Discovery of 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole, a novel CRF1receptor antagonist

Article abstract of DOI:10.1016/j.bmc.2016.11.011

Compound 1 exhibits potent binding inhibition activity against a corticotropin-releasing factor 1 (CRF₁) receptor (IC₅₀= 9.5 nM) and in vitro antagonistic activity (IC₅₀= 88 nM) but is rapidly metabolized by human hepatic microsomes (182 μL/min/mg). Here we identified metabolically stable compounds with potent CRF binding inhibitory activity. Structure–activity relationship (SAR) studies considering in vitro metabolic stability revealed that 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole 24d was more stable in human microsomes (87 μL/min/mg) than compound 1. Compound 24d demonstrated potent CRF binding inhibitory activity (IC₅₀= 4.1 nM), in vitro antagonistic activity (IC₅₀= 44 nM), and slow dissociation from the CRF₁receptor. Orally administered compound 24d (6–24 μmol/kg) showed ex vivo CRF₁receptor binding in the rat pituitary, olfactory bulb, and frontal cortex and suppressed stress-induced adrenocorticotropic hormone (ACTH) secretion. In this report, we discuss SAR studies on the metabolic stability as well as CRF binding inhibitory activity of the benzimidazole series as CRF₁receptor antagonists and the pharmacological profiles of compound 24d.

Full text of DOI:10.1016/j.bmc.2016.11.011

Accepted Manuscript
Discovery of 4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pen-
tan-3-yl)-1H-benzimidazole, a novel CRF1 receptor antagonist
Michiyo Mochizuki, Takuto Kojima, Katsumi Kobayashi, Etsuo Kotani, Yuji
Ishichi, Naoyuki Kanzaki, Hideyuki Nakagawa, Teruaki Okuda, Yohei Kosugi,
Takahiko Yano, Yuu Sako, Maiko Tanaka, Kazuyoshi Aso
PII:
S0968-0896(16)30811-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.011
BMC 13378
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 September 2016
4 November 2016
5 November 2016
Please cite this article as: Mochizuki, M., Kojima, T., Kobayashi, K., Kotani, E., Ishichi, Y., Kanzaki, N., Nakagawa,
H., Okuda, T., Kosugi, Y., Yano, T., Sako, Y., Tanaka, M., Aso, K., Discovery of 4-chloro-2-(2,4-dichloro-6-
methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole, a novel CRF1 receptor antagonist, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery of
4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-me
thyl-7-(pentan-3-yl)-1H-benzimidazole, a novel
CRF1 receptor antagonist
Michiyo Mochizuki a, Takuto Kojima a, Katsumi Kobayashi a, Etsuo Kotanib, Yuji Ishichi a,
Naoyuki Kanzaki a, Hideyuki Nakagawa a, Teruaki Okuda a, Yohei Kosugia, Takahiko
Yano c, Yuu Sako a, Maiko Tanaka a, and Kazuyoshi Aso a
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1,
Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
b CMC Center, Takeda Pharmaceutical Company Ltd., 17-85, Jusohonmachi 2-chome,
Yodogawa-ku, Osaka 532-8686, Japan
c Taisho Pharmaceutical Company Ltd., 403, Yoshino-cho 1-chome, Kita-ku, Saitama-shi,
Saitama 331-9530, Japan

ABSTRACT
Compound
1
exhibits potent binding inhibition activity against
a
corticotropin-releasing factor 1 (CRF1) receptor (IC50 = 9.5 nM) and in vitro antagonistic
activity (IC50 = 88 nM) but is rapidly metabolized by human hepatic microsomes (182
L/min/mg). Here we identified metabolically stable compounds with potent CRF binding
inhibitory activity. Structure–activity relationship (SAR) studies considering in vitro
metabolic
stability
revealed
that
4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole
24d was more stable in human microsomes (87 L/min/mg) than compound 1. Compound
24d demonstrated potent CRF binding inhibitory activity (IC50 = 4.1 nM), in vitro
antagonistic activity (IC50 = 44 nM), and slow dissociation from the CRF1 receptor. Orally
administered compound 24d (6–24 mol/kg) showed ex vivo CRF1 receptor binding in the
rat pituitary, olfactory bulb, and frontal cortex and suppressed stress-induced
adrenocorticotropic hormone (ACTH) secretion. In this report, we discuss SAR studies on
the metabolic stability as well as CRF binding inhibitory activity of the benzimidazole
series as CRF1 receptor antagonists and the pharmacological profiles of compound 24d.
Keywords
corticotropin-releasing factor, CRF1 receptor antagonist, 7-alkylbenzimidazole, metabolic
stability
Abbreviations
CRF, Corticotropin-releasing factor; HPA, hypothalamus-pituitary-adrenocortical; ACTH,
adrenocorticotropic
hormone;
WSC,
SAR,
structure-activity-relationship;
CDI,
N,N′-carbodiimidazole;
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride; HOBt, 1-hydroxybenzotriazole; CHO, Chinese hamster ovary; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; PMSF, phenylmethylsulfonyl
fluoride; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; FCS, fetal calf
serum.
Corresponding Author
E-mail: michiyo.mochizuki@takeda.com

1. Introduction
Corticotropin-releasing factor (CRF) is a 41-amino-acid neuropeptide that mediates its
actions through two Gs-coupled G protein-coupled receptor subtypes, CRF1 and CRF2.1,2
CRF is believed to be the main regulator of the hypothalamus–pituitary–adrenocortical
(HPA) axis via CRF1 receptors.3,4 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 In healthy
individuals not suffering from life-threatening events, a negative feedback system against
the activation of the HPA axis is operating; in contrast, the system collapses in patients with
stress-related disorders. Moreover, CRF plays an important role in the brain as a
neurotransmitter that mediates stress-related behaviors.4,7
Over the last 20 years, the efficacies of numerous non-peptide, small-molecule CRF1
receptor antagonists against stress-related disorders have been determined using animal
models; however, no drugs have been developed. The compounds described in Figure 1
were studied in clinical trials for their effects on stress-related disorders such as depression
and
3-(6-(dimethylamino)-4-methylpyridin-3-yl)-2,5-dimethyl-N,N-dipropylpyrazolo[1,5-a]pyri
midin-7-amine and
(R121919)8
anxiety.
Among
them,
2-(2,4-dichlorophenyl)-6-(heptan-4-yl)-4-methyl-7,8-dihydro-6H-1,3,6,8a-tetraazaacenapht
hylene (NBI-34041)9 were efficient in phase II studies. In contrast,
2-(mesityloxy)-3,6-dimethyl-4-(pentan-3-yloxy)pyridine
(CP-316,311),10
4-(2-chloro-4-methoxy-5-methylphenyl)-N-((1S)-2-cyclopropyl-1-(3-fluoro-4-methylpheny
l)ethyl)-5-methyl-N-(prop-2-yn-1-yl)-1,3-thiazol-2-amine (SSR125543),11 pexacerfont,12,13
16
emicerfont,14 and vercerfont15, failed to treat major depression, general anxiety disorder,
ost-traumatic stress disorder, and irritable bowel syndrome. On the other hand, a clinical
trial of verucerfont has been conducted by National Institute on Alcohol Abuse and
Alcoholism plans to conduct a trial for alcohol dependence.17 Furthermore, CRF1 receptor
antagonists serve as a target for other stress-related diseases such as pain and anorexia
caused by elevated levels of CRF.18,19 Therefore, CRF1 receptor antagonists are an
attractive target for drug discovery.
Figure 1. Reported CRF1 receptor antagonists

In our previous study, we designed unique benzazole derivatives with a one-atom linker
between the core and the pendant phenyl group as novel CRF1 receptor antagonists.20 Initial
structure–activity relationship (SAR) studies revealed that the benzimidazole derivative 1
exhibits potent CRF1 receptor-binding activity in vitro (IC50 = 9.5 nM).20 Compound 1
inhibited CRF-stimulated cAMP accumulation in Chinese hamster ovary (CHO) cells
expressing human CRF1 receptors (IC50 = 88 nM). Furthermore, compound 1 penetrated the
brain after oral administration in ex vivo CRF-binding tests in mice.20 However, compound
1 was found to be easily metabolized according to the results of in vitro analysis of
metabolites in human hepatic microsomes (Figure 2). The clearance of compound 1 was
182 L/min/mg, and the metabolites, depropylated (1a, 1b and 1d) and oxidized (1e-f) on
the 7-amino group and hydroxylated on the 2-anilino group (1c-d), were predicted using
LC/MS/MS. Acceleration of metabolism typically decreases the exposure period of a parent
compound in vivo and diminishes the efficacy of the parent compound. Therefore,
modifications of the dipropylamino group at the 7-position and the para position on the
2-anilino group are planned to identify metabolically stable compounds with potent in vitro
and ex vivo CRF1 receptor-binding activity and in vivo efficacy.
Figure 2. In vitro analysis of metabolites of compound 1 in human hepatic microsomes

b
aIn vitro metabolic clearance in human hepatic microsomes. The structures of the
metabolites were elucidated using LC/MS/MS.
Here we describe the syntheses and biological activities of the benzimidazole series as
well as the SAR associated with metabolic stability.
2. Results and Discussion
2.1.
Chemistry.
Synthesis of the 7-alkylaminobenzimidazole and 7-alkylbenzimidazole derivatives is
described in Schemes 1–4. Scheme 1 shows the synthetic route for the introduction of
substituents at the 2-, 4-, and 7-positions of the 7-aminobenzimidazole core.
Monoalkylamino benzimidazolones 3a–d and dialkylamino benzimidazolones 4a–b were
synthesized from 7-aminobenzimidazol-2-one 220 by reductive alkylation. The key
intermediates 8c–d for the target compounds were prepared from the dialkylamino
derivatives 4a–b by halogenation at the 4-position of the core using NBS and chlorination
at the 2-position using POCl3. The 3-pentyl analog 8a was derived from a corresponding
benzimidazol-2-one 3a by the reverse order of chlorination, that is, the first and second
chlorinations were performed at the 2- and 4-positions of the core to give 6a and 8a,
respectively. The acylamino analog 8b was obtained by acylation of
2,4-dichloro-7-isopropylaminobenzimidazole 7, prepared from 3b using a method similar
to that used for the synthesis of the 3-pentyl analog 8a. Chlorination at the 4-position of
2-chloro-7-monoalkylbenzimidazoles 6a–b produced low yields because of the production
of 6-chloro analogs.21 Finally, the target compounds 9a–d were obtained by heating the

reaction of 2-chlorobenzimidazoles 8a–d and 4-chloro-2-methoxy-6-methylaniline.20 All
reaction conditions from 7-aminobenzimidazol-2-one 2 to the targets 9a–d were efficient.
Scheme 1. Synthesis of 7-alkylaminobenzimidazoles 9a–da
aReagents and conditions: (a) 3-pentanone or acetone or CF3CH2CHO or EtO2CCHO,
NaBH3CN, AcOH, MeOH, rt; (b) MeCHO, NaBH3CN, AcOH, MeOH or EtOH, rt; (c)
NBS, MeCN, rt; (d) POCl3, 90°C–100°C; (e) 4-chloro-2-methoxy-6-methylaniline, NMP,
110°C; (f) POCl3, 70°C; (g) NCS, MeCN, rt; (h) i) NaH, DMF, 60°C, ii) propionyl
chloride, rt.

Methods for converting the amino group at the 7-position of the benzimidazole core are
shown in Scheme 2. Ethyl acetate 9d was reduced using lithium borohydride to give the
hydroxyethyl analog 10 in good yield. The hydroxyl group of compound 10 was treated
under Swern oxidation conditions, followed by the Grignard reaction with
methylmagnesium bromide to afford the 2-hydroxypropyl analog 12. Alkaline hydrolysis of
ethyl acetate 9d generated the acetic acid analog 13, which condensed with corresponding
amines to give amide analogs 14a–c.
Scheme 2. Conversion of the amino group at the 7-position of the benzimidazole corea
aReagents and conditions; (a) LiBH4, THF, reflux, 90%; (b) (COCl)2, DMSO, Et3N,
CH2Cl2, −65°C, 51%; (c) MeMgBr, Et2O, rt, 31%; (d) NaOH aq, EtOH, rt, 93%; (e)
HOBt·NH3, WSC, Et3N, DMF, rt, 52%; (f) MeNH2 or Me2NH, WSC, HOBt, THF, DMF,
rt, 70% or 62%, respectively.
Synthesis of 7-alkylbenzimidazoles 24a–f from a commercially available benzoic acid 15
is shown in Scheme 3. Benzoic acid 15 was esterified using oxalyl chloride and methanol to
give methyl 2-chlorobenzoate 16, followed by methylamination of 16 to produce
quantitative yields of compound 17. Nitro benzene 17 was hydrogenated using a palladium
catalyst to generate excellent yields of aniline 18. A common benzimidazolone intermediate
19 was prepared by ring construction of diamine 18 with CDI. The ethyl groups of 20b as
R7a were introduced by Grignard reaction with ethylmagnesium bromide with good yield,
whereas n-propylmagnesium bromide generated a mixture of dialkylated 20a and

monoalkylated compound. Therefore, n-propyllithium prepared from n-propylbromide and
lithium was used instead of a Grignard reagent and gave the target compound 20a. An
acid-catalyzed dehydration reaction of 20a–b gave olefins, which were hydrogenated with a
palladium catalyst to afford 7-alkylbenzimidazol-2-ones 21a–b. Selective halogenation at
the 4-position of compounds 21a–b using NCS and subsequent chlorination at the
2-position of 22a–b with POCl3 were performed similarly for 7-alkylaminobenzimidazoles.
Finally, replacement of the chlorine atom at the 2-position with the corresponding anilines
and phenols in neutral or basic heating conditions produced the target
2-anilinobenzimidazoles 24a–c and the 2-phenoxybenximidazoles 24d–e, respectively.
Scheme 3. Synthesis of 7-alkylbenzimidazoles 24a–ea
aReagents and conditions: (a) i) (COCl)2, DMF, CH2Cl2, rt; ii) MeOH, rt, >99%; b)
MeNH2, THF, rt, >99%; c) H2, Pd–C, MeOH, rt, 99%; (d) CDI, THF, rt to 50°C, 78%; (e)
n-PrBr, Li, Et2O, THF, −78°C to rt, or EtMgBr, Et2O, THF, 35°C–40°C; (f) i) HCl, H2O,

EtOH, 50°C–70°C; ii) H2, Pd–C, EtOH, rt; (g) NCS, (AIBN,) CCl4, 70°C; (h) POCl3, 90°C;
(i) ArNH2, NMP, 130°C; or ArOH, K2CO3, DMF, 100°C.
Conversion of substituents at the para position on the phenoxy group is described in
Scheme 4. A bromo group of 24e was replaced with a cyano group using copper (I) cyanide
and microwave irradiation to give cyanide 25. The carboxamide analog 26 was obtained
from 24e by lithiation, incorporation of a carbonyl group using carbon dioxide, and
condensation of ammonia.
Scheme 4. Conversion of substituents at the para position on the benzyl ring of the
phenoxy groupa
aReagents and conditions: (a) CuCN, NMP, 150°C, microwave, 21%; (b) i) n-BuLi,
n-hexane, THF, −78°C; ii) CO2, rt; (iii) WSC, HOBt·NH3, DMF, rt, 23%.
2.2 Biology.
Each compound was tested for its ability to inhibit ovine 125I-CRF binding to human
CRF1 receptors expressed on the membranes of CHO cells as well as its metabolic stability
in human hepatic microsomes. Our initial efforts focused on analyzing the substituents at
the 7-position of the benzimidazole core, because cleavage of carbon–nitrogen bonds and
oxidation of the alkyl group of 1 progressed in human hepatic microsomes as described
above. A branched alkyl group introduced on nitrogen and oxygen or fluorine was
incorporated into the alkyl groups to prevent their degradation by the cleavage of
carbon–nitrogen bonds and the oxidation of alkyl groups, respectively.
The results of analyses of human CRF1 receptor-binding activities and metabolic
clearance by human hepatic microsomes are listed in Table 1. The isopropylacylamino

analog 9b was 10-fold less potent as an inhibitor of CRF1 receptor-binding activity and
exhibited increased metabolic clearance (217 l/min/mg) compared with the dipropylamino
analog 1, although branched alkyl and amide groups were introduced to prevent cleavage of
a carbon–nitrogen bond. The monoalkylated and branched 3-pentyl analog 9a was
metabolically more stable than compound 1, with a clearance of 75 l/min/mg.
Unfortunately, the binding activity of 9a decreased. Blocking the terminal position of the
alkyl group using fluorine had no effect on clearance as exhibited by the trifluoropropyl
analog 9c, which was potently active as the propyl analog 1. Among compounds 9b, 10, 12,
and 14a–c with a hydrophilic group such as a carbonyl or a hydroxy group on the alkyl
group to prevent oxidation, clearance of the carboxamide analog 14a was lower than that of
the dipropyl analog 1, whereas the binding activity was 15-fold less potent than that of
compound 1. The binding activities of these oxidative analogs were inversely proportional
to that of the bulky terminal hydrophilic groups. Thus, the hydroxyethyl analog 10 was the
most potent and the N,N-dimethylcarboxamide analog 14c was the least potent. The alkyl
analog 24a markedly decreased metabolic clearance and demonstrated the highest binding
activity (IC50 = 7.4 nM). A carbon–carbon bond of the 7-alkyl moiety was expected to
exhibit increased resistance to cleavage than a nitrogen–carbon bond of 7-dialkylamino
moieties. The smaller alkyl analog 24b exhibited increased metabolic stability without
decreased binding activity compared with the corresponding alkylamino analog 27.
Table 1. Effects of substituents at the 7-position of 2-anilino-4-halogenobenzimidazole
derivatives on hCRF1 receptor-binding activities and metabolic stabilities in human hepatic
microsomes
Compound R7
No.
R4 Binding
(IC50, nM)a
Clearance
(l/min/mg, @ 1
M)b
Cl 9.5 (6.0–15)
182
1

Cl 110 (87–140)
Cl 110 (93–140)
Br 18 (9.8–32)
Br 48 (34–67)
Br 53 (42–67)
75
9a
217
189
227
202c
9b
9c
10
12
Br 140 (110–170) 155
Br 150 (110–210) 230
Br 270 (220–330) 244
14a
14b
14c
27
Cl 24 (20–30)
Cl 7.4 (4.5–12)
Cl 15 (10–21)
222
132
198
24a
24b
aIC50 values and 95% confidence intervals were calculated from the
concentration–response curves. bIn vitro metabolic clearance in human hepatic microsomes.
c40%–70% of the collection rate.
To analyze penetration into the brain, the alkyl analogs 24a and 24b were evaluated by ex
vivo CRF-binding studies in the rat pituitary, olfactory bulb, and frontal cortex, which
express high levels of CRF1 receptors (Table 2).22 The more potent binding activity of a test
compound in the olfactory bulb and frontal cortex indicate more penetration into the brain,
which was caused by increasing the amounts of a compound binding to the relative number
of CRF1 receptors in each tissue. Both compounds exhibited equal displacement of
125I-CRF in the pituitary at 46 and 49 mol/kg (20 mg/kg), respectively, 1 h after oral

administration, whereas the 4-chloro-7-(3-pentyl) analog 24b exhibited the highest
inhibition of 125I-CRF binding in the frontal cortex. These results indicate that compounds
24a and 24b were efficiently absorbed orally and that higher levels of the 3-pentyl analog
24b penetrated into the brain compared with the 3-heptyl analog 24a. Therefore, the
3-pentyl group was fixed in the following SAR studies of the 2-position of the core.
Table 2. Ex vivo 125I-CRF binding inhibitory activities of 7-alkylbenzimidazole
derivativesa
Compound R7
No.
CRF binding inhibition
(%)
Pit
61
OB
32
Ctx
15
24a
24b
69
41
60
aThe values represent the average % inhibition of ovine 125I-CRF binding to homogenates
of rat pituitary (Pit), olfactory bulb (OB), and frontal cortex (Ctx). Tissues were collected
after decapitating the rats 1 h after oral administration of 46 and 49 mol/kg of 24a and 24b
(20 mg/kg) (n = 3), respectively, and homogenates of each brain tissue were prepared from
three brains for each compound.
The effects of substituents at the 2-position of 4-chloro-7-(3-pentyl)benzimidazole
derivatives are shown in Table 3. Replacement of a methoxy group of 24b with a chloro
group at the ortho position of the anilino group decreased metabolic clearance.23 Additional
conversion of the anilino group of 24c to a phenoxy group decreased metabolic clearance
further using 87 l/min/mg of 24d. Moreover, the binding activity of 24d was more potent
than that of 24b. The chloro group of 24d at the para position of the phenoxy group was
replaced, because in vitro analysis of metabolites of 1 (Figure 2) indicated nucleophilic
substitution at this position (1c–d). Thus, hydrophilic groups were introduced at the para

position to increase metabolic stability. Introduction of a cyano group (25) contributed little
to metabolic stability compared with 24d, whereas a carboxamide group in compound 26
increased stability more than a chlorine atom in 24d. Unfortunately, compound 26 was
5-fold less potent than 24d.
Table 3. Effects of substituents at the 2-position of 4-chloro-7-(3-pentyl)benzimidazole
derivatives on hCRF1 receptor-binding activities and metabolic stabilities in human hepatic
microsomes
Compound No.
X2
Ra
Rb
Binding
(IC50, nM)a
Clearance
(l/min/mg, @ 1 M)b
NH
NH
O
OMe Cl
15 (10–21)
7.9 (5.5–11)
4.1 (2.7–6.1)
8.6 (5.7–13)
22 (17–28)
198
120
87
24b
24c
24d
25
Cl
Cl
Cl
Cl
Cl
Cl
O
CN
CO
NH2
84
O
29
26
aIC50 values and 95% confidence intervals were calculated from the
concentration–response curves. bIn vitro metabolic clearance in human hepatic microsomes.
SAR studies showed that 7-alkyl analogs enhanced metabolic stability with equally
potent binding activity compared with 7-dialkylamino analogs of the corresponding length
at the 7-position. The alkyl group containing only carbon–carbon bonds had no effect on
cleavage, unlike a dialkylamino group containing carbon–nitrogen bonds. Exchange of
linker atom X2 from nitrogen to oxygen contributed to improve metabolic stability.
Furthermore, the carboxamide group at the para position of the phenoxy group also
enhanced the stability, which suggests that it is effective to increase resistance against

oxidative metabolism derived from lower electron density on this phenyl ring as well as
that against nucleophilic substitution.
The metabolically stable compound 24d24 with the most potent binding activity was
tested in human hepatic microsomes in a similar way as compound 1 (Figure 3). No
cleavage of a carbon–carbon bond at the 7-position was detected, unlike that of a
carbon–nitrogen bond. Moreover, the para position of the phenoxy group was not
metabolized, and clearance (87 L/min/mg) was decreased compared with that of
compound 1.
Figure 3. In vitro analysis of metabolites of compound 24d in human hepatic microsomesa
aIn vitro analysis was carried out in a manner similar to that of compound 1 (Figure 2).
Compound 24d inhibited human CRF-stimulated cAMP accumulation in CHO cells
expressing human CRF1 receptors with an IC50 value of 44 nM (21–90 nM, 95%
confidence interval) and did not bind human CRF2α and CRF2β at 10 M. Moreover, the
affinity of compound 24d for more than 100 other receptors, channels, enzymes, and
transporters was not observed at 10 M (data not shown). These results demonstrate that
compound 24d antagonized ligand binding to the CRF1 receptor with high selectivity. In
addition, compound 24d exhibited good oral bioavailability (AUCpo: 94.6 ng·h/mL, F:
30.8%) at a dose of 1 mg/kg in rats.
To better understand the pharmacological profile of CRF1 receptor antagonists, the
correlation between the ligand dissociation rate and in vitro and in vivo efficacy has been
investigated.25,26,27 One study found that slow ligand dissociation from a CRF1 receptor
prolongs efficacy.27 Therefore, slowly dissociating compounds may serve as drugs with
increased efficacy. Inhibition of CRF1 receptor binding by compound 24d, R121919, and
pexacerfont with and without washing is described in Table 4. Compounds 24d and
R121919 showed approximately equally potent binding inhibition under washout and

no-washout conditions, whereas pexacerfont demonstrated much less inhibition under
washout conditions than under no-washout conditions. These results indicate that
compounds 24d and R121919 may dissociate more slowly than pexacerfont, suggesting
that the level and persistence of activity of compound 24d are greater than those of
pexacerfont.
Table 4. Comparison of hCRF1 receptor dissociation kinetics of compound 24d, R121919,
and pexacerfont
Compound
Washout
No washout
(IC50, nM)a
22 (12–40)
Ratio
(IC50, nM)a
32 (23–45)
67 (42–110)
>10000
(washout/no washout)
1.5
3.9
>29
24d
R121919
17 (9.8–29)
340 (170–670)
Pexacerfont
aIC50 values and 95% confidence intervals were calculated from the
concentration–response curves.
Penetration of the brain by compound 24d was evaluated according to ex vivo CRF
binding at 6, 12, and 24 mol/kg (2.5, 5, and 10 mg/kg) after 2 h of oral administration in
rats (Figure 4). Compound 24d inhibited the binding of ovine 125I-CRF to the pituitary,
olfactory bulb, and frontal cortex in a dose-dependent manner, suggesting that compound
24d was absorbed orally in quantities sufficient to penetrate the blood–brain barrier.
Figure 4. Ex vivo binding activity of compound 24d in ratsa

aInhibition of ovine 125I-CRF binding by compound 24d in homogenates of rat pituitary
(Pit), olfactory bulb (OB), and frontal cortex (Ctx). Tissues were collected after
decapitation of rats 2 h after oral administration of 6, 12, or 24 mol/kg (2.5, 5, or 10
mg/kg) of compound 24d. Data represent the mean ± standard error of the mean (SEM) (n
= 3). Statistical analysis was performed using the Williams test (#: p < 0.025).
Compound 24d was evaluated for its ability to normalize the HPA axis in a
stress-induced ACTH secretion test in rats. Oral administration of compound 24d at 6, 12,
and 24 mol/kg (2.5, 5, and 10 mg/kg) significantly reduced ACTH secretion in forced
swim stressed rats compared with vehicle-treated rats in a dose-dependent manner (Figure
5). These effective doses are quite reasonable because the binding to CRF1 receptors was
confirmed in the ex vivo test described above. The results of this in vivo test indicate that
compound 24d acts as a CRF1 receptor antagonist under conditions of unusual stress.
Compound 24d suppressed CRF- or stress-induced anxious/depressive behavior after oral
administration to rats at effective doses in an ex vivo binding test,28 and critical toxicity was
not observed in rat and dog studies lasting 13 weeks (data not shown).
Figure 5. Effect of compound 24d on the inhibition of ACTH secretion induced in rats
administered the acute swim stress testa

aCompound 24d [3–24 mol/kg (1.25–10 mg/kg)] was orally administered 2 h before the
90-s forced swim stress test. Blood samples were collected by decapitating the rats 5 min
after the test was terminated. The results are expressed as the mean ACTH (pg/mL)
concentration. Data are indicated as the mean ± SEM (n = 10–12). #: p < 0.025, parametric
Williams test, and ***: p<0.001, Student’s t-test, compared with vehicle.
3. Conclusion
SAR studies on binding inhibition activity against a CRF1 receptor and metabolic
stability
indicate
that
4-chloro-2-(2,4-dichloro-6-methylphenoxy)-1-methyl-7-(pentan-3-yl)-1H-benzimidazole
24d was more potent and metabolically stable than compound 1 and was therefore
identified as a selective CRF1 receptor antagonist. There was no detectable cleavage of a
carbon–carbon bond at the 7-position of compound 24, unlike that of a carbon–nitrogen
bond, and the para position of the benzene ring was not detected in human hepatic
microsomes. A rat ex vivo binding test indicated that compound 24d was efficiently
absorbed orally and penetrated the brain. Significant suppression of stress-induced ACTH
secretion was observed after oral administration of compound 24d at 6–24 mol/kg.
Furthermore, compound 24d dissociated slowly in vitro from the CRF1 receptor. These

results suggest that compound 24d may serve as an effective CRF1 receptor antagonist,
with a potential to treat patients with stress-related disorders.
4. Experimental section
4.1.
General
All reactions were performed using commercially available starting materials, reagents,
and solvents without further purification. Reactions were monitored using thin-layer
chromatography with silica gel 60 F254 plates (Merck) or liquid chromatography–mass
spectrometry (LC/MS). LC/MS analysis was performed using three methods. The first
method was performed using an HP-1100 (Agilent Technologies) separation module
[CAPCELL PAK UG-120 ODS (2.0 × 50 mm I.D., Shiseido Co., Ltd., Japan); 0.1% TFA
in a distilled H2O/MeCN gradient; UV detection at 220 nm or 254 nm]. MS spectra were
recorded using a Micromass ZMD with electrospray ionization. The second method was
performed using a Shimadzu LC-20AD separation module [L-column 2 ODS (3.0 × 50 mm
I.D., CERI, Japan); 5 mM AcONH4 in an ultrapure H2O/MeCN gradient; UV detection at
220 nm or 254 nm]. MS spectra were recorded using a Shimadzu LCMS-2020 with
electrospray ionization. The third method was performed using a Waters 2795 separation
module [L-column 2 ODS (3.0 × 50 mm I.D., CERI, Japan); 0.05% TFA in an ultrapure
H2O/MeCN gradient; UV detection at 220 nm or 254 nm]. MS spectra were recorded using
a Waters ZQ2000 with electrospray ionization. Magnesium sulfate or sodium sulfate was
used as a desiccant for organic extracts. Chromatographic purification was performed using
a silica gel column (Kieselgel 60, 0.063–0.22 mm, Merck) or Purif-Pack (SI 60 μm or NH
60 μm, Fuji Silysia, Ltd.). Preparative HPLC was performed using a Gilson pumping
system with a photodiode array detector (Hewlett Packard 1100 series) [YMC ODS-A (20
× 50 mm I.D.); 0.1% TFA in a distilled H2O/MeCN gradient; UV detection at 220 nm].
Synthesized compounds were analyzed as described below. LC/MS analysis for the
detection of mass ion peaks was performed as described above. Proton nuclear magnetic
resonance (1H-NMR) spectra were recorded using a Varian Mercury-300 (300 MHz) or a
Bruker DPX-300 (300 MHz) NMR system. Chemical shifts are shown in parts per million
(ppm), and tetramethylsilane was used as an internal standard. Abbreviations are as
follows: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, sxt = sextet, m =

multiplet, dd = doublets of a doublet, br s = broad singlet, and br = broad. Coupling
constants (J values) are given in hertz (Hz). The acidic protons of diketones, carboxylic
1
acids, alcohols, or anilines were not frequently observed in the H-NMR spectra. Carbon
nuclear magnetic resonance (13C-NMR) spectra were recorded on a Bruker Avance II+ 600
(600 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
using elemental analyses, analytical HPLC, or LC/MS. Elemental analyses were performed
by Takeda Analytical Research Laboratories, Ltd. HPLC analyses were performed using a
Shimadzu UFLC instrument [L-column 2 ODS (3.0 × 50 mm, I.D.); 0.1% TFA in a
distilled H2O/MeCN gradient; UV detection at 220 nm]. LC/MS analyses were performed
as described above. HPLC and LC/MS analyses revealed main peaks that were >95% of the
area under the curve.
4.2.
Synthesis
4.2.1 7-Isopropylamino-1-methyl-1,3-dihydro-2H-benzimidazol-2-one 3b. To
a
suspension of compound 213 (6.06 g, 37.1 mmol) in MeOH (60 mL) were added AcOH
(2.12 mL, 37.1 mmol), acetone (8.18 mL, 111 mmol) and NaBH3CN (6.98 g, 111 mmol),
and the mixture was stirred at rt for 15 h. The solvent was evaporated in vacuo. The residue
was suspended in H2O. The resulting solid was collected by filtration and washed with H2O
to give the title compound (1.25 g, 16%). H2O was added to the filtrate, and the solid was
collected by filtration and washed with H2O to give the title compound (4.95 g, 65%). total:
6.20 g, 81%. MS Calcd.: 205; Found: 206 (M+H). 1H NMR (DMSO-d6)  1.07 (6H, d, J =
6.3 Hz), 3.43–3.54 (1H, m), 3.55 (3H, s), 4.47 (1H, d, J = 7.2 Hz), 6.38–6.41 (1H, m),
6.77–6.93 (2H, m), 10.63 (1H, s).
4.2.2 1-Methyl-7-[(3,3,3-trifluoro)propylamino]-1,3-dihydro-2H-benzimidazol-2-one
3c. Compound 3c (1.95 g, 94%) was prepared from compound 2 (1.30 g, 7.97 mmol) and
3,3,3-trifluoropropionaldehyde (3.43 mL, 39.8 mmol) in a manner similar to that described
in compound 3b. Colorless solid. MS Calcd.: 259; Found: 260 (M+H). 1H NMR (CDCl3) 
2.41–2.57 (2H, m), 3.47 (2H, q, J = 6.3 Hz), 3.69 (3H, s), 3.95–4.00 (1H, br), 6.44 (1H, dd,
J = 8.1, 0.9 Hz), 6.62 (1H, dd, J = 8.1, 0.9 Hz), 6.95 (1H, t, J = 8.1 Hz), 8.83 (1H, s).

4.2.3 Ethyl N-(3-methyl-2-oxo-2,3-dihydro-1H-benzimidazol-4-yl)glycinate 3d.
Compound 3d (1.83 g, 24%) was prepared from compound 2 (50% in toluene, 4.97 g, 30.5
mmol) and ethyl glyoxylate (12.1 mL, 60.9 mmol) in a manner similar to that described in
1
compound 3b. Colorless solid. MS Calcd.: 249; Found: 250 (M+H). H NMR (CDCl3) 
1.20 (3H, t, J = 7.2 Hz), 3.56 (3H, s), 3.92 (2H, d, J = 6.0 Hz), 4.13 (2H, q, J = 7.2 Hz),
5.46 (1H, t, J = 6.0 Hz), 6.17 (1H, d, J = 7.8 Hz), 6.40 (1H, d, J = 7.8 Hz), 6.77 (1H, t, J =
7.8 Hz).
4.2.4
7-[Ethyl(3,3,3-trifluoropropyl)amino]-1-methyl-1,3-dihydro-2H-benzimidazol-2-one
4a. To a solution of compound 3c (0.058 g, 3.31 mmol) in MeOH (11 mL) were added
acetaldehyde (0.93 mL, 16.6 mmol), NaBH3CN (0.624 g, 9.93 mmol) and AcOH (0.38 mL,
6.62 mmol), and the mixture was stirred at rt for 15 h. The reaction mixture was quenched
with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed
with brine, dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluted with a 25–60% EtOAc/n-hexane gradient mixture to give (0.462 g,
1
49%) of the title compound as a colorless oil. MS Calcd.: 287; Found: 288 (M+H). H
NMR (CDCl3)  1.03 (3H, t, J = 6.9 Hz), 2.15–2.31 (2H, m), 3.05 (2H, q, J = 6.9 Hz),
3.20–3.35 (2H, br), 3.71 (3H, s), 6.89–6.94 (2H, m), 7.02 (1H, t, J = 7.8 Hz), 9.70 (1H, s).
4.2.5 Ethyl N-ethyl-N-(3-methyl-2-oxo-2,3-dihydro-1H-benzimidazol-4-yl)glycinate 4b.
To a suspension of 3d (7.24 g, 29.0 mmol) in EtOH (50 mL) were added AcOH (1.66 mL,
29.0 mmol), acetaldehyde (4.89 mL, 87.1 mmol) and NaBH3CN (3.64 g, 58.0 mmol), and
the mixture was stirred at rt. The reaction mixture was quenched with saturated aqueous
NaHCO3 and extracted with EtOAc-THF. The organic layer was washed with brine, dried
and concentrated in vacuo. The residue was purified by silica gel column chromatography
eluting with a 35–100% EtOAc/n-hexane gradient mixture to give the title compound (10.6
1
g, >99%) as a yellow oil. MS Calcd.: 277; Found: 278 (M+H). H NMR (CDCl3)  1.03
(3H, t, J = 7.2 Hz), 1.20 (3H, t, J = 7.2 Hz), 3.16 (2H, q, J = 7.2 Hz), 3.79 (3H, s), 3.81 (2H,
s), 4.12 (2H, q, J = 7.2 Hz), 6.85–7.01 (3H, m), 8.75–8.90 (1H br).
4.2.6
4-Bromo-7-[ethyl(3,3,3-trifluoropropyl)amino]-1-methyl-1,3-dihydro-2H-benzimidazo

l-2-one 5a. To a solution of compound 4a (0.460 g, 1.60 mmol) in MeCN (5 mL) was
added NBS (0.285 g, 1.60 mmol), and the mixture was stirred at rt for 15 h. The reaction
mixture was diluted with saturated aqueous NaHCO3 and extracted with EtOAc. The
organic layer was washed with brine, dried and concentrated in vacuo. The residue was
purified by silica gel column chromatography eluting with a 10–25% EtOAc/n-hexane
gradient mixture to give the title compound (0.491 g, 84%) as a colorless solid. MS Calcd.:
1
365; Found: 366 (M+H). H NMR (CDCl3)  1.02 (3H, t, J = 7.0 Hz), 2.14–2.30 (2H, m),
3.03 (2H, q, J = 7.0 Hz), 3.23–3.27 (2H, m), 3.68 (3H, s), 6.81 (1H, d, J = 8.8 Hz), 7.13
(1H, d, J = 8.8 Hz), 8.40 (1H, br s).
4.2.7.
Ethyl
7-bromo-N-ethyl-N-(3-methyl-2-oxo-2,3-dihydro-1H-benzimidazol-4-yl)glycinate 5b.
Compound 5b (3.46 g, 56%) was prepared from compound 4b (4.80 g, 17.3 mmol) in a
manner similar to that described in compound 5a. Colorless solid. MS Calcd.: 355; Found:
356 (M+H). 1H NMR (CDCl3)  1.03 (3H, t, J = 7.2 Hz), 1.21 (3H, t, J = 7.2 Hz), 1.50 (2H,
q, J = 7.2 Hz), 3.77 (3H, s), 3.79 (2H, s), 4.12 (2H, q, J = 7.2 Hz), 6.88 (1H, d, J = 8.7 Hz),
7.09 (1H, d, J = 8.7 Hz).
4.2.8 2-Chloro-N-isopropyl-1-methyl-1H-benzimidazol-7-amine 6b. A mixture of
compound 3b (4.94 g, 24.1 mmol) and POCl3 (25 mL) was stirred at 70oC for 20 h. After
cooling, POCl3 was evaporated in vacuo. The residue was neutralized with saturated
aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with brine,
dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 5–25% EtOAc/n-hexane gradient mixture. The resulting
solid was washed with n-hexane to give the title compound (2.53 g, 47%) as a colorless
1
solid. MS Calcd.: 223; Found: 224 (M+H). H NMR (CDCl3)  1.26 (6H, d, J = 6.0 Hz),
3.44 (1H, br s), 3.53–3.66 (1H, m), 4.08 (3H, s), 6.59 (1H, dd, J = 7.5, 1.0 Hz), 7.09 (1H,
dd, J = 8.1, 7.5 Hz), 7.16 (1H, dd, J = 8.1, 1.0 Hz).
4.2.9. 2-Chloro-1-methyl-N-(3-pentyl)-1H-benzimidazol-7-amine 6a. Compound 3a
(0.831 g, 49%) was prepared from compound 2 (1.18 g, 7.23 mmol) and 3-pentanone (2.10
mL, 19.8 mmol) in a manner similar to that described in compound 3b. Compound 6a
(0.128 g, 15%) was prepared from compound 3b (0.820 g, 3.51 mmol) in a manner similar

to that described in compound 6b. Colorless solid. MS Calcd.: 251; Found: 252 (M+H). 1H
NMR (CDCl3)  0.97 (6H, t, J = 7.5 Hz), 1.50–1.70 (4H, m), 3.22–3.34 (1H, m), 3.50–3.80
(1H, br), 4.07 (3H, s), 6.47–6.54 (1H, m), 7.04–7.10 (2H, m).
4.2.10. 2,4-Dichloro-N-isopropyl-1-methyl-1H-benzimidazol-7-amine 7. To
a
suspension of compound 6a (2.57 g, 11.5 mmol) in MeCN (25 mL) was added NCS (1.53
g, 11.5 mmol), and the mixture was stirred at rt for 4 h. The reaction mixture was diluted
with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed
with brine, dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 10–50% EtOAc/n-hexane gradient mixture to give the title
compound (1.10 g, 37%) as a solid. MS Calcd.: 257; Found: 258 (M+H). 1H NMR (CDCl3)
 1.18–1.90 (6H, m), 3.20–3.40 (2H, m), 4.06 (3H, s), 7.22 (1H, d, J = 8.4 Hz), 7.28 (1H, d,
J = 8.4 Hz).
4.2.11 2,4-Dichloro-1-methyl-N-(3-pentyl)-1H-benzimidazol-7-amine 8a. Compound 8a
(0.040 g, 28%) was prepared from compound 6a (0.125 g, 0.497 mmol) in a manner similar
1
to that described in compound 7. Solid. MS Calcd.: 285; Found: 286 (M+H). H NMR
(CDCl3)  0.97 (6H, t, J = 7.5 Hz), 1.49–1.70 (4H, m), 3.21–3.28 (1H, m), 3.57 (1H, br s),
4.08 (3H, s), 6.43 (1H, d, J = 8.7 Hz), 7.07 (1H, d, J = 8.7 Hz).
4.2.12 N-(2,4-Dichloro-1-methyl-1H-benzimidazol-7-yl)-N-isopropylpropamide 8b. To
a solution of compound 7 (0.202 g, 0.708 mmol) in DMF (2 mL) was added sodium
hydride (0.028 g, 0.779 mmol), and the mixture was stirred at 60oC for 30 minutes.
Addition of propionyl chloride (0.31 mL, 3.54 mmol) at 0oC was followed by stirring at rt
for 2 h. The reaction mixture was diluted with saturated aqueous NaHCO3 and extracted
with EtOAc. The combined organic layer was washed with brine, dried and concentrated in
vacuo. The residue was purified by silica gel column chromatography eluting with a
20–60% EtOAc/n-hexane gradient mixture to give the title compound (0.196 g, 88%) as a
1
solid. MS Calcd.: 313; Found: 314 (M+H). H NMR (CDCl3)  0.81 (3H, d, J = 6.9 Hz),
1.03 (3H, t, J = 7.3 Hz), 1.33 (3H, d, J = 6.6 Hz), 1.78–2.05 (2H, m), 3.78 (3H, s),
4.84–4.96 (1H, m), 6.98 (1H, d, J = 8.2 Hz), 7.32 (1H, d, J = 8.2 Hz).

4.2.13.
2-Chloro-4-bromo-N-ethyl-1-methyl-N-(3,3,3-trifluoropropyl)-1H-benzimidazol-7-ami
ne 8c. A mixture of compound 5a (0.452 g, 1.23 mmol) and POCl3 (2 mL) was stirred at
100oC for 2 h. After cooling, POCl3 was evaporated in vacuo. The residue was neutralized
with saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed
with brine, dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 5–20% EtOAc/n-hexane gradient mixture to give (0.410 g,
1
87%) as a colorless solid. MS Calcd.: 383; Found: 384 (M+H). H NMR (CDCl3)  1.03
(3H, t, J = 7.2 Hz), 2.11–2.35 (2H, m), 3.00–3.20 (2H, br), 3.20–3.40 (2H, br), 4.09 (3H, s),
6.96 (1H, d, J = 8.4 Hz), 7.40 (1H, d, J = 8.4 Hz).
4.2.14. Ethyl N-(4-bromo-2-chloro-1-methyl-1H-benzimidazol-7-yl)-N-ethylglycinate
8d. A mixture of compound 5b (1.84 g, 5.17 mmol) and POCl3 (10 mL) was stirred at 90oC
for 3 h. After cooling, POCl3 was evaporated in vacuo. The residue was neutralized with
saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with
brine, dried and concentrated in vacuo. The residual solid was recrystallized from
EtOAc-n-hexane to give (1.69 g, 87%) of the title compound as a colorless solid. MS
Calcd.: 373; Found: 374 (M+H). 1H NMR (CDCl3)  1.02 (3H, t, J = 7.2 Hz), 1.20 (3H, t, J
= 7.2 Hz), 3.10–3.25 (2H, br), 3.84 (2H, s), 4.11 (2H, q, J = 7.2 Hz), 4.19 (3H, s), 7.01 (1H,
d, J = 8.4 Hz), 7.36 (1H, d, J = 8.4 Hz).
4.2.15.
4-Chloro-N2-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N7-(pentan-3-yl)-1H-ben
zimidazole-2,7-diamine 9a. A mixture of compound 8a (0.035 g, 0.122 mmol),
4-chloro-2-methoxy-6-methylaniline (0.063 g, 0.367 mmol) and NMP (0.15 mL) was
stirred at 110oC for 3.5 d. After cooling, the reaction mixture was diluted with saturated
aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with brine,
dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 5–35% EtOAc/n-hexane gradient mixture, and the pure
fractions were concentrated in vacuo. The residual solid was washed with i-Pr2O to give the
o
title compound (0.020 g, 39%) as a pale yellow solid. mp: 211–214 C.MS Calcd.: 420;
1
Found: 421 (M+H). H NMR (CDCl3)  0.95 (6H, t, J = 7.4 Hz), 1.47–1.67 (4H, m), 2.14

(3H, s), 3.07–3.30 (1H, m), 3.20–3.50 (1H, br), 3.80 (3H, s), 3.86 (3H, s), 6.02 (1H, br s),
6.35 (1H, d, J = 8.4 Hz), 6.80 (1H, s), 6.86 (1H, s), 6.95 (1H, d, J = 8.4 Hz).
4.2.16.
N-(4-Chloro-2-(4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimidaz
ol-7-yl)-N-isopropylpropanamide 9b. Compound 9b (0.201 g, 74%) was prepared from
compound 8b (0.191 g, 0.608 mmol) and 4-chloro-2-methoxy-6-methylaniline (0.313 g,
1.82 mmol) in a manner similar to that described in compound 9a. A colorless solid. mp:
217–218 oC. MS Calcd.: 448; Found: 449 (M+H). 1H NMR (CDCl3)  0.86 (3H, d, J = 7.2
Hz), 1.04 (3H, d, J = 7.2 Hz), 1.31 (3H, d, J = 6.6 Hz), 1.86–2.12 (2H, m), 2.23 (3H, s),
3.56 (3H, s), 3.79 (3H, s), 4.81–4.94 (1H, m), 6.07 (1H, s), 6.73 (1H, d, J = 8.2 Hz), 6.78
(1H, d, J = 2.1 Hz), 6.91 (1H, d, J = 2.1 Hz), 7.13 (1H, d, J = 8.2 Hz). Anal. Calcd for
C22H26N4O2Cl2: C, 58.80; H, 5.83; N, 12.47. Found: C, 58.85; H, 5.85; N, 12.44.
4.2.17.
4-Bromo-N2-(4-chloro-2-methoxy-6-methylphenyl)-N7-ethyl-1-methyl-N7-(3,3,3-trifluo
ropropyl)-1H-benzimidazole-2,7-diamine 9c. Compound 9c (0.061 g, 31%) was prepared
from compound 8c (0.143 g, 0.372 mmol) and 4-chloro-2-methoxy-6-methylaniline (0.191
g, 1.12 mmol) in a manner similar to that described in compound 9a. A colorless crystal.
1
mp: 180–182 oC. MS Calcd.: 518; Found: 519 (M+H). H NMR (CDCl3)  1.04 (3H, t, J =
7.2 Hz), 2.15–2.32 (2H, m), 2.23 (3H, s), 3.00–3.15 (2H, br), 3.18–3.35 (2H, br), 3.80 (3H,
s), 3.88 (3H, s), 6.09 (1H, br s), 6.75 (1H, d, J = 8.4 Hz), 6.77 (1H, d, J = 2.4 Hz), 6.90 (1H,
d, J = 2.4 Hz), 7.23 (1H, d, J = 8.4 Hz). Anal. Calcd for C21H23N4OBrClF3: C, 48.52; H,
4.46; N, 10.78. Found: C, 48.64; H, 4.60; N, 10.89.
4.2.18.
Ethyl
N-(4-bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimidaz
ol-7-yl)-N-ethylglycinate 9d. Compound 9d (0.586 g, 71%) was prepared from compound
8d (0.609 g, 1.63 mmol) and 4-chloro-2-methoxy-6-methylaniline (0.837 g, 4.88 mmol) in
a manner similar to that described in compound 9a. A colorless crystal. MS Calcd.: 508;
1
Found: 509 (M+H). H NMR (CDCl3)  1.03 (3H, t, J = 7.2 Hz), 1.21 (3H, t, J = 7.2 Hz),
2.23 (3H, s), 3.10–3.25 (2H, m), 3.81 (3H, s), 3.83 (2H, s), 4.00 (3H, s), 4.12 (2H, q, J =

7.2 Hz), 6.09 (1H, s), 6.77 (1H, d, J = 2.1 Hz), 6.80 (1H, d, J = 8.1 Hz), 6.90 (1H, d, J = 2.1
Hz), 7.20 (1H, d, J = 8.1 Hz).
4.2.19.
2-((4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimidaz
ol-7-yl)(ethyl)amino)ethanol 10. To a solution of compound 9d (1.18 g, 2.31 mmol) in
THF (10 mL) was added lithium tetrahydroborate (0.151 g, 6.94 mmol), and the mixture
was at 50oC for 15 h and refluxed for 24 h. After cooling, the reaction mixture was diluted
with ice-cold H2O and EtOAc, and the resulting solid was collected by filtration and
washed with H2O and EtOAc to give the title compound (0.331 g) as a colorless solid. The
filtrate was extracted with EtOAc. The combined organic layer was washed with brine,
dried and concentrated in vacuo. The resulting solid was washed with Et2O–n-hexane to
give the title compound (0.645 g) as a colorless crystal. total: 0.976 g, 90%. mp: 178–180
1
oC. MS Calcd.: 466; Found: 467 (M+H). H NMR (DMSO-d6) 0.93 (3H, t, J = 6.3 Hz),
2.13 (3H, s), 3.02–3.09 (4H, m), 3.44 (2H, q, J = 6.3 Hz), 3.76 (3H, s), 3.95 (3H, s), 4.49
(1H, J = 5.1 Hz), 6.75 (1H, dd, J = 8.0, 1.8 Hz), 6.98 (1H, s), 7.02 (1H, s), 7.07 (1H, dd, J =
8.0, 1.8 Hz), 8.09 (1H, s).
4.2.20.
((4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimidazol
-7-yl)(ethyl)amino)acetaldehyde 11. To a solution of oxalyl chloride (0.27 mL, 3.12
mmol) in CH2Cl2 (2 mL) was added DMSO (0.22 mL, 3.12 mmol) at −65oC, and the
mixture was stirred at the same temperature for 2 minutes. Addition of a solution of
o
compound 10 (0.584 g, 1.25 mmol) in CH2Cl2 (30 mL) was followed by stirring at –65 C.
After 15 minutes, triethylamine (1.74 mL, 12.5 mmol) was added to the mixture, and the
mixture was stirred for 2 h with the temperature gradually raised to rt. The reaction mixture
was diluted with ice-cold H2O and extracted with CH2Cl2. The organic layer was washed
with brine, dried and concentrated in vacuo. The residue was purified by silica gel column
chromatography eluting with a 20–70% EtOAc/n-hexane gradient mixture to give the title
compound (0.294 g, 51 %) as an amorphous. MS Calcd.: 464; Found: 465 (M+H). 1H NMR
(CDCl3)  1.04 (3H, t, J = 7.2 Hz), 2.23 (3H, s), 3.12 (2H, q, J = 7.2 Hz), 3.79 (3H, s), 3.87
(2H, s), 3.98 (3H, s), 6.15 (1H, br s), 6.72 (1H, d, J = 8.4 Hz), 6.76 (1H, d, J = 1.8 Hz), 6.89
(1H, d, J = 1.8 Hz), 7.19 (1H, d, J = 8.4 Hz), 9.70 (1H, s).

4.2.21.
1-((4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimidaz
ol-7-yl)(ethyl)amino)propan-2-ol 12. To a suspension of compound 11 (0.281 g, 0.603
mmol) in Et2O (3 mL) was added 3 M solution of methylmagnesium bromide in Et2O (1.00
mL, 3.02 mmol) at 0oC, and the mixture was stirred at rt for 2 h. Addition of 3 M solution
of methylmagnesium bromide in Et2O (0.50 mL, 1.51 mmol) was followed by stirring at rt
for 2.5 d. The reaction mixture was quenched by ice-cold H2O and extracted with EtOAc.
The combined organic layer was washed with brine, dried and concentrated in vacuo. The
residue was purified by silica gel column chromatography eluting with a 25–60%
EtOAc/n-hexane gradient mixture to give the title compound as an oil. The oil was
crystallized from EtOAc-n-hexane to give the title compound (0.089 g, 31%) as a colorless
o
1
crystal. mp: 103–105 C. MS Calcd.: 480; Found: 481 (M+H). H NMR (CDCl3) 
0.95–1.08 (3H, br), 1.16 (3H, d, J = 6.0 Hz), 1.26 (1H, s), 2.16 (1H, s), 2.23 (3H, s),
2.70–3.05 (1H, br), 3.10 (2H, q, J = 7.2 Hz), 3.10-3.40 (1H, br), 3.81 (3H, s), 3.93 (3H, s),
6.10 (1H, s), 6.77 (1H, d, J = 1.8 Hz), 6.79 (1H, d, J = 8.4 Hz), 6.90 (1H, d, J = 1.8 Hz),
7.22 (1H, d, J = 8.4 Hz).
4.2.22.
1-((4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimidaz
ol-7-yl)(ethyl)amino)acetic acid 13. To a solution of compound 9d (0.496 g, 1.65 mmol)
in EtOH (4 mL) was added 8N aqueous sodium hydroxide (0.5 mL), and the mixture was
stirred at rt for 2 h. The reaction mixture was adjusted to pH 5 with 6N hydrochloric acid,
and H2O was added. The resulting solid was collected by filtration and washed with H2O to
give the title compound (0.435 g, 93%) as a colorless solid. MS Calcd.: 480; Found: 481
1
(M+H). H NMR (DMSO-d6)  0.96 (3H, t, J = 7.2 Hz), 2.13 (3H, s), 3.11 (2H, q, J = 7.2
Hz), 3.77 (3H, s), 3.80 (2H, s), 3.99 (3H, s), 6.79 (1H, d, J = 8.4 Hz), 6.99 (1H, d, J = 2.0
Hz), 7.04 (1H, d, J = 2.0 Hz), 7.07 (1H, d, J = 8.4 Hz), 8.13 (1H, s), 12.38 (1H, s).
4.2.23.
N2-(4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimida
zol-7-yl)-N2-ethylglycinamide 14a. To a solution of compound 13 (0.292 g, 0.606 mmol)
in DMF (3 mL) were added WSC (0.139 g, 0.727 mmol), Et3N (0.211 mL, 1.52 mmol) and

HOBt·NH3 (0.111 g, 0.727 mmol), and the mixture was stirred at rt for 15 h. The reaction
mixture was diluted with H2O, and extracted with EtOAc. The combined organic layer was
washed with brine, dried and concentrated in vacuo. The residue was purified by silica gel
column chromatography eluting with a 0–4% MeOH/EtOAc gradient mixture, and the
desired fractions were concentrated in vacuo. The residual solid was washed with i-Pr2O to
o
give the title compound (0.152 g, 52%) as a colorless solid. mp: 208–210 C. MS Calcd.:
1
479; Found: 480 (M+H). H NMR (CDCl3)  1.05 (3H, t, J = 7.2 Hz), 2.23 (3H, s), 3.14
(2H, q, J = 7.2 Hz), 3.75 (2H, s), 3.80 (3H, s), 3.92 (3H, s), 5.74 (1H, br s), 6.10 (1H, br s),
6.26 (1H, s), 6.75 (1H, d, J = 8.4 Hz), 6.78 (1H, d, J = 2.4 Hz), 6.90 (1H, d, J = 2.4 Hz),
7.22 (1H, d, J = 8.4 Hz). Anal. Calcd for C20H23N5O2BrCl: C, 49.96; H, 4.82; N, 14.57.
Found: C, 50.15; H, 4.93; N, 14.51.
4.2.24.
N2-(4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimida
zol-7-yl)-N2-ethyl-N-methylglycinamide 14b. To a solution of compound 13 (0.067 g,
0.139 mmol) in DMF (0.7 mL) were added HOBt (0.023 g, 0.153 mmol), 2M solution of
o
dimethylamine in THF (0.348 mL, 0.695 mmol) and WSC (0.035 g, 0.181 mmol) at 0 C,
and the mixture was stirred at rt for 3 h. The reaction mixture was diluted with H2O, and the
resulting solid was collected by filtration and washed with H2O to give the title compound
(0.055 g, 70%) as a colorless solid. Decomposition point: 255 oC. MS Calcd.: 493; Found:
494 (M+H). 1H NMR (CDCl3)  1.03 (3H, t, J = 7.2 Hz), 2.23 (3H, s), 2.80 (3H, d, J = 1.8
Hz), 3.11 (2H, q, J = 7.2 Hz), 3.73 (2H, s), 3.80 (3H, s), 3.93 (3H, s), 6.05–6.15 (1H, br),
6.73 (1H, d, J = 8.2 Hz), 6.77 (1H, d, J = 2.1 Hz), 6.90 (1H, d, J = 2.1 Hz), 7.21 (1H, d, J =
8.2 Hz). Anal. Calcd for C21H25N5O2BrCl: C, 50.97; H, 5.09; N, 14.15. Found: C, 51.12; H,
5.23; N, 13.77.
4.2.25.
N2-(4-Bromo-2-((4-chloro-2-methoxy-6-methylphenyl)amino)-1-methyl-1H-benzimida
zol-7-yl)-N2-ethyl-N,N-dimethylglycinamide 14c. Compound 14c (0.037 g, 62%) was
prepared from compound 13 (0.057 g, 0.118 mmol) and 2M solution of dimethylamine in
THF (0.295 mL, 0.590 mmol) in a manner similar to that described in compound 14b. A
colorless amorphous. MS Calcd.: 507; Found: 508 (M+H). 1H NMR (CDCl3)  1.01 (3H, t,
J = 7.2 Hz), 2.23 (3H, s), 2.93 (3H, s), 2.94 (3H, s), 3.16 (2H, q, J = 7.2 Hz), 3.80 (3H, s),

3.85 (2H, s), 4.04 (3H, s), 6.08 (1H, s), 6.76 (1H, s), 6.79 (1H, d, J = 8.4 Hz), 6.88 (1H, s),
7.19 (1H, d, J = 8.4 Hz). Anal. Calcd for C22H27N5O2BrCl·0.5H2O: C, 51.03; H, 5.38; N,
13.29. Found: C, 51.26; H, 5.38; N, 13.29.
4.2.26. Methyl 2-chloro-3-nitrobenzoate 16. A slurry of compound 15 (20 g, 99 mmol) in
CH2Cl2 (800 mL) was cooled in an ice bath. DMF (0.40 mL) was added to the reaction
followed by drop wise addition of oxalyl chloride (13.85 g, 109 mmol). The reaction was
allowed to warm to rt and was stirred for 6 h. MeOH (200 mL) was added drop wise and
the reaction was stirred overnight. The reaction mixture was concentrated in vacuo. The
residue was dissolved in CH2Cl2 and passed through a plug of silica gel eluting with a 50%
EtOAc/n-hexane mixture. The filtrate was concentrated in vacuo to give the title compound
(21.5 g, >99%). 1H NMR (CDCl3)  3.98 (3H, s), 7.48 (1H, t, J = 7.8 Hz), 7.84 (1H, d, J =
8.2 Hz,), 7.95 (1H, d, J = 7.8 Hz).
4.2.27. Methyl 2-methylamino-3-nitrobenzoate 17. A solution of (21.5 g, 99.5 mmol) of
compound 16 in THF (300 mL) was treated with drop wise addition of 2M solution of
methylamine in THF (300 mL, 597 mmol), and the mixture was stirred overnight at rt. The
reaction was concentrated to dryness, dissolved in CH2Cl2, and washed with saturated
aqueous NaHCO3 and H2O. The organic layer was dried, filtered, and concentrated in vacuo
1
to give the title compound (20.8 g, >99%). MS Calcd.: 210; Found: 211 (M+H). H NMR
(CDCl3)  2.82 (3H, d, J = 5.5 Hz), 3.9 (3H, s), 6.65 (1H, t, J = 7.8 Hz), 7.97 (1H, d, J = 8.2
Hz), 8.04 (1H, J = 7.8 Hz), 8.57 (1H, s).
4.2.28. Methyl 3-methyl-2-oxo-2,3-dihydro-1H-benzimidazole-4-carboxylate 19. A
solution of compound 17 (20.7 g, 98 mmol) in MeOH (1200 mL) was inerted with nitrogen.
To this solution was added 10% palladium on carbon (50% wet; 5 g, 2.3 mmol). The
reaction was purged with hydrogen and stirred under balloon pressure hydrogen for 7 h.
The catalyst was removed by filtration and the filtrate was concentrated in vacuo to give
compound 18 (17.5 g, 99%). MS Calcd.: 180; Found: 181 (M+H). To a solution of
compound 18 (17.5 g, 97 mmol) in THF (550 mL) was added CDI (20.5 g, 146 mmol) and
the reaction was stirred overnight at rt. The reaction was heated at 50°C for 2 h and allowed
to cool to rt overnight. The reaction mixture was concentrated in vacuo. The residue was
dissolved in EtOAc and washed with H2O. The organic layer was dried and concentrated in

vacuo. The residue was purified by flash chromatography eluting with a solution of 50%
EtOAc/CH2Cl2 to give the title compound (7.22 g, 78%). MS Calcd.: 206; Found: 207
(M+H). 1H NMR (CDCl3)  3.59 (3H, s), 3.95 (3H, s), 7.08 (1H, t, J = 7.8 Hz), 7.27 (1H, d,
J = 7.8 Hz), 7.52 (1H, d, J = 8.2 Hz).
4.2.29 7-(1-Hydroxy-1-propylbutyl)-1-methyl-1,3-dihydro-2H-benzimidazol-2-one 20a.
To a suspension of lithium (3.15 g, 15.3 mmol) in Et2O (60 mL) was added dropwise a
solution of 1-bromopropane (9.40 g, 76.4 mmol) in Et2O (30 mL) at −78oC under nitrogen
o
atmosphere, and the mixture was stirred at 0 C for 2 h. The lithium reagent was added
dropwise to a suspension of compound 19 (3.15 g, 15.3 mmol) in THF (90 mL) at −78oC.
The reaction temperature was gradually raised to rt with stirring. The reaction mixture was
quenched with H2O and extracted with EtOAc/THF. The organic layer was washed with
brine, dried and concentrated in vacuo. The resulting solid was recrystallized from
MeOH–Et2O to give the title compound (2.83 g, 71%) as a colorless crystal. MS Calcd.:
1
262; Found: 263 (M+H). H NMR (CDCl3)  0.93 (6H, t, J = 7.5 Hz), 1.23–1.41 (4H, m),
1.85–2.04 (4H, m), 3.82 (3H, s), 6.87–6.98 (3H, m), 8.78 (1H, br s).
4.2.30.
7-(1-Ethyl-1-hydroxypropyl)-1-methyl-1,3-dihydro-2H-benzimidazol-2-one
20b. 3M solution of ethylmagnesium bromide in Et2O (32 mL, 96 mmol) was added
dropwise to a suspension of compound 19 (5.00 g, 24.2 mmol) in THF (50 mL) at 0°C. The
mixture was stirred at 40°C overnight. The reaction was quenched with H2O and 1N HCl,
and extracted with EtOAc. The organic layer was washed with brine, dried and
concentrated in vacuo. The residue was crystallized from MeOH/Et2O to give the title
1
compound (3.39 g, 70%) as a colorless crystal. MS Calcd.: 234; Found: 235 (M+H). H
NMR (CDCl3)  0.90 (6H, t, J = 7.5 Hz), 1.90–2.20 (5H, m), 3.84 (3H, s), 6.90–7.05 (3H,
m), 9.10–9.30 (1H, m).
4.2.31. 1-Methyl-7-(1-propylbutyl)-1,3-dihydro-2H-benzimidazol-2-one 21a. A mixture
of compound 20a (3.24 g, 12.3 mmol) and 6N HCl (10 mL) in EtOH (50 mL) was stirred at
70°C for 6 h. The reaction mixture was concentrated in vacuo. The residue was neutralized
with aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with
brine, dried and concentrated in vacuo to give a colorless oil, which was used in the next
reaction without further purification. The crude material was dissolved in EtOH (80 mL).