Tricyclic pharmacophore-based molecules as novel integrin αvβ3 antagonists. Part 2: Synthesis of potent αvβ3/αIIbβ3 dual antagonists

Article abstract of DOI:10.1016/j.bmc.2005.10.061

We synthesized 4-aminopiperidine derivatives of our prototype integrin α_vβ₃ antagonist 1 in an attempt to increase the activity and water solubility. Introduction of one or two hydrophilic moieties into the central aromatic ring and/or the benzene ring at the C-terminus of 1 increased water solubility and enhanced inhibition of cell adhesion. The results of a structure-activity relationships (SAR) study indicated that the torsion angle between the central aromatic ring and the piperidine ring, and the acidity at the sulfonamide moiety, might be important for α_vβ ₃ receptor binding activity. Some of these compounds are novel and potent α_vβ₃/α_IIbβ ₃ dual antagonists with acceptable water solubility and a satisfactory early absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile.

Full text of DOI:10.1016/j.bmc.2005.10.061

Bioorganic & Medicinal Chemistry 14 (2006) 2109–2130
Tricyclic pharmacophore-based molecules as novel integrin
avb3 antagonists. Part 2: Synthesis of potent
avb3/aIIbb3 dual antagonists
Minoru Ishikawa, Dai Kubota, Mikio Yamamoto, Chizuko Kuroda, Maki Iguchi,
Akihiro Koyanagi, Shoichi Murakami and Keiichi Ajito*
Pharmaceutical Research Department, 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—We synthesized 4-aminopiperidine derivatives of our prototype integrin avb3 antagonist 1 in an attempt to increase the
activity and water solubility. Introduction of one or two hydrophilic moieties into the central aromatic ring and/or the benzene ring
at the C-terminus of 1 increased water solubility and enhanced inhibition of cell adhesion. The results of a structure–activity
relationships (SAR) study indicated that the torsion angle between the central aromatic ring and the piperidine ring, and the acidity
at the sulfonamide moiety, might be important for avb3 receptor binding activity. Some of these compounds are novel and potent
avb3/aIIbb3 dual antagonists with acceptable water solubility and a satisfactory early absorption, distribution, metabolism, excretion,
and toxicity (ADMET) profile.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
fragment of the human-murine monoclonal antibody
Abciximab,4 which binds to the avb3 receptor and aIIbb3
receptor, has clinical efficacy in the treatment of ische-
mic diseases. Therefore, an injectable avb3/aIIbb3 dual
antagonist would be a candidate drug for acute ischemic
diseases. We have already discovered highly constrained
avb3/aIIbb3 dual antagonists possessing a tricyclic phar-
macophore.5 Although a prototype dual antagonist 1
was successfully obtained, it shows poor water solubili-
ty. Herein, we describe the synthesis of 4-aminopiperi-
dine derivatives of 1 as novel and potent avb3/aIIbb3
dual antagonists exhibiting improved water solubility.
We also explored the structure–activity relationships
of the derivatives. The potency of these compounds is
discussed in terms of not only receptor binding affinity,
but also activity in the cellular context.
The vitronectin receptor, integrin avb3, is a member of
the integrin superfamily of cell adhesion molecules,
and is expressed in many cell types, including osteo-
clasts, leukocytes, vascular smooth muscle cells, and
endothelial cells. Integrin avb3 binds a number of pro-
teins, including vitronectin, fibrinogen, and osteopontin,
through recognition of the tripeptide RGD sequence,1
and is involved in osteoporosis, cancer growth and
metastasis, diabetic retinopathy, rheumatoid arthritis,
and restenosis.2 Therefore, avb3 is considered as a poten-
tial drug target, and several groups are searching for
orally active, small-molecular antagonists as candidate
drugs to treat these chronic diseases.3
We are interested in integrin avb3 antagonists because
avb3 is associated with adhesion and migration of vascu-
lar smooth muscle cells and leukocytes. Further, Fab
2. Chemistry
Antagonists possessing a piperidine tricyclic pharmaco-
phore were generally synthesized as shown in Scheme 1.
Nucleophilic substitution6 of ethyl 4-fluorobenzoates
2a–e with 4-hydroxypiperidine gave the bicyclic com-
pounds 3a–e. The alcohols 3a–e were converted to
amines 4a–e, then introduction of a pyrimidine moiety
followed by basic hydrolysis gave compounds 5a–e
Keywords: Integrin avb3 antagonist; Integrin aIIbb3 antagonist; Acute
ischemic disease; 4-Aminopiperidine derivatives.
*
Corresponding author at present address: R&D Strategy, R&D
Planning & Management, Pharmaceutical, Meiji Seika Kaisha, Ltd.,
4-16, Kyobashi 2-Chome, Chuo-ku, Tokyo 104-8002, Japan. Tel.: +81
3 3273 3346; fax: +81 3 3273 3380; e-mail: keiichi_ajito@meiji.co.jp
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.061

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M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
Scheme 1. Reagents: (a) 4-hydroxypiperidine, DMSO; (b) (i) phthalimide, DEAD, THF; (ii) hydrazine; (c) (i) MsCl, Et3N, CH2Cl2; (ii) NaN3, DMF;
(iii) Pd/C, H2, dioxane; (d) 2-bromopyrimidine, i-Pr2EtN, DMSO; (e) NaOH, THF, MeOH, H2O; (f) N-Boc-2-isothiocyanatoethylamine, THF (for
15), N-Boc-2-isothiocyanatobutylamine, THF (for 19); (g) (i) EtBr, EtOH; (ii) TFA, H2O; (iii) NaOEt, EtOH; (h) (i) 2-chlorobenzimidazole (for 24),
2-chloroimidazo[4,5-b]pyridine (for 28); (ii) HBr, H2O; (i) ethyl 4-fluorobenzoate, DMSO; (j) (i) 6a, EDC, HOBT, N-methylmorpholine, DMF; (k)
TFA, CH2Cl2; (l) 10% Pd/C, H2, dioxane, H2O.
containing the tricyclic pharmacophore. The carboxylic
acids 5a–e were coupled with a diaminopropionate7 6a
(Fig. 1) to afford amides 7a–e. Removal of the t-butyl
group with TFA followed by hydrogenolysis of the
pyrimidine ring finally gave the desired molecules 1, 8,
9, 10, and 11. A hydroxyl derivative 12 (Table 2) was ob-
tained from the methoxyl derivative 7e by treatment
with BBr3. Synthesis of the antagonists 13 and 14 pos-
sessing an imidazolidinyl group required an alternative
Figure 1. Chemical structures of C-termini.

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
Table 1. Structure–activity relationships of the N-terminus
2111
H
N
N-terminus
N
NHSO2Ph
CO2H
H
N
O
Compound
N-terminus
IC50 (nM)
aIIbb3/avb3 ratio
Solubilityc
avb3
aIIbb3
VSMCa
hPRPb
N
1
13
18
22
27
31
1.3
3.0
190
52
290
63
2.31
0.08
0.36
1.58
1.03
1.11
<1
1
NH
N
0.66
0.59
1.2
0.051
0.21
1.9
NH
N
79
88
NT
<0.5
NT
1
7
NH
N
530
770
1300
400
>1000
65
NH
N
0.35
0.99
0.36
1.1
NH
N
HN
H2N
a Human vascular smooth muscle cell.
b Human platelet aggregation.
c Maximum concentration as the free form in 10% aq DMSO (mg/mL).
Table 2. Structure–activity relationships of the central aromatic ring
H
H
N
N
3
N
N
2
NHSO 2Ph
CO2H
H
N
O
Compound
Central aromatic ring
IC50 (nM)
VSMC
aIIbb3/avb3 ratio
Solubilitya
avb3
aIIbb3
hPRP
1
8
Unsubstituted
3-F
3-Cl
1.3
3
190
48
290
37
2.31
0.58
0.14
0.60
2.23
2.32
<1
1.5
0.5
0.5
>2
>2
0.36
0.17
0.16
0.44
0.19
0.21
0.023
0.096
0.98
0.44
9
72
90
94
10
12
11
2, 3-Di-F
3-OH
3-OMe
120
530
110
170
130
a Maximum concentration as the free form in 10% aq DMSO (mg/mL).
method, because the coupling reaction of the amine 4a
and 2-thiomethylimidazolidine was unsuccessful. After
several trials, N-Boc-2-isothiocyanatoethylamine8 was
found to couple with the amines 4a,b to afford thioureas
15a,b, which were cyclized to furnish the imidazolidines
16a,b. Compound 18 possessing a 4,5,6,7-tetrahydro-
1H-[1,3]diazepinyl group was analogously synthesized
with N-Boc-2-isothiocyanatobutylamine. In order to
synthesize benzimidazole 22, we tried to couple 2-chlo-
robenzimidazole with the amine 4a, but the reaction
did not proceed. Then, 2-chlorobenzimidazole was cou-
pled with the amine 23, followed by deprotection to
afford a tricyclic amine 24. The amine 24 was coupled
with ethyl 4-fluorobenzoate to construct the key inter-
mediate 25. 2-Chloroimidazo[4,5-b]pyridine9 was also
coupled with the amine 23 in order to prepare an aza-
benzimidazolyl derivative 27. For synthesis of the guani-
dine 31, the N-terminal functionality was introduced at
the final stage of the synthetic route shown in Scheme
2. After N-Boc protection of the amine 4a, hydrolysis
of the ester and amidation with 6a furnished an amide.
After selective deprotection of the Boc group with
HCl, the guanidine 31 was finally prepared by reaction
with the pyrazole reagent10 and then TFA.

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M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
were further evaluated in avb3-mediated cell adhesion
assay using VSMC (human vascular smooth muscle cell)
and human vitronectin, and in hPRP (human platelet
aggregation) inhibition assay, and their solubility was
measured. Although an acid or a base salt shows better
water solubility than its free form in general, the pH
range of solubility of salts of molecules which can form
zwitterions, such as our compounds, may be outside the
acceptable pH range for an injectable medicine. There-
fore, we initially checked the solubility of the free form
in 10% aq DMSO. The aqueous solubility of several
promising
quantitatively.
compounds
was
then
evaluated
Scheme 2. Reagents: (a) Boc2O, NaOH, dioxane, H2O; (b) NaOH,
THF, MeOH, H2O; (c) 6a, EDC, HOBT, N-methylmorpholine, DMF;
(d) HCl, MeOH; (e) 1H-pyrazole-1-carboxyamidine, i-Pr2EtN; (f)
TFA, CH2Cl2.
3.1. Structure–activity relationships (SAR)
Chemical modification of the N-terminus gave impor-
tant SAR data, as shown in Table 1. The cyclic guani-
dine analogues 1, 13, and 18 exhibited strong
inhibitory activity towards cell adhesion. Although the
imidazolidine analogue 13 showed the most potent
activity and the greatest solubility among these three
compounds, we selected tetrahydropyrimidine as the
N-terminus for further SAR studies, because compound
1 could be synthesized in fewer steps. On the other hand,
the benzimidazole 22 and the azabenzimidazole 27
exhibited only moderate activity in cell adhesion assay
in spite of their strong receptor binding inhibition.
Moreover, the benzimidazole 22 showed poor water sol-
ubility. The guanidine derivative 31 also exhibited only
moderate activity in cell assay. Thus, appropriate lipo-
philicity may be required for strong inhibition of cell
adhesion.
Next, the general procedure for compound 1 mentioned
above was applied to modification of the C-terminal moi-
ety. Thus, compounds 34–38 (Table 3) were synthesized
through amide bond formation of the carboxylic acid 5
or 25 with amines 6b–f (Fig. 1), respectively. The amines
6d and 6e possessing a hydroxyl group at the a-position
were prepared through reaction with the t-butyl ester of
L-isoserine or 4-amino-(2S)-hydroxybutyric acid11 that
was used in the synthesis of an MRSA-active aminoglyco-
side antibiotic, arbekacin.12 Hydrogenolysis of the pyrim-
idine ring in acetic acid as the final step in the synthesis of
compound 36 also gave 39 as a by-product. The cyclic
amino ester 6f was prepared as shown in Scheme 3. A 2-
chloroethyl moiety was introduced into the primary
amine 6a and then N-trifluoroacetylation afforded the tri-
substituted amine 40. Intramolecular cyclization and
deprotection of the trifluoroacetyl group finally gave the
desired cyclic amine 6f.
The SAR at the central aromatic ring is summarized in
Table 2. In the preceding paper of this report,5 deriva-
tives halogenated at the aromatic C-3 position exhibited
a stronger aVb3-inhibitory activity than C-2 analogues,
so we chose the C-3 position for modification. Introduc-
ing an electron-withdrawing group or an electron-do-
nating group at the C-3 position increased the activity
for receptor binding inhibition (compounds 8, 9, 12,
and 11). Therefore, enhancement of activity might be ex-
plained by the steric substitution effect. The crystallo-
graphic structure of compound 8 (Fig. 2) revealed a
substantial torsion angle between the central aromatic
ring and the piperidine ring. Therefore, the torsion an-
gles of various compounds substituted on the central
aromatic ring might be related to the inhibitory activity
on avb3 receptor binding. As an alternative possibility,
the substituent at the 3-position might fill a cavity of
the receptor site. The fluoro derivative 8 and the
methoxyl derivative 11 showed not only strong inhibi-
tion of cell adhesion, but also improved solubility. Fur-
thermore, selectivity for aIIbb3 was somewhat changed
by substitution at this position. The electron-withdraw-
ing chlorine group (9) increased aIIbb3-selectivity by
approximately 10 times.
In order to clarify precisely the SAR at the C-terminus
moiety, the convergent method was used (Scheme 4).
At first, the carboxylic acid 25 and the amine 6c were
coupled to afford an amide. Introducing a Boc group
onto the basic group was next planned to afford a pro-
tected compound. The Boc group was not introduced
at the 4-amino group of the piperidine moiety, but at
the imidazole ring, based on examination of the 1H
NMR spectrum of the product. The compound was
selectively reduced to the key intermediate, the a-amine
41, by mild hydrogenolysis using THF as a solvent. The
obtained amine 41 was reacted with several sulfonyl
chlorides to give derivatives 42–45. Hydrogenolysis of
the nitro group of 45 gave 46. The carboxylic acid 5b
was analogously converted to an amine intermediate
47, and the tetrahydropyrimidine derivatives 48–52 were
similarly synthesized. The dihydroxy derivative 53 was
also synthesized from the carboxylic acid 5e using a sim-
ilar methodology.
3. Results and discussion
Third, the SAR of the C-terminus was investigated. The
fundamental SAR of piperazine-containing molecules
was disclosed in the first paper of this series.5 (1) Mod-
ification of the amide bond decreased activity.13 (2) A
All novel compounds were initially evaluated by means
of avb3 and aIIbb3 receptor binding assay. Selected com-
pounds exhibiting strong inhibition in binding assay

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
Table 3. Structure–activity relationships of the C-terminus
2113
H
H
N
N
N
N
C-terminus
a
Compound
C-terminus
IC50 (nM)
pKa
avb3
aIIbb3
NHSO2Ph
H
N
1
1.3
3.0
12.1
—
CO2H
O
Me
H
N
NSO2Ph
CO2H
55
11
2.4
3.8
O
O
NHSO2Ph
CO2H
H
N
34b
35c
42c
48b
54c
39
12
12.1
12.8
11.6
17.2
—
NHCO2Bn
CO2H
H
N
1.3
0.35
O
O
O
O
O
NHSO2n-Bu
CO2H
H
N
0.53
0.59
NHAc
CO2H
H
N
29
39
NH2
H
N
280
260
9.1
CO2H
OAc
H
N
430
—
CO2H
OH
H
N
36
17
9.8
15.8
16.7
—
CO2H
O
O
H
N
CO2H
OH
37
23,000
8800
5000
NSO2Ph
CO2H
38c
1100
N
O
a Acidity at N–H or O–H.
b 3-Fluorobenzoyl derivative.
c Benzimidazole at the N-terminus.
substituent at the a-position of C-terminus was required
for strong activity. Therefore, we broadly surveyed the
scope of suitable a substituents, including hydrophilic
functionalities. As shown in Table 3, benzenesulfona-
mide-containing 1 was favorable among the compounds,
including 54 and 36, with a hydrophilic functionality.
Because the a-substituent has been considered as car-
boxylic acid-mimetic, corresponding to aspartic acid of
RGD, its acidity might be important for the activity.
The calculated pKa value14 of the a-substituent of each
molecule is shown in Table 3. The results support the
idea that the acidic proton of the sulfonamide is impor-
tant for avb3-inhibitory activity. The relationships can
be clearly seen by comparing 48 with 39, and 54 with
36, which have similar molecular size. Thus, N-methyla-
tion of the sulfonamide (55) dramatically decreased its
activity, as expected. On the other hand, electrostatic
interaction of the acidic N–H center with the exosite
has been proposed in the case of aIIbb3 antagonists.15
Because sulfonamide was considered most suitable as
the a substituent, substitution of the benzenesulfonyl

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M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
group by convergent synthesis was planned. Benzimid-
azole was fixed as the N-terminus because of it being
convenient for facile synthesis. As shown in Table 4,
compounds bearing an electron-withdrawing group (43
or 45) or an electron-donating group (46) with para
orientation exhibited enhanced inhibitory activity on
receptor binding. Replacement of the benzenesulfonyl
group with a bulky substituent (44) also increased the
activity. Next, further modifications were systematically
continued (Table 5) in order to improve water solubility.
Compounds having a hydrophilic functionality (an ami-
no group 49, a carboxyl group 50, and a hydroxyl group
52 and 53) showed good inhibitory activity in receptor
binding assay. This result indicates that a hydrophilic
functionality may be acceptable to the exosite, which
has been hypothesized to be a hydrophobic binding site
in the case of the aIIbb3 receptor.15 Furthermore, the
solubility of these compounds was improved. However,
compounds 50 and 53 showed decreased inhibitory
activity on avb3-mediated cell adhesion. The reason
why these water-soluble compounds showed weaker
activity at the cellular level is not clear, but appropriate
lipophilicity of the molecule might be required for inter-
action with cells.
Scheme 3. Reagents: (a) (i) 2-chloroacetaldehyde, NaB(CN)H3,
AcOH, CH2Cl2, MeOH; (ii) (CF3CO)2O, NaHCO3, dioxane; (b) (i)
DBU, DMF; (ii) concd NH4OH, dioxane.
3.2. Early absorption, distribution, metabolism, excretion,
and toxicity (ADMET) profile
Based on the SAR analysis, we selected compounds 8,
11, and 52, possessing both strong inhibition of avb3-
mediated cell adhesion and good water solubility
(>1.5 mg/mL). The water solubility was measured
quantitatively, and studies of the pharmacokinetics in
rats, acute toxicity in mice, and mutagenic activity were
conducted with these compounds. Water solubility was
evaluated at pH 4 and pH 8. All compounds showed
satisfactory pharmacokinetic parameters after intrave-
nous infusion drug and exhibited no toxicity.16 Com-
pound 52 showed insufficient water solubility, because
crystals were observed in water, although the other
two compounds showed acceptable water solubility
(Table 6).
Scheme 4. Reagents: (a) 6c, EDC, HOBT, N-methylmorpholine,
DMF; (b) Boc2O, Et3N, CH2Cl2; (c) (i) Pd/C, H2, THF; (d) R3Cl,
i-Pr2EtN, DMF; (e) TFA, CH2Cl2; (f) 10% Pd/C, H2, dioxane, H2O.
The selected avb3/aIIbb3 dual antagonists17 11 and 8
showed significant efficacy compared with a selective
avb3 antagonist or a selective aIIbb3 antagonist in a canine
Figure 2. X-ray crystallographic data of compound 8.
Table 4. Structure–activity relationships of the sulfonamide moiety, Part 1
H
H
N
N
N
N
NHR
H
N
CO2H
O
Compound
R
IC50 (nM)
avb3
aIIbb3
VSMC
hPRP
22
43
44
45
46
–SO2Ph
–SO2C6H4–4-F
1.2
1.9
530
300
480
190
620
400
550
0.26
0.13
0.35
0.49
0.28
0.35
0.60
0.53
–SO2C6H2–2,4,6-Tri-Me
–SO2C6H4–4-NO2
–SO2C6H4–4-NH2
>1000
>1000
>1000

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
Table 5. Structure–activity relationships of the sulfonamide moiety, Part 2
2115
H
H
N
N
R1
N
N
( )n
NHSO2R2
CO2H
H
N
O
Compound
R1
n
R2
IC50 (nM)
aIIbb3/avb3 ratio
Solubilitya
avb3
aIIbb3
VSMC
hPRP
8
49
50
51
52
14
53
F
1
1
1
1
1
0
1
–Ph
0.36
0.29
0.77
0.38
0.14
0.17
0.30
0.21
0.087
1.2
48
44
37
110
930
110
230
45
0.58
0.30
1.56
0.82
1.29
3.29
3.13
1
1
F
–C6H4–4-NH2
–C6H4–4-CO2H
–C6H4–4-OMe
–C6H4–4-OH
–Ph
F
660
150
53
<2
<1.5
>2
1
F
0.31
0.18
0.56
0.94
F
F
OH
88
390
–C6H4–4-OH
510
>2
a Maximum concentration as the free form in 10% aq DMSO (mg/mL).
Table 6. Quantitative water solubility and pharmacokinetics in ratsa of selected compounds
Compound
H2O
(mg/mL)
pH 4 Mcllvaine
(mg/mL)
pH 8 Mcllvaine
(mg/mL)
t1/2
CL
(mL/min/kg)
AUC
(lg min/mL)
Vss
(min)
(mL/kg)
1
8
<0.1
0.6
0.7
4.3
0.8
2.9
0.1
26
46
30
55
47
49
9.17
10.78
10.22
510
559
689
11
52
1.3
0.1
0.08
a Dosed at 0.5 mg/kg iv.
acute coronary syndrome (ACS) model.18 Furthermore,
compounds 11 and 8 showed no prolongation of the
bleeding time at the effective dose in canines.19 These data
indicated that the avb3/aIIbb3 dual antagonists 11 and 8
might be effective for treatment of reperfusion injury.
Recently, an avb3/aIIbb3 dual inhibitor was shown to be
effective against restenosis after PCTA in mice.20 There-
fore, compounds 11 and 8 might also be effective.
5. Experimental
1H NMR spectra were recorded on JNM-LA400 spec-
trometers with chemical shifts reported in ppm with
internal tetramethylsilane as a standard. Electron ioni-
zation (EI) mass spectra were recorded on a Hitachi
M-80B instrument. Fast-atom bombardment (FAB)
mass spectra were recorded on a JEOL JMS-700 instru-
ment. Thermospray (TSP) mass spectra were recorded
on a Hewlett-Packard 5989A instrument. Electrospray
ionization (ESI) mass spectra were recorded on a Hew-
lett-Packard 5989A instrument. Atmospheric pressure
chemical ionization (APCI) mass spectra were recorded
on a Hewlett-Packard 5989A instrument. High-resolu-
tion mass spectra (HRMS) were recorded under FAB
conditions. Optical rotations were obtained on a JASCO
DIP-370 polarimeter.
4. Conclusions
In summary, we identified a series of 4-aminopiperidine-
based molecules as novel avb3/aIIbb3 dual antagonists
that might be suitable for use as injectable drugs. Mod-
ifications of the prototype 1 indicated that cyclic guani-
dine at the N-terminus might be the most acceptable
structure from the viewpoints of both cell adhesion inhi-
bition and solubility. Introduction of one or two hydro-
philic moieties at the central aromatic ring and/or
benzene ring of the exosite-binding region led to im-
proved water solubility and enhanced cell adhesion inhi-
bition. The SAR suggested that the torsion angle
between the central aromatic ring and the piperidine
ring, and the acidity at the sulfonamide moiety, might
be important factors for inhibitory activity on avb3
receptor binding. Based on evaluation of early ADMET
profile and water solubility, the methoxyl derivative 11
and the fluoro derivative 8 were selected as avb3/aIIbb3
dual antagonists that might have utility in the treatment
of acute ischemic diseases. Further studies of these com-
pounds are ongoing.
5.1. Ethyl 4-(4-hydroxypiperidin-1-yl)benzoate (3a)
A mixture of 2a (7.20 mL, 49.5 mmol), 4-hydroxypiperi-
dine (5.00 g, 49.5 mmol), and DMSO (10 mL) was
warmed up to 120 °C and stirred for 3 days. The cooled
mixture was poured into H2O (200 mL) with stirring vig-
orously. The resulting precipitate was rinsed with H2O
(50 mL) twice and hexane (50 mL) to provide 3a
(9.60 g, 38.4 mmol, 77%) as a faint yellow solid: 1H
NMR (400 MHz, CDCl3) d 1.37 (3H, t, CH2CH3),
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, CH2CH3), 6.87
(2H, d, C6H4), 7.91 (2H, d, C6H4); EIMS m/z 249 (M)+.

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M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
Using the procedures described for preparing 3a from
2a, the following compounds were prepared.
br d, piperidine), 2.62 (2H, dq, piperidine), 2.96 (2H,
dt, piperidine), 4.01 (2H, br d, piperidine), 4.34 (2H, q,
CH2CH3), 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)+.
5.2. Methyl 3-fluoro-4-(4-hydroxypiperidin-1-yl)benzoate
(3b)
The compound 2b (33.2 g, 193 mmol) afforded 3b (42.3 g,
167 mmol, 87%) as a faint yellow solid: 1H NMR
(400 MHz, CDCl3) d 1.74 (2H, ddt, piperidine), 2.04
(2H, m, piperidine), 2.96 (2H, ddd, piperidine), 3.50
(2H, m, piperidine), 3.89 (4H, m, CO2Me and piperidine),
6.91 (1H, t, C6H3CO), 7.64 (1H, dd, C6H3CO), 7.72 (1H,
dd, C6H3CO); TSPMS m/z 254 (M+H)+.
To a suspension of this phthalimide (848 mg, 2.24 mmol)
in MeOH (56 mL) hydrazine monohydrate (4.5 mL) was
added. The mixture was stirred for 16 h at room temper-
ature and the precipitate was filtered by glass filter. The
filtrate was concentrated and the residue was purified
by flash column chromatography (CHCl3/MeOH/concd
NH4OH = 30:10:1) to afford 4a (551 mg, 2.22 mmol,
99%) as a colorless solid: 1H NMR (400 MHz, CD3OD)
d 1.35 (3H, t, CH2CH3), 1.43 (2H, dq, piperidine), 1.90
(2H, br d, piperidine), 2.84 (1H, m, piperidine), 2.89
(2H, br t, piperidine), 3.92 (2H, br d, piperidine), 4.28
(2H, q, CH2CH3), 6.94 (2H, d, C6H4), 7.84 (2H, d,
C6H4); TSPMS m/z 249 (M+H)+.
5.3. Methyl3-chloro-4-(4-hydroxypiperidin-1-yl)benzoate(3c)
The compound 2c (6.20 g, 32.9 mmol) afforded 3c (5.80 g,
21.6 mmol, 66%) as a colorless solid: 1H NMR (400 MHz,
CDCl3) d 1.74 (2H, ddt, piperidine), 2.04 (2H, m, piperi-
dine), 2.89 (2H, ddd, piperidine), 3.40 (2H, m, piperidine),
3.88 (1H, m, piperidine), 3.89 (3H, s, CO2Me), 7.02 (1H, d,
C6H3CO), 7.85 (1H, dd, C6H3CO), 8.01 (1H, d, C6H3CO);
TSPMS m/z 270 (M+H)+.
5.7. Methyl4-(4-aminopiperidin-1-yl)-3-fluorobenzoate(4b)
To a mixture of 3b (24.8 g, 97.8 mmol), Et3N (70.0 mL,
502 mmol), and CH2Cl2 (700 mL) MsCl (11.5 mL,
149 mmol) was added dropwise. The mixture was stir-
red for an additional 1 h. The mixture was poured into
water (1000 mL) and CHCl3, and extracted with
CHCl3 twice. The combined organic layer was dried
over anhydrous MgSO4, filtered, and evaporated. The
residue was added water (500 mL) and extracted with
organic layers (hexane/AcOEt/CH2Cl2 = 1:1:1) (1 L)
twice. The combined organic layer was dried over
anhydrous MgSO4, filtered, and evaporated to provide
5.4. Methyl 2,3-difluoro-4-(4-hydroxypiperidin-1-yl)ben-
zoate (3d)
The compound 2d (5.00 g, 26.3 mmol) afforded 3d (4.73 g,
17.5 mmol, 67%) as a colorless solid, which was purified
by flash column chromatography (hexane/AcOEt = 2:3):
1H NMR (400 MHz, CDCl3) d 1.73 (2H, m, piperidine),
2.04 (2H, m, piperidine), 3.03 (2H, ddd, piperidine), 3.54
(2H, m, piperidine), 3.90 (3H, s, Me), 3.91 (1H, m, piper-
idine), 6.68 (1H, ddd, Ar), 7.61 (1H, ddd, Ar); EIMS m/z
271 (M)+.
methyl 3-fluoro-4-{4-(methanesulfonyloxy)piperidin-
1-yl}benzoate (29.9 g, 90.2 mmol, 92%).
5.5. Methyl 4-(4-hydroxypiperidin-1-yl)-3-meth-
oxybenzoate (3e)
To this (10.0 g, 27.6 mmol) DMF (50 mL) and NaN3
(3.87 g, 59.5 mmol) were added. The mixture was heated
at 80 °C for 5 h. The cooled mixture was poured into
water (1 L) and extracted with AcOEt twice. The com-
bined organic layer was washed with brine (500 mL)
twice, dried over anhydrous MgSO4, filtered, and evapo-
rated. The residue was added water (1 L) and extracted
with hexane twice. The combined organic layer was dried
over anhydrous MgSO4, filtered, and evaporated to pro-
vide methyl 4-(4-azidopiperidin-1-yl)-3-fluorobenzoate
The compound 2e (903 mg, 4.90 mmol) afforded 3e
(605 mg, 2.28 mmol, 47%) as a colorless solid: 1H NMR
(400 MHz, CDCl3) d 1.76 (2H, ddt, piperidine), 2.04
(2H, br ddd, piperidine), 2.86 (2H, ddd, piperidine),
3.47 (2H, tt, piperidine), 3.87 (1H, m, piperidine), 3.88
(3H, s, CO2Me), 3.92 (3H, s, C6H3OMe), 6.92 (1H, d,
C6H3CO), 7.51 (1H, d, C6H3CO), 7.62 (1H, dd,
C6H3CO); TSPMS m/z 266 (M+H)+.
1
(7.75 g, 27.8 mmol, 100%) as a brown syrup: H NMR
(400 MHz, CDCl3) d 1.82 (2H, ddt, piperidine), 2.05
(2H, m, piperidine), 2.99 (2H, ddd, piperidine), 3.47
(2H, m, piperidine), 3.62 (1H, tt, piperidine), 3.88 (3H,
s, CO2Me), 6.92 (1H, t, C6H3CO), 7.66 (1H, dd, C6H3
CO), 7.74 (1H, dd, C6H3CO); TSPMS m/z 279 (M+H)+.
5.6. Ethyl 4-(4-aminopiperidin-1-yl)benzoate (4a)
A mixture of 3a (2.00 g, 8.02 mmol), phthalimide (2.36 g,
16.0 mmol), and P(n-Bu)3 (4.00 mL, 16.0 mmol) in ben-
zene was cooled in an ice bath. To this 1,10-(azodicarbon-
yl)dipiperidine (4.04 g, 16.0 mmol) was added. The
cooling bath was removed and the mixture was stirred
for an additional 23 h. After dilution with H2O
(300 mL), the mixture was extracted with CH2Cl2 three
times. The combined organic layer was dried over anhy-
drous Na2SO4, filtered, and evaporated. The resulting
residue was purified by flash column chromatography
(CHCl3/acetone = 50:1) to give the phthalimide (1.57 g,
4.09 mmol, 51%) as a colorless solid: 1H NMR
(400 MHz, CDCl3) d 1.38 (3H, t, CH2CH3), 1.82 (2H,
To a solution of the azide (7.50 g, 26.9 mmol), 1,4-diox-
ane (190 mL), AcOH (27 mL), and water (54 mL) 10%
Pd/C (750 mg) was added and the mixture was hydroge-
nated under H2 for 4 h at room temperature. The mix-
ture was filtered through Celite, and solids were
washed with MeOH. The filtrate was concentrated.
The residue was dissolved in 0.5 M HCl (1 L) and
washed with AcOEt (500 mL) twice. The aqueous layer
was cooled to 0 °C, alkalized to pH 14 using concd
NH4OH, warmed to room temperature, saturated with

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2117
NaCl, and extracted with CH2Cl2 (1 L) three times. The
combined organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated to provide 4b (5.78 g,
22.9 mmol, 85%) as a faint yellow solid: 1H NMR
(400 MHz, CDCl3) d 1.54 (2H, dq, piperidine), 1.94
(2H, br d, piperidine), 2.85 (3H, m, piperidine), 3.58
(2H, dt, piperidine), 3.88 (3H, s, CO2Me), 6.92 (1H, t,
C6H3CO), 7.65 (1H, dd, C6H3CO), 7.73 (1H, dd,
C6H3CO); TSPMS 253 (M+H)+.
Step2:Toasolutionofthisbenzoate(100 mg, 0.306 mmol)
in a mixture of THF (9.0 mL) and MeOH (3.0 mL)
1 M NaOH (3.0 mL) was added. The reaction mixture
was stirred for 8 h at 40 °C, and concentrated. The
residue was purified using silica gel flash column chroma-
tography (CHCl3/MeOH/concd NH4OH = 30:10:1) to
give 5a (75.0 mg, 0.251 mmol, 82%) as a colorless
1
solid: H 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)+.
Using the procedures described for preparing 4b from
3b, the following compounds were prepared.
5.8. Methyl 4-(4-aminopiperidin-1-yl)-3-chlorobenzoate (4c)
Using the procedures described for preparing 5a from
4a, the following compounds were prepared.
The compound 3c afforded 4c (595 mg, 2.22 mmol, 10% in
three steps) as a colorless solid: H NMR (400 MHz,
1
CD3OD) d 1.84 (2H, dq, piperidine), 2.12 (2H, m, piperi-
dine), 2.86 (2H, dt, piperidine), 3.28 (1H, m, piperidine),
3.56 (2H, br d, piperidine), 3.88 (3H, S, CO2Me), 7.19
(1H, d, C6H3CO), 7.90 (1H, dd, C6H3CO), 7.98 (1H, d,
C6H3CO); TSPMS m/z 269 (M+H)+.
5.12. 3-Fluoro-4-{4-(pyrimidin-2-ylamino)piperidin-1-
yl}benzoic acid (5b)
The amine 4b (5.78 g, 22.9 mmol) afforded 5b (4.96 g, 15.7
mmol, 78% in two steps) as a colorless solid: H NMR
1
(400 MHz, DMSO-d6) d 1.63 (2H, br q, piperidine), 1.96
(2H, br d, piperidine), 2.88 (2H, br t, piperidine), 3.53
(2H, br d, piperidine), 3.89 (1H, m, piperidine), 6.54
(1H, t, pyrimidine), 7.08 (1H, t, C6H3CO), 7.54 (1H, dd,
C6H3CO), 7.66 (1H, dd, C6H3CO), 8.26 (2H, d, pyrimi-
dine); TSPMS m/z 317 (M+H)+.
5.9. Methyl 4-(4-aminopiperidin-1-yl)-2,3-difluorobenzo-
ate (4d)
The alcohol 3d afforded 4d (2.10 g, 7.78 mmol, 49% in
three steps) as a colorless solid: H NMR (400 MHz,
1
CDCl3) d 1.52 (2H, m, piperidine), 1.93 (2H, m, piperi-
dine), 2.83–2.93 (3H, m, piperidine), 3.62 (2H, br d,
piperidine), 3.90 (3H, s, Me), 6.66 (1H, ddd, Ar), 7.60
(1H, ddd, Ar); ESIMS m/z 271 (M+H)+.
5.13. 3-Chloro-4-{4-(pyrimidin-2-ylamino)piperidin-1-
yl}benzoic acid (5c)
The amine 4c afforded 5c (109 mg, 0.328 mmol, 34% in
two steps) as a colorless solid: 1H NMR (400 MHz,
DMSO-d6) d 1.67 (2H, dq, piperidine), 1.97 (2H, m,
piperidine), 2.81 (2H, br t, piperidine), 3.41 (2H, br d,
piperidine), 3.89 (1H, br d, piperidine), 6.54 (1H, t,
pyrimidine), 7.19 (1H, t, C6H3CO), 7.81 (1H, dd,
C6H3CO), 7.85 (1H, d, C6H3CO), 8.26 (2H, d, pyrimi-
dine); FABMS m/z 333 (M+H)+.
5.10. Methyl 4-(4-aminopiperidin-1-yl)-3-methoxybenzo-
ate (4e)
The alcohol 3e afforded 4e as a colorless solid: 1H NMR
(400 MHz, CDCl3) d 1.57 (2H, dq, piperidine), 1.95 (2H,
br d, piperidine), 2.70 (2H, dt, piperidine), 2.82 (1H, tt,
piperidine), 3.57 (2H, br d, piperidine), 3.88 (3H, s,
OMe), 3.92 (3H, s, OMe), 6.91 (1H, d, C6H3), 7.50
(1H, d, C6H3), 7.65 (1H, dd, C6H3).
5.14. 2,3-Difluoro-4-{4-(pyrimidin-2-ylamino)piperidin-1-
yl}benzoic acid (5d)
5.11. 4-{4-(Pyrimidin-2-ylamino)piperidin-1-yl}benzoic
acid (5a)
The amine 4d afforded 5d (402 mg, 1.20 mmol, 44% in two
steps) as a colorless solid: 1H NMR (400 MHz, DMSO-
d6) d 1.63 (2H, m, piperidine), 1.97 (2H, br d, piperidine),
2.97 (2H, br t, piperidine), 3.59 (2H, br d, piperidine), 3.92
(1H, m, piperidine), 6.56 (1H, t, pyrimidine), 6.90 (1H, br
t, Ar), 7.58 (1H, br t, Ar), 8.28 (2H, d, pyrimidine); ESIMS
m/z 335 (M+H)+.
Step 1: To a solution of 4a (250 mg, 1.01 mmol) in DMF
(10 mL) 2-bromopyrimidine (240 mg, 1.51 mmol) and i-
Pr2NEt (0.90 mL, 5.05 mmol) were added. The reaction
mixture was heated at 125 °C for 10 h. The cooled mix-
ture was added CH2Cl2 and brine, and extracted three
times. The organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated. The resulting residue
was purified by silica gel flash column chromatography
(hexane/AcOEt = 1:1) to give ethyl 4-{4-(pyrimidin-2-
ylamino)piperidin-1-yl}benzoate (212 mg, 0.649 mmol,
5.15. 3-Methoxy-4-{4-(pyrimidin-2-ylamino)-piperidin-1-
yl}benzoic acid (5e)
The amine 4e afforded 5e (261 mg, 0.796 mmol, 44%
from 3e) as a colorless solid: 1H NMR (400 MHz,
DMSO-d6) d 1.64 (2H, dq, piperidine), 1.93 (2H, br d,
piperidine), 2.70 (2H, br t, piperidine), 3.50 (2H, br d,
piperidine), 3.83 (3H, s, C6H3OMe), 3.85 (1H, m, piper-
idine), 6.55 (1H, t, pyrimidine), 6.93 (1H, d, C6H3CO),
7.41 (1H, d, C6H3CO), 7.49 (1H, dd, C6H3CO), 8.27
(2H, d, pyrimidine); ESIMS m/z 329 (M+H)+.
1
64%) as a colorless solid: H NMR (400 MHz, CDCl3)
d 1.37 (3H, t, CH2CH3), 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, CH2CH3), 6.55 (1H, t, pyrimidine), 6.89
(2H, d, C6H4), 7.92 (2H, d, C6H4), 8.28 (2H, d, pyrimi-
dine); EIMS m/z 326 (M)+.

2118
M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
5.16. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(pyrimi-
din-2-ylamino)piperidin-1-yl}benzoylamino]propionate
(7a)
(400 MHz, CDCl3) d 1.29 (9H, s, t-Bu), 1.75 (2H, br q,
piperidine), 2.21 (2H, br d, piperidine), 2.91 (2H, br t,
piperidine), 3.47 (2H, br d, piperidine), 3.56 (1H, ddd,
CONHCH2CH), 3.90 (2H, m, CONHCH2CH), 4.03
(1H, m, piperidine), 6. 55 (1H, t, pyrimidine), 7.07 (1H,
d, C6H3CO), 7.51 (2H, m, C6H5), 7.58 (1H, m, C6H5),
7.65 (1H, dd, C6H3CO), 7.84 (1H, dd, C6H3CO), 7.86
The carboxylic acid 5a (6.00 mg, 0.0201 mmol), i-Pr2NEt
(5.3 lL, 0.0302 mmol), and (benzotriazol-1-yloxy)tris
(dimethylamino)phosphonium
hexafluorophosphate
(BOP) (13.3 mg, 0.0302 mmol) were dissolved in a mixture
of CH2Cl2 (1.0 mL) and DMF (1.0 mL), and stirred for 3 h
at room temperature. To the mixture, a solution of hydro-
chloride salt of t-butyl (2S)-N-benzenesulfonyl-2,3-diami-
nopropionate 6a7 (8.10 mg, 0.0241 mmol) in CH2Cl2
(1.0 mL) was added at ꢀ10 °C. The solution was added i-
Pr2NEt (5.3 lL, 0.0302 mmol) and stirred an additional
2 h at the same temperature. The solvent was removed in
vacuo and the residue was purified by silica gel preparative
TLC (CHCl3/MeOH = 10:1) to give 7a (7.00 mg, 0.0121
(2H, br d, C6H5), 8.29 (2H, d, pyrimidine); TSPMS m/z
25
615 (M+H)+; ½aꢁ +49° (c 1.0, CH2Cl2).
D
5.19. t-Butyl (2S)-benzenesulfonylamino-3-[2,3-difluoro-
4-{4-(pyrimidin-2-ylamino)piperidin-1-yl}benzoylami-
no]propionate (7d)
The carboxylic acid 5d (130 mg, 0.389 mmol) afforded
7d (239 mg, 0.388 mmol, 100%) as a colorless solid: H
1
NMR (400 MHz, CDCl3) d 1.29 (9H, s, t-Bu), 1.70
(2H, br q, piperidine), 2.19 (2H, br s, piperidine), 3.01
(2H, br t, piperidine), 3.59 (2H, br d, piperidine),
3.67–3.86 (2H, m, CONHCH2CH), 3.97–4.07 (2H, m,
CONHCH2CH and piperidine), 6.54 (1H, t, pyrimi-
dine), 6.74 (1H, br t, C6H2F2), 7.46 (2H, br t, C6H5),
7.53 (1H, br t, C6H5), 7.68 (1H, br t, C6H2F2), 7.85
1
mmol, 60%) as a colorless solid: H 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, piperi-
dine), 3.90 (2H, m, CONHCH2CH), 4.05 (1H, m, piper-
idine), 6.55 (1H, t, pyrimidine), 6.91 (2H, d, C6H4), 7.49
(2H, m, C6H5), 7.57 (1H, m, C6H5), 7.70 (2H, d, C6H4),
(2H, m, C6H5), 8.30 (2H, d, pyrimidine); ESIMS m/z
25
617 (M+H)+; ½aꢁ +39° (c 1.0, CHCl3).
7.86 (2H, m, C6H5), 8.29 (2H, d, pyrimidine); TSPMS
D
25
m/z 581 (M+H)+; ½aꢁ +24° (c 0.35, MeOH).
D
5.20. t-Butyl (2S)-benzenesulfonylamino-3-[3-methoxy-4-
{4-(pyrimidin-2-ylamino)piperidin-1-yl}benzoylamino]pro-
pionate (7e)
5.17. t-Butyl (2S)-benzenesulfonylamino-3-[3-fluoro-4-{4-
(pyrimidin-2-ylamino)piperidin-1-yl}benzoylamino]propio-
nate (7b)
The carboxylic acid 5e (50.0 mg, 0.152 mmol) afforded 7e
(92.5 mg, 0.152 mmol, 100%) as a colorless solid: 1H
NMR (400 MHz, CDCl3) d 1.29 (9H, s, t-Bu), 1.76 (2H,
br dq, piperidine), 2.20 (2H, br d, piperidine), 2.85 (2H,
br t, piperidine), 3.53 (3H, m, piperidine and CON-
HCH2CH), 3. 93 (1H, m, CONHCH2CH), 3.95 (3H, s,
C6H3OMe), 4.02 (1H, m, piperidine), 6.54 (1H, t, pyrim-
idine), 6.95 (1H, d, C6H3CO), 7.33 (1H, dd, C6H3CO),
7.41 (1H, d, C6H3CO), 7.50 (2H, br t, C6H5), 7. 58 (1H,
Hydrochloride salts of the propionate 6a (1.17 g,
3.48 mmol) and 5b (1.00 g, 3.16 mmol) were suspended
in DMF (30 mL). HOBt (512 mg, 3.79 mmol), NMM
(1.04 mL, 9.48 mmol), and EDC (727 mg, 3.79 mmol)
were added and the reaction mixture was stirred for
13 h. The mixture was added a mixture of saturated solu-
tion of NaHCO3 (300 mL) and aqueous K2CO3 (100 mL)
and extracted with CH2Cl2 (200 mL) three times. The
organic layer was washed with brine (500 mL), dried over
anhydrous Na2SO4, filtered, and evaporated. The residue
was purified by silica gel flash column chromatography
(CH2Cl2/MeOH = 25:1) to give 7b (1.77 g, 2.95 mmol,
93%) as a colorless solid: 1H NMR (400 MHz, CDCl3) d
1.23 (9H, s, t-Bu), 1.74 (2H, br q, piperidine), 2.12 (2H,
br d, piperidine), 2.94 (2H, br t, piperidine), 3.49 (1H,
dd, CONHCH2CH), 3.59 (2H, br d, piperidine), 3.66
(1H, dd, CONHCH2CH), 3.95 (1H, m, piperidine), 4.12
(1H, br t, CONHCH2CH), 6.59 (1H, t, pyrimidine),
7.08 (1H, t, C6H3CO), 7.47 (3H, m, C6H3CO and
C6H5), 7.54 (2H, m, C6H3CO and C6H5), 7.83 (2H, d,
br t, C6H5), 7.86 (2H, br d, C6H5), 8.29 (2H, d, pyrimi-
22
dine); FABMS m/z 611 (M+H)+; ½aꢁ +22° (c 1.0,
D
MeOH).
5.21. (2S)-Benzenesulfonylamino-3-[4-{4-(1,4,5,6-tetrahy-
dropyrimidin-2-ylamino)piperidin-1-yl}benzoylamino]pro-
pionic acid (1)
Step 1: The compound 7a (7.00 mg, 0.0121 mmol) was
dissolved in CH2Cl2 (0.3 mL) and cooled in an ice bath,
to which TFA (0.3 mL) was added. The mixture was stir-
red at room temperature for 2 h. The solvent was evapo-
rated in vacuo and the residue was purified by silica gel
preparative TLC (CHCl3/MeOH/concd NH4OH =
30:10:1), and then LH-20 (MeOH) to give (2S)-benze-
nesulfonylamino-3-[4-{4-(pyrimidin-2-ylamino)piperidin-
1-yl}benzoylamino]propionic acid (6.30 mg, 0.0120
C6H5), 8.27 (2H, d, pyrimidine); TSPMS m/z 599
25
D
(M+H)+; ½aꢁ +26° (c 0.52, MeOH).
Using the procedures described for preparing 7b from
5b, the following compounds were prepared.
1
mmol, 99%) as a colorless solid: H NMR (400 MHz,
5.18. t-Butyl (2S)-benzenesulfonylamino-3-[3-chloro-4-{4-
(pyrimidin-2-ylamino)piperidin-1-yl}benzoylamino]propi-
onate (7c)
CD3OD) d 1.56 (2H, dq, piperidine), 1.99 (2H, br d, piper-
idine), 2.90 (2H, br t, piperidine), 3.46 (1H, dd, CON-
HCH2CH), 3.56 (1H, dd, CONHCH2CH), 3.63 (1H,
dd, CONHCH2CH), 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
The carboxylic acid 5c (107 mg, 0.321 mmol) afforded 7c
(168 mg, 0.274 mmol, 85%) as a colorless solid: 1H NMR

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2119
(2H, d, C6H4), 7.75 (2H, m, C6H5), 8.16 (2H, d, pyrimi-
173.4; TSPMS m/z 547 (M+H)+; FAB-HMS (M+H)+
calcd for C25H31FN6O5S: 547.2139, found: 547.2148;
½aꢁ +79° (c 0.56, DMSO).
23
dine); FABMS m/z 525 (M+H)+; ½aꢁ +65° (c 0.36,
D
26
D
MeOH).
Step 2: To a solution of the above compound (3.60 mg,
0.00686 mmol), concd HCl (0.5 mL), and AcOH
(5.0 mL), 10% Pd/C (1.8 mg) was added and the mixture
was hydrogenated under H2 (50 psi) in a Parr shaker for
2.5 h at room temperature. The mixture was filtered
through Celite, and solids were washed with MeOH twice
and H2O twice. The filtrate was concentrated. Toluene
was added and the mixture was again concentrated in vac-
uo. The residue was purified by silica gel preparative TLC
(CH2Cl2/EtOH/concd NH4OH/H2O = 8:8:1:1), and then
LH-20 (MeOH) to yield 1 (3.20 mg, 0.00605 mmol, 88%)
as a colorless solid: 1H NMR (400 MHz, CD3OD) d 1.49
(2H, br q, piperidine), 1.85 (4H, m, piperidine and tetra-
hydropyrimidine), 2.84 (2H, br t, piperidine), 3.26 (4H,
t, tetrahydropyrimidine), 3.45 (2H, m, CONHCH2CH
and piperidine), 3.53 (1H, dd, CONHCH2CH), 3.63
(1H, dd, CONHCH2CH), 3.73 (2H, br d, piperidine),
6.85 (2H, d, C6H4), 7.37 (2H, m, C6H5), 7.44 (1H, m,
Using the procedures described for preparing 8 from 7b,
the following compounds were prepared.
5.23. (2S)-Benzenesulfonylamino-3-[3-chloro-4-{4-(1,4,5,6-
tetrahydropyrimidin-2-ylamino)piperidin-1-yl}benzoylami-
no]propionic acid (9)
The compound 7c afforded 9 (15.5 mg, 0.0267 mmol,
36% in two steps) as a colorless solid: 1H NMR
(400 MHz, CD3OD) d 1.73 (2H, m, piperidine), 1.97
(2H, quintet, tetrahydropyrimidine), 2.04 (2H, m, piper-
idine), 2.84 (2H, br t, piperidine), 3.37 (4H, br t, tetrahy-
dropyrimidine), 3.43 (2H, br d, piperidine), 3.48 (1H, m,
piperidine), 3.56 (1H, dd, CONHCH2CH), 3.66 (1H, dd,
CONHCH2CH), 3.74 (1H, dd, CONHCH2CH), 7.15
(1H, d, pyrimidine), 7.47 (2H, m, C6H5), 7.53 (1H, m,
C6H5), 7.72 (1H, dd, C6H3CO), 7.85 (3H, m, C6H3CO
and C6H5); TSPMS m/z 563 (M+H)+; FAB-HMS
C6H5), 7.59 (2H, d, C6H4), 7.75 (2H, m, C6H5); FABMS
(M+H)+ calcd for C25H31ClN6O5S: 563.1843, found:
20
m/z 529 (M+H)+; ½aꢁ +69° (c 0.16, MeOH).
25
D
563.1830; ½aꢁ +64° (c 0.20, MeOH).
D
5.22. (2S)-Benzenesulfonylamino-3-[3-fluoro-4-{4-(1,4,5,6-
tetrahydropyrimidin-2-ylamino)piperidin-1-yl}benzoylami-
no]propionic acid (8)
5.24. (2S)-Benzenesulfonylamino-3-[2,3-difluoro-4-{4-(1,4,5,6-
tetrahydropyrimidin-2-ylamino)piperidin-1-yl}benzoylami-
no]propionic acid (10)
Step 1: The t-butyl ester 7b (600 mg, 1.00 mmol) was dis-
solved in CH2Cl2 (10 mL), added TFA (10 mL), and
stirred at room temperature for 4 h. The solvent was
evaporated in vacuo to give the TFA salt of (2S)-benze-
nesulfonylamino-3-[3-fluoro-4-{4-(pyrimidin-2-ylamino)pip-
eridin-1-yl}benzoylamino]propionic acid as a colorless
solid.
The compound 7d afforded 10 (87.2 mg, 0.155 mmol,
77% in two steps) as a colorless solid: 1H NMR
(400 MHz, DMSO-d6) d 1.54 (2H, m, piperidine), 1.81
(2H, quintet, tetrahydropyrimidine), 1.87 (2H, br d,
piperidine), 2.73 (2H, br t, piperidine), 3.08 (1H, br t,
CONHCH2CH), 3.21 (4H, br t, tetrahydropyrimidine),
3.43 (2H, m, piperidine), 3.80 (1H, m, CONHCH2CH),
6.87 (1H, br t, C6H2F2), 7.51 (1H, br t, C6H2F2), 7.56
(2H, m, C6H5), 7.63 (1H, m, C6H5), 7.84 (2H, m,
C6H5); ESIMS m/z 565 (M+H)+; FAB-HMS (M+H)+
Step 2: To a solution of TFA salt of this compound, 1,4-
dioxane (10 mL), and H2O (1.0 mL) 10% Pd/C (120 mg)
were added, and the mixture was hydrogenated under
H2 for 24 h at room temperature. The mixture was fil-
tered through Celite, and solids were washed with 1,4-
dioxane/H2O (10:1). The filtrate was concentrated. The
residue was purified by flash column chromatography
(CH2Cl2/EtOH/concd NH4OH/ H2O = 8:8:1:1), and
then LH-20 (MeOH) to give 8 (451 mg, 0.820 mmol,
82% in two steps) as a colorless solid: 1H NMR
(400 MHz, CD3OD) d 1.68 (2H, br ddd, J = 11.8 Hz,
11.8 Hz, 12.7 Hz, piperidine), 1.96 (2H, quintet,
J = 5.8 Hz, tetrahydropyrimidine), 2.02 (2H, br d,
J = 12.7 Hz, piperidine), 2.87 (2H, br dd, J = 11.8 Hz,
12.3 Hz, piperidine), 3.37 (4H, t, J = 5.8 Hz, tetrahydro-
pyrimidine), 3.48 (1H, m, piperidine), 3.52 (2H, br d,
J = 12.3 Hz, piperidine), 3.57 (1H, dd, J = 7.6 Hz,
13.9 Hz, CONHCH2CH), 3.65 (1H, dd, J = 5.1 Hz,
13.9 Hz, CONHCH2CH), 3.73 (1H, dd, J = 5.1 Hz,
7.6 Hz, CONHCH2CH), 7.04 (1H, t, J = 8.6 Hz,
C6H3CO), 7.53 (5H, m, C6H3CO and C6H5), 7.86 (2H,
br d, J = 7.3 Hz, C6H5); 13C NMR (DMSO-d6) d 19.8,
31.7, 31.8, 37.8, 42.6, 47.1, 48.9, 55.4, 114.5 (d,
JCF = 21.5 Hz), 118.4 (d, JCF = 2.5 Hz), 123.6, 126.6,
127.9 (d, JCF = 6.6 Hz), 128.9, 132.2, 140.8, 142.2 (d,
JCF = 8.3 Hz), 152.1, 153.8 (d, JCF = 245.6 Hz), 164.2,
calcd for C25H30F2N6O5S: 565.2045, found: 565.2033;
25
D
½aꢁ +92° (c 0.60, DMSO).
5.25. (2S)-Benzenesulfonylamino-3-[3-methoxy-4-{4-(1,4,
5,6-tetrahydropyrimidin-2-ylamino)piperidin-1-yl}benzoyl-
amino]propionic acid (11)
The compound 7e afforded 11 (16.6 mg, 0.0297 mmol,
63% in two steps) as a colorless solid: 1H NMR
(400 MHz, DMSO-d6) d 1.39–1.43 (2H, m, piperidine),
1.73–1.80 (4H, m, piperidine and tetrahydropyrimidine),
2.39–2.47 (2H, m, piperidine), 3.08–3.15 (2H, m, CON-
HCH2CH and piperidine), 3.19 (4H, m, tetrahydropyr-
imidine), 3.28 (2H, m, piperidine), 3.48 (1H, m,
CONHCH2CH), 3.60 (1H, m, CONHCH2CH), 3.68
(3H, s, C6H3OMe), 6.77 (1H, d, J = 8.7 Hz, C6H3CO),
7.26 (1H, d, J = 8.7 Hz, C6H3CO), 7.28 (1H, s,
C6H3CO), 7.53–7.57 (2H, m, C6H5), 7.61 (1H, m,
C6H5), 7.83 (1H, d, J = 6.8 Hz, C6H5), 7.84 (1H, d,
J = 6.8 Hz, C6H5), 8.31 (1H, br t, J = 5.0 Hz,
CONHCH2CH), 8.71 (1H, br s, NH), 9.42 (1H, br d,
J = 7.6 Hz, NH); 3C NMR (DMSO-d6) 19.8, 31.8,
32.1, 37.8, 42.6, 47.3, 48.9, 49.0, 55.3, 55.4, 110.5,
117.2, 119.6, 126.6, 128.3, 129.0, 132.3, 140.8, 143.9,

2120
M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
151.3, 152.0, 165.4, 173.5; FABMS m/z 559 (M+H)+;
5.28. Ethyl 4-[4-[3-{2-(t-butoxycarbonylamino)ethyl}thio-
ureido]piperidin-1-yl]-3-fluorobenzoate (15b)
FAB-HMS (M+H)+ calcd for C26H34N6O6S: 559.2339,
25
D
found: 599.2343; ½aꢁ +76° (c 0.83, MeOH/concd
NH4OH (10:1)).
The title compound was synthesized from 4b following
the general procedure for 15a: FABMS m/z 455 (M+H)+.
5.26. (2S)-Benzenesulfonylamino-3-[3-hydroxy-4-{4-(1,4,5,
6-tetrahydropyrimidin-2-ylamino)piperidin-1-yl}benzoyla-
mino]propionic acid (12)
5.29. 4-{4-(4,5-Dihydro-1H-imidazol-2-ylamino)piperi-
din-1-yl}benzoic acid (16a)
To a solution of 7e (23.7 mg, 0.0388 mmol) and
dichloroethane (3.6 mL) BBr3 (1.0 M in CH2Cl2,
0.70 mL, 0.700 mmol) was added. The mixture was
warmed to 40 °C and stirred for 7 h. A mixture of
1,4-dioxane (1.0 mL), water (0.1 mL), and NH4OH
(1.0 mL) was added to the mixture. The concentrated
residue was added water (20 mL) and washed with
CH2Cl2 twice. The aqueous layer was evaporated
and the residue was purified by CHP-20P (Mitsubishi
Chemical Corporation) (MeOH:water = 3:7) to prepare
(2S)-benzenesulfonylamino-3-[3-hydroxy-4-{4-(pyrimi-
din-2-ylamino)piperidin-1-yl}benzoylamino]propionic
acid (8.40 mg, 0.0155 mmol, 40%) as a colorless sol-
id: 1H NMR (400 MHz, CD3OD) d 1.79 (2H, dq,
piperidine), 2.10 (2H, br d, piperidine), 2.81 (2H, br
t, piperidine), 3.46 (2H, br d, piperidine), 3.54 (1H,
dd, CONHCH2CH), 3.66 (1H, dd, CONHCH2CH),
3.77 (1H, dd, CONHCH2CH), 3.93 (1H, m, piperi-
dine), 6.59 (1H, t, pyrimidine), 7.05 (1H, d,
C6H3CO), 7.27 (2H, m, C6H3CO), 7.45 (2H, br t,
C6H5), 7.52 (1H, br t, C6H5), 7.85 (2H, br d, C6H5),
Compound 15a (14.2 mg, 0.0315 mmol) was dissolved in
a solution of bromoethane (0.5 mL) and EtOH (0.5 mL).
The mixture was heated at reflux for 13 h under argon.
The solvent was removed in vacuo. The residue was puri-
fied by silica gel preparative TLC (CH2Cl2/MeOH = 7:1)
to afford the crude compound.
The resulting compound was dissolved in TFA (1.0 mL)
and water (0.5 mL), and stirred at room temperature for
3 h. The residue after concentration was dissolved in
EtOH (1.0 mL) and added dropwise over 3 h to a solu-
tion of sodium ethoxide (18.0 mg, 0.261 mmol) in EtOH
(1.0 mL). The system was stirred under argon for an
additional 16 h, at which point the solvent was removed
in vacuo. The residue was purified by silica gel prepara-
tive TLC (CH2Cl2/MeOH = 7:1) to yield ethyl 4-{4-(4,5-
dihydro-1H-imidazol-2-ylamino)piperidin-1-yl}benzoate
(6.10 mg, 0.0193 mmol, 61% from 15a) as a colorless oil:
1H NMR (400 MHz, CDCl3) d 1.37 (3H, t, CO2Et), 1.76
(2H, br q, piperidine), 2.02 (2H, br d, piperidine),
2.95 (2H, ddd, piperidine), 3.53 (1H, m, piperidine),
3.70 (4H, s, CH2CH2), 3.76 (2H, br d, piperidine), 4.32
(2H, q, CO2Et), 6.81 (2H, d, C6H4CO), 7.89 (2H, d,
C6H4CO); TSPMS m/z 317 (M+H)+.
8.27 (2H, d, pyrimidine); APCIMS m/z 541 (M+H)+;
26
D
½aꢁ +22° (c 0.42, MeOH).
The title compound 12 (3.50 mg, 0.00643 mmol, 43%) was
synthesized from this compound following the general
procedure for 8 (Step 2): 1H NMR (400 MHz, CD3OD)
d 1.74 (2H, br dq, piperidine), 1.99 (4H, m, piperidine
and tetrahydropyrimidine), 2.74 (2H, br t, piperidine),
3.37 (4H, t, tetrahydropyrimidine), 3.44 (3H, m, piperi-
dine), 3.56 (1H, dd, CONHCH2CH), 3.64 (1H, dd, CON-
HCH2CH), 3.74 (1H, dd, CONHCH2CH), 6.98 (1H, d,
C6H3CO), 7.27 (2H, m, C6H3CO), 7.47 (2H, br t, C6H5),
7.54 (1H, br t, C6H5), 7.86 (2H, br d, C6H5); ESIMS m/z
The ester (1.27 g, 4.00 mmol) afforded 16a (1.15 g,
4.00 mmol, 100%) as a colorless solid following the gen-
eral procedure for 5a (Step 2): 1H NMR (400 MHz,
CD3OD) d 1.64 (2H, dq, piperidine), 2.03 (2H, br d,
piperidine), 2.96 (2H, br t, piperidine), 3.53 (1H, m,
piperidine), 3.72 (4H, s, CH2CH2), 3.88 (2H, br d, piper-
idine), 6.95 (2H, d, C6H4CO), 7.86 (2H, d, C6H4CO);
FABMS m/z 289 (M+H)+.
545 (M+H)+; FAB-HMS (M+H)+ calcd for
5.30. 3-Fluoro-4-{4-(4,5-dihydro-1H-imidazol-2-yl-
amino)piperidin-1-yl}benzoic acid (16b)
23
D
C25H32N6O6S: 545.2182, found: 545.2178; ½aꢁ +40° (c
0.18, MeOH).
The title compound (49.0 mg, 0.153 mmol, 74% from 4b)
was synthesized from 15b and N-Boc-4-isothio-
cyanatobutylamine following the general procedure for
5.27. Ethyl 4-[4-[3-{2-(t-butoxycarbonylamino)ethyl}thio-
ureido]piperidin-1-yl]benzoate (15a)
1
16a: H NMR (400 MHz, CD3OD) d 1.74 (2H, br dq,
N-Boc-2-isothiocyanatoethylamine (29.0 mg, 0.145 mmol)
was added to a solution of 4a (30.0 mg, 0.121 mmol) in
THF (0.5 mL). The solution was stirred for 19 h under ar-
gon. The solvent was removed in vacuo. The product was
purified by silica gel preparative TLC (CH2Cl2/MeOH =
7:1), and then LH-20 (MeOH) to yield 15a (50.1 mg,
0.111 mmol, 92%) as a colorless solid: 1H NMR
(400 MHz, CDCl3) d 1.37 (3H, t, CO2Et), 1.42 (9H, s, t-
Bu), 1.62 (2H, m, piperidine), 2.15 (2H, m, piperidine),
3.03 (2H, br t, piperidine), 3.31 (2H, m, CH2CH2), 3.55
(2H, m, CH2CH2), 3.85 (2H, br d, piperidine), 4.33 (2H, q,
CO2Et), 4.98 (1H, m, piperidine), 6.97 (2H, d, C6H4CO),
7.92 (2H, d, C6H4CO); TSPMS m/z 451 (M+H)+.
piperidine), 2.06 (2H, br d, piperidine), 2.92 (2H, br t,
piperidine), 3.51 (1H, m, piperidine), 3.61 (2H, br d,
piperidine), 3.73 (4H, s, CH2CH2), 7.07 (1H, t,
C6H3CO), 7.62 (1H, d, C6H3CO), 7.75 (1H, d, C6H3CO).
5.31. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(4,5-
dihydro-1H-imidazol-2-ylamino)piperidin-1-yl}benzoyl-
amino]propionate (17a)
The carboxylic acid 16a (28.0 mg, 0.100 mmol), and 1,10-
carbonyldiimidazole (CDI) (16.0 mg, 0.100 mmol) were
dissolved in DMF (2.0 mL) and stirred for 1.5 h at room
temperature. The mixture was added hydrochloride salt

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2121
of t-butyl (2S)-N-benzenesulfonyl-2,3-diaminopropio-
5.35. 4-[4-[3-{2-(t-Butoxycarbonylamino)butyl}thio-
ureido]piperidin-1-yl]-3- fluorobenzoate (19a)
nate 6a (60.0 mg, 0.200 mmol) and warmed up to
60 °C. After 15 h, the solvent was removed in vacuo
and the residue was purified by silica gel preparative
TLC (CHCl3/MeOH = 8:2) to give 17a (16.6 mg,
0.0291 mmol, 29%) as a colorless solid: 1H NMR
(400 MHz, CD3OD) d 1.23 (9H, s, t-Bu), 1.63 (2H, m,
piperidine), 2.02 (2H, br d, piperidine), 2.96 (2H, br t,
piperidine), 3.48 (1H, dd, CONHCH2CH), 3.53 (1H,
m, piperidine), 3.64 (1H, dd, CONHCH2CH), 3.73
(4H, s, CH2CH2), 3.89 (2H, br d, piperidine), 4.11 (1H,
dd, CONHCH2CH), 6.98 (2H, d, C6H4CO), 7.47 (2H,
The title compound was synthesized from 4a and N-
Boc-4-isothiocyanatobutylamine following the general
procedure for 15a: FABMS m/z 479 (M+H)+.
5.36. 4-{4-(4,5,6,7-Tetrahydro-1H-[1,3]diazepin-2-yl-
amino)piperidin-1-yl}benzoic acid (20a)
The title compound (4.50 mg, 0.0129 mmol, 35% from
4a) was synthesized from 19a following the general pro-
cedure for 16a: FABMS m/z 317 (M+H)+.
t, C6H5), 7.54 (1H, t, C6H5), 7.67 (2H, d, C6H4CO),
27
D
7.83 (2H, br d, C6H5); ½aꢁ +20° (c 0.40, MeOH).
5.37. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(4,5,6,7-
tetrahydro-1H-[1,3] diazepin-2-ylamino)piperidin-1-
yl}benzoylamino]propionate (21a)
5.32. t-Butyl (2S)-benzenesulfonylamino-3-[3-fluoro-4-{4-
(4,5-dihydro-1H-imidazol-2-ylamino)piperidin-1-yl}-
benzoylamino]propionate (17b)
The title compound (2.00 mg, 0.00334 mmol, 26%) was
synthesized from 20a (4.5 mg, 0.0129 mmol) following
the general procedure for 17a: 1H NMR (400 MHz,
CDCl3) d 1.26 (9H, s, t-Bu), 1.55 (2H, m, piperidine), 1.61
(4H, m, CH2CH2CH2CH2), 1.94 (2H, br d, piperidine),
2.85(2H,brt,piperidine),3.22(4H,m,CH2CH2CH2CH2),
3.52 (2H, m, piperidine), 3.70 (1H, m, CONHCH2CH),
3.78 (1H, m, CONHCH2CH), 3.88 (1H, m, piperidine),
4.09 (1H, dd, CONHCH2CH), 6.59 (2H, d, C6H4CO),
7.41 (2H, br t, C6H5), 7.49 (1H, br t, C6H5), 7.66 (2H, d,
C6H4CO),7.85(2H,brd,C6H5);TSPMSm/z599(M+H)+.
The title compound (8.20 mg, 0.0139 mmol, 4%) was syn-
thesized from 16b (100 mg, 0.312 mmol) following the
general procedure for 17a: 1H NMR (400 MHz, CD3OD)
d 1.23 (9H, s, t-Bu), 1.72 (2H, m, piperidine), 2.06 (2H, br
d, piperidine), 2.89 (2H, br t, piperidine), 3.47 (1H, dd,
CONHCH2CH), 3.49 (1H, m, piperidine), 3.57 (2H, br
d, piperidine), 3.66 (1H, dd, CONHCH2CH), 3.73 (4H,
s, CH2CH2), 4.12 (1H, dd, CONHCH2CH), 7.06 (1H, t,
C6H3CO), 7.48 (2H, m, C6H5), 7.54 (2H, m, C6H5 and
C6H3CO), 7.67 (1H, ddt, C6H3CO), 7.83 (2H, br d,
C6H5); TSPMS m/z 589 (M+H)+.
5.38. (2S)-Benzenesulfonylamino-3-[4-{4-(4,5,6,7-tetra-
hydro-1H-[1,3]diazepin-2-ylamino)piperidin-1-yl}ben-
zoylamino]propionic acid (18)
5.33. (2S)-Benzenesulfonylamino-3-[4-{4-(4,5-dihydro-1H-
imidazol-2-ylamino)piperidin-1-yl}benzoylamino]propionic
acid (13)
The title compound (1.70 mg, 0.00314 mmol, 94%) was
synthesized from 21a following the general procedure
for 1 (Step 1): H NMR (400 MHz, CD3OD) d 1.61
The compound 17a (28.0 mg, 0.0490 mmol) afforded 13
(11.0 mg, 0.0214 mmol, 44%) as a colorless solid follow-
ing the general procedure for 1 (Step 1): 1H NMR
(400 MHz, DMSO-d6) d 1.42 (2H, br q, piperidine),
1.85 (2H, m, piperidine), 2.72 (2H, m, piperidine), 3.08
(1H, br t, CONHCH2CH), 3.35 (2H, m, CONHCH2CH
and piperidine), 3.56 (4H, s, CH2CH2), 3.64 (3H, m,
piperidine and CONHCH2CH), 6.89 (2H, d, C6H4CO),
7.54 (2H, br t, C6H5), 7.60 (3H, m, C6H5 and
C6H4CO), 7.81 (2H, br d, C6H5); TSPMS m/z 515
1
(2H, br q, piperidine), 1.70 (4H, m, CH2CH2CH2CH2),
1.98 (2H, br d, piperidine), 2.94 (2H, ddd, piperidine),
3.28 (4H, m, CH2CH2CH2CH2), 3.60 (3H, m, CON-
HCH2CH and piperidine), 3.73 (1H, dd, CON-
HCH2CH), 3.86 (2H, br d, piperidine), 6.96 (2H, d,
C6H4CO), 7.48 (2H, br t, C6H5), 7.54 (1H, br t,
C6H5), 7.71 (2H, d, C6H4CO), 7.86 (2H, br d, C6H5);
FABMS m/z 543 (M+H)+; FAB-HMS (M+H)+ calcd
25
D
(M+H)+; FAB-HMS (M+H)+ calcd for C24H30N6O S:
for C26H34N6O5S: 543.2390, found: 543.2401; ½aꢁ
5
23
D
+53° (c 0.24, MeOH/concd NH4OH = 10:1).
515.2077, found: 515.2070; ½aꢁ +31° (c 0.16, DMSO).
5.34. (2S)-Benzenesulfonylamino-3-[3-fluoro-4-{4-(4,5-dihy-
dro-1H-imidazol-2-ylamino)piperidin-1-yl}benzoylamino]-
propionic acid (14)
5.39. 4-(1H-Benzimidazol-2-ylamino)piperidine (24)
2-Chlorobenzimidazole (5.00 g, 32.8 mmol) and 23
(11.2 mL, 65.5 mmol) were combined and heated at
170 °C for 5 h. The mixture was cooled to room temper-
ature and added heated CHCl3 (200 mL). The resulting
solution was added aqueous NaHCO3 (500 mL). After
extraction twice, the combined organic layer was dried
over anhydrous Na2SO4, filtered, and evaporated. The
residue was added AcOEt (100 mL) and the resulting
precipitate was filtered by glassfilter. The solids were
rinsed with AcOEt (100 mL) and dried to give the cou-
pled compound (6.80 g, 23.5 mmol, 72%) as a faint
brown solid: 1H NMR (400 MHz, CD3OD) d 1.27
(3H, t, CH2CH3), 1.47 (2H, ddd, piperidine), 2.07 (2H,
The title compound (7.40 mg, 0.0139 mmol, 48%) was
synthesized from 17b following the general procedure
for 1 (Step 1): 1H NMR (400 MHz, CD3OD) d 1.74 (2H,
dd, piperidine), 2.06 (2H, br d, piperidine), 2.89 (2H, br t,
piperidine), 3.47 (1H, m, piperidine), 3.49 (1H, dd, CON-
HCH2CH),3.57(2H,brd,piperidine),3.70(1H,dd,CON-
HCH2CH), 3.73 (4H, s, CH2CH2), 4.07 (1H, dd,
CONHCH2CH),7.05(1H,t,C6H3CO),7.49(5H,m,C6H5
and C6H3CO), 7.83 (2H, br d, C6H5); TSPMS m/z 533
(M+H)+; FAB-HMS (M+H)+ calcd for C24H29FN6O5S:
23
D
533.1982,found:533.1988;½aꢁ +37°(c0.06,MeOH).

2122
M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
br d, piperidine), 3.03 (2H, m, piperidine), 3.79 (1H, tt,
piperidine), 4.13 (4H, m, piperidine and CH2CH3),
7.03 (2H, dd, benzimidazole), 7.23 (2H, dd, benzimid-
azole); TSPMS m/z 289 (M+H)+.
azole), 7.17 (1H, m, benzimidazole), 7.28 (1H, m,
benzimidazole), 7.37 (2H, t, C6H5), 7.48 (1H, t, C6H5),
7.58 (2H, d, C6H4), 7.77 (2H, br d, C6H5); TSPMS m/z
24
619 (M+H)+; ½aꢁ +27° (c 0.97, MeOH).
D
This compound (150 mg, 0.520 mmol) was dissolved in
47% HBr (2.5 mL) and heated at reflux. After 7.5 h,
the solvent was removed in vacuo. The residue was
purified by silica gel flash column chromatography
(CHCl3/MeOH/concd NH4OH = 30:10:1) to give 24
(91.0 mg, 0.421 mmol, 81%) as a colorless solid: 1H NMR
(400 MHz, CD3OD) d 1.47 (2H, dq, piperidine), 2.07 (2H,
m,piperidine),2.74(2H,dt,piperidine),3.10(2H,dt,piper-
idine), 3.72 (1H, tt, piperidine), 6.95 (2H, dd, benzimid-
azole),7.17(2H,dd,benzimidazole);EIMSm/z216(M)+.
5.42. (2S)-Benzenesulfonylamino-3-[4-{4-(1H-
benzimidazol-2-ylamino)piperidin-1-yl}benzoylamino]-
propionic acid (22)
The title compound (36.3 mg, 0.0645 mmol, 67%) was
synthesized from the amide 26 (60.0 mg, 0.0970 mmol)
following the general procedure for 1 (Step 1): 1H
NMR (400 MHz, CD3OD) d 1.54 (2H, br q, piperidine),
2.01 (2H, br d, piperidine), 2.95 (2H, br t, piperidine),
3.34 (2H, m, CONHCH2CH), 3.85 (2H, m, piperidine
and CONHCH2CH), 3.86 (2H, br d, piperidine), 6.89
(2H, m, benzimidazole), 6.96 (2H, d, C6H4), 7.14 (2H,
dd, benzimidazole), 7.47 (2H, m, C6H5), 7.54 (1H, br
t, C6H5), 7.62 (2H, d, C6H4), 7.76 (2H, br d, C6H5);
5.40. 4-{4-(1H-Benzimidazol-2-ylamino)piperidin-1-
yl}benzoic acid (25)
A mixture of 2a (0.770 mL, 5.23 mmol), 24 (1.13 g, 5.23
mmol), and DMSO (5.2 mL) was heated to 140 °C and
stirred for 6.5 h, then cooled. The residue was partitioned
between CH2Cl2 and brine (300 mL). The aqueous phase
was further extracted with CH2Cl2. The organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated.
The resulting residue was purified by silica gel flash
column chromatography (CH2Cl2/MeOH = 25:1) to
provide ethyl 4-{4-(1H-benzimidazol-2-ylamino)piperi-
din-1-yl}benzoate (941 mg, 2.58 mmol, 49%) as a col-
orless solid: 1H NMR (400 MHz, CD3OD) d 1.36
(3H, t, CH2CH3), 1.63 (2H, m, piperidine), 2.13 (2H,
br d, piperidine), 3.07 (2H, br t, piperidine), 3.84
(1H, tt, piperidine), 3.92 (2H, br d, piperidine), 4.30
(2H, q, CH2CH3), 6.97 (2H, br d, C6H4), 7.00 (2H,
dd, benzimidazole), 7.21 (2H, dd, benzimidazole),
7.87 (2H, br d, C6H4); TSPMS m/z 365 (M+H)+.
TSPMS m/z 563 (M+H)+; FAB-HMS (M+H)+ calcd
26
D
for C28H30N6O5S: 563.2077, found: 563.2072; ½aꢁ
+80° (c 1.1, MeOH/concd NH4OH = 9:1).
5.43. 4-{(1H-Imidazo[4,5-b]pyridin-2-yl)amino}piperidine (28)
The title compound (226 mg, 1.04 mmol, 24% in two steps)
was synthesized from 2-chloro-1H-imidazo[4,5-b]pyri-
dine9 following the general procedure for 24, except that
28 was purified by Amberlyst 15 (MeOH/concd NH4OH/
water = 4:3:1): 1H NMR (400 MHz, CD3OD) d 1.50
(2H, dq, piperidine), 2.07 (2H, br d, piperidine), 2.74
(2H, dt, piperidine), 3.10 (2H, dt, piperidine), 3.80 (1H,
tt, piperidine), 6.96 (1H, dd, C6H3N), 7.46 (1H, dd,
C6H3N), 7.92 (1H, dd, C6H3N); TSPMS m/z 218 (M+H)+.
5.44. 4-[4-{(1H-Imidazo[4,5-b]pyridin-2-yl)amino}piperi-
din-1-yl]benzoic acid (29)
To a solution of this benzoate (103 mg, 0.283 mmol) in a
mixture of THF (9.0 mL) and MeOH (3.0 mL) 1 M
NaOH (3.0 mL) was added. The reaction mixture was
stirred for 3.5 h at 40 °C and concentrated. The residue
was purified using silica gel flash column chromatogra-
phy (CHCl3/MeOH/concd NH4OH = 30:10:1) to give
25 (92.3 mg, 0.274 mmol, 97%) as a faint yellow solid:
1H NMR (400 MHz, DMSO-d6) d 1.53 (2H, br q, piper-
idine), 2.02 (2H, br d, piperidine), 3.00 (2H, br t, piper-
idine), 3.83 (1H, m, piperidine), 3.89 (2H, br d,
piperidine), 6.84 (2H, m, benzimidazole), 6.97 (2H, d,
C6H4), 7.11 (2H, m, benzimidazole), 7.75 (2H, d,
C6H4); TSPMS m/z 337 (M+H)+.
The title compound was synthesized from 28 following
the general procedure for 25: FABMS m/z 338 (M+H)+.
5.45. t-Butyl (2S)-benzenesulfonylamino-3-[4-[4-{(1H-
imidazo[4,5-b]pyridin-2-yl)amino}piperidin-1-yl]benzoyl-
amino]propionate (30)
The title compound (83.2 mg, 0.134 mmol, 23% from 28)
was synthesized from 29 following the general procedure
for 7b: 1H NMR (400 MHz, DMSO-d6) d 1.12 (9H, s, t-
Bu), 1.56 (2H, br q, piperidine), 2.01 (2H, br d, piperi-
dine), 2.95 (2H, m, piperidine), 3.31 (1H, m, CONHC
H2CH), 3.44 (1H, m, CONHCH2CH), 3.87 (3H, m, piper-
idine), 4.20 (1H, br t, CONHCH2CH), 6.88 (1H, m,
C6H3N), 6.96 (2H, d, C6H4CO), 7.36 (1H, m, C6H3N),
7.51 (2H, t, C6H5), 7.57 (1H, t, C6H5), 7.64 (2H, d,
5.41. t-Butyl (2S)-benzenesulfonylamino-3-[4-{4-(1H-
benzimidazol-2-ylamino)piperidin-1-yl}benzoylamino]-
propionionate (26)
C6H4CO), 7.76 (2H, br d, C6H5), 8.17 (1H, t, C6H3N);
24
TSPMS m/z 620 (M+H)+; ½aꢁ +29° (c 0.85, MeOH).
The title compound (62.2 mg, 0.101 mmol, 67%) was
synthesized from 25 (50.0 mg, 0.149 mmol) following
the general procedure for 7b: 1H NMR (400 MHz,
CDCl3) d 1.23 (9H, s, t-Bu), 1.26 (2H, m, piperidine),
1.93 (2H, br d, piperidine), 2.72 (2H, t, piperidine),
3.46 (2H, br d, piperidine), 3.64 (1H, m, piperidine),
3.81 (2H, m, CONHCH2CH), 4.07 (1H, dd, CON-
HCH2CH), 6.59 (2H, d, C6H4), 7.00 (2H, dd, benzimid-
D
5.46. (2S)-Benzenesulfonylamino-3-[4-[4-{(1H-imi-
dazo[4,5-b]pyridin-2-yl)amino}piperidin-1-yl]benzoyl-
amino]propionic acid (27)
The title compound (38.0 mg, 0.0675 mmol, 99%) was
synthesized from 30 (42.0 mg, 0.0679 mmol) following

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2123
the general procedure for 1 (Step 1): 1H NMR (400 MHz,
5.49. (2S)-Benzenesulfonylamino-3-{4-(4-guanigino-
piperidin-1-yl)benzoylamino}propionic acid (31)
DMSO-d6) d 1.57 (2H, br q, piperidine), 2.01 (2H, br d,
piperidine), 2.96 (2H, t, piperidine), 3.33 (1H, m, CON-
HCH2CH), 3.45 (1H, m, CONHCH2CH), 3.89 (3H, m,
piperidine), 3.96 (1H, t, CONHCH2CH), 6.85 (1H, br t,
C6H3N), 6.96 (2H, d, C6H4CO), 7.38 (1H, dd, C6H3N),
7.46 (2H, m, C6H5), 7.54 (1H, br t, C6H5), 7.62 (2H, d,
C6H4CO), 7.75 (2H, m, C6H5), 8.18 (1H, br t, C6H3N);
To a suspension of the amine 33 (18.8 mg, 0.0374 mmol)
in a mixture of 1,4-dioxane (0.080 mL) and water
(0.080 mL) i-Pr2NEt (27 lL, 0.150 mmol) and 1H-pyra-
zole-1-carboxamidine nitrate (22.0 mg, 0.150 mmol) were
added. The mixture was stirred for 31 h at room temper-
ature. After removing the solvent, the residue was purified
by silica gel preparative TLC (CHCl3/MeOH/concd
NH4OH = 30:10:1) to give the guanidine (17.1 mg,
0.0314 mmol, 84%) as a colorless solid: 1H NMR
(400 MHz, CD3OD) d 1.23 (9H, s, t-Bu), 1.62 (2H, dq,
piperidine), 2.04 (2H, br d, piperidine), 2.99 (2H, ddd,
piperidine), 3.48 (1H, dd, CONHCH2CH), 3.63 (2H, m,
CONHCH2CH and piperidine), 3.88 (2H, br d, piperi-
dine), 4.12 (1H, dd, CONHCH2CH), 6.98 (2H, br d,
C6H4CO), 7.47 (2H, br t, C6H5), 7.54 (1H, br t, C6H5),
FABMS m/z 564 (M+H)+; FAB-HMS (M+H)+ calcd
23
D
for C29H29N7O5S: 564.2029, found: 564.2022; ½aꢁ +53°
(c 0.35, MeOH/concd NH4OH = 10:1).
5.47. 4-[4-{(t-Butoxycarbonyl)amino}piperidin-1-yl]ben-
zoic acid (32)
A mixture of 4a (154 mg, 0.621 mmol), 1,4-dioxane
(2.0 mL), H2O (1.0 mL), 1 M NaOH (1.0 mL), and
Boc2O (170 mg, 0.778 mmol) was stirred for 0.5 hr and
concentrated. To this THF (9.0 mL), MeOH (3.0 mL),
and 1 M NaOH (6.0 mL) were added. The mixture
was stirred for 4.5 h at 50 °C, then cooled to room tem-
perature and acidified to pH 6 using 1 M aqueous HCl.
The mixture was added H2O (100 mL) and extracted
with AcOEt three times. The organic layer was dried
over anhydrous MgSO4, filtered, and evaporated to af-
ford 32 (168 mg, 0.527 mmol, 85% in two steps) as a col-
orless solid: 1H NMR (400 MHz, CD3OD) d 1.44 (9H, s,
t-Bu), 1.51 (2H, m, piperidine), 1.93 (2H, br d, piperi-
dine), 2.95 (2H, dt, piperidine), 3.55 (1H, m, piperidine),
3.88 (2H, br d, piperidine), 6.94 (2H, d, C6H4), 7.85 (2H,
d, C6H4); EIMS m/z 320 (M)+.
7.68 (2H, d, C6H4CO), 7.83 (2H, m, C6H5); FABMS
25
D
m/z 544 (M+H)+; ½aꢁ +24° (c 0.85, MeOH).
The ester (17.0 mg, 0.0312 mmol) afforded 31 (10.2 mg,
0.0209 mmol, 67%) following the general procedure for
1 (Step 1): 1H NMR (400 MHz, CD3OD) d 1.61 (2H, br
q, piperidine), 2.01 (2H, br d, piperidine), 2.96 (2H, t,
piperidine), 3.60 (3H, m, CONHCH2CH and piperidine),
3.74 (1H, dd, CONHCH2CH), 3.85 (2H, br d, piperidine),
6.96 (2H, br d, C6H4CO), 7.47 (2H, br t, C6H5), 7.54 (1H,
br t, C6H5), 7.70 (2H, br d, C6H4CO), 7.85 (2H, dd, C6H5);
TSPMS m/z 489 (M+H)+; FAB-HMS (M+H)+ calcd for
23
D
C22H28N6O5S: 489.1920, found: 489.1924; ½aꢁ +126° (c
0.05, DMSO).
5.48. t-Butyl 3-{4-(4-aminopiperidin-1-yl)benzoylamino}-
(2S)-(benzenesulfonylamino)propionate (33)
5.50. t-Butyl 3-amino-(2S)-hydroxypropionate (6d)
The carboxylic acid 32 (81.0 mg, 0.254 mmol) afforded t-
butyl (2S)-benzenesulfonylamino-3-[4-[4-{(t-butoxycar-
bonyl)amino}piperidin-1-yl]benzoylamino]propionate
(111 mg, 0.184 mmol, 73%) following the procedure for
In a Fisher–Porter tube, to a mixture of L-isoserine
(4.00 g, 38.1 mmol) and DME (76 mL) concd H2SO4
(3.2 mL) was added. The mixture was cooled to
ꢀ78 °C and added isobutylene (38 mL). The tube was
sealed and the cooling bath was removed. After 48 h,
isobutylene was removed at room temperature. The mix-
ture was poured into ice/water (128 mL) and washed
with Et2O twice. The aqueous phase was alkalized to
pH 7 using 6 M aqueous NaOH at 0 °C, saturated with
NaCl, and extracted with CHCl3 (100 mL) four times.
The combined extracts were dried over anhydrous
Na2SO4, filtered, and evaporated. The residue was puri-
fied by silica gel flash column chromatography (CHCl3/
MeOH/concd NH4OH = 400:20:1 to 100:10:1) to pre-
pare 6d (191 mg, 1.19 mmol, 3%) as a colorless crystal:
1H NMR (400 MHz, CDCl3) d 1.50 (9H, s, t-Bu), 2.91
(1H, dd, CH2NH2), 3.02 (1H, dd, CH2NH2), 4.05 (1H,
dd, COCH); FABMS m/z 162 (M+H)+.
1
7b: H NMR (400 MHz, CD3OD) d 1.28 (9H, s, t-Bu),
1.45 (9H, s, t-Bu), 1.48 (2H, br d, piperidine), 2.05
(2H, br d, piperidine), 2.95 (2H, br t, piperidine), 3.55
(1H, ddd, CONHCH2CH), 3.68 (1H, m, piperidine),
3.77 (2H, br d, piperidine), 3.89 (2H, m, CON-
HCH2CH), 6.89 (2H, d, C6H4), 7.50 (2H, m, C6H5),
7.57 (1H, m, C6H5), 7.69 (2H, d, C6H4), 7.84 (2H, m,
C6H5); TSPMS m/z 603 (M+H)+.
The amide (10.0 mg, 0.0166 mmol) and saturated HCl/
MeOH were combined and stirred at room temperature
for 4 h. The mixture was poured into a mixture of
MeOH (5.0 mL) and concd NH4OH (5.0 mL). The sol-
vent was evaporated in vacuo and the residue was puri-
fied by silica gel preparative TLC (CHCl3/MeOH/concd
NH4OH = 30:10:1) to give 33 (7.00 mg, 0.0139 mmol,
84%) as a colorless solid: 1H NMR (400 MHz, CD3OD)
d 1.22 (9H, s, t-Bu), 1.47 (2H, dq, piperidine), 1.93 (2H,
br d, piperidine), 2.87 (3H, m, piperidine), 3.49 (1H, dd,
CONHCH2CH), 3.64 (1H, dd, CONHCH2CH), 3.88
(2H, br d, piperidine), 4.11 (1H, dd, CONHCH2CH),
6.95 (2H, br d, C6H4CO), 7.47 (2H, br t, C6H5), 7.54
(1H, br t, C6H5), 7.65 (2H, br d, C6H4CO), 7.83 (2H,
m, C6H5); TSPMS m/z 502 (M+H)+.
5.51. t-Butyl 4-amino-(2S)-t-butoxybutylate (6e)
The title compound (161 mg, 0.697 mmol, 2%) was syn-
thesized from 4-amino-(2S)-hydroxybutylic acid11
(4.00 g, 33.6 mmol) following the general procedure 6d;
1H NMR (400 MHz, CDCl3) d 1.20 (9H, s, t-Bu), 1.46
(9H, s, CO2-t-Bu), 1.75 (2H, q, CH2CH2NH2), 2.76
(1H, ddd, CH2NH2), 2.83 (1H, ddd, CH2NH2), 3.95
(1H, dd, COCH); TSPMS m/z 232 (M+H)+.

2124
M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
5.52. (2R)-Benzenesulfonylamino-3-[3-fluoro-4-{4-(1,4,5,6-
tetrahydropyrimidin-2-ylamino)piperidin-1-yl}benzoyl-
amino]propionic acid (34)
dine), 3.74 (1H, dd, CONHCH2), 3.78 (2H, br d, piper-
idine), 3.80 (1H, dd, CONHCH2), 5.06 (1H, dd,
CONHCH2CH), 6.92 (2H, d, C6H4), 7.69 (2H, d,
C6H4); TSPMS m/z 432 (M+H)+; FAB-HMS (M+H)+
25
D
The title compound (53.4 mg, 0.0978 mmol, 58% in
three steps) was synthesized from 5b and 6b following
the general procedure for 8: 1H NMR (400 MHz,
CD3OD) d 1.68 (2H, br q, piperidine), 1.96 (2H, quintet,
tetrahydropyrimidine), 2.02 (2H, br d, piperidine), 2.87
(2H, br t, piperidine), 3.37 (4H, t, tetrahydropyrimi-
dine), 3.48 (1H, m, piperidine), 3.52 (2H, br d, piperi-
dine), 3.57 (1H, dd, CONHCH2CH), 3.65 (1H, dd,
CONHCH2CH), 3.73 (1H, dd, CONHCH2CH), 7.04
(1H, t, C6H3CO), 7.53 (5H, m, C6H3CO and C6H5),
7.86 (2H, br d, C6H5); TSPMS m/z 547 (M+H)+;
calcd for C21H29N5O5: 432.2247, found: 432.2245; ½aꢁ
ꢀ23° (c 0.10, MeOH).
5.56. (2S)-Hydroxy-4-[4-{4-(1,4,5,6-tetrahydropyrimidin-
2-ylamino)piperidin-1-yl}benzoylamino]butylic acid (37)
The title compound was synthesized from 5a and 6e fol-
lowing the general procedure for 1: 1H NMR (400 MHz,
CD3OD–20% ND4OD/D2O = 5:1) d 1.60 (2H, m, piper-
idine), 1.86 (1H, ddd, CONHCH2CH2), 1.96 (2H, quin-
tet, tetrahydropyrimidine), 2.03 (2H, br d, piperidine),
2.07 (1H, m, CONHCH2CH2), 2.98 (2H, br t, piperi-
dine), 3.38 (4H, t, tetrahydropyrimidine), 3.44–3.62
(3H, m, piperidine, CONHCH2), 3.83 (2H, br d, piperi-
dine), 4.00 (1H, dd, CONHCH2CH2CH), 7.00 (2H, d,
C6H4), 7.73 (2H, d, C6H4); TSPMS m/z 404 (M+H)+;
FAB-HMS (M+H)+ calcd for C25H31FN6O5S:
22
D
547.2139, found: 547.2148; ½aꢁ ꢀ100° (c 0.32, MeOH/
CH2Cl2 = 1:1).
5.53. 3-[4-{4-(1H-Benzimidazol-2-ylamino)piperidin-1-yl}-
benzoylamino]-(2S)-{(benzyloxycarbonyl)amino}propionic
acid (35)
FAB-HMS (M+H)+ calcd for C20H29N5O4: 404.2298,
25
D
found: 404.2290; ½aꢁ ꢀ7° (c 1.0, MeOH/H2O/concd
NH4OH = 8:1:1).
The title compound (7.00 mg, 0.0124 mmol, 20%) was
synthesized from 25 and 6c following the general proce-
dure for 1 (Step 1): H NMR (400 MHz, DMSO-d6) d
5.57. t-Butyl (2S)-benzenesulfonylamino-3-{N-(2-chloro-
ethyl)-2,2,2-trifluoroacetamido}propionate (40)
1
1.54 (2H, br q, piperidine), 2.01 (2H, br d, piperidine),
2.96 (2H, br t, piperidine), 3.56 (2H, m, CONHCH2CH),
3.83 (3H, m, piperidine and CONHCH2CH), 4.18 (1H, m,
piperidine), 5.02 (2H, s, CH2C6H5), 6.97 (2H, d,
C6H4CO), 7.11 (4H, m, benzimidazole and CH2C6H5),
7.32 (5H, m, benzimidazole and CH2C6H5), 7.38 (2H, d,
C6H4CO); FABMS m/z 557 (M+H)+; FAB-HMS
The compound 6a (400 mg, 1.33 mmol) was dissolved
in MeOH (20 mL). 2-Chloroacetaldehyde (105 mg,
1.33 mmol) was added, and then CHCl3 (20 mL) and a
drop of AcOH were added. NaBCNH3 (251 mg,
3.99 mmol) was added, and the reaction solution was kept
at room temperature for 4 h. The solution was evaporated
to afford a residue. Saturated NaHCO3 (40 mL) was
added, and a desired compound was extracted with
CHCl3 (40 mL) twice. The organic layer was concentrated
to afford crude 2-chloroethyl derivative of 6a.
(M+H)+ calcd for C30H32N6O5: 557.2512, found:
23
D
557.2504; ½aꢁ ꢀ9.5° (c 0.35, MeOH/concd NH4OH =
10:1).
5.54. (2S)-Hydroxy-3-[4-{4-(1,4,5,6-tetrahydropyrimi-
din-2-ylamino)piperidin-1-yl}benzoylamino]propionic
acid (36)
To the above crude compound, NaHCO3 (469 mg) and
1,4-dioxane (17.5 mL) were added. Trifluoromethane-
sulfonic anhydride (588 mg, 2.08 mmol) was added,
and the mixture was gently stirred at room temperature
for 16 h. Concd NH4OH (0.20 mL) was added and the
mixture was evaporated. Saturated NaHCO3 (150 mL)
was added, and the crude compound was extracted with
CHCl3 (150 mL) twice. The organic layer was dried and
concentrated. The residue was purified by silica gel flash
column chromatography (toluene/AcOEt = 5:1) to pre-
pare 40 (379 mg, 0.944 mmol, 71% in two steps) as an
The title compound was synthesized from 5a and 6d
following the general procedure 1: 1H NMR
(400 MHz, D2O) (as hydrochloride) d 1.65 (2H, m,
piperidine), 1.77 (2H, quintet, tetrahydropyrimidine),
2.03 (2H, br d, piperidine), 3.17 (4H, t, tetrahydropyrim-
idine), 3.18 (2H, br t, piperidine), 3.51 (1H, m, piperi-
dine), 3.57 (2H, dd, CONHCH2), 3.61 (2H, br d,
piperidine), 4.26 (1H, t, CONHCH2CH), 7.25 (2H, d,
C6H4), 7.66 (2H, d, C6H4); FABMS m/z 390 (M+H)+;
1
amorphous: H NMR (400 MHz, CDCl3) d 1.25 (9H,
FAB-HMS (M+H)+ calcd for C19H27N5O4: 390.2141,
s, t-Bu) 3.80 (7H, m, CH2CH2 and CH2CH), 7.51 (2H,
dd, C6H5), 7.59 (1H, t, C6H5), 7.83 (2H, br d, C6H5);
FABMS m/z 459 (M+H)+.
25
D
found: 390.2143; ½aꢁ ꢀ3° (c 1.2, H2O).
5.55. (2S)-Acetoxy-3-[4-{4-(1,4,5,6-tetrahydropyrimi-
din-2-ylamino)piperidin-1-yl}benzoylamino]propionic
acid (39)
5.58. t-Butyl 1-benzenesulfonylpiperazine-(2S)-carboxyl-
ate (6f)
The compound 39 was generated as a by-product in the
course of the final synthetic step of 36: 1H NMR
(400 MHz, CD3OD) d 1.57 (2H, br q, piperidine), 1.95
(2H, quintet, tetrahydropyrimidine), 1.96 (2H, br d,
piperidine), 2.09 (3H, s, Ac), 2.90 (2H, br t, piperidine),
3.36 (4H, t, tetrahydropyrimidine), 3.49 (1H, m, piperi-
DMF (5.9 mL) was added to 40 (294 mg, 0.641 mmol).
DBU (97.5 mg, 0.641 mmol) was added and the solution
was kept at room temperature for 1 h. The mixture was
extracted with AcOEt (100 mL). The organic layer was
washed with aqueous 5% KHSO4, saturated NaHCO3,
and brine. Driedorganic layerwas evaporated. Theresidue

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2125
was purified by silica gel flash column chromatography
(toluene/AcOEt = 5:1) to prepare trifluoroacetoamido of
6f (234 mg, 0.554 mmol, 91%) as an amorphous.
1.96 (2H, br s, piperidine), 2.27 (2H, br dd, piperidine), 3.12
(2H, br t, piperidine), 3.80 (4H, m, CONHCH2CH and piper-
idine), 4.19 (1H, m, piperidine), 4.44 (1H, dd, CON-
HCH2CH), 5.11 (2H, s, CH2C6H5), 6.89 (2H, d, C6H4CO),
7.03 (1H, dt, benzimidazole), 7.19 (1H, dt, benzimidazole),
7.33 (5H, m, CH2C6H5), 7.39 (1H, d, benzimidazole), 7.59
(1H, d, benzimidazole), 7.67 (2H, d, C6H4CO); FABMS
m/z 713 (M+H)+.
The above compound (50.0 mg, 0.118 mmol) was dis-
solved in 1,4-dioxane (4.0 mL) with concd NH4OH
(2.0 mL). The solution was kept at room temperature
for 16 h. Evaporated solution was purified by silica gel
preparative TLC (CHCl3/MeOH = 19:1) to give 6f
(35.0 mg, 0.107 mmol, 91%) as a colorless syrup: 1H
NMR (400 MHz, CD3OD) d 1.37 (9H, s, t-Bu), 2.59
(1H, dt, piperidine), 2.84 (1H, dd, piperidine), 2.90
(1H, br d, piperidine), 3.34 (2H, m, piperidine),
q3.56 (1H, dt, piperidine), 4.39 (1H, br d, piperidine),
7.53 (2H, br t, C6H5), 7.62 (1H, br t, C6H5), 7.81 (2H,
m, C6H5); TSPMS m/z 327 (M+H)+.
To a solution of the above compound (50.0 mg,
0.0701 mmol), THF (17.5 mL), water (5.0 mL), and
AcOH (2.5 mL), 10% Pd/C (50 mg) was added. The mix-
ture was hydrogenated under H2 for 14 h at room temper-
ature. The mixture was filtered through Celite, and solids
were washed with EtOH. The filtrate was neutralized
using 1 M aqueous NaOH, and the THF and EtOH were
removed in vacuo at 30 °C. The resulting aqueous solu-
tion was saturated by NaCl and extracted with CH2Cl2
three times. The organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated. The residue was puri-
fied by silica gel preparative TLC (CH2Cl2/MeOH = 7:1)
to give 41 (26.1 mg, 0.0451 mmol, 64%) as a colorless oil:
1H NMR (400 MHz, CDCl3) d 1.47 (9H, s, t-Bu), 1.70
(9H, s, t-Bu), 1.72 (2H, br dq, piperidine), 2.28 (2H, br
dd, piperidine), 3.12 (2H, ddd, piperidine), 3.47 (1H, m,
CONHCH2CH), 3.61 (1H, dd, CONHCH2CH), 3.81
(3H, m, piperidine and CONHCH2CH), 4.20 (1H, m,
piperidine), 6.92 (2H, d, C6H4CO), 7.04 (1H, ddd, benz-
imidazole), 7.20 (1H, dt, benzimidazole), 7.39 (1H, d,
5.59. 1-Benzenesulfonyl-4-[4-{4-(1H-benzimidazol-2-yl-
amino)piperidin-1-yl}benzoyl]piperazine-(2S)-carboxylic
acid (38)
The title compound was synthesized from 25 and 6f fol-
lowing the general procedure 22: H NMR (400 MHz,
1
CD3OD) d 1.72 (2H, m, piperidine), 2.09 (2H, br d, piper-
idine), 2.91 (2H, br t, piperidine), 3.33 (4H, m, piperidine
and piperazine), 3.53 (1H, m, piperidine), 3.72 (2H, m,
piperazine), 3.84 (2H, m, piperazine), 4.49 (1H, m, piper-
azine), 6.92 (2H, br d, C6H4), 7.22 (5H, m, C6H5 and benz-
imidazole), 7.42 (2H, m, benzimidazole), 7.52 (2H, m,
C6H5), 7.90 (2H, br d, C6H4); FABMS m/z 589
benzimidazole), 7.60 (1H, d, benzimidazole), 7.70 (2H,
23
d, C6H4CO); TSPMS m/z 579 (M+H)+; ½aꢁ +14° (c
(M+H)+; FAB-HMS (M+H)+ calcd for C30H32N6O5S:
D
27
D
589.2233, found: 589.2224; ½aꢁ ꢀ28° (c 0.45, MeOH/
0.97, MeOH).
CHCl3 = 1:1).
5.61. (2S)-(Butane-1-sulfonyl)amino-3-[4-{4-(1H-
benzimidazol-2-ylamino)piperidin-1-yl}benzoylamino]-
propionic acid (42)
5.60. t-Butyl (2S)-amino-3-[4-[4-{(1-t-butoxycarbonyl-
1H-benzimidazol-2-yl)amino}piperidin-1-yl]benzoyl-
amino]propionate (41)
Step 1: To a solution of the amine 41 (10.5 mg,
0.0181 mmol) in DMF (0.5 mL), i-Pr2NEt (6.5 ll,
0.0362 mmol) and n-butanesulfonyl chloride (2.4 lL,
0.0181 mmol) were added. The mixture was stirred at
room temperature for 1.5 h. Excess sulfonyl chloride
was trapped by piperazine. After dilution with aqueous
NaHCO3 (30 mL), the mixture was extracted with AcOEt
three times. The organic layer was dried over anhydrous
Na2SO4, filtered, and evaporated. The residue was puri-
fied by silica gel preparative TLC (hexane/AcOEt = 1:2)
to give t-butyl (2S)-(butane-1-sulfonyl)amino-3-[4-[4-
{(1-t-butoxycarbonyl-1H-benzimidazol-2-yl)amino}pip-
eridin-1-yl]benzoylamino]propionate (10.5 mg, 0.0150
mmol, 83%) as a colorless oil: 1H NMR (400 MHz,
CDCl3) d 0.92 (3H, t, CH2CH2CH2CH3) 1.42 (2H, sextet,
CH2CH2CH2CH3), 1.50 (9H, s, t-Bu), 1.70 (9H, s, t-Bu),
1.70 (2H, m, piperidine), 1.78 (2H, sextet,
CH2CH2CH2CH3), 2.28 (2H, br dd, piperidine), 3.03
(2H, ddd, CH2CH2CH2CH textsubscript3), 3.13 (2H,
ddd, piperidine), 3.68 (1H, ddd, CONHCH2CH), 3.79
(2H, br d, piperidine), 3.90 (1H, ddd, CONHCH2CH),
4.19 (1H, dt, CONHCH2CH), 4.21 (1H, m, piperidine),
6.92 (2H, d, C6H4CO), 7.13 (1H, dt, benzimidazole),
7.20 (1H, dt, benzimidazole), 7.39 (1H, d, benzimidazole),
The carboxylic acid 25 (42.0 mg, 0.125 mmol) and 6c
afforded the desired amide (58.8 mg, 0.0950 mmol,
77%) as a colorless solid following the general procedure
1
for 7b: H NMR (400 MHz, CD3OD) d 1.42 (9H, s,
t-Bu), 1.67 (2H, br q, piperidine), 2.15 (2H, m, piperi-
dine), 3.06 (2H, br t, piperidine), 3.72 (2H, d, CON-
HCH2CH), 3.85 (3H, m, piperidine), 4.36 (1H, t,
CONHCH2CH), 5.08 (2H, dd, CH2C6H5), 6.99 (2H,
d, C6H4CO), 7.02 (2H, m, benzimidazole), 7.23 (2H,
m, benzimidazole), 7.31 (5H, m, CH2C6H5), 7.70 (2H,
23
d, C6H4CO); TSPMS m/z 613 (M+H)+; ½aꢁ ꢀ11° (c
D
1.0, MeOH).
The amide (50.0 mg, 0.0816 mmol) dissolved in CH2Cl2
(1.0 mL) were added Et3N (34 lL, 0.245 mmol) and
Boc2O (47 lL, 0.204 mmol) at room temperature. After
1 h, the mixture was added aqueous NaHCO3 (30 mL)
and extracted with CH2Cl2 three times. The organic layer
was dried over anhydrous Na2SO4, filtered, and evaporat-
ed. The residue was purified by silica gel preparative TLC
(CH2Cl2/MeOH = 7:1) to give t-butyl (2S)-benzyloxycar-
bonylamino-3-[4-[4-{(1-t-butoxycarbonyl-1H-benzimidazol-
2-yl)amino}piperidin-1-yl]benzoylamino]propionate (60.0
mg, 0.0816 mmol, 100%) as a colorless oil: 1H NMR
(400 MHz, CDCl3) d 1.46 (9H, s, t-Bu), 1.70 (9H, s, t-Bu),
7.59 (1H, d, benzimidazole), 7.70 (2H, d, C6H4CO);
23
D
TSPMS m/z 699 (M+H)+; ½aꢁ ꢀ1.5° (c 0.65, MeOH).

2126
M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
Step 2: The above compound (15.2 mg, 0.0217 mmol)
afforded 42 (7.50 mg, 0.138 mmol, 64%) following the
general procedure for 1 (Step 1): H NMR (400 MHz,
HCH2CH and piperidine), 6.91 (2H, d, C6H4CO), 6.96
(2H, dd, benzimidazole), 7.20 (2H, dd, benzimidazole),
7.55 (2H, d, C6H4CO), 8.00 (2H, dt, C6H4NO2),
8.14 (2H, dt, C6H4NO2); TSPMS m/z 608 (M+H)+;
1
CD3OD) d 0.89 (3H, t, CH2CH2CH2CH3) 1.39 (2H, sex-
tet, CH2CH2CH2CH3), 1.75 (4H, m, piperidine and
CH2CH2CH2CH3), 2.12 (2H, br d, piperidine), 3.02
(4H, m, CH2CH2CH2CH3 and piperidine), 3.63 (1H,
ddd, CONHCH2CH), 3.75 (2H, m, piperidine and CON-
HCH2CH), 3.88 (2H, br d, piperidine), 4.04 (1H, dd,
CONHCH2CH), 6.98 (2H, d, C6H4CO), 7.23 (2H, dd,
benzimidazole), 7.36 (2H, dd, benzimidazole), 7.75 (2H,
d, C6H4CO); TSPMS m/z 543 (M+H)+; FAB-HMS
FAB-HMS (M+H)+ calcd for C28H29N7O7S: 608.1927,
28
D
found: 608.1916; ½aꢁ +24° (c 0.20, MeOH/concd
NH4OH = 10:1).
5.65. (2S)-(4-Aminobenzenesulfonyl)amino-3-[4-{4-(1H-
benzimidazol-2-ylamino)piperidin-1-yl}benzoylamino]pro-
pionic acid (46)
(M+H)+ calcd for C26H34N6O5S: 543.2390, found:
To a solution of 45 (15.0 mg, 0.0196 mmol) and EtOH
(5.0 mL), 10% Pd/C (15 mg) was added. The mixture
was hydrogenated under H2 for 3 h at room tempera-
ture. The mixture was filtered through Celite, and solids
were washed with MeOH. The filtrate was concentrated.
The residue was purified by preparative TLC (CH2Cl2/
MeOH = 7:1), and then LH-20 (MeOH/concd
NH4OH = 10:1) to give 46 (2.50 mg, 0.00433 mmol,
22%) as a colorless solid: 1H NMR (400 MHz, CD3OD)
d 1.66 (2H, dq, piperidine), 2.16 (2H, br d, piperidine),
3.06 (2H, ddd, piperidine), 3.52 (1H, dd, CON-
HCH2CH), 3.63 (1H, dd, CONHCH2CH), 3.69 (1H,
dd, CONHCH2CH), 3.85 (1H, ddd, piperidine), 3.91
(2H, br d, piperidine), 6.62 (2H, br d, C6H4NH2), 6.96
(2H, dd, benzimidazole), 7.01 (2H, d, C6H4CO), 7.19
(2H, dd, benzimidazole), 7.53 (2H, br d, C6H4NH2),
7.73 (2H, d, C6H4CO); TSPMS m/z 578 (M+H)+;
23
D
NH4OH = 10:1).
543.2401; ½aꢁ
+5.9° (c 0.38, MeOH/concd
5.62. 3-[4-{4-(1H-Benzimidazol-2-ylamino)piperidin-1-
yl}benzoylamino]-(2S)-{(4-fluorobenzenesulfonyl)ami-
no}propionic acid (43)
The title compound (10.0 mg, 0.0172 mmol, 38% in two
steps) was synthesized from 41 and 4-fluorobenzenesulfo-
1
nyl chloride following the general procedure for 42: H
NMR (400 MHz, CD3OD) d 1.72 (2H, br q, piperidine),
2.15 (2H, br d, piperidine), 3.04 (2H, br t, piperidine),
3.57 (1H, dd, CONHCH2CH), 3.64 (1H, dd, CON-
HCH2CH), 3.78 (1H, dd, CONHCH2CH), 3.80 (1H, m,
piperidine), 3.92 (2H, br d, piperidine), 6.99 (2H, d,
C6H4CO), 7.17 (4H, m, benzimidazole and C6H4F), 7.29
(2H, dd, benzimidazole), 7.70 (2H, d, C6H4CO), 7.89
(2H, dd, C6H4F); FABMS m/z 581 (M+H)+; FAB-
FAB-HMS (M+H)+ calcd for C28H31N7O5S: 578.2186,
23
D
found: 578.2184; ½aꢁ +101° (c 0.14, MeOH).
HMS (M+H)+ calcd for C28H29FN6O5S: 581.1982,
27
D
found: 581.1978; ½aꢁ +36° (c 0.50, MeOH/concd
5.66. t-Butyl (2S)-amino-3-[3-fluoro-4-{4-(pyrimidin-2-yl-
amino)piperidin-1-yl}benzoylamino]propionate (47)
NH4OH = 1:1).
5.63. 3-[4-{4-(1H-Benzimidazol-2-ylamino)piperidin-1-yl}ben-
zoylamino]-(2S)-{(2,4,6-trimethylbenzenesulfonyl)amino}pro-
pionic acid (44)
t-Butyl (2S)-(benzyloxycarbonyl)amino-3-[3-fluoro-4-
{4-(pyrimidin-2-ylamino)piperidin-1-yl}benzoylamino]-
propionate was synthesized from 5b and 6c following the
general procedure for 7b: 1H NMR (400 MHz, CD3OD)
d 1.43 (9H, s, t-Bu), 1.74 (2H, dq, piperidine), 2.11 (2H, br
d, piperidine), 2.94 (2H, dt, piperidine), 3.58 (2H, br d,
piperidine), 3.71 (2H, d, CONHCH2CH), 3.95 (1H, tt,
piperidine), 4.37 (1H, t, CONHCH2CH), 5.08 (2H, dd,
CH2C6H5), 6.59 (1H, t, pyrimidine), 7.07 (1H, t,
C6H3CO), 7.30 (5H, m, CH2C6H5), 7.49 (1H, dd,
C6H3CO), 7.55 (1H, dd, C6H3CO), 8.26 (2H, d, pyrimi-
dine); TSPMS m/z 593 (M+H)+.
The title compound (5.00 mg, 0.00828 mmol, 28% in
two steps) was synthesized from 41 and 2,4,6-trim-
ethylbenzenesulfonyl chloride following the general pro-
cedure for 42: 1H NMR (400 MHz, CD3OD) d 1.70 (2H,
br q, piperidine), 2.15 (2H, br d, piperidine), 2.22 (3H, s,
C6H2Me), 2.63 (6H, s, C6H2Me), 3.05 (2H, br t, piperi-
dine), 3.62 (3H, m, CONHCH2CH), 3.81 (1H, m, piper-
idine), 3.92 (2H, br d, piperidine), 6.92 (2H, s, C6H2Me),
6.99 (2H, d, C6H4CO), 7.10 (2H, dd, benzimidazole),
7.27 (2H, dd, benzimidazole), 7.69 (2H, d, C6H4CO);
To a solution of the above amide (500 mg, 0.844 mmol)
and THF (10 mL), 10% Pd/C (100 mg) was added. The
mixture was hydrogenated under H2 for 12 h at room
temperature. The mixture was filtered through Celite,
and solids were washed with MeOH. The filtrate was
concentrated. The residue was purified by silica gel flash
column chromatography (CH2Cl2/MeOH = 7:1) to give
47 (334 mg, 0.729 mmol, 86%) as a colorless amorphous:
1H NMR (400 MHz, CD3OD) d 1.43 (9H, s, t-Bu), 1.74
(2H, dq, piperidine), 2.11 (2H, br d, piperidine), 2.93
(2H, ddd, piperidine), 3.60 (5H, m, piperidine and CON-
HCH2CH), 3.95 (1H, tt, piperidine), 6.59 (1H, t, pyrim-
idine), 7.09 (1H, t, C6H3CO), 7.54 (1H, dd, C6H3CO),
7.60 (1H, dd, C6H3CO), 8.26 (2H, d, pyrimidine);
TSPMS m/z 459 (M+H)+.
TSPMS m/z 605 (M+H)+; FAB-HMS (M+H)+ calcd
27
D
for C31H36N6O5S: 605.2546, found: 605.2546; ½aꢁ
+55° (c 0.25, MeOH/concd NH4OH = 1:1).
5.64. 3-[4-{4-(1H-Benzimidazol-2-ylamino)piperidin-1-
yl}benzoylamino]-(2S)-{(4-nitrobenzenesulfonyl)-
amino}propionic acid (45)
The title compound (15.1 mg, 0.0248 mmol, 30% in two
steps) was synthesized from 41 and 4-nitrobenzenesulfo-
1
nyl chloride following the general procedure for 42: H
NMR (400 MHz, CD3OD) d 1.68 (2H, br q, piperidine),
2.18 (2H, br d, piperidine), 3.07 (2H, br t, piperidine),
3.58 (2H, m, CONHCH2CH), 3.91 (4H, m, CON-

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2127
5.67. (2S)-Acetylamino-3-[3-fluoro-4-{4-(1,4,5,6-tetra-
hydropyrimidin-2-ylamino)piperidin-1-yl}benzoyl-
amino]propionic acid (48)
rahydropyrimidine and piperidine), 2.96 (2H, br t, piper-
idine), 3.37 (4H, t, tetrahydropyrimidine), 3.55 (5H, m,
CONHCH2CH and piperidine), 3.84 (1H, dd, CON-
HCH2CH),6.97(1H,t,C6H3CO),7.29(1H,dd,C6H3CO),
7.45 (1H, dd, C6H3CO), 7.76 (2H, d, C6H4CO2H), 7.88
(2H, d, C6H4CO2H); TSPMS m/z 591 (M+H)+; FAB-
The title compound was synthesized from 47 and acetyl
chloride following the general procedure for 42 (Step 1)
1
and then for 8: H NMR (400 MHz, CD3OD) d 1.68
HMS(M+H)+ calcdforC26H31FN6O7S:591.2037,found:
26
D
(2H, br q, piperidine), 1.96 (3H, s, Ac), 1.97 (2H, m, tet-
rahydropyrimidine), 2.02 (2H, br d, piperidine), 2.87 (2H,
br t, piperidine), 3.37 (4H, t, tetrahydropyrimidine), 3.47
(1H, m, piperidine), 3.53 (2H, br d, piperidine), 3.66 (1H,
dd, CONHCH2CH), 3.74 (1H, dd, CONHCH2CH), 4.47
(1H, dd, CONHCH2CH), 7.05 (1H, t, C6H3CO), 7.51
(1H, dd, C6H3CO), 7.56 (1H, dd, C6H3CO); FABMS m/z
591.2013;½aꢁ +36°(c0.76,MeOH/concdNH4OH = 10:1).
5.70. 3-[3-Fluoro-4-{4-(1,4,5,6-tetrahydropyrimidin-2-
ylamino)piperidin-1-yl}benzoylamino]-(2S)-{(4-meth-
oxybenzenesulfonyl)amino}propionic acid (51)
The title compound (25.0 mg, 0.0434 mmol, 90% in
three steps) was synthesized from 47 and 4-meth-
oxybenzenesulfonyl chloride following the general pro-
cedure for 42 (Step 1) and then for 8: 1H NMR
(400 MHz, CD3OD) d 1.69 (2H, br dq, piperidine),
1.96 (2H, quintet, tetrahydropyrimidine), 2.03 (2H, br
d, piperidine), 2.90 (2H, br t, piperidine), 3.37 (4H, t, tet-
rahydropyrimidine), 3.51 (4H, m, CONHCH2CH and
piperidine), 3.66 (1H, dd, CONHCH2CH), 3.79 (1H,
m, CONHCH2CH), 3.80 (3H, s, C6H4OMe), 6.92 (2H,
br d, C6H4OMe), 7.03 (1H, t, C6H3CO), 7.46 (1H, dd,
C6H3CO), 7.53 (1H, dd, C6H3CO), 7.75 (2H, br d,
C6H4OMe); TSPMS m/z 577 (M+H)+; FAB-HMS
449(M+H)+;FAB-HMS(M+H)+calcdforC21H29FN6O4:
25
D
449.2313, found: 449.2309; ½aꢁ +9.3° (c 0.17, MeOH).
5.68. (2S)-(4-Aminobenzenesulfonyl)amino-3-[3-fluoro-4-{4-
(1,4,5,6-tetrahydropyrimidin-2-ylamino)piperidin-1-yl}ben-
zoylamino]propionic acid (49)
The title compound was synthesized from 47 and 4-
nitrobenzenesulfonyl chloride following the general pro-
cedure for 42 (Step 1) and then for 8: 1H NMR
(400 MHz, CD3OD) (as hydrochloride) d 1.67 (2H, br
q, piperidine), 1.96 (2H, quintet, tetrahydropyrimidine),
2.00 (2H, m, piperidine), 2.86 (2H, br t, piperidine), 3.37
(4H, t, tetrahydropyrimidine), 3.48 (5H, m, piperidine
and CONHCH2CH), 3.64 (1H, dd, CONHCH2CH),
3.69 (1H, m, CONHCH2CH), 6.63 (2H, br d,
C6H4NH2), 7.04 (1H, t, C6H3CO), 7.53 (3H, m,
C6H3CO and C6H4NH2), 7.58 (1H, dd, C6H3CO);
TSPMS m/z 562 (M+H)+; FAB-HMS (M+H)+ calcd
for C25H32FN7O5S: 562.2248, found: 562.2253.
(M+H)+ calcd for C26H33FN6O6S: 577.2245, found:
23
D
577.2251; ½aꢁ +60° (c 0.085, DMSO).
5.71. 3-[3-Fluoro-4-{4-(1,4,5,6-tetrahydropyrimidin-2-
ylamino)piperidin-1-yl}benzoylamino]-(2S)-{(4-hydrox-
ybenzenesulfonyl)amino}propionic acid (52)
To a solution of t-butyl 3-[3-fluoro-4-{4-(pyrimidin-2-yla-
mino)piperidin-1-yl}benzoylamino]-(2S)-{(4-methoxyben-
zenesulfonyl)amino}propionate (see experimental for 51)
(66.8 mg, 0.106 mmol) and dichloroethane (5.0 mL), BBr3
(1.0 M in CH2Cl2, 1.10 mL, 1.10 mmol) was added. The
mixture was warmed to 40 °C and stirred for 2.5 h. A mix-
ture of 1,4-dioxane (1.0 mL), water (0.2 mL), and Et3N
(1.0 mL) was added to the mixture. The concentrated resi-
due was purified by silica gel preparative TLC (CHCl3/
MeOH/concd NH4OH = 30:10:1) and then LH-20
(MeOH) to prepare 3-[3-fluoro-4-{4-(pyrimidin-2-ylami-
no)piperidin-1-yl}benzoylamino]-(2S)-{(4-hydroxybenzene-
sulfonyl)amino}propionic acid (35.1 mg, 0.0523 mmol,
5.69. (2S)-(4-Carboxybenzenesulfonyl)amino-3-[3-fluoro-
4-{4-(1,4,5,6-tetrahydropyrimidin-2-ylamino)piperidin-1-
yl}benzoylamino]propionic acid (50)
A mixture of 47 (20.0 mg, 0.0436 mmol), DMAP
(1.10 mg, 0.00872 mmol), and pyridine (0.5 mL) was
added 4-(chlorosulfonyl)benzoic acid21 (9.60 mg,
0.0436 mmol) over 3 h. After an additional 1 h, the mix-
ture was concentrated. The residue was purified by silica
gel preparative TLC (CHCl3/MeOH/concd NH4OH =
30:10:1) to give t-butyl (2S)-(4-carboxybenzenesulfo-
nyl)amino-3-[3-fluoro-4-{4-(pyrimidin-2-ylamino)piperi-
din-1-yl}benzoylamino]propionate (13.0 mg, 0.0201
1
59%) as a colorless solid: H NMR (400 MHz, CDCl3) d
1.75 (2H, dq, piperidine), 2.12 (2H, br d, piperidine), 2.94
(2H, br t, piperidine), 3.49 (1H, dd, CONHCH2CH), 3.59
(2H, br d, piperidine), 3.69 (1H, dd, CONHCH2CH),
3.86 (1H, dd, CONHCH2CH), 3.95 (1H, tt, piperidine),
6.60 (1H, t, pyrimidine), 6.78 (2H, br d, C6H4OH),
7.08 (1H, t, C6H3CO), 7.48 (1H, dd, C6H3CO), 7.53
1
mmol, 46%) as a colorless solid: H NMR (400 MHz,
CD3OD) d 1.23 (9H, s, t-Bu), 1.74 (2H, dq, piperidine), 2.11
(2H, br d, piperidine), 2.94 (2H, br t, piperidine), 3.50
(1H, dd, CONHCH2CH), 3.59 (2H, br d, piperidine),
3.66 (1H, dd, CONHCH2CH), 3.95 (1H, tt, piperidine),
4.13 (1H, dd, CONHCH2CH), 6.59 (1H, t, pyrimidine),
7.08 (1H, t, C6H3CO), 7.48 (1H, dd, C6H3CO), 7.49
(1H, dd, C6H3CO), 7.83 (2H, d, C6H4CO2H), 8.03 (2H,
(1H, dd, C6H3CO), 7.66 (2H, br d, C6H4OH), 8.27 (2H, d,
26
pyrimidine); TSPMS m/z 559 (M+H)+; ½aꢁ +36° (c 0.46,
D
MeOH).
d, C6H4CO2H), 8.26 (2H, d, pyrimidine); TSPMS m/z
23
643 (M+H)+; ½aꢁ +185° (c 0.33, MeOH).
The title compound (10.7 mg, 0.0125 mmol, 29%) was
synthesized from this compound following the general
procedure for 8 (Step 2): 1H NMR (400 MHz, CD3OD)
d 1.69 (2H, dq, piperidine), 1.97 (2H, quintet, tetrahy-
dropyrimidine), 2.03 (2H, br d, piperidine), 2.89 (2H,
br t, piperidine), 3.37 (4H, t, tetrahydropyrimidine),
D
The title compound (15.1 mg, 0.0256 mmol, 60% in two
steps) was synthesized from this compound following
the general procedure for 8: 1H NMR (400 MHz,
CD3OD) d 1.71 (2H, br q, piperidine), 1.96 (4H, m, tet-

2128
M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
3.51 (4H, m, CONHCH2CH and piperidine), 3.68 (1H,
dd, CONHCH2CH), 3.78 (1H, dd, CONHCH2CH),
6.80 (2H, br d, C6H4OH), 7.05 (1H, t, C6H3CO), 7.51
(1H, dd, C6H3CO), 7.56 (1H, dd, C6H3CO), 7.67 (2H,
br d, C6H4OH); ESIMS m/z 563 (M+H)+; FAB-HMS
The compound (52.9 mg, 0.0891 mmol) afforded 55
(14.1 mg, 0.0260 mmol, 29% in two steps) following
1
the general procedure for 1: H NMR (400 MHz, 77%
CD3OD/D2O) d 1.60 (2H, br q, piperidine), 1.96 (2H,
quintet, tetrahydropyrimidine), 2.02 (2H, br d, piperi-
dine), 2.89 (3H, s, NMe), 2.98 (2H, br t, piperidine),
3.36 (4H, t, tetrahydropyrimidine), 3.55 (1H, m, piperi-
dine), 3.71 (2H, m, CONHCH2CH), 3.81 (2H, br d,
piperidine), 6.59 (1H, t, pyrimidine), 6.99 (2H, d,
C6H4), 7.41 (2H, m, C6H5), 7.48 (1H, m, C6H5), 7.66
(2H, d, C6H4), 7.83 (2H, m, C6H5); FABMS m/z 543
(M+H)+.
(M+H)+ calcd for C25H31FN6O6S: 563.2088, found:
26
D
NH4OH = 10:1).
563.2085; ½aꢁ
+108° (c 0.54, MeOH/concd
5.72. (2S)-(4-Hydroxybenzenesulfonyl)amino-3-[3–
hydroxy-4-{4-(1,4,5,6- tetrahydropyrimidin-2-yl-
amino)piperidin-1-yl}benzoylamino]propionic acid (53)
The title compound was synthesized from 5e and 4-meth-
oxybenzenesulfonyl chloride following the general
procedure 52: 1H NMR (400 MHz, CD3OD) d 1.70
(2H, br q, piperidine), 1.97 (4H, m, piperidine and tetra-
hydropyrimidine), 2.71 (2H, br t, piperidine), 3.37 (4H,
t, tetrahydropyrimidine), 3.42 (3H, m, piperidine), 3.60
(3H, m, CONHCH2CH), 6.75 (2H, br d, C6H4OH),
6.96 (1H, d, C6H3CO), 7.27 (2H, m, C6H3CO), 7.63
(2H, d, C6H4OH); TSPMS m/z 561 (M+H)+;
5.75. Integrin-binding assays
Compounds were evaluated for their inhibitory activities
in avb3 and aIIbb3-ELISA (enzyme-linked immunosor-
22
bent assay). avb3 was purified from human placenta,
using RGDSPK-Sepharose CL-4B chromatography, fol-
22
lowed by mono Q chromatography (Pharmacia). aIIbb3
was purified from human platelet by RGDSPK-Sephar-
ose CL-4B. avb3 and aIIbb3 binding assays were per-
formed according to the modified method of Kouns
et al.23 EIA plates (Nunc) were coated with avb3 or aIIbb3,
and blocked with bovine serum albumin. In each reaction,
the reaction mixture (20 mM Tris–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 incubated for 4 h at 25 °C.
Thereafter the ligand binding was measured using anti-
vitronectin rabbit antibody (Calbiochem) and peroxi-
dase-conjugated anti-rabbit IgG antibody (Capell) for
avb3, or peroxidase-conjugated anti-fibrinogen antibody
(Capell) for aIIbb3, and 2,20-azino-bis(3-ethylbenzthiazo-
line-6-sulfonic acid) (Sigma) as the substrate of peroxi-
FAB-HMS (M+H)+ calcd for C25H32N6O7S: 561.2131,
23
D
found: 561.2125; ½aꢁ
+88° (c 1.0, MeOH/concd
NH4OH = 10:1).
5.73. (2S)-Amino-3-[4-{4-(1H-benzimidazol-2-ylami-
no)piperidin-1-yl}benzoylamino]propionic acid (54)
The ester 41 (30.7 mg, 0.0439 mmol) was dissolved in
CH2Cl2 (1.0 mL), added TFA (0.1 mL), and stirred at
room temperature for 24 h. The solvent was evaporated
in vacuo and the residue was purified by silica gel
preparative TLC (CH2Cl2/EtOH/concd NH4OH/H2O =
8:8:1:1), and then LH-20 (MeOH/concd NH4OH =
10:1) to give 54 (8.70 mg, 0.00206 mmol, 47%) as a
colorless solid: 1H NMR (400 MHz, CD3OD) d 1.71
(2H, br dq, piperidine), 2.17 (2H, br d, piperidine), 3.06
(2H, br ddd, piperidine), 3.82 (4H, m, CONHCH2CH,
and piperidine), 3.95 (2H, br d, piperidine), 7.02 (2H, d,
C6H4CO), 7.13 (2H, dd, benzimidazole), 7.29 (2H, dd,
benzimidazole), 7.77 (2H, d, C6H4CO); TSPMS m/z 423
dase. The IC50 values were determined from
measurement of absorbance at 415 nm.
a
5.76. Adhesion of human aorta smooth muscle cells to
vitronectin
The adhesion of human aorta smooth muscle cells to
vitronectin was measured as described before.24 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 were 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 measuring
the absorbance at 405 nm after the cytolysis by SDS.
The IC50 values were determined graphically from two
or more independent experiments.
(M+H)+; FAB-HMS (M+H)+ calcd for C22H26N6O3:
23
D
423.2145, found: 423.2145; ½aꢁ +2.8° (c 0.44, MeOH/
concd NH4OH = 1:1).
5.74. (2S)-(N-Benzenesulfonyl-N-methyl)amino-3-[4-{4-
(1,4,5,6-tetrahydropyrimidin-2-ylamino)piperidin-1-
yl}benzoylamino]propionic acid (55)
DMF (1.2 mL) was added to 7a (60.0 mg, 0.103 mmol)
to prepare a solution, to which methyl iodide (73.3 mg,
0.516 mmol) and diazabicycloundecene (94.4 mg) were
added. The mixture was stirred at room temperature
for 16 hr. The reaction solution was concentrated under
reduced pressure. The residue was extracted with AcOEt
(20 mL). The extract was washed once with distilled
water and once with saturated saline in that order, dried
over Na2SO4, and concentrated under reduced pressure.
The residue was purified by preparative thin-layer chro-
matography on silica gel (CHCl3/MeOH/concd
NH4OH = 400:20:1) to prepare the methylated com-
pound (53.1 mg, 0.0894 mmol, 87%).
5.77. Platelet aggregation assay
Platelet aggregation was determined according to the
previous method.23 Human platelet-rich plasma ob-
tained from healthy volunteers was prepared and the
aggregation was induced with 5lM ADP. The IC50 val-
ues were determined from two independent experiments.

M. Ishikawa et al. / Bioorg. Med. Chem. 14 (2006) 2109–2130
2129
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5.78. Aqueous solubility
Aqueous solubility of compounds was determined in
water, pH 4 Mcllvaine and pH 8 Mcllvaine at room tem-
perature. An excess of the compound was added to solu-
tions (5.0 mL), and the suspensions were kept in an
ultrasound bath for 10 min and vigorously shaken for
10 min. Suspensions were left for 30 min and filtered
(MiLLex-GV, 0.22 lm). The filtered solutions were ana-
lyzed by HPLC.
5.79. Single dose rat pharmacokinetic study
Intravenous formulation was prepared by dissolving in
DMSO (5 mg/mL) and diluting to a final concentration
of 0.5 mg/mL with 5% injectable glucose. Compounds
were intravenously administered at 0.5 mg/kg (dosing
volume: 1 mL/kg) to non-fasted 9- to 10-week-old male
Wistar rats (n = 2–3). 0.5 milliliter aliquots of blood
samples were taken from the polyethylene tube cannu-
lated in the femoral artery at 2, 5, 15, 30, 45, 60, 90,
120, and 180 min after administration, and centrifuged
to obtain plasma at about 6000g for 10 min at 4 °C,
which was preserved at ꢀ20 °C in a freezer. Plasma sam-
ples were extracted by solid-phase chromatography
(OASISTMHLB; 60 mg/3 mL) and plasma concentra-
tions ware determined by LC–MS/MS. Pharmacokinetic
parameters (t1/2), Cltot, AUC0–inf, and Vss) were calculat-
ed using the observed data by noncompartmental anal-
ysis (WinNonlin; Ver.3.1 Pharsight Corporation).
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concentration curve from hour 0 to infinity; Vss, steady-
state distribution volume.
7. Askew, B. C.; Bednar, R. A.; Bednar, B.; Claremon, D.
A.; Cook, J. J.; McIntyre, C. J.; Hunt, C. A.; Gould, R. J.;
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Acknowledgments
The authors thank Shigeko Miki and Takako Miyara
for mass spectral analysis, Shuji Ozaki for toxicological
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studies, and
crystallography.
Takashi
Watanabe
for
X-ray
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