Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2018.01.020

A new class of corticotropin releasing factor 1 (CRF₁) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF₁ receptor antagonists based on con

Full text of DOI:10.1016/j.bmc.2018.01.020

Accepted Manuscript
Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class
of corticotropin releasing factor 1 receptor antagonists
Takuto Kojima, Michiyo Mochizuki, Takafumi Takai, Yasutaka Hoashi, Sachie
Morimoto, Masaki Seto, Minoru Nakamura, Katsumi Kobayashi, Yuu Sako,
Maiko Tanaka, Naoyuki Kanzaki, Yohei Kosugi, Takahiko Yano, Kazuyoshi
Aso
PII:
S0968-0896(17)32337-4
https://doi.org/10.1016/j.bmc.2018.01.020
BMC 14174
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 December 2017
22 January 2018
24 January 2018
Please cite this article as: Kojima, T., Mochizuki, M., Takai, T., Hoashi, Y., Morimoto, S., Seto, M., Nakamura, M.,
Kobayashi, K., Sako, Y., Tanaka, M., Kanzaki, N., Kosugi, Y., Yano, T., Aso, K., Discovery of 1,2,3,4-
tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.01.020
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Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1
receptor antagonists
Takuto Kojima,* Michiyo Mochizuki, Takafumi Takai, Yasutaka Hoashi, Sachie Morimoto, Masaki Seto, Minoru
Nakamura, Katsumi Kobayashi,a Yuu Sako, Maiko Tanaka, Naoyuki Kanzaki,b Yohei Kosugi, Takahiko Yano,c and
Kazuyoshi Asod
Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa
251-0012, Japan
* Corresponding author
Tel: +81 466 32 1116; Fax: +81 466 29 4449; E-mail: takuto.kojima@takeda.com (T. Kojima)
aPresent address: ChromaJean Ltd. 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-0012, Japan
bPresent address: Seedsupply Ltd. 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-0012, Japan
cPresent address: Taisho Pharmaceutical Company Ltd. Taisho Pharmaceutical Company Ltd., 403, Yoshino-cho
1-chome, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
dPresent address: Axcelead Drug Discovery Partners, Inc. 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa
251-0012, Japan
Abstract
A new class of corticotropin releasing factor 1 (CRF1) receptor antagonists characterized by a tricyclic core
ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF1 receptor antagonists based on
conformation analysis of our original 2-anilinobenzimidazole CRF1 receptor antagonist. The synthesized tricyclic
derivatives 2a−e showed CRF1 receptor binding activity with IC50 values of less than 400 nM, and the
1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro
CRF1 receptor binding activity (IC50 = 7.1 nM). To optimize the pharmacokinetic profiles of lead compound 2e, we
explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which
exhibited potent CRF1 receptor binding activity (IC50 = 58 nM) with good oral bioavailability (F = 68% in rats).
Compound 42c-R exhibited dose-dependent inhibition of [125I]-CRF binding in the frontal cortex (5 and 10 mg/kg, p.o.)
as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10
mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF1 receptors in the brain and exhibits the
potential to be further examined for clinical studies.
Key words: CRF1 receptor antagonists, 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole, tricyclic ring system,
stress-related disorders

Introduction
Corticotropin releasing factor (CRF), a 41-amino acid neuropeptide secreted in the hypothalamic
paraventricular nucleus,1 acts as a primary regulator of the hypothalamus-pituitary-adrenocortical axis and stimulates the
secretion of adrenocorticotropic hormone from the pituitary.2,3 CRF exerts its physiological functions via three subtypes
(CRF1, CRF2α, and CRF2β) belonging to the class B subfamily of Gs-coupled G-protein coupled receptors.4-6 CRF1
receptor knockout mice exhibit decreased anxiety-like behavior and impaired stress responses.7 Furthermore,
intracerebroventricular (i.c.v.) administration of CRF induces anxiety-like behavior in rats, while the behavioral
responses to CRF can be reversed by CRF1 receptor antagonists.8,9 These findings indicate that CRF1 receptor antagonists
are promising targets for stress-related disorders.
Small-molecule CRF1 receptor antagonists, shown in Figure 1, have been evaluated in several clinical studies
in patients with anxiety, depression, irritable bowel syndrome, post-traumatic stress disorder, and alcohol
dependence;10-16 however, these studies were not conclusive in defining the clinical utility of CRF1 receptor antagonists.
We hypothesized that these ambiguous results are attributable to the properties of the new investigational drugs rather
than the mechanism of action. The reported CRF1 receptor antagonists displayed in Figure 1 show higher cLogP values
−8.2). We planned to optimize our compounds with considering logD which would be a key parameter not only for
(3.4
lipophilicity of the molecule but also for improvement in absorption, distribution, metabolism, and elimination
(ADME) profiles.17 Therefore, identification of novel drug candidates with potent binding activity and desirable ADME
properties is still an attractive area of drug discovery research. In this article, we describe the design and synthesis of a
new class of CRF1 receptor antagonists characterized by a tricyclic core ring, as well as its optimization toward acquiring
drug-like properties.
Figure 1. Reported CRF1 receptor antagonists.
We previously reported the discovery of 2-anilinobenzimidazole derivatives, which have a unique structure
among the numerous reported CRF1 receptor antagonists. These benzimidazole CRF1 receptor antagonists are

characterized by a nitrogen linker between the benzimidazole core and the pendant aryl group, which allows flexibility of
the pendant aryl group. In contrast, most reported compounds contain a rigid pendant aryl moiety. The representative
compound 1a demonstrated potent CRF1 receptor binding activities in vitro and in vivo.18 In the benzimidazole series,
there was a remarkable difference in the binding activity between compound 1b, a 2,4,6-trisubstituted phenyl derivative
(IC50 = 18 nM), and that of compound 1c, a 2,4-disubstituted phenyl derivative (IC50 = 200 nM) (Figure 2). To
understand the reason for the difference in binding activity, we examined the dihedral angle ϕ as a key parameter
controlling torsional conformation between the benzimidazole core and the pendant aryl group (Figure 2(B). The
calculated dihedral angle ϕ19 of compound 1b, with potent binding activity, exhibited an appropriate torsional angle (ϕ =
45), while compound 1c, with a moderate binding activity, had a nearly planar relation between the benzimidazole core
and the pendant aryl group (ϕ = 3). The relationship between binding activity and dihedral angle ϕ indicates that an
active conformation of the benzimidazole derivatives requires a certain range in dihedral angle. Further, the
2,4,6-trisubstituted phenyl group induces enough steric repulsion of ortho-substituents to maintain the torsional active
conformation. Following this rationale, we expected that stabilizing the torsional conformation of benzimidazole
derivatives could lead to a novel chemotype. Since we expected that ring-closing at the 1-position and 2-position of the
benzimidazole derivative could fix the restrained conformation by inducing a rotation barrier, we designed compound 2a,
a novel tricyclic, which predictably caused a torsional conformation with a dihedral angle (ϕ) of 48 degrees between the
tricyclic core and the 2,4-dichlorophenyl group to exhibit potent binding activity with the CRF1 receptor.
As described above, the dihedral angle ϕ is a key parameter for determining binding activity. Furthermore, we
examined the electron density (E value20) of the hydrogen bond acceptor (HBA) nitrogen atom because its importance
has been reported in the general pharmacophore.21 Based on these considerations, we designed four tricyclic derivatives,
−d
2a , which stabilized the torsional conformation (46 < ϕ < 83), with the various electron densities (E). Compounds 2c
and 2d, with a C1 sp3 carbon instead of a sp2 nitrogen, were designed to investigate the diversity of a ring system. During
this work, we explored a novel class of CRF1 receptor antagonists, focusing on the torsional conformation (ϕ value) and
electron density (E value) of the HBA nitrogen.
During the scaffold hopping process, target compounds without a substituent at the 9-position were designed,
attributable to chemical accessibility; the chlorine atom was important for the binding activity as previously described for
benzimidazole CRF1 antagonists.18 In addition, substituents at the 1- and 6-position were also fixed by a
2,4-dichlorophenyl and diethylamino group, respectively. After selecting the best ring system, we optimized the 1- and
6-position with a chlorine atom at the 9-position. Our optimization strategy included decreasing the lipophilicity through
pursuing polar and metabolically stable substituents at the 1- and 6-position, while maintaining potent binding activity.

−c
Figure 2. (A) Representative benzimidazole derivative for compounds 1a and (B) ϕ value indicating the dihedral angle
between planes, including the benzimidazole core and a plane with the pendant aryl ring. E values show electron density
−d
of the hydrogen bond acceptor (HBA) nitrogen. (C) Design of the novel tricyclic ring system 2a to stabilize torsional
conformation.
Chemistry
Synthesis of the newly designed tricyclic compounds 2a and 2b is described in Scheme 1. Amination of
2-chloro-1,3-dinitrobenzene 3 with β-alanine ethyl ester hydrochloride and the subsequent hydrogenation of nitro groups
of compound 4 afforded triaminophenyl derivative 5. Thiourea derivative 6 was prepared by reacting compound 5 with
phenylisothiocyanate, and then benzimidazole ring was constructed by intramolecular cyclization in the presence of
1-(3-dimethylaminopropyl)3-ethylcarbodimide (EDCI). Sequential reductive alkylation of compound
7 with
acetaldehyde provided a diethylaminobenzimidazole 8, which was converted to hydroxypropylbenzimidazole derivative

9 via reduction of the ester group with lithium borohydride. After mesylation of the hydroxyl group of compound 9,
intramolecular cyclization afforded the desired 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2a. The
3,4-dihydropyrimido[1,2-a]benzimidazol-2(1H)-one derivative 2b was synthesized by hydrolysis of the ester 8 and
subsequent intramolecular amidation.
Scheme
1.
Synthesis
of
1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole
derivative
2a
and
3,4-dihydropyrimido[1,2-a]benzimidazol-2(1H)-one derivative 2ba
aReagents and conditions: (a) β-alanine ethylester hydrochloride, Et3N, THF, rt, quant; (b) H2, Pd/C, THF, rt, 91%; (c)
2,4-dichlorophenyl isothiocyanate, THF, rt, 56%; (d) EDCI, THF, 50 °C, 83%; (e) MeCHO, NaBH(OAc)3, AcOH, THF,
0 °C to rt, quant; (f) LiBH4, THF, 0 °C to rt, 25%; (g) MsCl, pyridine, 0 °C to rt, 86%; (h) NaOH, H2O, MeOH, THF,
0 °C to rt, 78% (i) EDCI, Et3N, DMF, rt, 82%.
Synthesis of
triaminophenyl derivative
2-(2,4-dichlorophenyl)-2-hydroxyethanimidate
a C-linked 1,2,3,4-tetrahydropyrido[1,2-a]benzimidazole derivative 2c started from
5
(Scheme
2).
Condensation
12,
of
compound
5
with
methyl
from
hydrochloride
which
was easily
prepared
2,4-dichlorobenzaldehyde 11, directly afforded benzimidazole derivative 13. Reductive alkylation of the 7-amino group
of compound 13, followed by reduction of the ester group with lithium aluminum hydride provided a diol derivative 15.
Selective oxidation of the secondary alcohol of compound 15 using manganese dioxide afforded
1-(3-hydroxypropyl)bezimidazole 16, which was converted to bromide 17 by reaction with triphenylphosphine and

tetrabromomethane. After preparation of ylide intermediate from bromide 17, intramolecular Wittig reaction under basic
condition constructed 1,2-dihydropyrido[1,2-a]benzimidazole 18, and following hydrogenation gave the desired
1,2,3,4-tetrahydropyrido[1,2-a]benzimidazole derivative 2c.
Scheme 2. Synthesis of 1,2,3,4-tetrahydropyrido[1,2-a]benzimidazole derivative 2ca
aReagents and conditions: (a) (i) TMSCN, DMAP, MeCN, rt; (ii) AcCl, MeOH, 0 °C to rt, 90% in 2 steps. (b) 12, EtOH,
rt, 90%; (c) MeCHO, NaBH(OAc)3, AcOH, THF, 0 °C, 92%; (d) LiAlH4, THF, -5 °C, 78%; (e) MnO2, THF, rt, 99%; (f)
PPh3, CBr4, MeCN, 0 °C, 88%; (g) PPh3, MeCN, 80 °C then KOt-Bu, tolene, THF, rt, 23% in 2 steps; (h) H2, Pd/C,
MeOH, rt, 47%.
Synthesis of 3,4-dihydro-1H-[1,4]oxazino[4,3-a]benzimidazole derivative 2d is shown in Scheme 3.
Amination of 2-chloro-1,3-dinitrobenzene 3 with ethanolamine, and the subsequent reduction of nitro groups of
compound 19 gave 2-[(2,6-diaminophenyl)amino]ethanol derivative 20. After key intermediate 21 was prepared by
condensation of the compound 20 with the amidate 12, the following reductive alkylation provided compound 22.
Intramolecular cyclization of diol 22 under Mitsunobu reaction condition afforded the desired
3,4-dihydro-1H-[1,4]oxazino[4,3-a]benzimidazole derivative 2d.
Scheme 3. Synthesis of 3,4-dihydro-1H-[1,4]oxazino[4,3-a]benzimidazole derivative 2da

aReagents and conditions: (a) ethanol amine, Et3N, THF, rt, 99%; (b) H2, Pd/C, rt, THF, 89%; (c) 12, EtOH, rt, 99%; (d)
MeCHO, NaBH(OAc)3, AcOH, THF, 0 °C, 99%; (e) PPh3, DEAD, THF, rt, 59%.
Since 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole 2a was identified as the best ring system among the
designed compound 2a−d, we subsequently introduced a chlorine atom on the 9-position. Synthesis of 9-chloro
substituted 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e is shown in Scheme 4. Condensation of
2-chloro-5-nitroaniline 23 with formic acid, and following chlorination under mixture of sulfuryl chloride and thionyl
chloride provided dichloride derivative 24, which was treated with 1,3-propandiamine to yield guanidine derivative 25.
Intramolecular cyclization of the compound 25 in the presence of potassium tert-butoxide in dimethylsulfoxide
successfully afforded tricyclic derivative 26. Arylation of amine 26 with 1,3-dichloro-4-iodobenzene gave the desired
compound 27. Reduction of the nitro group of compound 27 gave aniline derivative 28, which was converted to the target
compound 2e by reductive alkylation.
Scheme 4. Synthesis of 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative with Cl atom on the 4-position 2ea
aReagents and conditions: (a) HCO2H, rt; (b) SO2Cl2, SOCl2, 55 °C, 67% in 2 steps; (c) 1,3-propandiamine, THF, 5-10 °C,
98%; (d) KOt-Bu, DMSO, 60 °C, 73%; (e) CuI, 2,2'-bipyridyl, 1,3-dichloro- 4-iodobenzene, 150 °C, 5%; (f) H2, Pd/C,
AcOH, rt, 45%; (g) MeCHO, NaBH(OAc)3, AcOH, MeOH, 0 °C to rt, 98%.

Next, we focused on the optimization of the new lead 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole 2e
with chlorine atom on the 9-position. The synthesis of key intermediate 36 for derivative synthesis is illustrated in
Scheme 5. Methylation of 2-chloro-3-nitrobenzoic acid 16, substituent reaction with 3-hydroxylpropylamine, and
reduction of the nitro group yielded compound 32. Sequential chlorination by NCS gave tetrasubstituted benzene
derivative 33 regioselectively. The reaction of compound 33 with 2,4-dichlorothioisocyanate yielded thiourea 34, which
was treated with water soluble condensing agent (WSC) to provide benzimidazole derivative 35. Finally, the tricyclic
structure was constructed by mesylation of the alcohol of the compound 35 and successive intramolecular cyclization
under basic condition to afford the key intermediate 36.
Scheme 5. Synthesis of the key intermediate 36a
aReagents and conditions: (a) (COCl)2, THF, 0 °C, then MeOH, rt, 96%; (b) 3-hydroxypropyllamine, NEt3, THF, 50 °C,
74%; (c) Pd/C, H2, rt, THF, 78%; (d) NCS, CH3CN, rt, 16%; (e) 2,4-dichloro-1-isothiocyanatobenzene, THF, rt; (f)
WSC, NEt3, THF, 50 °C, 71% in 2 steps; (g) 1) MsCl, pyridine, THF, 0 °C; 2) K2CO3, DMF, 70 °C, 87% in 2 steps.
Synthesis of compounds with variety of substituents at the 6-position is shown in Scheme 6. Reduction of ester
of compound 36 by lithium borohydride was followed by oxidation of the alcohol by SO3-pyridine to give aldehyde
derivative 38. Addition of ethylmagnesium bromide to aldehyde 38 provided 1-propanol derivative 39a, which was
converted to ketone derivative 39b by oxidation with Dess-Martin reagent. After chlorination of compound 24 with
SOCl2 and successive substitution reaction by potassium cyanide to give compound 40, treatment of ethyl iodide in the
presence of potassium tert-butoxide afforded compound 39c. Compound 39a was converted to compound 39d by the
reaction with 1,1'-carbonyldiimidazole (CDI) to install imidazole and also converted to compound 39e by methylation
with iodomethane. Addition of trifluoromethyl anion generated by (trimethylsilyl)trifuluoromethane and
trin-butylammonium fluoride (TBAF) to aldehyde derivative 38 provided 2,2,2-trifluoroethaol derivative 41, which was
converted to compound 39f by alkylation with difluoromehyl chloride gas under basic condition.

Scheme 6. Synthesis 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole analogs with various substituents at the 6-position
a
aReagents and conditions: (a) LiBH4, THF, rt, 89%; (b) SO3-py, NEt3, DMSO, CH2Cl2, rt, 82%; (c) EtMgBr, THF, 0 °C,
92%; (d) Dess-Martin reagent, CH3CN, DMSO, 0 °C to rt, 88%; (e) 1) SOCl2, pyridine, THF, 0 °C to rt; 2) KCN, DMSO,
H2O, rt, 96% in 2 steps; (f) KOt-Bu, EtI, THF, 0 °C, 38%; (g) CDI, THF, 50 °C, 94%; (h) NaH, MeI, DMF, rt°C, 76%; (i)
TMSCF3, TBAF, THF then HCl aq., 0 °C, 99%; (j) CHF2Cl, BnEt3N, THF, NaOH aq., 50 °C, 44%.
Synthesis of the compounds with variety of substituents on the 1-position are shown in Scheme 7. Starting
from the compound 33, 2,2,2-trifluoroethaol derivative 41a−e were obtained by the same procedure described in Scheme
5 and 6. The compound 41a with 4-bromo-2-methylphenyl group at 1-position was converted to compound 41f by
Ullmann reaction to replace bromo atom with methoxy group. Then, 2,2,2-trifluoroethaol derivative 41b−f were treated
with difluoromehyl chloride gas to synthesize the target compounds 42b−f.
Scheme 7. Synthesis 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole analogs with various substituents at the 1-position

a
aReagents and conditions: (a) ArNCS, THF, 65−70 °C; (b) WSC, NEt3, THF, 50−60 °C, 6-64% in 2 steps; (c) 1) MsCl,
NEt3, THF, 0 °C to rt; 2) K2CO3, DMF, 70−80 °C, 76−90%; (d) LiBH4, THF, 0 °C to 40−50 °C, 98−quant; (e)
Dess-Martin reagent, DMSO, CH3CN, 0 °C-rt, 79−93%; (f) TMSCF3, TBAF, THF, 0 °C, then HCl, 0 °C-rt, 61−96%; (g)
NaOMe, CuI, DMF, 100 °C, 94%; (h) CHF2Cl, BnEt3NCl, THF, NaOH aq., rt to 50 °C, 30−51%; (i) POCl3, 100 °C,
42%; (j) for 42g, MeMgBr, NiCl2(dppp), THF, 0 °C to rt, 32%; for 42h, EtMgBr, NiCl2(dppp), THF, 0 °C to rt, 9%.
Results and discussion
Novel tricyclic compounds with a torsional conformation between the core ring and the pendant aryl group
were synthesized and evaluated for their inhibitory activity against the binding of ovine [125I]-CRF to human CRF1
receptors expressed on Chinese hamster ovary (CHO) cellular membrane.
The relationship between binding activity and each parameter is discussed below. The most active
tetrahydropyrimidobenzimidazole derivative 2a (IC50 = 11 nM) was as potent as the benzimidazole derivative 1b with a
2,4,6-trisubstituted phenyl group (IC50 = 18 nM), even though compound 2a had a 2,4-disubstituted phenyl group.
Comparisons between the tetrahydropyrimidobenzimidazole derivative 2a and the benzimidazole derivative 1c with a
2,4-disubstituted phenyl group provided useful information regarding the relationship between torsional angle and

binding activity. The binding activity of compound 2a (IC50 = 11 nM) was 20-fold more potent than that of compound 1c
(IC50 = 200 nM). E values of compound 2a (E = -0.77) and compound 1c (E = -0.77) were equivalent, while there was
large difference in the dihedral angles between compound 2a (ϕ = 48) and compound 1c (ϕ = 3). These data indicate that
a torsional conformation between the core and pendant aryl group is better than a planar conformation to afford
compounds exhibiting potent binding activity with similar E value ranges.
The dihydropyrimidonebenzimidazole derivative 2b (IC50 = 39 nM) was 4-fold less active than compound 2a
(IC50 = 11 nM), while compound 2b possessed similar ϕ value (ϕ = 46) to that of compound 2a (ϕ = 48). This is likely
attributable to the decreased electron density of the HBA by an inductive effect of the carbonyl group (2b; E = -0.72, 2a;
E = -0.77), which consequently resulted in the binding activity of 2b being lower than that of compound 2a. Compounds
2c and 2d, with a C1 sp3 benzyl carbon instead of a sp2 nitrogen, were synthesized and evaluated as racemate. The
pendant aryl group conformation of compounds 2c and 2d was nearly orthogonal because their dihedral angles were
more than 69 degrees. However, the tetrahydropyridobenzimidazole derivative 2c (IC50 = 36 nM) was as potent as
compound 2b (IC50 = 39 nM). Since the electron density of compounds 2b and 2c was nearly equivalent (2b; E = -0.72,
2c; E = -0.74), differences in their dihedral angles (2b; ϕ = 46, 2c; ϕ = 69) did not impact binding activity. In other words,
only torsional conformation was required to critically impact binding activity, and there were no differences between
torsional conformation and orthogonal conformation. The dihydrooxazinobenzimidazole derivative 2d (IC50 = 270 nM)
had a 24-fold lower binding activity than that of compound 2a. We attributed this marked reduction in the binding
activity of compound 2d to the low electron density of its HBA nitrogen atom (2d; E = -0.67), although the dihedral
angle appeared to be sufficient. These results indicate that both the torsional conformation and electron density of the
HBA nitrogen atom affect binding activity.
To confirm our hypothesis, a docking study was conducted using GOLD software and MOE22 based on the
reported crystal structure of the CRF1 receptor with CP-376395.23 The dihedral angle of compound 2a in the model was
74 degrees, revealing that the torsional conformation was required for compounds to fit in the receptor pocket. In
addition, the electron density of the HBA nitrogen atom impacted binding activity, attributable to the HBA nitrogen of
compound 2a in the model having an obvious interaction with Asn283 of the receptor. This model also showed that
compounds 2c and 2d, with a C1 sp3 benzyl carbon, had decreased activity, attributable to a loss of energy resulting from
folding the pendant aryl group into a suitable position from their most stable conformation, as well as a decrease in
activity of the HBA nitrogen atom. These analyses matched and supported our hypothesis and results.
Table 1. hCRF1 receptor binding activities of novel tricyclic ring derivatives

aAr is 2,4-dichlorophenyl group. bIC50 values and 95% confidential intervals were calculated from the
concentration-response curve (n = 1).

Figure 3. Docking pose for compound 2a (magenta) in the CRF1 receptor; its crystal structure was identified with
CP376395 (yellow).23 This docking study indicates that the torsional conformation was necessary to occupy the binding
pocket in a suitable manner and the HBA nitrogen on the tricyclic ring clearly interacted with Asn283.
Eventually, our efforts to explore novel tricyclic ring systems resulted in identification of
pyrimidobenzimidazole derivative 2a, as the best scaffold to show the most potent binding activity among the designed
compounds 2a−2d. Our previous study indicated that the introduction of a small substituent, especially a chlorine atom,
enhances the activity.18 Further, the docking model suggested the presence of a small space around the 9-position.
Therefore, we introduced a chlorine atom to the 9-position to identify compound 2e with the most potent binding activity
(IC50 = 7.1 nM). Compound 2e also inhibited human CRF-stimulated cAMP accumulation with an IC50 value of 4.4 nM
in CHO cells expressing hCRF1 receptors, indicating that it was a promising lead compound for further optimization.
Figure 4. Profiles of compound 2e.
aIC50 values and 95% confidential intervals were calculated from the concentration-response curve (n = 1). b10 mmol/L
solution of the compound in DMSO was evaluated using JP2 buffer (pH 6.8), as described in the Japanese
Pharmacopoeia. cMetabolic stability was determined based on the disappearance of parent compound after incubation
with human or rat liver microsmes for 20 min. dResults from the rat cassette dosing test (mean, n = 3, 0.1 mg/kg, i.v., 1

mg/kg, p.o.) are shown.
Although lead compound 2e exhibited potent binding activity (IC50 = 7.1 nM), its bioavailability (BA) was
poor (cassette dosing in rats, BA = 4.1%, 1 mg/kg, p.o. and 0.1 mg/kg, i.v.), attributable to low metabolic stability and
low solubility resulting from its high lipophilicity (cLogP = 8.1, logDpH7.4 = 5.25). Therefore, we aimed to reduce
lipophilicity by replacing the diethylamino group on the 6-position and the 2,4-dichlorophenyl group on the 1-position
with polar substituents. Since it was expected that the diethylamino group would be easily metabolized, we further
explored which substituents provided resistance against metabolism.
First, we explored substituents at the 6-position, which was expected to maintain binding activity and reduce
lipophilicity. The synthesized compounds, along with binding activities, logD measured at pH 7.4, and in vitro metabolic
clearance in human hepatic microsomes are shown in Table 2. According to the docking model and our previous
structure-activity relationship (SAR) studies of the benzimidazole derivatives,18 we intensively designed compounds with
a 2-forked chain structure at the 6-position. IC50 values in the order of 10-8 M were maintained after introducing a
hydroxyl group (39a, IC50 = 89 nM), cyano group (39c, IC50 = 66 nM), and methoxy group (39e, IC50 = 20 nM), while
the introduction of an oxo group (39b, IC50 = 210 nM) and imidazolyl group (39d, IC50 = 130 nM) resulted in the binding
activity being 18–30 fold lower than that of compound 2e. Compound 39f, a 2,2,2-trifluoroethyl-1-difluoromethoxy
derivative (IC50 = 50 nM) that was designed to complement the metabolic stability of compound 39e, exhibited potent
binding activity with excellent metabolic stability. Compound 39f was more stable in human hepatic microsomes than
compound 39d, which possessed the lowest logD among test compounds 39a−39f. These data suggest that drastic
improvements in the metabolic stability of compound 39f were dependent on decreasing logD values as well as blockage
of the metabolic site by fluoride atoms. While modifying the 6-position, decreasing the logD by installing several polar
substituents tended to improve metabolic stability; compound 39f was identified as the next lead compound, with potent
binding activity and magnificent metabolic stability.
Table 2. Effect of substituents at the 6-position of compound 2e on hCRF1 receptor binding activities, logDpH7.4, and
metabolic stability in human

b
aIC50 values and 95% confidential intervals were calculated from the concentration-response curve (n = 1). Metabolic
stability was determined based on the disappearance of parent compound after incubation with human liver microsmes
for 20 min.
Since the solubility of compound 39f was poor (< 0.09 µg/ml), further reduction of its lipophilicity was
required. We aimed to improve the solubility of compound 39f by optimizing the lipophilic 2,4-dichlorophenyl group on
the 1-position. Introduction of a methoxy on the phenyl group at the 2-position decreased the logD and increased
solubility, while maintaining binding activity (42f; IC50 = 48 nM, logDpH7.4 = 3.76, and solubility = 1.5 µg/ml).
Replacement of the phenyl group compound 42f with a pyridine group further improved solubility and resulted in potent
binding activity (42b; IC50 = 48 nM, logDpH7.4 = 3.43, and solubility = 17 µg/ml), indicating that a pyridine group
nitrogen atom at the 1-position successfully reduced lipophilicity and had ignorable impact on CRF1 receptor binding.

These results encouraged us to introduce a pyrimidine group at the 1-position, and pyrmidine derivative 42c exhibited
moderate binding activity and excellent solubility (42c; IC50 = 110 nM, logDpH7.4 = 3.08, and solubility = 54 µg/ml).
Pyrimidine derivatives with compact substituents were synthesized and evaluated, as our previous SAR studies
for benzimidazole derivatives revealed that only small substituents were tolerated on the aryl group at the 1-position.21
The binding activity of compound 42g (IC50 = 320 nM) was 3-fold lower than that of compound 42c. Compound 42h
(IC50 = 210 nM) was also 2-fold less potent than compound 42c, suggesting that the methoxy group oxygen atom on the
pyrimidine group affected binding activity. However, based on results of the docking model, we did not expect
interactions with the CRF1 receptor as shown in Figure 3. The effect of ortho substituents on binding activity was
investigated using compounds 42d (IC50 = 100 nM) and 42e (IC50 = 46 nM) relative to compound 42g (IC50 = 320 nM).
These results show that larger substituents are preferable on the point of binding activity, which was consistent with our
torsion conformation hypothesis. However, the solubility of compounds 42d and 42e was lower and the metabolic
stability was less optimal than those of compound 42c due to increased lipophilicity. From these results, we concluded
that lipophilicity could be successfully reduced without a significant loss in binding activity. Further, compound 42c was
identified as the best compound with potent binding activity, excellent solubility and metabolic stability.
Table 3. Effect of substituens at the 1-position of compound 39f on hCRF1 receptor binding activities, logDpH7.4,
metabolic stability in human, and solubility

aIC50 values and 95% confidential intervals were calculated from the concentration-response curve (n = 1). All
b
compounds shown in this table were synthesized and evaluated as racemates. Metabolic stability was determined based
on the disappearance of parent compound after incubation with human liver microsmes for 20 min. c10 mmol/L solution
of the compound in DMSO was evaluated using JP2 buffer (pH 6.8), as described in the Japanese Pharmacopoeia.

Optical resolution of compound 42c was carried out to identify compound 42c-R as a eutomer whose absolute
configuration was determined by X-ray crystal analysis. Compound 42c-R (IC50 = 58 nM) was approximately 2-fold
more potent than its racemate 42c. In contrast, compound 42c-S, the distomer (IC50 = 350 nM), was 3-fold less potent
than its racemate 42c. Since some reported CRF1 receptor antagonists, such as pexacerfont and vercerfont, also have a
chiral center on the superposition of the 6-position of compound 42c-R, the CRF1 receptor should strictly recognize
ligand conformation around this position. Further, we asymmetrically synthesized the 41c-R precursor (Scheme 8), which
was converted to the 42c-R eutomer by the same procedure described above. After preparing ketone 44c from a racemic
alcohol 41c by oxidation with etrapropylammonium perruthenate (TPAP) and N-methylmorpholine N-oxide (NMO),
asymmetric hydrogenation with ruthenium (II) complexes24 under 0.7 MPa of hydrogen pressure successfully afforded
target R-configurations with excellent enantioselectivity (99.4%ee after recrystallization).
Compound 42c-R also inhibited human CRF-stimulated cAMP accumulation with an IC50 value of 55 nM in
CHO cells expressing hCRF1 receptors. In addition, compound 42c-R was inactive against CRF2α and CRF2β receptors
(IC50 > 10 µM). Therefore, we selected compound 42c-R for further evaluation of its pharmacological properties.
Figure 5. Profiles of chiral 42c-R and 42c-S
aIC50 values and 95% confidential intervals were calculated from the concentration-response curve (n = 1).
Scheme 8. Asymmetric synthesis alcohol derivative 41c-Ra
aReagents and conditions: (a) TPAP, NMO, MS4A, CH3CN, 0 °C−rt, 86%; (b) H2 (0.7 MPa), KOt-Bu,
[RuCl2{(R)-xylbinap}{(R)-daipen}], 2-propanol, toluene, rt, 75%, 99.4%ee.

The oral bioavailability of compound 42c-R was 68% in rats (i.v. 1 mg/kg, p.o. 5 mg/kg), which was
drastically improved from the initial lead compound, 2e. The oral bioavailability of 42c-R is likely attributable to its
metabolic stability, which enabled it to withstand oxidative metabolism in hepatic microsomes (rat: 24 µl/min/mg,
human: < 1 µl/min/mg) and improved its kinetic solubility in phosphate buffer saline (55 µg/ml, logD = 3.18). To confirm
brain penetration, we conducted an ex vivo binding test in rats. After orally administering compound 42c-R (5 and 10
mg/kg) to rats, [125I]-CRF binding in the frontal cortex was measured over time. Compound 42c-R inhibited [125I]-CRF
binding in the frontal cortex in a dose-dependent manner. In addition, maximum inhibition of [125I]-CRF binding was
observed 2 h after oral administration, and the inhibition diminished 24 h after administration. These results indicate that
compound 42c-R penetrates the brain in rats. Finally, we examined CRF1 receptor antagonist activity in rats. After oral
administration of compound 42c-R (2.5, 5, and 10 mg/kg) followed by i.c.v. administration of CRF (1 µg) 2 h later, we
monitored locomotor activity for 1 h. Results show that administration of compound 42c-R suppresses CRF-induced
increases in locomotor activity in a dose-dependent manner, suggesting that compound 42c-R exerts in vivo CRF1
receptor antagonist activity.
Figure 6. [125I]-Corticotropin-releasing factor (CRF) binding in membranes of the frontal cortex 1, 2, 4, 8, 16, and 24 h
after oral administration of 5 and 10 mg/kg compound 42c-R. Binding of [125I]-CRF in rat brain tissues under drug-free
conditions was defined as 100% binding. Data are expressed as means + S.E.M (n = 3).

Figure 7. Effect of orally administering 2.5, 5, and 10 mg/kg compound 42c-R on corticotropin-releasing factor
(CRF)-induced locomotor activation by i.c.v. administration in rats. Locomotor activity was measured as the mean
number of counts for 60 min after i.c.v. administration of CRF. Data are expressed as means + S.E.M. (n = 9–17). **p <
0.001 vs vehicle group via the Aspin-Welchtest; # p < 0.025 vs vehicle-h/rCRF group via the Shirley-Williams-test.
Conclusion
In the course of exploration of a novel class of CRF1 receptor antagonists, we hypothesized that stabilization of
torsional conformation between the core ring and pendant aryl group is important to determine binding activity based on
analyses of the 2-anilinobenzimidazole derivative pharmacophore. The relationship between binding activity and electron
−e
density of a HBA nitrogen atom was also examined. Based on these considerations, the tricyclic compounds 2a with
torsional conformations were designed and synthesized. The results suggested that both the dihedral angle and electron
density played important roles for determining binding activity. The torsional conformation between the core and pendant
aryl group was better than a planar conformation for exhibiting potent binding activity. Moreover, the binding activity
critically depended on the electron density of a HBA nitrogen atom. These considerations were supported by a docking
study using reported CRF1 receptor crystal structure information. Among the synthesized derivatives (2a−e),
1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole derivative 2e, was identified as the most potent ring system, and then we
moved to optimization to improve its metabolic stability and solubility. The exploration for a suitable substituent at the
6-position resulted in the identification of 2,2,2-trifluoroethyl-1-difluoromethoxy derivative 39f, with excellent metabolic
stability. We further aimed to reduce lipophilicity by optimizing the 1-position. Consequently,
2,6-dimethyl-4-methoxypyrimidine derivative 42c, was identified and determined to be compatible with potent binding
activity and good solubility. Finally, optical resolution of compound 42c led to the discovery of compound 42c-R, with

potent binding activity and a good pharmacokinetic profile. Compound 42c-R dose-dependently inhibited [125I]-CRF
binding in the frontal cortex (5 and 10 mg/kg, p.o.) and suppressed locomotor activation induced by i.c.v. administration
of CRF in rats (10 mg/kg, p.o.), suggesting that compound 42c-R successfully binds CRF1 receptors in the brain.
Compound 42c-R was selected as a candidate for further investigation to examine the clinical usefulness of CRF1
receptor antagonists.
Acknowledgements
The authors thank Dr. Yoshiro Tomimatsu, Mr. Katsuya Sakimura, and Dr. Yoshikazu Ohtani for their assistance
with pharmacological tests and helpful discussions during the preparation of the manuscript; Mr. Akihiro Yokota for
calculating the ϕ and Ε values, performing the docking study, and helpful discussions during preparation of the
manuscript; Mr. Yuji Shimizu and Mr. Kenichi Kuroshima for performing the in vitro CRF1 receptor binding assays,
human CRF-stimulated cAMP accumulation assays, and reviewing the data; Mr. Mitsuyoshi Nishitani for X-ray structure
analysis; Ms. Miki Hara for conducting optical resolution of compound 42, measuring the optical purity of compound
42c-R, and performing chiral resolution and analysis of enantiomer excess rate; and Mr. Masayuki Yamashita for
conducting the asymmetric hydrogenation of 44c.
Experimental section
Chemistry
General. In the following experimental, reactions were run using the commercially available starting materials and
solvents without further purification. Melting points were determined on a Yanaco micro melting point apparatus and
were uncorrected. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on Varian Mercury-300 (300
MHz). Chemical shifts are given in parts per million (ppm) with tetramethylsilane as an internal standard. Abbreviations
are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, dd = doublets of
doublet, brs = broad singlet. Coupling constants (J values) are given in hertz (Hz). Mass spectra (MS) were acquired
using an Agilent LC/MS system (Agilent1200SL/Agilent6130MS), Shimadzu LC/MS system (LC-10ADvp high pressure
gradient system/LCMS-2010A) or Shimadzu UFLC/MS (Prominence UFLC high pressure gradient system/LCMS-2020)
operating in electron spray ionization mode (ESI+). The column used was an L-column 2 ODS (3.0 x 50 mm I.D., 3 µm,
CERI, Japan) with a temperature of 40 °C and a flow rate of 1.2 or 1.5 mL/min. Mobile phase A was 0.05% TFA in
ultrapure water. Mobile phase B was 0.05% TFA in acetonitrile which was increased linearly from 5% to 90% over 2
minutes, 90% over the next 1.5 minutes, after which the column was equilibrated to 5% for 0.5 minutes. Elemental
analyses and Optical rotation analysis were carried out by Takeda Analytical Research Laboratories, Ltd.
Chromatographic purification was carried out on silica gel columns (Kieselgel 60, 0.063-0.22 mm, Merck) or on
Purif-Pack (SI 60 µm or NH 60 µm, Fuji Silysia, Ltd.). Each compound was confirmed to be ≥95% purity by either
LC/MS or elemental analysis. Yields were not optimized.

Ethyl N-(2,6-dinitrophenyl)-β-alaninate (4). A mixture of β-alanine ethyl ester hydrochloride (911 mg, 5.39 mmol),
2-chloro-1,3-dinitrobenzene (1.00 g, 4.94 mmol), triethylamine (2.07 mL, 14.9 mmol) and THF (50 mL) was stirred at
room temperature for 2 h. To the reaction mixture were added β-alanine ethyl ester hydrochloride (455 mg, 2.96 mmol)
and triethylamine (1.03 mL, 7.39 mmol) at room temperature and the resultant mixture was stirred at room temperature
for 2 h. The reaction mixture was diluted with EtOAc, washed with water and brine, dried over Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with a 0-20%
EtOAc/n-hexane gradient mixture to give 4 as an oil (1.39 g, 4.92 mmol, quant). 1H NMR (CDCl3, 300 MHz) δ 1.26 (t, J
= 7.1 Hz, 3H), 2.66 (t, J = 5.8 Hz, 2H), 3.23 - 3.31 (m, 2H), 4.17 (q, J = 7.1 Hz, 2H), 6.78 (t, J = 8.2 Hz, 1H), 8.17 (d, J =
8.2 Hz, 2H), 8.53 (brs, 1H).
Ethyl N-(2,6-diaminophenyl)-β-alaninate (5). Under hydrogen gas atmosphere, a mixture of compound 4 (1.39 g, 4.92
mmol), 10% palladium on carbon (50% wet, 280 mg) and THF (50 mL) was stirred at room temperature for 6 h. The
1
reaction mixture was filtered and concentrated in vacuo to give 5 as an oil (1.00 g, 4.50 mmol, 91%). H NMR (CDCl3,
300 MHz) δ 1.30 (t, J = 7.2 Hz, 3H), 2.56 - 2.62 (m, 2H), 3.12 - 3.17 (m, 2H), 3.95 (brs, 4H), 4.21 (q, J = 7.2 Hz, 2H),
6.19 (d, J = 7.8 Hz, 2H), 6.75 (t, J = 7.8 Hz, 1H).
Ethyl N-[2-amino-6-({[(2,4-dichlorophenyl)amino]carbonothioyl}amino)phenyl]-β-alaninate (6). To a solution of
compound 5 (500 mg, 2.24 mmol) in THF (22 mL) was added 2,4-dichlorophenyl isothiocyanate (503 mg, 2.46 mmol) at
room temperature. The resultant mixture was stirred at room temperature for 1 h and concentrated in vacuo. The residue
was purified by column chromatography on silica gel eluting with a 10-40% EtOAc/n-hexane gradient mixture and
NH-silica gel eluting with a 10-90% EtOAc/n-hexane gradient mixture to give 6 as a solid (533 mg, 1.25 mmol, 56%).
1H NMR (CDCl3, 300 MHz) δ 1.26 (t, J = 7.1 Hz, 3H), 2.66 (t, J = 5.8 Hz, 2H), 3.23 - 3.31 (m, 2H), 4.17 (q, J = 7.1 Hz,
2H), 6.78 (t, J = 8.2 Hz, 1H), 8.17 (d, J = 8.2 Hz, 2H), 8.53 (brs, 1H).
Ethyl 3-{7-amino-2-[(2,4-dichlorophenyl)amino]-1H-benzimidazol-1-yl}propanoate (7). To a solution of compound
6 (159 mg, 0.372 mmol) in THF (4 mL) was added EDCI (214 mg, 1.12 mmol) at room temperature. The resultant
mixture was stirred at 50 °C for 2 h. The reaction mixture was diluted with EtOAc, washed with water and brine, dried
over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel
eluting with EtOAc to give 7 as an oil (122 mg, 0.309 mmol, 83%). 1H NMR (CDCl3, 300 MHz) δ 1.21 (t, J = 7.2 Hz,
3H), 3.06 - 3.14 (m, 2H), 3.70 (brs, 2H), 4.14 (q, J = 7.2 Hz, 2H), 4.48 - 4.53 (m, 2H), 6.53 (d, J = 7.8 Hz, 1H), 6.98 (t, J
= 7.8 Hz, 1H), 7.12 - 7.23 (m, 2H), 7.36 (d, J = 2.2 Hz, 1H), 8.08 (brs, 1H), 8.16 - 8.27 (m, 1H).
Ethyl 3-[2-[(2,4-dichlorophenyl)amino]-7-(diethylamino)-1H-benzimidazol-1-yl]propanoate (8). To a solution of the
compound 7 (214 mg, 0.544 mmol) in MeOH (5.5 mL) and AcOH (0.11 mL) was added acetoaldehyde (0.204 mL, 3.27
mmol) at 0 °C. The resultant mixture was stirred at 0 °C for 30 min. To the reaction mixture was added sodium
triacetoxyborohydride (692 mg, 3.27 mmol) at 0 °C. After the resultant mixture was stirred at room temperature for 12 h,
the mixture was diluted with NaHCO3 aq. and extracted with EtOAc. The combined organic layer was washed with brine,

dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on NH-silica
gel eluting with EtOAc to give 8 as a solid (255 mg, 0.567 mmol, quant). 1H NMR (CDCl3, 300 MHz) δ 1.06 (t, J = 7.1
Hz, 6H), 1.21 (t, J = 7.2 Hz, 3H), 3.00 (t, J = 5.9 Hz, 2H), 3.07 (q, J = 7.1 Hz, 4H), 4.14 (q, J = 7.2 Hz, 2H), 4.62 (t, J =
5.9 Hz, 2H), 7.01 (dd, J = 1.1, 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.20 - 7.25 (m, 1H), 7.37 - 7.42 (m, 2H), 8.16 (s, 1H),
8.30 (d, J = 9.1 Hz, 1H).
3-[2-[(2,4-Dichlorophenyl)amino]-7-(diethylamino)-1H-benzimidazol-1-yl]propan-1-ol (9). To a suspension of
lithium borohydride (22.5 mg, 1.03 mmol) in THF (3.5 mL) was added compound 8 (155 mg, 0.345 mmol) at 0 °C. After
the resultant mixture was stirred at room temperature for 20 h, the mixture was diluted with NH4Cl aq. at 0 °C and stirred
at 0 °C for 30 min. The resultant mixture was diluted with NaHCO3 aq. and extracted with EtOAc. The combined organic
layer was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column
chromatography on silica gel eluting with a 25% EtOAc/n-hexane mixture to give 9 as a solid (35.7 mg, 0.0876 mmol,
1
25%). H NMR (CDCl3, 300 MHz) δ 1.06 (t, J = 7.2 Hz, 6H), 1.25 (s, 1H), 2.03 - 2.14 (m, 2H), 3.09 (q, J = 7.2 Hz, 4H),
3.56 (t, J = 5.5 Hz, 2H), 4.56 (t, J = 6.2 Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 7.22 - 7.28 (m, 2H),
7.32 - 7.40 (m, 2H), 8.40 (brs, 1H).
1-(2,4-Dichlorophenyl)-N,N-diethyl-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazol-6-amine (2a). To a solution of
compound 9 (35.7 mg, 0.0876 mmol) in pyridine (1 mL) was added methanesulfonyl chloride (0.0339 mL, 0.438 mmol)
at 0 °C. After the resultant mixture was stirred at room temperature for 2 h, the mixture was diluted with NaHCO3 aq. and
extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated
in vacuo. The residue was purified by column chromatography on silica gel eluting with a 10-40% EtOAc/n-hexane
gradient mixture to give 2a as a solid (29.2 mg, 0.0750 mmol, 86%). Analytically pure material was obtained by
recrystallization from EtOAc/n-hexane. mp 131-133 °C (EtOAc/n-hexane). 1H NMR (CDCl3, 300 MHz) δ 1.01 (t, J = 7.0
Hz, 6H), 2.27 - 2.38 (m, 2H), 3.01 - 3.12 (m, 4H), 3.63 - 3.75 (m, 2H), 4.59 - 4.67 (m, 2H), 6.86 (dd, J = 7.9, 1.1 Hz, 1H),
7.01 (t, J = 7.9 Hz, 1H), 7.21 (dd, J = 7.9, 1.1 Hz, 1H), 7.31 (dd, J = 8.5, 2.5 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.51 (d, J
= 2.5 Hz, 1H). LC-MS (ESI): m/z Calcd.: 388.1; Found: 388.9 (M+H). Anal. Calcd for C20H22N4Cl2: C,61.70; H,5.70;
N,14.39. Found: C,61.81; H,5.68; N,14.43.
3-[2-[(2,4-Dichlorophenyl)amino]-7-(diethylamino)-1H-benzimidazol-1-yl]propanoic acid (10). To a solution of
compound 8 (100 mg, 0.223 mmol) in a mixture of THF (2 mL) and MeOH (1 mL) was added 1N NaOH (0.446 mL,
0.446 mmol) at 0 °C. After the resultant mixture was stirred at room temperature for 1 h, the mixture was neutralized
with 1N HCl and extracted with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered
and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with EtOAc to give
10 as a solid (73.3 mg, 0.174 mmol, 78%). 1H NMR (DMSO-d6, 300 MHz) δ 0.98 (t, J = 7.0 Hz, 6 H), 2.70 - 2.84 (m, 2
H), 3.01 (q, J = 7.0 Hz, 4 H), 4.51 - 4.67 (m, 2 H), 6.89 - 7.29 (m, 3 H), 7.31 - 7.47 (m, 1 H), 7.60 (brs, 1 H), 8.07 (brs, 1
H), 8.74 (brs, 1 H), 12.57 (brs, 1 H).

1-(2,4-Dichlorophenyl)-6-(diethylamino)-3,4-dihydropyrimido[1,2-a]benzimidazol-2(1H)-one (2b). A mixture of
compound 10 (57.0 mg, 0.135 mmol), EDCI hydrochlroide (31.1 mg, 0.162 mmol), triethylamine (0.0226 mL, 0.162
mmol) and DMF (3 mL) was stirred at room temperature for 1 day. The reaction mixture was diluted with EtOAc,
washed with water and brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column
chromatography on silica gel eluting with a 20-50% EtOAc/n-hexane gradient mixture to give 2b as a solid (44.8 mg,
0.111 mmol, 82%). Analytically pure material was obtained by recrystallization from EtOAc/n-hexane. mp 113-114 °C
1
(EtOAc/n-hexane). H NMR (CDCl3, 300 MHz) δ 1.02 (t, J = 7.1 Hz, 6H), 3.05 - 3.22 (m, 6H), 4.73 - 5.01 (m, 2H), 7.03
(dd, J = 7.9, 0.9 Hz, 1H), 7.13 (t, J = 7.9 Hz, 1H), 7.36 (dd, J = 7.9, 0.9 Hz, 1H), 7.39 - 7.46 (m, 2H), 7.59 (d, J = 1.9 Hz,
1H). LC-MS (ESI): m/z Calcd.: 402.1; Found: 403.0 (M+H). Anal. Calcd for C20H20N4OCl2: C,59.56; H,5.00; N,13.89.
Found: C,59.62; H,4.98; N,13.93.
Methyl 2-(2,4-dichlorophenyl)-2-hydroxyethanimidoate hydrochloride (12). To
a
stirred solution of
2,4-dichlorobenzaldehyde (11) (18.3 g, 105 mmol) and 4-dimethylaminopyridine (128 mg, 1.05 mmol) in CH3CN (200
mL) was added trimethylsilyl cyanide (13.7 mL, 110 mmol) at room temperature. After 2 h, the reaction mixture was
concentrated in vacuo. To a stirred MeOH (150 mL) was added acetyl chloride (100 mL) at 0 °C, and the mixture was
warmed up to room temperature. After 30 min, the residue was added. After 2 h, the reaction mixture was concentrated in
vacuo. The resulting solid was washed with diethyl ether to afford 12 as colorless solid (25.5 g, 94.3 mmol, 90%), which
was used next step without further purification.
Ethyl 3-{7-amino-2-[(2,4-dichlorophenyl)(hydroxy)methyl]-1H-benzimidazol-1-yl}propanoate (13). To a stirred
solution of 5 (4.11 g, 18.4 mmol) in EtOH (36.8 mL) was added 12 (5.59 g, 20.7 mmol) at room temperature. After 12 h,
the reaction mixture was poured into water (120 mL). The resulting solid was collected by filtration and washed with
EtOAc to afford 13 as a colorless solid (6.77 g, 16.6 mmol, 90 %). 1H NMR (CDCl3, 300 MHz) δ 1.20 (t, J = 7.2 Hz, 3H),
2.55 - 2.70 (m, 1H), 2.76 - 2.90 (m, 1H), 3.94 (brs, 2H), 4.09 (q, J = 7.1 Hz, 2H), 4.36 - 4.61 (m, 2H), 4.93 (brs, 1H),
6.36 (s, 1H), 6.59 (d, J = 7.8 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 7.19 - 7.30 (m, 2H), 7.34 - 7.46 (m, 2H).
Ethyl 3-{2-[(2,4-dichlorophenyl)(hydroxy)methyl]-7-(diethylamino)-1H-benzimidazol-1-yl}propanoate (14). To a
suspension of 13 (6.00 g, 14.7 mmol) and AcOH (7.4 mL) in MeOH (147 mL) was added acetaldehyde (4.95 mL, 88.2
mmol) at 0 °C. After 30 min, sodium triacetoxyborohydride (18.7 g, 88.2 mmol) was added. After 2 h, the reaction
mixture was quenched with water, concentrated in vacuo, diluted with EtOAc, washed with 1N NaOH and brine, dried
over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel
eluting with a 10-30% EtOAc/n-hexane gradient mixture to give 14 as a colorless amorphous (6.30 g, 13.6 mmol, 92%).
1H NMR (CDCl3, 300 MHz) δ 0.92 - 1.11 (m, 6H), 1.23 (t, J = 7.1 Hz, 3H), 2.24 - 2.41 (m, 1H), 2.65 - 2.84 (m, 1H), 2.87
- 3.17 (m, 4H), 4.11 (q, J = 7.1 Hz, 2H), 4.44 - 4.68 (m, 2H), 5.13 (brs, 1H), 6.42 (s, 1H), 7.05 (d, J = 7.7 Hz, 1H), 7.13 -
7.30 (m, 2H), 7.33 - 7.54 (m, 3H).

3-{2-[(2,4-Dichlorophenyl)(hydroxy)methyl]-7-(diethylamino)-1H-benzimidazol-1-yl}propan-1-ol (15). To
a
suspension of lithium aluminum hydride (327 mg, 8.61 mmol) in THF (38 mL) was added a solution of 14 (2.00 g, 4.31
mmol) in THF (5 mL) at -5 °C. After the addition, Na2SO4-10H2O (3.3 g) was added. After 1 h, the resultant mixture was
filtrated and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with a
1
30-70% EtOAc/n-hexane gradient mixture to give 15 as a colorless solid (1.42 g, 3.36 mmol, 78%). H NMR (CDCl3,
300 MHz) δ 0.88 - 1.15 (m, 6H), 1.71 - 1.85 (m, 1H), 1.87 - 2.01 (m, 1H), 2.98 - 3.19 (m, 4H), 3.31 - 3.56 (m, 2H), 4.23 -
4.39 (m, 1H), 4.49 - 4.65 (m, 1H), 4.92 (brs, 1H), 6.41 (s, 1H), 7.04 (d, J = 7.4 Hz, 1H), 7.15 - 7.25 (m, 2H), 7.38 - 7.47
(m, 2H), 7.51 (d, J = 8.0 Hz, 1H).
(2,4-Dichlorophenyl)[7-(diethylamino)-1-(3-hydroxypropyl)-1H-benzimidazol-2-yl]methanone (16). A suspension of
15 (1.18 g, 2.79 mmol) and manganese dioxide (2.43 g, 27.9 mmol) in THF (14 mL) was stirred for 1.5 h at room
temperature. The reaction mixture was filtrated, and the filtrate was concentrated in vacuo to afford 16 as a yellow solid
(1.16 g, 2.76 mmol, 99%). 1H NMR (CDCl3, 300 MHz) δ 1.10 (t, J = 7.0 Hz, 6H), 2.02 - 2.14 (m, 2H), 2.55 (t, J = 5.9 Hz,
1H), 3.07 - 3.28 (m, 4H), 3.57 (q, J = 5.9 Hz, 2H), 5.21 (t, J = 6.9 Hz, 2H), 7.18 (dd, J = 7.7, 1.1 Hz, 1H), 7.23 - 7.31 (m,
1H), 7.38 (dd, J = 8.2, 1.9 Hz, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.56 - 7.65 (m, 2H).
[1-(3-Bromopropyl)-7-(diethylamino)-1H-benzimidazol-2-yl](2,4-dichlorophenyl)methanone (17). To a stirred
suspension of 16 (404 mg, 0.961 mmol) and tetrabromomethane (637 mg, 1.92 mmol) in CH3CN was added
triphenylphosphine (504 mg, 1.92 mmol). After 15 min, the reaction mixture was diluted with EtOAc, washed with
NaHCO3 aq. and brine, dried over Na2SO4, filtrated, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel eluting with a 0-30% EtOAc/n-hexane gradient mixture to afford 17 as yellow solid (407
mg, 0.842 mmol, 88%). 1H NMR (CDCl3, 300 MHz) δ 1.09 (t, J = 7.1 Hz, 6 H), 2.33 - 2.46 (m, 2H), 3.02 - 3.26 (m, 4H),
3.47 (t, J = 6.6 Hz, 2H), 5.19 - 5.28 (m, 2H), 7.20 (d, J = 7.8 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.38 (dd, J = 8.2, 1.9 Hz,
1H), 7.49 (d, J = 1.9 Hz, 1H), 7.56 - 7.64 (m, 2H).
4-(2,4-Dichlorophenyl)-N,N-diethyl-1,2-dihydropyrido[1,2-a]benzimidazol-9-amine (18). A solution of 17 (360 mg,
0.745 mmol) and triphenylphosphine (586 mg, 2.23 mmol) in CH3CN (5 mL) was stirred for 60 h at 80 °C. The reaction
mixture was concentrated in vacuo. Toluene (5 mL), THF (1 mL), and KOt-Bu (85%, 83.6 mg, 0.745 mmol) was added
at room temperature. After 1 h, the reaction mixture was diluted with EtOAc, washed with brine, dried over Na2SO4,
filtrated, and concentrated in vacuo. The residue was purified by column chromatography on silica gel eluting with a
1
5-25% EtOAc/n-hexane gradient mixture to afford 18 as pale yellow solid (65.5 mg, 0.170 mmol, 23%). H NMR
(CDCl3, 300 MHz) δ 1.03 (t, J = 7.0 Hz, 6H), 2.72 - 2.87 (m, 2H), 2.94 - 3.24 (m, 4H), 4.81 (t, J = 7.3 Hz, 2H), 6.37 (t, J
= 4.7 Hz, 1H), 7.04 (d, J = 7.8 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 7.27 - 7.33 (m, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.43 - 7.56
(m, 2H).
4-(2,4-Dichlorophenyl)-N,N-diethyl-1,2,3,4-tetrahydropyrido[1,2-a]benzimidazol-9-amine (2c).
A
solution of
compound 18 (53.0 mg, 0.137 mml) in MeOH was stirred in the presence of 1.9% palladium on fibroin (10 mg) under

hydrogen atmosphere at room temperature for 7 days. The catalyst was removed and the filtrate was concentrated in
vacuo. The residue was purified by column chromatography on silica gel eluting with a 0-30% EtOAc/n-hexane gradient
mixture to afford 2c as pale yellow oil (24.8 mg, 0.0640 mmol, 47%). 1H NMR (CDCl3, 300 MHz) δ 1.03 (t, J = 6.9 Hz,
6H), 1.92 - 2.20 (m, 3H), 2.28 - 2.41 (m, 1H), 2.96 - 3.21 (m, 4H), 4.45 - 4.55 (m, 1H), 4.70 - 4.90 (m, 2H), 6.90 (d, J =
8.2 Hz, 1H), 7.05 (dd, J = 7.7, 1.0 Hz, 1H), 7.09 - 7.21 (m, 2H), 7.41 (d, J = 1.9 Hz, 1H), 7.46 (dd, J = 7.7, 1.0 Hz, 1H).
LC-MS (ESI): m/z Calcd.: 387.1; Found: 387.9 (M+H).
2-[(2,6-Dinitrophenyl)amino]ethanol (19).
A
mixture of ethanolamine (5.96 mL, 49.4 mmol) and
2-chloro-1,3-dinitrobenzene (3) (5.00 g, 24.7 mmol) in THF (50 mL) was stirred at room temperature for 2 h. The
reaction mixture was diluted with EtOAc, washed with aqueous sodium hydrogen carbonate and brine, dried over
1
Na2SO4, filtered and concentrated in vacuo to give 19 as a brown solid (5.54 g, 24.4 mmol, 99%). H NMR (CDCl3, 300
MHz) δ 3.12 - 3.19 (m, 2 H), 3.85 - 3.90 (m, 2 H), 6.77 (t, J=8.2 Hz, 1 H), 8.19 (d, J=8.2 Hz, 2 H), 8.71 (brs., 1 H).
2-[(2,6-Diaminophenyl)amino]ethanol (20). A mixture of 19 (5.54 g, 24.4 mmol) and 10% palladium on carbon (50%
wet, 1.1 g) in THF (240 mL) was stirred at room temperature under H2 atmosphere for 2 h. The reaction mixture was
1
filtered and concentrated in vacuo to give 20 as a brown oil (3.63 g, 21.7 mmol, 89%). H NMR (CDCl3, 300 MHz) δ
3.09 - 3.15 (m, 2 H), 3.55 - 3.60 (m, 2 H), 3.83 (brs., 4 H), 6.24 (d, J=7.8 Hz, 2 H), 6.75 (t, J=7.8 Hz, 1 H).
2-{7-Amino-2-[(2,4-dichlorophenyl)(hydroxy)methyl]-1H-benzimidazol-1-yl}ethanol (21). To a stirred solution of 20
(3.63 g, 21.7 mmol) in EtOH (43 mL) was added 12 (7.04 g, 26.0 mmol) at room temperature. After 12 h, the reaction
mixture was diluted with NaHCO3 aq.. The resultant mixture was extracted with EtOAc. The combined organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel eluting with EtOAc to give 21 as an orange amorphous solid (7.58 g, 21.5 mmol, 99%). 1H
NMR (CDCl3, 300 MHz) δ 3.55 - 3.71 (m, 2 H), 4.10 - 4.21 (m, 1 H), 4.23 (brs., 2 H), 4.43 - 4.54 (m, 1 H), 6.01 (s, 1 H),
6.48 (dd, J=6.5, 2.2 Hz, 1 H), 6.89 - 6.98 (m, 2 H), 7.00 (dd, J=8.5, 2.2 Hz, 1 H), 7.25 - 7.32 (m, 2 H).
2-{2-[(2,4-Dichlorophenyl)(hydroxy)methyl]-7-(diethylamino)-1H-benzimidazol-1-yl}ethanol (22). To a solution of
21 (7.58 g, 21.5 mmol) in MeOH (220 mL) and AcOH (11 mL) was added acetoaldehyde (8.04 mL, 129 mmol) at 0 °C.
The resultant mixture was stirred at 0 °C for 30 min. To the reaction mixture was added sodium triacetoxyborohydride
(27.3 g, 129 mmol) at 0 °C. After the resultant mixture was stirred at room temperature for 2 h, the mixture was diluted
with NaHCO3 aq. and water, and extracted with EtOAc. The combined organic layer was washed with 1N NaOH and
brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on
silica gel eluting with EtOAc to afford a crude solid, which was washed with diisopropylether to give 22 as a colorless
solid (8.70 g, 21.3 mmol, 99%). 1H NMR (CDCl3, 300 MHz) δ 0.94 - 1.09 (6 H, m), 2.91 - 3.13 (4 H, m), 3.62 - 3.82 (2
H, m), 4.38 - 4.56 (2 H, m), 4.71 - 4.81 (1 H, m), 5.61 (1 H, brs.), 6.31 (1 H, s), 6.98 (1 H, dd, J=7.8, 0.8 Hz), 7.08 (1 H, t,
J=7.8 Hz), 7.25 (1 H, dd, J=8.3, 2.1 Hz), 7.32 - 7.37 (2 H, m), 7.63 (1 H, d, J=8.3 Hz).
1-(2,4-Dichlorophenyl)-N,N-diethyl-3,4-dihydro-1H-[1,4]oxazino[4,3-a]benzimidazol-6-amine (2d). To a stirred

solution of compound 22 (200 mg, 0.490 mmol) and triphenylphosphine (193 mg, 0.736 mmol) in THF (5 mL) was
added a solution of diethyl azodicarboxylate in toluene (40%, 0.335 mL, 0.736 mmol) at room temperature. The mixture
was stirred at room temperature for 12 h, concentrated in vacuo, and purified by column chromatography on silica gel
eluting with a 5-15% EtOAc/n-hexane gradient mixture to give 2d as a colorless solid (112 mg, 0.287 mmol, 59%).
Analytically pure material was obtained by recrystallization from n-hexane. mp 130-131 °C (n-hexane). 1H NMR (CDCl3,
300 MHz) δ 1.03 (t, J=7.1 Hz, 6 H), 3.11 (q, J=7.1 Hz, 4 H), 4.08 - 4.20 (m, 1 H), 4.29 - 4.38 (m, 1 H), 4.53 - 4.65 (m, 1
H), 4.82 - 4.92 (m, 1 H), 6.34 (s, 1 H), 7.07 (dd, J=7.7, 0.8 Hz, 1 H), 7.18 (t, J=7.7 Hz, 1 H), 7.22 - 7.23 (m, 2 H), 7.45 -
7.50 (m, 2 H). LC-MS (ESI): m/z Calcd.: 389.1; Found: 389.9 (M+H). Anal. Calcd for C20H21N3OCl2: C,61.55; H,5.42;
N,10.77. Found: C,61.65; H,5.48; N,10.80.
(2-Chloro-5-nitrophenyl)carbonimidic dichloride (24). 2-Chloro-5-nitroaniline (23) (43.1 g, 250 mmol), formic acid
(250 mL) was stirred at room temperature for 5 h. The reaction mixture was concentrated in vacuo. The solid residue was
o
taken up in water (350 mL), filtered, washed with water. The solid residue was dried at 50 C under vacuum to give a
crude formanilide as a light brown amorphous. A mixture of the crude formanilide, thionyl chloride (164 mL) and
o
sulfuryl chloride (58 mL) was stirred at 55 C for 48 h. The mixture was concentrated in vacuo. The residue was
dissolved in petroleum ether, decanted from a precipitate and the clear solution was evaporated in vacuo to give 24 as a
light brown amorphous (42.2 g, 166 mmol, 67%). 1H-NMR(CDCl3, 300 MHz) δ 7.62 (1H, d, J = 8.7 Hz), 7.83 (1H, d, J
= 2.7 Hz), 8.04 (1H, dd, J = 2.7, 8.7 Hz).
2-Chloro-5-nitro-N-(tetrahydropyrimidin-2(1H)-ylidene)aniline (25). A solution of 24 (35.5 g, 140 mmol) in THF
(135 mL) was added dropwise over 30 min to a solution of 1,3-propanediamine (58 mL, 700 mmol) in THF (335 mL)
o
with ice bath cooling. The reaction mixture was stirred for 1 h at 5-10 C, and stirred 2 hr at room temperature. The
insoluble material was removed by filtration, and the filtrate was concentrated in vacuo. The residue was washed with
water and diisopropyl ether. After drying at 50 oC under vacuum, 25 was obtained as yellow crystals (35.0 g, 137.4 mmol,
1
98%). H-NMR(CDCl3, 300 MHz) δ 1.95-2.03 (2H, m), 3.35 (4H, t, J = 6.0 Hz), 4.67 (2H, brs), 7.47 (1H, d, J = 8.7 Hz),
7.68 (1H, dd, J = 2.7, 8.7 Hz), 7.85 (1H, d, J = 8.7 Hz).
9-Chloro-6-nitro-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole (26). A mixture of 25 (5.0 g, 19.6 mmol) and
KOt-Bu (440 mg, 3.93 mmol) in DMSO (100 mL) was stirred at 60 oC for 24 h. After cooling to room temperature, to the
reaction mixture was directly purified by silica gel column chromatography eluting with a 50-100% EtOAc/n-hexane
1
gradient mixture to give 26 as an yellow amorphous (3.63 g, 14.3 mmol, 73 %). H-NMR(DMSO-d6, 300 MHz) δ
1.91-2.01 (2H, m), 3.03-3.40 (2H, m), 4.12 (2H, t, J = 6.0 Hz), 7.16 (1H, d, J = 9.0 Hz), 7.47 (1H, d, J = 9.0 Hz), 8.13
(1H, brs).
9-Chloro-1-(2,4-dichlorophenyl)-6-nitro-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole (27). A mixture of 26
(10.0 g, 39.6 mmol), 2,4-dichloro-1-iodobenzene (54.0g, 197.9 mmol), copper(I) iodide (7.57 g, 39.6 mmol),
2,2’-bipyridyl (12.3 g, 79.2 mmol) and Cs2CO3 (25.8 g, 79.2 mmol) in DMF (600 mL) was stirred at 150 °C for 12 h. The

mixture was duluted with EtOAc, washed with water, dried over Na2SO4 and concentrated in vacuo. The residue was
purified by flash chromatography on silica gel eluting with a 10-25% EtOAc/n-hexane gradient mixture to give 27 as a
light yellow amorphous (750 mg, 1.88 mmol, 5%). 1H-NMR(CDCl3, 300 MHz) δ 2.30-2.42 (2H, m), 3.70-3.85 (2H, m),
4.36 (2H, t, J = 6.0 Hz), 7.14 (1H, d, J = 9.0 Hz), 7.34 (1H, dd, J = 2.4, 8.4 Hz), 7.47 (1H, d, J = 8.4 Hz), 7.53 (1H, d, J =
2.4 Hz), 7.61 (1H, d, J = 9.0 Hz).
9-Chloro-1-(2,4-dichlorophenyl)-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazol-6-amine (28). A mixture of 27 (123
mg, 0.308 mmol) and 10% palladium on carbon (12 mg) in AcOH (5.0 mL) was stirred under H2 atmosphere at room
temperature for 8 h. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo. The residue was
purified by flash chromatography on silica gel eluting with a 50-100% EtOAc/n-hexane gradient mixture to give 28 (51
mg, 0.139 mmol, 45%) as an oil. 1H-NMR(CDCl, 300 MHz) δ 2.33-2.42 (2H, m), 3.56 (2H, brs), 3.62-3.75 (2H, m), 4.58
(2H, t, J = 6.0 Hz), 6.32 (1H, d, J = 9.0 Hz), 6.86 (1H, d, J = 9.0 Hz), 7.29 (1H, dd, J = 2.4, 8.4 Hz), 7.47 (1H, d, J = 2.4
Hz), 7.52 (1H, d, J = 8.4 Hz).
9-Chloro-1-(2,4-dichlorophenyl)-N,N-diethyl-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazol-6-amine (2e). To a
solution of 28 (45 mg, 0.122 mmol) in MeOH (5.0 mL) and AcOH (1.0 mL) was added acetaldehyde (0.10 mL, 1.64
mmol) at 0 °C. The resultant mixture was stirred at 0 °C for 30 min. To the reaction mixture was added sodium
triacetoxyborohydride (346 mg, 1.64 mmol) at 0 °C. After stirring at room temperature for 1 h, the mixture was
concentrated in vacuo, diluted with water, and extracted with EtOAc. The organic layer was dried over MgSO4, filtered
and concentrated in vacuo. The residue was purified by column chromatography on a silica gel eluting with a 10-50%
1
EtOAc/n-hexane gradient mixture to give 2e (50 mg, 0.120 mmol, 98%) as a colorless amorphous. H NMR (CDCl3, 300
MHz) δ 1.00 (6H, t, J = 7.2 Hz), 2.28-2.35 (2H, m), 3.02 (4H, q, J = 7.2 Hz), 3.69 (2H, brs), 4.61 (2H, t, J = 6.0 Hz), 6.78
(1H, d, J = 8.4 Hz), 7.01 (1H, d, J = 8.4 Hz), 7.30 (1H, dd, J = 2.4, 8.7 Hz), 7.49 (1H, d, J = 2.4 Hz), 7.52 (1H, d, J = 8.7
Hz). LC-MS (ESI): m/z Calcd.: 422.1; Found: 423.0 (M+H).
Methyl 2-chloro-3-nitrobenzoate (30). To a solution of 2-chloro-3-nitrobenzoic acid (29) (100 g, 0.496 mol) in THF
(1000 mL) was added dropwise oxalyl chloride (46.8 mL, 0.546 mol) at 0 °C, and the mixture was stirred at room
temperature for 3 h. To the mixture was added dropwise MeOH (300 mL) at 0 °C, and the mixture was stirred at room
temperature for 14 h. The mixture was concentrated in vacuo and the residue was neutralized with NaHCO3 aq., and
extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated
in vacuo to give the solid, which was washed with n-hexane to give 30 as an yellow powder (103 g, 0.478 mol, 96%). 1H
NMR (CDCl3, 300 MHz) δ 3.98 (s, 3H), 7.47 (t, J = 8.7 Hz, 1H), 7.83 (dd, J = 1.8 Hz, 8.7 Hz, 1H), 7.94 (dd, J = 1.8 Hz,
8.7 Hz, 1H).
Methyl 2-[(3-hydroxypropyl)amino]-3-nitrobenzoate (31). To a solution of 30 (100 g, 0.463 mol) in MeOH (800 mL)
and triethylamine (129 mL) was added 3-amino-1-propanol (52.2 g, 0.696 mol), and the mixture was stirred at 50 °C for
6 h. The mixture was concentrated in vacuo and the residue was diluted with NH4Cl aq., and extracted with EtOAc. The

combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography on silica gel eluting with a 50% EtOAc/n-hexane mixture to give 31 as a
1
brown oil (86.6 g, 0.341 mol, 74%). H NMR (CDCl3, 300 MHz) δ 1.61 (s, 1H), 1.86-1.95 (m, 2H), 3.04-3.12 (m, 2H),
3.77 (q, J = 6.0 Hz, 2H), 3.91 (s, 3H), 6.66 (t, J = 7.8 Hz, 1H), 7.95 (dd, J = 2.4 Hz, 8.7 Hz, 1H), 8.04 (dd, J = 2.4 Hz, 8.7
Hz, 1H), 8.52 (s, 1H).
Methyl 3-amino-2-[(3-hydroxypropyl)amino]benzoate (32). To a solution of 31 (86.6 g, 0.341 mol) in THF (1000 mL)
was added 10% palladium on carbon (50% wet; 8.70 g), and the mixture was purged with H2 and stirred under balloon
pressure hydrogen at room temperature for 23 h. The catalyst was removed by filtration, and the filtrate was concentrated
1
in vacuo to give 32 as a brown oil (59.8 g, 0.267 mol, 78%). H NMR (CDCl3, 300 MHz) δ 1.75 (s, 1H), 1.81-1.86 (m,
2H), 3.15 (t, J = 6.3 Hz, 2H), 3.86 (t, J = 6.3 Hz, 2H), 3.87 (s, 3H), 3.99 (s, 2H), 6.10 (s, 1H), 6.84-6.86 (m, 2H),
7.35-7.39 (m, 1H).
Methyl 3-amino-4-chloro-2-[(3-hydroxypropyl)amino]benzoate (33). N-Chlorosuccinimide (74.1 g, 555 mmol) was
added to a stirred solution of 32 (81.9 g, 370 mmol) in CH3CN (1480 mL) at room temperature, and the mixture was
stirred at room temperature for 16 h. Additional N-chlorosuccinimide (9.88 g, 74.0 mmol) was added to the mixture, and
the mixture was stirred at room temperature for 90 min. The mixture was diluted with NaHCO3 aq., concentrated in
vacuo, and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by flash column chromatography on silica gel eluting with a 20-45%
1
EtOAc/n-hexane gradient mixture to give 33 as a brown oil (15.7 g, 60.8 mmol, 16%). H NMR (CDCl3, 300 MHz) δ
1.79-1.87 (m, 2H), 2.33 (s, 1H), 3.15 (t, J = 6.3 Hz, 2H), 3.81-3.93 (m, 2H), 3.86 (s, 3H), 4.41 (s, 2H), 6.22 (s, 1H), 6.95
(d, J = 9.0 Hz, 1H), 7.29 (d, J = 9.0 Hz, 1H).
Methyl 4-chloro-2-[(2,4-dichlorophenyl)amino]-1-(3-hydroxypropyl)-1H-benzimidazole-7-carboxylate (35).
A
mixture of 33 (13.1 g, 50.6 mmol) and 2,4-dichloro-1-isothiocyanatobenzene (13.4 g, 65.8 mmol) in THF (150 mL) was
stirred at room temperature for 3 days. The mixture was diluted with NaHCO3 aq., concentrated in vacuo, and extracted
with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo.
The residue was washed with diisopropyl ether to give 34 as a colorless powder (19.6 g). A mixture of 34 (19.6 g),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (8.12 g, 42.3 mmol), and triethylamine (6.5 mL) in THF
(200 mL) was stirred at 50°C for 3 h. The mixture was concentrated in vacuo, diluted with NaHCO3 aq., and extracted
with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated in vacuo.
The residue was washed with diisopropyl ether to give 35 as a colorless powder (15.4 g, 35.9 mmol, 71% in 2 steps). 1H
NMR (CDCl3, 300 MHz) δ 2.00-2.11 (m, 3H), 3.54-3.61 (m, 2H), 3.95 (s, 3H), 4.64 (t, J = 6.3 Hz, 2H), 7.22 (d, J = 8.7
Hz, 1H), 7.28 (dd, J = 2.4 Hz, 9.0 Hz, 1H), 7.35 (d, J = 2.4 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 8.00 (s, 1H), 8.52 (d, J =
9.0 Hz, 1H).
Methyl
9-chloro-1-(2,4-dichlorophenyl)-1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazole-6-carboxylate
(36).