Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.5b01715

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF₁) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF₁ receptor antagonists. In a structure-activity relationship study, 4-chloro-N²-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N⁷,N⁷-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF₁ receptor and the antagonistic activity (IC₅₀ = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF₁ receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF₁ receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF₁ receptor antagonist drug discovery research.

Full text of DOI:10.1021/acs.jmedchem.5b01715

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Article
Design and synthesis of benzimidazoles as novel
corticotropin-releasing factor 1 receptor antagonists
Michiyo Mochizuki, Masakuni Kori, Katsumi Kobayashi, Takahiko Yano, Yuu Sako, Maiko Tanaka, Naoyuki
Kanzaki, Albert C. Gyorkos, Christopher P Corrette, Suk Young Cho, Scott A Pratt, and Kazuyoshi Aso
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01715 • Publication Date (Web): 22 Feb 2016
Downloaded from http://pubs.acs.org on February 24, 2016
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Design and synthesis of benzimidazoles as novel
corticotropinꢀreleasing factor 1 receptor antagonists
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,
†, #
†, #
†
†, §
†
Michiyo Mochizuki,* Masakuni Kori, Katsumi Kobayashi, Takahiko Yano, Yuu Sako,
†
†
‡
‡
Maiko Tanaka, Naoyuki Kanzaki, Albert C. Gyorkos, Christopher P. Corrette, Suk Young
‡
‡
†, #
Cho, Scott A. Pratt, and Kazuyoshi Aso
†
Takeda Pharmaceutical Company Ltd., 26ꢀ1, Muraokahigashi 2ꢀchome, Fujisawa, Kanagawa
51ꢀ8555, Japan
2
‡
Array BioPharma Inc., 3200 Walnut Street, Boulder, Colorado 80301, United States
Abstract
Benzazole derivatives with a flexible aryl group bonded through a oneꢀatom linker as a new
scaffold for a corticotropinꢀreleasing factor 1 (CRF ) receptor antagonist were designed,
1
synthesized and evaluated. We expected that structural diversity could be expanded beyond that
2
of reported CRF receptor antagonists. In a structure–activity relationship study, 4ꢀchloroꢀN ꢀ(4ꢀ
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7
7
chloroꢀ2ꢀmethoxyꢀ6ꢀmethylphenyl)ꢀ1ꢀmethylꢀN ,N ꢀdipropylꢀ1Hꢀbenzimidazoleꢀ2,7ꢀdiamine
9g had the most potent binding activity against a human CRF receptor and the antagonistic
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activity (IC = 9.5 nM and 88 nM, respectively) without concerns regarding cytotoxicity at 30
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0
ꢀM. Potent CRF receptorꢀbinding activity in brain in an ex vivo test and supression of stressꢀ
1
induced activation of the hypothalamus–pituitary–adrenocortical (HPA) axis were also observed
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at 138 ꢀmol/kg of compound 29g after oral administration in mice. Thus, the newly designed
benzimidazole 29g showed in vivo CRF receptor antagonistic activity and good brain
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penetration, indicating that it is a promising lead for CRF receptor antagonist drug discovery
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research.
Introduction
Corticotropinꢀreleasing factor (CRF) is a 41ꢀaminoꢀacid neuropeptide that mediates its actions
1
,2
through two G ꢀcoupled G proteinꢀcoupled receptor subtypes, CRF and CRF . CRF is
s
1
2
believed to be the main regulator of the hypothalamus–pituitary–adrenocortical (HPA) axis via
CRF receptors and has an important role as a neurotransmitter in the mediation of stressꢀrelated
1
3
,4
behaviors. After exposure to stress, secretion of CRF increases in the neurons of the
paraventricular nucleus of the hypothalamus and stimulates the release of adrenocorticotropic
hormone (ACTH) from the anterior pituitary gland.5,6 ACTH subsequently induces the secretion
of cortisol from adrenal glands. In a healthy individual, cortisol stimulates and controls the
hypothalamic secretion of CRF, which indicates that a negative feedback system against the
activation of the HPA axis is operating. On the other hand, the negative feedback system
collapses in patients with stressꢀrelated disorders. CRF also activates CRF receptors in the brain,
1
and such activation appears to be directly related to disease symptoms. In fact, mice lacking
CRF receptors exhibit decreased anxietyꢀlike behavior and impaired stress responses, and
1
neutralization of CRF receptor expression by central administration of CRF antisense
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7
oligonucleotides attenuates stress responses in rats. Moreover, intracerebroventricular (icv)
infusion of CRF induces anxietyꢀlike behavior, but behavioral responses to CRF or to a stressor
7
can be ameliorated by CRF receptor antagonists.
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Over the last 20 years, numerous nonꢀpeptides and smallꢀmolecule CRF receptor antagonists
1
have been reported to demonstrate their effectiveness in animal models for stressꢀrelated
disorders. However, no drugs have been launched. The structures in clinical trials in the past
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were exhibited in Figure 1. In fact, 1a (R121919) significantly decreased Hamilton Depression
and Anxiety Rating Scale (HAMꢀD and HAMꢀA) scores after 10 days of oral administration in
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patients with major depression in an openꢀlabel, smallꢀscale, phase IIa clinical trial. Another
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trial of 1a showed liver enzyme elevation, which has been reported to depend on the structure of
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1a but not on the mechanism of action (CRF antagonism). Subchronic treatment with 2ꢀ(2,4ꢀ
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dichlorophenyl)ꢀ6ꢀ(heptanꢀ4ꢀyl)ꢀ4ꢀmethylꢀ7,8ꢀdihydroꢀ6Hꢀ1,3,6,8aꢀtetraazaacenaphthylene
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0
(
NBIꢀ34041) also attenuated the neuroendocrine response to psychosocial stress in a placeboꢀ
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controlled clinical study. The results of both compounds suggest that nonꢀpeptide CRF1
receptor antagonists could be useful therapeutics in the treatment of stressꢀrelated disorders such
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as depression and anxiety. However, 1b (CPꢀ316,311) , 4ꢀ(2ꢀchloroꢀ4ꢀmethoxyꢀ5ꢀ
methylphenyl)ꢀNꢀ((1S)ꢀ2ꢀcyclopropylꢀ1ꢀ(3ꢀfluoroꢀ4ꢀmethylphenyl)ethyl)ꢀ5ꢀmethylꢀNꢀ(propꢀ2ꢀ
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ynꢀ1ꢀyl)ꢀ1,3ꢀthiazolꢀ2ꢀamine(SSR125543) , and 1c (verucerfont) failed to demonstrate efficacy
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1–13
14,15
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in the treatment of major depression.
Pexacerfont
and emicerfont also failed in clinical
trials for major depression, general anxiety disorder, and irritable bowel syndrome. On the other
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hand, a clinical trial of 1c has been conducted by an Emory University group for postꢀtraumatic
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stress disorder, and the National Institute on Alcohol Abuse and Alcoholism plans to conduct a
trial for alcohol dependence. In addition, a CRF receptor antagonist is considered to be a
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9,20
relevant target for other stressꢀrelated diseases, such as pain and anorexia,
resulting from
elevated CRF levels. Therefore, CRF receptor antagonists are an attractive drug discovery target
1
for the treatment of stressꢀrelated diseases.
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Figure 1. Reported CRF receptor antagonists.
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A variety of typical nonꢀpeptide CRF receptor antagonists reported by many companies have
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,2,21
common structural features, as shown in I in Figure 2.
Generally, A monoꢀ, diꢀ, or triꢀcyclic
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hetero aromatic core containing a ring A with an sp basic nitrogen as a hydrogenꢀbonding
acceptor (HBA) is substituted with a pendant aromatic group (Ar) at the bottom that has an
x
y
orthogonal relationship to the core. The heteroaromatic core also has alkyl moieties, R , R , and
z
x
y
R . R and R are located at the top region of the core and appear to fit into a large lipophilic
z
2
pocket of a CRF receptor, and R is a small alkyl group adjacent to the sp nitrogen. Numerous
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reported CRF receptor antagonists can be classified into two subgroups: one in which the
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pendant aryl group (Ar) directly bonds to the heteroaromatic core and the other in which the aryl
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2
group bonds through a oneꢀatom linker Z without a ring B, such as 1b. Historically, CRF
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receptor antagonists have been associated with less optimal physicochemical drug properties, and
it has been assumed that these properties are a major obstacle in developing CRF receptor
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2
antagonists in the clinic. We focused on designing the latter type of scaffolds with a more
flexible Ar group, with the expectation that the resulting improved physicochemical properties,
2
3,24
such as solubility, would provide better biological profiles
than those of the former CRF1
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receptor antagonist subgroup. We designed a 6ꢀ5 fused type of scaffold with ring C rather than
ring D or E, which was unique among the numerous reported CRF receptor antagonists.
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x
y
Requisite lipophilic groups, such as R and R , were placed on ring C. Identification of a new
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type of scaffold would allow a wider design space range for future CRF antagonists.
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Cytotoxicity was also closely monitored during our discovery efforts because 1a showed both
liver enzyme elevation in the clinical trial described above and cytotoxicity (vide infra).
Although it is unclear if the liver enzyme elevation by 1a was due to cytotoxicity, identification
of a new scaffold with reduced cytotoxicity is one of the goals of our continuing research.
The unique structure II has been never reported as a CRF receptor antagonist, and we
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proposed new benzoxazole, benzothiazole, and benzimidazole central cores. Flexible alignment
of 7ꢀdipropylaminobenzoxazole analog 5 to a typical type of reported CRF receptor antagonist
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25
1
a was performed using MOE. It was found that the key functional groups, an HBA, a
dialkylamino group, and pendant aryl groups, of benzoxazole analog 5 overlapped well with the
corresponding key groups of 1a, as illustrated in Figure 3. This superimposition study suggested
that the new compound designs should exhibit potent CRF receptor antagonism.
1
Figure 2. Design of a benzazole core for a novel type of CRF receptor antagonists.
1
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Figure 3. Superimposition of 1a (gray) on benzoxazole analog 5 (orange).
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N
N
N
N
O
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NH
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1a
5
In this report, we describe the synthesis and structure–activity relationships (SARs) as well as
the biological activities of a novel series of benzazoles as CRF receptor antagonists.
1
Results and Discussion
Chemistry. Syntheses of the 7ꢀalkylaminoꢀ2ꢀanilinobenzazole series are illustrated in Schemes
1–5. A benzoxazole derivative 5 was synthesized according to Scheme 1. Thiourea 3 was
prepared from commercially available aminophenol 2 and (2,4,6ꢀtrimethylphenyl)isothiocyanate.
Cyclization of thiourea 3 with mercury(II) chloride provided 7ꢀnitrobenzoxazole 4, and
subsequent hydrogenation and reductive alkylation with propionadehyde afforded the target
compound 5.
a
Scheme 1. Synthesis of a benzoxazole analog 5
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a
Reagents and conditions: (a) (2,4,6ꢀtrimethylphenyl)isothiocyanate, Na CO , EtOH, reflux,
2
3
8
0%; (b) HgCl , MeCN, rt, 90%; (c) (i) H , Pd/C, MeOH, rt; (ii) EtCHO, NaBH CN, AcOH, rt,
2 2 3
9
0%.
A benzothiazole analog 9 was synthesized from (3ꢀnitrophenyl)isothiocyanate 6. Condensation
of compound 6 and mesitylamine was followed by cyclization with bromine to give 7ꢀ
nitrobenzothiazole 7. After reduction of the nitro group of 7 with iron to give
aminobenzothiazole 8, reductive alkylation of 8 afforded the target benzothiazole 9 (Scheme 2).
a
Scheme 2. Synthesis of a benzothiazole analog 9
a
Reagents and conditions: (a) (i) mesitylamine, MeOH, rt; (ii) Br , AcOH, reflux, 11%; (b) Fe,
2
AcOH, EtOH, reflux, 55%; (c) EtCHO, NaBH(OAc) , AcOH, ClCH CH Cl, 50°C, 10%.
3
2
2
The synthetic route of the benzimidazole analog 13 is similar to that of benzoxazole 5 and
benzothiazole 9 described in Schemes 1 and 2, respectively (Scheme 3). 3ꢀNitroꢀ1,2ꢀ
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phenylenediamine 10 was treated with (2,4,6ꢀtrimethylphenyl)isothiocyanate and subsequent
DIC to give 7ꢀnitro benzimidazole 11. The nitro group of 11 was hydrogenated with a palladium
catalyst, followed by reductive alkylation to give the target compound 13.
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Scheme 3. Synthesis of a benzimidazole analog 13
a
Reagents and conditions: (a) (i) (2,4,6ꢀtrimethylphenyl)isothiocyanate, Na CO , EtOH,
2
3
reflux; (ii) DIC, reflux, 54%; (b) H , PdꢀC, MeOH, rt, 86%; (c) (i) EtCHO, MPBH CN, AcOH,
2
3
MeOH, rt; (ii) conversion to HCl salt, 16%.
The synthesis of 7ꢀdialkylaminobenzimidazoles with a substituent at the 1ꢀposition, 22a–e and
3, is described in Scheme 4. The intermediates 21a–d were prepared from commercially
2
available 2ꢀchloroꢀ1,3ꢀdinitrobenzene 14 by substitution of the chloro group with a
corresponding amine, hydrogenation, condensation of the corresponding isothiocyanate, and
cyclization, respectively, in good yields. Reductive alkylation of the intermediates 21a–d with
propionaldehyde or nꢀbutylaldehyde was performed to give the 7ꢀdipropyl or 7ꢀ
dibutylaminobenzimidazoles 22a–d. The 7ꢀisopropylamino analog 22f was also prepared by
reductive alkylation of 21d with acetone. Preparation of methoxyethylamino analog 22e was
performed by reductive alkylation of 21d with methoxyacetaldehyde prepared from 1,1,2ꢀ
trimethoxyethane in the presence of iron(III) chloride. The ethyl(isopropyl)amino analog 23 was
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obtained by additional reductive alkylation of 22f with acetaldehyde. The isothiocyanate 19 for
reaction with triamine 16a was prepared by bromination of aniline 17 and subsequent treatment
with carbon disulfide to convert the amino group of (4ꢀbromoꢀ2ꢀmethoxyꢀ6ꢀmethyl)aniline 18 to
isothiocyanate.
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Scheme 4. Synthesis of benzimidazole analogs with various substituents at the 1ꢀ and 7ꢀ
a
positions, 22a–e and 23
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NH2 R1
N
NH2 NHR1
NO2
NO2
NH2
R2a
R2b
a
NHR1
NO2
b
NHR1
NH2
e
f
Cl
R2a
S
NH
N
N
N
H
R2c
NO2
H
R2c
R2b
R1 yield (%)
5a Me 99
5b iPr 77
R1 yield (%)
R1 R2a R2b R2c yield (%)
R1 R2a R2b R2c yield (%)
21a Me Me Me Me 55
1
4
1
1
16a Me 75
20a Me Me Me Me 60
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16b iPr 91
16c Ph 98
20b iPr Me Me Me
20c Ph Me Me Me
-
21b iPr Me Me Me 77 (2 steps)
21c Ph Me Me Me 45 (2 steps)
21d Me OMe Br Me 49
15c Ph 70
-
2
0d Me OMe Br Me 38 (2 steps)
NH2
NCS
NH2
OMe
OMe
O
c
d
Br
Br
1
7
1
8
19
R7a
R7b
R1
N
N
g (h)
i, h
N
N
N
NH R2a
NH OMe
N
R2c
HCl
Br
R2b
R1 R7a
2a Me nPr
22b
iPr nPr
R7b
R2a R2b R2c salt
yield (%)
70
23
2
nPr
nPr
nPr
Me Me Me
Me Me Me
-
-
59
2
2
2
2
2c Ph nPr
2d Me nBu
Me Me Me HCl
OMe Br Me HCl
30
nBu
32
2e Me MeOCH CH2 MeOCH CH2 OMe Br Me
-
-
18
2
2
2f
Me iPr
H
OMe Br Me
21
a
1
Reagents and conditions: (a) R NH , MeOH or THF, rt or reflux; (b) H or cyclohexene, Pdꢀ
2
2
C, MeOH, rt or reflux; (c) Br , AcOH, MeOH, rt, 53%; (d) CS , Et N, DCC, pyridine, −10°C,
2
2
3
>
99%;
(e)
(2,4,6ꢀtrimethylphenyl)isothiocyanate
or
(4ꢀbromoꢀ2ꢀmethoxyꢀ6ꢀ
methylphenyl)isothiocyanate 19, (Na CO ), EtOH or MeOH, reflux; (f) HgCl , (Et N), MeCN, rt
2
3
2
3
or DIC, EtOH reflux; (then, PSBH CN, MeOH, rt); (g) EtCHO or nꢀPrCHO or acetone,
3
NaBH CN or NaBH(OAc) or MPBH CN, (AcOH), MeOH or CH Cl , rt to 55°C; or
3
3
3
2
2
MeOCH CH(OMe) , FeCl , MPBH CN, AcOH, MeOH, rt; (h) 2N HCl in Et O; (i)
2
2
3
3
2
acetaldehyde, NaBH(OAc) , AcOH, CH Cl , 50°C, 98%.
3
2
2
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Page 11 of 57
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
Syntheses of compounds with and without various substituents at the 2ꢀ and 4ꢀpositions 29a–o
are described in Scheme 5. An alternative route without using phenyl thiourea was effective for
introducing 2ꢀanilino groups at the final step. The benzimidazole core 24 was constructed by
condensation of CDI and triamine 16a in good yield. The common intermediates 25a and 25b
were obtained by reductive alkylation of the prepared 7ꢀaminobenzimidazolꢀ2ꢀone 24. Target
compounds without substituents at the 4ꢀposition 29a–f were provided by chlorination of 25a or
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25b using phosphorus oxychloride and subsequent treatment with various anilines. Chlorination
of 25a was performed using NCS to afford a mixture of 4ꢀchloro (26a) and 4,6ꢀdichloro (26b)
derivatives and a single isomer of 6ꢀchloro analog 26c. 4ꢀChloro (29g), 4,6ꢀdichloro (29h), and
6ꢀchloro (29i) were obtained from 26a–c via 2ꢀchlorobenzimidazoles 28c–e using a method
similar to those used for the nonꢀsubstituted targets 29a–f. The next two steps from the mixture
of 4ꢀchloro (26a) and 4,6ꢀdichlorobenzimidazolꢀ2ꢀone (26b) derivatives were carried out via
compounds 28c and 28d, and the targets 29g and 29h, respectively, were separated by silica gel
column chromatography in the final step. On the other hand, 4ꢀchloroꢀ7ꢀ
diethylaminobenzimidazolꢀ2ꢀone 26d was isolated as a single isomer after chlorination of the
diethylamino derivative 25b and converted to the target compound 29k using methods similar to
those described above. Bromination of 25a and 25b and subsequent substitution of the bromo
group with a cyano group using copper cyanide afforded the 4ꢀcyanobenzimidazolꢀ2ꢀones 27a
and 27b. The 4ꢀmethyl 27c and 4ꢀphenyl 27d derivatives were synthesized using a coupling
reaction of the 4ꢀbromo analog 26f with a corresponding tin reagent. A coupling reaction of the
4ꢀbromo analog 26f with sodium methoxide in the presence of copper iodide also afforded 4ꢀ
methoxybenzimidazolꢀ2ꢀone 27e. The targets 29j and 29l-o were obtained using methods similar
to those described above.
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Journal of Medicinal Chemistry
Page 12 of 57
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 5. Synthesis of benzimidazole analogs with various substituents at the 4ꢀ and 6ꢀpositions,
a
29a–o
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R7
R7
R7
R7
R7
R7
NH2
NH2
N
N
N
a
b
c
R6
d (e)
R6
NHMe
NH2
6a
N
N
N
N
N
N
O
Cl
NH R2a
O
N
H
N
H
2
R2c
R4
R4
6
1
24
25
R7 yield (%)
5a nPr 73
28
29
4
R2b
R7 R4 R6 yield (%)
2
2
28a nPr H
28b Et
8c nPr Cl
28d nPr Cl
8e nPr H
H
H
H
Cl
69
47
-
R4 = Cl
c
5b Et
91
H
c
2
f
-
2
Cl 34
R7
R7
R7
R7
28f nPr CN
28g Et Cl
28h Et CN
H
H
H
H
H
62
>99
43
-
R4 = Br
N
N
R6
g
N
N
O
O
2
2
8i Et Me
8j Et Ph
N
N
H
67 (2 steps)
H
R4
R4
28k Et OMe H 98
2
6
27
R7 R4 yield (%)
27a nPr CN 75
27b Et CN 82
7c Et Me 51
27d Et Ph
7e Et OMe 74
R7 R4 R6 yield (%)
R7 R4 R6 R2a R2b R2c salt yield (%)
2
2
2
2
2
6a nPr Cl
H
-
-
6b nPr Cl Cl
29a nPr H
29b nPr H
H
H
H
Me Me
Me
OMe Cl Me
H
TFA 60
Me TFA 45
9.9
6c nPr H Cl 33
6d Et Cl
28
6e nPr Br H 33
H
2
H
-
29c
29d
nPr H
nPr H
-
2
H
H
H
H
OMe Br Me HCl 42
26f Et Br H 67
2
2
2
2
2
2
2
2
9e nPr H
9f Et
iPr Cl Me HCl 21
H
OMe Br Me HCl 35
9g nPr Cl
9h nPr Cl
9i nPr H
9j nPr CN
9k Et Cl
9l Et CN
OMe Cl Me
-
-
-
-
-
-
-
-
-
7 (3steps)
Cl OMe Cl Me
Cl OMe Cl Me
1.8 (3 steps)
31
H
H
H
H
H
OMe Cl Me
OMe Cl Me
OMe Cl Me
OMe Cl Me
OMe Cl Me
4.3
52
48
2
9mEt Me
36 (2 steps)
29n Et Ph
57
45
29o Et OMe H OMe Cl Me
a
Reagents and conditions: (a) CDI, THF, rt, 81%; (b) EtCHO or MeCHO, NaBH CN or
3
NaBH(OAc) , AcOH, MeOH or CH Cl , rt; (c) POCl , (C H NMe ), 100°C–120°C; (d) ArNH ,
3
2
2
3
6
5
2
2
(NMP), 70°C–130°C; (e) conversion to HCl salt; (f) NCS or NBS, (AIBN,) CCl or MeCN, rt to
4
reflux; (g) CuCN, NMP, 180°C or 170°C, microwave; or Me Sn or Ph Sn, Pd(PPh ) , HMPA,
4
4
3 4
reflux; or NaOMe, CuI, MeOH, DMF, 100°C.
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Page 13 of 57
Journal of Medicinal Chemistry
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4
5
6
7
8
9
Biology. The synthesized compounds 5, 9, 13, 22a–e, 23, and 29a–o were screened for their
1
25
inhibitory activity against ovine
IꢀCRF binding to human CRF receptors expressed on
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Chinese hamster ovary (CHO) cellular membranes, and cytotoxicity was indicated by the ATP
content in HepG2 cells. Compound 1a was selected as a reference compound because it was used
in clinical and the results were reported. Initial efforts were focused on the investigation of core
scaffolds. The results of the benzazole series are listed in Table 1. Benzoxazole 5 showed
submicromolar CRF receptorꢀbinding inhibition activity as expected by superimposition with a
1
reported CRF receptor antagonist 1 as described in Figure 3. Benzothiazole 9 also exhibited
1
activity with an IC value of 77 nM, whereas benzimidazole 13 showed 30ꢀfold less potent
5
0
activity than benzothiazole 9. These results suggested that hetero atoms at the 1ꢀposition of the
core affected CRF receptorꢀbinding activity. In fact, flexible alignment of three compounds (5,
1
25
9
, and 13) with MOE indicated that the sulfur atom of benzothiazole 9 was possibly out of
alignment with the oxygen and nitrogen atoms in the benzoxazole (5) and benzimidazole (13)
rings, respectively (Figure 4). Therefore, introduction of a substituent on the 1ꢀN-position of the
benzimidazole core should be effective for occupying the space of a CRF receptor surrounding
1
1ꢀposition. As expected, Nꢀmethylbenzimidazole 22a markedly improved the binding activity,
with an IC value of 15 nM, which was as potent as that of 1a. Compound 22a also showed less
5
0
cytotoxicity than benzoxazole 5, benzothiazole 9, deꢀmethyl benzimidazole 13, and 1a. Thus, we
selected Nꢀsubstituted benzimidazoles for research as pharmacophores of a 6ꢀ5 fused scaffold
with a oneꢀatom linker Z between the core and pendant aryl group. The analogs 22b and 22c
with a bulkier group at the 1ꢀNꢀposition (isopropyl and phenyl, respectively), resulted in
considerable reduction of the binding activity relative to that of the Nꢀmethyl analog 22a as well
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Journal of Medicinal Chemistry
Page 14 of 57
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
as severe cytotoxicity at 30 ꢀM. The isopropyl and phenyl groups appeared to be too bulky to
accommodate a CRF receptor and too lipophilic to exhibit low cytotoxicity for this series. The
1
2
6
compounds 22a, 22b, and 22c had log D values at pH 7.4 of 5.23, 5.90, and 6.31, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. hCRF receptorꢀbinding activities and cytotoxicity of the benzazole derivatives
1
Compound
No.
X
Salt Binding
(IC , Cytotoxicity
50
a
b
nM)
(%@30 ꢀM)
5
O
ꢀ
ꢀ
990 (710–1400)
65
75
9
S
77 (61–98)
13
NH
NMe
NiꢀPr
NPh
ꢀ
HCl 2500 (1500–4200) 41
22a
22b
22c
ꢀ
ꢀ
15 (11–21)
89
10
97 (77–120)
HCl 2200 (1500–3200) 32
1a
ꢀ
8.5 (5.7–13)
51
a
IC values and 95% confidential intervals were calculated from the concentration–response
5
0
b
curves (n = 1). The values are rates of ATP content relative to that for 100% with only DMSO
and no compound (n = 3).
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Page 15 of 57
Journal of Medicinal Chemistry
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4
5
6
7
8
9
Figure 4. Superimposition of 5 (orange), 9 (aqua), and 13 (green).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Anilino groups of the 1ꢀNꢀmethylbenzimidazole series were investigated (Table 2).
Disubstituted phenyl analogs at the 2,4ꢀ and 2,6ꢀpositions (29a and 29b, respectively) showed
reduced CRF receptorꢀbinding activity relative to that of the 2,4,6ꢀtrisubstituted lead compound
1
2
2a. As a result of the binding assay for various anilino groups and in consideration of diversity,
a 4ꢀchloroꢀ2ꢀmethoxyꢀ6ꢀmethylphenyl group in 29c was found to bring activity equal to that of
the trimethylphenyl group in 22a. Replacement of the 4ꢀchloro group of 29c with a bromo group
(
29d) maintained potent activity. A bulkier isopropyl analog 29e was also as potent as a 2ꢀ
methoxy analog 29c, suggesting that a branched substituent is acceptable at the orthoꢀposition of
a CRF receptor. Unfortunately, the cytotoxicity of the isopropyl analog 29e was worse than that
1
of the methoxy analog 29d. The lipophilicity of compound 29e was higher than that of 29d, with
logDs at pH 7.4 of 5.96 and 5.39, respectively.
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Journal of Medicinal Chemistry
Page 16 of 57
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Effects of anilino groups at the 2ꢀposition of 1ꢀNꢀmethylbenzimidazole derivatives on
hCRF receptorꢀbinding activity and cytotoxicity
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
a
2b
2c
Compound R
No.
R
R
Salt
Binding (IC , Cytotoxicity
50
a
b
nM)
(%@30 ꢀM)
2
2a
9a
Me
Me
Me
OMe
OMe
iꢀPr
ꢀ
Me
Me
H
Me
H
ꢀ
15 (11–21)
89
79
2
TFA
TFA
ꢀ
84 (67–100)
29b
Me
Me
Me
Me
ꢀ
140 (100–200) 72
c
29c
29d
29e
Cl
Br
Cl
ꢀ
14 (10–19)
12 (7.7–17)
22 (16–31)
8.5 (5.7–13)
ND
HCl
HCl
ꢀ
82
28
51
1a
a
IC values and 95% confidential intervals are calculated from the concentration–response
5
0
b
curves (n =1). The values are rates of ATP content relative to that for 100% with only DMSO
and no compound (n = 3).
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Page 17 of 57
Journal of Medicinal Chemistry
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7
8
9
The effects on conversion of dialkylamino groups at the 7ꢀposition are summarized in Table 3.
The diethylamino (29f) and dibutylamino (22d) analogs were as potent as the dipropylamino
analog 29d. Furthermore, the (isopropyl)ethylamino analog 23 also had potent activity. For CRF1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
receptorꢀbinding activity, these results suggested that a CRF receptor loosely recognizes their
1
length and size of the alkyl groups at this position On the other hand, conversion of the butyl
group of 22d to a methoxyethyl group (22e) was found to reduce activity, which indicated that
oxygen at this position was less preferable for a CRF receptor because of the lower lipophilicity.
1
7
a
These results suggested that this site of CRF receptors is lipophilic and the alkyl groups R and
1
7b
27
R are required for lipophilicity, as reported in previous SAR studies. These structural changes
in Table 3 had no effect on cytotoxicity.
Table 3. Effects of dialkylamino groups at the 7ꢀposition of 1ꢀNꢀmethylbenzimidazole
derivatives on hCRF receptorꢀbinding activity and cytotoxicity
1
7
a
7b
Compound R
No.
R
Salt
Binding (IC , Cytotoxicity
50
a
b
nM)
(%@30 ꢀM)
29d
nꢀPr
Et
nꢀPr
Et
HCl
HCl
HCl
12 (7.7–17)
13 (11–17)
14 (11–19)
82
93
90
29f
22d
nꢀBu
nꢀBu
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Journal of Medicinal Chemistry
Page 18 of 57
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
3
iꢀPr
Et
HCl
11 (8.4–15)
87
92
51
2
2e
MeOCH
2
CH
2
MeOCH
ꢀ
2
CH
2
ꢀ
ꢀ
65 (53–79)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1a
ꢀ
8.5 (5.7–13)
a
IC values and 95% confidential intervals are calculated from the concentration–response
5
0
b
curves (n = 1). The values are rates of ATP content relative to that for 100% with only DMSO
and no compound (n = 3).
Substituents at the 4ꢀ and 6ꢀpositions of benzimidazole were investigated (Table 4).
Introduction of a chloro group at the 4ꢀposition maintained the binding activity similar to that of
2
9g. The 4,6ꢀdichloro analog 29h showed diminished activity, and the 6ꢀchloro analog 29i was
inactive at 10 ꢀM. Based on these results, we considered that a substituent at the 6ꢀposition of
the benzimidazole core prevented good binding to the pocket of a CRF receptor. The area of the
1
6
ꢀposition of the core is outside the 1a molecule (1), as shown in Figure 3. In addition,
introduction of a chloro group at the 6ꢀposition may change the conformation of alkyl groups at
2
3
the 7ꢀposition. SAR studies that led to 1b have been reported to give similar results.
Replacement of a chloro group at the 4ꢀposition in 29g with a more electronꢀwithdrawing cyano
group (29j) maintained CRF receptorꢀbinding activity. The methyl (29m) and methoxy (29o)
1
analogs demonstrated activity comparable to that of the cyano analog 29l; however, the phenyl
analog 29n was much less potent than was 29m. On the other hand, the methoxy analog 29o was
shown to be highly cytotoxic. These results indicated that both electronꢀwithdrawing and
electronꢀdonating properties did not greatly influence the binding activity but had a significant
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Page 19 of 57
Journal of Medicinal Chemistry
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4
5
6
7
8
9
28,29
effect upon cytotoxicity
and that a large substituent at the 4ꢀposition reduced CRF receptorꢀ
1
binding affinity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. hCRF receptorꢀbinding activities and cytotoxicity of 1ꢀNꢀmethylbenzimidazole
1
derivatives substituted at the 4 and 6 positions
7
a
7b
4
6
Compound
No.
R
R
R
R
Binding (IC , Cytotoxicity
50
a
b
nM)
(%@30 ꢀM)
c
29c
nꢀPr
nꢀPr
nꢀPr
nꢀPr
nꢀPr
Et
nꢀPr
nꢀPr
nꢀPr
nꢀPr
nꢀPr
Et
H
H
H
Cl
Cl
H
H
H
H
H
14 (10–19)
ND
29g
29h
29i
Cl
9.5 (6.0–15)
83
Cl
170 (110–280) 72
H
>10000
86
83
76
81
72
92
29j
CN
Cl
14 (11–18)
24 (20–30)
13 (8.4–19)
7.6 (5.4–11)
>10000
2
2
2
2
9k
9l
Et
Et
CN
Me
Ph
9 m
9n
Et
Et
Et
Et
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Journal of Medicinal Chemistry
Page 20 of 57
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
9o
Et
ꢀ
Et
ꢀ
OMe
ꢀ
H
ꢀ
13 (8.3–20)
8.5 (5.7–13)
5
1a
51
a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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IC values and 95% confidential intervals are calculated from the concentration–response
50
b
curves (n = 1). The values are rates of ATP content relative to that for 100% with only DMSO
c
and no compound (n = 3). ND: no data.
The SAR study showed that this new type of benzazole series had potent CRF receptorꢀ
1
binding activity and the pharmacophore was similar to that of reported CRF receptor
1
antagonists. Removal of ring B and addition of the flexible aryl group provided analogs with
acceptable binding activity when a methyl group at the 1ꢀposition of the benzimidazole core was
introduced. It was also found that the binding activity was undisturbed by incorporation of ring
C, the benzene ring of the benzimidazole core. Compound 29g exhibited potent CRF receptorꢀ
1
binding inhibition activity and no cytotoxicity issues at 30 ꢀM and was therefore selected for
further evaluation.
Compound 29g inhibited human CRFꢀstimulated cAMP accumulation in CHO cells expressing
human CRF receptors, with an IC value of 88 nM (95% confidential interval : 39–198 nM). In
1
50
addition, no binding of compound 29g to human CRF and CRF was observed up to 10 ꢀM
2
α
2β
(
data not shown). This compound showed acceptable physicochemical properties, such as
solubility for oral administration (33 ꢀg/ml with bile acid in pH 6.8 phosphate buffer). Therefore,
brain penetration and CRF receptorꢀbinding activity of compound 29g after oral administration
1
were also evaluated by ex vivo testing in mice (Table 5). Compound 29g almost entirely
1
25
inhibited the IꢀCRF binding in the frontal cortex, olfactory bulb, and pituitary gland in mice
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after 1 h of oral administration at 138 ꢀmol/kg (60 mg/kg). This result indicated that compound
29g was well absorbed orally and penetrated the blood–brain barrier in mice as well as 1a at 53
ꢀmol/kg (20 mg/kg).
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a
Table 5. Ex vivo IꢀCRF binding inhibitory activity
Compound No. Frontal
cortex
Olfactory
bulb
Pituitary
29g
86
96
83
101
98
1a
95
a
125
The values are % inhibition of ovine IꢀCRF binding to mouse frontal cortex, olfactory bulb,
and pituitary homogenates. Tissues were collected by decapitation 1 h after oral administration
of 138 ꢀg/kg (60 mg/kg) or 53 ꢀg/kg (20 mg/kg) of compound 29g or 1a (n = 5), respectively.
Homogenates of each brain area were prepared from 5 brains for each compound.
Compound 29g was also evaluated against HPA axis activation in mice by measuring
increased plasma ACTH levels in response to stress. As shown in Figure 5, significant reduction
of HPA axis activation was observed after 1 h of oral administration at 138 ꢀmol/kg (60 mg/kg),
which was a dose that was effective in the ex vivo test described in Table 5. These results
revealed that compound 29g also antagonized CRFꢀrelated responses in mice.
a
Figure 5. Effect of compound 29g on the suppression of ACTH secretion
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a
ACTH concentration in blood was measured 1 h after oral administration of mice with 138
ꢀmol/kg (60 mg/kg) of compound 29g or 53 ꢀmol/kg (20 mg/kg) of compound 1a. Data are
indicated as the mean ± standard error of the mean (SEM) (n = 10). *p < 0.05, parametric
Dunnett’s test compared with vehicle.
Conclusion
We designed a novel type of CRF receptor antagonist using a 6ꢀ5 fused benzazole series with
1
an anilino group substituted on the scaffold via one atom. Ring A, which includes HBA, is the
z
core structure and is fused with ring C, which contains a small alkyl group R , and the compound
does not have a ring B, which distinguishes this novel type of CRF receptor antagonist from
1
numerous others that have been reported. Among the designed benzazole series consisting of
benzoxazole 5, benzothiazole 9, and benzimidazole 13 derivatives, benzothiazole 9 had the most
potent CRF receptorꢀbinding activity. We believe that the outermost sulfur atom on the
1
benzothiazole ring illustrated in Figure 4 contributed to the CRF receptorꢀbinding activity. As
1
expected, the binding activity of the 1ꢀNꢀmethylbenzimidazole analog 22a was improved relative
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to that of benzothiazole 9. The results of SAR studies at the other positions on the 1ꢀNꢀ
methylbenzimidazole core suggested that the newly designed 6ꢀ5 fused benzazole series was
quite effective at binding to a CRF receptor. Identification of new types of scaffolds will allow a
1
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wide variety of CRF receptor antagonist designs in the future. Furthermore, our benzimidazole
1
series has greater structural flexibility than series with scaffolds directly bonding to the
heteroaromatic core and is expected to have better physicochemical properties after further
optimization to develop a clinical candidate. Among the synthesized compounds in this series, 4ꢀ
2
7
7
chloroꢀN ꢀ(4ꢀchloroꢀ2ꢀmethoxyꢀ6ꢀmethylphenyl)ꢀ1ꢀmethylꢀN ,N ꢀdipropylꢀ1Hꢀbenzimidazoleꢀ
2
,7ꢀdiamine 29g showed CRF receptorꢀbinding inhibition activity as potent as compound 1, and
1
it was less cytotoxic at 30 ꢀM than compound 1. Compound 29g also exhibited inhibitory
activity of human CRFꢀstimulated cAMP accumulation and ex vivo CRF binding inhibitory
activity in the brain after oral administration in mice. In addition, oral treatment of compound
29g demonstrated efficacy for suppression of stressꢀinduced HPA axis activation in mice. These
results suggested that compound 29g is a promising lead compound for the development of a
novel CRF receptor antagonist. Additional optimization studies for this benzimidazole series
1
continue and will be reported in due course.
Experimental section
All reactions were performed using commercially available starting materials, reagents, and
solvents without further purification. In the following experimental, reaction progress was
determined by thin layer chromatography (TLC) analysis on silica gel 60 F254 plates (Merck) or
by liquid chromatography–mass spectrometry (LC/MS) analysis. LC/MS analysis was performed
using five methods. The first method was performed on a HPꢀ1100 (Agilent Technologies)
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separations module [CAPCELL PAK UGꢀ120 ODS (2.0 × 50 mm I.D., Shiseido Co., Ltd.,
Japan); 0.1% TFA in distilled H O/MeCN gradient; UV detection at 220 nm or 254 nm]. MS
2
spectra were recorded using a Micromass ZMD with electrospray ionization. The second method
was performed on a Thermo Surveyor highꢀperformance liquid chromatography (HPLC)
separations module [YMC ODSꢀAQ (4.6 × 50 mm I.D.); 1% isopropyl alcohol and 0.01%
0
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9
0
heptafluorobutyric acid in distilled H O/MeCN gradient; UV detection at 220 nm or 254 nm].
2
MS spectra were recorded using a Finnigan LCQ Duo Ion Trap MS with atmospheric pressure
chemical ionization in the positive mode. The third method was performed on a Thermo
Surveyor HPLC separations module [YMC ODSꢀAQ (4.6 × 50 mm I.D.); 1% isopropyl alcohol
and 10 mM NH OAc in a distilled H O/MeCN gradient; UV detection at 220 nm or 254 nm].
4
2
MS spectra were recorded using a Finnigan LCQ Duo Ion Trap MS with atmospheric pressure
chemical ionization in the negative mode. The fourth method was performed on a Shimadzu LCꢀ
2
0AD separations module [Lꢀcolumn2 ODS (3.0 × 50 mm I.D., CERI, Japan); 5 mM AcONH4
in an ultrapure H O/MeCN gradient; UV detection at 220 nm or 254 nm]. MS spectra were
2
recorded using a Shimadzu LCMSꢀ2020 system with electrospray ionization. The fifth method
was performed on a Waters 2795 separations module [Lꢀcolumn2 ODS (3.0 × 50 mm I.D.,
CERI, Japan); 0.05% TFA in an ultrapure H O/MeCN gradient; UV detection at 220 nm or 254
2
nm]. MS spectra were recorded using a Waters ZQ2000 with electrospray ionization. Magnesium
sulfate or sodium sulfate was used for desiccants of organic extracts. Chromatographic
purification was performed on a silica gel column (Kieselgel 60, 0.063–0.22 mm, Merck) or on
PurifꢀPack (SI 60 ꢁm or NH 60 ꢁm, Fuji Silysia, Ltd.). Preparative HPLC was performed on a
Gilson pumping system used with a photodiode array detector (Hewlett Packard 1100 series)
[
YMC ODSꢀA(20 × 50 mm I.D.); 0.1% TFA in a distilled H O/MeCN gradient; UV detection at
2
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20 nm]. Hydrochlorides were prepared from a corresponding free form using 4 M hydrogen
chloride in EtOAc, 10% in MeOH, 2 M in Et O, or 4 M in 1,4ꢀdioxane unless noted otherwise.
2
Data of synthesized compounds were measured and shown as described below. LC/MS analysis
for the detection of mass ion peaks was performed as described above. Proton nuclear magnetic
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1
resonance ( HꢀNMR) spectra were recorded on a Varian Mercuryꢀ300 (300 MHz), Bruker DPXꢀ
3
00 (300 MHz), or Varian INOVAꢀ400 (400 MHz). Chemical shifts are given in parts per
million (ppm), with tetramethylsilane used as an internal standard. The following abbreviations
are used: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, sxt = sextet, m =
multiplet, dd = doublets of doublet, br s = broad singlet, br = broad. Coupling constants (J
values) are given in hertz (Hz). The acidic protons of diketones, carboxylic acids, alcohols, or
1
anilines were not frequently observed in the H NMR spectra. Carbon nuclear magnetic
13
resonance ( CꢀNMR) spectra was recorded on a Bruker Avance III 400 (400 MHz). Chemical
shifts are given in ppm, with tetramethylsilane used as an internal standard. The purities of all
compounds tested in the biological systems were assessed by elemental analyses, analytical
HPLC, or LC/MS. Elemental analyses were performed by Takeda Analytical Research
Laboratories, Ltd. HPLC analyses were performed on a Varian ProStar [YMC ODSꢀAQ (4.6 ×
1
50 mm I.D.); 1% iꢀPrOH and 10 mM NH OAc in a distilled H O/MeCN gradient; UV detection
4 2
at 220 nm or 254 nm] or a Shimadzu UFLC instrument [Lꢀcolumn2 ODS (3.0 × 50 mm, I.D.);
0.1% of TFA in a distilled H O/MeCN gradient; UV detection at 220 nm]. LC/MS analyses were
2
performed as described above. All compounds were ≥95% pure as determined by HPLC and
LC/MS analyses, unless noted otherwise.
N-Mesityl-7-nitro-1,3-benzoxazol-2-amine 4. To a mixture containing compound 2 (0.10 g,
0
.65 mmol) and sodium carbonate (0.14 g, 1.3 mmol) in EtOH was added (2,4,6ꢀ
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trimethylphenyl)isothiocyanate (0.14 g, 0.78 mmol), and the mixture was refluxed overnight.
After cooling, the reaction mixture was filtered. The filtrate was concentrated in vacuo.
Purification of the residue via Biotage chromatography eluting with 20% EtOAc/CH Cl gave 1ꢀ
2
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0
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(
2ꢀhydroxyꢀ3ꢀnitrophenyl)ꢀ3ꢀmesitylthiourea 3 (0.17 g, 80%). MS Calcd.: 331; Found: 332
(M+H). To a solution containing compound 3 (0.06 g, 0.18 mmol) in MeCN was added mercury
II) chloride (0.10 g, 0.36 mmol), and the mixture was then stirred for 1 h. The reaction mixture
(
was diluted with EtOAc (2 mL) and filtered through a prepacked celite column. The filtrate was
concentrated in vacuo. The residue was purified via Biotage chromatography eluting with 20%
EtOAc/CH Cl to afford the title compound (0.047 g, 90%). MS Calcd.: 297; Found: 298
2
2
1
(
M+H). H NMR (CDCl , 400 MHz) δ 2.29 (6H, s), 2.32 (3H, s), 6.99 (2H, s), 7.30 (1H, t, J =
3
8.2 Hz), 7.77 (1H, d, J = 8.1 Hz), 7.78 (1H, d, J = 8.6 Hz).
2
7
7
N -Mesityl-N ,N -dipropyl-1,3-benzoxazole-2,7-diamine 5. To a flask was added compound
4
(0.10 g, 0.34 mmol) and MeOH (40 mL). The flask was purged with nitrogen followed by the
addition of palladium on carbon (10%, 0.01 g). The flask was evacuated and pressurized to 2ꢀ3
psig hydrogen and stirred for 1 h. After completion as determined by HPLC, the reaction was
filtered. To the filtrate were added propionaldehyde (0.1 mL, 1.7 mmol), NaBH CN (0.1 g, 1.7
3
mmol) and AcOH (1 mL). The mixture was stirred overnight, then diluted with EtOAc and
washed with water. The organic layer was dried and concentrated in vacuo. The residue was
purified by Biotage chromatography eluting with 5% MeOH/CH Cl gave the title compound
2
2
1
(
0.11 g, 90%). MS Calcd.: 351; Found: 352 (M+H). H NMR (CDCl , 400 MHz) δ 0.74 (6H, t, J
3
=
7.2 Hz), 1.47–1.53 (4H, m), 2.27 (6H, s), 2.29 (3H, s), 3.18 (4H, t, J = 7.8 Hz), 6.34 (1H, d, J =
8.1 Hz), 6.70 (1H, d, J = 7.0 Hz), 6.93 (2H, s), 6.98 (1H, t, J = 8.1 Hz).
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N -Mesityl-N ,N -dipropyl-1,3-benzothiazole-2,7-diamine
9.
A
mixture
of
3ꢀ
nitrophenylisothiocyanate 6 (2.25 g, 12.5 mmol) and mesityl amine (1.4 mL, 10 mmol) in MeOH
(
10 mL) was stirred at rt for 2 h. The resulting precipitate was collected by filtration to give 1ꢀ
mesityl)ꢀ3ꢀ(3ꢀnitrophenyl)thiourea. To the suspension of the thiourea (1.26 g, 4.0 mmol) in
10
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(
AcOH (20 mL) was added bromine (0.22 mL, 4.2 mmol), and the mixture was refluxed for 1 h.
After cooling, the reaction mixture was concentrated in vacuo. The resulting solid was solved in
MeOH and the insoluble material was filtered off. The filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography eluting with 25% EtOAC/hexane to
2
give N ꢀmesitylꢀ7ꢀnitroꢀ1,3ꢀbenzothiazolꢀ2ꢀamine 7 (0.14 g, 11%). MS Calcd.: 313; Found: 314
(M+H). To a solution of compound 7 (1.8 g, 5.7 mmol) in glacial AcOH (7.2 mL) and EtOH (25
mL) was added iron powder (1.8 g, 32 mmol). The resulting solution was refluxed for 18 h. After
cooling, the slurry was filtered and the filtrate was concentrated in vacuo to give a brown solid.
The solid was slurried in water, collected by filtration and purified by flash chromatography
2
eluting with a 33% hexane/EtOAc mixture to give N ꢀmesitylꢀ1,3ꢀbenzothiazoleꢀ2,7ꢀdiamine 8
(0.9 g, 55%) as a tan powder. MS Calcd.: 283; Found: 284 (M+H). To compound 8 (0.125 g,
0.44 mmol) and propionaldehyde (0.16 mL, 2.2 mmol) in 1,2ꢀdichloroethane (5 mL) was added
glacial AcOH (one drop) and NaBH(OAc) (0.28 g. 1.3 mmol). The mixture was stirred at 50°C
3
for 1 h and concentrated in vacuo. The residue was purified by flash chromatography eluting
with a 2% MeOH/CH Cl mixture to give the title compound (0.016 g, 10%) as a tan powder.
2
2
1
MS Calcd.: 367; Found: 368 (M+H). H NMR (CDCl , 400 MHz) δ 0.73 (6H, t, J = 7.4 Hz),
3
1.31–1.40 (4H, m), 2.23 (6H, s), 2.26 (3H, s), 2.94–2.98 (4H, m), 6.67 (1H, t, J = 2.7 Hz), 6.92
(2H, s), 7.14–7.17 (2H, m).
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N -Mesityl-N ,N -dipropyl-1H-benzimidazole-2,7-diamine hydrochloride 13. To a mixture
containing 3ꢀnitroꢀoꢀphenylenediamine 10 (5.0 g, 33 mmol) and sodium carbonate (10.0 g, 98
mmol) in EtOH was added (2,4,6ꢀtrimethylphenyl)isothiocyanate (5.79 g, 32.6 mmol), and the
mixture was refluxed for 12 h. DIC was added at this temperature, and the reaction mixture was
refluxed for 48 h. After cooling, the solvent was removed in vacuo. Purification of the residue
via Biotage chromatography eluting with 30% EtOAc/CH Cl gave Nꢀmesitylꢀ7ꢀnitroꢀ1Hꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
1
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4
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0
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2
3
4
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2
2
benzimidazoleꢀ7ꢀamine 11 (5 g, 54 %). MS Calcd.: 296; Found: 297 (M+H). Compound 11
(0.078 g, 0.263 mmol) was dissolved in MeOH and hydrogenated with palladium on carbon
(
0.028 g) at rt under balloon pressure for 0.5 h. The catalyst was filtered off and the filtrate was
2
concentrated in vacuo to give N ꢀmesitylꢀ1Hꢀbenzimidazoleꢀ2,7ꢀdiamine 12 (0.06 g, 86%). MS
Calcd.: 266; Found: 267 (M+H). A mixture of compound 12 (0.089 g, 0.334 mmol),
propionaldehyde (0.097 g, 1.70 mmol) and 5% AcOH/MeOH was stirred at rt for 10 min.
MPBH CN was added to the mixture, followed by stirring at rt for 2 h. The reaction mixture was
3
filtrated, and the filtrate was washed with saturated aqueous NaHCO and concentrated in vacuo.
3
The residue was purified by flash column chromatography to give the free form of the title
compound (0.05 g, 43%). The residue was transformed to the hydrogen chloride (0.02 g, 16%).
1
MS Calcd.: 350; Found: 351 (M+H). H NMR (DMSOꢀd , 00 MHz) δ 0.82 (6H, t, J = 7.4 Hz),
6
1
.42 (4H, d, J = 7.6 Hz), 2.02–2.23 (6H, m), 2.23–2.38 (3H, m), 3.25 (4 H, br s), 6.97 (2H, br s),
.02–7.25 (3H, m), 9.70 (1H, br s), 12.30 (1H, br s).
7
N-Methyl-2,6-dinitroaniline 15a. To a suspension of 2ꢀchloroꢀ1,3ꢀdinitrobenzene 14 (200 g,
987 mmol) in MeOH (300 mL) was added methylamine (40% solution in MeOH; 314 mL, 2.96
mol), and the mixture was stirred at rt for 3 h. The solvent was evaporated in vacuo, and the
residue was dissolved in EtOAc and saturated aqueous NaHCO . The aqueous layer was
3
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separated and extracted with EtOAc. The organic layer was washed with water, brine, passed
through Celite, dried and concentrated in vacuo to give the title compound (192 g, 99%) as a
1
yellow solid. H NMR (CDCl , 300 MHz) δ 2.89 (3H, d, J = 5.4 Hz), 6.76 (1H, t, J = 8.1 Hz),
3
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11
12
13
14
15
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8
.18 (2H, d, J = 8.1 Hz), 8.49 (1H, br s).
2
N -Methylbenzene-1,2,3-triamine 16a. A mixture of compound 15a (12.2 g, 0.122 mmol)
and palladium on carbon (10%, 4.30 g) in MeOH (450 mL) was stirred at rt under hydrogen
atmosphere for 2 h. The catalyst was removed by filtration and the filtrate was concentrated in
vacuo. The residue was purified by silica gel column chromatography eluting with 50–100%
1
EtOAc/hexane gradient mixture to give the title compound (12.6 g, 75%). H NMR (CDCl , 300
3
MHz) δ 2.71 (3H, br s), 3.73 (5H, br s), 6.22 (2H, d, J = 7.8 Hz), 6.75 (1H, d, J = 7.8 Hz).
4-Bromo-2-methoxy-6-methylaniline 18. To a solution of 2ꢀmethoxyꢀ6ꢀmethylaniline 17
(
25.0 g, 182 mmol) in AcOH (30 mL) and MeOH (60 mL) was added a solution of bromine
9.34 mL, 182 mmol) in AcOH (60 mL) at 0°C, and the mixture was stirred at rt for 2 h. The
(
precipitate was collected by filtration, washed with Et O and dissolved in EtOAc and saturated
2
aqueous NaHCO . The organic layer was separated, and the aqueous layer was extracted with
3
EtOAc. The combined organic layer was washed with brine, dried and concentrated in vacuo to
1
give the title compound (20.7 g, 53%) as a brown solid. MS Calcd.: 215; Found: 216 (M+H). H
NMR (CDCl , 300 MHz) δ 2.14 (3H, s), 3.83 (3H, s), 6.80 (1H, s), 6.84 (1H, s).
3
(4-Bromo-2-methoxy-6-methylphenyl)isothiocyanate 19. To a solution of Et N (0.65 mL,
3
4
.63 mmol) in pyridine (3 mL) were added dropwise carbon disulfide (0.70 mL 11.6 mmol) and
o
a solution of compound 18 (1.00 g, 4.63 mmol) in pyridine (5 mL) at −10 C, and the mixture
o
o
was stirred at −10 C for 1 h. DCC (0.955 g, 4.63 mmol) was added to the mixture at ꢀ10 C,
o
followed by stirring at −10 C for 3 h and at rt overnight. The reaction mixture was concentrated
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