Tricyclic pharmacophore-based molecules as novel integrin αvβ3 antagonists. Part 1: Design and synthesis of a lead compound exhibiting αvβ3/ αIIbβ3 dual antagonistic activity

Article abstract of DOI:10.1016/j.bmc.2005.10.060

In order to generate novel compounds with integrin α _vβ₃-antagonistic activity together with antiplatelet activity, tricyclic pharmacophore-based molecules were designed and synthesized. Although piperazine-containing compounds initially prepared were selective α_IIbβ₃ antagonists, replacement of piperazine with piperidine furnished a potent α_vβ₃/ α_IIbβ₃ dual antagonist. Structure-activity relationship (SAR) studies provided clues for further development of tricyclic pharmacophore-based integrin antagonists.

Full text of DOI:10.1016/j.bmc.2005.10.060

Bioorganic & Medicinal Chemistry 14 (2006) 2089–2108
Tricyclic pharmacophore-based molecules as novel integrin
avb3 antagonists. Part 1: Design and synthesis of a lead
compound exhibiting avb3/aIIbb3 dual antagonistic activity
Dai Kubota, Minoru Ishikawa, Mikio Yamamoto, Shoichi Murakami, Mitsugu Hachisu,
Kiyoaki Katano and Keiichi Ajito*
Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
Received 6 August 2005; revised 31 October 2005; accepted 31 October 2005
Available online 23 November 2005
Abstract—In order to generate novel compounds with integrin avb3-antagonistic activity together with antiplatelet activity, tricyclic
pharmacophore-based molecules were designed and synthesized. Although piperazine-containing compounds initially prepared were
selective aIIbb3 antagonists, replacement of piperazine with piperidine furnished a potent avb3/aIIbb3 dual antagonist. Structure–
activity relationship (SAR) studies provided clues for further development of tricyclic pharmacophore-based integrin antagonists.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ment of coronary thrombosis. Our research program has
been focused on the functions of integrin avb3 in vascu-
lar smooth muscle cells and leukocytes. Further, Fab
fragment of the human-murine monoclonal antibody
Abciximab,6 which binds to the avb3 receptor and aIIbb3
receptor, has clinical efficacy in the treatment of ische-
mic diseases. In 1996, we started a search for novel inte-
grin avb3 antagonists with antiplatelet activity as
candidate drugs for the treatment of acute ischemic dis-
eases. In this paper, we describe the synthesis of a lead
compound exhibiting avb3/aIIbb3 dual antagonistic
activity, and we present basic SAR data concerning
the selectivity for avb3 over aIIbb3.
Integrins make up a family of heterodimeric transmem-
brane proteins that mediate essential cell–cell and cell–
matrix interactions involved in many physiological and
pathological processes.1,2 Among them, integrin avb3 is
expressed on a variety of cell types, including osteo-
clasts, leucocytes, vascular smooth muscle cells, and
endothelial cells. And integrin avb3 binds a number of
proteins, including vitronectin, fibrinogen, and osteo-
pontin, through recognition of the tripeptide RGD
sequence. And antagonists of avb3 are considered as
candidate drugs for treatment of osteoporosis, cancer
growth and metastasis, inflammation, diabetic retinopa-
thy, and restenosis.3 Another member of the family,
integrin aIIbb3, serves as the final common pathway
leading to platelet aggregation via its binding to fibrino-
gen. Blockade of aIIbb3 binding to fibrinogen provides a
potent antiplatelet activity, and two aIIbb3 antagonists,
tirofiban4 and integrelin,5 are currently used in the treat-
2. Background
When we started our integrin research program, three ma-
jor pharmaceutical companies had already reported their
original avb3 antagonists, including an aromatic-based
antagonist7–9 and a 1,4-benzodiazepine-type antago-
nist10,11 (see Fig. 1). For the molecular design of novel
non-peptide integrin avb3 antagonists, we focused on
mimetics of the RGD tripeptide sequence, which is a
key recognition site for binding/adhesion. In order to ob-
tain potent antagonists, high affinity between the receptor
and antagonist at the molecular level is crucial. Since the
spatial arrangement of the ligand molecule is important,
we selected a linear, unfused-tricyclic pharmacophore.
Keywords: Non-peptide; Integrin avb3 antagonist; Integrin aIIbb3
antagonist; Acute ischemic disease.
*
Corresponding author at Present address: R&D Strategy, R&D
Planning and Management, Pharmaceutical, Meiji Seika Kaisha,
Ltd., 2-4-16 Kyobashi Chuo-ku, Tokyo 104-8002, Japan. Tel.: +81 3
3273 3346; fax: +81 3 3273 3380; e-mail: keiichi_ajito@meiji.co.jp
Present address: Pharmaceutical Research Department.
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.060

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D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
hydrolysis of the ethyl ester, the C-terminal building
block13 (8) was attached to the tricyclic moiety using
water-soluble carbodiimide. Deprotection of the t-butyl
group with trifluoroacetic acid, followed by hydrogenol-
ysis of pyrimidine, furnished the desired compound 4.
This molecule exhibited weak antagonistic activity to
avb3,14,15 but strong aIIbb3-antagonistic activity. Thus,
we attempted to enhance the avb3-antagonistic activity
by chemical modification.
Compound 15 was generated through compound 13, in
which the secondary amine at piperazine was protected
with a Boc group, as shown in Scheme 3.
For the synthesis of benzylamine-type compounds (23–
25), reductive amination was utilized (Scheme 4). Thus,
ethyl benzoate was converted into benzaldehyde in two
steps, and coupling with the C-terminal moiety (8) fol-
lowed by further elaboration gave 23–25.
First, the SAR of the N-terminus and the amide bond
was examined, using integrin avb3 and aIIbb3 receptor
binding assays (Tables 1 and 2). Regarding the N-termi-
nus, the formimidoyl and benzimidazole derivatives (15,
16) showed moderate inhibition, like the tetrahydropyr-
imidine analogue (4), but we did not proceed with fur-
ther modification of 16 because of its extremely low
water solubility. Transformation of the amide portion
failed to improve the activity.
Figure 1. Reported integrin avb3 antagonists.
Second, the effect of C-terminal structure was investigat-
ed (see Schemes 5 and 6). The importance of an acidic
proton at a benzenesulfonylamide group for avb3-antag-
onistic activity was confirmed (Table 3).
Scheme 1. Design of a tricyclic pharmacophore-based molecule.
Thus, we designed compound 4 possessing a cyclic N-
terminus and a phenyl piperazine moiety as shown in
Scheme 1. At almost at the same time, structurally relat-
ed antagonists were synthesized by Nicolaou et al.12
3. Results and discussion
Scheme 2. Reagents and conditions: (a) Ethyl 4-fluorobenzoate,
DMSO, 120 °C, 20 h; (b) 2-bromopyrimidine, N,N-diisopropylethyl-
amine, DMF, 120 °C, 4.5 h; (c) NaOH, H2O/MeOH/THF, 40 °C, 5 h;
(d) HOBT, N-methylmorpholine, EDC, DMF, rt, 24 h; (e) TFA,
CH2Cl2, rt, 3 h; (f) H2/Pd-C, 3 atm, HCl/aq AcOH, rt, 4 h.
Piperazine was coupled with ethyl 4-fluorobenzoate un-
der heated condition, followed by reaction with 2-
bromopyrimidine in the presence of a tertiary amine,
to afford a tricyclic moiety (Scheme 2). After basic

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2091
Table 1. Effect of replacement of the N-terminus on receptor binding
inhibition
N-terminus
N
N
NHSO2Ph
H
N
CO2H
O
Compound
N-terminus
IC50 (nM)
avb3 aIIbb3
avb3/aIIbb3
0.0046
0.002
N
4
10
15
16
17
160
0.73
N
H
N
N
4,700
220
9.3
NH
0.57
1.2
0.0026
0.019
H2N
N
64
N
H
Boc—
4,900
18
0.0037
Scheme 3. Reagents and conditions: (a) Boc2O, NaOH, H2O/1,4-
dioxane, rt, 1 h; (b) NaOH, H2O/MeOH/THF, 50 °C, 8 h; (c) HOBT,
N-methylmorpholine, EDC, DMF, rt, 18 h; (d) TFA, H2O rt, 1 h; (e)
3,5-dimethylpyrazole-1-carboxamide nitrate, pyridine, 90 °C, 20 h.
Table 2. Effect of replacement of the amide portion on receptor
binding inhibition
Compound
IC50 (nM)
aIIbb3
0.73
76
avb3/aIIbb3
avb3
160
4
23
24
25
0.0046
0.011
1.4
6,700
7,000
4,900
10,000
8,100
1.7
Scheme 5. Reagents and conditions: (a) MeI, DBU, DMF, rt, 16 h; (b)
TFA, anisole, CH2Cl2, 0 °C, 16 h; (c) H2/Pd-C, 3 atm, HCl/aq AcOH, rt,
3 h.
Scheme 4. Reagents and conditions: (a) DIBAH, CH2Cl2 ꢀ78 °C, 1 h;
(b) MnO2 AcOEt, rt, 2 h; (c) NaBH3 AcOH, MeOH/CH2Cl2 rt,
30 min; (d) TFA, CH2Cl2 rt, 16 h; (e) HCHO, AcOH, NaBH3CN,
MeOH/CH2Cl2 rt, 30 min; (f) PhCHO, AcOH, NaBH3CN, MeOH/
CH2Cl2 rt, 1.5 h; (g) H2/Pd-C, 3 atm, AcOH/HCl, rt, 3 h.
but this approach alone failed to yield a dual antagonist.
Several antagonists that exhibited potent avb3 receptor
binding inhibition were further evaluated for avb3-med-
iated cell adhesion inhibitory activity using vascular
smooth muscle cells (VSMC).16
Third, substitutions were made in the central aromatic
ring. As shown in Table 4, introduction of a halogen
atom generally enhanced avb3-antagonistic activity,

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D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
Table 3. Effect of replacement of the C-terminus on receptor binding
inhibition
N
N
N
H
N
H
N
C-terminus
O
Compound
C-terminus
IC50 (nM)
aIIbb3
avb3/aIIbb3
avb3
NHSO2Ph
4
27
31
32
33
34a
160
0.73
0.0046
0.0055
0.076
0.13
CO2
H
Me
NSO2Ph
CO2H
14,000
17,000
35
77
NH 2
1300
CO2H
NHCbz
CO2H
4.4
NHB oc
CO2H
120
8.5
0.071
0.0055
NHEt
CO2H
49,000
270
NHSO2Ph
CO2H
10b
4,700
46,000
9.3
7,900
NT
0.002
0.17
NT
35b,c
36b,d
Scheme 6. Reagents and conditions: (a) HOBT, N-methylmorpholine,
EDC, DMF, rt, 24 h; (b) TFA, CH2Cl2, rt, 5.5 h; (c)H2/Pd-C, 3 atm,
HCl/aq AcOH, rt, 4 h; (d) CbzCl, K2CO3, acetone/H2O, rt, 6.5 h; (e)
Boc2O NaOH, H2O, rt, 2 h.
CO2
H
180,000
CO2H
a 3-Fluorobenzoyl instead of the central benzoyl.
b Pyrimidine at the N-terminus.
Finally, piperazine was replaced as shown in Table 5.
Although introduction of a methyl group into the pipera-
zine ring increased avb3-antagonistic activity, replace-
ment of piperazine with 4-aminopiperidine drastically
enhanced the avb3 activity, providing compound 47 with
avb3/aIIbb3 dual antagonistic activity as the first example
in this series. This dual activity is expected to exhibit clin-
ical efficacy as well as tirofiban,17 which is used as remedy
for acute coronary syndrome. Moreover, compound 47
showed strong inhibition of avb3-mediated cell adhesion.
c Prepared by using b-alanine ethyl ester.
d Prepared by using ethyl (3S)-ethynyl-3-aminopropionate.
Table 4. Effect of substitution of the central aromatic ring on receptor
binding inhibition
N
N
N
3
H
N
2
NHSO2Ph
CO2H
H
N
O
4. Conclusion
Compound 2 or 3 position
IC50 (nM)
avb3/aIIbb3
As a design principle for novel avb3 antagonists, we
focused on mimetics of the RGD tripeptide sequence,
which is a key recognition site for binding/adhesion.
Thus, we designed and synthesized piperazine-based
non-peptide integrin avb3 antagonists with a linear, un-
fused-tricyclic pharmacophore. Compound 4 exhibited
potent aIIbb3-antagonistic activity. SAR studies aimed
at improving selectivity for avb3 over aIIbb3 were con-
ducted, and replacement of piperazine with 4-aminopi-
peridine afforded compound 47, with potent avb3/
aIIbb3 dual antagonistic activity.
avb3 aIIbb3 VSMCb
4
37
38
39
40
41a
Unsubstituted 160
0.73 NT
0.0046
3,500 0.045
3-F
2-F
22
31
1.0
0.41
5,500 0.013
4,200 0.033
3-Cl
2-Cl
3-NO2
3.6 0.12
19
220
0.14 15,000 0.0071
1.1 NT 0.0050
a Pyrimidine at the N-terminus.
b avb3-mediated cell adhesion assay: vascular smooth muscle cell-
vitronectin.
tetramethylsilane as a standard. Electron ionization
(EI) mass spectra were recorded on a Hitachi M-80B
instrument. Fast-atom bombardment (FAB) mass spec-
tra were recorded on a JEOL JMS-700 instrument.
Thermospray (TSP) mass spectra were recorded on a
5. Experimental
1H NMR spectra were recorded on JNM-LA400 spec-
trometers with chemical shifts in ppm with the internal

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2093
Table 5. Effect of replacement of the hetero ring on receptor binding
inhibition
chromatography on silica gel (CH2Cl2/MeOH/concd
NH4OH = 950:50:1) to prepare compound 6 (142 mg,
1
95%) as a colorless solid; H NMR (400 MHz, CDCl3)
N
N
d: 1.37 (3H, t, Et), 3.43 (4H, m, piperazine), 3.99 (4H,
m, piperazine), 4.33 (2H, q, Et), 6.54 (1H, t, pyrimidine),
6.90 (2H, d, C6H4), 7.94 (2H, d, C6H4), 8.34 (2H, d, pyrim-
idine); TSPMS m/z 313 (M+H)+.
H
hetero ring
NHSO2Ph
H
N
CO2H
O
5.1.3. Compound 7. MeOH (5.0 ml) and THF (25 ml)
were added to compound 6 (127 mg, 0.41 mmol) to pre-
pare a solution, and 1 N NaOH (25 ml) was added to the
solution at room temperature. The mixture was stirred
at 40 °C for 5.0 h. The reaction mixture was returned
to room temperature, neutralized by 1 N HCl (pH 7),
and then concentrated under reduced pressure. The res-
idue was purified by column chromatography on silica
gel (CH2Cl2/EtOH/H2O/concd NH4OH = 8:8:1:1) to
prepare compound 7 (128 mg, 100%) as a colorless solid;
1H NMR (400 MHz, CD3OD) d: 3.35 (4H, m, pipera-
zine), 3.95 (4H, m, piperazine), 6.60 (1H, t, pyrimidine),
6.97 (2H, d, C6H4), 7.87 (2H, d, C6H4), 8.33 (2H, d,
pyrimidine); TSPMS m/z 285 (M+H)+.
Compound Hetero rings
IC50 (nM)
avb3/aIIbb3
avb3 aIIbb3 VSMCb
4
160
67
0.73
1.3
NT
0.0046
0.019
N
N
42
43
3,700
Me
N
N
N
24
0.73
1.7
1,600
0.030
Me
Me
N
44
45
120
NT
NT
0.014
Me
N
N
280
270
0.77
4.9
0.0028
5.1.4. Compound 9. DMF (60 ml) was added to compound
7 (734 mg, 2.6 mmol) to prepare a solution. Compound 8
(860 mg, 2.9 mmol) was added to the solution. Further,
1-hydroxybenzotriazole (523 mg, 3.9 mmol), N-meth-
ylmorpholine (1.5 ml, 14 mmol), and 1-ethyl-3-(3-dim-
ethylaminopropyl)carbodiimide hydrochloride (758 mg,
4.0 mmol) were added thereto, and the mixture was stir-
red at room temperature for 24 h. An aqueous NaHCO3
solution (500 ml) was added to stop the reaction, and
the mixture was extracted with CH2Cl2 (500 ml). The
organic layer was dried over anhydrous Mg2SO4 and con-
centrated under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (n-hexane/
ethyl acetate = 1:2) to prepare compound 9 (1.0 g, 68%)
as a colorless solid; 1H NMR (400 MHz, CDCl3) d: 1.27
(9H, s, t-Bu), 3.39 (4H, m, piperazine), 3.57 (1H, m, CON-
HCH2CH), 3.89 (2H, m, CONHCH2CH), 3.99 (4H, m,
piperazine), 6.53 (1H, t, pyrimidine), 6.93 (2H, d, C6H4),
7.49 (2H, m, C6H5), 7.57 (1H, m, C6H5), 7.73 (2H, d,
C6H4), 7.86 (2H, m, C6H5), 8.33 (2H, d, pyrimidine);
TSPMS m/z 567 (M+H)+.
46a
47
NT
190
0.018
2.4
N
N
1.3 3.1
HN
N
Me
48
76
0.19
9,000
0.0025
N
N
a Benzimidazole at the N-terminus.
b avb3-mediated cell adhesion assay: vascular smooth muscle cell-
vitronectin.
Hewlett–Packard 5989A instrument. Atmospheric pres-
sure chemical ionization (APCI) mass spectra were
recorded on a Hewlett–Packard 5989A instrument.
High-resolution mass spectra (HRMS) were recorded
under FAB conditions. Optical rotations were obtained
on a JASCO DIP-370 polarimeter.
5.1. Preparation of compound 4
5.1.1. Compound 5. DMSO (10 ml) was added to pipera-
zine (5.2 g, 60 mmol) to prepare a suspension, to which
ethyl 4-fluorobenzoate (2.5 g, 15 mmol) was added at
room temperature. The reaction mixture was stirred at
120 °C for 20 h and then concentrated under reduced
pressure. The residue was purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH/concd NH4OH =
900:100:1) to prepare compound 5 (3.3 g, 94%) as a color-
less solid; 1H NMR (400 MHz, CDCl3) d: 1.37 (3H, t, Et),
3.02 (4H, m, piperazine), 3.28 (4H, m, piperazine), 4.32
(2H, q, Et), 6.86 (2H, d, C6H4), 7.92 (2H, d, C6H4);
TSPMS m/z 235 (M+H)+.
5.1.5. Compound 10. CH2Cl2 (4.0 ml) was added to com-
pound 9 (51 mg, 0.090 mmol) to prepare a solution. Tri-
fluoroacetic acid (2.0 ml) was added at room
temperature to the solution. The reaction mixture was
stirred at that temperature for 3.0 h and then concen-
trated under reduced pressure. The residue was purified
by preparative thin-layer chromatography (CH2Cl2/
EtOH/H2O/concd NH4OH = 8:8:1:1) and Sephadex
LH-20 chromatography (MeOH) to prepare compound
10 (46 mg, 100%) as a colorless solid; 1H NMR
(400 MHz, CD3OD) d: 3.30 (4H, m, piperazine), 3.46
(1H, dd, CONHCH2CH), 3.57 (1H, dd, CON-
HCH2CH), 3.64 (1H, dd, CONHCH2CH), 3.87 (4H,
m, piperazine), 6.52 (1H, t, pyrimidine), 6.92 (2H, d,
C6H4), 7.36 (2H, m, C6H5), 7.42 (1H, m, C6H5), 7.63
(2H, d, C6H4), 7.75 (2H, m, C6H5), 8.25 (2H, d, pyrim-
5.1.2. Compound 6. DMF (4.0 ml) was added to com-
pound 5 (114 mg, 0.48 mmol) to prepare a solution, and
2-bromopyrimidine (76 mg, 0.48 mmol) and N,N-diiso-
propylethylamine (0.4 ml, 2.3 mmol) were added to the
solution at room temperature. The reaction mixture was
stirred at 120 °C for 4.5 h and then concentrated under
reduced pressure. The residue was purified by column
idine); FAB-HRMS (M+H)+ calcd for C24H26N6O5S
25
D
511.1764, found 511.1759; ½aꢁ +63° (c 0.055, MeOH).

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D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
5.1.6. Compound 4. Acetic acid (100 ml) and concentrat-
ed hydrochloric acid (10 ml) were added to compound
10 (378 mg, 0.74 mmol) to prepare a solution. 10% Pd/
C (200 mg) was added to the solution, and the mixture
was vigorously shaken at room temperature for 4.0 h
under a hydrogen pressure of 3 atm. The insolubles were
filtered and then washed twice with water. The filtrate
was combined with the washings, followed by concen-
tration under reduced pressure. The residue was purified
by preparative thin-layer chromatography (CH2Cl2/
EtOH/H2O/concd NH4OH = 8:8:1:1) and Sephadex
LH-20 chromatography (MeOH) to prepare compound
4 (198 mg, 52%) as a colorless solid; 1H NMR
(400 MHz, CD3OD) d: 1.95 (2H, quintet, J = 5.8, tetra-
hydropyrimidine), 3.37 (4H, m, piperazine), 3.40 (4H, t,
J = 5.8, tetrahydropyrimidine), 3.52 (4H, m, piperazine),
3.57 (1H, dd, J = 8.0, 13.2, CONHCH2CH), 3.64 (1H,
dd, J = 5.0, 13.2, CONHCH2CH), 3.72 (1H, dd,
J = 5.0, 8.0, CONHCH2CH), 6.91 (2H, d, J = 9.0,
C6H4), 7.47 (2H, m, C6H5), 7.53 (1H, m, C6H5), 7.72
(2H, d, J = 9.0, C6H4), 7.85 (2H, m, C6H5); FAB-
Trifluoroacetic acid (2.0 ml) and H2O were added to com-
pound 13 (31 mg, 0.053 mmol) to prepare a solution, and
the mixture was stirred at room temperature for 1 h. The
reaction mixture was concentrated under reduced pres-
sure. The residue was purified by column chromatogra-
phy on silica gel (CH2Cl2/EtOH/H2O/concd NH4OH =
8:8:1:1) and Sephadex LH-20 chromatography (MeOH/
concd NH4OH = 8:1) to prepare compound 14 (7.9 mg,
34%) as a colorless solid; 1H NMR (400 MHz, CD3OD)
d: 3.30 (5H, m, piperazine, CONHCH2CH), 3.45 (4H, t,
piperazine), 3.62 (1H, dd, CONHCH2CH), 4.04 (1H,
dd, CONHCH2CH), 6.94 (2H, d, C6H4), 7.21 (3H, m,
C6H5), 7.40 (2H, d, C6H4), 7.61 (2H, d, C6H5); TSPMS
m/z 433 (M+H)+.
5.2.3. Compound 15. Pyridine (10 ml) was added to com-
pound 14 (23 mg, 0.052 mmol) and 3,5-dimethylpyraz-
ole-1-carboxamidine nitrate (22 mg, 0.11 mmol). The
reaction mixture was stirred at 90 °C for 20 h and then
returned to room temperature. It was concentrated un-
der reduced pressure, and the residue was purified by
column chromatography on silica gel (CH2Cl2/EtOH/
H2O/concd NH4OH = 8:8:1:1) and Sephadex G-10
chromatography (0.05 N HCl) to prepare compound
HRMS (M+H)+ calcd for C24H30N6O5S 515.2077,
25
D
found 515.2076; ½aꢁ +112° (c 0.048, MeOH).
1
5.2. Preparation of compound 15
15 hydrochloride (5.3 mg, 17%) as a colorless solid; H
NMR (400 MHz, CD3OD) (hydrochloride salt) d: 3.49
(1H, dd, CONHCH2CH), 3.51 (4H, m, piperazine),
3.71 (4H, m, piperazine), 3.72 (1H, dd, CONHCH2CH),
4.18 (1H, dd, CONHCH2CH), 7.06 (2H, d, C6H4), 7.43
(2H, m, C6H5), 7.50 (1H, m, C6H5), 7.73 (2H, d, C6H4),
5.2.1. Compound 12. 1,4-Dioxane (10 ml) and H2O
(5.0 ml) were added to compound 5 (500 mg, 2.1 mmol)
to prepare a solution. 1 N NaOH (3.0 ml) and di-t-butyl
dicarbonate (465 mg, 2.1 mmol) were added to the solu-
tion at room temperature. After 1.0 h stirring, the reac-
tion mixture was concentrated under reduced pressure
to prepare compound 11.
7.82 (2H, m, C6H5); FAB-HRMS (M+H)+ calcd for
25
D
C21H26N6O5S 475.1764, found 475.1766; ½aꢁ +45°
(c 0.26, MeOH).
THF (20 ml) and MeOH (5.0 ml) were added to com-
pound 11 to prepare a solution. 1 N NaOH (20 ml)
was added to the solution, and the mixture was stirred
at 50 °C for 8.0 h. The reaction mixture was returned
to room temperature, neutralized by 1 N HCl (pH 7),
and then concentrated under reduced pressure. The res-
idue was purified by column chromatography on silica
gel (CH2Cl2/EtOH/H2O/concd NH4OH = 8:8:1:1) to
prepare compound 12 (400 mg, 62%) as a colorless solid;
1H NMR (400 MHz, CD3OD) d: 1.48 (9H, s, t-Bu), 3.26
(4H, br t, piperazine), 3.57 (4H, br t, piperazine), 6.94
(2H, d, C6H4), 7.87 (2H, d, C6H4); TSPMS m/z 307
(M+H)+.
5.3. Preparation of compound 16
5.3.1.Ethyl4-{4-(1H-benzimidazol-2-yl)piperazin-1-yl}ben-
zoate. DMF (2.0 ml) was added to compound 5 (93 mg,
0.40 mmol)toprepareasolution,and2-chlorobenzimidaz-
ole (37 mg, 0.24 mmol) was added to the solution. The
mixture was stirred at 140 °C for 28 h and cooled to
room temperature, and water (20 ml) and CH2Cl2
(20 ml) were added thereto. The organic layer was sep-
arated, dried over anhydrous MgSO4, and concentrat-
ed under reduced pressure. The residue was purified
by column chromatography on silica gel (n-hexane/eth-
yl acetate = 1:2) to prepare the title compound (37 mg,
1
44%) as a colorless solid; H NMR (400 MHz, CDCl3)
5.2.2. Compound 14. DMF (2.0 ml) was added to com-
pound 12 (63 mg, 0.21 mmol) to prepare a solution.
Compound 8 (61 mg, 0.20 mmol) was added to the solu-
tion. Further, 1-hydroxybenzotriazole (42 mg, 0.31
mmol), N-methylmorpholine (0.50 ml, 4.5 mmol), and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (81 mg, 0.42 mmol) were added thereto, and
the mixture was stirred at room temperature for 18 h.
An aqueous NaHCO3 solution (20 ml) was added to
stop the reaction, and the mixture was extracted with
CH2Cl2 (20 ml). The organic layer was dried over anhy-
drous MgSO4 and concentrated under reduced pressure.
The residue was purified by column chromatography on
silica gel (n-hexane/ethyl acetate = 2:3) to prepare com-
pound 13 as a colorless solid.
d: 1.38 (3H, t, Et), 3.44 (4H, m, piperazine), 3.73 (4H,
m, piperazine), 4.34 (2H, q, Et), 6.88 (2H, br d,
C6H4), 7.09 (2H, dd, benzimidazole), 7.34 (2H, m,
benzimidazole), 7.95 (2H, br d, C6H4); TSPMS m/z
351 (M+H)+.
5.3.2. 4-{4-(1H-Benzimidazol-2-yl)piperazin-1-yl}benzoic
acid. The title compound was prepared from ethyl 4-{4-
(1H-benzimidazol-2-yl)piperazin-1-yl}benzoate by the
same procedure as employed for compound 7 as a color-
less solid. Yield: 20 mg, 92%; 1H NMR (400 MHz,
CD3OD) d: 3.38 (4H, m, piperazine), 3.60 (4H, m, piper-
azine), 6.93 (4H, m, benzimidazole and C6H4), 7.17 (2H,
dd, benzimidazole), 7.80 (2H, d, C6H4); TSPMS m/z 323
(M+H)+.

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2095
5.3.3. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(1H-
benzimidazol-2-yl)piperazin-1-y1}benzoylamino]propionate.
The title compound was prepared from 4-{4-(1H-ben-
zimidazol-2-yl)piperazin-1-yl}benzoic acid by the same
salt solution (50 ml) were added thereto, followed by
vigorous stirring at room temperature for 15 min. The
organic layer was separated, washed twice with saturat-
ed saline (50 ml), dried over anhydrous Na2SO4, and
then concentrated under reduced pressure to prepare
compound 18 (158 mg).
procedure as employed for compound
9
as
a
1
colorless solid. Yield: 25 mg, 92%; H NMR (400 MHz,
CDCl3) d: 1.21 (9H, s, t-Bu), 3.21 (4H, m, piperazine),
3.50 (1H, m, CONHCH2CH), 3.64 (4H, m, piperazine),
3.86 (2H, m, CONHCH2CH), 6.82 (2H, d, C6H4), 7.03
(2H, dd, benzimidazole), 7.30 (2H, m, benzimidazole),
7.42 (2H, m, C6H5), 7.50 (1H, m, C6H5), 7.66 (2H, d,
C6H4), 7.77 (2H, d, C6H5); FABMS m/z 605 (M+H)+.
Compound 18 (143 mg) was dissolved in ethyl acetate
(14 ml), and active manganese dioxide (358 mg) was
added to the solution. The mixture was stirred vigor-
ously at room temperature for 2 h. The insolubles
were filtered and then washed twice with MeOH.
The filtrate was combined with the washings, followed
by concentration under reduced pressure to prepare
compound 19 (142 mg). 75% MeOH/CH2Cl2 (14 ml)
was added to compound 19 and compound 8 hydro-
chloride (142 mg, 0.43 mmol) to dissolve them in the
MeOH/CH2Cl2. The reaction solution was adjusted
to pH 3–4 by the addition of a small amount of acetic
acid. Sodium cyanoborohydride (150 mg) was added
thereto, followed by a reaction at room temperature
for 30 min. The reaction solution was concentrated
under reduced pressure. The residue was purified by
column chromatography on silica gel (CHCl3/MeOH/
concd NH4OH = 1000:30:1) to prepare t-butyl (2S)-
benzenesulfonylamino-3-[4-{4-(pyrimidin-2-yl)piperazin-
1-yl}benzylamino]propionate (288 mg) as a colorless
solid.
5.3.4. Compound 16. CH2Cl2 (2.0 ml) was added to t-bu-
tyl (2S)-benzenesulfonylamino-3-[4-{4-(1H-benzimidazol-
2-yl)piperazin-1-y1}benzoylamino]propionate (10 mg,
0.018 mmol) to prepare a solution. Trifluoroacetic acid
(0.20 ml) was added at room temperature to the solu-
tion, and the mixture was stirred at that temperature
for 8 h before the reaction solution was concentrated
under reduced pressure. The residue was purified by
preparative thin-layer chromatography on silica gel
(EtOH/H2O/concd NH4OH = 8:1:1) and then purified
by Sephadex LH-20 (MeOH/concd NH4OH = 9:1) to
prepare the compound 16 (4.5 mg, 47%) as a colorless
1
solid; H NMR (400 MHz, CD3OD) d: 3.21 (4H, m,
piperazine), 3.47 (1H, dd, CONHCH2CH), 3.57 (1H,
dd, CONHCH2CH), 3.60 (4H, m, piperazine), 3.64
(1H, dd, CONHCH2CH), 6.92 (2H, dd, benzimidazole),
6.96 (2H, d, C6H4), 7.17 (2H, dd, benzimidazole), 7.37
(2H, m, C6H5), 7.43 (1H, br t, C6H5), 7.65 (2H, d,
CH2Cl2 (6.0 ml) was added to t-butyl (2S)-benze-
nesulfonylamino-3-[4-{4-(pyrimidin-2-yl)piperazin-1-yl}-
benzylamino]propionate (269 mg, 0.49 mmol) to
prepare a solution, and anisole (0.26 ml) was added to
the solution. The mixture was cooled to 0 °C. Trifluoro-
acetic acid (6.0 ml) was added at that temperature to
the mixture, and the reaction was allowed to proceed
at room temperature for 16 h. The reaction solution
was concentrated under reduced pressure. The residue
was subjected to azeotropic distillation twice with tolu-
ene, followed by purification by column chromatogra-
phy on silica gel (CHCl3/MeOH/concd NH4OH =
90:20:1) to prepare compound 20 (224 mg, 0.45 mmol)
as a colorless solid; 1H NMR (400 MHz, CD3OD/
CDCl3 = 1:1) d: 3.18 (1H, dd, ArCH2NHCH2), 3.28
(1H, dd, ArCH2NHCH2), 3.33 (4H, m, piperazine),
3.58 (1H, dd, PhSO2NHCH), 3.98 (4H, m, piperazine),
4.08 (2H, ABq, ArCH2), 6.62 (1H, t, pyrimidine), 7.02
(2H, d, C6H4), 7.34 (2H, d, C6H4), 7.54 (2H, m,
SO2C6H5), 7.62 (1H, m, SO2C6H5), 7.88 (2H, m,
C6H4), 7.76 (2H, m, C6H5); FAB-HRMS (M+H)+ calcd
25
D
for C27H28N6O5S 549.1920, found 549.1929; ½aꢁ +89°
(c 0.068, MeOH).
5.3.5. Compound 17. To a solution of compound 14
(86 mg, 0.20 mmol) in 1,4-dioxane/H2O (2.0 ml/1.0 ml),
1 N NaOH (1.0 ml) and di-t-butyl dicarbonate (57 mg,
0.26 mmol) were added at room temperature. After
1.5 h stirring, the reaction mixture was concentrated un-
der reduced pressure. The residue was purified by silica
gel column chromatography (CH2Cl2/EtOH/H2O/concd
NH4OH = 8:8:1:1) and Sephadex LH-20 chromatogra-
phy (MeOH) to prepare compound 17 (85 mg, 80%) as
1
a colorless solid; H NMR (400 MHz, CD3OD) d: 1.48
(9H, s, t-Bu), 3.29 (4H, m, piperazine), 3.53 (1H, dd,
CONHCH2CH), 3.57 (4H, m, piperazine), 3.66 (1H,
dd, CONHCH2CH), 3.91 (1H, dd, CONHCH2CH),
6.98 (2H, d, C6H4), 7.44 (2H, m, C6H5), 7.51 (1H, m,
C6H5), 7.69 (2H, d, C6H4), 7.85 (2H, m, C6H5); FAB-
HRMS (M+H)+ calcd for C25H32N4O7S 533.2070,
found 533.2061.
SO2C6H5), 8.35 (2H, d, pyrimidine); TSPMS (neg.) m/
25
z 495 (MꢀH)ꢀ; ½aꢁ +99° (c 0.40, MeOH/CHCl3 = 1:1).
D
5.4.2. Compound 23. Compound 23 was prepared from
compound 20 by the same procedure as employed for
compound 4 as a colorless solid. Yield: 27 mg, 61%;
1H NMR (400 MHz, CD3OD) d: 1.90 (2H, quintet, tet-
rahydropyrimidine), 2.97 (2H, m, ArCH2NHCH2), 3.21
(4H, m, piperazine), 3.34 (4H, t, tetrahydropyrimidine),
3.46 (4H, m, piperazine), 3.60 (1H, dd, PhSO2NHCH),
3.86 (2H, ABq, ArCH2), 6.92 (2H, d, C6H4), 7.26 (2H,
d, C6H4), 7.47 (2H, m, SO2C6H5), 7.54 (1H, m,
5.4. Preparation of compound 23
5.4.1. Compound 20. Compound 6 (200 mg, 0.64 mmol)
was dissolved in CH2Cl2 (5.6 ml), and 1 M toluene solu-
tion of diisobutyl aluminum hydride (1.6 ml) was added
dropwise to the solution under cooling at ꢀ78 °C over a
period of 10 min. The reaction was allowed to proceed
at that temperature for 1 h. MeOH (0.8 ml) was added
thereto at that temperature and heated to room temper-
ature. CH2Cl2 (50 ml) and a saturated aqueous Rochelle
SO2C6H5), 7.82 (2H, m, SO2C6H5); TSPMS m/z 501
25
D
(M+H)+; ½aꢁ +74° (c 1.0, MeOH).

2096
D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
5.5. Preparation of compound 24
C6H4), 7.33 (5H, m, C6H5CH2N), 7.46 (2H, m, SO2C6H5),
7.54 (1H, m, SO2C6H5), 7.75 (2H, m, SO2C6H5); FABMS
25
m/z 591 (M+H)+; ½aꢁ +36° (c 0.5, MeOH).
5.5.1. Compound 21. 50% MeOH/CH2Cl2 (12 ml) was
added to compound 20 (80 mg, 0.16 mmol) to prepare
a solution. A 37% aqueous formaldehyde solution
(131 mg) was added to the solution, and the reaction
solution was then adjusted to pH 3–4 by the addition
of a minor amount of acetic acid. Sodium cyanoborohy-
dride (60 mg) was added thereto, and a reaction was
allowed to proceed at room temperature for 30 min.
The reaction solution was concentrated under reduced
pressure. The residue was purified by preparative thin-
layer chromatography on silica gel (CHCl3/MeOH/con-
cd NH4OH = 90:15:1) to prepare compound 21 (66 mg,
81%) as a colorless solid; 1H NMR (400 MHz, CD3OD/
CDCl3 = 1:1) d: 2.70 (3H, s, NMe), 3.22 (1H, dd,
ArCH2NMeCH2), 3.34 (4H, m, piperazine), 3.64 (1H,
dd, PhSO2NHCH), 3.98 (4H, m, piperazine), 4.13 (2H,
ABq, ArCH2), 6.62 (1H, t, pyrimidine), 7.02 (2H, d,
C6H4), 7.34 (2H, d, C6H4), 7.55 (2H, m, SO2C6H5),
D
5.7. Preparation of compound 27
5.7.1. Compound 26. The compound
9
(40 mg,
0.071 mmol) was dissolved in DMF (0.80 ml). Methyl
iodide (50 mg, 0.35 mmol) and 1,8-diazabicyclo[5.4.0]-
7-undecene (65 mg, 0.43 mmol) were added to the
solution. The reaction was allowed to proceed at room
temperature for 16 h. The reaction solution was concen-
trated under reduced pressure. The residue was extract-
ed with ethyl acetate (8.0 ml), followed by washing with
water and saturated saline in that order. The organic
layer was dried over anhydrous MgSO4 and then con-
centrated under reduced pressure. The residue was puri-
fied by preparative thin-layer chromatography on silica
gel (CHCl3/MeOH/concd NH4OH = 200:10:1) to pre-
pare compound 26 (41 mg, 100%) as a colorless solid;
1H NMR (400 MHz, CDCl3) d: 1.30 (9H, s, t-Bu),
2.89 (3H, s, NMe), 3.39 (4H, m, piperazine), 3.76 (1H,
ddd, CONHCH2CH), 3.88 (1H, ddd, CONHCH2CH),
3.99 (4H, m, piperazine), 4.73 (1H, dd, CONHCH2CH),
6.54 (1H, t, pyrimidine), 6.95 (2H, d, C6H4), 7.50 (2H,
m, C6H5), 7.57 (1H, m, C6H5), 7.77 (2H, d, C6H4),
7.87 (2H, m, C6H5), 8.34 (2H, d, pyrimidine); TSPMS
m/z 581 (M+H)+.
7.62 (1H, m, SO2C6H5), 7.89 (2H, m, SO2C6H5), 8.35
25
D
(2H, d, pyrimidine); TSPMS m/z 511 (M+H)+; ½aꢁ
+77° (c 0.8, CHCl3).
5.5.2. Compound 24. Compound 24 was prepared from
compound 21 by the same procedure as employed for
compound 4 as a colorless solid. Yield: 33 mg, 40%;
1H NMR (400 MHz, CD3OD) d: 1.90 (2H, quintet, tet-
rahydropyrimidine), 2.21 (3H, s, NMe), 2.76 (2H, m,
ArCH2NMeCH2), 3.19 (4H, m, piperazine), 3.35 (4H,
t, tetrahydropyrimidine), 3.48 (4H, m, piperazine), 3.56
(1H, br d, ArCH2), 3.66 (1H, br d, ArCH2), 3.70 (1H,
dd, PhSO2NHCH), 6.88 (2H, d, C6H4), 7.20 (2H, d,
5.7.2. 2-(N-Benzenesulfonyl-N-methylamino)-3-[4-{4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic
acid.
The title compound was prepared from compound 26 by
the same procedure as employed for compound 10. Yield:
25 mg, 33% as a colorless solid; H NMR (400 MHz,
1
C6H4), 7.46 (2H, m, SO2C6H5), 7.53 (1H, m, SO2C6H5),
25
D
7.83 (2H, m, SO2C6H5); TSPMS m/z 515 (M+H)+; ½aꢁ
CD3OD) d: 2.90 (3H, s, NMe), 3.39 (4H, m, piperazine),
3.71 (1H, dd, CONHCH2CH), 3.79 (1H, dd, CON-
HCH2CH), 3.96 (4H, m, piperazine), 6.61 (1H, t, pyrimi-
dine), 7.01 (2H, d, C6H4), 7.38 (2H, m, C6H5), 7.46 (1H,
m, C6H5), 7.70 (2H, d, C6H4), 7.81 (2H, m, C6H5), 8.35
(2H, d, pyrimidine); TSPMS m/z 525 (M+H)+.
+27° (c 1.0, MeOH).
5.6. Preparation of compound 25
5.6.1. Compound 22. Compound 22 was prepared from
compound 20 by the similar procedure as employed
for compound 21 using 5.0 equiv of benzaldehyde in-
stead of the formaldehyde solution as a colorless solid.
Yield: 8.3 mg, 71%; 1H NMR (400 MHz, CDCl3) d:
3.12 (1H, dd, ArCH2NBzlCH2), 3.27 (1H, dd,
ArCH2NBzlCH2), 3.32 (4H, m, piperazine), 3.52 (1H,
dd, PhSO2NHCH), 3.87 (1H, d, ArCH2), 3.92 (1H, d,
ArCH2), 3.99 (1H, d, ArCH2), 3.99 (4H, m, piperazine),
4.03 (1H, d, ArCH2), 6.54 (1H, t, pyrimidine), 6.96 (2H,
d, C6H4), 7.27 (2H, d, C6H4), 7.40 (5H, m, C6H5CH2N),
7.48 (2H, br t, SO2C6H5), 7.55 (1H, br t, SO2C6H5), 7.77
5.7.3. Compound 27. Compound 27 was prepared from 2-
(N-benzenesulfonyl-N-methylamino)-3-[4-{4-(pyrimidin-
2-yl)piperazin-1-yl}benzoylamino]propionic acid by the
same procedure as employed for compound 4 as a color-
less solid. Yield: 13 mg, 53%; 1H NMR (400 MHz,
CD3OD) d: 1.96 (2H, quintet, tetrahydropyrimidine),
2.89 (3H, s, NMe), 3.38–3.43 (8H, m, tetrahydropyrimi-
dine and piperazine), 3.56 (4H, m, piperazine), 3.72 (2H,
d, CONHCH2CH), 4.72 (1H, t, CONHCH2CH), 6.95
(2H, d, C6H4), 7.38 (2H, m, C6H5), 7.46 (1H, m,
C6H5), 7.70 (2H, d, C6H4), 7.85 (2H, m, C6H5); FAB-
HRMS (M+H)+ calcd for C25H32N6O5S 529.2233,
found 529.2223.
(2H, br d, SO2C6H5), 8.35 (2H, d, pyrimidine); FABMS
25
D
(+NaI) m/z 609 (M+Na)+; ½aꢁ +118° (c 0.8, CHCl3).
5.6.2. Compound 25. The title compound was prepared
from compound 22 by the same procedure as employed
for compound 4. Yield: 5.1 mg, 34%; 1H NMR
(400 MHz, CD3OD) d: 1.92 (2H, m, tetrahydropyrimi-
dine), 2.92 (1H, dd, ArCH2NBzlCH2), 3.06 (1H, dd,
ArCH2NBzlCH2), 3.24 (4H, m, piperazine), 3.36 (4H, t,
tetrahydropyrimidine), 3.49 (4H, m, piperazine), 3.54
(1H, dd, PhSO2NHCH), 3.83 (2H, ABq, ArCH2), 3.91
(2H, ABq, ArCH2), 6.92 (2H, d, C6H4), 7.25 (2H, d,
5.8. Preparation of compound 31
5.8.1. Compound 29. DMF (6.0 ml) was added to com-
pound 7 (101 mg, 0.35 mmol) to prepare a solution,
and compound 28 (107 mg, 0.35 mmol) was added to
the solution. Further, 1-hydroxybenzotriazole (63 mg,
0.47 mmol), N-methylmorpholine (0.70 ml), and 1-eth-
yl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2097
ride (121 mg, 0.63 mmol) were added to the mixture,
and the mixture was stirred at room temperature for
24 h. An aqueous NaHCO3 solution (60 ml) was added
to the reaction solution to stop the reaction, and CH2Cl2
(60 ml) was added thereto. The organic layer was then
separated, dried over anhydrous MgSO4, and concen-
trated under reduced pressure. The residue was purified
by column chromatography on silica gel (CH2Cl2/
MeOH/concd NH4OH = 950:50:1) to prepare com-
to room temperature. The mixture was adjusted to pH 8
by the addition of 1 N hydrochloric acid and then con-
centrated under reduced pressure. The residue was puri-
fied by Sephadex LH-20 (MeOH) to prepare compound
33 (12 mg, 43%) as a colorless solid; 1H NMR
(400 MHz, CD3OD) d: 1.40 (9H, s, t-Bu), 1.97 (2H,
quintet, tetrahydropyrimidine), 3.41 (8H, m, piperazine
and tetrahydropyrimidine), 3.54 (4H, m, piperazine),
3.67 (2H, m, CONHCH2CH), 4.20 (1H, m, CON-
HCH2CH), 6.95 (2H, d, C6H4), 7.74 (2H, d, C6H4);
1
pound 29 (53 mg, 27%) as a colorless solid; H NMR
(400 MHz, CDCl3) d: 1.46 (9H, s, t-Bu), 3.40 (4H, m,
piperazine), 3.81 (2H, m, CONHCH2CH), 4.01 (4H,
m, piperazine), 4.45 (1H, m, CONHCH2CH), 5.12
(2H, s, CH2C6H5), 6.57 (1H, t, pyrimidine), 6.92 (2H,
d, C6H4), 7.31 (5H, m, CH2C6H5), 7.69 (2H, d, C6H4),
8.38 (2H, d, pyrimidine); TSPMS m/z 561 (M+H)+.
FAB-HRMS (M+H)+ calcd for C23H34N6O5 475.2669,
25
D
found 475.2669; ½aꢁ +5.7° (c 0.58, MeOH).
5.11. Preparation of compound 34
5.11.1.
Methyl
3-fluoro-4-(piperazin-1-yl)benzoate.
DMSO (1.0 ml) was added to piperazine (344 mg,
4.0 mmol) to prepare a suspension, to which methyl 3,4-
difluorobenzoate (367 mg, 2.1 mmol) was added. The
mixture was stirred at 80 °C for 4.5 h. The temperature
of the system was then returned to room temperature.
The reaction solution was concentrated under reduced
pressure. The residue was purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH/concd NH4OH =
900:100:1) to prepare the title compound (392 mg, 78%)
as a colorless solid; 1H NMR (400 MHz, CDCl3) d: 3.05
(4H, m, piperazine), 3.17 (4H, m, piperazine), 3.88 (3H,
s, CH3), 6.91 (1H, t, C6H3), 7.67 (1H, dd, C6H3), 7.75
(1H, dd, C6H3); EIMS m/z 238.
5.8.2. Compound 30. Compound 30 was prepared from
compound 29 by the same procedure as employed for
compound 10 as a colorless solid. Yield: 13 mg, 17%;
1H NMR (400 MHz, CD3OD) d: 3.29 (4H, t, pipera-
zine), 3.57 (1H, dd, CONHCH2CH), 3.65 (1H, dd,
CONHCH2CH), 3.86 (4H, t, piperazine), 4.17 (1H, dd,
CONHCH2CH), 4.96 (2H, dd, CH2C6H5), 6.52 (1H, t,
pyrimidine), 6.91 (2H, d, C6H4), 7.19 (5H, m,
CH2C6H5), 7.62 (2H, d, C6H4), 8.25 (2H, d, pyrimidine);
25
D
TSPMS m/z 505 (M+H)+; ½aꢁ ꢀ9.6° (c 0.072, MeOH).
5.8.3. Compound 31. Compound 31 was prepared by the
same procedure as employed for compound 4 as a color-
less solid. Yield: 4.9 mg, 65%; 1H NMR (400 MHz,
CD3OD) d: 1.97 (2H, quintet, tetrahydropyrimidine),
3.43 (8H, m, piperazine), 3.56 (4H, m, tetrahydropyrimi-
5.11.2. Methyl 3-fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoate. The title compound was prepared from
methyl 3-fluoro-4-(piperazin-1-yl)benzoate by the same
procedure as employed for compound 6 as a colorless
solid. Yield: 184 mg, 69%; 1H NMR (400 MHz, CDCl3)
d: 3.27 (4H, m, piperazine), 3.89 (3H, s, CH3), 4.00 (4H,
m, piperazine), 6.54 (1H, t, pyrimidine), 6.94 (1H, t,
C6H3), 7.70 (1H, dd, C6H3), 7.77 (1H, dd, C6H3), 8.36
(2H, d, pyrimidine); TSPMS m/z 317 (M+H)+.
dine), 3.81 (3H, m, CONHCH2CH), 6.99 (2H, d, C6H4),
25
D
7.79 (2H, d, C6H4); FABMS m/z 375 (M+H)+; ½aꢁ
ꢀ6.2° (c 0.11, MeOH).
5.9. Preparation of compound 32
5.9.1. Compound 32. Acetone (1.0 ml) and water (1.0 ml)
were added to the compound 31 (26 mg, 0.069 mmol) to
prepare a solution. Potassium carbonate (55 mg) was add-
ed to the solution. Benzyloxycarbonyl chloride (0.040 ml)
was added to the mixture, and the mixture was stirred for
6.5 h and then concentrated under reduced pressure. The
residue was purified by preparative thin-layer chromatog-
raphy on silica gel (EtOH/H2O/concd NH4OH = 6:1:1)
and then purified by Sephadex LH-20 (MeOH/concd
NH4OH = 9:1) to prepare compound 32 (2.3 mg, 6.5%)
5.11.3. 3-Fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}ben-
zoic acid. The title compound was prepared from methyl
3-fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzoate
by the same procedure as employed for compound 7 as a
colorless solid. Yield: 150 mg, 85%; 1H NMR (400 MHz,
CD3OD) d: 3.26 (4H, m, piperazine), 3.97 (4H, m, piper-
azine), 6.62 (1H, t, pyrimidine), 7.10 (1H, t, C6H3), 7.65
(1H, dd, C6H3), 7.77 (1H, dd, C6H3), 8.35 (2H, d, pyrim-
idine); TSPMS m/z 303 (M+H)+.
1
as a colorless solid; H NMR (400 MHz, CD3OD) d:
1.97 (2H, quintet, tetrahydropyrimidine), 3.40 (8H, m,
piperazine and tetrahydropyrimidine), 3.54 (4H, m, piper-
azine), 3.71 (2H, m, CONHCH2CH), 4.24 (1H, m, CON-
HCH2CH), 5.05 (2H, dd, CH2C6H5), 6.94 (2H, d, C6H4),
5.11.4. t-Butyl (2S)-benzyloxycarbonylamino-3-[3-fluoro-
4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzoyl amino]propio-
nate. The title compound was prepared from 3-fluoro-
4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzoic acid and
compound 28 by the same procedure as employed for
compound 29 as a colorless solid. Yield: 420 mg, 76%;
1H NMR (400 MHz, CDCl3) d: 1.46 (9H, s, t-Bu), 3.22
(4H, br t, piperazine), 3.78 (2H, m, CONHCH2CH),
4.00 (4H, br t, piperazine), 4.45 (1H, m, CONHCH2CH),
5.12 (2H, d, CH2C6H5), 6.53 (1H, t, pyrimidine), 6.92 (1H,
t, C6H3), 7.32 (5H, m, CH2C6H5), 7.45 (1H, d, C6H3), 7.50
7.28 (5H, m, CH2C6H5), 7.71 (2H, d, C6H4); TSPMS m/z
25
509 (M+H)+; ½aꢁ ꢀ3.4° (c 0.052, MeOH).
D
5.10. Preparation of compound 33
5.10.1. Compound 33. A 1 N aqueous NaOH (2.0 ml)
was added to the compound 31 (22 mg, 0.059 mmol),
and an excess of di-t-butyl dicarbonate was added to
the solution. The mixture was stirred for 2 h and cooled
(1H, d, C6H3), 8.34 (2H, d, pyrimidine); TSPMS m/z 579
25
D
(M+H)+; ½aꢁ ꢀ12° (c 0.98, CH2Cl2).

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D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
5.11.5. t-Butyl (2S)-ethylamino-3-[3-fluoro-4-{4-(pyrimi-
din-2-yl)piperazin-1-yl}benzoylamino]propionate. THF
(60 ml) and EtOH (30 ml) were added to t-butyl (2S)-
benzyloxycarbonylamino-3-[3-fluoro-4-{4-(pyrimidin-2-
yl)piperazin-1-yl}benzoylamino]propionate to (210 mg,
0.36 mmol) to prepare a solution, and 10% Pd/C
(210 mg) was added to the solution. The mixture was
stirred vigorously in a hydrogen atmosphere at room
temperature for 24 h. The insolubles were filtered and
then washed twice with THF. The filtrate was com-
bined with the washings, followed by concentration un-
der reduced pressure. The residue was purified by
preparative thin-layer chromatography on silica gel
(CH2Cl2/MeOH = 9:1) to prepare the title compound
(72 mg), N-methylmorpholine (0.18 ml), and 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride
(148 mg) were added thereto, and the mixture was stir-
red at room temperature for 24 h. An aqueous NaHCO3
solution (30 ml) was added to stop the reaction. Ethyl
acetate (300 ml) and water (100 ml) were added thereto.
The organic layer was then separated, dried over anhy-
drous MgSO4, and concentrated under reduced pres-
sure. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH = 15:1)
to prepare the title compound (110 mg, 84%) as a color-
1
less solid; H NMR (400 MHz, CDCl3) d: 1.27 (3H, t,
Et), 2.64 (2H, t, CONHCH2CH2), 3.38 (4H, br t, piper-
azine), 3.71 (2H, q, CONHCH2CH2), 3.99 (4H, m,
piperazine), 4.17 (2H, q, Et), 6.54 (1H, t, pyrimidine),
6.93 (2H, d, C6H4), 7.70 (2H, d, C6H4), 8.34 (2H, d,
pyrimidine); TSPMS m/z 384 (M+H)+.
1
(56 mg, 33%) as a colorless solid; H NMR (400 MHz,
CDCl3) d: 1.12 (3H, t, Et), 1.47 (9H, s, t-Bu), 2.59 (1H,
dq, Et), 2.71 (1H, dq, Et), 3.22 (4H, br t, piperazine),
3.34 (1H, dd, CONHCH2CH), 3.44 (1H, ddd, CON-
HCH2CH), 3.77 (1H, ddd, CONHCH2CH), 4.00 (4H,
br t, piperazine), 6.53 (1H, t, pyrimidine), 6.95 (1H, t,
5.12.2. Compound 35. MeOH (0.20 ml) and THF
(0.60 ml) were added to ethyl 3-[4-{4-(pyrimidin-2-
C6H3), 7.50 (2H, m, C6H3), 8.34 (2H, d, pyrimidine);
yl)piperazin-1-yl}benzoylamino]propionate
(40 mg,
25
FABMS m/z 473 (M+H)+; ½aꢁ +7.3° (c 1.0, CH2Cl2).
0.10 mmol) to prepare a solution, and a 1 N aqueous
NaOH (0.20 ml) was added to the solution. The mixture
was stirred at 60 °C for 5.0 h and cooled to room tem-
perature, and the system was adjusted to pH 7 by the
addition of 1 N hydrochloric acid. The resultant precip-
itate was collected by filtration on a glass filter and then
dried to prepare compound 35 (21 mg, 57%) as a color-
D
5.11.6. (2S)-Ethylamino-3-[3-fluoro-4-{4-(pyrimidin-2-yl)pip-
erazin-1-yl}benzoylamino]propionic acid. The title compound
was prepared from t-butyl (2S)-ethylamino-3-[3-fluoro-4-
{4-(pyrimidin-2-yl)piperazin-1- yl}benzoylamino]propio-
nate by the same procedure as employed for compound 10
1
1
as a pale yellow solid. Yield: 64 mg; H NMR (400 MHz,
less solid; H NMR (400 MHz, CD3OD) d: 2.60 (2H, t,
CDCl3) d: 1.36 (3H, t, Et), 3.25 (6H, m, piperazine and
Et), 3.83 (1H, dd, CONHCH2CH), 3.98 (4H, br t, pipera-
zine), 4.07 (1H, dd, CONHCH2CH), 4.20 (1H, dd, CON-
CONHCH2CH2), 3.38 (4H, br t, piperazine), 3.60 (2H,
t, CONHCH2CH2), 3.95 (4H, m, piperazine), 6.62
(1H, t, pyrimidine), 7.02 (2H, d, C6H4), 7.73 (2H, d,
C6H4), 8.34 (2H, d, pyrimidine); FAB-HRMS (M+H)+
calcd for C18H21N5O3 356.1723, found 356.1710.
HCH2CH), 6.66 (1H, t, pyrimidine), 7.11 (1H, t, C6H3),
25
7.61 (1H, dd, C6H3); TSPMS m/z 417 (M+H)+; ½aꢁ +1.9°
D
(c 0.84, MeOH).
5.13. Preparation of compound 36
5.11.7. Compound 34. 1,4-Dioxane (10 ml) and water
(5.0 ml) were added to (2S)-ethylamino-3-[3-fluoro-4-
{4-(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propi-
onic acid (64 mg) to prepare a solution, and 10% Pd/C
(52 mg) was added to the solution. The mixture was vig-
orously stirred in a hydrogen atmosphere at room tem-
perature for 8.0 h. The insolubles were filtered and then
washed twice with 1,4-dioxane. The filtrate was com-
bined with the washings, followed by concentration un-
der reduced pressure, to prepare compound 34 (50 mg,
99%) as a colorless solid; 1H NMR (400 MHz, CD3OD)
d: 1.29 (3H, t, Et), 1.97 (2H, quintet, tetrahydropyrimi-
dine), 3.06 (2H, q, Et), 3.25 (4H, m, piperazine), 3.42
(4H, br t, tetrahydropyrimidine), 3.55 (4H, br t, pipera-
zine), 3.60 (1H, dd, CONHCH2CH), 3.74 (1H, dd, CON-
HCH2CH), 3.84 (1H, dd, CONHCH2CH), 7.08 (1H, t,
C6H3), 7.60 (1H, d, C6H3), 7.64 (1H, d, C6H3); FAB-
5.13.1. Ethyl (3S)-[4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoylamino]pent-4-ynate. DMF (3.0 ml) was added
to compound 7 (24 mg, 0.084 mmol) to prepare a solu-
tion, and ethyl 3-amino (3S)-ethynyl-propionate hydro-
chloride (21 mg) was added to the solution. Further,
1-hydroxybenzotriazole (23 mg), N-methylmorpholine
(0.060 ml), and 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (33 mg) were added to the mix-
ture, and the mixture was stirred at room temperature for
23 h. An aqueous NaHCO3 solution (30 ml) was added to
the mixture to stop the reaction, and CH2Cl2 (30 ml) was
added thereto. The organic layer was then separated,
dried over anhydrous MgSO4, and concentrated under re-
duced pressure. The residue was purified by column chro-
matography on silica gel (n-hexane/ethyl acetate = 3:7) to
prepare the title compound (19 mg, 55%) as a colorless
1
HRMS (M+H)+ calcd for C20H29N6O3F 421.2363, found
solid; H NMR (400 MHz, CDCl3) d: 1.29 (3H, t, Et),
25
D
421.2363; ½aꢁ +23° (c 1.0, MeOH).
2.30 (1H, d, C (triple bond) CH), 2.78 (1H, dd, CON-
HCHCH2), 2.86 (1H, dd, CONHCHCH2), 3.39 (4H, m,
piperazine), 3.99 (4H, m, piperazine), 4.22 (2H, q, Et),
5.32 (1H, m, CONHCHCH2), 6.54 (1H, t, pyrimidine),
6.94 (2H, d, C6H4), 7.73 (2H, d, C6H4), 8.34 (2H, d, pyrim-
idine); TSPMS m/z 408 (M+H)+.
5.12. Preparation of compound 35
5.12.1. Ethyl 3-[4-{4-(pyrimidin-2-yl)piperazin-1-yl}ben-
zoylamino]propionate. DMF (10 ml) was added to com-
pound 7 (97 mg, 0.34 mmol) to prepare a solution, and
b-alanine ethyl ester hydrochloride (176 mg) was added
to the solution. Further, 1-hydroxybenzotriazole
5.13.2. Compound 36. The title compound was prepared
from ethyl (3S)-[4-{4-(pyrimidin-2-yl)piperazin-1-yl}ben-

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2099
zoylamino]pent-4-ynate by the same procedure as em-
ployed for compound 35 as a colorless solid. Yield:
4.5 mg, 47%; 1H NMR (400 MHz, CD3OD) d: 2.44
(1H, d, C (triple bond) CH), 2.51 (1H, d, CON-
HCHCH2), 2.53 (1H, d, CONHCHCH2), 3.31 (4H, m,
piperazine), 3.86 (4H, m, piperazine), 5.02 (1H, dt, CON-
HCHCH2), 6.51 (1H, t, pyrimidine), 6.92 (2H, d, C6H4),
7.66 (2H, d, C6H4), 8.25 (2H, d, pyrimidine); FAB-
HRMS (M+H)+ calcd for C20H21N5O3 380.1723, found
370.1718.
prepare a suspension, to which methyl 2,4-difluorobenzo-
ate (368 mg, 2.1 mmol) was added. The mixture was
stirred at 80 °C for 1 h and cooled to room temperature.
The reaction solution was concentrated under reduced
pressure. The residue was purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH/concd NH4OH =
900:100:1) to prepare the title compound (274 mg, 55%)
as a colorless solid; 1H NMR (400 MHz, CDCl3) d:
3.00 (4H, m, piperazine), 3.29 (4H, m, piperazine), 3.88
(3H, s, CH3), 6.51 (1H, dd, C6H3), 6.63 (1H, dd, C6H3),
7.82 (1H, t, C6H3); EIMS m/z 238.
5.14. Preparation of compound 37
5.15.2. Methyl 2-fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoate. The title compound was prepared from
methyl 2-fluoro-4-(piperazin-1-yl)benzoate by the same
procedure as employed for compound 6 as a colorless
solid. Yield: 120 mg, 50%; 1H NMR (400 MHz, CDCl3)
d: 3.44 (4H, br t, piperazine), 3.91 (3H, s, CH3), 3.98
(4H, br t, piperazine), 6.55 (1H, dd, C6H3), 6.55 (1H,
t, pyrimidine), 6.66 (1H, dd, C6H3), 7.85 (1H, t,
C6H3), 8.35 (2H, d, pyrimidine); TSPMS m/z 317
(M+H)+.
5.14.1. t-Butyl (2S)-benzenesulfonylamino-3-[3-fluoro-
4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propi-
onate. The title compound was prepared from 3-fluoro-
4-{4-(pyrimidin-2-yl)-piperazin-1-yl}benzoic acid by the
same procedure as employed for compound 9 as a color-
less solid. Yield: 218 mg, 76%; 1H NMR (400 MHz,
CDCl3) d: 1.29 (9H, s, t-Bu), 3.23 (4H, br t, piperazine),
3.54 (1H, m, CONHCH2CH), 3.90 (2H, m, CON-
HCH2CH), 4.01 (4H, br t, piperazine), 6.53 (1H, t,
pyrimidine), 6.95 (1H, t, C6H3), 7.53 (5H, m, C6H3
and C6H5), 7.85 (2H, m, C6H5), 8.33 (2H, d, pyrimi-
dine); TSPMS m/z 585 (M+H)+.
5.15.3. 2-Fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}ben-
zoic acid. The title compound was prepared from
methyl
2-fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}-
5.14.2. (2S)-Benzenesulfonylamino-3-[3-fluoro-4-{4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
CH2Cl2 (10 ml) was added to t-butyl (2S)-benzenesulfo-
nylamino-3-[3-fluoro-4-{4-(pyrimidin -2-yl)piperazin-1-
yl}benzoylamino]propionate (212 mg, 0.36 mmol) to
prepare a solution. Trifluoroacetic acid (3.0 ml) was add-
ed at room temperature to the solution. The mixture was
stirred at that temperature for 7 h before the reaction
solution was concentrated under reduced pressure to pre-
pare trifluoroacetate of the title compound (258 mg) as a
pale yellow solid; 1H NMR (400 MHz, CD3OD) (as triflu-
oroacetate) d: 3.28 (4H, m, piperazine), 3.47 (1H, dd,
CONHCH2CH), 3.71 (1H, dd, CONHCH2CH), 4.00
(4H, br t, piperazine), 4.18 (1H, dd, CONHCH2CH),
6.77 (1H, t, pyrimidine), 7.09 (1H, t, C6H3), 7.45 (4H,
m, C6H3 and C6H5), 7.53 (1H, dd, C6H3), 7.81 (2H, m,
benzoate by the same procedure as employed for
compound 7 as a colorless solid. Yield: 60 mg, 54%;
1H NMR (400 MHz, CD3OD) d: 3.47 (4H, m, piper-
azine), 4.11 (4H, m, piperazine), 6.62 (1H, t, pyrimi-
dine), 6.69 (1H, dd, C6H3), 6.80 (1H, dd, C6H3), 7.81
(1H, t, C6H3), 8.35 (2H, d, pyrimidine); TSPMS m/z
303 (M+H)+.
5.15.4. t-Butyl (2S)-benzenesulfonylamino-3-[2-fluoro-4-
{4-(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propio-
nate. The title compound was prepared from 2-fluoro-4-
{4-(pyrimidin-2-yl)piperazin-1-yl}benzoic acid by the
same procedure as employed for compound 9 as a
colorless solid. Yield: 64 mg, 58%; 1H NMR
(400 MHz, CDCl3) d: 1.29 (9H, s, t-Bu), 3.42 (4H, br
t, piperazine), 3.70 (1H, m, CONHCH2CH), 3.77 (1H,
m, CONHCH2CH), 4.11 (5H, m, piperazine and
CONHCH2CH), 6.54 (1H, dd, C6H3), 6.55 (1H, t,
pyrimidine), 6.74 (1H, dd, C6H3), 7.45 (2H, m, C6H5),
7.52 (1H, m, C6H5), 7.83 (2H, m, C6H5), 7.95 (1H, t,
C6H5), 8.43 (2H, d, pyrimidine); TSPMS m/z 529
25
(M+H)+; ½aꢁ +27° (c 1.0, MeOH).
D
5.14.3. Compound 37. Compound 37 was prepared from
(2S)-benzenesulfonylamino-3-[3-fluoro-4-{4-(pyrimidin-
2-yl)piperazin-1- yl}benzoylamino]propionic acid the
same procedure as employed for compound 4 as a color-
less solid. Yield: 44 mg, 23%; 1H NMR (400 MHz,
CD3OD) d: 1.97 (2H, quintet, tetrahydropyrimidine),
3.25 (4H, br t, piperazine), 3.42 (4H, br t, tetrahydropyr-
imidine), 3.55 (5H, m, piperazine and CONHCH2CH),
3.67 (1H, dd, CONHCH2CH), 3.77 (1H, dd, CON-
HCH2CH), 7.08 (1H, t, C6H3), 7.49 (3H, m, C6H5), 7.55
(1H, dd, C6H3), 7.60 (1H, dd, C6H3), 7.86 (2H, m,
C6H3), 8.35 (2H, d, pyrimidine); FABMS m/z 585
25
(M+H)+; ½aꢁ +40° (c 1.0, CH2Cl2).
D
5.15.5. (2S)-Benzenesulfonylamino-3-[2-fluoro-4-{4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
CH2Cl2 (3.0 ml) was added to t-butyl (2S)-benzenesulfo-
nylamino-3-[2-fluoro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoylamino]propionate (61 mg, 0.10 mmol) to
prepare a solution. Trifluoroacetic acid (1.0 ml) was
added at room temperature to the solution. The mixture
was stirred at that temperature for 2.5 h before the reac-
tion solution was concentrated under reduced pressure
to prepare trifluoroacetate of the title compound
(69 mg) as a pale yellow solid; 1H NMR (400 MHz,
CD3OD) (as trifluoroacetate) d: 3.46 (1H, dd, CON-
HCH2CH), 3.50 (4H, br t, piperazine), 3.77 (1H, dd,
CONHCH2CH), 3.99 (4H, br t, piperazine), 4.17 (1H, dd,
C6H5); FAB-HRMS (M+H)+ calcd for C24H29N6O5SF
25
D
533.1982, found 533.1981; ½aꢁ +24° (c 1.0, MeOH).
5.15. Preparation of compound 38
5.15.1. Methyl 2-fluoro-4-(piperazin-1-yl)benzoate. DMSO
(1.0 ml) was added to piperazine (544 mg, 6.3 mmol) to

2100
D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
CONHCH2CH), 6.71 (1H, dd, C6H3), 6.73 (1H, t,
pyrimidine), 6.84 (1H, dd, C6H3), 7.44 (3H, m, C6H5),
7.70 (1H, t, C6H3), 7.82 (2H, m, C6H5), 8.43 (2H, d,
din-2-yl)-piperazin-1-yl}benzoic acid by the same proce-
dure as employed for compound 9 as a colorless solid.
Yield: 368 mg, 90%; H NMR (400 MHz, CDCl3) d: 1.29
(9H, s, t-Bu), 3.18 (4H, br t, piperazine), 3.57 (1H, m,
CONHCH2CH), 3.90 (2H, m, CONHCH2CH), 4.02
(4H, br t, piperazine), 6.53 (1H, t, pyrimidine), 7.06 (1H,
d, C6H3), 7.51 (2H, m, C6H5), 7.58 (1H, m, C6H5), 7.66
1
25
pyrimidine); TSPMS m/z 529 (M+H)+; ½aꢁ +28° (c
D
1.0, MeOH).
5.15.6. Compound 38. The title compound was prepared
from (2S)-benzenesulfonylamino-3-[2-fluoro-4-{4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid
by the same procedure as employed for compound 4
as a colorless solid. Yield: 22 mg, 34%; 1H NMR
(400 MHz, CD3OD) (as hydrochloride) d: 1.97 (2H,
quintet, tetrahydropyrimidine), 3.43 (5H, dd, tetrahy-
dropyrimidine and CONHCH2CH), 3.49 (4H, m, piper-
azine), 3.56 (4H, m, piperazine), 3.78 (1H, dd,
CONHCH2CH), 4.18 (1H, dd, CONHCH2CH), 6.69
(1H, dd, C6H3), 6.80 (1H, dd, C6H3), 7.44 (3H, m,
C6H5), 7.71 (1H, t, C6H3), 7.82 (2H, m, C6H5); FAB-
(1H, dd, C6H3), 7.86 (3H, d, C6H5 and C6H3), 8.53 (2H,
25
d, pyrimidine); TSPMS m/z 601 (M+H)+; ½aꢁ +58° (c
D
1.0, CH2Cl2).
5.16.5. (2S)-Benzenesulfonylamino-3-[3-chloro-4-{4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
CH2Cl2 (25 ml) was added to t-butyl (2S)-benzenesulfo-
nylamino-3-[3-chloro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoylamino]propionate (308 mg, 0.51 mmol) to
prepare a solution. Trifluoroacetic acid (10 ml) was
added at room temperature to the solution. The mix-
ture was stirred at that temperature for 5.0 h before
the reaction solution was concentrated under reduced
pressure to prepare trifluoroacetate of the title com-
pound (340 mg) as a pale yellow solid; 1H NMR
(400 MHz, CD3OD) (as trifluoroacetate) d: 3.22 (4H,
br t, piperazine), 3.47 (1H, dd, CONHCH2CH), 3.71
(1H, dd, CONHCH2CH), 4.00 (4H, br t, piperazine),
4.19 (1H, dd, CONHCH2CH), 6.74 (1H, t, pyrimidine),
7.18 (1H, d, C6H3), 7.44 (3H, m, C6H5), 7.68 (1H, dd,
HRMS (M+H)+ calcd for C24H29N6O5SF 533.1982,
25
D
found 533.1989; ½aꢁ +29° (c 0.54, MeOH).
5.16. Preparation of compound 39
5.16.1.
Methyl
3-chloro-4-(piperazin-1-yl)benzoate.
DMSO (15 ml) was added to piperazine (4.5 g, 52 mmol)
to prepare a suspension, to which methyl 3-chloro-4-flu-
orobenzoate (1.1 g, 5.8 mmol) was added. The mixture
was stirred at 80 °C for 5.0 h. The temperature of the
system was then returned to room temperature. Ethyl
acetate (1000 ml) and water (500 ml) were added there-
to. The organic layer was separated, dried over anhy-
drous MgSO4, and concentrated under reduced
pressure. The residue was purified by column chroma-
tography on silica gel (CH2Cl2/MeOH = 5:1) to prepare
the title compound (905 mg, 61%) as a colorless solid;
1H NMR (400 MHz, CD3OD) d: 2.89 (4H, m, pipera-
zine), 3.01 (4H, m, piperazine), 3.78 (3H, s, CH3), 7.05
(1H, d, C6H3), 7.79 (1H, dd, C6H3), 7.86 (1H, d,
C6H3); TSPMS m/z 255 (M+H)+.
C6H3), 7.79 (1H, d, C6H3), 7.81 (2H, m, C6H5), 8.43
25
D
(2H, d, pyrimidine); TSPMS m/z 545 (M+H)+; ½aꢁ
+12° (c 0.95, MeOH).
5.16.6. Compound 39. Compound 39 was prepared from
(2S)-benzenesulfonylamino-3-[3-chloro-4-{4-(pyrimidin-
2-yl)piperazin-1-yl}benzoylamino]propionic acid by the
same procedure as employed for compound 4 as a color-
less solid. Yield: 69 mg, 85%; 1H NMR (400 MHz,
CD3OD) d: 1.97 (2H, quintet, tetrahydropyrimidine),
3.17 (4H, br t, piperazine), 3.42 (4H, br t, tetrahydropyr-
imidine), 3.55 (5H, dd, piperazine and CONHCH2CH),
3.70 (1H, m, CONHCH2CH), 3.76 (1H, m, CON-
HCH2CH), 7.16 (1H, d, C6H3), 7.47 (2H, m, C6H5),
7.53 (1H, m, C6H5), 7.75 (1H, dd, C6H3), 7.85 (3H, d,
5.16.2. Methyl 3-chloro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoate. The title compound was prepared from
methyl 3-chloro-4-(piperazin-1-yl)benzoate by the same
procedure as employed for compound 6 as a colorless sol-
id. Yield: 1.0 g, 86%; 1H NMR (400 MHz, CDCl3) d: 3.20
(4H, br t, piperazine), 3.90 (3H, s, CH3), 4.01 (4H, br t,
piperazine), 6.53 (1H, t, pyrimidine), 7.04 (1H, d, C6H3),
7.90 (1H, dd, C6H3), 8.05 (1H, d, C6H3), 8.34 (2H, d,
pyrimidine); TSPMS m/z 333 (M+H)+.
C6H5 and C6H3); FAB-HRMS (M+H)+ calcd for
25
D
C24H29N6O5SCl 549.1687, found 549.1696; ½aꢁ +66°
(c 0.53, MeOH).
5.17. Preparation of compound 40
5.17.1.
Methyl
2-chloro-4-(piperazin-1-yl)benzoate.
DMSO (15 ml) was added to piperazine (4.5 g,
52 mmol) to prepare a suspension, to which methyl 2-
chloro-4-fluorobenzoate (3.3 g, 17 mmol) was added.
The mixture was stirred at 80 °C for 1.0 h. The temper-
ature of the system was then returned to room temper-
ature. Ethyl acetate (1000 ml) and water (500 ml) were
added thereto. The organic layer was separated, dried
over anhydrous MgSO4, and concentrated under re-
duced pressure to prepare the title compound (3.7 g,
83%) as a colorless solid; 1H NMR (400 MHz,
CD3OD) d: 2.83 (4H, m, piperazine), 3.18 (4H, m,
piperazine), 3.73 (3H, s, CH3), 6.76 (1H, dd, C6H3),
6.85 (1H, d, C6H3), 7.70 (1H, d, C6H3); TSPMS m/z
255 (M+H)+.
5.16.3. 3-Chloro-4-{4-(pyrimidin-2-yl)-piperazin-1-yl}ben-
zoic acid. The title compound was prepared from methyl
3-chloro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzoate by
the same procedure as employed for compound 7 as a
colorless solid. Yield: 878 mg, 92%; 1H NMR
(400 MHz, CD3OD) d: 3.18 (4H, br t, piperazine), 3.97
(4H, br t, piperazine), 6.61 (1H, t, pyrimidine), 7.18
(1H, d, C6H3), 7.90 (1H, dd, C6H3), 7.98 (1H, d, C6H3),
8.34 (2H, d, pyrimidine); TSPMS m/z 319 (M+H)+.
5.16.4. t-Butyl (2S)-benzenesulfonylamino-3-[3-chloro-4-{4-
(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propionate. The
title compound was prepared from 3-chloro-4-{4-(pyrimi-

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2101
5.17.2. Methyl 2-chloro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoate. The title compound was from methyl 2-
chloro-4-(piperazin-1-yl)benzoate prepared by the same
procedure as employed for compound 6 as a colorless
3.68 (1H, dd, CONHCH2CH), 3.75 (1H dd, CON-
HCH2CH), 6.90 (1H, dd, C6H3), 6.96 (1H, d, C6H3),
7.55 (4H, d, C6H5 and C6H3), 7.87 (2H, m, C6H5); FAB-
HRMS (M+H)+ calcd for C24H29N6O5SCl 549.1687,
25
D
1
solid. Yield: 2.7 g, 90%; H NMR (400 MHz, CDCl3)
found 549.1695; ½aꢁ +68° (c 0.37, MeOH).
d: 3.42 (4H, br t, piperazine), 3.88 (3H, s, CH3), 3.98
(4H, br t, piperazine), 6.55 (1H, t, pyrimidine), 6.77
(1H, dd, C6H3), 6.91 (1H, d, C6H3), 7.86 (1H, d,
C6H3), 8.35 (2H, d, pyrimidine); TSPMS m/z 333
(M+H)+.
5.18. Preparation of compound 41
5.18.1. 3-Nitro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzo-
ic acid. DMSO (10 ml) was added to 1-(2-pyrimidyl)pi-
perazine dihydrochloride (2.3 g, 9.7 mmol) to prepare
5.17.3. 2-Chloro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}ben-
zoic acid. The title compound was prepared from methyl
2-chloro-4-{4-(pyrimidin-2-yl)piperazin-1-yl}benzoate by
the same procedure as employed for compound 7 as a col-
orless solid. Yield: 1.4 g, 98%; 1H NMR (400 MHz,
DMSO-d6) d: 3.42 (4H, br t, piperazine), 3.85 (4H, br t,
piperazine), 6.66 (1H, t, pyrimidine), 6.94 (1H, dd,
C6H3), 6.99 (1H, d, C6H3), 7.77 (1H, d, C6H3), 8.39 (2H,
d, pyrimidine); TSPMS m/z 319 (M+H)+.
a suspension. 4-Fluoro-3-nitrobenzoic acid (1.9 g,
10 mmol) was added to the suspension. N,N-Diisopro-
pylethylamine (1.0 ml) was added thereto. The mixture
was stirred at 120 °C for 17 h and cooled to room tem-
perature. Ethyl acetate (3000 ml) and water (1000 ml)
were added thereto. The organic layer was separated,
dried over anhydrous MgSO4, and concentrated under
reduced pressure to prepare the title compound
1
(570 mg, 18%) as a yellow solid; H NMR (400 MHz,
CD3OD) d: 3.27 (4H, br t, piperazine), 3.96 (4H, br t,
piperazine), 6.63 (1H, t, pyrimidine), 7.32 (1H, d,
C6H3), 8.11 (1H, dd, C6H3), 8.35 (2H, d, pyrimidine),
8.39 (1H, d, C6H3); FABMS m/z 330 (M+H)+.
5.17.4. t-Butyl (2S)-benzenesulfonylamino-3-[2-chloro-4-
{4-(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propionate.
The title compound was prepared from 2-chloro-4-{4-
(pyrimidin-2-yl)piperazin-1-yl}benzoic acid by the same
procedure as employed for compound 9 as a colorless sol-
5.18.2. t-Butyl (2S)-benzenesulfonylamino-3-[3-nitro-4-{4-
(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propionate.
The title compound was prepared from 3-nitro-4-{4-
(pyrimidin-2-yl)piperazin-1-yl}benzoic acid by the
same procedure as employed for compound 9 as a yel-
low solid. Yield: 493 mg, 89%; 1H NMR (400 MHz,
CDCl3) d: 1.30 (9H, s, t-Bu), 3.26 (4H, br t, piperazine),
3.53 (1H, m, CONHCH2CH), 3.91 (1H, m, CON-
HCH2CH), 3.96 (1H, d, CONHCH2CH), 4.01 (4H, br
t, piperazine), 6.55 (1H, t, pyrimidine), 7.15 (1H, d,
C6H3), 7.51 (2H, m, C6H5), 7.59 (1H, m, C6H5), 7.86
(2H, m, C6H5), 7.93 (1H, dd, C6H3), 8.31 (1H, d,
1
id. Yield: 355 mg, 92%; H NMR (400 MHz, CDCl3) d:
1.29 (9H, s, t-Bu), 3.37 (4H, br t, piperazine), 3.73 (1H,
dd, CONHCH2CH), 3.81 (1H, ddd, CONHCH2CH),
3.98 (5H, m, piperazine and CONHCH2CH), 6.55 (1H,
t, pyrimidine), 6.85 (2H, m, C6H3), 7.49 (2H, m, C6H5),
7.57 (1H, m, C6H5), 7.73 (1H, d, C6H3), 7.86 (2H, m,
C6H5), 8.35 (2H, d, pyrimidine); TSPMS m/z 601
25
(M+H)+; ½aꢁ +38° (c 1.0, CH2Cl2).
D
5.17.5. (2S)-Benzenesulfonylamino-3-[2-chloro-4-{4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
CH2Cl2 (25 ml) was added to t-butyl (2S)-benzenesulfo-
nylamino-3-[2-chloro-4-{4-(pyrimidin-2-yl)piperazin-1-
yl}benzoylamino]propionate (289 mg, 0.48 mmol) to
prepare a solution. Trifluoroacetic acid (10 ml) was
added at room temperature to the solution. The mix-
ture was stirred at that temperature for 6.0 h before
the reaction solution was concentrated under reduced
pressure to prepare trifluoroacetate of the title com-
pound (321 mg) as a pale yellow solid; 1H NMR
(400 MHz, CD3OD)(as trifluoroacetate) d: 3.41 (4H,
br t, piperazine), 3.48 (1H, dd, CONHCH2CH), 3.71
(1H, dd, CONHCH2CH), 3.97 (4H, m, piperazine),
4.18 (1H, dd, CONHCH2CH), 6.72 (1H, t, pyrimidine),
6.94 (1H, dd, C6H3), 7.00 (1H, d, C6H3), 7.45 (1H, d,
C6H3), 7.50 (2H, m, C6H5), 7.57 (1H, m, C6H5), 7.86
C6H3), 8.34 (2H, d, pyrimidine); TSPMS m/z 612
25
(M+H)+; ½aꢁ +58° (c 1.0, CH2Cl2).
D
5.18.3. Compound 41. CH2Cl2 (25 ml) was added to t-bu-
tyl (2S)-benzenesulfonylamino-3-[3-nitro-4-{4-(pyrimi-
din-2-yl)piperazin-1-yl}benzoylamino]propionate (233 mg,
0.38 mmol) to prepare a solution, and trifluoroacetic acid
(10 ml) was added at room temperature to the solution.
The mixture was stirred at that temperature for 5.5 h
before the reaction solution was concentrated under re-
duced pressure to prepare trifluoroacetate of compound
41 (259 mg, 76%) as a yellow solid; 1H NMR (400 MHz,
CD3OD) (as trifluoroacetate) d: 3.29 (4H, br t, pipera-
zine), 3.47 (1H, dd, CONHCH2CH), 3.73 (1H, dd, CON-
HCH2CH), 3.98 (4H, br t, piperazine), 4.20 (1H, d,
CONHCH2CH), 6.72 (1H, t, pyrimidine), 7.32 (1H, d,
C6H3), 7.43 (3H, m, C6H5), 7.81 (2H, m, C6H5), 7.92
(1H, dd, C6H3), 8.18 (1H, d, C6H3), 8.41 (2H,
(2H, m, C6H5), 8.41 (2H, d, pyrimidine); TSPMS m/z
25
545 (M+H)+; ½aꢁ +18° (c 0.56, DMSO).
D
5.17.6. Compound 40. Compound 40 was prepared from
(2S)-benzenesulfonylamino-3-[2-chloro-4-{4-(pyrimidin-
2-yl)piperazin-1- yl}benzoylamino]propionic acid by the
same procedure as employed for compound 4 as a
d, pyrimidine); FAB-HRMS (M+H)+ calcd for C24H25
25
D
N7O7S 556.1614, found 556.1607; ½aꢁ +25° (c 1.1,
MeOH).
1
colorless solid. Yield: 58 mg, 69%; H NMR (400 MHz,
5.19. Preparation of compound 42
CD3OD) d: 1.97 (2H, quintet, tetrahydropyrimidine),
3.41 (8H, br t, piperazine and tetrahydropyrimidine),
3.54 (4H, m, piperazine), 3.59 (1H, dd, CONHCH2CH),
5.19.1. Ethyl 4-{(3R)-methyl-(piperazin-1-yl)}benzoate.
DMSO (2.0 ml) was added to (2R)-methylpiperazine

2102
D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
1
solid. Yield: 30 mg, 77%; H NMR (400 MHz, CD3OD)
(295 mg, 2.9 mmol) to prepare a suspension, to which
ethyl 4-fluorobenzoate (420 mg, 2.5 mmol) was added.
The mixture was stirred at 120 °C for 6.0 h. The temper-
ature of the system was then returned to room tempera-
ture, and the reaction solution was concentrated under
reduced pressure. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH/concd
NH4OH = 900:100:1) to prepare the title compound
(410 mg, 66%) as a pale yellow solid; 1H NMR
(400 MHz, CD3OD) d: 0.90 (3H, d, CH3), 1.26 (3H, t,
Et), 2.36 (1H, m, piperazine), 2.70 (1H, m, piperazine),
2.81 (2H, m, piperazine), 2.98 (1H, m, piperazine),
3.66 (2H, m, piperazine), 4.20 (2H, q, Et), 6.86 (2H, d,
C6H4), 7.77 (2H, d, C6H4); FABMS m/z 249 (M+H)+.
d: 1.21 (3H, d, CH3), 2.88 (1H, dt, piperazine), 3.08 (1H,
dd, piperazine), 3.36 (1H, ddd, piperazine), 3.47 (1H, dd,
CONHCH2CH), 3.56 (1H, dd, CONHCH2CH), 3.63
(1H, dd, CONHCH2CH), 3.68 (1H, m, piperazine), 3.76
(1H, m, piperazine), 4.42 (1H, dt, piperazine), 4.79 (1H,
m, piperazine), 6.51 (1H, t, pyrimidine), 6.89 (2H, d,
C6H4), 7.35 (2H, m, C6H5), 7.43 (1H, m, C6H5), 7.63
(2H, d, C6H4), 7.76 (2H, m, C6H5), 8.25 (2H, d, pyrimi-
25
dine); TSPMS m/z 525 (M+H)+; ½aꢁ +55° (c 0.073,
D
MeOH).
5.19.6. Compound 42. Compound 42 was prepared from
(2S)-benzenesulfonylamino-3-[4-{(3R)-methyl-4-(pyrim-
idin-2-yl)piperazin-1-yl}benzoylamino]propionic acid by
the same procedure as employed for compound 4 as a col-
5.19.2. Ethyl 4-{(3R)-methyl-4-(pyrimidin-2-yl)piperazin-
1-yl}benzoate. The title compound was prepared from
ethyl 4-{(3R)-methyl-(piperazin-1-yl)}benzoate by the
same procedure as employed for compound 6 as a pale
1
orless solid. Yield: 2.5 mg, 25%; H NMR (400 MHz,
CD3OD) d: 1.34 (3H, d, CH3), 1.96 (2H, quintet, tetrahy-
dropyrimidine), 2.99 (1H, dt, piperazine), 3.18 (1H, dd,
piperazine), 3.42 (4H, br t, tetrahydropyrimidine), 3.47
(1H, m, piperazine), 3.57 (1H, dd, CONHCH2CH), 3.60
(1H, m, piperazine), 3.65 (1H, dd, CONHCH2CH), 3.71
(1H, dd, CONHCH2CH), 3.73 (1H, m, piperazine), 3.80
(1H, m, piperazine), 4.09 (1H, m, piperazine), 6.95 (2H,
d, C6H4), 7.48 (2H, m, C6H5), 7.55 (1H, m, C6H5), 7.74
1
yellow solid. Yield: 290 mg, 63%; H NMR (400 MHz,
CDCl3) d: 1.31 (3H, d, CH3), 1.37 (3H, t, Et), 3.08
(1H, ddd, piperazine), 3.28 (1H, dd, piperazine), 3.50
(1H, ddd, piperazine), 3.68 (1H, dt, piperazine), 3.79
(1H, ddt, piperazine), 4.33 (2H, q, Et), 4.52 (1H, dt,
piperazine), 4.95 (1H, m, piperazine), 6.52 (1H, t, pyrim-
idine), 6.86 (2H, d, C6H4), 7.94 (2H, d, C6H4), 8.34 (2H,
d, pyrimidine); TSPMS m/z 327 (M+H)+.
(2H, d, C6H4), 7.86 (2H, m, C6H5); FABMS m/z 529
25
D
(M+H)+; ½aꢁ +81° (c 0.060, MeOH).
5.19.3. 4-{(3R)-Methyl-4-(pyrimidin-2-yl)piperazin-1-yl}ben-
zoic acid. The title compound was prepared from ethyl 4-
{(3R)-methyl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoate by
the same procedure as employed for compound 7 as a col-
orless solid. Yield: 52 mg, 47%; 1H NMR (400 MHz,
CDCl3) d: 1.31 (3H, d, CH3), 3.14 (1H, ddd, piperazine),
3.34 (1H, dd, piperazine), 3.53 (1H, ddd, piperazine),
3.77 (1H, m, piperazine), 3.83 (1H, m, piperazine), 4.51
(1H, dt, piperazine), 4.95 (1H, m, piperazine), 6.54 (1H,
t, pyrimidine), 6.88 (2H, d, C6H4), 8.00 (2H, d, C6H4),
8.36 (2H, d, pyrimidine); EIMS m/z 298.
5.20. Preparation of compound 43
5.20.1. Ethyl 4-{(3S)-methyl-4-(pyrimidin-2-yl)piperazin-
1-yl}benzoate. DMSO (2.0 ml) was added to (2S)-methyl-
piperazine (310 mg, 3.1 mmol) to prepare a suspension, to
which ethyl 4-fluorobenzoate (530 mg, 3.2 mmol) was
added. The mixture was stirred at 120 °C for 21 h. The
temperature of the system was then returned to room tem-
perature, and the reaction solution was concentrated un-
der reduced pressure. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH/concd
NH4OH = 1900:100:1) to prepare ethyl 4-{(3S)-methyl-
(piperazin-1-yl)}benzoate (450 mg) as a pale yellow solid.
5.19.4. t-Butyl (2S)-benzenesulfonylamino-3-[4-{(3R)-
methyl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoyl amino]-
propionate. The title compound was prepared from 4-
{(3R)-methyl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoic acid
by the same procedure as employed for compound 9 as
DMF (10 ml) was added to ethyl 4-{(3S)-methyl-(pipera-
zin-1-yl)}benzoate (450 mg, 1.8 mmol) to prepare a solu-
tion, and 2-bromopyrimidine (480 mg, 3.0 mmol) was
added to the solution. N,N-Diisopropylethylamine
(1.5 ml) was added thereto. The mixture was stirred at
120 °C for 15 h and cooled to room temperature, followed
by concentration under reduced pressure. The residue was
purified by column chromatography on silica gel (n-hex-
ane/ethyl acetate = 1:2) to prepare the title compound
(330 mg, 56%) as a pale yellow solid; 1H NMR
(400 MHz, CDCl3) d: 1.31 (3H, d, CH3), 1.37 (3H, t,
Et), 3.08 (1H, ddd, piperazine), 3.28 (1H, dd, piperazine),
3.50 (1H, ddd, piperazine), 3.68 (1H, dt, piperazine), 3.79
(1H, ddt, piperazine), 4.33 (2H, q, Et), 4.52 (1H, dt, piper-
azine), 4.95 (1H, m, piperazine), 6.52 (1H, t, pyrimidine),
6.86 (2H, d, C6H4), 7.94 (2H, d, C6H4), 8.34 (2H, d, pyrim-
idine); FABMS m/z 327 (M+H)+.
a
colorless solid. Yield: 85 mg, 86%; 1H NMR
(400 MHz, CDCl3) d: 1.28 (9H, s, t-Bu), 1.32 (3H, d,
CH3), 3.03 (1H, dt, piperazine), 3.23 (1H, dd, piperazine),
3.47 (1H, ddd, piperazine), 3.59 (1H, ddd, CON-
HCH2CH), 3.65 (1H, m, piperazine), 3.76 (1H, m, piper-
azine), 3.89 (1H, ddd, CONHCH2CH), 3.94 (1H, dt,
CONHCH2CH), 4.54 (1H, dt, piperazine), 4.96 (1H, m,
piperazine), 6.53 (1H, t, pyrimidine), 6.88 (1H, d, C6H4),
7.49 (2H, m, C6H5), 7.56 (1H, m, C6H5), 7.73 (2H, d,
C6H4), 7.86 (2H, m, C6H5), 8.35 (2H, d, pyrimidine);
FABMS m/z 581 (M+H)+.
5.19.5.
(2S)-Benzenesulfonylamino-3-[4-{(3R)-methyl-4-
(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
The title compound was prepared from t-butyl (2S)-benze-
nesulfonylamino-3-[4-{(3R)-methyl-4-(pyrimidin-2-yl)pip-
erazin-1-yl}benzoyl amino]propionate by the same
procedure as employed for compound 10 as a colorless
5.20.2. 4-{(3S)-Methyl-4-(pyrimidin-2-yl)piperazin-1-yl}ben-
zoic acid. The title compound was prepared from ethyl 4-
{(3S)-methyl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoate by

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2103
the same procedure as employed for compound 7 as a col-
orless solid. Yield: 155 mg, 81%; H NMR (400 MHz,
azine (3.6 g, 32 mmol) to prepare a suspension, to which
ethyl 4-fluorobenzoate (2.5 g, 15 mmol) was added. The
mixture was stirred at 80 °C for 24 h. The temperature
of the system was then returned to room temperature
and water (100 ml) was added thereto. The mixture
was extracted three times with ethyl acetate (100 ml).
The combined organic layer was dried over anhydrous
MgSO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on sil-
ica gel (CH2Cl2/MeOH/concd NH4OH = 900:100:1) to
prepare the title compound (1.5 g, 38%) as a colorless
solid; 1H NMR (400 MHz, CD3OD) d: 1.16 (6H, d,
CH3), 1.35 (3H, t, Et), 2.38 (2H, dd, piperazine), 2.95
(2H, m, piperazine), 3.78 (2H, dd, piperazine), 4.29
(2H, q, Et), 6.95 (2H, d, C6H4), 7.86 (2H, d, C6H4);
TSPMS m/z 263 (M+H)+.
1
CD3OD) d: 1.19 (3H, d, CH3), 2.92 (1H, ddd, piperazine),
3.12 (1H, dd, piperazine), 3.37 (1H, ddd, piperazine), 3.71
(1H, dt, piperazine), 3.78 (1H, ddt, piperazine), 4.40 (1H,
dt, piperazine), 4.81 (1H, m, piperazine), 6.51 (1H, t,
pyrimidine), 6.86 (2H, d, C6H4), 7.79 (2H, d, C6H4), 8.25
(2H, d, pyrimidine); TSPMS m/z 299 (M+H)+.
5.20.3. t-Butyl (2S)-benzenesulfonylamino-3-[4-{(3S)-meth-
yl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoyl amino]propio-
nate. The title compound was prepared from 4-{(3S)-
methyl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoic acid by
the same procedure as employed for compound 9 as a col-
orless solid. Yield: 98 mg, 99%; 1H NMR (400 MHz,
CDCl3) d: 1.28 (9H, s, t-Bu), 1.33 (3H, d, CH3), 3.04
(1H, dt, piperazine), 3.23 (1H, dd, piperazine), 3.47 (1H,
ddd, piperazine), 3.58 (1H, ddd, CONHCH2CH), 3.66
(1H, br d, piperazine), 3.77 (1H, br d, piperazine), 3.91
(2H, m, CONHCH2CH), 4.53 (1H, dt, piperazine), 4.96
(1H, m, piperazine), 6.53 (1H, t, pyrimidine), 6.89 (2H,
d, C6H4), 7.49 (2H, t, C6H5), 7.57 (1H, t, C6H5), 7.73
(2H, d, C6H4), 7.86 (2H, br d, C6H5), 8.35 (2H, d, pyrim-
idine); APCIMS m/z 581 (M+H)+.
5.21.2. Ethyl 4-{cis-3,5-dimethyl-4-(pyrimidin-2-yl)pip-
erazin-1-yl}benzoate. The title compound was prepared
from ethyl 4-{cis-3,5-dimethyl-(piperazin-1-yl)}benzoate
by the same procedure as employed for compound 6 as a
colorless solid. Yield: 26 mg, 4.8%; 1H NMR (400 MHz,
CDCl3) d: 1.36 (6H, d, CH3), 1.38 (3H, t, Et), 3.15 (2H,
dd, piperazine), 3.74 (2H, dd, piperazine), 4.34 (2H, q,
Et), 4.89 (2H, m, piperazine), 6.53 (1H, t, pyrimidine),
6.92 (2H, d, C6H4), 7.95 (2H, d, C6H4), 8.36 (2H, d,
pyrimidine); ESIMS m/z 341 (M+H)+.
5.20.4.
(2S)-Benzenesulfonylamino-3-[4-{(3S)-methyl-4-
(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
The title compound was prepared from t-butyl (2S)-benze-
nesulfonylamino-3-[4-{(3S)-methyl-4-(pyrimidin-2-yl)pip-
erazin-1-yl}benzoyl amino]propionate by the same
procedure as employed for compound 10 as a colorless
5.21.3. 4-{cis-3,5-Dimethyl-4-(pyrimidin-2-yl)piperazin-1-
yl}benzoic acid. The title compound was prepared from
ethyl 4-{cis-3,5-dimethyl-4-(pyrimidin-2-yl)piperazin-1-
yl}benzoate by the same procedure as employed for
compound 7 as a colorless solid. Yield: 22 mg, 92%;
1H NMR (400 MHz, CD3OD) d: 1.33 (6H, d, CH3),
3.11 (2H, dd, piperazine), 3.89 (2H, d, piperazine),
4.87 (2H, m, piperazine), 6.61 (1H, t, pyrimidine), 7.02
(2H, d, C6H4), 7.90 (2H, d, C6H4), 8.36 (2H, d, pyrimi-
dine); ESIMS m/z 313 (M+H)+.
1
solid. Yield: 39 mg, 44%; H NMR (400 MHz, CD3OD)
d: 1.21 (3H, d, CH3), 2.89 (1H, dt, piperazine), 3.08 (1H,
dd, piperazine), 3.38 (2H, m, piperazine and CON-
HCH2CH), 3.59 (1H, dd, CONHCH2CH), 3.68 (1H, br
d, CONHCH2CH), 3.76 (1H, br d, piperazine), 3.95
(1H, m, piperazine), 4.41 (1H, dt, piperazine), 4.82 (1H,
m, piperazine), 6.51 (1H, t, pyrimidine), 6.87 (2H, d,
C6H4), 7.33 (2H, t, C6H5), 7.40 (1H, t, C6H5), 7.58 (2H,
d, C6H4), 7.74 (2H, d, C6H5), 8.25 (2H, d, pyrimidine);
25
TSPMS m/z 525 (M+H)+; ½aꢁ +36° (c 0.073, MeOH).
5.21.4. t-Butyl (2S)-benzenesulfonylamino-3-[4-{cis-3,5-di-
methyl-4-(pyrimidin-2-yl)piperazin-1-yl} benzoylamino]pro-
pionate. The title compound was prepared from 4-{cis-
3,5-dimethyl-4-(pyrimidin-2-yl)piperazin-1-yl}benzoic acid
by the same procedure as employed for compound 9 as
D
5.20.5. Compound 43. Compound 43 was prepared from
(2S)-benzenesulfonylamino-3-[4-{(3S)-methyl-4-(pyrim-
idin-2-yl)piperazin-1-yl}benzoylamino]propionic acid by
the same procedure as employed for compound 4 as a col-
orless solid. Yield: 2.2 mg, 32%; 1H NMR (400 MHz,
CD3OD) d: 1.33 (3H, d, CH3), 1.96 (2H, quintet, tetrahy-
dropyrimidine), 2.97 (1H, ddd, piperazine), 3.16 (1H, dd,
piperazine), 3.42 (4H, br t, tetrahydropyrimidine), 3.45
(1H, m, piperazine), 3.58 (1H, dd, CONHCH2CH), 3.59
(1H, m, piperazine), 3.65 (1H, dd, CONHCH2CH), 3.70
(1H, m, piperazine), 3.72 (1H, dd, CONHCH2CH), 3.79
(1H, m, piperazine), 4.09 (1H, m, piperazine), 6.93 (2H,
d, C6H4), 7.48 (2H, m, C6H5), 7.55 (1H, m, C6H5), 7.73
(2H, d, C6H4), 7.86 (2H, m, C6H5); FAB-HRMS
a
colorless solid. Yield: 25 mg, 66%; 1H NMR
(400 MHz, CDCl3) d: 1.29 (9H, s, t-Bu), 1.38 (6H, d,
CH3), 3.13 (2H, dd, piperazine), 3.56 (1H, m, CON-
HCH2CH), 3.70 (2H, d, piperazine), 3.91 (2H, m,
CONHCH2CH), 4.89 (2H, m, piperazine), 6.52 (1H, t,
pyrimidine), 6.96 (2H, d, C6H4), 7.50 (2H, m, C6H5),
7.58 (1H, m, C6H5), 7.74 (2H, d, C6H4), 7.87 (2H, m,
C6H5), 8.37 (2H, d, pyrimidine); TSPMS m/z 595
(M+H)+.
5.21.5. (2S)-Benzenesulfonylamino-3-[4-{cis-3,5-dimethyl-4-
(pyrimidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid.
CH2Cl2 (2.0 ml) was added to t-butyl (2S)-benzenesulfo-
nylamino-3-[4-{cis-3,5-dimethyl-4-(pyrimidin-2-yl)pipera-
zin-1-yl}benzoylamino]propionate (24 mg, 0.040 mmol)
to prepare a solution. Trifluoroacetic acid (1.0 ml) was
added at room temperature to the solution. The mixture
was stirred at that temperature for 6.0 h before the reac-
(M+H)+ calcd for C25H32N6O5S 529.2233, found
25
D
529.2239; ½aꢁ +96° (c 0.085, MeOH).
5.21. Preparation of compound 44
5.21.1. Ethyl 4-{cis-3,5-dimethyl-(piperazin-1-yl)}benzo-
ate. DMSO (10 ml) was added to cis-2,6-dimethylpiper-

2104
D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
tion solution was concentrated under reduced pressure to
prepare the trifluoroacetate of title compound (25 mg) as
a yellow solid; 1H NMR (400 MHz, CD3OD) (as trifluo-
roacetate) d: 1.37 (6H, d, CH3), 3.11 (2H, m, piperazine),
3.48 (1H, dd, CONHCH2CH), 3.70 (1H, dd, CON-
HCH2CH), 3.85 (2H, br d, piperazine), 4.17 (1H, dd,
CONHCH2CH), 4.84 (2H, m, piperazine), 6.71 (1H, t,
pyrimidine), 7.02 (2H, d, C6H4), 7.42 (2H, m, C6H5),
7.49 (1H, m, C6H5), 7.67 (2H, d, C6H4), 7.82 (2H, m,
3.73 (2H, m, homopiperazine), 4.01 (2H, m, homopiper-
azine), 6.49 (1H, t, pyrimidine), 6.73 (2H, d, C6H4), 7.95
(2H, d, C6H4), 8.30 (2H, d, pyrimidine); TSPMS m/z 299
(M+H)+.
5.22.4. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(pyr-
imidin-2-yl)-[1,4]diazepan-1-yl}benzoylamino]propionate.
The title compound was prepared from 4-{4-(pyrimidin-
2-yl)-[1,4]diazepan-1-yl}benzoic acid by the same proce-
dure as employed for compound 9 as a colorless solid.
Yield: 172 mg, 95%; 1H NMR (400 MHz, CDCl3) d:
1.29 (9H, s, t-Bu), 2.10 (2H, quintet, homopiperazine),
3.55 (2H, t, homopiperazine), 3.58 (1H, ddd, CON-
HCH2CH), 3.68 (4H, m, homopiperazine), 3.86 (1H,
ddd, CONHCH2CH), 3.93 (1H, m, CONHCH2CH),
4.01 (2H, m, homopiperazine), 6.47 (1H, t, pyrimidine),
6.71 (2H, d, C6H4), 7.47 (2H, m, C6H5), 7.54 (1H, m,
C6H5), 7.68 (2H, d, C6H4), 7.86 (2H, m, C6H5), 8.29
(2H, d, pyrimidine); APCIMS m/z 581 (M+H)+.
C6H5), 8.43 (2H, d, pyrimidine); TSPMS m/z 539
25
(M+H)+; ½aꢁ +46° (c 0.072, MeOH).
D
5.21.6. Compound 44. Compound 44 was prepared from
(2S)-benzenesulfonylamino-3-[4-{cis-3,5-dimethyl-4-(pyr-
imidin-2-yl)piperazin-1-yl}benzoylamino]propionic acid
by the same procedure as employed for compound 4
as a colorless solid. Yield: 6.9 mg, 32%; 1H NMR
(400 MHz, CD3OD) d: 1.40 (6H, d, CH3), 1.98 (2H,
quintet, tetrahydropyrimidine), 3.12 (2H, m, pipera-
zine), 3.44 (4H, br t, tetrahydropyrimidine), 3.57 (1H,
dd, CONHCH2CH), 3.66 (1H, dd, CONHCH2CH),
3.73 (2H, m, piperazine), 3.83 (2H, m, piperazine),
3.98 (1H, m, CONHCH2CH), 7.01 (2H, d, C6H4), 7.48
(2H, m, C6H5), 7.55 (1H, m, C6H5), 7.75 (2H, d,
5.22.5. (2S)-Benzenesulfonylamino-3-[4-{4-(pyrimidin-2-
yl)-[1,4]diazepan-1-yl}benzoylamino]propionic acid. The
title compound was prepared from t-butyl (2S)-benze-
nesulfonylamino-3-[4-{4-(pyrimidin-2-yl)-[1,4]diazepan-
1-yl}benzoylamino]propionate by the same procedure
as employed for compound 10 as a colorless solid.
C6H4), 7.85 (2H, m, C6H5); FAB-HRMS (M+H)+ calcd
25
D
for C26H34N6O5S 543.2390, found 543.2380; ½aꢁ +79°
1
(c 0.28, MeOH).
Yield: 210 mg, 58%; H NMR (400 MHz, CD3OD) d:
1.96 (2H, quintet, homopiperazine), 3.39 (1H, dd,
CONHCH2CH), 3.50 (2H, m, homopiperazine), 3.56
(1H, dd, CONHCH2CH), 3.60 (2H, m, homopiper-
azine), 3.66 (2H, m, homopiperazine), 3.91 (2H, m,
homopiperazine), 3.99 (1H, dd, CONHCH2CH), 6.43
(1H, t, pyrimidine), 6.67 (2H, d, C6H4), 7.27 (2H, m,
C6H5), 7.32 (1H, m, C6H5), 7.48 (2H, d, C6H4), 7.70
5.22. Preparation of compound 45
5.22.1. Ethyl 4-([1,4]diazepan-1-yl)benzoate. Ethyl 4-flu-
orobenzoate (1.2 g, 7.1 mmol) was added to homopiper-
azine (1.4 g, 14 mmol) to prepare a suspension. The
suspension was stirred at 120 °C for 7.5 h and cooled
to room temperature, followed by purification by col-
umn chromatography on silica gel (CH2Cl2/MeOH/con-
cd NH4OH = 900:100:1) to prepare the title compound
(2H, m, C6H5), 8.17 (2H, d, pyrimidine); TSPMS m/z
25
525 (M+H)+; ½aꢁ +37° (c 0.068, MeOH).
D
1
(1.1 g, 62%) as a colorless solid; H NMR (400 MHz,
5.22.6. Compound 45. Compound 45 was prepared from
(2S)-benzenesulfonylamino-3-[4-{4-(pyrimidin-2-yl)-[1,4]-
diazepan-1-yl}benzoylamino]propionic acid by the same
procedure as employed for compound 4 as a pale yellow
CDCl3) d: 1.36 (3H, t, Et), 1.89 (2H, m, homopiper-
azine), 2.82 (2H, m, homopiperazine), 3.03 (2H, m,
homopiperazine), 3.61 (4H, m, homopiperazine), 4.31
(2H, q, Et), 6.66 (2H, d, C6H4), 7.89 (2H, d, C6H4);
TSPMS m/z 249 (M+H)+.
1
solid. Yield: 34 mg, 41%; H NMR (400 MHz, CD3OD)
(as hydrochloride) d: 1.73 (2H, quintet, tetrahydropyrim-
idine), 1.97 (2H, quintet, homopiperazine), 3.25 (4H, m,
tetrahydropyrimidine), 3.45 (1H, dd, CONHCH2CH),
3.49 (2H, m, homopiperazine), 3.65 (2H, m, homopiper-
azine), 3.70 (2H, m, homopiperazine), 3.72 (1H, dd,
CONHCH2CH), 3.82 (2H, m, homopiperazine), 4.18
(1H, dd, CONHCH2CH), 6.80 (2H, d, C6H4), 7.43 (2H,
m, C6H5), 7.50 (1H, m, C6H5), 7.68 (2H, d, C6H4), 7.82
5.22.2. Ethyl 4-{4-(pyrimidin-2-yl)-[1,4]diazepan-1-yl}benzo-
ate. The title compound was prepared from ethyl 4-
([1,4]diazepan-1-yl)benzoate by the same procedure as em-
ployed for compound 6 as a colorless solid. Yield: 200 mg,
61%; 1H NMR (400 MHz, CDCl3) d: 1.36 (3H, t, Et), 2.10
(2H, quintet, homopiperazine), 3.56 (2H, t, homopiper-
azine), 3.65 (2H, t, homopiperazine), 3.70 (2H, dd, homo-
piperazine), 3.99 (2H, dd, homopiperazine), 4.32 (2H, q,
Et), 6.47 (1H, t, pyrimidine), 6.70 (2H, d, C6H4), 7.90
(2H, d, C6H4), 8.28 (2H, d, pyrimidine); APCIMS m/z
327 (M+H)+.
(2H, m, C6H5); FAB-HRMS (M+H)+ calcd for
25
D
C25H32N6O5S 529.2233, found 529.2239; ½aꢁ +38° (c
0.14, MeOH).
5.23. Preparation of compound 46
5.22.3. 4-{4-(Pyrimidin-2-yl)-[1,4]diazepan-1-yl}benzoic
acid. The title compound was prepared from ethyl 4-
{4-(pyrimidin-2-yl)-[1,4]diazepan-1-yl}benzoate by the
same procedure as employed for compound 7 as a color-
less solid. Yield: 94 mg, 100%; 1H NMR (400 MHz,
CDCl3) d: 2.12 (2H, quintet, homopiperazine), 3.58
(2H, t, homopiperazine), 3.67 (2H, t, homopiperazine),
5.23.1. Ethyl 4-[4-{(1H-benzimidazol-2-yl)methyl}pipera-
zin-1-yl]benzoate. DMF (5.0 ml) and acetone (10 ml)
were added to compound 5 (250 mg, 1.1 mmol) to pre-
pare a solution, and 2-(chloromethyl)benzimidazole
(180 mg, 1.1 mmol) and K2CO3 (300 mg) were added
to the solution. The mixture was stirred at room
temperature for 25 h and then water (200 ml) was added

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2105
thereto. After extracting with CH2Cl2 (100 ml) four
times, the organic layer was collected. The organic layer
was dried over anhydrous MgSO4 and concentrated
under reduced pressure. The residue was purified by
column chromatography on silica gel (CH2Cl2/MeOH/
concd NH4OH = 90:10:1) to prepare the title compound
CDCl3) d: 1.37 (3H, t, Et), 1.65 (2H, m, piperidine), 1.99
(2H, br d, piperidine), 3.09 (2H, ddd, piperidine), 3.72
(2H, dt, piperidine), 3.92 (1H, tt, piperidine), 4.32 (2H,
q, Et), 6.87 (2H, d, C6H4), 7.91 (2H, d, C6H4); EIMS m/
z 249 (M)+.
1
(274 mg, 70%) as a colorless solid; H NMR (400 MHz,
5.24.2. Ethyl 4-(4-phthalimidopiperidin-1-yl)benzoate. Ben-
zene (80 ml) was added to ethyl 4-(4-hydroxypiperidin-1-
yl)benzoate (2.0 g, 8.1 mmol) to prepare a solution. Phthal-
imide (2.4 g, 16 mmol) and tributylphosphine (4.0 ml,
16 mmol) were added to the solution. The mixture was
cooled to 0 °C. 1,10-(Azodicarbonyl)-dipiperidine (4.0 g,
16 mmol) was added thereto at that temperature, and
the mixture was then stirred at room temperature for
23 h. Water (300 ml) was added to stop the reaction. The
mixture was extracted three times with CH2Cl2 (300 ml).
The combined organic layer was then dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on
silica gel (CHCl3/acetone = 50:1) to prepare the title
CDCl3) d: 1.37 (3H, t, Et), 2.73 (4H, br t, piperazine),
3.37 (4H, br t, piperazine), 3.90 (2H, s, CH2), 4.33
(2H, q, Et), 6.87 (2H, d, C6H4), 7.45 (2H, m, benzimid-
azole), 7.75 (2H, m, benzimidazole), 7.94 (2H, d, C6H4);
TSPMS m/z 365 (M+H)+.
5.23.2. 4-[4-{(1H-Benzimidazol-2-yl)methyl}piperazin-1-
yl]benzoic acid. The title compound was prepared from
ethyl 4-[4-{(1H-benzimidazol-2-yl)methyl}piperazin-1-
yl]benzoate by the same procedure as employed for com-
pound 7 as a colorless solid. Yield: 36 mg, 39%; 1H
NMR (400 MHz, CD3OD) d: 2.61 (4H, br t, piperazine),
3.29 (4H, br t, piperazine), 3.77 (2H, s, CH2), 6.85 (2H,
d, C6H4), 7.13 (2H, dd, benzimidazole), 7.44 (2H, dd,
benzimidazole), 7.77 (2H, m, C6H4); TSPMS m/z 337
(M+H)+.
1
compound (1.6 g, 52%); H NMR (400 MHz, CDCl3) d:
1.38 (3H, t, Et), 1.82 (2H, br d, piperidine), 2.62 (2H,
dq, piperidine), 2.96 (2H, dt, piperidine), 4.01 (2H, br d,
piperidine), 4.34 (2H, q, Et), 4.35 (1H, m, piperidine),
6.90 (2H, d, C6H4), 7.72 (2H, dd, phthalimide), 7.83 (2H,
dd, phthalimide), 7.93 (2H, d, C6H4); TSPMS m/z 379
(M+H)+.
5.23.3. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(1H-ben-
zimidazol-2-yl)methyl}piperazin-1-y1]benzoyl amino]propio-
nate. The title compound was prepared from 4-[4-{(1H-
benzimidazol-2-yl)methyl}piperazin-1-yl]benzoic acid by
the same procedure as employed for compound 9 as a col-
orless solid. Yield: 31 mg, 56%; 1H NMR (400 MHz,
CDCl3) d: 1.28 (9H, s, t-Bu), 2.73 (4H, br t, piperazine),
3.33 (4H, br t, piperazine), 3.56 (1H, m, CONHCH2CH),
3.90 (4H, m, CONHCH2CH and CH2), 6.89 (2H, d,
C6H4), 7.26 (2H, m, benzimidazole), 7.52 (5H, m, benz-
imidazole and C6H5), 7.72 (2H, d, C6H4), 7.85 (2H, m,
C6H5); FABMS m/z 619 (M+H)+.
5.24.3. Ethyl 4-(4-aminopiperidin-1-yl)benzoate. MeOH
(56 ml) was added to ethyl 4-(4-phthalimidopiperidin-
1-yl)benzoate (850 mg, 2.2 mmol) to prepare a suspen-
sion. Hydrazine monohydrate (4.5 ml) was added to
the suspension, and the mixture was stirred at room
temperature for 16 h. The reaction solution was filtered
through a glass filter, and the filtrate was concentrated
under reduced pressure. The residue was purified by col-
umn chromatography on silica gel (CHCl3/MeOH/concd
NH4OH = 30:10:1) to prepare the title compound
5.23.4. Compound 46. Compound 46 was prepared from
t-butyl (2S)-benzenesulfonylamino-3-[4-{4-(1H-benzimidazol-
2-yl)methyl}piperazin-1-y1]benzoyl amino]propionate by
the same procedure as employed for compound 10 as a
colorless solid. Yield: 0.64 mg, 7.0%; 1H NMR
(400 MHz, CD3OD) d: 2.72 (4H, br t, piperazine), 3.37
(4H, br t, piperazine), 3.53 (1H, dd, CONHCH2CH),
3.65 (1H, dd, CONHCH2CH), 3.82 (1H, dd, CON-
HCH2CH), 3.88 (2H, s, CH2), 6.96 (2H, d, C6H4), 7.23
(2H, dd, benzimidazole), 7.43 (2H, m, C6H5), 7.50 (1H,
m, C6H5), 7.55 (2H, dd, benzimidazole), 7.68 (2H, d,
C6H4), 7.83 (2H, m, C6H5); FABMS m/z 563 (M+H)+.
1
(551 mg, 100%); H NMR (400 MHz, CD3OD) d: 1.35
(3H, t, Et), 1.43 (2H, dq, piperidine), 1.90 (2H, br d, piper-
idine), 2.84 (1H, m, piperidine), 2.89 (2H, br t, piperidine),
3.92 (2H, br d, piperidine), 4.28 (2H, q, Et), 6.94 (2H, d,
C6H4), 7.84 (2H, d, C6H4); TSPMS m/z 249 (M+H)+.
5.24.4. Ethyl 4-{4-(pyrimidin-2-ylamino)piperidin-1-yl}ben-
zoate. DMF (10 ml) was added to ethyl 4-(4-aminopiperi-
din-1-yl)benzoate (250 mg, 1.0 mmol) to prepare
a
solution, and 2-bromopyrimidine (240 mg, 1.5 mmol)
was added to the solution. Further, N,N-diisopropylethyl-
amine (0.10 ml) was added thereto. The mixture was heat-
ed to 125 °C, stirred for 10 h, and then cooled to room
temperature. Saline (150 ml) was then added thereto, fol-
lowed by extraction three times with CH2Cl2 (150 ml).
The combined organic layer was dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The
residue was purified by column chromatography on silica
gel (n-hexane/ethyl acetate = 1:1) to prepare the title com-
5.24. Preparation of compound 47
5.24.1. Ethyl 4-(4-hydroxypiperidin-1-yl)benzoate. DMSO
(10 ml) was added to 4-hydroxypiperidine (5.0 g, 49 mmol)
to prepare a solution, and ethyl 4-fluorobenzoate (7.2 ml,
49 mmol) was added to the solution. The mixture was
stirred at 120 °C for 3 days and cooled to room tempera-
ture. The reaction solution was cooled to room tempera-
ture. The reaction solution was then poured into water
(200 ml) with vigorous stirring. The insolubles were collect-
ed by filtration and washed twice with water (50 ml) and
then once with hexane (50 ml). The solid was dried to pre-
pare the title compound (9.6 g, 79%); 1H NMR (400 MHz,
1
pound (212 mg, 65%); H NMR (400 MHz, CDCl3) d:
1.37 (3H, t, Et), 1.61 (2H, br q, piperidine), 2.17 (2H, br
d, piperidine), 3.08 (2H, br t, piperidine), 3.84 (2H, br d,
piperidine), 4.06 (1H, m, piperidine), 4.33 (2H, q, Et),
6.55 (1H, t, pyrimidine), 6.89 (2H, d, C6H4), 7.92 (2H, d,
C6H4), 8.28 (2H, d, pyrimidine); EIMS m/z 326 (M)+.

2106
D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
23
dine); FABMS m/z 525 (M+H)+; ½aꢁ +65° (c 0.36,
5.24.5. 4-{4-(Pyrimidin-2-ylamino)piperidin-1-yl}benzoic
acid. THF (9.0 ml) and MeOH (3.0 ml) were added to
ethyl 4-{4-(pyrimidin-2-ylamino)piperidin-1-yl}benzo-
ate (100 mg, 0.31 mmol) to prepare a solution, and a
1 N aqueous NaOH (3.0 ml) was added to the solution.
The mixture was stirred at 40 °C for 8 h and then con-
centrated under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (CHCl3/
MeOH/concd NH4OH = 30:10:1) to prepare the title
compound (75 mg, 81%); 1H NMR (400 MHz,
DMSO-d6) d: 1.53 (2H, br q, piperidine), 1.91 (2H, br
d, piperidine), 2.88 (2H, br t, piperidine), 3.82 (2H, br
d, piperidine), 3.90 (1H, m, piperidine), 6.54 (1H, t,
pyrimidine), 6.89 (2H, br d, C6H4), 7.73 (2H, br d,
C6H4), 8.25 (2H, d, pyrimidine); EIMS m/z 298 (M)+.
D
MeOH).
5.24.8. Compound 47. Acetic acid (5.0 ml) and concentrat-
ed hydrochloric acid (0.50 ml) were added to (2S)-benze-
nesulfonylamino-3-[4-{4-(pyrimidin-2-ylamino)piperidin-
1-yl}benzoylamino]propionic acid (3.6 mg, 0.0069 mmol)
to prepare a solution. 10% Pd/C (1.8 mg) was added to
the solution, and the mixture was vigorously shaken un-
der a hydrogen pressure of 3 atm at room temperature
for 2.5 h. The insolubles were filtered and washed twice
with water and twice with MeOH. The filtrate was com-
bined with the washings, followed by concentration under
reduced pressure. The residue was subjected to azeotropic
distillation twice each with toluene, followed by purifica-
tion by preparative thin-layer chromatography on silica
gel (CH2Cl2/EtOH/H2O/concd NH4OH = 8:8:1:1) and
then by Sephadex LH-20 (MeOH) to prepare compound
47 (3.2 mg, 88%); 1H NMR (400 MHz, CD3OD) d: 1.49
(2H, br q, J = 10.8, piperidine), 1.85 (4H, m, piperidine
and tetrahydropyrimidine), 2.84 (2H, ddd, J = 2.6, 10.8,
13.5, piperidine), 3.26 (4H, t, J = 5.8, tetrahydropyrimi-
dine), 3.45 (2H, m, CONHCH2CH and piperidine), 3.53
(1H, dd, J = 5.0, 13.4, CONHCH2CH), 3.63 (1H, dd,
J = 5.0, 8.0, CONHCH2CH), 3.73 (2H, br d, J = 13.5,
piperidine), 6.85 (2H, d, J = 9.0, C6H4), 7.37 (2H, m,
C6H5), 7.44 (1H, m, C6H5), 7.59 (2H, d, J = 9.0, C6H4),
7.75 (2H, m, C6H5); 13C NMR (DMSO-d6) d: 19.8, 31.2,
31.9, 37.8, 42.4, 46.6, 47.1, 55.2, 114.1, 123.9, 126.6,
5.24.6. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(pyrim-
idin-2-ylamino)piperidin-1-yl}benzoylamino]propionate.
CH2Cl2 (1.0 ml) and DMF (1.0 ml) were added to 4-{4-
(pyrimidin-2-ylamino)piperidin-1-yl}benzoic acid (6.0 mg,
0.020 mmol) to prepare a solution. N,N-Diisopropylethyl-
amine (6 ll) and benzotriazol-1-yloxy tri(dimethylami-
no)phosphonium hexafluorophosphate (13 mg) were
added to the solution. The mixture was stirred at room
temperature for 3 h. The reaction solution was added to a
solution of t-butyl (2S)-N-benzenesulfonyl-2,3-diamino-
propionate hydrochloride (8.1 mg) in CH2Cl2 (1.0 ml)
cooled to ꢀ10 °C. Further, N,N-diisopropylethylamine
(6 ll) was added thereto, followed by stirring at that tem-
perature for 2 h. The reaction solution was concentrated
under reduced pressure. The residue was purified by pre-
parative thin-layer chromatography on silica gel (CHCl3/
MeOH = 10:1) to prepare the title compound (7.0 mg,
60%); 1H NMR (400 MHz, CDCl3) d: 1.28 (9H, s, t-Bu),
1.60 (2H, m, piperidine), 2.18 (2H, br d, piperidine), 3.06
(2H, br t, piperidine), 3.57 (1H, ddd, CONHCH2CH),
3.81 (2H, br d, piperidine), 3.90 (2H, m, CONHCH2CH),
4.05 (1H, m, piperidine), 6.55 (1H, t, pyrimidine), 6.91
(2H, d, C6H4), 7.49 (2H, m, C6H5), 7.57 (1H, m, C6H5),
126.8, 128.1, 129.0, 129.2, 132.3, 140.6, 152.1, 152.8,
20
165.0, 173.1; FABMS m/z 529 (M+H)+; ½aꢁ +69° (c
D
0.16, MeOH).
5.25. Preparation of compound 48
5.25.1. Ethyl 4-[4-[{N-methyl-N-(pyrimidin-2-yl)}ami-
no]piperidin-1-yl]benzoate. DMF (3.6 ml) was added to
ethyl 4-{4-(pyrimidin-2-ylamino)piperidin-1-yl}benzo-
ate (179 mg, 0.55 mmol) to prepare a solution. Methyl
iodide (156 mg) was added to the solution. 60% sodium
hydride in oil (43.9 mg, 1.1 mmol) was added thereto,
and a reaction was allowed to proceed at 45 °C for
11 h. The reaction solution was extracted with ethyl ace-
tate (200 ml). The extract was washed with distilled
water, an aqueous NaHCO3 solution, and saline, and
then dried over anhydrous Na2SO4, concentrated under
reduced pressure, and dried to prepare the title com-
7.70 (2H, d, C6H4), 7.86 (2H, m, C6H5), 8.29 (2H, d,
25
pyrimidine); TSPMS m/z 581 (M+H)+; ½aꢁ +24° (c 0.35,
D
MeOH).
5.24.7. (2S)-Benzenesulfonylamino-3-[4-{4-(pyrimidin-2-yl-
amino)piperidin-1-yl}benzoylamino]propionic acid. CH2Cl2
(0.30 ml) was added to the t-butyl (2S)-benzenesulfonyla-
mino-3-[4-{4-(pyrimidin-2-ylamino)piperidin-1-yl}ben-
zoylamino]propionate (7.0 mg, 0.012 mmol) to prepare a
solution. Trifluoroacetic acid (0.3 ml) was added at 0 °C
to the solution. The mixture was stirred at room temper-
ature for 2 h. The reaction solution was concentrated un-
der reduced pressure. The residue was purified by
preparative thin-layer chromatography on silica gel
(CHCl3/MeOH/acetic acid = 10:2:1) and then purified
by Sephadex LH-20 (MeOH) to prepare the title com-
pound (6.3 mg, 100%); 1H NMR (400 MHz, CD3OD)
d: 1.56 (2H, dq, piperidine), 1.99 (2H, br d, piperidine),
2.90 (2H, br t, piperidine), 3.46 (1H, dd, CONHCH2CH),
3.56 (1H, dd, CONHCH2CH), 3.63 (1H, dd, CON-
HCH2CH), 3.80 (2H, br d, piperidine), 3.89 (1H, m,
piperidine), 6.49 (1H, t, pyrimidine), 6.89 (2H, d,
C6H4), 7.36 (2H, m, C6H5), 7.43 (1H, m, C6H5), 7.60
(2H, d, C6H4), 7.75 (2H, m, C6H5), 8.16 (2H, d, pyrimi-
1
pound (186 mg, 99%); H NMR (400 MHz, CDCl3) d:
1.37 (3H, t, Et), 1.80 (2H, br d, piperidine), 1.90 (2H,
dq, piperidine), 3.01 (3H, s, Me), 3.04 (2H, br dt, piper-
idine), 3.99 (2H, br d, piperidine), 4.33 (2H, q, Et), 4.90
(1H, dddd, piperidine), 6.48 (1H, t, pyrimidine), 6.90
(2H, d, C6H4), 7.93 (2H, d, C6H4), 8.32 (2H, d, pyrimi-
dine); TSPMS m/z 341 (M+H)+.
5.25.2. 4-[4-[{N-Methyl-N-(pyrimidin-2-yl)}amino]piperi-
din-1-yl]benzoic acid. The title compound was prepared
from ethyl 4-[4-[{N-methyl-N-(pyrimidin-2-yl)}ami-
no]piperidin-1-yl]benzoate by the same procedure as em-
1
ployed for compound 7. Yield: 102 mg, 55%; H NMR
(400 MHz, 67% CD3OD/CDCl3) d: 1.80 (2H, br d,
piperidine), 1.94 (2H, dq, piperidine), 3.01 (3H, s, Me),
3.03 (2H, br t, piperidine), 4.06 (2H, br d, piperidine),

D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
2107
4.86 (1H, dddd, piperidine), 6.57 (1H, t, pyrimidine),
6.96 (2H, d, C6H4), 7.91 (2H, d, C6H4), 8.32 (2H, d,
pyrimidine); TSPMS m/z 313 (M+H)+.
HCl, 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2,
pH 7.4, 100 ll) including vitronectin (Calbiochem) or
fibrinogen, added to the receptor-coated plate, was incu-
bated for 4 h at 25°C. Thereafter the ligand binding was
measured using anti-vitronectin rabbit antibody (Cal-
biochem) and peroxidase-conjugated anti-rabbit IgG
antibody (Capell) for avb3, or peroxidase-conjugated
anti-fibrinogen antibody (Capell) for aIIbb3, and 2, 20-
azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma)
as the substrate of peroxidase. The IC50 values were
determined from measurement of absorbance at 415 nm.
5.25.3. t-Butyl (2S)-benzenesulfonylamino-3-[4-[4-[{N-meth-
yl-N-(pyrimidin-2-yl)}amino]piperidin-1-yl] benzoylamino]-
propionate. The title compound was prepared from 4-[4-
[{N-methyl-N-(pyrimidin-2-yl)}amino]piperidin-1-yl]benzoic
acid by the same procedure as employed for compound 9.
1
Yield: 303 mg, 98%; H NMR (400 MHz, CDCl3) d: 1.29
(9H, s, t-Bu), 1.80 (2H, br d, piperidine), 1.92 (2H, dq,
piperidine), 3.02 (3H, s, Me), 3.02 (2H, br t, piperidine),
3.58 (1H, ddd, CONHCH2CH), 3.85-4.00 (4H, m, CON-
HCH2CH, CONHCH2CH and piperidine), 4.89 (1H,
dddd, piperidine), 6.48 (1H, t, pyrimidine), 6.92 (2H, d,
C6H4), 7.49 (2H, br t, C6H5), 7.57 (1H, br t, C6H5), 7.71
(2H, d, C6H4), 7.86 (2H, m, C6H5), 8.32 (2H, d, pyrimi-
5.27. Adhesion of human aorta smooth muscle cells to
vitronectin
The adhesion of human aorta smooth muscle cells to
vitronectin was measured as described before.18 Briefly,
EIA plates (Nunc) were coated with human vitronectin
(Calbiochem) and blocked with bovine serum albumin.
The cell suspension of human aorta smooth muscle cells
(50,000 cells/100 ll, Clonetics) in DulbeccoÕs modified
EagleÕs basal medium containing 0.1% bovine serum
albumin was added to the vitronectin-coated plates
and incubated for 1.5 h at 37 °C in the presence or ab-
sence of the test compounds. The adherent cells were
stained with toluidine blue and calculated by the mea-
suring of absorbance at 405 nm after the cytolysis by
SDS. The IC50 values were determined graphically from
two or more independent experiments.
dine); TSPMS m/z 595 (M+H)+; ½aꢁ25 +55° (c 2.0, CH2Cl2).
D
5.25.4. (2S)-Benzenesulfonylamino-3-[4-[4-[{N-methyl-N-
(pyrimidin-2-yl)}amino]piperidin-1-yl]benzoyl amino]pro-
pionic acid. The title compound was prepared from t-butyl
(2S)-benzenesulfonylamino-3-[4-[4-[{N-methyl-N-(pyrim-
idin-2-yl)}amino]piperidin-1-yl]benzoylamino]propionate
by the same procedure as employed for compound 10.
1
Yield: 262 mg, 99%; H NMR (400 MHz, CD3OD) d:
1.77 (2H, br d, piperidine), 1.95 (2H, dq, piperidine), 2.96
(2H, br t, piperidine), 3.00 (3H, s, Me), 3.55 (1H, dd, CON-
HCH2CH), 3.66 (1H, dd, CONHCH2CH), 3.81 (1H, dd,
CONHCH2CH), 4.03 (2H, br d, piperidine), 6.57 (1H, t,
pyrimidine), 7.00 (2H, d, C6H4), 7.45 (2H, m, C6H5), 7.52
5.28. Platelet aggregation assay
(1H, m, C6H5), 7.69 (2H, d, C6H4), 7.85 (2H, m, C6H5),
25
D
8.32 (2H, d, pyrimidine); TSPMS m/z 539 (M+H)+; ½aꢁ
Platelet aggregation was determined according to the
previous method.15 Human platelet-rich plasma ob-
tained from healthy volunteers was prepared and the
aggregation was induced with 5 lM ADP. The IC50 val-
ues were determined from two independent experiments.
+78° (c 0.50, MeOH/CHCl3 = 1:1).
5.25.5. Compound 48. Compound 48 was prepared from
(2S)-benzenesulfonylamino-3-[4-[4-[{N-methyl-N-(pyrim-
idin-2-yl)}amino]piperidin-1-yl]benzoyl amino]propionic
acid by the same procedure as employed for compound
1
4. Yield: 29 mg, 78%; H NMR (400 MHz, CD3OD) d:
1.74 (2H, br d, piperidine), 1.86 (2H, dq, piperidine),
1.93 (2H, quintet, tetrahydropyrimidine), 2.78 (3H, s,
Me), 2.86 (2H, br t, piperidine), 3.38 (4H, t, tetrahydro-
pyrimidine), 3.57 (1H, dd, CONHCH2CH), 3.65 (1H,
dd, CONHCH2CH), 3.73 (1H, dd, CONHCH2CH),
3.92 (2H, br d, piperidine), 6.92 (2H, d, C6H4), 7.47
Acknowledgments
We thank Miss Shigeko Miki and Mrs. Takako Miyara
for mass spectral analysis.
(2H, m, C6H5), 7.53 (1H, m, C6H5), 7.70 (2H, d, C6H4),
References and notes
25
D
7.86 (2H, m, C6H5); FABMS m/z 543 (M+H)+; ½aꢁ
+83° (c 1.0, MeOH).
1. Hynes, R. O. Cell 1992, 69, 11.
2. Byzova, T. V.; Rabbani, R.; DÕSouza, S. E.; Plow, E. F.
Thromb. Haemost. 1998, 80, 726.
5.26. Integrin-binding assays
3. Brooks, P. C. Drug News Perspect. 1997, 10, 456, and
references cited therein.
Compounds were evaluated for their inhibitory activities
in avb3 and aIIbb3-ELISA (enzyme-linked immunosor-
bent assay). avb3 was purified from human placenta,
using RGDSPK-Sepharose CL-4B chromatography,
4. Egbertson, M. S.; Chang, C. T.-C.; Duggan, M. E.;
Gould, R. J.; Halczenko, W.; Hartman, G. D.; Laswell,
W. L.; Lynch, J. J.; Lynch, R. J., Jr.; Manno, P. D.;
Naylor, A. M.; Prugh, J. D.; Ramjit, D. R.; Sitko, G. R.;
Smith, R. S.; Turchi, L. M.; Zhang, G. J. Med. Chem.
1994, 37, 2537.
14
followed by mono Q chromatography (Pharmacia).
14
aIIbb3
was purified from human platelet by
RGDSPK-Sepharose CL-4B. avb3 and aIIbb3 binding
assays were performed according to the modified meth-
od of Kouns et al.15 EIA plates (Nunc) were coated with
avb3 or aIIbb3 and blocked with bovine serum albumin.
In each reaction, the reaction mixture (20 mM Tris–
5. Goa, K. L.; Noble, S. Drugs 1999, 57, 439.
6. Susan, H. T.; Patricia, M. S.; Robert, E. J.; Marian, T. N.
Circulation 1998, 98, 1085.
7. Hartman, G. D.; Duggan, M. E.; Ihle, N. C.; Hoffman, W.
F. WO95/32710, 1995.

2108
D. Kubota et al. / Bioorg. Med. Chem. 14 (2006) 2089–2108
8. Duggan, M. E.; Duong, L. T.; Fisher, J. E.; Hamill, T. G.;
13. Askew, B. C.; Bednar, R. A.; Bednar, B.; Claremon, D.
A.; Cook, J. J.; McIntyre, C. J.; Hunt, C. A.; Gould, R. J.;
Lynch, R. J.; Lynch, J. J., Jr.; Gaul, S. L.; Stranieri, M. T.;
Sitko, G. R.; Holahan, M. A.; Glass, J. D.; Hamill, T.;
Gorham, L. M.; Prueksaritanont, T.; Baldwin, J. J.;
Hartman, G. D. J. Med. Chem. 1997, 40, 1779.
Hoffman, W. F.; Huff, J. R.; Ihle, N. C.; Leu, C.-T.; Nagy,
R. M.; Perkins, J. J.; Rodan, S. B.; Wesolowski, G.;
Whitman, D. B.; Zartman, A. E.; Rodan, G. A.; Hartman,
G. D. J. Med. Chem. 2000, 43, 3736.
9. Ruminski, P. G.; Lindmark, R. J.; Russell, M. A.; Clare,
M.; Collins, P. W.; Rico, J. G.; Rogers, T. E.; Desai, B. N.
WO9708145, 1997.
14. Pytela, R.; Pierschbacher, M. D.; Argraves, S.; Suzuki, S.;
Rouslahti, E. Methods Enzymol. 1987, 144, 475.
10. Keenan, R. M.; Miller, W. H.; Kwon, C.; Ali, F. E.;
Callahan, J. F.; Calvo, R. R.; Hwang, S.; Kopple, K.
D.; Peishoff, C. E.; Samanen, J. M.; Wong, A. S.;
Yuan, C.; Huffman, W. F. J. Med. Chem. 1997, 40,
2289.
15. Kouns, W. C.; Kirchhofer, D.; Hadvary, P.; Edenhofer,
A.; Weller, T.; Pfenninger, G.; Baumgartner, H. R.;
Jennings, L. K.; Steiner, B. Blood 1992, 80, 2539.
16. Wong, A.; Hwang, S. M.; McDevitt, P.; McNulty, D.;
Stadel, J. M.; Johanson, K. Mol. Pharmacol. 1996, 50, 529.
17. Tam, S. H.; Sassoli, P. M.; Jordan, R. E.; Nakada, M. T.
Circulation 1998, 98, 1085.
11. Wells, A. S.; Walsgrove, T. C.; Hayes, J.; Etridge, S. K.
WO9724336, 1997.
12. Nicolaou, K. C.; Trujillo, J. I.; Jandeleit, B.; Chibale, K.;
Rosenfeld, M.; Diefenbach, B.; Cheresh, D. A.; Good-
man, S. L. Bioorg. Med. Chem. 1998, 6, 1185.
18. Liaw, L.; Almeida, M.; Hart, C. E.; Schwartz, S. M.;
Giachelli, C. M. Circ. Res. 1994, 74, 214, and references
cited therein.