Post on 19-Feb-2020
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
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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
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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