135 35 56.0

34a_A115 15 -24.0

34a_A141 41 80.0

34a_A120 20 -4.0

Figure 34: Different ring conformations along the scan of dihedral angle C4-C5-C6-N1 (D1) for

compound 34a.


74

Conformer

name

Scan

Point

No.

D2 Ring conformer

Conformer

name

Scan

Point

No.

D2 Ring

conformer

34b_A201 01 −80.0

34b_A225 25 16.0

34b_A205 05 −64.0

34b_A230 30 36.0

34b_A210 10 −44.0

34b_A235 35 56.0

34b_A215 15 −24.0

34b_A241 41 80.0

34b_A220 20 −4.0

Figure 35: Different ring conformations along the scan of dihedral angle C2-N1–C6–C5 (D2) for

compound 34b.

As may be noted 34a is present in chair (C) and half chair (HC) forms and 34b exhibits

twist boat (TB) and twist chair (TC) ring conformations. Furthermore 34a conformers

with the BC values >5% in water possesses 1C4 ring conformation, whereas 2TB5

conformation is noticed in case of 34b. The conformers 34a_A112, 34a_A119,

34a_A211 and 34a_D111 reveal similar structure except for orientation of the hydroxyl

group. Similar inferences may be drawn for 34b_A206, 34b_B206, and 34b_D207

conformers. Lowest energy conformers 34a_A119 and 34b_A206 are shown in Figure

35 where both views top and side along the C2-C3 bond are displayed along with the


75

ring conformations. Hydrogen bonding patterns in hydroxyl groups of 34a and 34b are

displayed in Figure 36 and Figure 37 respectively.

(i) (ii) (iii)

34a_

AD

112

34a_

C23

2

34b_

AD

206

Figure 36: Optimized geometries of 34a and 34b in water. Views from (i) top, (ii) along C2-C3 bond are

shown (iii) ring conformations (1C4, 3HC and 2TB5 respectively) are displayed.

Depending on the different hydrogen bonding patterns in the constrained form the 4

conformers “A”, “B”, “C”, and “D” have been considered. In “A” and “B” conformers

the C4-OH is directed towards the lone pair of the nitrogen whereas it is accepting the

hydrogen bond from the C2O-H. The conformer “A” differ in the orientation of the –OH

at C3 position compared to “B”. The reverse hydrogen bonding pattern is considered in

“C” and “D” conformers.


76

A B

C D

Figure 37: Hydrogen bonding patterns in hydroxyl groups of 34a.

34b d(C2-N1 –C6 –C5) d(C4-C5-C6-N1)

Figure 38: The selected angles in 34b are scanned to check the minimum energy structure these conformers.

In order to obtain minimum energy conformers the two dihedral angles (C2-N1–C6 –C5

and C4-C5-C6-N1) were scanned. These dihedral angle (the atoms highlighted in Figure

38) are scanned from –80° to +80° at HF/3-21G(d) level of theory. The energy profiles

for the scans of aforementioned dihedral angles have been shown in 34b_X1 and


77

34b_X2 (X = “A”, “B”, “C”, or “D”), respectively. The energy of the lowest energy

conformers has been chosen as a reference.

Thus 1C4 conformation of 34a is transparent; 34b on the other hand exhibits 2TB5

ring conformation. It should be noted here that the configuration around the C2-C3 bond

is different in 34a and 34b diastereomers. The dihedral angle H-C3-C4-H turns out to be

67o here compared to 98o in 34b with near eclipsed conformer. Furthermore, the

conformer 34a_C232 is predicted to possess the half chair ring conformation (3HC) as

inferred from the experiments. The calculated coupling constants for the minimum

energy 34a_A112 conformer and that inferred from experiments 34a_C232 and

34b_A206 have been given in Table 5.

Table 5: Calculated coupling constants (in Hz) of the 34a and 34b protons.

34a 34b

Expt Calc. A112 (C232)

Expt Calc. A206

H-2a 11.0 1.37 (9.11) 6.0 3.45

H-2e 5.0 3.27 (4.20) 3.3 1.41

H-3 3.68, 3.27, 1.37

(9.11, 4.20,7.52) 3.45, 1.41, 2.43

H-4 6.9, 6.9 3.51, 3.68

(7.52,7.33) 8.2, 4.8 5.68, 2.43

H-5 6.9, 6.0 4.19, 3.51

(7.33,7.64) 9.51, 5.68

H-6 1.26, 7.28, 4.19

(7.64, 6.01, 9.61) 7.72, 2.66, 9.51

H-7a 1.41, 7.62, 1.26

(9.61, 8.97, 8.51) 6.52, 8.45, 7.72

H-7e 7.58, 6.52, 7.28

(6.01, 1.04, 5.49) 9.56, 3.18, 2.66

H-8a 10.0, 4.7 7.62, 6.51

(8.97, 1.04) 8.45, 3.18

H-8e 10.0, 8.8 7.58, 1.41

(8.51, 5.49) 6.52, 9.56


78

In case of conformer 34a_C232, coupling constants for H-2a and H-2e (J2a,3 and J2e,3)

are 9.11 and 4.20 Hz which matches well with 11.0 and 5.0 Hz, respectively, as observed

in the experimental NMR spectra of 34a. While for 34a_A112, the calculated values of

coupling constant were found to be 1.37 and 3.27 Hz and thus deviate largely from

experimental values. In addition, for conformer 34a_C232 the calculated J3,4 = 7.52 Hz

and J4,5 = 7.33 Hz values for the H-4 were matching well with the experimentally

observed value of 6.9 Hz. The large coupling constant values suggested that the H-3 and

H-5 protons are trans to H-4 (dihedral angle H-C3-C4-H and H-C4-C5-H being ~180°).

Thus, the 1H NMR data of 34a_C232 in 3HC ring conformation matches well with the

experimental values corresponding to half chair conformation of 34a.

Table 6a: Calculated 1H NMR spectra of conformers of 34a.

34a

A103 A112 A119 A211 B103 C104 D111 D208

H-2a 2.76 2.55 2.35 2.45 2.75 2.72 2.35 2.65

H-2e 2.65 3.00 2.97 2.90 2.74 2.79 3.07 2.81

H-3 3.71 4.02 4.07 3.61 3.55 3.91 3.96 3.52

H-4 4.03 4.12 4.24 4.25 4.02 4.04 4.23 3.93

H-5 3.64 3.59 3.57 4.02 3.84 3.51 3.71 3.68

H-6 3.62 3.88 3.86 3.87 3.55 3.38 3.77 3.43

H-7a 1.77 2.16 2.16 2.30 1.77 1.64 2.17 1.70

H-7e 2.17 2.22 2.22 1.81 2.18 2.28 2.23 2.18

H-8a 3.21 3.79 3.85 3.91 3.21 3.19 3.84 3.16

H-8e 3.05 3.35 3.33 3.34 3.06 3.15 3.33 3.05

In case of compound 34b, the experimental coupling constants for the H-2 were

observed to be J2a,3 = 6.0 Hz and J2e,3 = 3.3 Hz. The similar trend has been observed for

the calculated coupling constant of 34b_A206 conformer for which the coupling

constants for these protons are predicted to be 3.45 Hz and 1.41 Hz, respectively. The


79

lower coupling constant for the J2e,3 is in agreement with the experiments. Similar

observation may also be made for the coupling constant of H-4. Thus, coupling constant

data suggests that the compound 34b (34b_A206) present in twisted boat (2TB5)

conformation.

Table 7b: Calculated 1H NMR spectra of conformers of 34b.

34b

A140 A206 B206 C113 D113 D207

H-2a 2.46 2.84 2.53 2.85 2.81 2.87

H-2e 3.25 2.67 3.06 3.25 3.47 2.63

H-3 4.50 3.83 4.11 4.29 4.10 3.67

H-4 3.73 3.55 3.92 4.13 4.08 3.64

H-5 3.84 3.88 4.18 3.49 3.66 3.90

H-6 3.45 3.38 3.36 4.21 4.17 3.34

H-7a 2.05 2.03 1.97 3.12 3.09 1.96

H-7e 2.07 2.35 1.95 2.06 2.08 2.36

H-8a 2.96 3.68 2.96 3.69 3.66 3.65

H-8e 3.19 3.34 3.19 2.63 2.64 3.33

Table 8: Atomic coordinates in selected conformers of 34a optimized in water using SCRF_PCM model.

34a_A103

N 0.970884 0.787949 0.001569

C 2.420501 0.881045 _0.277956

C 2.503590 _0.652221 _0.008764

C 0.994045 _0.644253 _0.346929

C _0.164904 _1.303391 0.380913

C _1.453205 _0.663917 _0.224722

C _1.439859 0.891737 _0.195126

C _0.099045 1.514045 _0.662003

O _0.101910 _1.054427 1.787622

O _1.615659 _1.050180 _1.587056

O _1.819580 1.369907 1.096101

H 2.641799 1.116154 _1.332008

34a_A112

N 0.963080 0.667547 _0.957918

C 2.073873 0.888556 _0.003636

C 2.377524 _0.630452 _0.072481

C 0.957503 _0.792525 _0.673764

C _0.123185 _1.273777 0.326072

C _1.478439 _0.593562 0.026440

C _1.341879 0.942707 0.061832

C _0.282377 1.423655 _0.936855

O 0.218674 _1.077883 1.700626

O _1.964508 _0.928598 _1.269684

O _0.964710 1.379029 1.382731

H 2.848043 1.577191 _0.367300


80

H 2.978900 1.563795 0.371541

H 2.706734 _0.875362 1.042378

H 3.155001 _1.245758 _0.652596

H 0.860400 _0.806627 _1.430681

H _0.202008 _2.392298 0.234227

H _2.315482 _0.984279 0.380855

H _2.228384 1.228056 _0.879914

H _0.020239 1.446733 _1.763051

H _0.092927 2.573107 _0.381314

H 0.442986 _0.246582 1.889355

H _1.680434 _2.029795 _1.616556

H _1.312080 0.876544 1.760962

H 1.761409 1.211519 0.999125

H 2.553943 _1.133135 0.878146

H 3.168145 _0.875087 _0.785850

H 0.869448 _1.401344 _1.585085

H _0.239943 _2.360394 0.197931

H _2.193054 _0.898897 0.809750

H _2.309580 1.387658 _0.213940

H _0.740855 1.369757 _1.933333

H _0.055203 2.477728 _0.740705

H 0.013061 _0.143865 1.896489

H _2.090035 _1.901808 _1.298921

H _1.740715 1.249842 1.974089

34a_A119

N 0.947131 0.685758 _0.956793

C 2.062602 0.880760 _0.002490

C 2.369168 _0.634428 _0.121884

C 0.940702 _0.780872 _0.707195

C _0.128146 _1.286763 0.294725

C _1.483204 _0.586045 0.042953

C _1.338735 0.945033 0.108402

C _0.297049 1.440657 _0.906790

O 0.239006 _1.150591 1.669651

O _1.998812 _0.879089 _1.250463

O _0.991259 1.255488 1.467758

H 2.832553 1.584295 _0.346047

H 1.752843 1.167474 1.011986

H 2.562966 _1.166382 0.809154

H 3.148557 _0.852512 _0.855895

H 0.838673 _1.366579 _1.632100

H _0.258835 _2.366357 0.127014

H _2.180872 _0.901977 0.835291

H _2.313361 1.394073 _0.141270

H _0.774235 1.399684 _1.895744

H _0.063393 2.495802 _0.712632

H 0.025872 _0.232313 1.922850

H _2.149699 _1.847396 _1.298418

H _0.794494 2.215378 1.531464

34a_A211

N 0.932376 0.775484 _0.897219

C 2.055764 0.913763 0.055766

C 2.378512 _0.582863 _0.182722

C 0.942147 _0.703623 _0.760826

C _0.124253 _1.294506 0.191339

C _1.483805 _0.590431 _0.007732

C _1.355421 0.933462 0.190785

C _0.318063 1.517859 _0.784635

O 0.174884 _1.139470 1.588181

O _1.974053 _0.798938 _1.327697

O _1.078170 1.254630 1.556339

H 2.814672 1.648760 _0.243369

H 1.755707 1.118723 1.092387

H 2.625671 _1.183064 0.695637

H 3.143121 _0.734904 _0.947912

H 0.843081 _1.223310 _1.724553

H _0.241773 _2.366330 _0.038507

H _2.184096 _0.980762 0.747678

H _2.330160 1.384369 _0.042691

H _0.787261 1.559874 _1.777687

H _0.089923 2.548351 _0.489297

H 0.943576 _1.700748 1.824859

H _2.103901 _1.764290 _1.450269

H _0.465923 0.573202 1.887831


81

34a_B103

N 0.967637 0.788834 0.006485

C 2.415858 0.893820 _0.276308

C 2.508273 _0.642020 _0.025659

C 0.998200 _0.638243 _0.361908

C _0.155495 _1.314217 0.361249

C _1.446601 _0.676191 _0.227496

C _1.442931 0.882918 _0.169841

C _0.109822 1.518294 _0.640273

O _0.087279 _1.078168 1.770216

O _1.545859 _1.143057 _1.569827

O _1.815065 1.337728 1.133612

H 2.633506 1.143576 _1.327731

H 2.972043 1.571318 0.380626

H 2.714435 _0.876447 1.022376

H 3.161699 _1.223896 _0.678025

H 0.866695 _0.787235 _1.447732

H _0.191990 _2.398764 0.197456

H _2.304532 _1.014478 0.374548

H _2.240748 1.234666 _0.838882

H _0.040762 1.463020 _1.742692

H _0.104813 2.574228 _0.348061

H 0.457431 _0.270834 1.878970

H _2.406494 _0.839618 _1.930701

H _1.309185 0.824435 1.783856

34a_C104

N 0.997137 0.756482 0.040224

C 2.452627 0.774755 _0.204993

C 2.410906 _0.780754 _0.096129

C 0.922330 _0.620308 _0.476060

C _0.299636 _1.349959 0.068965

C _1.532378 _0.459327 _0.285699

C _1.334130 1.031391 0.097137

C _0.037252 1.625480 _0.488255

O _0.236741 _1.626328 1.465818

O _1.798802 _0.480680 _1.684253

O _1.307586 1.140422 1.530095

H 2.722796 1.099661 _1.223660

H 3.042285 1.340330 0.524843

H 2.534532 _1.119063 0.935507

H 3.048480 _1.355529 _0.770557

H 0.831313 _0.619341 _1.579839

H _0.422445 _2.320692 _0.433328

H _2.393552 _0.833931 0.290792

H _2.206890 1.589226 _0.266259

H _0.080076 1.639282 _1.591362

H 0.086742 2.654984 _0.131217

H _0.496992 _0.813580 1.928691

H _2.029196 _1.401735 _1.931649

H _0.366728 1.250027 1.783390

34a_D111

N 0.940188 0.699463 _0.953676

C 2.056974 0.894782 _0.000006

C 2.377071 _0.615999 _0.137421

C 0.947669 _0.769687 _0.717547

C _0.111660 _1.293616 0.284385

C _1.473136 _0.611096 0.044017

C _1.344464 0.925807 0.121817

C _0.312806 1.439775 _0.893785

O 0.257321 _1.154196 1.658469

O _1.931645 _1.008255 _1.243173

34a_D208

N 0.943572 0.794086 0.032914

C 2.394640 0.920395 _0.225940

C 2.481269 _0.631109 _0.111223

C 0.975388 _0.596451 _0.460106

C _0.167321 _1.370406 0.177916

C _1.473147 _0.613153 _0.217449

C _1.427279 0.934734 _0.014582

C _0.131621 1.573743 _0.556555

O _0.026716 _1.495093 1.587822

O _1.695911 _0.923065 _1.593748


82

O _0.999025 1.235099 1.482985

H 2.818505 1.610719 _0.336059

H 1.746892 1.164468 1.018889

H 2.587270 _1.158297 0.783978

H 3.151445 _0.814950 _0.882398

H 0.844042 _1.349111 _1.646126

H _0.231455 _2.371842 0.111654

H _2.163263 _0.942402 0.836143

H _2.325052 1.370561 _0.121226

H _0.791982 1.396999 _1.881591

H _0.090896 2.496635 _0.695371

H 0.027627 _0.240975 1.913741

H _2.836538 _0.647201 _1.364863

H _0.768999 2.188175 1.543953

O _1.623745 1.276157 1.357887

H 2.623940 1.261702 _1.248570

H 2.944916 1.535585 0.494224

H 2.665558 _0.957397 0.915758

H 3.145098 _1.155413 _0.801006

H 0.851114 _0.635538 _1.557549

H _0.246229 _2.389596 _0.221786

H _2.289383 _0.995576 0.415062

H _2.290181 1.350838 _0.554852

H _0.116975 1.544225 _1.660969

H _0.086194 2.621109 _0.236716

H 0.348520 _0.656938 1.921977

H _2.546265 _0.511941 _1.860061

H _0.749535 1.259834 1.796038

Table 9: Atomic coordinates in selected conformers of 34b optimized in water using SCRF_PCM model.

34b_A140

N 1.081830 _0.706474 0.123807

C 2.519921 _0.580312 _0.186925

C 2.340043 0.960982 _0.060524

C 0.844163 0.671029 _0.344097

C _0.391862 1.260916 0.351201

C _1.274032 0.062139 0.829455

C _1.282343 _1.080210 _0.215752

C 0.144093 _1.689923 _0.390038

O _1.116087 2.154857 _0.490918

O _0.859981 _0.424584 2.101843

O _1.766435 _0.476452 _1.420724

H 3.196374 _1.093637 0.504917

H 2.763037 _0.875800 _1.220974

H 2.874542 1.593452 _0.771760

H 2.513175 1.311063 0.961129

H 0.672861 0.702504 _1.436351

H _0.114770 1.834123 1.245476

H _2.298808 0.434052 0.947859

H _1.975399 _1.864919 0.120223

34b_A206

N _1.203831 _0.943275 0.050591

C _2.455293 _0.499084 _0.617090

C _2.387758 0.850087 0.142336

C _0.967797 0.436097 0.596688

C 0.211192 1.132623 _0.080089

C 1.485796 0.275917 0.054148

C 1.186864 _1.247147 _0.038952

C _0.127230 _1.494327 _0.779109

O 0.408716 2.466603 0.381937

O 2.425545 0.597710 _0.968434

O 1.146032 _1.836889 1.262245

H _2.347407 _0.391229 _1.706518

H _3.322186 _1.139836 _0.417553

H _3.105190 0.938165 0.961580

H _2.436166 1.751588 _0.473459

H _0.804688 0.434046 1.686356

H _0.000070 1.234747 _1.154004

H 1.920672 0.450191 1.053409

H 2.022367 _1.709308 _0.580698


83

H 0.234342 _2.626142 0.173068

H 0.302751 _1.922180 _1.459380

H _1.465506 1.608755 _1.217144

H 0.084666 _0.654545 1.987182

H _1.819945 _1.159693 _2.122424

H _0.072154 _1.027882 _1.778244

H _0.288897 _2.569358 _0.927208

H 0.595627 2.422494 1.345858

H 2.538852 1.571241 _0.968635

H 0.209074 _1.906320 1.531398

34b_B206

N _1.191938 _0.970778 0.054544

C _2.444605 _0.542463 _0.619656

C _2.417615 0.789217 0.170320

C _0.980187 0.409557 0.606654

C 0.179051 1.137425 _0.073697

C 1.467471 0.310731 0.057845

C 1.214266 _1.218662 _0.059097

C _0.105943 _1.505909 _0.772929

O 0.433194 2.416853 0.506616

O 2.409170 0.672238 _0.951412

O 1.243429 _1.850739 1.222149

H _2.322278 _0.405181 _1.704447

H _3.300076 _1.206839 _0.449666

H _3.129146 0.829549 0.998335

H _2.510799 1.702599 _0.424023

H _0.802370 0.414052 1.692467

H _0.035052 1.246373 _1.149340

H 1.882960 0.492268 1.061940

H 2.049677 _1.634765 _0.636078

H _0.074404 _1.057341 _1.781603

H _0.237961 _2.587687 _0.900518

H _0.219508 3.055412 0.151213

H 2.551797 1.639904 _0.883265

H 0.329747 _1.879095 1.567718

34b_C113

N _1.085936 0.773113 _0.718042

C _2.493688 0.584601 _0.222800

C _2.158947 _0.836929 0.283461

C _0.905151 _0.730195 _0.613292

C 0.458925 _1.287251 _0.193001

C 1.205884 _0.383280 0.811440

C 1.162767 1.079799 0.346293

C _0.280928 1.562678 0.231942

O 1.290528 _1.523110 _1.334578

O 0.628565 _0.413964 2.112034

O 1.873958 1.123046 _0.897660

H _2.816773 1.324940 0.517091

H _3.203801 0.589374 _1.058514

H _2.867341 _1.642888 0.067326

H _1.903382 _0.847461 1.345779

H _1.114114 _1.155392 _1.604725

H 0.296168 _2.271574 0.270927

H 2.255789 _0.714521 0.834570

H 1.672126 1.703207 1.097970

H _0.690716 1.545002 1.254567

H _0.289737 2.613499 _0.092020

H 1.573705 _0.637923 _1.630648

H 0.698850 _1.333752 2.446494

H 1.713200 1.991380 _1.325151

34b_D113

N _1.113924 0.692757 _0.727106

C _2.516540 0.493113 _0.218885

C _2.124614 _0.864452 0.408744

C _0.889025 _0.794763 _0.515401

C 0.500715 _1.287130 _0.087304

34b_D207

N _1.160943 _1.000229 0.042652

C _2.394826 _0.588377 _0.674320

C _2.420566 0.732580 0.134451

C _0.985581 0.382779 0.594892

C 0.152887 1.154628 _0.073541


84

C 1.272511 _0.279545 0.785729

C 1.127659 1.143414 0.207677

C _0.339845 1.544428 0.195026

O 1.297148 _1.625690 _1.228614

O 0.763994 _0.350524 2.113017

O 1.720641 1.145396 _1.105277

H _2.871877 1.284604 0.449251

H _3.218305 0.392841 _1.055078

H _2.806319 _1.710442 0.278044

H _1.856321 _0.770800 1.464243

H _1.107308 _1.293401 _1.469285

H 0.371206 _2.214891 0.486559

H 2.334368 _0.569556 0.754649

H 1.682419 1.848873 0.846907

H _0.689133 1.485222 1.237513

H _0.448843 2.589512 _0.123962

H 1.600898 _0.772569 _1.590620

H 1.283158 0.270702 2.669677

H 1.084101 1.526766 _1.744688

C 1.471629 0.363263 0.017920

C 1.248397 _1.179845 0.007812

C _0.038590 _1.532902 _0.734946

O 0.260971 2.481456 0.431276

O 2.265922 0.776807 _1.091954

O 1.234596 _1.704718 1.337551

H _2.232177 _0.432501 _1.751531

H _3.242699 _1.271834 _0.547709

H _3.150950 0.737396 0.947005

H _2.518729 1.655501 _0.442141

H _0.823413 0.387822 1.684738

H _0.071907 1.272339 _1.143035

H 1.965929 0.605812 0.973164

H 2.109676 _1.627931 _0.507702

H 0.016150 _1.110869 _1.753860

H _0.140824 _2.620971 _0.835508

H 0.452325 2.416516 1.392423

H 3.165783 0.406182 _0.970013

H 0.305429 _1.836975 1.607012

1.3.3 Computational Method

The conformations of 34a and 34b possessing different intramolecular hydrogen bonded

network were optimized at the Hartree-Fock (HF/3-21G) level of theory using the

Gaussian03 package.99 The minimum energy conformers for compound 34a possessing

unique hydrogen bonding pattern were labeled with notations A, B, C, and D (Figure

37). Other ring conformations viz. chair, twist chair, half chair, twist boat, and boat

forms, were generated by scanning the C5-C6-N1-C2 and C4-C5-C6-N1 dihedral angle

from –80.0° to 80.0°. Minima on the scanned potential energy surface of both the

dihedral angles were identified and further optimized at B3LYP/6-31G(d,p) level of

theory by relaxing all the geometrical constraints.100 Relative stabilization energies

(ΔERel) were calculated by subtracting the energy of lowest energy conformer from those

of rest of the conformers. Along with the conformers having different ΔERel only one


85

conformer those possessing equal ΔERel values and same geometries are considered for

the calculations hereafter. Self consistent reaction field theory was utilized to investigate

the influence of solvent on the conformational energies of 34a and 34b. The gas phase

structures were optimized in water employing the polarizable continuum model

(PCM)101 for this purpose. This is termed as the geometry relaxation in the presence of

solvent. NMR chemical shifts were calculated from the gauge invariant atomic orbital

method by subtracting the nuclear magnetic shielding tensors of protons in conformers of

34a and 34b from that of the protons in TMS.102 DFT investigations revealed that the

calculated 1H NMR chemical shifts in solvent using the SCRF-PCM model agree better

with the experimental ones than those from the gas phase.103 In case of glucose, it has

been pointed out that the accuracy of 1H chemical shifts is affected significantly by the

solute geometry and the SCRF models suffice to describe the effect of solvent on the

shielding constants. Boltzmann contributions (BC) of these conformers were estimated

from the relative stabilization energy values in the gas phase and water. The calculated

BC values provide the valuable information regarding relative population of different

conformers.


86

CHAPTER 1


Section D: Glycosidase inhibitory activity of conidine iminosugars

1.4.1 -Glycosidase Inhibition

The glycosidase inhibitory activity was studied with different glycosidases.104 As

shown in Table 9, glycosidase inhibition studies of the target compounds revealed that

compound 34a showed no inhibition with -galactosidase (E.C. 3.2.1.22) and moderate

inhibition with both -mannosidase (E.C. 3.2.1.24) and -glucosidase (E.C. 3.2.1.20)

while 34b showed selective inhibition against -mannosidase and moderate inhibition

against -galactosidase and -glucosidase in millimolar range. As a reference, porcine

pancreatic -amylase with 0.21 Umin-1 was taken as 100% enzymatic activity. The

corresponding IC50 values of 34a and 34b for these enzymes are given in Table 10.

Table 10: Percentage inhibition of 34a and 34b on various glycosidases.

Compounds -Glycosidase inhibitory activity (%)

-Galactosidase -Mannosidase -Glucosidase

34a NI 4.65 ± 0.12 3.22 ± 0.22

34b 25 ± 0.03 44.62 ± 0.09* 5.55 ± 0.06

Table 11: IC50 values of 34a and 34b in mM

Compounds -Galactosidase -Mannosidase -Glucosidase -Amylase

34a NI 322.6 ± 0.12 465.8 ± 0.22 110.6 ± 0.03

34b 60 ± 0.03 33.6 ± 0.09* 270.3 ± 0.06 126.5 ± 0.02

NI = No Inhibition under assay conditions, The data is indicated as the mean ± SEM;

(n=3). (*) denoting more significant value (P<0.05),


87

1.4.2 Molecular Docking

Biological activity was supported by computational study which involved molecular

docking in order to understand the interactions between compound 34b and amino acid

residues of mannosidase. Mannosidases are lectin binding proteins and N-glycans of -

mannosidase have unique topologies, important functions in protein folding,

oligomerization or enzyme activity.105 The complete sequence of jack bean mannosidase

is yet to be reported. Therefore, it is difficult to compare the active sites of the

mannosidase from jack bean and Endoplasmic Reticulum (ER). Hence, for this study, we

have chosen human mannosidase (PDB: 1FO3) as our target structure to perform the

molecular docking studies. The template structure of human mannosidase is used to dock

the ligand 34b into the binding pocket of mannosidase. This might provide an insight

into the interaction of 34b with mannosidase from Jack Bean. The active site was

predicted by using WHAT IF software. The AUTODOCK 3.0106 program was used to

perform an automatic docking exploration for different conformations of the ligand in

the model. AUTODOCK 3.0 uses three search methods [a genetic algorithm, a local

search method, and an adaptive global– local search method based on Lamarckian

genetics (LGA)] in conjunction with an empirical force field that allows the prediction of

binding free energies for docked ligand. The optimized autodocking run parameters are

as follows: the maximum number of energy evaluations was increased to 2,500,000 per

run; the maximum number of generation in the genetic algorithm was increased to

1,00,000; and the number of GA run was 100. The binding pocket consists mainly of

glutamate residues that make the site very active. Distances between polar atoms less

than 3.6 Å may be regarded as hydrogen bonds and interactions less than 4.5 Å may be

taken as Van der Waals interactions. Such interactions were observed between 34b and

Ser158, Thr446, Lys229, Ser222, Glu225 (Figure 39). It shows that 34b may have

significant interaction with mannosidase and may act as an inhibitor. The total binding


88

energies are given in Table 10. The total energy of the complex is 133.5 kcal/mol with

the total electrostatic energy being −27.07 kcal/mol and total Van der Waals interaction

energy −106.44 kcal/mol.

Table 12: The total energy (Etotal), Van der Waals energy (Evdw) and electrostatic energy (Eele) between

interacting residues of compound 34b and target enzyme.

Residue Etotal (kcal/mol) Evdw kcal/mol) Eelec (kcal/mol)

Ser158 −50.101 −1.45 −48.65

Thr446 25.131 −12.27 24.06

Lys229 254.120 −15.71 257.61

Ser222 92.476 −6.65 82.41

Glu225 256.171 −20.30 262.41

Figure 39: Binding of 34b to mannosidase active pocket. A docking grid of 60 Å (0.375 spacing) was

computed around the binding site of apo-mannosidase (PDB: 1FO3). 34b placed in different locations

outside the grid consistently docked well into the binding site.


89

1.4.3 -Amylase Inhibition

Compounds 34a and 34b were tested for -amylase inhibition studies. As a reference,

porcine pancreatic -amylase (E.C. 3.2.1.1)107 with 0.21 Umin-1 was taken as 100%

enzymatic activity. As shown in Figure 40, compound 34a inhibited activity of porcine

pancreatic amylase up to 13.56 % at a concentration of 150 mM while compound 34b

showed inhibition up to 11.86 % at the same concentration of 150 mM as compared to

acarbose.

Figure 40: The percent relative enzyme activity after inhibition with 34a and 34b on porcine pancreatic -

amylase. Acarbose is taken as standard -amylase inhibitor. Pure porcine pancreatic -amylase serves as

control.

1.4.4 Murine Pancreatic, Liver and Intestinal Glucosidase Inhibition

In order to check the inhibitory effects of 34a and 34b on the crude murine glucosidases

pancreatic tissue weighing 1.0 g was used as a source of enzyme.108 Pancreatic enzyme

activity exhibiting 4U/ml was taken as 100% enzymatic activity. 1 U/ml was taken as

100% enzyme activity for liver and intestinal glucosidase. Murine pancreatic tissue

weighing 1 g was used as the source of the enzyme. 34a showed 13% inhibition while

34b showed no inhibition with the enzyme. No significant inhibitory activity of the

34a 34b


90

glucosidase from the murine small intestine as well as liver was observed with either of

the synthesized compounds.

1.4.5 Experimental Section

Chemicals

-Galactosidase from Escherichia coli, -glucosidase from Bacillus stearothermophilus

-Mannosidase from Canavalia ensiformis, 4-nitrophenyl--D-galactopyranoside, 4-

nitrophenyl--D-mannopyranoside and 4-nitrophenyl--D-glucopyranoside were

purchased from Sigma Aldrich. DNSA (dinitrosalicylic acid), porcine pancreatic -

amylase and chloroform were obtained from SRL Pvt. Ltd (Mumbai, India). Di-

potassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4),

methanol, sodium potassium tartarate, sodium hydroxide (NaOH), were obtained from

Merck Chemicals Ltd (Mumbai, India). Sodium chloride (NaCl) was obtained from

HiMedia Laboratories Mumbai, India). Acarbose was obtained from Bayer

Pharmaceuticals Pvt. Ltd. (Mumbai, India). All the chemicals and reagents procured

were of analytical reagent grade.

General procedure for glycosidase inhibition assay

Glycosidase inhibition assay of conidine iminosugars 34a and 34b was carried out by

mixing 0.1 U/ml each of -galactosidase, -mannosidase and -glucosidase with the

samples and incubated for 1 hour at 37˚C. Enzyme action for -galactosidase was

initiated by addition of 10 mM p-nitrophenyl--D-galactopyranoside (pNPG) as a

substrate in 200 mM sodium acetate buffer. The reaction was incubated at 37˚C for 10

min and stopped by adding 2 mL of 200 mM borate buffer of pH 9.8. -Mannosidase

activity was initiated by addition of 10 mM p-nitrophenyl--D-mannopyranoside as a

substrate in 100 mM citrate buffer of pH 4.5. The reaction was incubated at 37˚C for 10

min and stopped by adding 2 mL of 200 mM borate buffer of pH 9.8. Initiation of -


91

glucosidase activity was done by addition of 10 mM p-nitrophenyl--D-glucopyranoside

in 100 mM phosphate buffer of pH 6.8 and stopped by adding 2 mL of 0.1M Na2CO3

after an incubation of 10 minutes at 37˚C. -Glycosidase activity was determined by

measuring absorbance of the p-nitrophenol released from pNPG at 420 nm using

Shimadzu Spectrophotometer UV-1601. One unit of glycosidase activity is defined as

the amount of enzyme that hydrolyzed 1 µM of p-nitrophenyl pyranoside per minute

under assay condition.

General procedure for -amylase inhibition assay

Amylase activity was assayed using a modified Bernfeld method (1955) using starch as

substrate. 50 µg ml-1 (O. D. adjusted to 0.4 at 280nm) of porcine pancreatic -amylase

was incubated with 20 mg ml-1 samples at 37˚C for 10 minutes. One percent starch was

used as substrate. The samples without -amylase were used as controls and the test

reading were subtracted from the absorbance of these controls. The reducing sugar was

estimated using DNSA assay at 540 nm and the enzyme units were expressed as micro-

molar per minute. One unit of enzyme was defined as the amount of enzyme required to

liberate 1 µM of maltose under assay conditions. The final inhibition shown by different

samples were compared with the standard inhibitor, acarbose.

General procedure for glucosidase inhibition assay with murine pancreatic, liver and

small intestinal extracts:

The 10-week-old Swiss male mice weighing 20 gm were procured from National

Toxicology Centre. The entire procedure was carried out with guidelines of Institutional

Animal Ethical Committee. The mouse was starved for 12 h. Pancreas; liver and small

intestine tissues were excised and homogenized with 10 mM ice cold phosphate buffer

containing 6 mM NaCl (1:10 w/v) and appropriate amount of phorbol-12-myristate-13-

acetate as protease inhibitors. The homogenate was centrifuged for 10 min at 10,000


92

r.p.m. and the supernatant was taken as a source of the enzyme. Supernatant obtained

from pancreas, liver and small intestine tissues were taken as a source of enzyme and

were diluted so as to get an absorbance of 0.4 (at 280 nm). The enzyme inhibition assay

was carried out as described above. The percentage inhibition of the samples against

pancreatic, small intestinal and liver glucosidases was calculated.

Statistical Analysis:

The statistical analysis was performed using one way analysis of variance (ANOVA) and

two tailed t-test (P<0.05). Results are expressed as means ± SEM.


93

1.4.6 Conclusion

We have synthesized the new class of iminosugars viz. conidine iminosugars 34a and

34b with an overall yield of 41% and 37%, respectively, from D-glucose. Reduction of

sugar derived β-lactam to azetidine was achieved which is less common in sugar moiety

due to acid labile 1,2-acetonide protection. Hydrogenolysis was achieved without

destruction of constrained azetidine ring which is otherwise already reported in the

literature. The experimental and DFT calculated 1H coupling constant data suggests that

the compound 34a was assigned the half chair conformation whereas compound 34b as

twist boat conformation. Compound 34b was found to have inhibitory activity against -

mannosidase at mM range whereas compound 34a was found to be moderate inhibitor of

-mannosidase and -glucosidase at the same concentration. Molecular docking study

could successfully correlate interaction energies calculated from the docked poses of the

ligands with experimental binding affinities which offer a possible structural rationale for

the inhibition mechanism of the conidine iminosugars, 34a and 34b. It is noteworthy that

34a and 34b in spite of being diastereomers of the same conidine iminosugar showed the

differential inhibition against -mannosidase that supports that stereochemistry of ring

fusion proton in conidine iminosugars is significant in glycosidase inhibition.


94

1.4.7 References and Notes:

1 (a) Fellows, L. E. Pestic. ScL, 1986, 17, 602; (b) Fellows, L. E.; Evans, S. V.;

Nash, R. J.; Bell, E. A. ACS Symp. Ser. 1986, 296, 72; (c) Fellows, L. E.; Kite, G.

C.; Nash, R. J.; Simmonds, M. S. J.; Scofield A. M. in Plant Nitrogen

Metabolism (Eds: J. E. Poulton, J. T. Romeo, E. E. Conn), 1989, Plenum Press,

New York, p. 395-427; (d) Simmonds, M. S. J.; Fellows, L. E.; Blaney W. M. in

New Crops for Food and Industry (Eds.: G. Wickens, N. Haq, P. Day), 1989,

Groom Helm, England, 365-377.

2 (a) Elbein, A. D.; Molyneux, R. J. in Alkaloids: Chem. Biol Perspect. (Ed.: S. W.

Pelletier), Elsevier, Oxford, 1987, 5, 1-56; (b) Nash, R. J.; Watson, A. A.; Asano,

N. in Alkaloids: Chem. Biol. Perspect. II (Ed.: S.W. Pelletier), Elsevier, Oxford,

1996, 345-376.

3 Manning, K. S.; Lynn, D. G.; Shabanowitz, J.; Fellows, L. E.; Singh, M.; Schrire,

B. D. J. Chem. Soc. Chem. Comm. 1985, 127.

4 Fleet, G. W. J. in Topics in Medicinal Chemistry (Ed.: P. R. Leeming),

Cambridge. University Press, Cambridge, 1987, 149-161.

5 Fleet, G. W. J.; Fellows, L. E.; Winchester B. in Bioactive Compounds from

Plants (Eds.: D. J. Chadwick, J. Marsh), John Wiley Sons, Chichester, 1990, p.

112-125.

6 (a) Fellows, L. E.; Nash, R. J. ScL Procress Oxford, 1990, 245-261; (b) Stütz, A.

E. (ed.), 1999 Iminosugars as Glycosidase Inhibitors: Nojirimycin and Beyond

Wiley-VCH, New York; for an historical background, see Chapter 1 by Paulsen,

H., The early days of monosaccharides containing nitrogen in the ring.

7 (a) Iminosugars: From Synthesis to Therapeutic Applications; Compain, P.,

Martin, O. R. Eds.; John Wiley: Chichester, UK, 2007; (b) Paulsen, H. Angew.

Chem., Int. Ed. Engl. 1966, 5, 495.

8 Aerts, J. M. F. G. 2006 Proc. European Working Group for the Study of Gaucher

Disease; http://www.ewggd.co.uk/

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