Discovery of 5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide (TAK-259) as a Novel, Selective, and Orally Active α1D Adrenoceptor Antagonist with Antiurinary Frequency Effects: Reducing Human Ether-a-go-g

Article abstract of DOI:10.1021/acs.jmedchem.5b01528

A novel structural class of iminopyridine derivative 1 was identified as a potent and selective human α_1D adrenoceptor (α_1D adrenergic receptor; α_1D-AR) antagonist against α_1A- and α_1B-AR through scre

Full text of DOI:10.1021/acs.jmedchem.5b01528

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Article
Discovery of 5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-
dihydropyridine-3-carboxamide (TAK-259), as a Novel, Selective, and
Orally Active #1D Adrenoceptor Antagonist with Anti-urinary Frequency
Effects: Reducing Human Ether-a-go-go-Related Gene (hERG) Liabilities
Nobuki Sakauchi, Yasuhisa Kohara, Ayumu Sato, Tomohiko Suzaki, Yumi Imai, Yuichi Okabe, Shigemitsu
Imai, Reiko Saikawa, Hiroshi Nagabukuro, Haruhiko Kuno, Hisashi Fujita, Izumi Kamo, and Masato Yoshida
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01528 • Publication Date (Web): 08 Mar 2016
Downloaded from http://pubs.acs.org on March 10, 2016
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Page 1 of 43
Journal of Medicinal Chemistry
Discovery
of
5ꢀChloroꢀ1ꢀ(5ꢀchloroꢀ2ꢀ(methylsulfonyl)benzyl)ꢀ2ꢀiminoꢀ1,2ꢀ
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dihydropyridineꢀ3ꢀcarboxamide (TAKꢀ259), as a Novel, Selective, and Orally
Active α1D Adrenoceptor Antagonist with Anti−urinary Frequency Effects:
Reducing Human Ether−a−go−go−Related Gene (hERG) Liabilities
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Authors:
Nobuki Sakauchi,† Yasuhisa Kohara,† Ayumu Sato,† Tomohiko Suzaki,† Yumi Imai,† Yuichi
Okabe,† Shigemitsu Imai,† Reiko Saikawa, † Hiroshi Nagabukuro,† Haruhiko Kuno,† Hisashi Fujita,†
Izumi Kamo,† and Masato Yoshida*,†
Author Affiliations:
†Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26ꢀ1, Muraokaꢀ
Higashi 2ꢀchome, Fujisawa, Kanagawa 251ꢀ8555, Japan
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ABSTRACT
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A novel structural class of iminopyridine derivative 1 was identified as a potent and selective
human α1D adrenoceptor (α1D adrenergic receptor; α1DꢀAR) antagonist against α1Aꢀ and α1BꢀAR
through screening of an inꢀhouse compound library. From initial structureꢀactivity relationship
studies, we found lead compound 9m with hERG K+ channel liability. To develop analogues with
reduced hERG K+ channel inhibition, a combination of siteꢀdirected mutagenesis and docking
studies was employed. Further optimization led to the discovery of (R)ꢀ9s and 9u, which showed
antagonistic activity by a bladder strip test in rats with bladder outlet obstruction, as well as
ameliorated cystitisꢀinduced urinary frequency in rats. Ultimately, 9u was selected as a clinical
candidate. This is the first study to show the utility of iminopyridine derivatives as selective α1DꢀAR
antagonists and evaluate their effects in vivo.
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KEYWORDS iminopyridine, α1D adrenergic receptor antagonist, hERG K+ channel inhibition
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Introduction
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Overactive bladder (OAB) is defined as urinary urgency, typically with urinary frequency
and with or without urinary incontinence.1 More than 10% of the population over 40 years old in
Japan, the U.S., and Europe suffers from OAB, with worldwide numbers steadily increasing.2
Antimuscarinic agents that inhibit detrusor muscle contraction are widely used to treat OAB.3
However, these agents have several issues, including mechanism basedꢀadverse events, such as dry
mouth, constipation, stomach discomfort, and voiding difficulty in patients with benign prostatic
hyperplasia.4 Alternative therapeutic approaches are sought to both avoid side effects and provide
more potent therapeutic options, leading to the development of new drug classes with different
mechanisms.5
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The α1 adrenergic receptors (α1 adrenoceptors, α1ꢀARs) are widely distributed in many
organs, including the cardiovascular system and lower urinary tract, and are involved in sympathetic
responses. Moreover, α1ꢀARs have been an active target of drug discovery research for more than
three decades because of their significant relationship with diseases, including hypertension,
cardiomegaly, and dysuria. α1ꢀARs are classified into three subtypes, α1A, α1B, and α1DꢀARs, which
have different tissue distribution profiles.6 Among these, α1DꢀAR is expressed at higher levels in the
bladder and parasympathetic nucleus of the sacral cord, as compared to other subtypes.7,8
Furthermore, it is known to be involved in urinary tract function. For example, α1DꢀAR knockout
mice show significantly increased bladder capacity and single urine volume per void than wild type
mice.9 Αdditionally, an α1Aꢀ and α1DꢀAR antagonist, tamsulosin, improves OAB symptoms in
patients with benign prostatic hyperplasia.10ꢀ12 Naftopidil monotherapy, which has greater affinity for
α1DꢀAR than for α1AꢀAR, is more effective for storage symptoms, whereas tamsulosin monotherapy
is more effective for voiding symptoms.13 Thus, selective α1DꢀAR antagonists could represent a
novel therapeutic treatment for OAB.
The phenylpiperazine derivatives 3 (BMY 7378) and 4 (SNAP 8719), as well as
phenoxyethylamine derivative 5, are reported to be selective α1DꢀAR antagonists (Figure 1).14, 15
Although 3 is a reference selective α1DꢀAR ligand, it has high binding affinity for the serotonin type
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1A (5ꢀHT1A) receptor. Furthermore, 4 showed better selectivity for α1DꢀAR, with lower binding
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affinity for the 5ꢀHT1A receptor. Nevertheless, there are no selective α1DꢀAR antagonists in clinical
trials. Hence, the identification of selective α1DꢀAR antagonists would provide a useful treatment
strategy and clarify the functional roles of α1DꢀAR in disease pathogenesis. Accordingly, we sought
to identify novel and selective α1DꢀAR antagonists.
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Figure 1. Structures and biological activity of reported α1ꢀARs antagonists (literature data).14, 15
In order to develop novel and selective α1DꢀAR antagonists, we conducted a highꢀthroughput
screen of our chemical library. This led to the identification of the iminopyridine derivative 1, a
known compound for which biological activity has not been described previously in the literature
(Figure 2).16 Compound 1 has a different structure from the known α1ꢀAR antagonists shown in
Figure 1. Additionally, 1 showed low binding affinity for 5ꢀHT1A receptors, while the reported
selective α1DꢀAR antagonist, 3, showed high affinity. Hence, iminopyridine derivative 1 was
considered to be an attractive starting point for lead optimization as a selective α1DꢀAR antagonist.
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Journal of Medicinal Chemistry
In this report, we describe the discovery of a highly potent, selective, and orally active α1DꢀAR
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antagonist, 9u (TAKꢀ259), as a clinical development candidate with antiꢀurinary frequency effects.
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1
1D: Ki = 30 nM
1A: Ki = >2700 nM, 1B: Ki = >1200 nM
5-HT1A: IC50 = >1000 nM
Figure 2. Structure of 1. Ki and IC50 values shown are inꢀhouse data.
Chemistry
The synthesis of various iminopyridine derivatives was conducted by two main routes,
Method A and B, as shown in Scheme 1. Cyclization is a key reaction in Method A, using the
appropriate amine and dihydrofuran 8.16 Acetamide 6aꢀc was treated with mucochloric acid or
mucobromic acid 7 in the presence of aqueous sodium hydroxide (NaOH) to afford dihydrofuranꢀ2ꢀ
one 8aꢀd, followed by amine treatment to obtain iminopyridine derivatives 9bꢀu. Hydrolysis of
iminopyridine 116 with 6 M hydrochloric acid (HCl) afforded pyridone 10. Alkylation is a key
reaction in Method B, using 2ꢀaminopyridine 12 or 14 and appropriate benzylbromide.
Iminopyridine 13 was prepared by acidic hydrolysis of cyanopyridine 11 to afford pyridine
carboxamide 12, followed by alkylation with benzylbromide. The other method used to synthesize
iminopyridine derivatives 15aꢀi is oxidative chlorination of 12 with 30% hydrogen peroxide in the
presence of concentrated HCl, which afforded aminopyridine 14. Iminopyridine derivatives 15aꢀi
were then prepared from 14 by alkylation with the corresponding benzyl bromide.
Scheme 1. Synthesis of iminopyridine derivativesa
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aReagents and conditions: (a) aqueous NaOH, MeOH; (b) amine, Et3N, EtOH, or THF, rt, then
DMSO, 80 °C; (c) amine, K2CO3, EtOH, 80 °C; (d) 6 M HCl, reflux; (e) conc. H2SO4, 100 °C; (f)
benzylbromide, DMF, 80 °C; (g) 30% H2O2, conc. HCl, 0 to 60 °C; (h) R3CH2Br, DMF, 100 °C.
Benzylamines 18 and 21, which contain methylsulfonyl groups for the synthesis of 9u and 9v
in Method A, were prepared as depicted in Scheme 2. Substitution of benzonitrile derivative 16 with
sodium thiomethoxide, followed by reduction with lithium aluminum hydride and treatment with 4
M HCl in ethyl acetate (AcOEt) gave methylsulfanyl derivative 17. Benzylamine 18 was obtained by
oxidation of the sulfanyl group with mꢀchloroperoxybenzoic acid (mCPBA). 5ꢀChloroꢀ2ꢀ
fluorobenzonitrile 19 was converted to methylsulfonyl derivative 20 through the introduction of a
methylsulfanyl moiety, followed by oxidation with Oxone. Reduction of 20 with RaneyꢀCo,
followed by salt formation with 2 M HCl in methanol (MeOH), provided benzylamine hydrochloride
21.
Scheme 2. Synthesis of intermediates 18 and 21a
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Journal of Medicinal Chemistry
Br
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aReagents and conditions: (a) NaSMe, iꢀPr2NEt, Pd2(dba)3, Xantphos, toluene, 90 °C; (b) LiAlH4,
THF, 0 °C to rt; (c) 4 M HCl in AcOEt, MeOH, rt; (d) Boc2O, Et3N, THF, rt; (e) mCPBA, AcOEt,
rt; (f) 4 M HCl in AcOEt, MeOH, 60 °C; (g) NaSMe, DMSO, rt; (h) Oxone, CH3CN, H2O, rt; (i)
RaneyꢀCo, NH4OH, H2, EtOH, MeOH, rt; (j) 2 M HCl in MeOH, EtOH, rt.
Benzylbromides 23, 26, and 28, which contained a carboxamide moiety for the synthesis of
15b, 15d, and 15e in Method B, were synthesized as follows (Scheme 3). Hydrolysis of the cyano
group with concentrated sulfuric acid for benzonitrile 22 afforded benzylbromide 23.
Methylbenzoate 25 was obtained by selective monoꢀhydrolysis of dimethyl ester group for dimethyl
isophtalate 24 and amidation of the carboxyl group. Benzylbromide 26 was prepared by reduction of
the remaining methyl ester to an alcohol and bromination. Hydrolysis of the cyano group with
concentrated sulfuric acid for benzonitrile 27, followed by bromination of the methyl group with Nꢀ
bromosuccinimide, afforded benzylbromide 28.
Scheme 3. Synthesis of intermediate 23, 26 and 28a
aReagents and conditions: (a) conc. H2SO4, 50 °C; (b) 1 M NaOH, MeOH, rt; (c) (COCl)2, DMF,
THF, 0 °C; (d) 8 M NH3 in MeOH, EtOH, 0 °C; (e) LiAlH4, THF, EtOH, rt to 60 °C; (f) Br2, PPh3,
CH3CN, rt; (g) conc.H2SO4, 80 °C; (h) NBS, AIBN, AcOtꢀBu, 80 °C.
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Results and Discussion
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All compounds were evaluated for their affinity for cloned human α1DꢀAR in binding studies,
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and the results are expressed as Ki values. The selectivity for α1DꢀAR over α1Aꢀ and α1BꢀAR was
also evaluated. Inhibition of the human etherꢀaꢀgoꢀgo related gene (hERG) channel was evaluated
using an in vitro Ion Works Quattro study.17
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We conducted structureꢀactivity relationship (SAR) studies for various substituents on the
dihydropyridine ring (Table 1). NꢀEthyl carboxamide derivative 9b, N,Nꢀdimethyl carboxamide
derivative 9c, and pyridineꢀ2ꢀone derivative 10 had low binding affinity for cloned human α1DꢀAR.
The Xꢀray crystal structure of 1, shown in Figure 3, revealed that hydrogen bond formation between
the hydrogen atom of the carboxamide moiety at 3ꢀposition on the dihydropyridine ring and the
nitrogen atom on the imino moiety at 2ꢀposition was observed. Immobilization between the 2ꢀ
position and 3ꢀposition due to the hydrogen bonding was found to be important for compound
potency, based on the SAR analysis. As for the 5ꢀposition on the iminopyridine ring, 1, which
contains chlorine, was found to be more potent than 9d, containing bromine, and 13, which contains
hydrogen. Next, the effects of modifications as the 1ꢀposition on the iminopyridine ring were
investigated. 9e, which has a tertꢀbutylmethyl moiety at this position, showed decreased potency,
whereas 9g (2ꢀfurylmethyl), 9h (2ꢀpyridylmethyl), and 9i (3ꢀpyridylmethyl) exhibited a 5 to 14ꢀfold
decrease in the potency versus 1. Regarding the length of the methylene linker, compound 9j
(phenethyl) and 9k (3ꢀphenylpropyl) showed inhibitory activity equipotent to 1, although
introduction of a phenyl group in 9f, which did not contain a methylene linker, resulted in decreased
potency. These results indicate that a phenyl ring with a methyleneꢀbased linker (length: 1 to 3) was
indispensable for potency.
Table 1. Affinity of dihydropyridine derivatives for human α1DꢀAR
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aReceptor binding affinity of human α1DꢀAR. Ki value is presented as the mean of triplicate
experiments with 95% confidence intervals in parentheses.
Figure 3. Chemical structure and single crystal structure of compound 1
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Based on the SAR study summarized in Table 1, compounds 9j and 9k were found to
possess potency similar to 1. Among these, 1 was selected for further optimization, because it
demonstrated the highest ligand efficiency score (1: 0.570, 9j: 0.559, 9k: 0.495).18 The effects of
introducing substituents into the benzyl group at the iminopyridine 1ꢀposition on α1DꢀAR binding
affinity are displayed in Table 2. Substitution with a chlorine atom on the phenyl ring, 3ꢀchloro
derivative 9m, exhibited the highest inhibitory activity, as compared to the 2ꢀ or 4ꢀchloro derivatives
(9l, 9n). Furthermore, this compound was found to be 10ꢀfold more potent than 1. These results
indicate that the introduction of a substituent at 3ꢀposition on the phenyl ring was important to
increase potency. Next, we evaluated the effect of a substituent at 3ꢀposition. Introduction of methyl
(15a), methoxy (9o), carboxamide (15b), cyano (15c), or methylsulfonyl (9p) group resulted in
equipotent binding affinity compared to 1. These results indicated that introduction of a substituent
on the phenyl ring resulted in potency retention.
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Table 2. Affinity of 5ꢀchloroꢀ3ꢀcarboxamideꢀ2ꢀiminopyridine derivatives with a substituted benzyl
group for human α1DꢀAR
aReceptor binding affinity of human α1DꢀAR. Ki value is presented as the mean of triplicate
experiments with 95% confidence intervals in parentheses.
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Based on the results shown in Table 2, we selected 3ꢀchloro derivative 9m, which exhibited
the highest potency among the monoꢀsubstituted compounds, and evaluated its absorption,
distribution, metabolism, excretion, toxicity (ADMET) profile (Figure 4). Compound 9m displayed
favorable properties in studies investigating cytochrome P450 3A4 (CYP3A4) inhibition, parallel
artificial membrane permeability assay (PAMPA) permeability,19 and metabolic stability in human
liver microsomes. However, this compound exhibited moderate hERG inhibitory activity (47%
inhibition at 10 µM). Accordingly, we focused on the identification of analogues with reduced
hERG liability.
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Figure 4. ADMET profile of 9m.
Several reports indicate that hERG inhibition can be reduced by lowering compound
lipophilicity.20 An effective method to reduce the hERG K+ channel binding affinity might be to
lower lipophilicity by the introduction of hydrophilic substituents. Hence, we sought to find
appropriate substituents for reducing hERG inhibition. A docking study using 9m and the hERG K+
channel was carried out using the results of a tandem siteꢀdirected mutagenesis approach: a tandem
dimer of the hERG K+ channel was constructed, wherein mutation was introduced into one subunit
to determine the residues that bind to 9m. We have previously reported the use of this approach.21
The presumed 9m binding mode to the hERG K+ channels is shown in Figure 5. Our docking study
suggested that the terminal phenyl ring at the iminopyridine ring 1ꢀposition and the carboxamide
group at the 3ꢀposition adopted relative positions suitable for simultaneous interactions with both
Phe656 and the adjacent small pocket. The small pocket is surrounded by three amino acid residues
(Thr623, Ser624, and Val625), and is occupied by a carboxamide moiety. The initial SAR study
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using substituents on the dihydropyridine derivative (Table 1) indicated that the carboxamide group
at 3ꢀposition was requisite for potency. Thus, we decided to avoid this unfavorable interaction with
Phe656 by further modifying 1ꢀposition of the iminopyridine ring and/or lowering the overall
compound lipophilicity. Our approaches to attenuate 9m–induced hERG inhibition are described in
Figure 5: (i) steric changes through the introduction of a methyl group at the benzyl position to avoid
interaction with Phe656; (ii) changes in pi electron density on the phenyl ring by introduction of a
polar moiety into the phenyl ring of 3ꢀchlorobenzyl derivative 9m, as well as modification of the
overall compound lipophilicity, as needed.
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Ser624
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Figure 5. A) Presumed 9m (black) binding to both hERG K+ channel models and hypothetical
modifications to avoid hERG binding (blue). B) The binding mode for 9m shown by MOE.22
To attenuate hERG inhibition by 9m, we prepared the compounds shown in Table 3. We
first introduced a methyl group at the benzylic position, thereby inducing a steric change. The 3ꢀ
chlorophenethyl moiety 9q showed 4ꢀfold higher binding affinity for α1DꢀAR, but similar hERG
inhibitory activity to 9m. Interestingly, these results indicated that the introduction of a methyl group
at the benzyl position was effective at enhancing α1DꢀAR affinity, without increasing hERG
inhibition, despite the higher lipophilicity of 9q. Thus, we postulated that the introduction of a polar
substituent in place of chlorine on the phenyl ring of 9q would be result in retained potency and
altered hERG inhibition. Indeed, 3ꢀmethylsulfonylphenethyl derivative 9r and 3ꢀcyanophenethyl
derivative 9s (clogP = ꢀ0.25 for 9r, clogP = 0.82 for 9s)23 both exhibited a lower clogP values than
9q (clog P = 2.10), and tended to have low hERG inhibitory activity. Additionally, we evaluated the
difference between the two enantiomers 3ꢀcyanophenethyl derivative 9s. The eutomer, (R)ꢀ9s,
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showed retention of in vitro activity with decreased hERG inhibition. Other optically resolved chiral
derivatives tended to yield similar results (data not shown). On the other hand, the calculated pKa
values for these compounds did not correlate with the hERG inhibitory activity.23
Table 3. Affinity for human α1DꢀAR and hERG inhibitory activity of 1ꢀphenethyl derivatives
1
2
3
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5
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aReceptor binding affinity of human α1DꢀAR. Ki value is presented as the mean of triplicate
b
experiments with 95% confidence intervals in parentheses. Percent inhibition is represented as
mean ± standard deviation. ccLogP value determined using Daylight software. dpKa value calculated
e
by using Accelrys software. Percent inhibition is represented as the average of duplicate
measurements (n = 2).
A second approach to attenuate 9m–induced hERG inhibition was the introduction of a polar
moiety in the mꢀchlorophenyl ring to modulate the πꢀelectron density (Table 4). Carboxamide (15d,
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Journal of Medicinal Chemistry
15e) and methylsulfonyl derivatives (9t, 9u and 15f) showed reduced hERG inhibition, while the the
corresponding cyano derivatives demonstrated increased hERG inhibiton. These data indicated that
the type of polar substituent selected would have an effect on the hERG inhibitory activity of these
3ꢀchlorobenzyl derivatives. Furthermore this effect did not depend strongly on the substituted
position. In addition, the benzonitrile derivatives possessed potential hERG liabilities, despite the
clogP values being lower than 9m. Regarding the binding affinity, 15d (R3 = carboxamide) exhibited
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lower potency for α1DꢀAR and carboxamide derivative 15e (R2 = carboxamide) showed equipotent
affinity for α1DꢀAR compared to 9m. Compound 15h (R2 = cyano) showed higher potency than 9m,
whereas 15g (R3 = cyano) or 15i (R1 = cyano) possessed lower binding affinity for α1DꢀAR.
Methylsulfonyl groups at R2 or R3 (9t and 15f, respectively) resulted in decreased binding affinity,
while compound 9u (R1 = methylsulfonyl) showed α1DꢀAR potency retention.
Table 4. Affinity for α1DꢀAR and hERG inhibitory activity of 2ꢀiminopyridine derivatives containing
a substituted 3ꢀchlorobenzyl moiety
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aReceptor binding affinity of human α1DꢀAR. Ki value is presented as the mean of triplicate
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2
3
b
experiments with 95% confidence intervals in parentheses. Percent inhibition is represented as
4
5
mean ± standard deviation. ccLogP value determined using Daylight software.
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Based on the in vitro study results shown in Tables 3 and 4, we selected (R)ꢀ9s, 9u, and 15e
for further evaluation. The ARꢀsubtype selectivity and pharmacokinetic parameters were
investigated, and the results are shown in Table 5. These compounds showed higher selectivity for
α1DꢀAR over α1Aꢀ and α1BꢀARs. Indeed, the selectivity of each compound over both α1Aꢀ and α1Bꢀ
ARs was more than 200ꢀfold. In terms of pharmacokinetic parameters, the 3ꢀcyanophenethyl
derivative (R)ꢀ9s and 2ꢀmethylsulfonylbenzyl derivative 9u showed higher plasma concentrations
and better oral bioavailability at a dose of 1 mg/kg than 15e. In particular, 9u exhibited the longest
mean residence time (MRT)po value and would be promising in terms of duration of action.
Table 5. Selectivity and pharmacokinetic profiles for (R)ꢀ9s, 9u and 15e
comp. Bindinga
Selectivity
Pharmacokinetic parameters in ratb, c
b
c
c
c
Ki
Vdssb
CLtotal
Cmax
AUCpo
MRTpo
(h)
Fc
(nM)
1A/1D
1B/1D
>750
800
(mL/kg) (mL/h/kg) (ng/mL) (ng·h/mL)
(%)
1.6
(R)ꢀ9s
>1700
200
4676
4432
2350
2395
142
368
358
3.14
4.53
86.0
84.5
(1.4–1.9)
1.1
60.2
9u
(0.91–1.3) (190–250) (690–1100)
3.7
>730
>320
3212
2830
3.20
10.8
2.47
3.0
15e
(3.0–4.5)
aReceptor binding affinity of human α1DꢀAR. Ki value is presented as the mean of triplicate
experiments with 95% confidence intervals in parentheses.
b0.1 mg/kg, iv. Solvent: DMA/1,3ꢀbutanediol = 1:1 (v/v).
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Journal of Medicinal Chemistry
c1 mg/kg, po (fed). Solvent: 0.5% methylcellulose suspension. All values are the average of
three rats.
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9
Given the selectivity and pharmacokinetic profiles (Table 5), 9u and (R)ꢀ9s were chosen for
in vitro and in vivo evaluation (Table 6). The effects of these compounds on phenylephrineꢀinduced
bladder contraction in isolated bladder strips taken from rats with bladder outlet obstruction (BOO)
were measured, and the results were expressed as IC30 values. Phenylephrineꢀinduced bladder
contractions were obvious in strips from rats with BOO, but not in normal rats. Both 9u and (R)ꢀ9s
doseꢀdependently inhibited bladder contractions, with IC30 values of 12 nM and 15 nM, respectively.
In rats with BOO, the nonꢀvoiding bladder contractions observed during urinary storage phase were
doseꢀdependently decreased by both 9u and (R)ꢀ9s, with ID50 values of 1.3 µg/kg, i.v. and 4.4 µg/kg,
i.v., respectively. In rats with cyclophosphamideꢀinduced cystitis, which induces significantly higher
urinary frequency, the compounds increased voiding intervals, with similar minimum effective doses
(10 µg/kg, p.o.). These results demonstrated that 1) both the phenylephrineꢀinduced contractions of
rat bladder strips in vitro and the nonꢀvoiding bladder contractions in vivo were mediated by
stimulation of α1DꢀARs, and that 2) α1DꢀAR antagonists effectively reduced urinary frequency. As
phenylephrine induces similar contractions in bladder strips taken from patients with benign
prostatic hyperplasia24, and involuntary bladder contractions are observed during the urinary storage
phase in some OAB patient populations,25 α1DꢀAR antagonists may inhibit these contractions in
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patients. Thus, selective α1DꢀAR antagonists may alleviate the symptoms of OAB. However, as
OAB is a disease induced by various causes, the percentage of patients who could be treated by this
drug remains uncertain. Clinical studies with a selective α1DꢀAR antagonist are needed to clarify this
point.
Table 6. In vivo pharmacological data for 9u and (R)ꢀ9s
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Phenylephrineꢀinduced
Nonꢀvoiding contractionsb
ID50
Urinary frequencyc
MEDd
contractionsa
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4
compound
IC30
5
6
7
8
(nM)
(µg/kg, iv)
1.3
(µg/kg, po)
10
12
15
9u
[0.79ꢀ2.1]
4.4
9
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(R)ꢀ9s
10
[0.80ꢀ16]
aEffects on the phenylephrineꢀinduced bladder contractions in rats with BOO (n = 6 − 8).
bEffects on nonꢀvoiding bladder contractions in rats with BOO (n = 5). ID50 values are presented as
the dose required to induce a 50% reduction in nonꢀvoiding bladder contractions with 95%
confidence intervals in parentheses.
cCyclophosphamide induced urinary frequency in rats (n = 5 − 10).
dMED = minimum effective dose
Conclusion
We identified iminopyridine derivative 1 as a novel, selective α1DꢀAR antagonist. Initial
SAR studies revealed that the 2ꢀiminopyridineꢀ3ꢀcarboxamide structure was critical for binding
affinity, and that substitution of the benzyl moiety at the iminopyridine ring 1ꢀposition was tolerated
in terms of potency. In order to enhance α1DꢀAR binding activity, we modified the benzyl group, and
found that 3ꢀchlorobenzyl derivative 9m was more potent than 1. However, this compound showed
moderate hERG inhibitory activity. To attenuate the hERG inhibition, a docking study of compound
9m and the hERG K+ channel was performed to characterize the binding site and binding mode
using a tandem siteꢀdirected mutagenesis approach. The docking study indicated that the
carboxamide group at 3ꢀposition and phenyl group at the 1ꢀposition interacted with the hERG K+
channel. Because initial SARs suggested that the carboxamide group was important for inhibitory
activity against α1DꢀAR, we decided to weaken the interaction with the phenyl group at 1ꢀposition.
Thus, we introduced polar substituents into the phenyl ring to decrease hERG inhibitory activity. We
thus identified lead compounds 15e, 9u, and (R)ꢀ9s. 9u and (R)ꢀ9s showed higher selectivity for
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α1DꢀAR over α1Aꢀ and α1BꢀAR, preferable pharmacokinetic parameters, and in vitro and in vivo
effects in rats, suggesting potential efficacy in patients with urinary frequency. In particular, 9u
exhibited a larger MRTpo value and more potent antiꢀurinary frequency efficacy than (R)ꢀ9s. We
selected 9u as a clinical candidate and our results indicated that 9u represents a promising novel
therapeutic agent for the treatment of OAB symptoms.
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Experimental Section
Chemistry
Melting points were determined on a BÜCHI Bꢀ545 melting point apparatus and were
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker Ultra
Shieldꢀ300 (300 MHz) instruments. Carbon nuclear magnetic resonance (13C NMR) spectra were
recorded on Bruker Avance II 600 (600 MHz) instruments. Chemical shifts are given in parts per
million (ppm) with tetramethylsilane as an internal standard. Abbreviations are used as follows: s =
singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets, br = broad. Coupling
constants (J values) are given in hertz (Hz). Elemental analyses were carried out by Takeda
Analytical Laboratories, Ltd., and were within ±0.4% of the theoretical values unless otherwise
noted. Lowꢀresolution mass spectra (MS) were determined on a Shimadzu UFLC/MS (Prominence
UFLC high pressure gradient system/LCMSꢀ2020) with an Lꢀcolumn 2 ODS (3.0 x 50 mm I.D.,
CERI, Japan), and Waters Liquid Chromatography–Mass Spectrometer System (MS), using a
CAPCELL PAK UGꢀ120 ODS (Shiseido Co., Ltd.) column (2.0 mm i.d. × 50 mm) with aqueous
CH3CN (10–95%) containing 0.05% trifluoroacetic acid (TFA), or an HPꢀ1100 (Agilent
Technologies) apparatus for monitoring at 220 nm. All MS experiments were performed using
electrospray ionization (ESI) in positive ion mode. HPLC separation was carried out using a Gilson
system employing the following conditions; Column: Shiseido Capcelpak C18 UGꢀ120, Sꢀ5 ꢁM, 20
x 50 mm or YMC CombiPrep Hydrosphere C18 HSꢀ340ꢀCC, Sꢀ5 ꢁM, 20 x 50 mm; Mobile phase:
A: 0.1% trifluoroacetic acid in water, B: 0.1% trifluoroacetic acid in acetonitrile; gradient cycle:
0.00 min (A/B＝95/5), 1.10 min (A/B＝95/5), 5.00 min (A/B＝0/100), 6.40 min (A/B＝0/100),
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6.50 min (A/B＝95/5); flow rate; 20 ml/min; detection UV 200 nm. After purification by preparative
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9
HPLC, the solutions were neutralized by PLꢀHCO3 MP tube (200 mg cartridge, Polymer
Laboratories Ltd.). Reagents and solvents were obtained from commercial sources and used without
further purification. Reaction progress was determined by thin layer chromatography (TLC) analysis
on Merck Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic purification was
carried out on silica gel columns [(Merck Kieselgel 60, 70–230 mesh or 230–400 mesh, Merck) or
(Chromatorex NHꢀDM 1020, 100–200 mesh)] or on PurifꢀPack (SI ɸ 60 ꢁM or NH ɸ 60 ꢁM, Fuji
Silysia Chemical, Ltd.). Each compound was confirmed to be ≥95% purity by either LC/MS or
elemental analysis. Yields are not optimized.
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2ꢀCyanoꢀ2ꢀ(3,4ꢀdichloroꢀ5ꢀoxoꢀ2,5ꢀdihydrofuranꢀ2ꢀyl)acetamide (8a). To an iceꢀcooled
mixture of 2ꢀcyanoacetamide (6a) (25.3 g, 0.30 mol) and mucochloric acid (7) (50.4 g, 0.30 mol) in
MeOH (180 mL) was added a solution of sodium hydroxide (18.0 g, 0.45 mol) in water (180 mL) at
0 °C, and the resulting mixture was stirred at room temperature for 3 h. The mixture was poured into
ice and 1 M HCl (200 mL), concentrated in vacuo and extracted with AcOEt. The extract was
washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was crystallized from
1
EtOHꢀiPr2O to give the title compound (14.3 g, 20%) as pale brown solids. HꢀNMR (DMSOꢀd6) δ
4.84 (d, J = 3.2 Hz, 1H), 5.91 (d, J = 4.0 Hz, 1H), 7.85 (br. s., 1H), 8.03 (br. s., 1H). mp 162–163 °C.
2ꢀCyanoꢀ2ꢀ(3,4ꢀdichloroꢀ5ꢀoxoꢀ2,5ꢀdihydrofuranꢀ2ꢀyl)ꢀNꢀethylacetamide (8b). To an iceꢀ
cooled mixture of mucochloric acid (3.00 g, 17.8 mmol) and 2ꢀcyanoꢀNꢀethylacetamide (6b) (2.22 g,
19.8 mmol) in MeOH (10 mL) was added dropwise a solution of sodium hydroxide (1.08 g, 27.0
mmol) in water (10 mL), and the resulting mixture was stirred at room temperature for 18 h. The
mixture was poured into ice and 1 M HCl (20 mL), and concentrated in vacuo. The residue was
dissolved in AcOEt. The solution was washed with brine and dried over MgSO4. The solvent was
evaporated in vacuo to give the title compound as a pale brown amorphous solids. This product was
used for the next reaction without further purification.
2ꢀCyanoꢀ2ꢀ(3,4ꢀdichloroꢀ5ꢀoxoꢀ2,5ꢀdihydrofuranꢀ2ꢀyl)ꢀN,Nꢀdimethylacetamide (8c). To an
iceꢀcooled mixture of mucochloric acid (5.00 g, 29.6 mmol) and 2ꢀcyanoꢀN,Nꢀdimethylacetamide
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Journal of Medicinal Chemistry
(6c) (3.70 g, 33.0 mmol) in MeOH (18 mL) was added dropwise a solution of sodium hydroxide
(1.80 g, 45.0 mmol) in water (18 mL), and the resulting mixture was stirred at room temperature for
3 h. The mixture was poured into ice and 1 M HCl (20 mL), concentrated in vacuo and dissolved in
AcOEt. The solution was washed with brine and dried (MgSO4). The solvent was evaporated in
vacuo to give the title compound as a brown syrup. This material was used for the next reaction
without further purification.
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2ꢀCyanoꢀ2ꢀ(3,4ꢀdibromoꢀ5ꢀoxoꢀ2,5ꢀdihydrofuranꢀ2ꢀyl)acetamide (8d). To an iceꢀcooled
mixture of mucobromic acid (5.00 g, 19.4 mmol) and 2ꢀcyanoacetamide (6a) (1.63 g, 19.4 mmol) in
MeOH (18 mL) was added a solution of sodium hydroxide (1.16 g, 29.0 mmol) in water (18 mL),
and the resulting mixture was stirred at room temperature for 4 h. The mixture was poured into ice
and 1 M HCl (200 mL), concentrated in vacuo and extracted with AcOEt. The extract was washed
with brine and dried over MgSO4. The solvent was evaporated in vacuo to give the title compound
as a dark brown tar. This material was used for the next reaction without further purification.
1ꢀBenzylꢀ5ꢀchloroꢀNꢀethylꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9b). To a solution of
8b (1.00g, 3.80 mmol) in EtOH (10 mL) was added benzylamine (0.61 g, 5.69 mmol). After stirring
at room temperature for 8 h, Et3N (0.58 g, 5.73 mmol) was added. The resulting mixture was stirred
at room temperature for further 48 h and concentrated in vacuo. The residue was dissolved in DMSO
(10 ml) and stirred at 80 °C for 1 h. The reaction mixture was concentrated in vacuo, and the residue
was purified by preparative HPLC to afford the title compound (0.21 g, 19%) as a pale yellow solid.
1HꢀNMR (CDCl3): δ 1.13 (t, J = 7.3 Hz, 3H), 3.35–3.46 (m, 2H), 4.89 (s, 2H), 7.20 (d, J = 7.2 Hz,
13
2H), 7.24 (d, J = 2.6 Hz, 1H), 7.33–7.46 (m, 3H), 8.26 (d, J = 2.6 Hz, 1H), 11.1 (br. s., 1H). Cꢀ
NMR (DMSOꢀd6): δ 14.0, 34.2, 56.0, 117.4, 117.5, 127.4, 128.5, 128.8, 132.5, 140.8, 142.4, 151.8,
163.1. LCꢀMS (ESI): m/z [M + H+] calcd, 290.1; found, 290.2. purity: 100%. mp 140–143 °C.
1ꢀBenzylꢀ5ꢀchloroꢀ2ꢀiminoꢀN,Nꢀdimethylꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9c). To a
solution of 8c (3.95 g, 15.0 mmol) in EtOH (50 mL) was added benzylamine (4.82 g, 45.0 mmol).
After stirring at room temperature for 18 h, Et3N (4.55 g, 45.0 mmol) was added. The resulting
mixture was stirred at room temperature for further 5 h and concentrated in vacuo. A solution of the
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residue in DMSO (20 mL) was stirred at 60 °C for 1 h. The mixture was concentrated in vacuo, and
the residue was purified by preparative HPLC to afford the title compound (0.12 g, 3%) as a yellow
1
2
3
4
5
1
amorphous solid. HꢀNMR (CDCl3): δ 3.03 (br. s., 6H), 5.10 (s, 2H), 6.86 (d, J = 2.3 Hz, 1H), 7.02
6
7
8
9
(d, J = 2.3 Hz, 1H), 7.28–7.41 (m, 5H). 13CꢀNMR (DMSOꢀd6): δ 34.7, 37.8, 56.2, 118.2, 124.1,
127.4, 128.5, 128.9, 132.6, 138.7, 140.7, 149.5, 162.3. LCꢀMS (ESI): m/z [M + H+] calcd, 290.1;
found, 290.0. purity: 98.0%.
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1ꢀBenzylꢀ5ꢀbromoꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9d). To a solution of 8d
(15.0 mmol) in EtOH (50 mL) was added benzylamine (4.82 g, 45.0 mmol). After stirring at room
temperature for 18 h, Et3N (4.55 g, 45.0 mmol) was added. The resulting mixture was stirred at
room temperature for further 5 h and concentrated in vacuo. A solution of the obtained compound in
DMSO (20 mL) was stirred at 60 °C for 1 h. The mixture was concentrated in vacuo, and the residue
was purified by preparative HPLC to afford the title compound (10 mg, 0.34%) as a pale orange
1
amorphous solid. HꢀNMR (CDCl3): δ 4.90 (s, 2H), 5.84 (br. s., 1H), 7.21 (d, J = 7.2 Hz, 1H), 7.30–
7.46 (m, 5H), 8.33 (br. s., 1H), 10.8 (br. s., 1H). 13CꢀNMR (DMSOꢀd6): δ 55.8, 103.6, 116.7, 127.3,
128.5, 128.8, 132.6, 143.1, 145.0, 152.2, 165.6. LCꢀMS (ESI): m/z [M + H+] calcd, 306.0; found,
306.2 . purity: 98.4%.
5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(3ꢀchlorobenzyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
hydrochloride
(9m). To an iceꢀcooled solution of 8a (0.50 g, 2.10 mmol) in THF (10 mL) was added a mixture of
3ꢀchlorobenzylamine (0.45 g, 3.20 mmol) and Et3N (0.32 g, 3.20 mmol) in THF (3 mL). The
reaction mixture was stirred for 18 h at room temperature and concentrated in vacuo to give a
crystalline product. The product was added DMSO (5 mL) and water (5 mL) at room temperature.
The mixture was stirred for 1 h at 80 °C and concentrated in vacuo. The residue was purified by
preparative HPLC to give as a pale yellow amorphous. This amorphous was dissolved in MeOH (1
mL), and 4 M HCl in AcOEt (1 mL) was added at room temperature. The resulting precipitate was
collected by filtration and recrystallized from EtOH and AcOEt to give the title compound (48.2 mg,
7%) as a colorless solid. 1HꢀNMR (DMSOꢀd6) δ 5.61 (s, 2H), 7.23–7.30 (m, 1H), 7.43–7.47 (m, 2H),
7.50 (s, 1H), 8.19 (br. s., 1H), 8.67–8.74 (m, 2H), 8.83 (d, J= 1.8 Hz, 1H), 9.55 (br. s., 2H). 13Cꢀ
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NMR (DMSOꢀd6): δ 55.3, 116.9, 117.4, 125.9, 127.5, 128.4, 130.7, 133.4, 135.0, 141.3, 142.9,
152.3, 165.8. LCꢀMS (ESI): m/z [M + H+] calcd, 296.0; found, 296.0. purity: 100%. mp 160−163 °C.
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Compounds 9eꢀl and 9nꢀp were prepared in a manner similar to that described for the synthesis of
9m, and salt formation conducted as needed to provide crystalline solid.
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5ꢀChloroꢀ1ꢀ(2,2ꢀdimethylpropyl)ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9e). Yield
3%. 1HꢀNMR (CDCl3): δ 1.05 (s, 9H), 3.54 (s, 2H), 5.82 (br. s., 1H), 6.36 (br. s., 1H), 7.08 (d, J =
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2.6 Hz, 1H), 8.20 (d, J = 2.6 Hz, 1H), 11.1 (br. s., 1H). CꢀNMR (DMSOꢀd6): δ 26.5, 34.2, 61.6,
116.1, 116.2, 141.9, 142.7, 152.8, 165.9. LCꢀMS (ESI): m/z [M + H+] calcd, 242.1; found, 242.3.
purity: 99.2%.
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5ꢀChloroꢀ2ꢀiminoꢀ1ꢀphenylꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9f). Yield 10%. HꢀNMR
(CDCl3): δ 5.82 (br. s., 1H), 6.17 (br. s., 1H), 7.19 (d, J = 2.6 Hz, 1H), 7.31–7.36 (m, 2H), 7.53–7.65
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(m, 3H), 8.29 (d, J = 2.8 Hz, 1H), 10.9 (br. s., 1H). CꢀNMR (DMSOꢀd6): δ 103.5, 108.1, 117.2,
123.6, 127.5, 129.3, 129.7, 130.5, 139.2, 140.1, 154.0, 164.3. LCꢀMS (ESI): m/z [M + H+] calcd,
248.1; found, 248.2. purity: 98.6%.
5ꢀChloroꢀ1ꢀ(furanꢀ2ꢀylmethyl)ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9g). Yield 8%.
1HꢀNMR (CDCl3): δ 4.82 (s, 2H), 5.83 (br. s., 1H), 6.40–6.42 (m, 1H), 6.43–6.46 (m, 1H), 7.21 (d, J
= 2.6 Hz, 1H), 7.24–7.29 (m, 1H), 7.46 (d, J = 1.3 Hz, 1H), 8.18 (d, J = 2.4 Hz, 1H), 10.81 (br. s.,
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1H). CꢀNMR (DMSOꢀd6): δ 49.1, 110.3, 111.4, 115.9, 116.6, 140.3, 142.5, 144.1, 144.6, 151.2,
165.1. LCꢀMS (ESI): m/z [M + H+] calcd, 252.1; found, 252.2. purity: 99.5%.
5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(pyridinꢀ2ꢀylmethyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9h). Yield
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9%. HꢀNMR (CDCl3) δ 4.98 (s, 2H), 5.79 (br. s., 1H), 6.59 (br. s., 1H), 7.22 (d, J = 7.8 Hz, 1H),
7.30 (dd, J = 5.0, 7.5 Hz, 1H), 7.39 (d, J = 2.4 Hz, 1H), 7.70–7.78 (m, 1H), 8.24 (br. s., 1H), 8.63 (d,
J = 4.9 Hz, 1H), 10.94 (br. s., 1H). 13CꢀNMR (DMSOꢀd6): δ 55.9, 116.4, 116.9, 123.5, 124.4, 140.1,
142.1, 143.2, 147.0, 150.1, 152.7, 165.7. LCꢀMS (ESI): m/z [M + H+] calcd, 263.1; found, 262.7.
purity: 100%.
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5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(pyridinꢀ3ꢀylmethyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9i). Yield
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17%. HꢀNMR (CDCl3): δ 4.95 (s, 2H), 5.84 (br. s., 1H), 6.22 (br. s., 1H), 7.26–7.28 (m, 1H), 7.36
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(dd, J = 4.7, 7.9 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 8.23 (br. s., 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.62–
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8.65 (m, 1H), 10.84 (br. s., 1H). CꢀNMR (DMSOꢀd6): δ 53.1, 116.6, 117.5, 126.9, 132.6, 141.0,
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141.2, 141.5, 143.1, 145.0, 152.7, 165.7. LCꢀMS (ESI): m/z [M + H+] calcd, 263.1; found, 263.2.
purity: 100%.
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5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(2ꢀphenethyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9j). Yield 23%. Hꢀ
NMR (CDCl3): δ 3.03 (t, J = 7.2 Hz, 2H), 3.95 (t, J = 7.2 Hz, 2H), 5.86 (br.s., 1H), 6.80 (d, J = 2.6
Hz, 1H), 7.12–7.17 (m, 2H), 7.19–7.48 (m, 4H), 8.14 (d, J = 2.4 Hz, 1H), 10.96 (br.s., 1H). 13Cꢀ
NMR (DMSOꢀd6): δ 32.3, 54.6, 115.9, 116.5, 126.9, 128.3, 129.2, 136.1, 141.3, 142.4, 151.7, 165.8.
LCꢀMS (ESI): m/z [M + H+] calcd, 276.1; found, 276.2. purity: 99.5%.
5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(2ꢀphenylpropyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (9k). Yield 21%.
1HꢀNMR (CDCl3): δ 2.05–2.15 (m, 2H), 2.74 (t, J = 7.3 Hz, 2H), 3.67 (d, J = 7.0 Hz, 2H), 5.86 (br.
s., 1H), 7.06 (d, J = 2.6 Hz, 1H), 7.16–7.29 (m, 4H), 7.30–7.37 (m, 2H), 8.18 (d, J = 2.6 Hz, 1H),
10.97 (br. s., 1H). 13CꢀNMR (DMSOꢀd6): δ 28.4, 31.3, 54.0, 115.9, 116.8, 125.9, 128.1, 128.2, 140.6,
141.4, 142.3, 151.7, 165.8. LCꢀMS (ESI): m/z [M + H+] calcd, 290.1; found, 290.1. purity: 97.9%.
5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(2ꢀchlorobenzyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide trifluoroacetate
(9l). Yield 5%. To a solution of 8a (23.5 mg, 0.10 mmol) in EtOH (1 mL) was added a mixture of 2ꢀ
chlorobenzylamine (17.0 mg, 0.12 mmol) and triethylamine (25 µL, 0.18 mmol) in EtOH (0.5 mL)
and stirred at room temperature for 18 hr. The mixture was purified by preparative HPLC to give the
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title compound (2.1 mg, 5%). HꢀNMR (DMSOꢀd6): δ 5.53 (s, 2H), 6.79–6.86 (m, 1H), 7.28－7.38
(m, 1H), 7.44 (td, J = 7.7, 1.5 Hz, 1H), 7.58–7.65 (m, 1H), 8.25 (s, 1H), 8.62 (br.s., 1H), 8.66 (q, J =
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2.4 Hz, 2H), 9.22–9.90 (m, 2H). CꢀNMR (DMSOꢀd6): δ 54.8, 116.3, 117.0, 117.2, 118.3, 126.3,
127.6, 129.7, 130.2, 132.0, 141.1, 142.9, 152.7, 157.5, 165.8. LCꢀMS (ESI): m/z [M + H+ –
(CF3CO2H)] calcd, 296.0; found, 296.0. purity: 100%.
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5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(4ꢀchlorobenzyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide trifluoroacetate
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(9n). Yield 7%. HꢀNMR (DMSOꢀd6) δ ppm 5.52 (s, 2H), 7.30–7.36 (m, 2H), 7.47–7.52 (m, 2H),
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8.21 (s, 1H), 8.56–8.62 (m, 2H), 8.78 (d, J = 2.2 Hz, 1H), 9.21–9.60 (m, 2H). CꢀNMR (DMSOꢀ
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d6): δ 55.3, 113.3, 115.3, 117.0, 117.2, 117.3, 128.8, 129.2, 131.4, 133.2, 141.2, 142.8, 152.2, 157.9,
165.7. LCꢀMS (ESI): m/z [M + H+ – (CF3CO2H)] calcd, 296.0; found, 296.0. purity: 100%.
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5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(3ꢀmethoxybenzyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide hydrochloride
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(9o). Yield 16%. HꢀNMR (DMSOꢀd6): δ 3.76 (s, 3H), 5.53 (s, 2H), 6.79–6.87 (m, 1H), 6.92–7.00
(m, 2H), 7.28–7.39 (m, 1H), 8.19 (s, 1H), 8.66 (d, J = 1.9 Hz, 2H), 8.79 (d, J = 2.3 Hz, 1H), 9.47 (br.
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s., 2H). CꢀNMR (DMSOꢀd6): δ 55.1, 55.8, 113.6, 113.8, 116.7, 117.2, 119.2, 130.1, 134.0, 141.1,
142.9, 152.2, 159.4, 165.8. LCꢀMS (ESI): m/z [M + H+ – (HCl)] calcd, 292.1; found, 292.2. purity:
98.9%.
5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ[3ꢀ(methylsulfonyl)benzyl]ꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
hydrochloride (9p). Yield 10%. 1HꢀNMR (DMSOꢀd6): δ 3.24 (s, 3H), 5.66 (s, 2H), 7.57 (d, J = 8.0
Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.88–8.05 (m, 2H), 8.20 (s, 1H), 8.55–8.70 (m, 2H), 8.85 (d, J =
2.3 Hz, 1H), 9.33–9.68 (m, 2H). 13CꢀNMR (DMSOꢀd6): δ 43.4, 55.4, 116.9, 117.3, 126.2, 126.9,
130.0, 132.0, 134.1, 141.2, 141.4, 143.0, 152.4, 165.8. LCꢀMS (ESI): m/z [M + H+ – (HCl)] calcd,
340.0; found, 339.8. purity: 100%.
5ꢀChloroꢀ1ꢀ(1ꢀ(3ꢀchlorophenyl)ethyl)ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
(9q).
Yield 6%. 1HꢀNMR (CDCl3): δ 1.78 (d, J = 6.8 Hz, 3H), 5.32 (br. s., 1 H), 5.79 (br. s, 1H), 6.35 (br.
s., 1H), 7.08–7.12 (m, 1 H), 7.19 (d, J = 2.4 Hz, 1H), 7.22 (s, 1H), 7.33–7.38 (m, 2H), 8.16 (br.s.,
1H), 10.94 (br. s., 1H). 13CꢀNMR (DMSOꢀd6): δ 19.0, 59.2, 116.7, 117.9, 126.0, 127.4, 128.8, 130.8,
133.5, 137.1, 139.4, 142.7, 152.3, 165.9. LCꢀMS (ESI): m/z [M + H+] calcd, 310.0; found, 310.0.
purity: 97%.
5ꢀChloroꢀ2ꢀiminoꢀ1ꢀ(1ꢀ(3ꢀ(methylsulfonyl)phenyl)ethyl)ꢀ1,2ꢀdihydropyridineꢀ3ꢀ
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carboxamide hydrochloride (9r). Yield 40%. HꢀNMR (DMSOꢀd6): δ 1.95 (d, J = 6.7 Hz, 3H),
3.26 (s, 3H), 6.24 (q, 1H, J = 6.7 Hz), 7.69ꢀ7.76 (m, 2H), 7.94–8.00 (m, 1H), 8.04 (br. s., 1H), 8.23
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(br. s., 1H), 8.47 (d, J = 2.2 Hz, 1H), 8.59 (d, J = 2.2 Hz, 1H), 8.66 (br. s., 1H), 9.80 (br. s., 2H). 13Cꢀ
NMR (DMSOꢀd6): δ 19.1, 43.3, 59.13, 116.9, 117.7, 125.9, 127.3, 130.1, 132.3, 137.2, 138.5, 141.4,
142.6, 152.6, 166.0. LCꢀMS (ESI) m/z [M + H+ – (HCl)] calcd, 354.1; found, 354.0. purity: 100%.
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5ꢀChloroꢀ1ꢀ(1ꢀ(3ꢀcyanophenyl)ethyl)ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
hydrochloride (9s). Yield 8%. 1HꢀNMR (DMSOꢀd6): δ 1.92 (d, J = 6.7 Hz, 3H), 6.19 (q, J = 6.7 Hz,
1H), 7.61–7.69 (m, 1H), 7.71–7.79 (m, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.97 (s, 1H), 8.22 (s, 1H), 8.43
(d, J = 2.3 Hz, 1H), 8.59 (d, J = 2.3 Hz, 1H), 8.65 (s, 1H), 9.79 (br.s., 2H). 13CꢀNMR (DMSOꢀd6): δ
19.0, 58.9, 111.8, 116.9, 117.8, 118.4, 130.0, 131.2, 132.3, 132.5, 137.2, 138.5, 142.5, 152.7, 166.1.
LCꢀMS (ESI): m/z [M + H+] calcd, 301.1; found, 301.0. purity: 100%.
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5ꢀChloroꢀ1ꢀ((1R)ꢀ1ꢀ(3ꢀcyanophenyl)ethyl)ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
hydrochloride ((R)ꢀ9s). To an iceꢀcooled solution of 8a (6.42 g, 27.3 mmol) in MeOH (60 mL) was
added a mixture of (R)ꢀ3ꢀ(1ꢀaminoethyl)benzonitrile (8 g, 54.7 mmol) and Et3N (7.6 mL, 54.5
mmol) in MeOH (10 mL). The reaction mixture was stirred for 18 h at 45 °C and concentrated in
vacuo to give a crystalline product. To the residue was added acetic acid (50 mL) at room
temperature. The mixture was stirred for 4 h at 50 °C and concentrated in vacuo. The residue was
basified by 8 M NaOH and extracted with AcOEt. The extract was washed with brine, dried
(MgSO4) and concentrated in vacuo. The residue was purified by column chromatography on silica
gel (AcOEt:hexane = 7:3–1:0) to give a pale brown oil. To this oil in EtOH (3 mL) was added 4 M
HCl in AcOEt (3 mL) at room temperature. The resulting precipitate was collected by filtration,
recrystallized from EtOH, H2O and washed with AcOEt to give the title compound (5.32 g, 58%,
98.9% e.e.) as a colorless solid. 1HꢀNMR (DMSOꢀd6): δ 1.92 (d, J = 6.8 Hz, 3H), 6.24 (q, J = 6.7 Hz,
1H), 7.60–7.71 (m, 1H), 7.71–7.80 (m, 1H), 7.85–7.93 (m, 1H), 7.98 (s, 1H), 8.22 (s, 1H), 8.43 (d, J
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= 2.3 Hz, 1H), 8.62 (d, J = 2.3 Hz, 1H), 8.69 (s, 1H), 9.71–9.96 (m, 2H). CꢀNMR (DMSOꢀd6): δ
19.0, 59.0, 111.8, 116.9, 117.8, 118.4, 130.0, 131.2, 132.3, 132.4, 137.2, 138.6, 142.6, 152.7, 166.11.
LCꢀMS (ESI): m/z [M + H+] calcd, 301.1; found, 301.2. purity: 100%. Anal. Calcd for
C15H14Cl2N4O: C, 53.43; H, 4.18; N, 16.62. Found: C, 53.33; H, 4.39; N, 16.59. mp 191–193 °C.
[α]25D +157.0 (c 0.526, MeOH).
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5ꢀChloroꢀ1ꢀ((1S)ꢀ1ꢀ(3ꢀcyanophenyl)ethyl)ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
hydrochloride ((S)ꢀ9s). Optical resolution of 9s (174 mg, 0.516 mmol) was conducted by HPLC
(CHIRALCEL OJ (JL001), 50 mmID×500 mmL, solvent phase: hexane/EtOH/diethylamine =
700/300/1) after desalting. The desired fraction with larger retention time was separated and
concentrated in vacuo. The residue (66.9 mg, 98.3% e.e.) in EtOH (1 mL) was added 4 M HCl in
AcOEt (1 mL) at room temperature. The resulting precipitate was collected by filtration,
recrystallized from EtOH, H2O and washed with AcOEt to give the title compound (52.9 mg, 30%)
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as a colorless solid. HꢀNMR (DMSOꢀd6): δ 1.92 (d, J = 6.6 Hz, 3H), 6.12–6.27 (m, 1H), 7.60–7.69
(m, 1H), 7.71–7.78 (m, 1H), 7.86–7.93 (m, 1H), 7.97 (s, 1H), 8.24 (s, 1H), 8.44 (d, J = 2.1 Hz, 1H),
8.57–8.73 (m, 2H), 9.66–9.97 (m, 2H). 13CꢀNMR (DMSOꢀd6): δ 19.0, 58.9, 111.8, 116.9, 117.8,
118.4, 130.0, 131.2, 132.3, 132.5, 137.1, 138.5, 142.5, 152.7, 166.1. LCꢀMS (ESI): m/z [M + H+]
calcd, 301.1; found, 301.2. purity: 100%. mp 195–199 °C. [α]25D −145.4 (c 0.921, MeOH).
5ꢀChloroꢀ1ꢀ[3ꢀchloroꢀ5ꢀ(methylsulfonyl)benzyl]ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀ
carboxamide hydrochloride (9t). A mixture of 7a (1.68 g, 7.16 mmol), 1ꢀ[3ꢀchloroꢀ5ꢀ
(methylsulfonyl)phenyl]methanamine hydrochloride (20) (2.20 g, 8.59 mmol) and K2CO3 (2.97 g,
21.5 mmol) in EtOH (30 mL) was stirred overnight at 85 °C. To the reaction mixture was added 1 M
NaOH until the mixture was made basic and the product was extracted with AcOEt. The extract was
washed with brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by column
chromatography on NH silica gel (AcOEt:MeOH = 1:0ꢀ17:3) to give a pale yellow amorphous. This
amorphous was dissolved in MeOH, and 4 M HCl/AcOEt (3 mL) was added at room temperature.
The resulting precipitate was collected by filtration and recrystallized from EtOH and AcOEt to give
the title compound (630 mg, 21%) as a colorless solid. 1HꢀNMR (DMSOꢀd6): δ 3.31 (s, 3H), 5.65 (s,
2H), 7.78 (t, J = 1.7 Hz, 1H), 7.92 (t, J = 1.5 Hz, 1H), 8.03 (t, J = 1.7 Hz, 1H), 8.21 (s, 1H), 8.65 (s,
1H), 8.67 (d, J = 2.1 Hz, 1H), 8.83 (d, J = 2.1 Hz, 1H), 9.56 (s, 2H). 13CꢀNMR (DMSOꢀd6): δ 43.1,
55.0, 116.9, 117.3, 124.9, 126.8, 132.2, 134.3, 136.3, 141.4, 142.8, 143.0, 152.6, 165.8. LCꢀMS
(ESI): m/z [M + H+ – (HCl)] calcd, 374.0; found, 373.8. purity: 100%. Anal. Calcd for
C14H14Cl3N3O3S: C, 40.94; H, 3.44; N, 10.23. Found: C, 40.96; H, 3.46; N, 10.11. mp 276–277 °C.
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5ꢀChloroꢀ1ꢀ[5ꢀchloroꢀ2ꢀ(methylsulfonyl)benzyl]ꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀ
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carboxamide hydrochloride (9u). A mixture of 8a (7.65 g, 32.5 mmol), 1ꢀ[5ꢀchloroꢀ2ꢀ
(methylsulfonyl)phenyl]methanamine hydrochloride (21) (10.0 g, 39.0 mmol) and potassium
carbonate (11.9 g, 85.9 mmol) in EtOH (100 mL) was stirred overnight at 85 °C. To the reaction
mixture was added 1 M sodium hydroxide until the mixture was made basic and the product was
extracted with AcOEt and THF. The extract was washed with brine, dried (MgSO4) and
concentrated in vacuo. The residue was purified by column chromatography on NH silica gel
(AcOEt:MeOH = 4:1) to give a pale yellow amorphous solid. This amorphous solid was dissolved in
MeOH, and 4 M HCl in AcOEt (15 mL) was added at room temperature. The resulting precipitate
was collected by filtration and recrystallized from MeOH, H2O and AcOEt to give the title
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compound (3.80 g, 28%) as a colorless solid. HꢀNMR (DMSOꢀd6) δ 3.43 (s, 3H), 5.82 (br. s., 2H),
7.14 (d, J = 1.9 Hz, 1H), 7.78 (dd, J = 8.5, 2.1 Hz, 1H), 8.07 (d, J = 8.5 Hz, 1H), 8.22 (br. s., 1H),
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8.66 (br. s., 3H), 9.65 (br. s., 2H). CꢀNMR (DMSOꢀd6): δ 44.5, 54.5, 116.8, 117.3, 126.6, 129.3,
132.5, 134.2, 136.9, 139.3, 141.0, 143.0, 153.2, 166.0. LCꢀMS (ESI): m/z [M + H+ – (HCl)] calcd,
374.0; found, 374.2. purity: 99.4%. Anal. Calcd for C14H14Cl3N3O3S: C, 40.94; H, 3.44; N, 10.23.
Found: C, 40.93; H, 3.56; N, 10.15. mp 256 °C.
1ꢀBenzylꢀ5ꢀchloroꢀ2ꢀoxoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide (10). A mixture of 1 (0.25 g,
0.96 mmol) and 6 M HCl (10 mL) was stirred under reflux for 15 h. The mixture was adjusted to pH
5 with ammonium hydroxide. The resulting precipitate was collected by filtration and washed with
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AcOEt to give the title compound (0.21 g, 83.3%) as a pale brown solid. HꢀNMR (DMSOꢀd6): δ
5.43 (s, 2H), 7.22–7.29 (m, 2H), 7.33–7.46 (m, 3H), 8.34 (d, J = 2.5 Hz, 1H), 8.38–8.71 (m, 1H),
8.48 (d, J = 2.5 Hz, 1H). 13CꢀNMR (DMSOꢀd6): δ 55.0, 116.9, 123.6, 127.1, 128.3, 128.9, 133.0,
138.2, 142.7, 154.0, 162.9. LCꢀMS (ESI): m/z [M + H+] calcd, 263.1; found, 263.2. purity: 97.2%.
mp 216−219°C.
2ꢀAminopyridineꢀ3ꢀcarboxamide (12). A solution of 2ꢀaminopyridineꢀ3ꢀcarbonitrile (11) (0.50
g, 4.20 mmol) in concentrated sulfuric acid (5 mL) was stirred at 100 °C for 5 h. The mixture was
poured into iceꢀwater and adjusted to pH 8 with saturated sodium hydrogen carbonate solution. The
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Journal of Medicinal Chemistry
precipitate was collected by filtration and washed with water to give the title compound (0.29 g,
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50%) as a colorless solid. HꢀNMR (DMSOꢀd6): δ 6.55 (dd, J = 4.8, 7.6 Hz, 1H), 7.19 (br. s., 2H),
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7.32 (br. s., 1H), 7.92 (dd, J = 1.7, 7.6 Hz, 1H), 7.94 (br. s., 1H), 8.06 (dd, J = 1.7, 4.8 Hz, 1H). mp
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197−198 °C.
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1ꢀBenzylꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide hydrobromide (13). A mixture of 12
(0.10 g, 0.73 mmol) and benzylbromide (0.25 g, 1.46 mmol) in DMF (3 mL) was stirred at 80 °C for
18 h. After addition of toluene to the mixture, the resulting precipitate was collected by filtration and
washed with AcOEt to give the title compound (0.10 g, 45%) as a colorless solid. 1HꢀNMR (DMSOꢀ
d6): δ 5.56 (s, 2H), 7.13 (t, J = 7.1 Hz, 1H), 7.26 (d, J = 7.1 Hz, 2H), 7.34–7.47 (m, 3H), 8.10 (br. s.,
1H), 8.40 (d, J = 6.6 Hz, 1H), 8.49 (d, J = 7.6 Hz, 1H), 8.56 (br. s., 1H), 9.30 (br. s., 2H). 13CꢀNMR
(DMSOꢀd6): δ 55.5, 111.9, 115.8, 127.3, 128.4, 128.9, 132.8, 142.8, 143.5, 153.1, 166.9. LCꢀMS
(ESI): m/z [M + H+] calcd, 228.1; found, 228.2. purity: 100%. mp 226−228 °C.
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2ꢀAminoꢀ5ꢀchloropyridineꢀ3ꢀcarboxamide (14). To a mixture of 12 (2.0 g, 14.6 mmol) and
conc. HCl (12 mL) was added 30% hydrogen peroxide (1.2 mL) at 0°C. After stirring at 60°C for 1 h,
the mixture was diluted with water. The reaction mixture was basified with 1 M NaOH and
extracted with THF and AcOEt. The organic layer was washed with brine, dried (MgSO4) and
concentrated in vacuo. The residue was purified by column chromatography (AcOEt:hexane = 4:1
–
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1:0) to give the title compound (0.94 g, 38%) as colorless crystals. HꢀNMR (DMSOꢀd6) δ 7.35 (s,
2H), 7.46 (s, 1H), 7.97–8.07 (m, 2H), 8.09 (d, J = 2.7 Hz, 1H). mp 226–228 °C.
1ꢀ(3ꢀCarbamoylꢀ5ꢀchlorobenzyl)ꢀ5ꢀchloroꢀ2ꢀiminoꢀ1,2ꢀdihydropyridineꢀ3ꢀcarboxamide
hydrobromide (15e). A mixture of 14 (150 mg, 0.87 mmol) and 3ꢀcarbamoylꢀ5ꢀchlorobenzyl
bromide (258 mg, 1.04 mmol) in DMF (3 mL) was stirred at 100 °C for 3 h, cooled to room
temperature and diluted with AcOEt. The resulting precipitate was collected by filtration and
recrystallized from MeOH and AcOEt to give the title compound (110 mg, 30%) as a colorless solid.
1HꢀNMR (DMSOꢀd6): δ 5.57 (s, 2H), 7.61 (s, 2H), 7.67 (s, 1H), 7.94 (s, 1H), 8.14 (s, 1H), 8.20 (s,
1H), 8.59 (s, 1H), 8.63 (d, J = 1.5 Hz, 1H), 8.81 (d, J = 1.1 Hz, 1H), 9.42 (s, 2H). 13CꢀNMR
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