Design, synthesis, and structure-activity relationships of potent GPIIb/IIIa antagonists: Discovery of FK419

Article abstract of DOI:10.1016/j.bmc.2005.03.056

The discovery of the non-peptide antiplatelet injectable agent FK419 is reported. Based on the β-turn structure of RGD peptide sequences in the α chain of fibrinogen, which binds the glycoprotein IIb/IIIa (GPIIb/IIIa) on the surface of platelets to induce

Full text of DOI:10.1016/j.bmc.2005.03.056

Bioorganic & Medicinal Chemistry 13 (2005) 4343–4352
Design, synthesis, and structure–activity relationships of potent
GPIIb/IIIa antagonists: discovery of FK419
a,
a
a
Toshio Yamanaka,a,* Mitsuru Ohkubo, Satoru Kuroda, Hideko Nakamura,
*
Fumie Takahashi,a Toshiaki Aoki,b Kayoko Mihara,b Jiro Sekib and Masayuki Katoa
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6 Kashima, Yodogawa-Ku,
Osaka 532-8514, Japan
bMedicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6 Kashima, Yodogawa-Ku, Osaka 532-8514, Japan
Received 15 February 2005; revised 31 March 2005; accepted 31 March 2005
Available online 25 April 2005
Abstract—The discovery of the non-peptide antiplatelet injectable agent FK419 is reported. Based on the b-turn structure of RGD
peptide sequences in the a chain of fibrinogen, which binds the glycoprotein IIb/IIIa (GPIIb/IIIa) on the surface of platelets to
induce platelet aggregation, the prototype 2 was designed. After further substituent effects were investigated at the a-position of
the carboxylic acid in 2, we enhanced platelet aggregation inhibition, and discovered the useful feature of reduced prolongation
of bleeding time. Finally, the potent platelet aggregation inhibitor FK419 (3) could be discovered. FK419 shows a safe feature
of reduced prolongation of bleeding time, as well as potent inhibition of platelet aggregation.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
the RGD peptide, many researchers have attempted to
find non-peptide small molecules, which have high affin-
ity to GPIIb/IIIa,5 and Merck researchers discovered the
first marketed non-peptide GPIIb/IIIa antagonist, tirofi-
ban (Aggrastateꢁ) as an injectable agent.6 However, re-
sults from in vivo assays, and clinical trials of several
development candidates indicated that efficacy, and the
side effect of bleeding time prolongation could not be
separated.7 We therefore initiated efforts to find a safer
antiplatelet agent with reduced prolongation of bleeding
time. In order to stop bleeding, platelets have to bind von
Willbrand factor (vWF), which causes platelet adhesion
to subendothelium, however, earlier GPIIb/IIIa antago-
nists are assumed to inhibit platelets binding to vWF.8 In
the course of our study, we proposed that if a GPIIb/IIIa
antagonist did not inhibit vWF-platelet binding, that is,
platelet adhesion to subendothelium, we may discover a
new generation antiplatelet agent with superior safety.
We wish to report here the discovery of FK419 (3), which
has reduced prolongation of bleeding time, as well as po-
tent inhibition of platelet aggregation, and the structure–
activity relationships of related compounds.
Uncontrolled platelet aggregation and platelet adhesion
to the subendothelium by damaged blood vessels causes
critical diseases such as myocardial infarction, transient
ischemic attack and unstable angina.1 Platelet aggrega-
tion is thought to be induced by the following steps:
agonists such as ADP, thrombin, thromboxane A2, sero-
tonin, and collagen first induce glycoprotein IIb/IIIa
(GPIIb/IIIa) on the surface of platelets to adopt an active
form. Aggregation of the activated platelet then occurs
with fibrinogen mediation.2 In such processes, even if
antiplatelet agents inhibit one of the agonistic pathways
responsible for activation of GPIIb/IIIa, platelet aggre-
gation is not necessarily interrupted perfectly, because
GPIIb/IIIa may be activated by another agonistic path-
way. However, if an agent which antagonizes the
GPIIb/IIIa receptor can be developed, it may be a very
efficient antiplatelet agent, because GPIIb/IIIa is the
obligatory receptor in platelet aggregation. GPIIb/IIIa
binds the Arg-Gly-Asp (RGD) sequence in the fibrino-
gen a chain.3 On the basis of the b-turn structure4 of
2. Prototype drug design
Keywords: GPIIb/IIIa antagonist; FK419; RGD b-turn mimetics;
Reduced prolongation of bleeding time.
*
Initially, we opted to modify the RGD peptide sequence
of fibrinogen. Since the type II0 b-turn structure is
Corresponding authors. Tel.: +81 6 6390 1143; fax: +81 6 6304
5414; e-mail: toshio.yamanaka@jp.astellas.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.056

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T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
After the evaluation of the derivatives, which are incor-
porated in various kinds of ring moieties in 1,13–15 we se-
lected piperidine-containing derivative
prototype.13,16
2
as
a
3. Results and discussion
3.1. Chemistry
Prototype 2 was synthesized according to Scheme 1. 4-
Pyridinecarboxaldehyde (4) was treated with triethyl
phosphonoacetate and sodium hydride as base to give
the a,b-unsaturated ester 5. The pyridine ring and the
a,b-unsaturated bond in 5 were reduced simultaneously
under 1 atm of hydrogen atmosphere with catalytic plat-
inum(IV) oxide and 1 equiv of hydrochloric acid in
EtOH to give 6 as a hydrochloride. The resulting piper-
idine ring was N-protected with a Boc group to give 7,
and the ester in 7 was hydrolyzed by aqueous sodium
hydroxide solution to give 8. Acid 8 was coupled with
ethyl (R)-3-piperidinecarboxylate in the presence of
EDC and HOBT to give 9. After hydrolysis of the ethyl
ester in 9 with lithium hydroxide, the resulting acid 10
was coupled with b-alanine methyl ester hydrochloride
(11) in the presence of EDC and HOBT. The ester and
Boc groups were cleaved by treatment with lithium
hydroxide followed by hydrogen chloride in ethyl ace-
tate, to give 2 as a hydrochloride.
Figure 1. Design of prototype 2 from the structure of RGD b-turn
peptide, and the structure of FK419 (3).
Compound 13 was synthesized according to the proce-
dure for 2 except for using ethyl 3-amino-2-phenylpro-
panoate hydrochloride (12) instead of 11.
thought to be present in the RGD sequence of fibrino-
gen,9 we designed RGD b-turn mimetics 1 containing
N-heterocyclic b-amino acids based on the speculation
that the ring moiety of 1 could serve as the b-turn of
RGD peptide,10 and the structures of reported GPIIb/
IIIa antagonists11 (Fig. 1). In order for the mimetics 1
to act as GPIIb/IIIa antagonists, incorporation of two
functional groups, carboxylic acid and amine, at an
appropriate separation and angle must be essential.12
a-Substituted derivatives 3 and 20a–j were synthesized
as illustrated in Scheme 2. a-Substituted b-alanine
derivatives 14 and 15 were obtained from N-carbobenzyl-
oxy-L-asparagine and N-carbobenzyloxy-D-asparagine,
respectively, according to the literature procedures.17
Acid 10 was coupled with amine 14 and 15 using EDC
Scheme 1. Reagents and conditions: (a) (EtO)2POCH2CO2Et, NaH, THF, 78%; (b) PtO2, H2, HCl, EtOH, 88%; (c) (Boc)2O, Et3N, CH2Cl2, 81%; (d)
aq NaOH, THF, EtOH, 91%; (e) ethyl (R)-3-piperidinecarboxylate, EDC, HOBT, DMF, 86%; (f) aq LiOH, THF, EtOH, 90%; (g) EDC, HOBT,
DMF; (h) aq LiOH; (i) HCl, EtOAc, 89% for 2, 100% for 13 in three steps.

T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
4345
Scheme 2. Reagents and conditions: (a) 10, EDC, HOBT, DMF, 93% for 16, 96% for 17; (b) Pd–C/H2, MeOH; (c) aq LiOH, THF, 97% for 18, 95%
for 19 in two steps; (d) acylating reagent, MSA or aq NaOH (see Table 1); (e) HCl, EtOAc; (f) ODS column, then lyophilization (yields for 3, 20a–i;
see Table 1).
Table 1. Acylating reagents used and yields of 3 and 20a–i
Product
R
Acylating reagent
Stereo
Yield, %a
20a
20b
20c
20d
3
NH2
—
(S)
(S)
(S)
(R)
(S)
(R)
(S)
(S)
(S)
(R)
60
NHCOPh-pCN
NHCOPh-pOMe
NHCOPh-pOMe
NHAc
pCN-PhCOCl
pMeO-PhCOCl
pMeO-PhCOCl
Ac2O
90b
94b
94b
82c
85c
90c
86b
86b
79b
20e
20f
20g
20h
20i
NHAc
Ac2O
NHCOnHex
NHCOcHex
NHCOtBu
NHCOtBu
(nHexCO)2O
cHexCOCl
tBuCOCl
tBuCOCl
a Isolated yields from 18 or 19.
b 1.1 equiv of acid chloride and 12 equiv of MSA was used.
c 2.2 equiv of acid anhydride and 3.0 equiv of aq NaOH solution were used.
and HOBT to afford 16 and 17, respectively. The Cbz
group was deprotected under 1 atm of hydrogen with cat-
alytic palladium-charcoal, followed by ethyl ester hydro-
lysis with lithium hydroxide under ice cooling to give
a-amino acids 18 and 19. The amino groups in 18 and
19 were treated with various acylating reagents such as
acid chloride or acid anhydride, except in the case of
20a. Finally, the Boc group at the piperidine ring was
deprotected by hydrogen chloride in ethyl acetate. The
crude product was purified by ODS column and lyophi-
lized to give 3 and 20a–i. The acylating reagents and yields
from 18 or 19 are illustrated in Table 1.
efficacy for platelet aggregation inhibition. Namely, var-
ious kind of functional groups at the a-position of the
carboxylic acid were substituted. When a phenyl group
was introduced to the a-position of the carboxylic acid
(13), IC50A was enhanced compared to 2. On the other
hand, 20a, which has an amino substituent at the a-po-
sition of the carboxylic acid, was found to have a dimin-
ished IC50A value. But the a-amide derivatives (3, 20b–i)
improved IC50A values. These amide bonds correspond
to the RGD-CONH- amide bond in fibrinogenꢀs a-
chain. As a result, 20b–d, which have aromatic amides
at the a-position gave greatly enhanced IC50A. So it is
speculated that some interaction were generated be-
tween the amide carbonyl groups at the a-position of
carboxylic acid and the binding site in GPIIb/IIIa. The
configuration at the a-position did not affect IC50 A val-
ues (20c,d). Even if electron withdrawing groups or elec-
tron donating groups were substituted on the aromatic
ring, the IC50A values were not affected (20b,c). Next,
aliphatic amide derivatives were examined. The simple
acetamide derivative 3 showed good IC50A value
(53 nM). Compound 20e, the epimer of 3, showed some-
what lower IC50A value than 3. When the acyl carbon
chain was elongated, IC50A was much improved (20f).
3.2. In vitro assay
Initially, we measured the IC50 values for ADP induced
human platelet aggregation inhibition (termed IC50A in
this letter) of the prototype and optimized compounds
(Table 2). The IC50A of tirofiban was also measured
as a reference.6
Prototype 2 showed moderate efficacy for platelet aggre-
gation inhibition (IC50A = 241 nM). Based on com-
pound 2, optimization was investigated to improve the

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T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
Table 2. IC50A values of 2, 3, 13, and 20a–i
Compd
R
Stereoa
IC50 (inhibition of ADP
induced aggregation), nMb (IC50A)
2c
H
Ph
—
241
110
272
43
13c
Racemic
(S)
(S)
20a
20b
20c
NH2
NHCOPh-pCN
NHCOPh-pOMe
NHCOPh-pOMe
NHAc
(S)
32
33
20d
3 (FK419)
20e
(R)
(S)
53
80
NHAc
(R)
(S)
20f
NHCOnHex
NHCOcHex
NHCOtBu
NHCOtBu
26
34
20g
20h
20i
(S)
(S)
123
37
(R)
(Tirofiban)
(S)
46
a The configuration at the a-position of the carboxylic acid.
b Concentration required to reduce ADP-induced human platelet aggregation response by 50%.
c As HCl salt.
This substituent effect at the a-position was also ob-
served in the research of tirofiban and its analogues.6b
When a more bulky acyl substituents were introduced
at the a-position (20g,i), the IC50A values were raised.
It was noticeable that much difference in IC50A between
the epimer 20h and 20i was observed compared to the re-
sults with epimer pair of 20c and 20d, or 3 and 20e. Fur-
thermore, in the case of 20h and 20i, (R) configuration
derivative (20i) was more potent, but in the case of tirofi-
ban and its enantiomer, (S) configurationꢀs tirofiban was
more potent.6b It was interesting result that the more
effective configuration at the a-position of 20i was con-
trary to that of tirofiban. From these results, we chose
the effective compounds (3, 20b–d,f,g,i) for further eval-
uation. Next, we measured the IC50 values required to
inhibit vWF mediated adhesion by 50% (termed
IC50B). And the ratio of IC50B/IC50A was calculated
to evaluate the safety margin in vitro. These results are
showed in Table 3.
IC50B value, the more bulky amide derivatives (20g,i)
lowered IC50B value.
From these in vitro assay results, 3 and 20i were selected
for further study, and they were examined in an in vivo
and ex vivo assay, involving measurement of template
bleeding time to evaluate the safety margin.
3.3. In vivo and ex vivo assays
Various amounts of 3, 20i, and tirofiban were adminis-
trated by bolus injection, followed by continuous injec-
tion (3: 20 lg/kg + 7 lg/kg/h, 100 lg/kg + 30 lg/kg/h,
200 lg/kg + 70 lg/kg/h, and 500 lg/kg + 150 lg/kg/h;
20i: 3 lg/kg + 3 lg/kg/h, 10 lg/kg + 10 lg/kg/h, 30 lg/
kg + 30 lg/kg/h, and 60 lg/kg + 60 lg/kg/h; tirofiban:
39 lg/kg + 1.3 lg/kg/h, 75 lg/kg + 2.5 lg/kg/h, 150 lg/
kg + 5 lg/kg/h, and 300 lg/kg + 10 lg/kg/h) to dogs
(beagle; n = 2–4), respectively. Blood samples were
taken at 6 time-points for the platelet aggregation study
ex vivo and for measurement of drug concentrations in
plasma by HPLC analyses at 30 min intervals after dos-
ing. At the same time as blood sample collection, bleed-
ing time was assessed with an automated spring-loaded
device designed to produce a standardized incision ap-
plied to the inner side of the upper jowl. Blood from
the incision was blotted with filter paper every 30 s until
all bleeding had stopped. Prolongation of bleeding time
was determined by comparison with the time in the pre-
treatment period. Drug concentrations in plasma for
The IC50B/IC50A ratio of prototype 2 (= 1.39) was lar-
ger than that of tirofiban (= 0.15), namely prototype 2
may have the possibility of reducing the prolongation
of bleeding time. But all of a-aromatic amide derivatives
(20b–d) showed high IC50B values. And the IC50B/IC50A
ratio was decreased compared to the prototype 2. On the
other hand, a-acetamide derivative 3 showed weak
IC50B value, so the IC50B/IC50A ratio was obtained
superior value (5.45) to prototype 2 and tirofiban. While
the elongated amide chain derivative (20f) enhanced the

T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
Table 3. IC50B values and the IC50B/IC50A ratio of 2, 3, 20b–d,f,g,i
4347
Compd
R
Stereoa
IC50 (inhibition of vWF
mediated adhesion), nMb (IC50B)
Ratio (IC50B)/(IC50A)
2c
20b
H
—
335
21
1.39
0.49
0.41
0.64
5.45
0.81
2.26
2.54
0.15
NHCOPh-pCN
NHCOPh-pOMe
NHCOPh-pOMe
NHAc
NHCOnHex
NHCOcHex
NHCOtBu
(Tirofiban)
(S)
(S)
(R)
(S)
(S)
(S)
(R)
20c
20d
13
21
3 (FK419)
20f
20g
289
21
77
94
20i
6.7
a The configuration at the a-position of the carboxylic acid.
b Concentration required to inhibit vWF mediated human platelet adhesion by 50%.
c As HCl salt.
Table 4. In vivo and ex vivo potency of 3 and 20i
Compd
Concentration, ng/mL (C)a
Concentration, ng/mL (D)b
Ratio (D/C)
3 (FK419)
20i
(tirofiban)
46 4.0
31 3.0
57 1.0
770 20
244 12
148 5.8
17
7.8
3.0
a Concentration required to reduce ADP-induced dogꢀs platelet aggregation response by 50%.
b Plasma concentration of drug in dog when bleeding time was 2.5-fold prolonged.
2.5-fold prolongation were calculated (termed D). These
results and drug concentrations required to reduce
ADP-induced dog platelet aggregation by 50% (termed
C) are illustrated in Table 4. The ratio (D/C) was calcu-
lated to evaluate the safety margin.
because absorption from the intestine is quite poor (bio-
availability = 12.0% in dog; fasted). We are currently
searching for new orally active antiplatelet agents hav-
ing the key features of FK419.20
As a result, 3, 20i, and tirofiban displayed almost paral-
lel inhibition of platelet aggregation ex vivo in dog (as
shown by the value C), but have been shown to be dif-
ferent in terms of drug concentration in plasma when
bleeding time was 2.5-fold prolonged (value D). From
the ratio of D/C, 3 was the safest in terms of reduced
prolongation of bleeding time (D/C = 17). The ratio
(D/C) of 3 was about 6-fold larger than for tirofiban,
whereas 20i was not so good ratio compared to 3.
5. Experimental
5.1. General
}
Melting points were determined with a BUCHI 535. Pro-
ton NMR spectra were recorded on a Brucker BIOSPIN
AVANCE400 or DPX200. d Values in parts per million
relative to tetramethylsilane are given. IR spectra were
recorded with the compound (neat) on a sodium chloride
disk or as KBr pellets or Nujol suspension using HIT-
ACHI 260-10, or HORIBA FT-710. Mass spectra were
recorded with Hewlett Packard 1100LC/MSD. High
resolution mass spectra were recorded with Micromass
LCT. Results of elemental analysis were recorded with
PERKINELMER 2400II.
4. Conclusions
From the result showed above, compound 3 was se-
lected as a candidate compound, renamed as FK419,
which is now under clinical trials.18 The new injectable
GPIIb/IIIa antagonist FK419 has the virtue of reduced
prolongation of bleeding time.19 FK419 is anticipated to
be developed as an agent for prevention of cerebroscle-
rosis, because the risk of brain hemorrhage, which may
be caused by antiplatelet agents could be lower. Unfor-
tunately, FK419 is not suitable as an orally active drug
5.1.1. Ethyl (2E)-3-(4-pyridinyl)acrylate (5). To a suspen-
sion of sodium hydride (60% dispersion in mineral oil,
39.4 g, 1.02 mol) in THF (500 mL) was added dropwise
triethyl phosphonoacetate (230 g, 1.02 mol) in THF
(500 mL) over a period of 0.5 h at 15–20 ꢂC. The mix-
ture was stirred for 1 h at the same temperature. To

4348
T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
the reaction mixture was added isonicotinaldehyde (4)
(100 g, 934 mmol) in THF (40 mL) over a period of
20 min at room temperature, and the mixture was stirred
overnight at the same temperature. The resulting mix-
ture was poured into 1 N HCl, and extracted with
EtOAc twice. The combined organic layer was washed
with brine, dried over MgSO4, filtered, and concentrated
in vacuo. The residual solid was triturated with hexane,
filtered, washed with hexane, dried in vacuo to give 5
2H), 2.50–2.80 (m, 2H), 3.88–3.95 (m, 2H), 12.03 (s,
1H); MS (APCI) m/z 158 (M+H)+.
5.1.5. tert-Butyl 4-{3-[(3R)-3-(ethoxycarbonyl)-1-piperid-
inyl]-3-oxopropyl}-1-piperidinecarboxylate (9). To a solu-
tion of 8 (3.04 g, 11.8 mmol), ethyl (R)-3-piperidine-
carboxylate (1.86 g, 11.8 mmol) and HOBT (1.60 g,
11.8 mmol) in DMF (20 mL) was added EDC (1.83 g,
11.8 mmol) under ice cooling. The reaction mixture
was stirred at room temperature overnight. To the
resulting solution was added saturated NaHCO3 fol-
lowed by extraction with EtOAc. The organic layer
was washed with water (·3) and brine, dried over anhy-
drous MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on sil-
ica gel with CHCl3/MeOH (100:1). The eluate was con-
centrated in vacuo to give 9 (4.01 g, 86%) as a viscous
1
(129 g, 78%) as a solid: H NMR (DMSO-d6) d 1.28
(t, J = 7.1 Hz, 3H), 4.23 (q, J = 7.1 Hz, 2H), 6.90 (d,
J = 16.4 Hz, 1H), 7.64 (d, J = 16.4 Hz, 1H), 7.69 (dd,
J = 7.7, 4.5 Hz, 2H), 8.64 (dd, J = 7.7, 4.5 Hz, 2H);
MS (APCI) m/z 178 (M+H)+.
5.1.2. Ethyl 3-(4-piperidinyl)propanoate hydrochloride
(6). A solution of 5 (123 g, 747 mmol) in EtOH
(1500 mL) and 4 N HCl in 1,4-dioxane (187 mL,
748 mmol) was treated with PtO2 (12.3 g) with bubbling
H2 for 5 h at 45 ꢂC. After the catalyst was filtered off, the
filtrate was concentrated in vacuo. The residual solid
was triturated with Et2O, filtered, washed with Et2O,
1
oil: IR (Nujol) 1710, 1670, 1620, 1405 cmꢀ1; H NMR
(CDCl3, rotomer observed) d 1.00–1.20 (m, 1H), 1.28
(t, J = 7.1 Hz, 3H), 1.45 (s, 9H), 1.48–1.88 (m, 9H),
1.98–2.15 (m, 1H), 2.31–2.51 (m, 3H), 2.62–3.12 (m,
4H), 3.35–3.47 (m, 1/2H), 3.65–3.85 (m, 1H), 4.00–4.22
(m, 4H), 4.56–4.69 (m, 1/2H); MS (APCI) m/z 397
(M+H)+.
1
dried in vacuo to give 6 (146 g, 88%) as a solid: H
NMR (DMSO-d6) d 1.18 (t, J = 7.1 Hz, 3H), 1.20–1.60
(m, 5H), 1.65–1.85 (m, 2H), 2.31 (t, J = 7.3 Hz, 2H),
2.68–2.85 (m, 2H), 3.16–3.22 (m, 2H), 4.05 (q,
J = 7.1 Hz, 2H), 8.85–9.55 (br, 2H); MS (APCI) m/z
186 (M+H)+.
5.1.6. (3R)-1-{3-[1-(tert-Butoxycarbonyl)-4-piperidinyl]-
propanoyl}-3-piperidinecarboxylic acid (10). To a solu-
tion of 9 (3.09 g, 10.1 mmol) in THF (10 mL) and EtOH
(10 mL) was added dropwise 3.0 M aqueous LiOH solu-
tion (10 mL, 30.0 mmol) under ice cooling and the mix-
ture was stirred for 1 h at room temperature. The
resulting solution was adjusted to pH 3 with 10% aque-
ous KHSO4 solution, and extracted with EtOAc. The
organic layer was washed with water and brine, dried
over MgSO4, filtered, and concentrated in vacuo. The
residue was triturated with hexane, collected, and dried
in vacuo to give 10 (3.34 g, 90%) as a solid: IR (KBr)
2931, 2862, 1732, 1716, 1697, 1686, 1456, 1279,
5.1.3. tert-Butyl 4-(3-ethoxy-3-oxopropyl)-1-piperidine-
carboxylate (7). To a suspension of 6 (50 g, 225 mmol)
in CH2Cl2 (500 mL) were added Et3N (69 mL,
496 mmol) and di-tert-butyl dicarbonate (59 g,
271 mmol) in CH2Cl2 (200 mL) under ice cooling. The
mixture was stirred for 5 h at room temperature. To this
was added water, and extracted with CH2Cl2. The or-
ganic layer was washed with 10% aqueous KHSO4 solu-
tion, water, and brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by col-
umn chromatography on silica gel with CHCl3/MeOH
(100:1). The eluate was concentrated in vacuo to give
7 (52 g, 81%) as an oil: IR (neat) 2980, 2960, 2920,
1
1248 cmꢀ1; H NMR (DMSO-d6 rotomer observed) d
0.84–1.10 (m, 2H), 1.38 (s, 9H), 1.38–1.76 (m, 8H),
1.82–2.01 (m, 1H), 2.20–2.45 (m, 3H), 2.59–2.76 (m,
2H), 2.89–3.09 (m, 1H), 3.28–3.40 (m, 1H), 3.69–3.98
(m, 3+1/2H), 4.31–4.44 (m, 1/2H), 11.75–12.82 (br,
1H); MS (APCI) m/z 269 (M+HꢀBoc)+.
1
1730, 1690, 1160 cmꢀ1; H NMR (CDCl3) d 1.01–1.19
(m, 2H), 1.26 (t, J = 7.1 Hz, 3H), 1.39 (s, 9H), 1.41–
1.72 (m, 5H), 2.32 (t, J = 7.6 Hz, 2H), 2.61–2.72 (m,
2H), 4.04–4.18 (m, 2H), 4.13 (q, J = 7.1 Hz, 2H); MS
(APCI) m/z 186 (M+HꢀBoc)+.
5.1.7. 3-[({(3R)-1-[3-(4-Piperidinyl)propanoyl]-3-piperidi-
nyl}carbonyl)amino]propanoic acid hydrochloride (2). To
a solution of 10 (1.35 g, 3.66 mmol), b-alanine ethyl ester
hydrochloride (11) (560 mg, 3.66 mmol), and HOBT
(495 mg, 3.66 mmol) in DMF (10 mL) was added EDC
(570 mg, 3.66 mmol) under ice cooling. The reaction
mixture was stirred at room temperature overnight. To
the resulting solution was added saturated NaHCO3
solution, and extracted with EtOAc. The organic layer
was washed with water and brine, dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel
with CHCl3/MeOH (100/1). The eluate was concentrated
in vacuo to give the desired intermediate (1.70 g, 100%)
as an oil: IR (neat) 2920, 2850, 1730, 1670, 1620, 1535,
5.1.4. 3-[1-(tert-Butoxycarbonyl)-4-piperidinyl]propanoic
acid (8). To a solution of 7 (46.9 g, 164 mmol) in THF
(200 mL) and EtOH (200 mL) was added dropwise
1 N aqueous NaOH solution (247 mL, 247 mmol), and
the mixture was stirred for 2 h at room temperature.
Volatiles were evaporated off, and residual aqueous
solution was adjusted to pH 4 with 10% aqueous
KHSO4 solution, and extracted with EtOAc. The organ-
ic layer was washed with water and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue
was triturated with the mixture of Et2O and hexane, col-
lected and dried in vacuo to give 8 (38.5 g, 91%) as a so-
1
1
lid: IR (Nujol) 1730, 1660, 1260, 1160 cmꢀ1; H NMR
1160, 860 cmꢀ1; H NMR (CDCl3, rotomer observed)
(DMSO-d6) d 0.84–1.07 (m, 2H), 1.38 (s, 9H), 1.41–
1.50 (m, 3H), 1.55–1.65 (m, 2H), 2.22 (t, J = 7.4 Hz,
d 1.00–1.21 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H), 1.45 (s,
9H), 1.52–1.77 (m, 7H), 1.83–2.09 (m, 2H), 2.17–2.39

T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
4349
(m, 3H), 2.48–2.73 (m, 4H), 3.16–3.68 (m, 4+1/2H),
3.83–3.96 (m, 1/2H), 4.02–4.25 (m, 2+1/2H), 4.16 (q,
J = 7.1 Hz, 2H), 4.36–4.49 (m, 1/2H), 6.23–6.36 (m, 1/
3H), 6.55–6.66 (m, 2/3H); MS (APCI) m/z 468
(M+H)+. To a solution of this material (900 mg,
1.72 mmol) in THF (8 mL) and EtOH (8 mL) was added
LiOHÆH2O (240 mg, 5.77 mmol) solution in H2O (8 mL)
under ice cooling. The reaction mixture was stirred at
room temperature for 1 h. The resulting solution was
washed with Et2O. The pH of the aqueous layer was ad-
justed to 3 with 10% aqueous KHSO4 solution, and ex-
tracted with EtOAc. The organic layer was washed
with H2O and brine, dried over MgSO4, filtered, and
concentrated in vacuo to give the carboxylic acid inter-
mediate (850 mg, 100%) as an oil: IR (neat) 1720, 1610,
1434 cmꢀ1 1H NMR (CDCl3) d 0.95–1.15 (m, 2H),
;
1.20–1.80 (m, 9H), 1.46 (s, 9H), 2.15–2.50 (m, 4H),
2.56–2.67 (m, 2H), 3.15–3.45 (m, 3H), 3.45–3.75 (m,
2H), 3.69 (s, 3H), 3.95–4.20 (m, 4H), 4.25–4.55 (m,
1H), 5.12 (s, 2H), 6.41 (d, J = 8.8 Hz, 3H), 7.25–7.45
(m, 5H); MS (APCI) m/z 503 (M+HꢀBoc)+.
5.1.10. tert-Butyl 4-[3-((3R)-3-{[((2R)-2-{[(benzyloxy)car-
bonyl]amino}-3-methoxy-3-oxopropyl)amino]carbonyl}-1-
piperidinyl)-3-oxopropyl]-1-piperidinecarboxylate (17).
This compound was prepared from 10 and 15 using a
procedure similar to that employed for the preparation
of 16 (96%): IR (neat) 3309, 2974, 2935, 2860, 1726,
1689, 1535, 1435 cmꢀ1
; d
1H NMR (DMSO-d6)
0.80–1.15 (m, 2H), 1.15–1.90 (m, 9H), 1.38 (s, 9H),
2.00–2.80 (m, 6H), 2.80–4.00 (m, 6H), 3.61 (s, 3H),
4.10–4.45 (m, 2H), 5.04 (s, 2H), 7.36 (s, 5H), 7.64
(d, J = 8.1 Hz, 1H), 7.95–8.15 (m, 1H); MS (ESI) m/z
625.3 (M+Na)+.
1
1430, 1160 cmꢀ1; H NMR (DMSO-d6) d 0.84–1.09 (m,
2H), 1.32–1.83 (m, 9H), 1.38 (s, 9H), 2.26–2.40 (m,
5H), 2.55–2.75 (m, 3H), 2.84–3.27 (m, 4H), 3.71–3.98
(m, 3H), 4.11–4.38 (m, 1H), 7.90–8.02 (m, 1H); MS
(APCI) m/z 440 (M+H)+. To a solution of this carboxylic
acid (910 mg, 2.07 mmol) in EtOAc (9 mL) was added
4 N HCl solution in EtOAc (5.2 mL, 21 mmol) under
ice cooling. The reaction mixture was stirred at room
temperature for 2 h. The resulting precipitate was col-
lected, washed with Et2O, and dried under vacuum to
give 2 (690 mg, 89%) as a white solid: IR (Nujol) 1710,
5.1.11. (2S)-2-Amino-3-{[((3R)-1-{3-[1-(tert-butoxycar-
bonyl)-4-piperidinyl]propanoyl}-3-piperidinyl)carbonyl]ami-
no}propanoic acid (18). To a solution of 16 (9.70 g,
16.1 mmol) in MeOH (200 mL) was added 10% palla-
dium on charcoal (1.94 g). The reaction mixture was stir-
red under H2 atmosphere at room temperature for 3 h.
The catalyst was filtered off, and washed with MeOH.
The filtrate was concentrated in vacuo. The residue was
dissolved with THF (100 mL). To this was added 1 N
aqueous LiOH solution (48.3 mL, 48.3 mmol) dropwise
under ice cooling, and stirred for further 0.5 h at 5 ꢂC.
The pH of the resulting solution was adjusted to 7 with
20% aqueous KHSO4 solution, and evaporated under
vacuum to remove THF. The residual aqueous solution
was purified by ODS column chromatography with
MeCN/H2O (3:7). The eluate was concentrated in vacuo,
and lyophilized to give 18 (7.08 g, 97%) as an amorphous
1
1620, 1450 cmꢀ1; H NMR (DMSO-d6) d 1.21–1.65 (m,
7H), 1.62–1.83 (m, 3H), 2.29–2.41 (m, 4H), 2.56–3.07
(m, 4H), 3.15–3.26 (m, 4H), 3.70–3.85 (m, 1H), 4.13–
4.37 (m, 4H), 7.97–8.10 (m, 1H), 8.60–8.76 (br, 1H),
8.91–9.03 (br, 1H); HRMS (ESI) m/z calcd for
25
D
C17H30N3O4 (M+H)+: 340.2236, found: 340.2227; ½aꢁ
ꢀ24.3 (c 1.0, MeOH).
5.1.8. 2-Phenyl-3-[({(3R)-1-[3-(4-piperidinyl)propanoyl]-
3-piperidinyl}carbonyl)amino] propanoic acid hydrochlo-
ride (13). This compound was prepared from 10 and
ethyl 3-amino-2-phenylpropanoate hydrochloride (12)
using a procedure similar to that employed for the pre-
1
solid: IR (KBr) 1691, 1649, 1427, 1244, 1165 cmꢀ1; H
NMR (D2O) d 0.90–1.22 (m, 3H), 1.45 (s, 9H), 1.48–
1.60 (m, 2H), 1.61–1.80 (m, 4H), 1.80–2.10 (m, 1H),
2.35–2.55 (m, 4H), 2.70–2.88 (m, 2H), 2.95 (dd,
J = 13.0, 10.6 Hz, 1H), 3.12–3.38 (m, 1H), 3.62 (dd,
J = 14.9, 6.8 Hz, 1H), 3.77 (dd, J = 14.9, 3.4 Hz, 1H),
3.82–4.10 (m, 3H), 3.90 (dd, J = 6.8, 3.4 Hz, 1H), 4.15–
4.40 (m, 1H); MS (APCI) m/z 455 (M+H)+.
paration of 2 (100%): IR (Nujol) 1724, 1635, 1616 cmꢀ1
;
1H NMR (DMSO-d6) d 1.05–1.85 (m, 11H), 2.02–2.60
(m, 4H), 2.60–3.06 (m, 3H), 3.10–3.27 (m, 2H), 3.33–
3.68 (m, 3H), 3.70–3.90 (m, 2H), 4.10–4.40 (m, 1H),
7.18–7.40 (m, 5H), 7.95–8.20 (m, 1H), 8.60–9.20 (m,
2H); HRMS (ESI) m/z calcd for C23H34N3O4 (M+H)+:
416.2549, found: 416.2535.
5.1.12. (2R)-2-Amino-3-{[((3R)-1-{3-[1-(tert-butoxycar-
bonyl)-4-piperidinyl]propanoyl}-3-piperidinyl)carbonyl]ami-
no}propanoic acid (19). This compound was prepared
from 17 using a procedure similar to that employed
for the preparation of 18 (95%): 1H NMR (D2O) d
0.90–1.22 (m, 3H), 1.45 (s, 9H), 1.46–1.60 (m, 2H),
1.65–1.85 (m, 4H), 1.85–2.10 (m, 1H), 2.40–2.55 (m,
4H), 2.70–2.90 (m, 2H), 2.98 (dd, J = 12.8, 10.6 Hz,
1H), 3.12–3.35 (m, 1H), 3.53–3.67 (m, 1H), 3.76 (dd,
J = 3.7, 2.2 Hz, 1H), 3.82–3.95 (m, 2H), 4.18–4.38 (m,
1H); MS (ESI) m/z 455.3 (M+H)+.
5.1.9. tert-Butyl 4-[3-((3R)-3-{[((2S)-2-{[(benzyloxy)car-
bonyl]amino}-3-methoxy-3-oxopropyl)amino]carbonyl}-1-
piperidinyl)-3-oxopropyl]-1-piperidinecarboxylate (16).
To a solution of 10 (20.0 g, 54.3 mmol), 14 (17.2 g,
59.7 mmol), and HOBT (5.07 g, 59.7 mmol) in DMF
(10 mL) was added EDC (10.9 mL, 59.7 mmol) drop-
wise under ice cooling. The reaction mixture was stirred
at 4 ꢂC overnight. To the resulting solution was added
water followed by extraction with EtOAc twice. The or-
ganic layer was washed with saturated aqueous NaH-
CO3 solution, water and brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel with
EtOAc/hexane (1:1). The eluate was concentrated in
vacuo to give 16 (30.5 g, 93%) as a colorless oil: IR
(neat) 3307, 2974, 2933, 2855, 1724, 1689, 1535,
5.1.13. (2S)-2-Amino-3-[({(3R)-1-[3-(4-piperidinyl)propa-
noyl]-3-piperidinyl}carbonyl)amino] propanoic acid (20a).
To a solution 18 (200 mg, 0.44 mmol) in EtOAc (4 mL)
was added 4 N HCl solution in EtOAc (1.10 mL,
4.40 mmol) under ice cooling. The reaction mixture

4350
T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
was stirred at room temperature for 2 h. The resulting
precipitate was collected and washed with Et2O and
dried under vacuum to give a crude solid. It was dis-
solved in water, and the pH of the solution was adjusted
to 7.0 with saturated aqueous NaHCO3 solution. The
resulting solution was purified by ODS column chroma-
tography with MeCN/H2O (3:97). The eluate was con-
centrated in vacuo, and lyophilized to give 20a (94 mg,
60%) as an amorphous solid: IR (KBr) 1631, 1566,
7.80 (d, J = 9.1 Hz, 1H); HRMS (ESI) m/z calcd for
C25H37N4O6 (M+H)+: 489.2713, found: 489.2713.
5.1.16. (2R)-2-[(4-Methoxybenzoyl)amino]-3-[({(3R)-1-[3-
(4-piperidinyl)propanoyl]-3-piperidinyl}carbonyl)amino]pro-
panoic acid (20d). This compound was prepared from 19
and p-methoxybenzoyl chloride using a procedure similar
to that employed for the preparation of 20b (98%): IR
(KBr) 1647, 1608, 1254, 1180, 1024, 850 cmꢀ1 1H
;
1444, 1228, 1084 cmꢀ1
;
1H NMR (D2O) d 1.26–1.90
NMR (D2O) d 1.25–1.60 (m, 7H), 1.60–1.78 (m, 2H),
1.78–2.00 (m, 3H), 2.27–2.50 (m, 3H), 2.70–3.20 (m,
3H), 3.22 (dd, J = 13.6, 10.3 Hz, 1H), 3.30–3.49 (m,
2H), 3.53–3.88 (m, 2H), 3.91 (s, 3H), 4.05–4.30 (m, 1H),
4.60–4.72 (m, 1H), 7.10 (d, J = 9.0 Hz, 2H), 7.81 (d,
J = 9.0 Hz, 2H); HRMS (ESI) m/z calcd for
C25H37N4O6 (M+H)+: 489.2713, found: 489.2721.
(m, 9H), 1.90–2.08 (m, 3H), 2.35–2.58 (m, 3H), 2.75–
3.10 (m, 3H), 2.75–3.10 (m, 3H), 3.12–3.60 (m, 6H),
3.78–4.05 (m, 1H) 4.12–4.38 (m, 1H); HRMS (ESI) m/z
calcd for C17H31N4O4 (M+H)+: 355.2345, found:
355.2331.
5.1.14. (2S)-2-[(4-Cyanobenzoyl)amino]-3-[({(3R)-1-[3-(4-
piperidinyl)propanoyl]-3-piperidinyl} carbonyl)amino]pro-
panoic acid (20b). To a suspension of 18 (204 mg,
0.45 mmol) in MeCN (5 mL) was added N-(trimethylsil-
yl)acetamide (MSA; 700 mg, 5.3 mmol), and the reac-
tion mixture was stirred at 40 ꢂC for 30 min, and then
cooled to 0 ꢂC. To this solution was added p-cya-
nobenzoyl chloride (80 mg, 0.48 mmol) under ice cool-
ing, and the reaction mixture was stirred for a further
0.5 h at room temperature. The pH of the resulting solu-
tion was adjusted to 2.5 with 20% aqueous KHSO4 solu-
tion, and extracted with EtOAc, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was dis-
solved in EtOAc (6 mL). To this was added 4 N HCl
solution in EtOAc (2.1 mL, 8.4 mmol) under ice cooling.
The reaction mixture was stirred at room temperature
for 2 h. The resulting precipitate was collected, and
washed with Et2O and dried under vacuum, dissolved
in water, and the pH of the solution adjusted to 7 with
saturated aqueous NaHCO3 solution. The solution
was purified by ODS column chromatography with
MeCN/H2O (1:4). The eluate was concentrated in
vacuo, and lyophilized to give 20b (191 mg, 90%) as an
amorphous solid: IR (KBr) 1641, 1630, 1610, 1444,
5.1.17. (2S)-2-(Heptanoylamino)-3-[({(3R)-1-[3-(4-piperidi-
nyl)propanoyl]-3-piperidinyl}carbonyl)amino]propanoic acid
(20f). To a suspension of 18 (200 mg, 0.44 mmol) in THF
(4 mL) were added 1 N aqueous NaOH solution
(1.45 mL, 1.45 mmol) and heptanoic anhydride (0.26
mL, 0.97 mmol) under ice cooling. After 15 min, the
reaction mixture was stirred at room temperature for fur-
ther 1 h. The resulting mixture was washed with Et2O,
and the pH of the aqueous layer was adjusted to 2.0 with
20% KHSO4 solution, and extracted with a mixed sol-
vent of EtOAc/THF (1:1), dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was dissolved
in EtOAc (10 mL). To this was added 4 N HCl solution
in EtOAc (1.1 mL, 4.4 mmol) under ice cooling. The
reaction mixture was stirred at room temperature for
2 h. The resulting precipitate was collected, and washed
with Et2O, and dried under vacuum, dissolved in water,
and the pH of the solution was adjusted to 7 with satu-
rated aqueous NaHCO3 solution. The solution was then
purified by ODS column chromatography with MeCN/
H2O (1:3). The eluate was concentrated in vacuo, and
lyophilized to give 20f (184 mg, 90%) as an amorphous
1
solid: IR (KBr) 3430, 3413, 1648, 1456, 1392 cmꢀ1; H
1390 cmꢀ1
;
1H NMR (D2O) d 1.25–1.62 (m, 7H),
NMR (D2O) d 0.86 (t, J = 6.5 Hz, 3H), 1.20–1.80 (m,
17H), 1.80–2.05 (m, 3H), 2.29 (t, J = 7.2 Hz, 2H), 2.42–
2.55 (m, 2H), 2.73–3.06 (m, 3H), 3.07–3.34 (m, 1H),
3.35–3.50 (m, 3H), 3.66 (ddd, J = 13.8, 4.2, 3.0 Hz,
1H), 3.77–3.98 (m, 1H), 4.12–4.38 (m, 1H), 4.39 (dd,
J = 8.9, 4.2 Hz, 1H); HRMS (ESI) m/z calcd for
C24H43N4O5 (M+H)+: 467.3233, found: 467.3231.
1.62–1.82 (m, 2H), 1.82–2.02 (m, 3H), 2.25–2.52 (m,
3H), 2.70–3.12 (m, 3H), 3.23 (dd, J = 13.2, 10.6 Hz,
1H), 3.35–3.48 (m, 2H), 3.56–3.83 (m, 3H), 4.05–4.22
(m, 1H), 4.58–4.78 (m, 1H), 7.92 (d, J = 1.6 Hz, 4H);
HRMS (ESI) m/z calcd for C25H34N5O5 (M+H)+:
484.2560, found: 484.2577; Anal. Calcd for
C25H33N5O5Æ2H2O: C, 57.79; H, 7.17; N, 13.47. Found:
C, 57.65; H, 7.21; N, 13.42.
5.1.18. (2S)-2-(Acetylamino)-3-[({(3R)-1-[3-(4-piperidinyl)-
propanoyl]-3-piperidinyl}carbonyl)amino]propanoic acid
(3). This compound was prepared from 18 and acetic
anhydride using a procedure similar to that employed
for the preparation of 20f (82%). Purified amorphous
product was crystallized from H2O/EtOH (0.5:99.5):
5.1.15. (2S)-2-[(4-Methoxybenzoyl)amino]-3-[({(3R)-1-[3-
(4-piperidinyl)propanoyl]-3-piperidinyl}carbonyl)amino]pro-
panoic acid (20c). This compound was prepared from 18
and p-methoxybenzoyl chloride using a procedure simi-
lar to that employed for the preparation of 20b (94%):
IR (KBr) 1644, 1640, 1633, 1622, 1608, 1502 1254,
mp >210 ꢂC; IR (KBr) 1633, 1624, 1444, 1396 cmꢀ1
;
1H NMR (D2O) d 1.30–2.10 (m, 11H), 2.03 (s, 3H),
2.30–2.60 (m, 3H), 2.70–3.10 (m, 3H), 3.10–3.55 (m,
4H), 3.69 (dt, J = 13.9, 4.2 Hz, 1H), 3.80–4.05 (m, 1H),
4.10–4.35 (m, 1H), 4.38 (dd, J = 8.4, 4.2 Hz, 1H);
HRMS (ESI) m/z calcd for C19H33N4O5 (M+H)+:
397.2456, found: 397.2438; Anal. Calcd for
1
1180, 1022, 850 cmꢀ1; H NMR (D2O) d 1.20–1.60 (m,
7H), 1.60–1.75 (m, 2H), 1.75–2.00 (m, 3H), 2.15–2.30
(m, 1H), 2.30–2.50 (m, 2H), 2.65–2.85 (m, 1H), 2.85–
3.05 (m, 2H), 3.20 (dd, J = 13.4, 10.4 Hz, 1H), 3.40–
3.48 (m, 2H), 3.55–3.85 (m, 3H), 3.90 (s, 3H), 4.05–
4.20 (m, 1H), 4.55–4.71 (m, 1H), 7.09 (d, J = 8.8 Hz,
1H), 7.11 (d, J = 9.1 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H),
C19H32N4O5Æ3H2O: C, 50.65; H, 8.50; N, 12.44. Found:
26
D
C, 50.51; H, 8.54; N, 12.33; ½aꢁ ꢀ11.8 (c 1.00, MeOH).

T. Yamanaka et al. / Bioorg. Med. Chem. 13 (2005) 4343–4352
4351
5.1.19. (2R)-2-(Acetylamino)-3-[({(3R)-1-[3-(4-piperidinyl)-
propanoyl]-3-piperidinyl}carbonyl)amino]propanoic acid
(20e). This compound was prepared from 19 and acetic
anhydride using a procedure similar to that employed
for the preparation of 20f (85%). Purified amorphous
solid was crystallized from H2O/EtOH (0.5:99.5): mp
>124.0–124.5 ꢂC; IR (KBr) 1666, 1627, 1599, 1402,
was prepared by rapid centrifugation of whole blood.
PRP was incubated for 2 min in the aggregometer at
37 ꢂC. ADP (2.5 lM for humans and 20–40 lM for
dogs) was added as an agonist at which the full response
of platelet aggregation was obtained, and the change in
light transmittance was monitored with a PL500 recor-
der (Yokogawa, Japan). Percent inhibition of aggrega-
tion in the drug-treated sample was calculated by
comparison with the aggregation in absence of drug or
in the pretreatment period.
1
1144 cmꢀ1; H NMR (D2O) d 1.30–2.10 (m, 11H), 2.03
(s, 3H), 2.30–2.65 (m, 3H), 2.80–3.10 (m, 3H), 3.10–3.50
(m, 4H), 3.69 (dt, J = 13.9, 4.2 Hz, 1H), 3.80–4.00 (m,
1H), 4.10–4.35 (m, 1H), 4.38 (dd, J = 8.4, 4.2 Hz, 1H);
HRMS (ESI) m/z calcd for C19 H33N4O5 (M+H)+:
397.2456, found: 397.2451; Anal. Calcd for
5.3. Platelet adhesion to vWF coated plate
C25H33N5O5Æ3H2O: C, 50.65; H, 8.50; N, 12.44. Found:
C, 50.88; H, 8.51; N, 12.49; ½aꢁ ꢀ45.9 (c 1.00, MeOH).
Platelets were washed with modified HEPES-Tyrodeꢀs
buffer (129 mM NaCl, 2.8 mM KCl, 0.8 mM KH2PO4,
8.9 mM NaHCO3, 0.8 mM MgCl2, 10 mM HEPES,
5.5 mM glucose, 0.1% bovine serum albumin (BSA),
pH 7.4) containing 1 lM PGE 1. After washing, plate-
lets were suspended in modified HEPES-Tyrodeꢀs buffer
containing 1.0 mM CaCl2, and platelet count was ad-
justed. Adhesion assay protocol was performed as fol-
lows. Ninety-six-well microtiter plates were coated
with 1 lg/well of von Willebrand factor. The plates were
then blocked with 1% BSA. After the plates were
washed with buffer, washed platelets, which were acti-
vated by 20 lM ADP for 10 min were added to each well
in the presence of drugs or buffer, and incubate for
30 min at 37 ꢂC. The plates were then washed three
times with buffer. The number of adhered cells was
determined by the acid phosphatase activity of cells at
410 nm using a microplate reader.
26
D
5.1.20. (2S)-2-[(Cyclohexylcarbonyl)amino]-3-[({(3R)-1-
[3-(4-piperidinyl)propanoyl]-3-piperidinyl}carbonyl)amino]-
propanoic acid (20g). This compound was prepared from
18 and cyclohexanecarbonyl chloride using a procedure
similar to that employed for the preparation of 20b
(86%): IR (KBr) 1643, 1637, 1633, 1523, 1469, 1441,
1255, 1225 cmꢀ1 1H NMR (D2O) d 1.20–1.80 (m,
;
18H), 1.80–2.05 (m, 3H), 2.15–2.60 (m, 4H), 2.73–3.15
(m, 3H), 3.20–3.35 (m, 1H), 3.35–3.52 (m, 3H), 3.66
(dt, J = 14.0, 3.9 Hz, 1H), 3.78–3.95 (m, 1H), 4.20–4.34
(m, 1H), 4.38 (dd, J = 8.5, 4.4 Hz, 1H); HRMS (ESI)
m/z calcd for C24H41N4O5 (M+H)+: 465.3077, found:
465.3077.
5.1.21. (2S)-2-[(2,2-Dimethylpropanoyl)amino]-3-[({(3R)-
1-[3-(4-piperidinyl)propanoyl]-3-piperidinyl}carbonyl)amino]-
propanoic acid (20h). This compound was prepared from
18 and pivaloyl chloride using a procedure similar to
that employed for the preparation of 20b (86%): IR
Acknowledgments
1
(KBr) 1631, 1541, 1390, 1230 cmꢀ1; H NMR (D2O) d
We are greatly indebted to Dr. David Barrett, Medicinal
Chemistry Research Laboratories, for his help in the
preparation of this manuscript.
1.19 (s, 9H), 1.33–1.89 (m, 8H), 1.90–2.05 (m, 3H),
2.42–2.56 (m, 3H), 2.83–3.18 (m, 3H), 3.20–3.58 (m,
4H), 3.62–3.74 (m, 1H), 3.80–3.95 (m, 1H), 4.10–4.30
(m, 1H), 4.30–4.38 (m, 1H); HRMS (ESI) m/z calcd
for C22H39N4O5 (M+H)+: 439.2920, found: 439.2942.
References and notes
5.1.22. (2R)-2-[(2,2-Dimethylpropanoyl)amino]-3-[({(3R)-
1-[3-(4-piperidinyl)propanoyl]-3-piperidinyl}carbonyl)ami-
no]propanoic acid (20i). This compound was prepared
from 19 and pivaloyl chloride using a procedure similar
to that employed for the preparation of 20b (79%): IR
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1
(KBr) 1631, 1541, 1390, 1228 cmꢀ1; H NMR (D2O) d
1.19 (s, 9H), 1.28–1.88 (m, 8H), 1.90–2.05 (m, 3H),
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