理學博士學位 請求論文

생체활성물질 개발 및 방향족 친전자성

첨가반응에 관한 고찰

The Development of Biologically Active Compounds

and Electrophilic Aromatic Addition Reaction

2005년 2월

指導敎授 池 大 潤

이 논문을 박사학위 논문으로 제출함

Inha University

Chemistry (Organic Chemistry)

Ekaruth Srisook

理學博士學位 請求論文

생체활성물질 개발 및 방향족 친전자성

첨가반응에 관한 고찰

The Development of Biologically Active Compounds

and Electrophilic Aromatic Addition Reaction

2005년 2월

Inha University

Chemistry (Organic Chemistry)

Ekaruth Srisook

Contents

• Abstract (English) ….. 6

• Abstract (Korean) ….. 8 • Part 1. The Syntheses of 3-Substituted 4-(Pyridin-2-ylthio)indoles

via Leimgruber-Batcho Indole Synthesis ….. 10

Introduction …...11

Results and discussion ….. 22

Conclusions ….. 27

Experimental section ….. 28

References ….. 35

NMR spectra ….. 39

• Part 2. Structural Modification of Nitric Oxide Inhibitors ….. 50

Introduction ….. 51

Chemistry ….. 56

Biological results and discussion ….. 59

Conclusions ….. 62

Experimental section ….. 63

References ….. 70

NMR spectra ….. 74

• Part 3. Electrophilic Aromatic Addition Reaction: AdEAr ….. 84

Introduction ….. 85

Results and discussion ….. 91

1. Synthesis of precursors ….. 91

2. AdEAr reaction of 1-methoxynaphalene ….. 93 3. Reaction of 8-methoxyquinaldine under various conditions

….. 95

4. AdEAr reaction of some selected compounds ….. 97 5. Mechanistic studies ….. 99 6. Reaction and application of addition product …..102

Conclusions …..104

Experimental section …..105

References …..113

NMR spectra …..115

Acknowledgement …..137

6

Abstract

Development of Biologically Active Compounds and Electrophilic

Aromatic Addition Reaction

Compounds which are strongly binding to serotonin transporter or nitric

oxide synthases (NOS) would be new radiopharmaceuticals as serotonin

selective reuptake inhibitors (SSRIs) or NOS inhibitors, respectively.

Based on the structural diversities of selective serotonin reuptake

inhibitors recently published, new family of ligands, 3-(amino- and

hydroxymethyl)-4-(5-iodopyridin-2-ylthio)indoles have been designed for

SSRI. These target compounds have been designed by a combination of

characteristically distinct moieties proven to impart successful binding

ability. The syntheses of 3-substituted 4-(5-iodopyridin-2-ylthio)indoles

are described. Key intermediate 1-(5-iodopyridin-2-ylthio)-2-methyl-3-

nitrobenzene (6) was achieved by nucleophilic aromatic substitution of

chloropyridine 7 with thiophenol 8. A modified Leimgruber-Batcho indole

synthesis from 1-(5-iodopyridin-2-ylthio)-2-methyl-3-nitrobenzene was

used as a key step of this synthetic route. Unfortunately, these two

compounds as well as other derivatives showed low binding affinities

toward serotonin transporter. Nitric oxide (NO) or nitrogen monoxide, a

small free radical, is generated by nitric oxide synthases (NOS). The

overproductions of NO from iNOS, the inducible isoform of NOS, have

been implicated in the pathophysiology of ischemic neuronal death and

NOS inhibitors protected neurons in these animal models. A study of the

regulation of NO overproductions is important because it prevents cell

damage. It was reported that treatment with N-acetyl-O-methyldopamine

(NAMDA), a metabolite of dopamine in CNS, significantly protected CA1

7

neurons in rat ischemic hippocampus and inhibited LPS-induced NO

production in BV-2 microglia cells. In this work, NAMDA was used as a

lead compound for NO inhibitor and the structure modification was made

by elongation, cyclization and replacing of the acetamide group with other

function groups. Ten compounds were synthesized and their biological

effects were evaluated on NO production and cytotoxicity. Among all

compounds, four compounds showed some improvements of inhibitory

activity without increasing cytotoxicity. Compound 15 exhibited strikingly

as the most potent NO reducing agent and more significantly potent than

the lead compound, NAMDA.

Recently, it was found that the bromination of 8-

methoxyquinaldine under basic condition gave an unusual addition

product, the 5,7-dibromo-8,8-dimethoxy-7,8-dihydroquinaldine as the

major product via a new type reaction – electrophilic aromatic addition

reaction: AdEAr. Upon first extension of this methodology to 1-

methoxynaphthalene, the addition adduct was successfully obtained when

1 equivalent of pyridine was added. This is the first reported isolation of

an addition adduct during the electrophilic aromatic substitution of non-

heterocyclic aromatic compounds. The importance of condition reaction

was investigated on effect of bases and bromine sources. The reaction was

also applied on various aromatic substrates, which yielded addition

products as major products. Lastly, the study of mechanism indicated that

the AdEAr reaction was occurred in anti-addition manner via cation

intermediate. This result not only allows for the functionalization of

aromatic compounds via the addition adducts, but also introduces the

possibility of an alternate mechanism for electrophilic substitution

reactions.

8

요 약 문

생체활성물질 개발 및 아로마틱 친전자성 첨가반응에 관한

고찰

세로토닌 운반체나 nitric oxide synthases 에 선택적으로 강하게 결합을 하는 화합물은 선택적 세로토닌재흡수 저해제(SSRI) 역할을 하는 새로운 방사성의약품으로 혹은 NOS 저해제가 될 수 있다. 최근 발표된 선택적 세로토닌재흡수 저해제들의 다양한 구조를 바탕으로 3-(amino- and hydroxymethyl)-4-(5-iodopyridin-2-ylthio)indole 화합물들을 디자인 하였다. 이들은 세로토닌재흡수단계에서 좋은 결합을 할 것으로 예상되는 구조들을 합쳐서 디자인을 한 것이다. C3 에 치환된 4-(5-iodopyridin-2-ylthio)indoles 의 합성에 대해 기술을 하였으며, 또한 주요 중간체인 1-(5-iodopyridin-2-ylthio)-2-methyl-3-nitrobenzene (6)의 합성은 chloropyridine 7 에 thiophenol 8 로 아로마틱 친핵성치환반응에 의해서 이루어졌다. 1-(5-Iodopyridin-2-ylthio)-2-methyl-3-nitrobenzene 으로부터 개선된 Leimgruber-Batcho 인돌 합성법을 사용하여 인돌을 합성하였는데 이 과정이 목표화합물 합성 경로에서 가장 주요 반응과정이다. 불행하게도 목표화합물들과 그 유도체들은 세로토닌 운반체와 in vitro 실험에서 낮은 결합력을 가짐을 알았다. Nitric oxide (NO) 혹은 nitrogen monoxide 는 nitric oxide synthases (NOS)에 의해서 생성되는 작은 free radical 분자이다. 동물모델을 이용한 실험에서 NOS 중의 하나인 iNOS 로부터 NO 가 과생산이 될 수 있는데 이는 이스키믹상태로 인한 신경손상에 따른 생리학적 변화로 나타나며, 이는 iNOS 를 저해하므로 막을 수가 있다. NO 의 과생산을 조절하는 연구는 세포의 손상을 막을 수가 있기 때문에 매우 중요한 연구이다. 도파민의 대사물질중의 하나인 N-acetyl-O-methyldopamine (NAMDA)은 히포캠프스가 이스키믹 상태인 쥐의 CA1 neurons 의 손상을 막아주는 연구가 발표되었으며 또한 BV-2 microglia 세포에서 LPS 로 유도된 NO 의 과생산을 저해한다고 알려졌다. 이 논문에서 NAMDA 를 NO 의 생산을 저해할 수 있는 선도화합물로 사용하여, 탄소의 길이조정, 고리화 등 구조를 변화한

9

유도체를 합성하였다. 열개의 유도체를 합성하여 NO 의 생성 및 세포독성과 같은 생체활성조사를 하였다. 유도체중 네개는 세포독성의 증가없이 NO 의 생성 저해효과가 나타났으며, 그중 화합물 15 는 선도화합물 NAMDA 에 비해 가장 좋은 저해효과를 보여주고 있다. 최근 염기조건에서 8-methoxyquinaldine 을 브롬화시킬 때 치환반응대신에 첨가반응이 진행된 5,7-dibromo-8,8-dimethoxy-7,8-dihydroquinaldine 을 주 생성물로 분리할 수 있었으면 이는 새로운 형태의 반응 즉 아로마틱 친전자성 첨가반응 AdEAr 을 본 연구실에서 찾았다. 1-Methoxynaphthalene 에 이를 시도하였으며 1 당량의 피리딘을 넣어서 성공적으로 첨가반응이 진행된 생성물을 얻을 수 있었다. 이것은 헤테로원소가 없는 아로마틱 화합물에 친전자성 첨가 반응이 진행된 생성물에 대한 처음 보고이다. 이 반응에 대한 자세한 연구와 여러 가지 유도체에 대한 일반화 시키는 것에 대한 연구를 진행하였다. 그리고 반응메카니즘에 대한 연구로부터 anti-addition 에 의해 반응이 진행됨을 알았다. 첨가생성물로부터 다양한 유도체를 합성할 수가 있으며, 또한 친전자성 치환반응의 새로운 메커니즘을 제시할 가능성을 언급하였다.

1

PART 1

The Syntheses of 3-Substituted 4-(Pyridin-2-ylthio)indoles

via Leimgruber-Batcho Indole Synthesis

2

Introduction

Serotonin or 5-hydroxytryptamine is the baby-boomer of

neurotransmitters. Serotonin has been associated with, among other things,

anxiety, depression, schizophrenia, drug abuse, sleep, dreaming,

hallucinogenic activity, headache, cardiovascular disorders, appetite

control, and is now dabbling in acupuncture and transcendental

meditation.1 A review of the recent patent literature provides an indication

of some of the newer claim being made for novel serotonergic agents.

Tens of thousands of papers have been published on serotonin; much is

known –but an incredible amount remains to be learned.

NH

NH2

HO

Serotonin (5-HT)

Serotonin biosynthesis, catabolism and function as targets for drug

manipulation2

5-HT is biosynthesized from its dietary precursor L-tryptophan

(Scheme 1). Serotonergic neurons contain tryptophan hydroxylase (L-

trytophan-5-monooxygenase) that converts tryptophan to 5-

hydroxytryptophan (5-HTP) in what is the rate-limiting step in 5-HT

biosynthesis, and aromatic L-amino acid decarboxylase (previously called

5-HTP decarboxylase) that decarboxylates 5-HTP to 5-HT. This latter

enzyme is also responsible for the conversion of L-DOPA to dopamine.

3

The major route of metabolism for 5-HT is oxidative deamination by

monoamine oxidase (MAO-A) to the unstable 5-hydroxylindole-3-

acetaldehyde which is either reduced to 5-hydroxytryptophol (~15%) or

oxidized to 5-hydroxyindole-3-acetic acid (~85%). In the pineal gland, 5-

HT is acetylated by 5-HT N-acetyltransferase to N-acetylserotonin, which

undergoes O-methylation by 5-hydroxyindole-O-methyltransferase to

melatonin.

NH

NH2

HO

MAO

NH

HN

HO

Ac

NH

NH2HOOC

HO

NH

OHC

HO

NH

OH

HO

NH

HOOC

HO

NH

HN

H3CO

Ac

NH

NH2HOOC

+

Tryptophan 5-Hydroxytryptophan(5-HTP)

Serotonin(5-HT)

Melatonin N-acetyl-5-HT

5-Hydroxyindole-3-acetaldehyde

5-Hydroxyindole-3-acetic acid

5-Hydroxytryptophol

Tryptophan

Hydroxylase

Aromatic

amino aciddecarboxylase

5-HT N-acetyl-transferase

5-Hydroxyindole-

O-methyltransferase

Scheme 1. Biosynthesis and catabolism of serotonin.

4

Each of the steps in 5-HT biosynthesis, metabolism, and function

is a theoretical target for drug manipulation. Tryptophan depletion, by

reducing or eliminating dietary tryptophan, can result in decreased 5-HT

biosynthesis. Conversely, tryptophan “loading,” by increasing dietary

tryptophan, can result in the overproduction of 5-HT. This latter effect can

also occur in non-serotonergic neurons, such as dopaminergic neurons,

because of the non-selective nature of aromatic amino acid decarboxylase.

Inhibitors of trytophan hydroxylase such as para-chlorophenylalanine are

used as pharmacologic tools and are not used therapeutically.

Serotonin reuptake transporter

Transporter proteins are specific to their respective transmitter. In

the case of serotonin, a transporter protein found in the plasma membrane

of serotonergic neurons is responsible for re-uptake of the transmitter. The

transporter protein acts as a carrier of serotonin molecules across the

membrane. Unlike channels, which stay open or closed, transporters

undergo conformational changes (changes in their three dimensional

shape) and move one molecule in each cycle.

The serotonin transporter (SERT) is similar to other biogenic

amine transporters (e.g. norepinephrine and dopamine transporters) and is

part of a family of sodium (Na+) and chloride (Cl-) dependent transporters.

SERTs, with molecular weights of 60-80 kDA, have twelve

transmembrane domains (TM) with a large extracellular loop between

TMs 3 and 4. Both the N- and C-termini are located within the cytoplasm

(Figure 1). The large extracellular loop and the intracellular parts of the N-

5

and C-termini do not appear to be the significant sites that determine

interactions with 5-HT or transporter inhibitors. Rather, the areas

important for selective 5-HT affinity appear to be localized within TMs 1-

3 and TMs 8-12. It is believed that SERTs have a common binding site for

5-HT and many of its inhibitors. SERT activity, like serotonin, is seen

most often in the raphe nuclear complex. SERTs have also been seen in

the amygdale, thalamus, hypothalamus, substantial nigra, and locus

coeruleus. In addition to being found on neurons, SERTs are seen in the

placenta, lungs, and blood platelets. Blood platelets utilize SERTs to

obtain serotonin from the environment because they cannot synthesize it

themselves. In the placenta, SERTs may protect heavily vascularized

embryonic tissue from constricting too early due to maternal serotonin.

Figure 1. Structure of serotonin reuptake transporter

Serotonin reuptake transporters (SERTs) are dependent on

extracellular Na+ and extracellular Cl-. Unlike Na+, Cl- can be at least

partly substituted for by NO2-, Br-, and other anions. Intracellular

6

potassium (K+) is also used in the process but can be replaced by other

ions, most notably hydrogen (H+). The driving force for the energetically

unfavorable transport of serotonin is the Na+ influx down its concentration

gradient. The Na+/K+ pump (Na+/K+ ATPase) maintains the extracellular

Na+ concentration as well as the intracellular K+ concentration. Na+/K+

ATPase pumps three Na+ ions our for each two K+ ions pumped into the

cell. The electrical potential produced, in addition to creating the Na+

concentration used by the transporter protein, also leads to the loss of Cl-

ions from the cell, which is also used in transport.

According to the present model of SERT function, the first step

occurs when Na+ binds to the carrier protein. Serotonin, in its protonated

form (5-HT+), then binds to the transporter followed by Cl-. Chloride ions

are not required for 5-HT+ binding to occur but are necessary for net

transport to take place. The initial complex of serotonin, Na+, and Cl-

creates a conformational change in the transporter protein. The protein,

which began by facing the outside of the neuron, moves to an inward

position where the neurotransmitter and ions are released into the

cytoplasm of the neuron. Intracellular K+ then binds to the SERT to

promote reorientation of the carrier for another transport cycle. The

unoccupied binding site becomes, once again, exposed to the outside of

the cell and the K+ is released outside the cell.

Serotonin reuptake inhibitor

The reuptake process is susceptible to drug manipulation. By

blocking the action of serotonin reuptake transporter, the amount of

serotonin in the synaptic cleft increases. Selective serotonin reuptake

7

inhibitors (SSRIs) act primarily at the 5-HT transporter protein and have

limited, if any, reaction with other neurotransmitter systems. SSRIs bind

to the transporter protein directly and block the reuptake process.

Consequently, more serotonin remains in the cleft where it is free to travel

further to more distant receptors as well as continue to react with nearby

receptors. Like the binding of substrates, antagonist binding to SERTs is

also dependent on extracellular Na+ although ion dependency is different

for each SERT antagonist. It is unclear whether SSRIs bind to the same

SERT domain as serotonin or operate through more indirect mechanisms.

Recent evidence suggests that binding of SSRIs to SERTs occurs at the

same site as 5-HT binding, but it has not been determined conclusively.

In the late-1980s, the serotonin-selective reuptake inhibitor (SSRI)

fluoxetine became the mainstay of treatment for clinical depression-

replacing the more toxic tricyclic antidepressants (TCAs).3 SSRIs have a

more favorable adverse reaction profile in comparison to the TCAs and

are much easier to tolerate. SSRIs are the focus of extensive research into

finding beneficial pharmacological therapies. There is evidence that

serotonergic pathways are the most closely related systems to mood

disorders, especially depression, and thus SSRIs may lead to significant

therapy.4 Clinical depression is one of the most common psychiatric

disorders, with an incidence of about 4% and a life-time prevalence of 15-

20%. Despite significant research, the neurobiological dysfunctions of

major depression remain elusive. Findings implicate multiple

abnormalities in serotonergic pathways in the cause of depression.

Findings include 1) low concentrations of the major serotonin metabolite;

2) a low density of brain and platelet serotonin transporters in depressed

individuals; 3) a high density of brain and platelet serotonin binding sites;

8

and 4) a low concentration of tryptophan, which is used in serotonin

synthesis. Of these, the low level of SERTs in depressed patients has

received the most attention. Still, while the precise cause of depression

eludes neuroscientists, SSRIs have been shown to alleviate the mood

disorder and are a common therapy for depression. There are many SSRIs

either in the market or in development.5 SSRIs currently available include

citalopram, fluoxetine (Prozac), fluvoxamine, paroxetine (Paxil), and

sertraline (Zoloft). In addition, while not as selective as the above-

mentioned, drugs of abuse such as cocaine, fenfluramine, and (3,4-

methylenedioxy) methamphetamine (MDMA or ecstasy) are inhibitors of

serotonin uptake.

SSRI imaging

Imaging of SERT in humans would provide a useful tool to

understand how alterations of this system are related to depressive illness

and other psychiatric disorders; therefore, it potentially can benefit

millions of patients who are being treated with SSRIs. The first successful

radioligand was [11C](+)McN5652 (1) for positron emission tomography

(PET) imaging (Chart 1).6,7 It showed excellent inhibition of 5-HT

reuptake in rat brain synaptosomes (Ki = 0.40 nM for inhibition of SERT)

and moderate selectivity toward other monoamine transporters (DAT,

dopamine, and NET, norepinephrine transporters; Ki 23.5 and 1.82 nM,

respectively).8 Specific binding of [11C](+)McN5652 correlates well with

the known density of SERT sites in the human brain.6,9,10 Recent reports,

using [11C](+)McN5652 for imaging SERT as an indicator of serotonin

neurons, have suggested that MDMA (methylenedioxymethamphetamine,

“ecstasy”) may cause an irreversible decrease of SERT binding sites.11

9

(Despite its successful demonstration in imaging SERTs in humans, it has

been reported previously that [11C](+)McN5652 has several limitations.10)

The uptake in the specific binding area is slow requiring at least 120 min

of data acquisition. The nonspecific binding is relatively high, which

precludes the measurements of lower SERT density regions. The plasma

free fraction is very low (

10

SERT over NET and DAT (Ki ) 699 and 840 nM, for NET and DAT,

respectively). A preliminary imaging study of [123I]- ADAM in the brain

of a baboon by SPECT at 180-240 min post iv injection indicated a

specific uptake in the midbrain region rich in SERT. It is apparent that

[123I]- ADAM showed a significant improvement over [123I]- IDAM as a

SPECT imaging agent for SERT in the brain.17,20 Initial imaging studies in

humans suggest that the agent clearly localized in the region of

hypothalamus region of the brain where the concentration of SERT is the

highest.

N

O2NI

NNH

I

S

N

NH2

I

S

N

OH

N S

I

NH

R

N

SCH3

1, (+)McN5652 2, 5-iodoquipazine 3, ADAM

4, IDAM 5 Chart 1

Although these radioligands all serve as potent selective ligands of

SERT, they bear significant structural differences from one another. In

light of their combined success, and in an effort to optimize structural

contributions to SERT binding,21 we have designed a new type of

11

radioligand, 4-(pyridin-2-ylthio)indole 5 (Chart 1), as a combination of the

various structural moieties characteristic of these potent inhibitors by

computer designing from 3D structure of SERT binding site. The designed

target compound 5 is synthetically challenging due to the lack of available

methodology for the formation of heterodiaryl sulfides, specifically 4-

thioindoles. After attempt of various synthetic routes, the Leimgruber-

Batcho indole synthesis22 was finally selected as the key step in converting

precursor 6 to indole 5 because of its mild reaction conditions and

regioselectivity for 4-substituted indoles (see Figure 2 for retrosynthetic

analysis). The synthesis of key intermediate 6 occurs by nucleophilic

aromatic substitution of chloropyridine 7 with thiophenol 8. The synthesis

of 2-chloro-5-iodopyridine (7) by iodination23 and Sandmeyer reaction24

was reported whereas thiophenol 8 is a new compound. Although it is

possible to directly convert aniline derivative to thiolphenol by

Sandmeyer-type reaction, violently explosive diazo sulfides and related

compounds may be formed, and another less hazardous method for the

preparation of desired compound should be used, if possible.25 Therefore,

commercially available 2-methyl-3-nitroaniline would be diazotized to

phenol derivative and then finally converted to thiolphenol 8 by Newman

and Karnes’ method.26 Herein, we wish to report the synthesis of 4-

(pyridin-2-ylthio)indole analogues in detail.

12

N S

I

NH

R

5

N S

I

NO2

N Cl

I

NO2

SH

+

6

7 8

N NH2NO2

NH2

Figure 2. The retrosynthesis of 3-substituted 4-(5-iodopyridin-2-ylthio)indoles.

13

Results and Discussion

2-Chloro-5-iodopyridine (7) (Scheme 2) was prepared as previously

reported by conversion of commercially available 2-aminopyridine (9) to

2-amino-5-iodopyridine (1023) with periodic acid and iodine, followed by

halogenation via the diazonium salt to give 724 in 41% yield.

Preparation of 2-methyl-3-nitrothiophenol was successfully achieved

by the method outlined in Scheme 3. Aniline 11 was first diazotized to

phenol 12 in excellent yield. Treatment of 12 with dimethylthiocarbamoyl

chloride in the presence of KOH provided thionocarbamate 13, which was

then thermally converted to thiocarbamate 14 in a sealed tube by Newman

and Karnes’ condition.26 Hydrolysis of 14 afforded thiol 8 with a trace

amount of disulfide 15 as by-product.

N Cl

I

N NH2 N NH2

Ia b

9 10 7 Scheme 2. (a) I2, H5IO6, AcOH, H2SO4, 80 °C, 1 h, 65%; (b) NaNO2, HCl, CuCl,

0 °C-rt, overnight, 41%.

14

NO2

S 2

NH2

NO2

S

NO2

O

Me2N

OH

NO2

SH

NO2

O

NO2

S

Me2N

11 12 13

14 8 15

a b

c d+

Scheme 3. (a) NaNO2, H2SO4, H2O, 0-5 °C, 10 min, 120 °C, 5 min, 94%; (b)

N,N-dimethylthiocarbamoyl chloride, KOH, THF, H2O, 0 °C-rt, 30 min, 76%; (c)

220 °C in a sealed tube, 3 h, 80%; (d) KOH, H2O, MeOH, 80 °C, 1 h, (8, 95%)

(15, trace)

Chloropyridine 7 and thiol 8 were subsequently used as starting

materials in the preparation of 4-(pyridin-2-ylthio)indole analogues as

shown in Scheme 4. The iodo moiety of 7 precluded the use of metal

catalysts for the coupling reaction between 7 and 8, but the reaction

proceeded quite nicely when executed in a sealed tube under N2 to give

sulfide 6 in the absence of catalyst. Unfortunately, under these conditions

we also obtained a by-product disulfide 15, which proved difficult to

remove from the major product 6. Interestingly, the addition of

triphenylphosphine to the reaction mixture not only prevented disulfide

formation, but also permitted the use of the thiophenol/disulfide mixture

as a suitable starting material under these conditions.

15

S

NO2

N

I

6

a

SN

I

NH

CHO

19

b

SN

I

NH

NH2

SN

I

NH

OH

20

21

d

e

SH

NO28 (with trace amount 15)

c

SN

I

NH

+

16

SN

I

NH

O

17

+

18

NH2

S N

I

Scheme 4. (a) 7, PPh3, Et3N, DMF, 110 °C, 20 h, 71%; (b) (i)

dimethylformamide dimethyl acetal (DMF-DMA), pyrrolidine, DMF, 110 °C, 2

h, (ii) Fe, AcOH, 100 °C, 3h, (16, 28%, 17, 11%, 18, 13%); (c) POCl3, DMF,

40oC, 1 h, 40%; (d) NaCNBH3, NH4OAc, MeOH, rt, 3 days, 20%; (e) NaBH4,

EtOH, rt, 4 h, 52%.

16

The modified Leimgruber-Batcho indole synthesis (Scheme 4) was

attempted under a variety of conditions. The first effort, which utilized

acetic acid as solvent in the reduction step, afforded the desired indole 16

as well as acetyl derivative 17 and thiophene 18 as by-products. The

structure of 18 was confirmed by spectroscopic data, including 2D-

HETCOR NMR, which was assigned structurally in Figure 3, and mass

spectrometry. (A plausible mechanism for the formation of thiophene is

shown in Scheme 5). A subsequent attempt using HCl/EtOH as solvent

gave only trace amounts of target product 16 and some unidentified

residue. After a considerable number of trials, an eventual solvent mixture

of AcOH/EtOH (1:1) proved optimal, affording product 16 and

dramatically decreasing the amount of by-product formed. Formylation of

16 via the Vilsmeier reaction yielded aldehyde 19, which subsequently

underwent either reductive-amination or reduction to give 3-

aminomethylindole 20 and 3-hydroxymethylindole 21 respectively.

8.86

7.46 8.05

7.28

7.186.64

5.11NH2

S N

I

NH2

S N

I

7.40 127.0

145.2

154.3

126.0

126.0

112.5

110.0

Figure 3 Assignments of 1H and 13C NMR based on 2D-HETCOR:

17

SN

I

NO2

-H+ SN

I

NO2

N+MeO

SN

I

NO2

NMe2

S

NO2

NMe2

N

I

H

S

NO2

N

H NMe2

I

NO2

S N

I

NH2

S N

I

S

NO2

NMe2

NI

15

-HNMe2

reduction

18 Scheme 5. Plausible Mechanism of the Formation of Thiophene 18.

18

Conclusions

In conclusion, a series of 4-(pyridin-2-ylthio)indole derivatives have

been successfully synthesized for serotonin transporter imaging agent

evaluation. The conversion of 2-methyl-3-nitrophenol (12) to 2-methyl-2-

nitrothiophenol (8) was took place via thionocarbamate intermediate,

which was subjected to thermal rearrangement. Addition of

triphenylphosphine in the coupling reaction of 7 and 8 effectively

prevented disulfide formation. A modified Leimgruber-Batcho indole

synthesis was accomplished from the key intermediate 1-(5-iodopyridin-2-

ylthio)-2-methyl-3-nitrobenzene (6). The heterodiaryl sulfide chemistry

utilized in this synthesis could be useful for the preparation of other novel

bioactive compounds.

19

Experimental Section

2-Amino-5-iodopyridine (10). A mixture of 2-aminopyridine (2.06 g,

21.9 mmol), acetic acid (14 mL), water (3 mL), sulfuric acid (0.42 mL),

and H5IO6 (1.05 g, 4.6 mmol) was allowed to stir at 80 °C for 15 min.

Iodine crystals (2.28 g, 9.0 mmol) were added in portions. After it was

stirred for 1 h, the reaction mixture was poured into saturated sodium

thiosulfate solution and extracted with ethyl acetate. The organic layer was

separated, dried (Na2SO4), and evaporated to give 10 (3.13 g, 65%) as an

orange solid: 1H NMR (200 MHz, CDCl3) δ 4.85 (br, 2H), 6.36 (dd, J =

8.8, 0.8 Hz, 1H), 7.64 (dd, J = 8.8, 2.2 Hz, 1H), 8.18 (d, J = 2.0 Hz, 1H).

CAS No. 20511-12-0.

2-Chloro-5-iodopyridine (7). A mixture of aminopyridine 10 (1.05 g,

4.74 mmol) and concentrated HCl (10 mL) was stirred at 0 °C for 10 min.

Sodium nitrite (1.38 g, 20.0 mmol) was slowly added, then followed by

CuCl (0.50 g, 5.1 mmol) with stirring continued overnight. The mixture

was poured into 1:1 NH4OH:H2O, extracted with ethyl acetate, dried

(Na2SO4), and concentrated. The crude residue was purified by flash

column chromatography on silica gel using pure dichloromethane as an

eluant to yield 7 (0.47 g, 41%) as a colorless solid: 1H NMR (200 MHz,

DMSO-d6) δ 7.36 (d, J = 8.4 Hz, 1H), 8.18 (dd, J = 8.2, 2.2 Hz, 1H), 8.64

(d, J = 1.6 Hz, 1H); 13C NMR (50 MHz, DMSO-d6) δ 93.1, 126.8, 148.0,

150.2, 155.9; CAS No. 69045-79-0.

2-Methyl-3-nitrophenol (12). To a mixture of 11 (3.8 g, 25.0 mmol),

concentrated sulfuric acid (5.5 mL) and water (7.5 mL), 20 g of ice was

added and the solution was cooled to 0-5 °C. A solution of sodium nitrite

20

(1.8 g, 26 mmol) in 1.5 mL of water was added. After stirred for 10 min,

the mixture was allowed to stand at 0-5 °C for 5 min. To a boiling solution

of concentrated sulfuric acid (16.5 mL) and water (15 mL), the diazotized

solution was slowly added. After adding, the mixture was boiled for 5 min

and then poured to a beaker containing ice-water. The precipitate was

collected by suction filtration, washed with cold water and dried. The solid

was purified by flash column chromatography (EtOAc:hexane, 1:9) to

yield 3.63 g (94%) of 12 as a yellow solid: 1H NMR (200 MHz, DMSO-

d6) δ 2.23 (s, 3H), 7.12 (dd, J = 8.2, 1.8 Hz, 1H), 7.19 (dd, J = 8.2, 7.8 Hz,

1H), 7.44 (dd, J = 7.8, 1.8 Hz, 1H); 13C NMR (50 MHz, DMSO-d6) δ 11.8,

114.7, 119.3, 127.4, 151.5, 157.2; CAS No. 5460-31-1

2-Methyl-3-nitrophenyl N,N-Dimethylthionocarbamate (13). To a

powder of 12 (3.06 g, 20.0 mmol) was added a solution of potassium

hydroxide (1.12 g, 20.0 mmol) in 15 mL of H2O at rt. The mixture was

cooled below 5 °C in ice-water bath. A solution of N,N-

dimethylthiocarbamyl chloride (0.185 g, 1.5 mmol) in 5 mL of dry THF

was added with cooling. After the addition, the reaction mixture was

allowed to stir at rt for 30 min. The mixture made alkaline with 10%

potassium hydroxide and extracted with dichloromethane. The organic

layers are combined, washed with brine, and dried over. The residue was

purified by flash column chromatography (CH2Cl2:hexane, 8:2) to give 13

(3.67 g, 76%) as an yellow solid: 1H NMR (200 MHz, CDCl3) δ 2.36 (s,

3H), 3.40 (s, 3H), 3.48 (s, 3H), 7.26 (dd, J = 8.0, 1.4 Hz, 1H), 7.36 (t, J =

8.0 Hz, 1H), 7.83 (dd, J = 8.0, 1.4 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ

13.0, 39.0, 43.7, 122.3, 126.6, 127.5, 128.5, 150.7, 153.5, 186.4. MS (EI)

240 (M+), 225, 223, 194, 179, 151, 121, 88, 72 (100), 63, 51. HRMS (EI)

Calc. for C10H12N2O3S (M+) 240.0569. Found 240.0564.

21

2-Methyl-3-nitrophenyl N,N-Dimethylthiocarbamate (14). A powder of

13 (1.57 g, 6.5 mmol) was added into a sealed tube and purged with N2.

The tube was capped and heated at 215-220 °C for 3 h. The reaction was

cooled to rt. The residue was purified by flash column chromatography

(CH2Cl2) to provide 14 (1.24 g, 80%) as an orange solid: 1H NMR (200

MHz, CDCl3) δ 2.59 (s, 3H), 3.03 (br, 3H), 3.14 (br, 3H), 7.33 (dd, J = 7.8,

7.6 Hz, 1H), 7.75 (dd, J = 7.6, 1.4 Hz, 1H), 7.85 (dd, J = 8.0, 1.4 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 17.7, 37.3, 125.9, 126.7, 132.5, 137.7, 141.7,

151.5, 165.2; MS (EI) 240 (M+), 210, 168, 149, 121, 110, 72 (100), 56.

HRMS (EI) Calc. for C10H12N2O3S (M+) 240.0569. Found 240.0570.

2-Methyl-3-nitrothiophenol (8). A solution of 14 (1.24 g, 5.2 mmol) and

KOH (0.60 g, 10.7 mmol) in H2O (2 mL) and MeOH (10 mL) was heated

at 80 °C for 1 h under N2 atmosphere. The reaction mixture was cooled

and poured to 5 g of ice. The solution was washed with CH2Cl2 (20 mL x

2). The organic layer was discarded. The aqueous layer was acidified by 4

M HCl solution and extracted with CH2Cl2 (20 mL x 2). The organic

layers were combined, dried (Na2SO4) to give a mixture of 8 (0.84 g,

95%): 1H NMR (200 MHz, CDCl3) δ 2.44 (s, 3H), 3.58 (s, 1H), 7.17 (t, J

= 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.55 (dd, J = 8.0, 0.8 Hz, 1H); 13C

NMR (50 MHz, CDCl3) δ 17.2, 121.6, 126.9, 130.0, 133.7, 135.8, 151.6;

MS (EI) 169 (M+), 152 (100), 124, 121, 110, 97, 77, 63, 45, 39; HRMS

(EI) Calc. for C7H7NO2S (M+) 169.0198. Found 169.0195. Di(2-methyl-

3-nitrophenyl) Disulfide (15). (trace amount) 1H NMR (200 MHz,

CDCl3) δ 2.60 (s, 6H), 7.29 (dd, J = 8.2, 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz,

1H), 7.71 (dd, J = 8.2, 8.0 Hz, 1H); 13C NMR (50 MHz, CDCl3) δ 16.3,

123.6, 127.3, 131.7, 132.3, 138.4, 151.5; MS (EI) 336 (M+, 100), 320, 306,

22

259, 241, 168, 129, 121, 110, 77, 57. HRMS (EI) Calc. for C14H12O4N2S2

(M+) 336.0239. Found 336.0242.

1-(5-Iodopyridin-2-ylthio)-2-methyl-3-nitrobenzene (6). A mixture of 8

and 15 (0.84 g, 4.97 mmol), 7 (1.19 g, 4.97 mmol), PPh3 (0.13 g, 0.5

mmol) and Et3N (0.72 mL) in DMF (5 mL) was added into a sealed tube

and purged with N2. The reaction was heated at 110 °C for 20 h. The

mixture was cooled, added CH2Cl2 (20 mL) and washed with H2O and

brine. The organic layer was dried (Na2SO4) and evaporated. The residue

was purified by flash column chromatography (EtOAc:hexane, 8:2) to

give 6 (1.32 g, 71%) of as a yellow solid: 1H NMR (200 MHz, CDCl3) δ

2.56 (s, 3H), 6.77 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 8.0, 7.8 Hz, 1H), 7.76-

7.82 (m, 2H), 7.87 (d, J = 8.0 Hz, 1H), 8.59 (d, J = 2.2 Hz, 1H); 13C NMR

(50 MHz, CDCl3) δ 17.5, 89.1, 123.5, 125.8, 127.4, 133.8, 137.1, 140.5,

145.2, 151.9, 156.1, 158.3; MS (EI) 372 (M+), 357, 353, 327, 311, 227,

197, 154, 121, 89, 77, 63, 51, 39. HRMS (EI) Calc. for C12H9N2O2SI (M+)

371.9430. Found 371.9426.

4-(5-Iodopyridin-2-ylthio)indole (16). A mixture of 6 (1.72 g, 4.6 mmol),

dimethylformamide dimethyl acetal (1.4 mL, 10 mmol) and pyrrolidine

(0.4 mL, 5 mmol) in 8 mL of DMF was allowed to heat at 110 °C for 2 h

under N2 atmosphere. The reaction was cooled, added ether (20 mL) and

washed with H2O (20 mL x 2). The organic layer was dried over, and

evaporated. The red residue was dissolved in the mixture of AcOH (15

mL) and EtOH (15 mL) and added iron powder (2 g). The suspension was

heated at 100 °C for 3 h. The reaction was cooled, filtered and washed by

water. The filtrate was basified by 1 M NaOH solution and extracted with

ether, washed with H2O and brine. The extract was dried over and was

23

purified by flash column chromatography (ethyl acetate:hexane, 1:4) to

provide 16 (0.62 g, 36%) as a off-white solid: 1H NMR (200 MHz,

DMSO-d6) δ 6.27-6.29 (m, 1H), 6.43 (d, J = 8.4 Hz, 1H), 7.19 (dd, J = 7.6,

7.4 Hz, 1H), 7.32 (dd, J = 7.2, 1.0 Hz, 1H), 7.44 (dd, J = 3.0, 2.4 Hz, 1H),

7.59 (dd, J = 8.0, 1.0 Hz, 1H), 7.83 (dd, J = 8.4, 2.2 Hz, 1H), 8.60 (d, J =

2.2 Hz, 1H) 11.49 (br, 1H); 13C NMR (50 MHz, DMSO-d6) δ 88.7, 100.4,

113.9, 119.0, 121.8, 122.3, 126.7, 127.0, 130.5, 136.3, 144.8, 154.7,

160.2; MS (EI) 352 (M+), 224 (100), 207, 147, 104, 77, 73, 50. HRMS

(EI) Calc. for C13H9N2SI (M+) 351.9531. Found 351.9529.

3-Acetyl-4-(5-iodopyridin-2-ylthio)indole (17) and 3-(4-Iodopyridin-

2yl)-4-aminothiophene (18). Same procedure used as 16, pure AcOH (30

mL) was used instead of the mixture of AcOH and EtOH in the reduction

step to provide 16 (0.48 g, 28%), 17, and 18. 17 (0.20 g, 11%) as a white

solid: 1H NMR (200 MHz, CDCl3) δ 2.05 (s, 3H), 7.40 (dd, J = 8.2, 7.6 Hz,

1H), 7.52 (d, J = 8.4 Hz, 1H), 7.63 (dd, J = 8.0, 1.0 Hz, 1H), 7.62 (s, 1H),

8.12 (dd, J = 8.4, 2.2 Hz, 1H), 8.27 (d(br), J = 7.4 Hz, 1H), 8.88 (d, J = 2.2

Hz, 1H) 11.45 (br, 1H); 13C NMR (50 MHz, CDCl3) δ 25.0, 91.4, 118.5,

118.9, 126.0, 126.6, 127.2, 130.2, 135.2, 135.7, 142.6, 146.6, 153.6, 155.0,

168.4; MS (EI) 394 (100, M+), 379, 352, 281, 224, 209, 197, 121, 73, 50;

HRMS (EI) Calc. for C15H11N2OSI (M+) 393.9637. Found 393.9636. 18

(0.23 g, 13%) as a white solid: 1H NMR (400 MHz, CDCl3) δ 5.11 (br,

2H), 6.64 (dd, J = 7.6, 1.2 Hz, 1H), 7.18 (dd, J = 8.0, 7.6 Hz, 1H), 7.28

(dd, J = 8.0, 1.2 Hz, 1H), 7.40 (s, 1H), 7.46 (dd, J = 8.2, 0.8 Hz, 1H), 8.05

(dd, J = 8.2, 2.4 Hz, 1H), 8.86 (dd, J = 2.4, 0.8 Hz, 1H); 13C NMR (100

MHz, CDCl3) δ 91.3, 111.0, 112.5, 124.0, 126.0, 127.0 136.5, 142.9,

144.2, 145.3, 154.3, 155.2; MS (EI) 352 (M+, 100), 336, 281, 225, 224,

24

198, 121, 113, 99, 77, 57. HRMS (EI) Calc. for C13H9N2SI (M+) 351.9531.

Found 351.9532.

3-Formyl-4-(5-iodopyridin-2-ylthio)indole (19). Phosphorus oxychloride

(0.05 mL, 0.27 mmol) was added dropwise with stirring to DMF (0.5 mL)

at 0-5 °C. A solution of 16 (0.10 g, 0.28 mmol) in DMF (1 mL) was then

added dropwise. After addition, the mixture was stirred at 40 °C for 1 h.

The mixture was poured to 1 g of crushed ice and then made alkaline by 1

M NaOH solution. The resulting suspension was heated to the boiling

point and cooled to rt. The precipitate was filtered, washed by water, air-

dried. The solid was purified by flash column chromatography

(CH2Cl2:hexane, 6:4) to give 19 (42 mg, 40%) as a off-white solid: 1H

NMR (400 MHz, DMSO-d6) δ 6.56 (dd, J = 8.4, 0.8 Hz, 1H), 7.33 (dd, J =

8.0, 7.6 Hz, 1H), 7.53 (dd, J = 7.6, 0.8 Hz, 1H), 7.72 (dd, J = 8.0, 0.8 Hz,

1H), 7.88 (dd, J = 8.4, 2.0 Hz, 1H), 8.25 (d, J = 3.2 Hz, 1H), 8.59 (dd, J =

2.0, 0.8 Hz, 1H) 10.32 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δ 89.9,

116.0, 118.9, 120.1, 123.0, 124.0, 128.2, 131.7, 134.2, 138.4, 145.8, 155.8,

160.0, 186.0; MS (EI) 380 (M+), 351, 347 (100), 320, 224, 176, 148, 129,

104, 88, 72, 57. HRMS (EI) Calc. for C14H9N2OSI (M+) 379.9480. Found

379.9477.

3-(Aminomethyl)-4-(5-iodopyridin-2-ylthio)indole (20). To a solution of

19 (0.10 g, 0.26 mmol), ammonium acetate (0.20 g, 2.6 mmol) in 4 mL of

MeOH was added a solution of 1.0 M NaCNBH3 in THF (0.26 mL, 0.26

mmol) with stirring. The reaction was stirred at rt for 3 days. The reaction

was acidified by 2 M HCl solution until pH

25

(CH2Cl2:MeOH, 10:1) to give 20 (20 mg, 20%) of as a white solid: 1H

NMR (400 MHz, DMSO-d6) δ 4.27 (br, 2H), 6.37 (d, J = 8.4 Hz, 1H),

7.22 (dd, J = 8.0, 7.6 Hz, 1H), 7.28 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 8.0 Hz,

1H), 7.74 (d, J = 2.8 Hz, 1H), 7.81 (dd, J = 7.6, 2.4 Hz, 1H), 8.43 (d, J =

2.4 Hz, 1H), 9.22 (br, 1H), 12.04 (br, 1H); 13C NMR (100 MHz, DMSO-

d6) δ 42.0, 90.2, 105.8, 115.3, 119.6, 122.9, 123.1, 128.3, 130.2, 130.7,

137.8, 145.7,155.5, 161.0 MS (FAB) 382 (M+), 380, 365 (100), 332, 239,

207, 115. HRMS (FAB) Calc. for C14H13N3SI (M+H+) 381.9875. Found

3819871.

3-(Hydroxymethyl)-4-(5-iodopyridin-2-ylthio)indole (21). A suspension

of 19 (38 mg, 0.10 mmol), NaBH4 (4.0 mg, 0.1 mmol) in 0.6 mL of 95%

EtOH was stirred at rt for 4 h. The reaction mixture was added with water

and extracted with ethyl acetate. The organic layers were combined, then

washed by water and brine, and dried (Na2SO4). The crude was purified by

flash column chromatography (EtOAc:hexane, 3:7)to provide 21 (20 mg,

52%) as an off-white-off solid: 1H NMR (400, DMSO-d6) δ 4.63-4.67 (m,

3H), 6.35 (dd, J = 8.8, 0.4 Hz, 1H), 7.17 (dd, J = 8.0, 7.2 Hz, 1H), 7.25

(dd, J = 7.2, 1.2 Hz, 1H), 7.34 (dd, J = 1.6, 1.2 Hz, 1H), 7.55 (dd, J = 8.0,

2.0 Hz, 1H), 7.85 (dd, J = 8.8, 2.4 Hz, 1H), 8.59 (dd, J = 2.2, 0.4 Hz, 1H),

11.31 (d, J = 2.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 57.2, 89.1,

114.8, 117.6, 119.4, 122.4, 122.5, 126.0, 128.1, 129.0, 138.3, 145.6, 155.3,

162.6; MS (FAB) 383 (M+), 365 (100), 332, 239, 160, 117. HRMS (FAB)

Calc. for C14H12N2OSI (M+H+) 382.9715. Found 382.9713..

26

References

1. a) Williams, D. A.; Lemke, T. L. Foye’s Principles of Medicinal

Chemistry 5th ed.; Lippincott Williams&Wilkins: Baltimore, 2002.

b) Rapport, M. R. Perspect. Bio. Med. 1997, 40, 260-273.

2. Frazer, A.; Hensler, J. G. In Basic Neurochemistry; Siegel G. J.;

Agranoff, B. W.; Alber, R. W.; et al, Eds.; Raven Press: New York,

1993.

3. Richelson, E. M. Clin. Proc. 1994, 69, 1069.

4. a) Owens, M. J.; Nemeroff, C. B. Clin. Chem. 1994, 40, 288-295. b)

Frazer, A. J. Clin. Psychiatry 1997, 6, 9-25. c) Hyttel, J. Int. Clin.

Psychopharmacol. 1994, 1, 19-26.d) Staley, J. K.; Malison, R. T.;

Innis, R. B. Biol. Psychiatry 1998, 44, 534-549. e) Oh, S. J.; Ha, H.-

J.; Chi, D. Y.; Lee, H. K. Current Med. Chem. 2001, 8, 999-1034.

5. Rothman, R. B.; Baumann, M. H. Pharmacol Ther. 2002, 95, 73-88.

6. Szabo, Z.; Kao, P. F.; Scheffel, U.; Suehiro, M.; Mathews, W. B.;

Ravert, H. T.; Musachio, J. L.; Marenco, S.; Kim, S. E.; Ricaurte, G.

A.; Wong, D. F.; Wagner, H. N., Jr.; Dannals, R. F. Synapse 1995,

20, 37-43.

7. a) Suehiro, M.; Musachio, J. L.; Dannals, R. F.; Mathews, W. B.;

Ravert, H. T.; Scheffel, U.; Wagner, H. N. Nucl. Med. Biol. 1995, 22,

543-545. b) Suehiro, M.; Scheffel, U.; Dannals, R. F.; Ravert, H. T.;

Ricaurte, G. A.; Wagner, H. N. J. Nucl. Med. 1993, 34, 120-127. c)

Elfving, B.; Bjornholm, B.; Ebert, B.; Knudsen, G. M. Synapse 2001,

41, 203-211.

8. Maryanoff, E. M.; Vaught, J. L.; Shank, R. P.; McComsey, D. F.;

Costanzo, M. J.; Nortey, S. O. J. Med. Chem. 1990, 33, 2793-2797.

27

9. a) Szabo, Z.; Scheffel, U.; Mathews, W. B.; Ravert, H. T.; Szabo, K.;

Kraut, M.; Palmon, S.; Ricaurte, G. A.; Dannals, R. F. J. Cereb.

Blood Flow Metab. 1999, 19, 967-981. b) Szabo, Z.; Mohamadiyeh,

M.; Scheffel, U.; Matthews, W. B.; Ravert, H. T.; Szabo, K.; Palmon,

S.; Dannals, R. F. J. Nucl. Med. 1996, 37, 125.

10. Parsey, R. V.; Kegeles, L. S.; Hwang, D. R.; Simpson, N.; Abi-

Dargham, A.; Mawlawi, O.; Slifstein, M.; Van Heertum, R. L.;

Mann, J. J.; Laruelle, M. J. Nucl. Med. 2000, 41, 1465-1477.

11. a) Ricaurte, G. A.; McCann, U. D.; Szabo, Z.; Scheffel, U. Toxicol

Lett. 2000, 112-113, 143-146. b) McCann, U. D.; Szabo, Z.; Scheffel,

U.; Dannals, R. F.; Ricaurte, G. A. Lancet 1998, 352, 1433-1437.

12. a) Chumpradit, S.; Kung, M.-P.; Panyachotipun, C.; Prapansiri, V.;

Foulon, C.; Brooks, B. P.; Szabo, S. A.; Tejani-Butt, S.; Frazer, A.;

Kung, H. F. J. Med. Chem. 1992, 35, 4492-4497. b) Soudijn, W.; van

Wijngaarden, I. Serotonin Receptors and Their Ligands; Elsevier

Science: New York, 1997; pp 327-361. c) Suehiro, M.; Scheffel, U.

A.; Ravert, H. T.; Ricaurte, G. A.; Hatzidimitriou, G.; Dannals, R.

F.; Bogeso, K. P.; Wagner, H. N. Nucl. Med. Biol. 1994, 21, 1083-

1091. d) Kung, M.-P.; Chumpradit, S.; Billings, J. J.; Kung, H. F.

Life Sci. 1992, 51, 95-106.

13. a) Biegon, A.; Mathis, C. A.; Hanrahan, S. M.; Jagust, W. J. Brain

Res. 1993, 619, 236-246. b) Mathis, C. A.; Taylor, S. E.; Biegon, A.;

Enas, J. D. Brain Res. 1993, 619, 229-235. c) Mathis, C. A.; Biegon,

A.; Taylor, S. E.; Enas, J. D.; Hanrahan, S. M. Eur. J. Pharmacol.

1992, 210, 103-104.

14. Jagust, W. J.; Eberling, J. L.; Biegon, A.; Taylor, S. E.; Van-

Brocklin, H. F.; Jordan, S.; Hanrahan, S. M.; Roberts, J. A.; Brennan,

K. M.; Mathis, C. A. J. Nucl. Med. 1996, 37, 1207-1214.

28

15. Ferris, R. M.; Brieaddy, L.; Mehta, N.; Hollingsworth, E.; Rigdon,

G.; Wang, C.; Soroko, F.; Wastila, W.; Cooper, B. J. Pharm.

Pharmacol. 1995, 47, 775-781.

16. Oya, S.; Kung, M. P.; Acton, P. D.; Mu, M.; Hou, C.; Kung, H. J.

Med. Chem. 1999, 42, 333-335.

17. Oya, S.; Choi, S. R.; Hou, C.; Mu, M.; Kung, M. P.; Acton, P. D.;

Siciliano, M.; Kung, H. F. Nucl. Med. Biol. 2000, 27, 249-254.

18. Zhuang, Z. P.; Choi, S. R.; Hou, C.; Mu, M.; Kung, M. P.; Acton, P.

D.; Kung, H. F. Nucl. Med. Biol. 2000, 27, 169-175.

19. a) Acton, P. D.; Kung, M. P.; Mu, M.; Plossl, K.; Hou, C.; Siciliano,

M.; Oya, S.; Kung, H. F. Eur. J. Nucl. Med. 1999, 26, 854-861. b)

Kung, M. P.; Hou, C.; Oya, S.; Mu, M.; Acton, P. D.; Kung, H. F.

Eur. J. Nucl. Med. 1999, 26, 844-853.

20. a) Choi, S. R.; Hou, C.; Oya, S.; Mu, M.; Kung, M. P.; Siciliano, M.;

Acton, P. D.; Kung, H. F. Synapse 2000, 38, 403-412.b) Acton, P.

D.; Choi, S. R.; Hou, C.; Plossl, K.; Kung, H. F. J. Nucl. Med. 2001,

42, 1556-1562.

21. a) Lee, B. S.; Chu, S.; Lee, K. C.; Lee, B.-S.; Chi, D. Y.; Kim, S. E.;

Choe, Y. S.; Song, Y. S.; Jin, C. Bioorg. Med. Chem. 2003, 11,

4949-4958. b) Lee, B. S.; Chu, S.; Lee, B.-S.; Chi, D. Y.; Song, Y.

S.; Jin, C. Bioorg. Med. Chem. Lett. 2002, 12, 811-815. c) In, M. Y.;

Chi, D. Y.; Choi, S. J.; Park, K. B.; Cho, C. G. Bull. Korean Chem.

Soc. 2002, 23, 1439-1444. d) Lee, B. S.; Chu S.; Lee, I. Y.; Lee, B.-

S.; Song, C. E.; Chi, D. Y. Bull. Korean Chem. Soc. 2000, 21, 860-

866. e) Lee, B. S.; Chu, S.; Lee, B. C.; Chi, D. Y.; Choe, Y. S.;

Jeong, K. J.; Jin, C. Bioorg. Med. Chem. Lett. 2000, 10, 1559-1562.

22. a) Batcho, A. D.; Leimgruber, W. Org. Synth. 1985, 63, 214-220. b)

Clark, R. D.; Repke, D. B. Heterocycles 1984, 22, 195-221.

29

23. Hama, Y.; Nobuhara, Y.; Aso, Y.; Otsubo, T.; Ogura, F. Bull. Chem.

Soc. Jpn. 1988, 61, 1683-1686.

24. Clayton, S. C.; Regan, A. C. Tetrahedron Lett. 1993, 34, 7493-7496.

25. Vogel, A. I. .Vogel’s Text book of practical organic chemistry, 5th

ed.; revised by Furiss, B. S.; Hannaford, A. J.; Smith, P. W. G.;

Tatchell, A. R. Longman Group UK Limited: Harlow, England,

1989.

26. Newman, M. S.; Karnes, H. A. J. Org. Chem. 1966, 31, 3980-3984.

30

NMR spectra

2-Methyl-3-nitrophenyl N,N-Dimethylthionocarbamate (13). 1H NMR (200 MHz, CDCl3)

13C NMR (50 MHz, CDCl3)

O

NO2

S

Me2N

31

2-Methyl-3-nitrophenyl N,N-Dimethylthiocarbamate (14). 1H NMR (200 MHz, CDCl3)

13C NMR (50 MHz, CDCl3)

S

NO2

O

Me2N

32

2-Methyl-3-nitrothiophenol (8). 1H NMR (200 MHz, CDCl3)

13C NMR (50 MHz, CDCl3)

SH

NO2

33

1-(5-Iodopyridin-2-ylthio)-2-methyl-3-nitrobenzene (6). 1H NMR (200 MHz, CDCl3)

13C NMR (50 MHz, CDCl3)

S

NO2

N

I

34

4-(5-Iodopyridin-2-ylthio)indole (16). 1H NMR (200 MHz, DMSO-d6)

13C NMR (50 MHz, DMSO-d6)

SN

I

NH

35

3-Acetyl-4-(5-iodopyridin-2-ylthio)indole (17).

1H NMR (200 MHz, CDCl3)

13C NMR (50 MHz, CDCl3)

SN

I

NH

O

36

3-(4-Iodopyridin-2-yl)-4-aminobenzothiophene (18). 1H NMR (400 MHz, CDCl3)

13C NMR (100 MHz, CDCl3)

NH2

S N

I

37

3-(4-Iodopyridin-2yl)-4-aminothiophene (18)

2D-HETCOR. in CDCl3

NH2

S N

I

38

3-Formyl-4-(5-iodopyridin-2-ylthio)indole (19). 1H NMR (400 MHz, DMSO-d6)

13C NMR (100 MHz, DMSO-d6)

SN

I

NH

CHO

39

3-(Aminomethyl)-4-(5-iodopyridin-2-ylthio)indole (20) 1H NMR (400 MHz, DMSO-d6)

13C NMR (100 MHz, DMSO-d6)

SN

I

NH

NH2

40

3-(Hydroxymethyl)-4-(5-iodopyridin-2-ylthio)indole (21) 1H NMR (400 MHz, DMSO-d6)

13C NMR (100 MHz, DMSO-d6)

SN

I

NH

OH

41

PART 2

Structural Modification of Nitric Oxide Inhibitors

42

Introduction

Nitric oxide or nitrogen monoxide, a small free radical, is formed

by nitric oxide synthases (NOS; EC 1.14.13.39). In the presence of

oxygen and NADPH, NOS catalyses five-electron oxidation of the

terminal guanidino nitrogen atoms of L-arginine to generate L-citrulline

and NO (Scheme 1)1 using flavin adenine dinucleotide (FAD), flavin

mononucleotide (FMN), heme, and tetrahydrobiopterin (BH4) as

cofactors.2 Functional NOS has two bidomain structures. The reductase

domain of NOS at a C-terminus contains the binding sites for FAD, FMN,

NADPH and calmodulin, and a N-terminal oxygenase domain contains the

binding sites for heme, BH4 and L-arginine. The N-terminal oxygenase

domain is linked by a calmodulin recognition site to the C-terminal

reductase domain. Native NOS is a homodimer, which requires heme for

dimerization of monomers and full NOS activity.3

NH

COOH3N

NH2H2NNADPH+H+ NADP+

O2 H2O

NH

COOH3N

HNH2N OH

0.5 NADPH+H+

O2BH4

NADP +

H2O

NO

NH

COOH3N

OH2N

+

L-Arginine NG-Hydroxy-L-arginine Nitric oxide L-C itrulline Scheme 1. The biosynthesis of nitric oxide.

Three different NOS isoforms have been characterized in

mammalian tissues.3 Two constitutively expressed NOS isoforms which

are activated by stimulation dependent Ca2+ entry, are present mainly in

brain (neuronal NOS; nNOS or Type I NOS) and endothelial cell

(endothelial NOS; eNOS or Type III NOS). Nitric oxide produced by

43

nNOS functions mainly in neurotransmission while the NO produced by

eNOS functions in the maintenance of normal vascular homeostasis such

as regulation of blood pressure and prevention of platelet and leukocyte

adhesion and activation. A third isoform, cytokine-inducible and Ca2+-

independent NOS (inducible NOS; iNOS or Type II NOS) is expressed in

macrophages, neutrophils, hepatocytes and other cells. The expression of

iNOS is induced after stimulation with lipopolysaccharide (LPS) and pro-

inflammatory cytokines e.g. interleukin (IL), tumor nrcrosis factor-α

(TNF-α), and interferons (IFN).4 High amount of nitric oxide produced by

iNOS functions mainly in pathogen killing processes, where its toxicity

can be due to a combination of effects, including inhibition of target cell

respiration and cell division, reaction with redox active catalytic iron

centers, and indirect cytotoxicity through formation of toxic oxidants as

well as nitrating/nitrosating species.5 Moreover, BH4, an essential cofactor

for the activation of all isoforms of NOS, is synthesized from GTP via

sequential enzyme reactions catalyzed by GTPcyclohydrolase I (GTPCH),

6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase. It was

reported that cytokine-induced NO production requires GTPCH activation

in cardiac myocytes.6 Also, increased BH4 levels in murine fibroblasts,7

cardiac myocytes,8 and endothelial cells9 indicate that the availability of

the cofactor regulates NOS activity.

Nitric oxide (NO) and other free radicals have been implicated in

the pathophysiology of ischemic neuronal death.10 NO is an important

signaling molecule in normal synaptic transmission but can be a

neurotoxin under pathological conditions. Increases in NO generation,

NOS mRNA, and protein were reported in animal models of ischemia,11

and NOS inhibitors protected neurons in these animal models.12 The

44

mechanism by which NO contributes to ischemic neuronal death, either

through necrosis or apoptosis, is not known. However, NO-mediated

hydrolytic cleavage of poly(ADP-ribose)-polymerase, one of the key

substrates for activated cysteine protease,13 suggests that NO plays a role

in apoptotic cell death.

Figure 1. Current major approaches A-D for NO inhibition.

There are many approaches to inhibit NO production, not only at

transcription level but also at postranscription level, as shown in Figure 1.

NO production is blocked at DNA or mRNA level (approach A) related to

45

the production of cofactors for iNOS activity.14 In approach B, GTP

derivatives derived from GTP or related compounds were tested for the

inhibition of GTP cyclohydrolase (GTPCH),15 a rate-limiting enzyme for

the conversion of GTP to BH4. BH4 is an essential cofactor for the

production of NO16 and it also acts as glue to maintain NOS in dimeric

active form.16,17 BH4 derivatives were also synthesized to inhibit

dimerization of NOS (approach C).18 Recently, arginine mimetic

compounds19 and other small molecules20 were reported to inhibit NO

production by competitively blocking at substrate L-arginine binding site

(approach D).

The regulation of NO overproduction to prevent cell damage was

interested widely. Although there are many ways to inhibit NO production,

most of them also showed some side effect and toxicity.21 Recently, Cho

and coworkers reported that treatment with N-acetyl-O-methyldopamine

(NAMDA) (Chart 1), a metabolite of dopamine in CNS, significantly

protected CA1 neurons in rat ischemic hippocampus and inhibited LPS-

induced NO production in BV-2 microglia cells without direct inhibition

of iNOS activity.22 More recently, it was demonstrated that NAMDA act

as a neuroprotectant by repressing LPS-induced proinflammatory

cytokines gene expression via a cAMP-dependent protein kinase pathway

without preventing NF-κB nuclear translocation or its DNA binding

activity in microglia cells.23 They suggested that NAMDA as a potent

agent, which attenuated neuronal injury. Although, NAMDA is not toxic,

it showed good protective activity only at high concentration (5 mM).

Therefore, it was clear that structure modification was needed to improve

its activity and to develop as a potent therapeutic agent.

46

H3CO

HOHN

O1 Chart 1 The structure of NAMDA 1.

Figure 3 Strategies of structure modification

The structure modification was made by homologation,

cyclization and replacing of the acetamide group with other function

groups as shown in Figure 3. It was considered that the restrict

rotation of flexible carbon and changing of acetamide group would

provide some improvement on its inhibitory activity. Herein, several

compounds were synthesized and their biological effects were

evaluated on NO production and cytotoxicity.

H3CO

HOHN

OChanging of acetamide group

Homologation

Cyclization

47

Chemistry

3-O-methyldopamine hydrochloride (4) was firstly synthesized by

previously reported method.24 4-Hydroxy-3-methoxybenzyl alcohol (2)

was converted to (4-hydroxy-3-methoxyphenyl)acetonitrile (3) with

sodium cyanide, and then reduced to amine by hydrogenation using

palladium on charcoal as a catalyst. The treatment of 4 with S-2-

naphthylmethyl thioacetimidate hydrobromide25 in ethanol at room

temperature gave acetamidine hydrobromide 5 in a high yield (Scheme 2).

OH

HO

MeOCN

HO

MeO

HO

MeOHN

NHHO

MeO NH2.HCl

2 3

4

a

5

b

c

Scheme 2. (a) NaCN, DMF, 130°C, 20 h, 60%; (b) H2, Pd/C, HCl, EtOH, rt, 12h,

82%; (c) S-2-naphthylmethyl thioacetimidate hydrobromide, Et3N, EtOH, rt, 3 h,

70%.

In many attempt to synthesis of cyclic amide, the reactions were

failed due to double bond formation by fast elimination at α,β position.

The cyclic amine derivatives were, however, designed and synthesized as

shown in Scheme 3. The nitrile 3 was hydrolyzed under basic condition to

a carboxylic derivative 6. The coupling reaction of 6 with piperidine and

pyrrolidine by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

48

hydrochloride (EDCI) yielded amide 7 and 8, respectively. Subsequently,

the amide 7 and 8 were reduced using lithium aluminium hydride to

corresponding amine 9 and 10.

HO

COOHMeO MeO

HO

R

O

N

NMeO

HO

R

N

N

3

67, R =

8, R =

a b

c

9, R =

10, R =

Scheme 3. (a) KOH, EtOH, reflux, overnight, 74%; (b) piperidine or pyrrolidine,

EDCI, DMAP, DMF, rt, 3 h, 75% for 7, 51% for 8; (c) LAH, THF, reflux, 2 h,

69% for 9, 80% for 10.

MeO

HO

O

HMeO

HO

CN

MeO

HO

NH

OMeO

HO

NH2.HC l

a b

c

11

13 14

12

Scheme 4. (a) KOH, CH3CN, reflux, overnight, 83%; (b) H2, Pd/C, HCl, EtOH,

rt, 12 h, %; (c) Ac2O, Et3N, CH2Cl2, rt, 30 min, 76%.

49

To obtain a homologous chain derivative (Scheme 4), vanillin (11)

was refluxed with potassium hydroxide in acetonitrile and leaded to trans-

cinamonitrile 12 as a major product. The catalytic hydrogenation of 12

under acidic condition completely reduced both double bond and nitrile

group to give amine 13 as an ammonium hydrochloride salt. The amide 14

was consequently obtained by acetylation of 13 with acetic anhydride.

Cyclized derivatives, tetrahydroisoquinoline, were next

synthesized via the Pictet-Spengler synthesis26 as shown in Scheme 5. The

treatment of 4 with formaldehyde gave 15 by one-pot synthesis. N-

formylation of 15 with trimethyl orthoformate was performed in a sealed

tube under N2 atmosphere and need to add concentrated hydrochloride in a

catalytic amount to yield formamide 16. Lastly, tetrahydroisoquinoline 15

was simply treated with benzoic anhydride and lead to benzamide 17.

MeO

HONH

MeO

HON H

O

MeO

HON

O

.HCl

4a

b c

15

16 17

Scheme 5. (a) HCHO, H2O, HCl, 70°C, 1 h, 70%; (b) trimethylorthoformate,

conc.HCl (cat.), reflux, overnight, 90%; (c) Bz2O, Et3N, CH2Cl2, rt, 30 min, 65%

50

Biological Results and Discussion.

Table 1 Nitrite and LDH production assay: BV2 Cell (2.5x104 cells/well)a

% of LPS alone Compound

Nitrite a LDH cytotoxicity assay a

5 96.9 96.4

7 90.3 81.1

8 56.1 68.8

9 85.9 78.2

10 84.4 83.7

12 54.0 100.4

14 77.5 98.6

15 16.5 75.4

16 128.8 108.1

17 84.9 104.7

NAMDA (1) 84.0 73.4

LPS 100.0 100.0

control 37.8 77.2 aEach assay was treated at 100 µM concentration of tested compound.

51

HO

MeOHN

NHHO

MeO N

OHO

MeO N

O

HO

MeO N

HO

MeO N MeO

HO

CN

MeO

HO

NH

OMeO

HONH.HC l

MeO

HON H

O

MeO

HON

O

5 87

9 10 12

14 15 16

17 Chart 2 The structure of tested compounds.

The inhibitory activity and cytotoxicity of 11 compounds including NAMDA on NO production induced by lipopolysaccharide (LPS) in microgial (BV2) were shown in Table 1. Those compounds (Chart 2) were modified structurally at amide group or chain but remained catechol moiety untouched. Among open-chain compounds, three compounds 8, 12, 14 showed significantly more potent activity than that of NAMDA and all did not show cytotoxicity in LDH assay. Both amine 9, 10 and acetamidine 5 showed similar potency to NAMDA maybe because of their polarity, whereas less polar compound such as amide 8 and cinamonitrile 12 are more potent. Interestingly, the homologous compound 14 showed some improvement of inhibitory activity. This result indicated that less polar function group trended to show better potency. Next, three tetrahydroisoquinolines were tested. Compound 15 showed excellent activity, which could reduce NO production lower than that of control sample, whereas amide 16 increased NO production. Among all tested compounds, tetrahydroisoquinoline 15 showed the best result and could successfully inhibit NO

52

production induced by LPS in microgial cell at 100 µM concentration. This compound can be a new lead compound for development of NO inhibitor due to its much higher activity and non-cytotoxicity. The extensive study including mechanism and target enzyme would, however, be investigated and confirmed further.

53

Conclusions

Ten compounds were synthesized and evaluated its biological

activity for NO production. The structural modification was made by

homologation, cyclization and changing acetamide group to other

functional groups, acetamidine, amides, cyclic amines and cinamonitrile.

Among all compounds, four compounds showed some improvement of

inhibitory activity without increasing cytotoxicity. Compound 15

exhibited strikingly as the most potent NO reducing agent and more

significantly potent than the lead compound, NAMDA.

54

Experimental Section

4-Hydroxy-3-methoxyphenylacetonitrile (3). To a solution of 4-

hydroxy-3-methoxybenzly alcohol (2) (20 g, 0.13 mol) in DMF (300 mL)

was added potassium cyanide (0.16 mol, 10 g) and then set the

temperature at 130 °C under N2. After 20 h, reaction mixture was cooled

to room temperature and quenched by water. Brine was added and

extracted with chloroform. Organic layer was washed two times more with

Brine and water. Chloroform was evaporated, and dark brown mixture was

placed to vacuum distillation apparatus. Vacuum distillation was proceed

at 0.6 torr and 120 °C. 3 (12.7 g, 60%) was obtained as a white solid: 1H

NMR (CDCl3, 200 MHz) δ 6.88-6.73 (m, 3H), 5.89 (s, 1H), 3.84 (s, 3H),

3.64 (s, 3H); 13C NMR (CDCl3, 50 MHz) 146.9, 145.3, 121.4, 120.7,

144.7, 110.4, 55.8, 22.9. CAS No. 4468-59-1

3-O-Methyldopamine hydrochloride (4). To a solution of 3 (6.0 g, 37

mmol) in ethanol (60 mL) was added 10% wet palladium charcoal (1.0 g).

Round-bottom flask was sealed with septum and purged with H2 for 2 min,

then conc. HCl (6 mL) was added. Mixture was stirred for 12 h at room

temperature. Reaction mixture was filtered through celite and then solvent

was evaporated. Crystallization in methanol and ethyl acetate gave 4 (6.2

g, 82%) as a white solid: 1H NMR (DMSO-d6, 200 MHz)

δ 8.92 (s, 1Η), 8.19 (br s, 2H), 6.81 (d, J = 1.6 Hz, 1H), 6.74 (d, J = 8.0 Hz,

1H), 6.61 (dd, J = 8.0, 1.4 Hz, 1H), 3.75 (s, 3H), 3.05-2.83 (brm, 2H),

2.81-2.66 (br m, 2H); 13C NMR (DMSO-d6, 50 MHz)

δ 151.6, 149.3, 132.0, 124.8, 119.5, 116.9, 59.6, 44.1, 36.5. CAS No.

1477-68-5.

55

N-[2-(4-Hydroxy-3-methoxyphenyl)ethyl]acetamidine hydrobromide

(5) The suspension of 4 (0.50 g, 2.5 mmol) and triethylamine (0.7 mL) in

EtOH (7.5 mL) was stirred at 0°C in an ice-salt bath. S-2-naphthylmethyl

thioacetimidate hydrobromide25 was added and the reaction was allowed

to stir at rt for 3 hr. The reaction was reduced in vacuo and residue was

dissolved in water (20 mL). The aqueous solution was washed by ether

(2x20 mL) and evaporated. The residue was crystallized in MeOH-ethyl

acetate, giving 5 (0.50 g, 70%) as off-white solid. 1H NMR (DMSO-d6,

200 MHz) δ 9.78 (brs, 1H), 9.24 (brs, 1H), 8.86 (brs, 1H), 8.75 (brs, 1H),

6.92 (d, J = 1.4 Hz, 1H), 6.73 (d, J = 8.0 Hz, , 1H), 6.65 (dd, J =1.4 and

8.0 Hz, 1H), 3.76 (s, 3H), 3.44 (t, J = 7.4 Hz, 2H), 2.74 (t, J = 7.4 Hz, 2H),

2.16 (s, 3H); 13C NMR (DMSO-d6, 50 MHz) δ 164.5, 148.2, 145.9, 129.5,

121.7, 116.1, 113.9, 56.4, 44.2, 33.7, 19.2; MS (CI) : 210, 208 (M+), 191,

168, 151 (100), 138, 121.

(4-Hydroxy-3-methoxyphenyl)acetic acid (6). The mixture of nitrile 3

(4.00 g, 24.6 mmol) and KOH (10g, 178 mmol) in EtOH 200 mL was

reflux for overnight. After cooling, the reaction was acidified by 5% HCl

until pH

56

mixture was extracted with ethyl acetate (2x20 mL). The combined

organic layers were washed by sat. NH4Cl, then dried over and evaporated.

The residue was purified by column chromatography (EtOAc), yielding 7

as solid (0.37 g, 75%). 1H NMR (CDCl3, 200 MHz) δ 6.84 (d, J = 7.6 Hz,

1H), 6.80 (d, J = 1.8 Hz, 1H), 6.68 (dd, J = 1.6 and 8.0 Hz, 1H), 5.73 (s,

1H), 3.86 (s, 3H), 3.64 (s, 2H), 3.55 (dd, J = 4.4 and 5.8 Hz, 2H), 3.38 (dd,

J =5.4, 5.6Hz, 2H), 1.49-1.61 (m, 4H), 1.29 -1.40 (m, 2H); 13C NMR

(CDCl3, 50 MHz) δ 169.7, 146.8, 144.5, 127.3, 121.5, 114.45, 111.1, 56.0,

47.3, 43.0, 40.9, 26.3, 25.6, 24.5. CAS No. 53283-49-1.

2-(4-Hydroxy-3-methoxy-phenyl)-1-pyrrolidin-1-yl-ethanone (8) The

same procedure used as 7, 6 (0.85 g, 4.6 mmol), pyrrolidine (0.50 mL) in

DMF (10 mL), EDCI (1.10 g, mmol) and DMAP (0.12 g) were used. The

reaction gave 8 as off-white solid (0.55 g, 51%) 1H NMR (CDCl3, 200

MHz) δ 6.85 (s, 1H), 6.82 (d, J = 7.6 Hz, 1H), 5.96 (s, 1H), 3.84 (s, 3H),

3.56 (s, 2H), 3.39-3.50 (m, 4H), 1.78-1.93 (m, 4H); 13C NMR (CDCl3, 50

MHz) δ 170.0, 146.9, 144.7, 126.6, 121.8, 114.4, 11.6, 56.0, 47.0, 46.1,

42.0, 26.2, 24.4. CAS No. 131656-82-1.

2-Methoxy-4-(2-piperidin-1-yl ethyl)phenol (9) To solution of 7 (0.37 g,

1.49 mmol) in dry THF (7 mL), 1.0M LiAlH4 in THF (4 mL) was added.

The reaction was refluxed for 2 hr. After cooling, ethyl acetate (1 mL),

water (1 mL), and 2 M NaOH (2mL) were added respectively and stirred

at rt for 30 min. The suspension was filtered through celite and washed by

water and ethyl acetate. The filtrate extracted with ethyl acetate (2x10 mL).

The organic layers were combined, dried with anhydrous NaSO4 and

evaporated in vacuo, giving 9 (0.24 g, 69%) as off-white solid. 1H NMR

(CDCl3, 200 MHz) δ 6.79 (d, J = 8.0 Hz, 1H), 6.68 (s, 1H), 6.66 (dd, J =

57

1.4 and 8.0 Hz, 1H), 3.84 (s, 3H), 2.78-2.71 (m, 2H), 2.57-2.49 (m, 6H),

1.69-1.59 (m, 4H), 1.47-1.45 (m,2H); 13C NMR (CDCl3, 50 MHz) δ 146.9,

144.2, 132.3, 121.2, 114.7, 111.5, 61.8, 55.9, 54.6, 33.2, 25.9, 24.5 –MS

(FAB) 236 (M+H+), 234, 151, 98. HRMS (FAB) Calc. for C14H21NO2

(M+H+) 236.1651. Found 236.1649.

2-Methoxy-4-(2-pyrrolidin-1-yl ethyl)phenol (10) The same procedure

used as 9, 8 (0.40 g, 1.70 mmol) in dry THF (8 mL), 1.0M LiAlH4 in THF

(4 mL) were used. The reaction gave 8 as off-white solid (0.30 g, 80%). 1H NMR (CDCl3, 200 MHz) δ 7.4 (brs, 1H), 6.75 (d, J = 7.8 Hz, 1H), 6.67

(s, 1H), 6.65 (dd, J = 2.0 and 7.6 Hz, 1H), 3.84, s, 3H), 2.60-2.83 (m, 8H),

1.78-1.83 (m, 4H); 13C NMR (CDCl3, 50 MHz) δ 147.1, 144.4, 131.9,

121.1, 114.9, 111.5, 58.8, 55.8, 54.3 (2C), 35.3, 23.47 (2C); MS (FAB)

222 (M+H+, 100), 151, 84. HRMS (FAB) Calc. for C13H19NO2 (M+H+)

222.1494. Found 222.1495.

3-(4-Hydroxy-3-methoxyphenyl)acrylonitrile (12) The suspension of

KOH (1.12 g, 20 mmol) in acetonitrile 20 mL was stirred at rt for 30 min.

The solution of vanillin (1.52 g, 10 mmol) in acetonitrile 10 mL was

added and the reaction was refluxed for overnight. After cooling, the

reaction was added by 50 mL of water and then extracted by

dichloromethane (2x40 mL). The combined organic layer was washed by

5% HCl (40 mL) and brine. The residue was purified by column

chromatography (50% ethyl acetate-hexane), giving 12 as off-white solid

(1.12 g, 83%). 1H NMR (CDCl3, 200 MHz) δ 7.29 (d, J = 16.6 Hz, 1H),

6.90-7.01 (m, 2H), 6.15 (s, 1H), 5.71 (d, J = 16.8 Hz, 1H), 3.92 (s, 3H); 13C NMR (CDCl3, 50 MHz) δ 150.6, 148.9, 147.1, 126.3, 122.6, 118.9,

115.0, 108.9, 93.2, 56.2. CAS No. 71750-09-9.

58

4-(3-Amino-propyl)-2-methoxyphenol hydrochloride (13) The same

procedure used as 4, 12 (0.42 g, 3.11 mmol), 10% palladium on charcoal

(0.18 g), 95% EtOH (10 mL), conc.HCl 0.5 mL) were used. The reaction

gave 13 as off-white solid (0.32 g, 58%). 1H NMR (DMSO-d6, 200 MHz)

δ 8.0 (brs, 2H), 6.74 (d, J = 1.4 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.54 (dd,

J = 1.8 and 8.0 Hz, 1H), 3.70 (s, 3H), 2.68 (t, J = 7.6 Hz, 2H), 2.50 (t, J =

7.6 Hz, 1H), 1.75-1.83 (m, 2H); 13C NMR (DMSO-d6, 100 MHz) δ 148.1,

145.3, 132.2, 121.0, 116.0, 113.1, 56.2, 38.8, 32.0, 29.5. CAS No.

112798-57-9.

N-[3-(4-Hydroxy-3-methoxyphenyl)propyl]acetamide (14) The same

procedure used as 1, 13 (0.20 g,), acetic anhydride (92 mg, 0.92 mmol),

dichloromethane (2 mL) and triethylamine (0.38 mL) were used. The

reaction gave 14 as colorless liquid (0.16 g, 76%). 1H NMR (CDCl3, 200

MHz) δ 6.81 (d, J = 7.6 Hz, 1H), 6.65 (d, J = 1.8 Hz, 1H), 6.62 (dd, J =

1.8 and 8.0 Hz, 1H), 6.41 (brs, 1H), 6.19 (brs, 1H), 3.83 (s, 3H), 3.24 (dt, J

= 6.2 and 6.8 Hz, 2H), 2.55 (t, J = 7.6 Hz, 2H), 1.94 (s, 3H), 1.70-1.85 (m,

2H); 13C NMR (CDCl3, 50 MHz) δ 170.7, 146.8, 144.0, 133.4, 120.8,

114.6, 111.3, 56.0, 39.4, 33.0, 31.4, 23.3; MS (FAB) 224 (M+H+, 100),

182, 164, 136. HRMS (FAB) Calc. for C12H17NO3 (M+H+) 224.1287.

Found 224.1283.

6-Methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline hydrochloride

(15) To solution of 4 (1.0 g, 4.91 mmol) in 1N HCl was added 36%

formaldehyde (2.5 mL) under N2 atmosphere and heated at 70 °C for 1 h.

The solvent was removed by evaporator. The residue was crystallized in

EtOH-ether to yield 15 as pale yellow solid (0.74 g, 70%). 1H NMR

59

(DMSO-d6, 200 MHz) δ 9.39 (br, 2H), 9.08 (br, 1H), 6.75 (s, 1H), 6.60 (s,

1H), 4.07 (brs, 2H), 3.74 (s, 3H), 3.27-3.30 (brm, 2H), 2.88 (t, J= 9.8 Hz,

2H); 13C NMR (DMSO-d6, 50 MHz) δ 146.7, 145.2, 128.6, 125.6, 113.7,

113.3, 56.4, 47.7, 44.1, 28.7. CAS No. 1078-26-8.

7-Hydroxy-6-methoxy-3,4-dihydro-1H-isoquinoline-2-carbaldehyde

(16) Trimethyl orthoformate (2 mL) and conc.HCl (2 drops) were added to

pressure tube which contains 15 (260 mg, 1.2 mmol) under N2. After tube

was tightly capped, mixture had been refluxed for overnight. Mixture was

cooled to room temperature and remaining trimethyl orthoformate was

evaporated. Crude organic was separated by flash column chromatography

(2% methanol-dichloromethane). 16 (224 mg, 90%) was obtained as a off-

white solid: 1H NMR (CDCl3, 200 MHz, a mixture of two isomers) δ 8.23

(s, 1H), 8.17 (s, 1H), 6.67 (s, 1H), 6.64 (s, 1H), 6.60 (s, 1H), 6.58 (s, 1H),

6.11 (s, 1H), 4.57 (s, 1H), 4.42 (s, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.76 (t, J

= 6.4 Hz, 2H), 3.61 (t, J = 5.8 Hz, 2H), 2.74-2.84 (m, 6H); 13C NMR

(CDCl3, 50 MHz, a mixture of two isomers) δ 161.6, 161.2, 145.6, 144.7,

144.4, 125.4, 124.6, 124.1, 112.2, 111.6, 111.1, 110.9, 55.9, 46.9, 43.4,

41.8, 38.1, 29.3, 27.5; MS (EI) 207 (M+, 100), 192, 178, 163, 150, 135,

107, 91, 77, 67, 51. CAS No. 36646-95-4.

(7-Hydroxy-6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-phenyl-

methanone (17) To a solution of 15 (215 mg, 1.0 mmol) in

dichloromethane (8 mL) was added benzoic anhydride (0.9 mmol, 205

mg) and triethylamine (0.45 mL) slowly at room temperature. Reaction

mixture was stirred for 30 min. Reaction was diluted by dichloromethane

and then washed with saturated ammonium chloride solution and water.

Organic layer was dried over sodium sulfate. After then, solvent was

60

evaporated and flash column chromatography gave 17 (184 mg, 65%) as a

yellow solid: 1H NMR (CDCl3, 200 MHz, a mixture of two isomers) δ

7.43 (s, 5H), 6.45-6.74 (br(m), 2H), 5.72 (brs, 1H), 4.77 (brs, 1H), 4.46

(brs, 1H), 3.94 (brs, 1H), 3.86 (s, 3H), 3.60 (brs, 1H), 2.79 (brs, 2H); 13C

NMR (CDCl3, 100 MHz, a mixture of two isomers) δ 170.9, 170.4, 145.5,

144.5, 144.2, 136.1, 129.7, 128.5, 127.1, 126.8, 125.8, 125.3, 124.9, 112.4,

111.5, 110.9, 110.7, 55.9, 49.3, 45.4, 44.3, 40.6, 29.2, 27.8; MS (EI) 283

(M+), 268, 178, 163, 150, 131, 105 (100), 77, 51; HRMS (FAB) Calc. for

C17H17NO3 (M+H+) 284.1287. Found 284.1288.

61

References

1. Marletta, M. A. J. Biol. Chem. 1993, 268, 12231-12234.

2. MacMicking, J.; Xie, Q. W.; Nathan, C. Annu. Rev. Immunol. 1997,

15, 323-350.

3. Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001,

357, 593-615.

4. a) Jacobs, A. T.; Ignarro, L. J. J. Biol. Chem. 2001, 276, 47950-

47957. b) Nathan, C. FASEB J. 1992, 6, 3051-3064.

5. a) Coleman, J. W. Int. Immunopharmacol. 2001, 1, 1397-1406. b)

Eiserich, J. P.; Patel, R. P.; O’Donnell V. B. Molec. Aspects Med.

1998, 19, 221-357.

6. Oddis, C. V.; Finkel, M. S. Am. J. Physiol. 1996, 270, H1864–

H1868.

7. Werner-Felmayer, G.; Werner, E. R.; Fuchs, D.; Hausen, A.;

Reibnegger, G.; Wachter, H. J. Exp. Med. 1990, 172, 1599 –1607.

8. Kasai, K.; Hattori, Y.; Banba, N.; Hattori, S.; Motohashi, S.;

Shimoda, S.; Nakanishi, N.; Gross, S. S. Am. J. Physiol. 1997, 273,

H665–H672.

9. a) Werner-Felmayer, G.; Werne,r E. R.; Fuchs, D.; Hausen, A.;

Reibnegger, G.; Schmidt, K.; Weiss, G.; Wachter, H. J. Biol. Chem.

1993, 268, 1842–1846. b) Rosenkranz-Weiss, P.; Sessa, W. C.;

Milstien, S.; Kaufman, S.; Watson, C. A.; Pober, J. S. J. Clin. Invest.

1994, 93, 2236 –2243.

10. a) Patt, A.; Harken, A. H.; Burton, L. K.; Rodell T. C.; Piermattei D.;

Schorr, J.; Parker, N. B.; Berger, E. M.; Horesh, I. R.; Terada, L. S.;

Linas, S. L.; Cheronis, J. C.; Repine, J.E. J. Clin. Invest. 1988, 81,

62

1556 –1562. b) Kader, A.; Frazzini, V. I.; Solomon, R. A.; Trifiletti,

R. R. Stroke 1993, 24, 1709 –1716.

11. a) Zhang, Z. G.; Chopp, M.; Gautam, S.; Zaloga, C.; Zhang, L.;

Schmidt, H. H.; Pollock, J. S.; Forstermann, U. Brain Res. 1994,

654, 85–95. b) Iadecola, C. Trends Neurosci. 1997, 20, 132–139.

12. a) Buisson, A.; Plotkine, M.; Boulu, R. G. Br. J. Pharmacol. 1992,

106, 766 –767. b) Hamada, Y.; Hayakawa, T.; Hattori, H.; Mikawa,

H. Pediatr. Res. 1994, 35, 10 –14. c) Huang, Z.; Huang, P. L.;

Panahian, N.; Dalkara, T.; Fishman, M. C.; Moskowitz, M. A.

Science 1994, 265, 1883–1885.

13. a) Bonfoco, E.; Zhivotovsky, B.; Rossi, A. D.; Aguilar-Santelises,

M.; Orrenius, S.; Lipton, S. A.; Nicotera, P. Neuro. Report 1996, 8,

273–276. b) Messmer, U. K.; Reimer, D. M.; Reed, J. C.; Brune, B.

FEBS Lett. 1996, 384, 162–166.

14. Fujiwara, N.; Okado, A; Seo, H. G.; Fujii, J.; Kondo, K; Taniguchi,

N. FEBS Letter. 1996, 395, 299-303.

15. Xie, L.; Smith, J. A.; Gross, S. S. J. Biol. Chem. 1998, 273, 21091-

21098.

16. Kinoshita, H.; Tsutsui, M.; Milstien, S.; Katusic, Z. S. Prog. Neuro.

Biol. 1997, 52, 295-302. 17. Frohlich, L. G.; Kotsonis, P.; Traub, H.; Taghavi-Moghadam, S.; Al-Masoudi,

N.; Hofmann, H.; Strobel, H.; Matter, H.; Pfleiderer, W.; Schmidt, H. H. H.

W.; J. Med. Chem. 1999, 42, 4108-4121

18. a) Werner, E.R.; Pitters, E.; Schmidt, K.; Wachter, H.; Werner-

Felmayer, G.; Maye,r B. Biochem. J. 1996, 320, 193-6. b) Bommel,

H. M.; Reif, A.; Frohlich, L. G.; Frey, A.; Hofmann, H.; Marecak, D.

M.; Groehn, V.; Kotsonis, P.; La, M.; Koster, S.; Meinecke, M.;

Bernhardt, M.; Weeger, M.; Ghisla, S.; Prestwich, G. D.; Pfleiderer,

W.; Schmidt, H. H. J. Biol. Chem. 1998, 273, 33142-33149.

63

19. a) Huang, H.; Martasek, P.; Roman, L. J.; Silverman, R. B.; J. Med.

Chem. 2000, 43, 2938-2945. b) Huang, H.; Martasek, P.; Roman, L.

J.; Masters, B. S. S.; Silverman, R. B.; J. Med. Chem. 1999, 42,

3147-3153. c) Collins, J. L.; Shearer, B. G.; Oplinger, J. A.; Lee, S.;

Garvey, E. P.; Salter, M.; Duffy, C.; Burnette, T. C.; Furfine, E. S.; J.

Med. Chem. 1998, 41, 2858-2871.

20. a) Schumann, P.; Collot, V.; Hommet, Y.; Gsell, W.; Dauphin, F.;

Sopkova, J.; MacKenzie, E. T.; Duval, D.; Boulouard, M.; Rault, S.

Bioorg. Med. Chem. Lett. 2001, 11, 1153-6. b) Moormann, A. E.;

Metz, S.; Toth, M. V.; Moore, W. M.; Jerome, G.; Kornmeier, C.;

Manning, P.; Hansen, D. W. Jr.; Pitzele, B. S.; Webber, R. K. Bioorg.

Med. Chem. Lett. 2001, 11, 2651-3. c) Hagmann, W. K.; Caldwell, C.

G.; Chen, P.; Durette, P. L.; Esser, C. K.; Lanza, T. J.; Kopka, I. E.;

Guthikonda, R.; Shah, S. K.; MacCoss, M.; Chabin, R. M.; Fletcher,

D.; Grant, S. K.; Green, B. G.; Humes, J. L.; Kelly, T. M.; Luell, S.;

Meurer, R.; Moore, V.; Pacholok, S. G.; Pavia, T.; Williams, H. R.;

Wong, K. K. Bioorg. Med. Chem. Lett. 2000, 10, 1975-8.

21. Southan, G. J.; Szabo, C. Biochem. Pharmacol. 1996, 51, 383-94.

22. Cho, S.; Volpe, B. T.; Bae, Y.; Hwang, O.; Choi, H. J.; Gal, J.; Park,

L. C. H.; Chu, C. K.; Du, J.; Joh, T. H. J. Neurosci. 1999, 19, 878–

889.

23. Cho, S.; Kim, Y.; Cruz, M. O.; Park, E. M.; Chu, C. K.; Song, G. Y.;

Joh, T.H. GLIA 2001, 33, 324–333.

24. Schwartz, M. A; Zoda, M.; Vishnuvajjala, B.; Mami, I. J. Org. Chem.

1976; 41, 2502-2503.

25. Shearer, B.G.; Oplinger, J. A.; Lee, S. Tetrahedron Lett. 1997, 38,

179-182.

26. Whaley, W. M.; Govindachari, T. R. Org. React. 1951, 6, 151.

64

65

NMR Spectra

N-[2-(4-Hydroxy-3-methoxyphenyl)ethyl]acetamidine hydrobromide

(5) 1H NMR (DMSO-d6, 200 MHz)

13C NMR (DMSO-d6, 50 MHz)

HO

MeOHN

NH

66

2-(4-Hydroxy-3-methoxy-phenyl)-1-piperidin-1-yl-ethanone (7) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

HO

MeO N

O

67

2-(4-Hydroxy-3-methoxy-phenyl)-1-pyrrolidin-1-yl-ethanone (8) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

HO

MeO N

O

68

2-Methoxy-4-(2-piperidin-1-yl ethyl)phenol (9) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

HO

MeO N

69

2-Methoxy-4-(2-pyrrolidin-1-yl ethyl)phenol (10) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

HO

MeO N

70

3-(4-Hydroxy-3-methoxyphenyl)acrylonitrile (12) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

MeO

HO

CN

71

N-[3-(4-Hydroxy-3-methoxyphenyl)propyl]acetamide (14) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

MeO

HO

NH

O

72

6-Methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline hydrochloride

(15) 1H NMR (DMSO-d6, 200 MHz)

13C NMR (DMSO-d6, 50 MHz)

MeO

HONH.HCl

73

7-Hydroxy-6-methoxy-3,4-dihydro-1H-isoquinoline-2-carbaldehyde

(16) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

MeO

HON H

O

74

(7-Hydroxy-6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl)-phenyl-

methanone (17) 1H NMR (CDCl3, 200 MHz)

13C NMR (CDCl3, 50 MHz)

MeO

HON

O

75

PART 3

Electrophilic Aromatic Addition Reaction: AdEAr

76

Introduction

Although a wide variety of electrophilic species can attack

aromatic rings and effect substitution, a single broad mechanism (Figure

1) encompasses the large majority of electrophilic aromatic substitution

reactions through the reversible formation of π and σ complexes.1 In this

mechanism, a complexation of the electrophile with the π-electron system

of the aromatic ring is the first step. This species, called the π-complex,

may or may not be involved directly in the substitution mechanism. π-

Complex formation is, in general, rapidly reversible, and in many cases

the equilibrium constant is small. The π-complex is a donor-acceptor type

complex, with the π electrons of the aromatic ring donating electron

density to the electrophile. No position selectivity is associated with the π-

complex.

E ES

HH

E

a

b

c

d

E+

S

SEAr

HS

σ-complexcation intermediate

addition adduct

H+E

π-complex

e

E+

SH

HEf

and/or

g

Figure 1 The typical mechanism of aromatic substitution reaction

77

In order for a substitution to occur, a “σ-complex” must be formed.

The term σ-complex is used to describe an intermediate in which the

carbon at the site of substitution is bonded to both the electrophile and the

hydrogen is displaced. As the term implies, a σ bond is formed at the site

of substitution. The intermediate is a cyclohexadienyl cation. Its

fundamental structural characteristics can be described in simple MO

terms. The σ-complex is four-π-electron delocalized system that is

electronically equivalent to a pentadienyl cation. There is no longer cyclic

conjugation. The LUMO has nodes at C-2 and C-4 of the pentadienyl

structure, and these positions correspond to the position meta to site of

substitution on the aromatic ring. As result, the positive charge of the

cation is located at the positions ortho and para to the site of substitution.

In electrophilic substitution, the formation of the σ complex is

generally the rate-determining step, with the aromatization occurring

much faster than the addition of the nucleophile to the σ complex

carbocation, but there are exceptions. Some authors indicate that

nucleophile addition proceeds faster than deprotonation,2 but the inability

to isolate the intermediate adducts - due to their rapid rearomatization or

further reaction to multi-addition products - forces investigators to draw

conclusions regarding intermediate identity based solely on structural

information obtained from the products as shown in Scheme 1, 2 and 3.

78

NF

H

ClCH2Cl

-OAcN

CH2Cl2

NCH2Cl2

NF

H

C lNF

H

OAc

AcOF

-HF

N Cl N OAc+ +

F2, AcOH

Scheme 1. The substitution of pyridine via addition intermediate.2a

NCH3

NCS

NCH3

Cl N

O

O

-HCl

NCH3

N

O

O74%

NaHCO3,CHCl3

Scheme 2. The substitution of N-methylpyrrole via addition intermediate.2b

CH3

C l

CH3

C l HH

Cl+

NH2-

CH3

H

-H+

CH3

C l H

NH2H

-HCl

CH3

NH2

C lH2N AlC l3

NH2C l, AlC l3NH2C l + AlC l3

C l+[H2NAlC l3]-

+

67% 13%

Scheme 3. The chlorination and amination of toluene.2c

79

In cases where isolated adducts have been identified, the

intermediates sometimes imply significant mechanistic differences when

compared to the majority of known electrophilic aromatic substitutions.

One example of this is the series of adducts identified in the nitration of

furan (Scheme 4), whose mode of decomposition differs greatly from that

commonly seen in 6-membered systems.3

O

HNO3--AcOH

OO2NAcO-

OO2N OAc

46%

Scheme 4. The electrophilic aromatic addition of furan.

During preparation of 5,7-dibromo-8-methoxyquinaldine4 as a key

intermediate in the synt