Novel non-peptide fibrinogen receptor antagonists. 1. Synthesis and glycoprotein IIb-IIIa antagonistic activities of 1,3,4-trisubstituted 2- oxopiperazine derivatives incorporating side-chain functions of the RGDF peptide

Article abstract of DOI:10.1021/jm970235u

Based on the lead tetrapeptide RGDF, two possible non-peptide glycoprotein (GP) IIb-IIIa antagonists possessing an (S)-2-oxopiperazine-3- acetic acid moiety as a scaffold incorporating the indispensable Asp fragment were prepared, and (S)-4-[[trans-[4-(gu

Full text of DOI:10.1021/jm970235u

489
J . Med. Chem. 1998, 41, 489-502
Novel Non -P ep tid e F ibr in ogen Recep tor An ta gon ists. 1. Syn th esis a n d
Glycop r otein IIb-IIIa An ta gon istic Activities of 1,3,4-Tr isu bstitu ted
2-Oxop ip er a zin e Der iva tives In cor p or a tin g Sid e-Ch a in F u n ction s of th e RGDF
P ep tid e
Hirosada Sugihara,*,† Hideto Fukushi,† Toshio Miyawaki,† Yumi Imai,† Zen-ichi Terashita,† Masaki Kawamura,†
Yukio Fujisawa,† and Shunbun Kita‡
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 2-17-85, J usohonmachi, Yodogawa-ku, Osaka 532-8686,
J apan, and Discovery Research Division, Takeda Chemical Industries, Ltd., 10, Wadai, Tsukuba-shi, Ibaraki 300-42471,
J apan
Received April 7, 1997
Based on the lead tetrapeptide RGDF, two possible non-peptide glycoprotein (GP) IIb-IIIa
antagonists possessing an (S)-2-oxopiperazine-3-acetic acid moiety as a scaffold incorporating
the indispensable Asp fragment were prepared, and (S)-4-[[trans-[4-(guanidinomethyl)-
cyclohexyl]carbonyl]glycyl]-2-oxopiperazine-1,3-diacetic acid, 1a , was identified as a potential
lead. A series of 3-substituted 2-oxopiperazine-1-acetic acids bearing the Arg-Gly equivalent
at the 4-position were prepared and evaluated for their ability to prevent platelet aggregation
and for their binding affinity for the GP IIb-IIIa receptor purified from human HEL cells. (S)-
4-[(4-Amidinobenzoyl)glycyl]-3-[(methoxycarbonyl)methyl]-2-oxopiperazine-1-acetic acid, 9 (TAK-
029), inhibited in vitro human platelet aggregation with an IC50 value of 0.03 µM and GP
IIb-IIIa-fibrinogen binding with an IC50 value of 0.49 nM. The [4-(2-aminoethyl)benzoyl]glycyl
derivative 26 showed activity comparable to that of 9 (IC50 ) 0.093 µM, guinea pig platelet
aggregation assay). Compound 9 dose-dependently inhibited ex vivo platelet aggregation in
guinea pigs (0.03 and 0.1 mg/kg, iv), and long-lasting inhibition of platelet aggregation was
observed upon oral administration of 9 (3 mg/kg) to guinea pigs. On the other hand, the activity
of 26 disappeared within 1 h after a dose of 1 mg/kg (iv). Compound 9 may therefore be useful
in the clinical treatment of arterial thrombotic diseases.
In tr od u ction
clonal antibodies raised against GP IIb-IIIa,20,21 and
non-peptide GP IIb-IIIa receptor antagonists that mimic
the RGD tripeptide sequence.22-33
The activation, adhesion, and aggregation of platelets
are important processes in the initiation of thrombus
formation at sites showing high-grade stenosis, ruptured
atheromatous plaque, and endothelial damage within
arteries. Recent studies on the biochemical mechanisms
of platelet activation indicate that the final obligatory
step in aggregation, regardless of the initiating stimu-
lus, is the cross-linking of the dimeric plasma protein
fibrinogen between membrane glycoprotein IIb-IIIa (GP
IIb-IIIa) receptor complexes exposed on adjacent acti-
vated platelets.1-4 Antagonism of the fibrinogen-GP
IIb-IIIa interaction therefore represents an attractive
therapeutic target with potential utility in the treatment
and prevention of acute myocardial infarction, unstable
angina, or transient ischemic attacks (TIA).5-7
Fibrinogen interacts with binding sites on GP IIb-IIIa
through peptide domains present on the R- and γ-
chains: RGDF (R95-98), RGDS (R572-575), and HHLG-
GAKQAGDV (γ400-411).8-10 Most of the molecules
that produce antithrombotic activity via the inhibition
of the GP IIb-IIIa-fibrinogen interaction fall into four
categories: RGD-based peptides (small linear and cyclic
peptides containing the RGD sequence or its
equivalent),1,2,8,11-14 snake venom peptides,15-19 mono-
Previous structure-activity studies (SARs) on fibrino-
gen receptor antagonists have suggested that the sole
prerequisite for receptor recognition is a molecule which
contains critical guanidino (A) and carboxylate (B)
equivalents spaced appropriately.7,22-33 In the struc-
tural and stereochemical elucidation of RGD peptides
in cell adhesion, it has been demonstrated that substi-
tution of aspartic acid with D-Asp or Glu or of glycine
with Ala or Val resulted in a complete loss of activity
and that the tetrapeptide RGDF (FgR95-98) has 3-5-
fold more potent antiplatelet activity than RGDS
(FgR572-575).8,34,35 Besides the indispensable carbox-
ylate of aspartic acid, the presence of the C-terminal
negative charge appears to contribute to the effective
interaction with the receptor and to the antithrombic
activity.7,12,25
One approach to designing non-peptide and peptide
mimetics of bioactive peptides has been the use of
conformational constraints by various types of cycli-
zation.36-38 The resulting increase in the structural
rigidity of peptides has been found to provide some
useful properties including enhancement of affinity,
specificity, and enzymatic stability. We decided to use
this approach in our compounds, and in addition, we
postulated on the basis of the three-ligand concept39 that
the hydrophobic phenyl ring (C) or the carboxylate (D)
* Corresponding author. Phone: +81-6-300-6420. Fax: +81-6-300-
6576. E-mail: Sugihara_Hirosada@takeda.co.jp.
† Pharmaceutical Research Division.
‡ Discovery Research Division.
S0022-2623(97)00235-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/24/1998

490 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sugihara et al.
F igu r e 1. Two hypothetical candidates, 1a and 2, incorporating the functions of the RGDF peptide into a 2-oxopiperazine scaffold
as a dipeptide mimic.
of the C-terminal phenylalanine in the prototypical
RGDF molecule may be the third functional group
participating in ligand-receptor interaction. On trial
we designed two possible pharmacophores, (S)-1-(car-
boxymethyl)- and (S)-1-phenethyl-2-oxopiperazine-3-
acetic acids, as the Asp-Phe mimic and synthesized two
compounds (1a and 2, Figure 1) incorporating a [trans-
[4-(guanidinomethyl)cyclohexyl]carbonyl]glycyl group as
an Arg-Gly equivalent. Compound 1a exhibited signifi-
cant antiaggregatory activity and binding affinity for
GP IIb-IIIa, but 2 did not show any activity in either
biological test. Therefore, we chose 1a as a lead
compound and investigated optimization of the tentative
Arg-Gly moiety and modification of the substituents at
the 1- and/or 3-position on the 2-oxopiperazine ring. In
this paper, we describe the synthesis of novel 2-oxopip-
erazine derivatives and biological activities of com-
pounds thus obtained. Molecular modeling studies are
also described.
otetrahydropyrazine intermediate, which was hydroge-
nated in the presence of 10% Pd/C in MeOH to afford
crystalline tert-butyl (S)-[(3-tert-butyloxycarbonyl)meth-
yl]-2-oxopiperazine-1-acetate, 5a , as an oxalate. A simi-
lar procedure using N-(2,2-dimethoxyethyl)pheneth-
ylamine instead of 3 was used to prepare tert-butyl (S)-
1-phenethyl-2-oxopiperazine-3-acetate, 8, as a key in-
termediate (Scheme 1). Acylation of 5a and 8 with
N-Cbz-glycine using EDC in methylene chloride followed
by removal of the Cbz group, subsequent coupling of the
resulting glycyl derivative with the N-hydroxysuccin-
imide ester of trans-4-guanidinocyclohexanecarboxylic
acid activated by utilizing hydroxysuccinimide and
dicyclohexylcarbodiimide (DCC) in DMF, and finally
deprotection of the tert-butyl ester using trifluoroacetic
acid (TFA) in methylene chloride gave 1a and 2,
respectively. Compounds 1b-i were prepared in three
steps in a similar fashion by hydrogenolysis of di-tert-
butyl (S)-4-(N-Cbz-glycyl)-2-oxopiperazine-1,3-diacetate,
6, subsequent condensation with various kinds of acti-
vated N-hydroxysuccinimide esters of carboxylic acids
bearing a guanidino or amidino moiety, and finally
deprotection (Scheme 1, Table 1).
The synthesis of the 3-substituted 4-[(4-amidinoben-
zoyl)glycyl]-2-oxopiperazine-1-acetic acids 9-14 is il-
lustrated in Scheme 2. The tert-butyl 3-substituted
2-oxopiperazine-1-acetates 5b-g were afforded by ap-
plying the method described for the preparation of 5a .
Condensation of 3 with R-N-Cbz-protected R-amino
acids, acid-catalyzed annulation, and catalytic hydro-
genation furnished 5b-g. Standard acylation of 5b-g
with N-Cbz-glycine, subsequent condensation of amines
arising from deprotection of the N-Cbz group with
p-amidinobenzoyl chloride in the presence of sodium
bicarbonate in a 1:1 mixture of H2O and dioxane, and
finally treatment with TFA afforded 9-14. Enantio-
Ch em istr y
Synthesis of the chiral 1,3-disubstituted 2-oxopipera-
zine derivatives 5a -g and 6 as conformationally re-
stricted dipeptide mimetics was achieved in two steps
starting from N-substituted N-(2,2-dimethoxyethyl)-
amides of N-Cbz-protected R-amino acids by modifying
the method developed by DiMaio et al.40 (Schemes 1-3).
The synthesis of 1a and 2 is illustrated in Scheme 1.
Condensation of tert-butyl N-(2,2-dimethoxyethyl)gly-
cine, 3, and N-Cbz-Asp(O-t-Bu)-OH using 1-ethyl-3-[3-
(N,N-dimethylamino)propyl]carbodiimide hydrochloride
(EDC) in methylene chloride provided the intermediary
acetal amide 4. Intramolecular cyclization of 4 using a
catalytic amount of p-toluenesulfonic acid in toluene at
70 °C proceeded smoothly with an acetal exchange and
the following elimination of methanol to yield a 2-ox-

Novel Non-Peptide Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 491
Sch em e 1a
a
Reagents: (a) N-Cbz-Asp(O-t-Bu)-OH, EDC; (b) p-TsOH in toluene; (c) H2, Pd/C in MeOH; (d) N-Cbz-Gly-OH, EDC; (e) trans-
(guanidinomethyl)cyclohexanecarboxylic acid, HOSu, DCC in DMF; (f) TFA; (g) R2N(HNd)C-X-CO2H, HOSu, DCC.
Sch em e 2a
a
Reagents (a) 3, EDC; (b) p-TsOH in toluene; (c) H2, Pd/C in MeOH; (d) N-Cbz-Gly-OH, EDC; (e) 4-amidinobenzoyl chloride, NaHCO3
in dioxane/H2O; (f) TFA.
meric (R)-9 of 9 was prepared in the same manner as
that described for 9 except that N-Cbz-D-Asp(O-t-Bu)-
OH was used as the starting material. Optical purities
of 9 and (R)-9 were determined by chiral HPLC and
estimated to be over 99.3% for 9 and 98.3% for (R)-9
(Scheme 2, Table 2).
Modification of the substituent at the 1-position of the
(S)-3-(carboxymethyl)-2-oxopiperazine ring (17-21) was
carried out as shown in Scheme 3. The tert-butyl (S)-
1-substituted 2-oxopiperazine-3-acetates 16a -e as key
intermediates were similarly prepared in three steps
using the acetal amines 15a -e and N-Cbz-Asp(O-t-Bu)-
OH as starting materials. Acylation of 16a -e with
N-Cbz-glycine followed by removal of the Cbz group,
subsequent condensation with 4-amidinobenzoyl chlo-
ride using sodium bicarbonate as a base, and finally
treatment with TFA gave compounds 17-21 (Scheme
3, Table 2).
Acylation of 5a -c,f,g with N-Cbz-glycine followed by
removal of the Cbz group, condensation with 4-[(N-Cbz-
amino)alkyl]benzoic acid using diethylphosphoryl cya-
nide (DEPC) as a coupling agent in DMF, stepwise
deprotection by treatment with TFA, and subsequent
hydrogenolysis (Pd/C in MeOH) afforded the 4-(ω-
aminoalkyl)benzoyl derivatives 23-28. The 4-[2-(N,N-
dimethylamino)ethyl]benzoyl derivatives 29 and 30
were obtained by condensation of 5f,g with 4-[2-(N,N-

492 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sugihara et al.
Sch em e 3a
a
Reagents: (a) N-Cbz-Asp(O-t-Bu)-OH, EDC; (b) p-TsOH in toluene; (c) H2, Pd/C in MeOH; (d) N-Cbz-Gly-OH, EDC; (e) 4-amidinobenzoyl
chloride, NaHCO3 in dioxane/H2O; (f) TFA.
Sch em e 4a
side-chain mimic of arginine. Aromatization of the
cyclohexane ring of 1a had little effect on affinity in the
binding assay (1f, IC50 ) 18 nM). This implies the π
orbital of the phenyl ring contributes little to the
interaction with the receptor in this case. Deletion of
a single methylene of the p-(guanidinomethyl)phenyl
group of 1f provided the p-guanidinophenyl derivative
1e which showed a 4-fold improvement in binding
affinity (IC50 ) 4.8 nM). Further replacement of the
guanidino moiety of 1e with an amidino group (1d )
caused an additional 4.8-fold increase in affinity for the
GP IIb-IIIa receptor (IC50 ) 1.0 nM). After optimization
at the N-terminus of compound 1a (1a -g), the p-
amidinophenyl moiety of 1d was found to be the most
favorable surrogate for the arginine side chain, as has
been reported for known GP IIb-IIIa antago-
nists.23,27-29,31-33 On the other hand, replacement of the
phenyl ring of the p-amidinophenyl group of 1d with a
piperidine ring gave the N-amidinopiperidin-4-yl de-
rivative 1c which showed 21 times less binding affinity.
Transposition of amidino moiety to the m-position from
the p-position on the phenyl ring (1d f 1g) resulted in
a marked decrease in both activities. These results
suggest that a strict spatial arrangement of the posi-
tively charged site and the anionic carboxylate is
required for interaction with the GP IIb-IIIa receptor.
a
Reagents: (a) N-Cbz-Gly-OH, EDC; (b) H2, Pd/C in MeOH;
(c) (N-Cbz-aminoalkyl)benzoic acid, DEPC, Et3N in DMF; (d) TFA.
dimethylamino)ethyl]benzoic acid using DEPC and
finally treatment with TFA (Scheme 4, Table 3).
Resu lts a n d Discu ssion
The compounds synthesized in this study were evalu-
ated in vitro for their ability to inhibit ADP-induced
aggregation of human and guinea pig platelet-rich
plasma (PRP) and for their ability to inhibit the binding
of biotin-labeled human fibrinogen to immobilized GP
IIb-IIIa receptors purified from human erythroleukemia
(HEL) cells.41 In general, a good correlation was
observed between the order of potency in the platelet
aggregation assay and that in the binding assay. No
species differences between humans and guinea pigs
were observed in the platelet aggregation assay (Tables
1-3).
Compound 1a exhibited a significant inhibitory effect
on platelet aggregation (IC50 ) 1.1 and 4.2 µM for
human and guinea pig PRP, respectively) and sufficient
affinity for the GP IIb-IIIa receptor (IC50 ) 20 nM). On
the other hand, compound 2 did not show any activity
in the platelet aggregation assay even at a concentration
of 30 µM and was 130-fold less potent than 1a in the
binding affinity assay. In comparison with the proto-
typical peptide RGDF, compound 1a showed a 5.5-fold
increase in binding affinity for human GP IIb-IIIa.
These biological results indicate that, of the two com-
pounds we designed based on our hypothesis, compound
1a incorporating the dipeptide mimic lacking the phen-
ethyl side chain of the terminal phenylalanine in the
RGDF sequence might be promising. In the initial trial,
the use of the cyclohexylmethyl group (1a ) as a substi-
tute for the flexible n-butyl moiety (1b) of the Arg side
chain resulted in a 2.3-fold increase in the binding
affinity. We then examined further optimization of a
Next, to determine which carboxyl group of the two
carboxylic acids in 1d mimics the side-chain carboxylic
acid of Asp in the RGDF sequence, two monoesters (9
and 18) and two monoamides (11 and 19) were pre-
pared. Compounds possessing an acetate moiety at the
1-position, 9 and 11, exhibited full activities, but
compounds in which the 1-acetate was modified, 18 and
19, had markedly reduced activities. These SARs
indicate that the side-chain carboxylic acid of Asp in the
RGDF sequence does not correspond to the 3-acetate of
the 2-oxopiperazine ring in the compound we designed
but to the acetic acid at the 1-position. Further confir-
mation was accomplished as follows. Introduction of an
alanyl or phenylalanyl side chain into the R-carbon of
the acetic acid at the 1-position resulted in more than
a 20-fold decrease in the inhibitory potency in the
platelet aggregation assay (20 and 21). Homologation
of the 1-acetic acid by one methylene unit (17) caused
marked reduction of the activity. All these data dem-
onstrated that the acetic acid at the 1-position is a
crucial Asp function for manifestation of GP IIb-IIIa

Novel Non-Peptide Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 493
Ta ble 1. Modification of an n-Butyl Surrogate of the Arg Side Chain in the Lead Compound 1a
c
b
aThe concentrations of each compound inhibiting the ADP-induced aggregation of human and guinea pig platelets by 50%. The
concentrations of each compound inhibiting the binding of biotinated fibrinogen to immobilized human GP IIb-IIIa by 50%. IC50 values
were calculated using linear regression analysis, plotting the percent inhibition versus the logarithm of the concentration of the compound;
n ) 2-8. c nt, not tested.
Ta ble 2. Modification of the Substituent at the 3- and/or 1-Position of the 2-Oxopiperazine Derivatives 9-21 and Elucidation of the
Asp Carboxylate Equivalent
platelet aggregation IC50 (µM)a
GP IIb-IIIa-Fg
compd no.
R1
R2
R3
R4
n
human
guinea pig
binding IC50 (nM)b
9
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2CH3
CONH2
CO2H
CO2H
H
H
H
H
H
H
H
H
H
H
CH2CO2CH3
H
H
CH2CONH2
CH2Ph
CH2CH2CO2H
(CH2)2CO2CH3
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
CH2CO2H
H
0
0
0
0
0
0
0
1
0
0
0
0
0.03
0.58
0.094
0.74
0.11
0.052
0.09
30
2.6
30
1.5
4.1
0.053
2.81
0.21
0.11
0.16
0.066
0.11
ntc
0.49
16.4
0.42
0.92
1.95
0.21
0.29
63
(R)-9
10
11
12
13
14
17
18
19
20
21
CH2CO2CH3
H
H
H
H
H
H
H
H
H
H
1.8
20
ntc
330
CH3
CH2Ph
1.5
4.1
ntc
6.1
a-c See corresponding footnotes in Table 1.
antagonistic activity. Alig et al. showed that the
C-terminal dipeptide of RGDX could be replaceable with
a simple (piperidinyloxy)acetic acid moiety.23 They also
described compounds with two carboxyl termini and
found that one carboxyl group was necessary but the
other not. It is interesting that the two different
approaches reached similar simplifications; the two
carboxylate groups of the catechol-O,O′-diacetate part
or of the Asp and Phe fragments in RGDF were
replaceable with achiral oxyacetate or acetate moieties

494 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Ta ble 3. Modification of the Amidino Moiety
Sugihara et al.
platelet aggregation IC50 (µM)a
GP IIb-IIIa-Fg binding
compd no.
R1
R2
human
guinea pig
IC50 (nM)b
22
23
24
25
26
27
28
29
30
H2NCH2
CH2CO2H
CH2CO2H
CH2CO2H
H
CH2CO2CH3
(CH2)2CO2H
(CH2)2CO2CH3
(CH2)2CO2H
(CH2)2CO2CH3
2.8
2.9
0.23
1.1
0.18
0.093
0.15
0.11
4.2
18
2.5
15
1.6
0.91
1.0
0.89
45
H2N(CH2)2
H2N(CH2)3
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
H2N(CH2)2
(CH3)2N(CH2)2
(CH3)2N(CH2)2
0.13
1.03
0.15
0.078
0.11
0.12
2.5
2.3
7.3
37
a,b
See corresponding footnotes in Table 1.
attached to the six-membered heterocyclic ring. Modi-
fication of the substituent at the 3-position on the
2-oxopiperazine ring of 1d was then explored, while the
(p-amidinobenzoyl)glycyl moiety was used as the N-
terminus to serve as an Arg-Gly fragment surrogate
(Table 2). Replacement of the 3-acetic acid group with
nonacidic functions such as esters (9 and 14), an amide
(11), a benzyl group (12), and even a hydrogen atom (10)
did not affect the activities of 1d in the platelet
aggregation or binding assay. Slight discrepancies
between potency of physiological response and binding
affinity were observed for compounds 10, 13, and 14,
which showed rather higher affinity for the GP IIb-IIIa
receptor in comparison with the inhibitory effect on
platelet aggregation. Among our compounds (9-14),
3-(methoxycarbonyl)methyl derivatives (9) showed the
most potent activity in the human platelet aggregation
assay (IC50 ) 0.03 µM). To confirm the requirement for
chirality, we synthesized the enantiomeric compound
(R)-9, which exhibited 33 times less binding affinity and
19 times less activity in the human platelet aggregation
assay.
Finally, modification of the amidino moiety was
examined (Table 3). Primary [p-(ω-aminoalkyl)benzoyl]-
glycyl derivatives (22-24) were prepared to optimize
the methylene length between the terminal amino group
and the phenyl ring as a bioisostere of the p-amidi-
nophenyl group. A p-(2-aminoethyl) substituent on the
phenyl ring was optimum with regard to the methylene
length. From the SARs of p-(2-aminoethyl)benzoyl
derivatives (23, 25-28), the (methoxycarbonyl)methyl
moiety (26) proved to be the most preferable substituent
at the 3-position on the 2-oxopiperazine ring as well as
From the SARs, it has been suggested that the
distance between Arg and Asp surrogates plays an
important role in the GP IIb-IIIa antagonistic activity.
We therefore analyzed this distance for RGDF, 1a , 9,
and 18. As shown in Figure 2, RGDF adopted a variety
of conformations in which the distance was dispersed
in the range of 3-15 Å and the relative energy was
dispersed in a range of over 15 kcal/mol. In the case of
1a , the distance was distributed in the range of 3-18
Å, but a tendency toward clustering in a longer-distance
range than in RGDF was observed. Almost all confor-
mations of 1a were within 10 kcal/mol of the most stable
conformer. The conformers generated were less diverse
than those of RGDF, suggesting that the conformation
of 1a is more restricted than that of RGDF. In the case
of 9, the most potent antagonist we have obtained, the
conformations were localized in a narrower range in
terms of both the distance and relative energy. This
indicates that 9 adopts a small number of favorable
conformations. The conformation of 18 was also local-
ized, but the distribution pattern was different from that
of 9, 6-13 Å as compared with 8-15 Å. Figure 3 shows
the population of conformers plotted against the dis-
tance. RGDF had three peaks with similar height at
4, 7, and 11 Å. There was a single peak at 14 Å for 1a
and one at 11-13 Å for 9. With these compounds, the
peak positions were consistent with the region where
the compounds adopted stable conformations. On the
contrary, 18 showed a different profile from 1a and 9
with peaks at 10 and 12 Å, which is inconsistent with
the stable region of 6-8 Å. There have been some
reports that analyzed the distance between the sur-
rogates theoretically or experimentally.33,42-44 Zablocki
et al. reported that the interatomic distance between
the central carbon atoms of the carboxylate and amidine
moieties calculated by a theoretical method is greater
than 10 Å, and especially in the global minimum
conformation, the central carbon atoms of the key
pharmacophores were separated by 14 Å in the (ami-
nobenzamidino)succinyl series of fibrinogen receptor
antagonists.33 This value is not identical with but is
very similar to our value of 11-13 Å. As other reports
are concerned with the distances between CR or Câ
atoms of the Arg and Asp residues in cyclic peptides
including RGD,43,44 the results cannot be compared with
our results directly. As mentioned before, Alig et al.
also described non-peptidic antagonists with two car-
boxyl termini and found out that just one carboxyl group
in the series of p-amidinobenzoyl derivatives (IC50
)
0.91 nM for binding affinity). However, N,N-dimeth-
ylation of the primary amine caused a ca. 40-fold
decrease in binding affinity for the GP IIb-IIIa receptor.
The N,N-dimethylguanidino derivative (1i) was 210-fold
less potent than the corresponding guanidino derivative
(1d ). Thus, susceptibility of the N-terminal cationic site
to removal of a proton-donating hydrogen along with a
decrease in the basicity and an increase in steric
hindrance on interacting with the GP IIb-IIIa receptor
may indicate that the reinforced ionic mode of the ligand
binding function to the GP IIb-IIIa receptor27 is impli-
cated in the binding interaction between the N-terminal
cationic site of RGDX mimetics and the platelet GP IIb-
IIIa receptor.

Novel Non-Peptide Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 495
F igu r e 2. Relative energy of the model compounds plotted against the distance between the Arg and Asp surrogates.
was necessary for the activity.23 We had an interest in
that they reached the result similar to ours while the
two approaches are very different from each other in
modifying the C-terminus moiety. Comparison of our
compounds with their (piperidinyloxy)acetic acid deriva-
tives demonstrates that the distance distributions be-
tween the surrogates can be overlapped (data not
shown). With this result, we speculate that their
compounds may interact in a mode similar to that of 9.
In conclusion, the following can be said about the
relationship between the distance and the activity: (1)
RGDF, a linear peptide, is very flexible and can assume
a variety of conformations. RGDF would have the
lowest probability of adopting the active conformation,
and this is consistent with the fact that it has the
weakest activity of the four compounds described here.
(2) In the most potent antagonist, 9, the distance
between the surrogates in the major conformations was
within the range of 11-13 Å. This suggests that a
distance of 11-13 Å is important for GP IIb-IIIa
antagonistic activity. This assumption is supported by
the results that weaker antagonists such as RGDF and
1a have a significantly different peak distribution. (3)
As for 18, it was interesting to determine whether the
carboxymethyl group at the 3-position could be substi-
tuted for that of 9 at the 1-position. As shown in Figure
3, 18 showed a distance profile different from that of 9.
This indicates that its carboxymethyl group cannot play
the same role as in 9, being consistent with its weak
activity.

496 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sugihara et al.
F igu r e 5. Time course of the inhibitory effect of TAK-029 on
ex vivo platelet aggregation in guinea pigs. At various time
intervals after oral administration of TAK-029, ex vivo platelet
aggregation induced by ADP (final concentration: 0.5-1.0 µM)
was examined. Each point represents the mean for 6-13
animals. From ref 45 with permission.
other hand, the antiplatelet activity of compound 26 (1
mg/kg, iv) disappeared rapidly, and no activity was
observed 1 h after administration. The antiplatelet
effect of 9 over the first 24 h after oral administration
was examined at doses of 3 and 10 mg/kg. Compound
9 showed an antiplatelet effect for up to 8 h at a dose of
3 mg/kg. At 10 mg/kg, 9 caused more than 50%
inhibition even 24 h after administration (Figure 5).
Compound 9 (TAK-029) was selected as a candidate for
further detailed pharmacological evaluation. Com-
pound 9 inhibited the aggregation of human PRP
induced by ADP, collagen, arachidonic acid, and platelet-
activating factor (PAF) in vitro with IC50 values of 29-
38 nM and showed potent in vivo antithrombic effects
in guinea pigs without severely prolonging the bleeding
time.45 It was found that 9 is a specific antagonist for
interaction at GP IIb-IIIa from a comparison of the
inhibitory effects on the attachment of human umbilical
vein endotherial cells (HUVEC) to immobilized peptide
ligands containing the RGD sequence, fibrinogen, von
Willebrand factor, fibronectin, and vitronectin.46
F igu r e 3. Conformers population of the model compounds
plotted against the distance between the Arg and Asp sur-
rogates.
Con clu sion s
Starting with the lead tetrapeptide RGDF, (S)-4-
[[trans-[4-(guanidinomethyl)cyclohexyl]carbonyl]glycyl]-
2-oxopiperazine-1,3-diacetic acid, 1a , was chosen as a
potential lead. Optimization of 1a has led to the potent
and orally active GP IIb-IIIa antagonist 9. The in vivo
antiplatelet activity of the primary 4-(2-aminoethyl)-
benzoyl derivative 26 disappeared rapidly, despite the
compound’s high in vitro potency comparable to that of
9. The SAR study indicated the importance of the
spatial arrangement of the indispensable cationic site
and anionic site for receptor binding and of the p-
amidino moiety susceptible to modifications on the
antiplatelet effect. Further pharmacological studies on
9 are in progress, and the results will be reported in
due course.45-47
F igu r e 4. Time course of the inhibitory effect of TAK-029 and
26 on ex vivo platelet aggregation in guinea pigs. At various
time intervals after intravenous administration of drugs, ex
vivo platelet aggregation induced by ADP was examined. Each
point represents the mean for 3-6 animals.
There were marked species differences in the anti-
platelet effect of 9; its relative potencies on ADP-induced
platelet aggregation were as follows: human 1, guinea
pig 1/1.4, monkey 1/1.5, dog 1/8, mouse 1/21, hamster
1/280, rabbit 1/1100, and rat <1/13 000.45 Similar
relative potencies were obtained for 26 (human 1 and
guinea pig 1/1.2). Therefore, the in vivo antiplatelet
activities of 9 and 26 were evaluated by determining
the extent of inhibition of ex vivo aggregation after iv
administration to guinea pigs (Figure 4). Intravenous
administration of 9 resulted in dose-dependent inhibi-
tion of platelet aggregation at doses of 0.03 and 0.1 mg/
kg, and the activity lasted for 4 h at 0.1 mg/kg. On the
Exp er im en ta l Section
Melting points were determined on a Yanagimoto mi-
cromelting apparatus and are uncorrected. Infrared (IR)
spectra were recorded with a J ASCO IR-800 spectrophotom-

Novel Non-Peptide Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 497
eter. 1H NMR spectra were taken on a Varian XRD-200
spectrometer in CDCl3 unless otherwise noted. Chemical
shifts are given in ppm with tetramethylsilane (TMS) as an
internal standard, and coupling constants (J ) are given in hertz
(Hz). The following abbreviations are used: s ) singlet, d )
doublet, q ) quartet, t ) triplet, dd ) double doublet, m )
multiplet, b ) broad peak. Mass spectra (MS) were recorded
with a J EOL J MS-AX505W apparatus. Optical rotations were
recorded on a J ASCO DIP-370 digital polarimeter. The yields
obtained are not optimized. The following abbreviations are
used for reagents: EDC, 1-ethyl-3-[3-(N,N-dimethylamino)-
propyl]carbodiimide hydrochloride; TFA, trifluoroacetic acid;
DCC, dicyclohexylcarbodiimide; DEPC, diethylphosphoryl cya-
nide.
ter t-Bu tyl N-(2,2-Dim eth oxyeth yl)glycin e (3). tert-Bu-
tyl chloroacetate (200 g, 1.45 mol) was added dropwise to a
mixture of 2,2-dimethoxyethylamine (150 g, 1.43 mol), anhy-
drous K2CO3 (200 g, 1.45 mol), and DMF (1.4 L) over 1 h with
stirring at room temperature. The solution was stirred for
another 7 h, poured into ice-water, and extracted with EtOAc.
The organic layer was washed with saturated NaCl solution,
dried over anhydrous MgSO4, and concentrated in vacuo. The
residual oil was distilled under reduced pressure to give 3
(162.7 g, 51.9%) as a colorless oil: bp 80-85 °C, 0.4 mmHg;
IR (neat) cm-1 2970, 2900, 1735, 1455, 1240, 1045, 860; 1H
NMR 1.49 (9H, s), 2.75 (2H, d, J ) 5 Hz), 3.33 (2H, s), 3.39
(6H, s), 4.47 (1H, t, J ) 5.4 Hz).
of trans-4-(guanidinomethyl)cyclohexanecarboxylic acid (300
mg, 1.5 mmol), N-hydroxysuccinimide (174 mg, 1.5 mmol),
DCC (320 mg, 2 mmol), and DMF (5 mL) was stirred at room
temperature for 1 h and concentrated in vacuo. The residue
was dissolved in 1,4-dioxane (1 mL) (solution A). A solution
of 6 (520 mg, 1.0 mmol) in MeOH (10 mL) was treated with
10% Pd/C (50 mg) and hydrogenated under atmospheric
pressure at room temperature for 1 h. The reaction mixture
was filtered, and the filtrate was concentrated in vacuo. To a
mixture of the residual oil and NaHCO3 (420 mg, 5 mmol) in
H2O (5 mL) and 1,4-dioxane (5 mL) was added solution A
dropwise at room temperature. The resulting mixture was
stirred for 2 h and then concentrated in vacuo. The residue
was subjected to column chromatography (ODS, 1:9 CH3CN/
H2O) to give 1a (260 mg, 43%) as a colorless powder: [R]20
D
+65.8° (c ) 0.25, MeOH); IR (KBr) cm-1 3390, 2930, 1730,
1
1645, 1550, 1445, 1210, 970; H NMR (DMSO-d6) 0.82-1.08
(2H, m), 1.20-1.52 (3H, m), 1.68-1.88 (3H, m), 2.08-2.26 (1H,
m), 2.69 (2H, d, J ) 5.4 Hz), 2.97 (2H, dd, J ) 5.4, 5.6 Hz),
3.58-3.71 (1H, m), 3.81-4.35 (4H, m), 4.89 (1H, t, J ) 5.4
Hz), 7.64-8.06 (4H, m). Anal. (C19H30N6O7‚HCl) C, H, N.
ter t-Bu tyl (S)-3-[N-(Ben zyloxyca r bon yl)a m in o]-4-[N-
(2,2-dieth oxyeth yl)-N-ph en eth ylam in o]-4-oxobu tylate (7).
EDC (2.4 g, 12.5 mmol) was added to a solution of (2,2-
diethoxyethyl)phenethylamine (2.96 g, 12.5 mmol) and N-Cbz-
Asp(O-t-Bu)-OH (3.42 g, 10 mmol) in CH3CN (50 mL). The
mixture was stirred at room temperature for 1.5 h and
evaporated in vacuo. The residual oil was dissolved in EtOAc,
washed with 5% aqueous KHSO4 and then saturated NaHCO3,
dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was subjected to column chromatography (SiO2, 1:1
ter t-Bu tyl N-[N-(Ben zyloxyca r bon yl)-O-ter t-bu tyl-L-a s-
p a r tyl]-N-(2-d im eth oxyeth yl)a m in oa ceta te (4). EDC (5.7
g, 30 mmol) was added to a mixture of 3 (6.8 g, 28 mmol),
N-Cbz-Asp(O-t-Bu)-OH (8.1 g, 25 mmol), and CH3CN (100 mL)
with stirring at room temperature. After stirring for another
2 h, the reaction mixture was evaporated in vacuo. The
resulting oil was dissolved in EtOAc, washed successively with
5% KHSO4 and saturated NaHCO3, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was subjected
to column chromatography (SiO2, 1:4 EtOAc/n-hexane) to give
4 (6.9 g, 50%) as a colorless oil: IR (neat) cm-1 3300, 2975,
n-hexane/EtOAc) to give 7 (4.6 g, 78%) as a colorless oil: [R]20
D
-8.7° (c ) 0.63, MeOH); IR (neat) cm-1 2975, 1720, 1640, 1530,
1
1450, 1365, 1290, 970; H NMR 1.16 (3H, t, J ) 7 Hz), 1.19
(3H, t, J ) 7 Hz), 1.26 (3/2H, t, J ) 7.0 Hz), 1.42 (9H, s), 2.05
(3/2H, s), 2.30-3.05 (4H, m), 3.20-3.85 (8H, m), 4.12 (2/2H,
q, J ) 7.0 Hz), 4.61 (1H, t, J ) 5.3 Hz), 5.01 (1H, m), 5.10
(2H, s), 5.50 (1H, t, J ) 8.8 Hz), 7.15-7.40 (10H, m). Anal.
(C30H41N2O7‚1/2EtOAc) C, H, N.
1
2930, 1720, 1655, 1525, 1450, 1360, 1160, 640, 745, 690; H
NMR 1.43 (9H, s), 1.45 (9H, s), 2.60-2.94 (2H, m), 3.40-4.05
(4H, m), 3.66 (6H, s), 4.58 (1H, t, J ) 5.4 Hz), 5.12 (2H, s),
5.81 (1H, t, J ) 3 Hz), 7.34 (5H, s).
ter t-Bu tyl (S)-1-P h en eth yl-2-oxop ip er a zin e-3-a ceta te
(8). A solution of 7 (3.93 g, 7.25 mmol) and p-toluenesulfonic
acid monohydrate (0.14 g) in toluene (200 mL) was stirred at
80 °C for 4 h. After cooling, the mixture was washed with
saturated aqueous NaHCO3, dried over anhydrous Na2SO4,
and concentrated in vacuo. The residue was subjected to
column chromatography (3:1 hexane/EtOAc) to give a tetrahy-
dropyrazine intermediate as a colorless oil (1.5 g, 46%): IR
(neat) cm-1 1715, 1680, 1400, 1310, 1145, 1115, 695; 1H NMR
1.14 (9H, s), 2.55 (2H, d, J ) 6.4 Hz), 2.88 (2H, m), 3.72 (2H,
t, J ) 6.4 Hz), 5.00-5.55 (2H, m), 5.19 (2H, s), 6.10-6.35 (1H,
m), 7.15-7.35 (5H, m), 7.37 (5H, s).
Di-ter t-bu tyl (S)-2-Oxop ip er a zin e-1,3-d ia ceta te (5a ). A
mixture of 4 (20 g, 36.2 mmol), p-toluenesulfonic acid (680 mg,
3.5 mmol), and toluene (750 mL) was stirred at 70 °C for 2 h.
After cooling, the solution was washed with saturated NaH-
CO3, dried over anhydrous MgSO4, and concentrated in vacuo.
The residual oil was dissolved in methanol (500 mL), treated
with 10% Pd/C (10 g), and hydrogenated under atmospheric
pressure at room temperature for 12 h. The reaction mixture
was filtered, and the filtrate was condensed in vacuo to give
5a (12.7 g, 75%) as a colorless oil, which was converted into
an oxalate and recrystallized from MeOH-EtOAc-hexane to
The oil thus obtained (1.5 g, 3.2 mmol) was dissolved in
MeOH (10 mL), treated with 10% Pd/C (1.5 g), and then
hydrogenated under atmospheric pressure at room tempera-
ture for 1 h. The reaction mixture was filtered, and the filtrate
was concentrated in vacuo. The residue was subjected to
column chromatography (SiO2, 9:1 EtOAc/MeOH) to give 8
afford colorless crystals: mp 158-159 °C; IR (neat) cm-1
:
3450, 3300, 2830, 2810, 1740, 1645, 1500, 1450, 1370, 1240,
1
1160, 990; H NMR 1.48 (9H, s), 1.51 (9H, s), 2.75-3.95 (8H,
m), 4.03 (1H, t, J ) 4.5 Hz). Anal. (C16H28N2O5‚C2H2O4) C,
(0.76 g, 69%) as a colorless oil: [R]20 -57.7° (c ) 0.583,
H, N.
D
MeOH); IR (neat) cm-1 2975, 1720, 1640, 1490, 1450, 1360,
1150; 1H NMR 1.45 (9H, s), 2.50-3.74 (12H, m), 7.15-7.37
(5H, m). Anal. (C18H26N2O3‚1/2H2O) C, H, N.
Di-ter t-b u t yl (S)-4-[N-(Ben zyloxyca r b on yl)glycyl]-2-
oxop ip er a zin e-1,3-d ia ceta te (6). A mixture of 5a (3.7 g, 9
mmol), N-Cbz-Gly-OH (2.1 g, 9 mmol), EDC (2.5 g, 13 mmol),
and CH3CN (20 mL) was stirred at room temperature for 2 h
and then concentrated in vacuo. The residual oil was dissolved
in EtOAc, washed with aqueous 5% KHSO4 and then saturated
NaHCO3, dried over anhydrous MgSO4, and evaporated in
vacuo. The residue was subjected to column chromatography
(SiO2, 1:1 EtOAc/n-hexane) to give 6 (3.7 g, 79%) as a colorless
oil: IR (neat) cm-1 : 3420, 2975, 2900, 1720, 1650, 1495, 1450,
1360, 1240, 1205, 1155, 1040; 1H NMR 1.42 (9H, s), 1.44 (9H,
s), 2.50-3.68 (4H, m), 3.68-3.97 (4H, m), 4.85 (1H, t, J ) 5.2
Hz), 5.13 (2H, s), 5.66 (1H, b s), 7.35 (5H, s) Anal. (C26H37N3O8)
Calcd: 519.2581. Found: 519.2584..
(S)-4-[[tr a n s-[4-(Gu an idin om eth yl)cycloh exyl]car bon yl]-
glycyl]-3-p h en eth yl-2-oxop ip er a zin e-1-a cetic Acid (2). A
mixture of trans-4-(guanidinomethyl)cyclohexanecarboxylic
acid (600 mg, 3 mmol), N-hydroxysuccinimide (348 mg, 3
mmol), and DCC (830 mg, 4 mmol) in DMF (8 mL) was stirred
at room temperature for 1 h. The solution was filtered to give
a solution of an activated ester (solution A). EDC (0.48 g, 2.5
mmol) was added to a solution of 8 (0.69 g, 2.1 mmol) and
N-Cbz-Gly-OH (0.44 g, 2.1 mmol) in CH3CN (15 mL), and the
mixture was stirred at room temperature for 2 h. The reaction
mixture was evaporated in vacuo. The residue was dissolved
in EtOAc, washed with 5% aqueous KHSO4 and then saturated
NaHCO3, dried over anhydrous Na2SO4, and concentrated in
(S)-4-[[tr a n s-[4-(Gu an idin om eth yl)cycloh exyl]car bon yl]-
glycyl-2-oxop ip er a zin e-1,3-d ia cetic Acid (1a ). A mixture

498 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sugihara et al.
vacuo. A solution of the residual oil in MeOH (20 mL) was
treated with 10% Pd/C (500 mg) and hydrogenated under
atmospheric pressure at room temperature for 1 h. The
reaction mixture was filtered, and the filtrate was concentrated
in vacuo. To a mixture of the residual oil and NaHCO3 (840
mg, 10 mmol) in H2O (5 mL) and dioxane (5 mL) was added
solution A dropwise followed by stirring at room temperature
for 2 h. The mixture was evaporated in vacuo, and the residue
was treated with CH2Cl2 (10 mL)-TFA (10 mL) at room
temperature for 1 h. The mixture was evaporated in vacuo,
and the residue was then dissolved in 0.5 N HCl and subjected
to column chromatography (ODS, 1:4 CH3CN/H2O) to give 2
Piperazine derivatives 5c-g were prepared by the same
procedure as that described for the synthesis of 5b.
ter t-Bu t yl 2-oxop ip er a zin e-1-a cet a t e (5c): conversion
into a hydrochloride and recrystallization from MeOH-Et2O
gave colorless crystals (yield 65%); mp 156 °C. Anal.
(C10H18N2O3‚HCl) C, H, N.
ter t-Bu t yl (S)-3-[[N-(4,4′-d im et h oxyb en zh yd r yl)ca r -
ba m oyl]m eth yl]-2-oxop ip er a zin e-1-a ceta te (5d ): conver-
sion into a hydrochloride and recrystallization from EtOH-
Et2O gave colorless crystals (yield 60%); mp 120-123 °C; [R]20
D
-93.2° (c ) 0.91, MeOH). Anal. (C27H35N3O6‚HCl) C, H, N.
ter t-Bu t yl
(S)-3-b en zyl-2-oxop ip er a zin e-1-a cet a t e
(520 mg, 45%) as a colorless powder: [R]20 +87.4° (c ) 0.57,
D
(5e): conversion into a hydrochloride and recrystallization
from MeOH-Et2O gave colorless crystals (yield 83%); mp 206-
MeOH); IR (KBr) cm-1 3350, 2940, 1740, 1650, 1540, 1495,
1
208 °C; [R]23 -93.2° (c ) 0.95, MeOH). Anal. (C17H24N2O3‚
1455, 1360, 1280, 1210, 1165, 750, 700; H NMR (DMSO-d6)
D
0.85-1.05 (2H, m), 1.20-1.50 (3H, m), 1.69-1.87 (3H, m),
2.10-2.27 (1H, m), 2.69-2.83 (3H, m), 2.97 (2H, t, J ) 5.8
Hz), 3.05-4.40 (11H, m), 4.77 (1H, t, J ) 5.6 Hz), 7.20-7.34
(5H, m). Anal. (C25H36N6O5‚HCl‚1/2H2O) C, H, N.
Compounds 1b-i were synthesized by the same procedure
as that described for the synthesis of 2.
HCl) C, H, N.
ter t-Bu t yl (S)-3-[2-(ter t-b u t oxyca r b on yl)et h yl]-2-ox-
op ip er a zin e-1-a ceta te (5f): conversion into an oxalate and
recrystallization from MeOH-EtOAc-hexane gave colorless
crystals (yield 70%); mp 153-155 °C; [R]20 -18.7° (c ) 1.0,
D
MeOH). Anal. (C17H30N2O5‚C2H2O4) C, H, N.
(S)-4-[(4-Gu a n id in obu tyl)glycyl]-2-oxop ip er a zin e-1,3-
ter t-Bu tyl (S)-3-[2-(m eth oxyca r bon yl)eth yl]-2-oxop ip -
er a zin e-1-a ceta te (5g): conversion into an oxalate and re-
crystallization from MeOH-Et2O gave colorless crystals (yield
d ia cetic a cid (1b): amorphous powder (yield 34%); [R]20
D
+83.0° (c ) 0.28, MeOH). Anal. (C17H26O7‚HCl‚H2O) C, H,
65%); mp 139-141 °C; [R]20 -21° (c ) 0.75, MeOH). Anal.
N.
D
(C14H24N2O5‚C2H2O4) C, H, N.
(S)-4-[(N-Am idin oison ipecotyl)glycyl]-2-oxopiper azin e-
1,3-d ia cetic a cid (1c): amorphous powder (yield 59%); [R]20
ter t-Bu tyl (R)-3-[(m eth oxyca r bon yl)m eth yl]-2-oxop ip -
er a zin e-1-a ceta te ((R)-5b): conversion into an oxalate and
recrystallization from MeOH-Et2O gave colorless crystals: mp
D
+57.5° (c ) 0.50, MeOH). Anal. (C17H26N6O7‚H2O) C, H, N.
(S)-4-[(4-Am id in oben zoyl)glycyl]-2-oxop ip er a zin e-1,3-
d ia cetic a cid (1d ): recrystallization from H2O-methanol
140-141 °C; [R]20 +25.4° (c
) 0.60, MeOH). Anal.
D
gave colorless crystals (yield 53%); mp 254-258 °C; [R]20
(C13H22N2O5‚C2H2O4) C, H, N.
D
+90.1° (c ) 1.0, H2O). Anal. (C18H21N5O7‚1/2H2O) C, H, N.
(S)-4-[(4-Gu a n id in ob en zoyl)glycyl]-2-oxop ip er a zin e-
(S)-4-[(4-Am id in oben zoyl)glycyl]-3-[(m eth oxyca r bon -
yl)m eth yl]-2-oxop ip er a zin e-1-a cetic Acid (9). A mixture
of 5b (39 g, 136 mmol), N-Cbz-Gly-OH (28.4 g, 136 mmol), EDC
(27.8 g, 146 mmol), and DMF (200 mL) was stirred at room
temperature for 2 h and evaporated in vacuo. The residual
oil was dissolved in EtOAc, and the solution was washed with
aqueous 5% KHSO4 and saturated NaHCO3, dried over
anhydrous MgSO4, and concentrated in vacuo. A solution of
the residual oil in MeOH (15 mL) was treated with 10% Pd/C
(300 mg) and hydrogenated under atmospheric pressure at
room temperature for 1 h. The reaction mixture was filtered,
and the filtrate was evaporated in vacuo. To a mixture of the
residual oil and NaHCO3 (1.05 g, 12.5 mmol) in H2O (20 mL)
and 1,4-dioxane (20 mL) was added 4-amidinobenzoyl chloride
hydrochloride (1.67 g, 7.64 mmol) portionwise over 10 min with
stirring at room temperature. After stirring for 2 h, the
reaction mixture was neutralized with 1 N HCl and concen-
trated in vacuo. To a suspension of the residual powder in
CH2Cl2 (20 mL) was added TFA (20 mL) at 0 °C, and the
resulting mixture was gradually warmed to room temperature,
stirred for 1 h, and evaporated in vacuo. The crude product
was purified by column chromatography (CHP-20P, 5:95 CH3-
CN/H2O) to give 9 (1.9 g, 70%). Recrystallization from H2O-
1,3-d ia cetic a cid (1e): amorphous powder (yield 64%); [R]20
+78.6° (c ) 0.30, MeOH). Anal. (C18H22N6O7‚HCl) C, H, N.
D
(S)-4-[[4-(Gu a n id in om eth yl)ben zoyl]glycyl]-2-oxop ip -
er a zin e-1,3-d ia cetic a cid (1f): amorphous powder (yield
46%); [R]20 +58.5° (c ) 0.18, MeOH). Anal. (C19H24N6O7‚
D
HCl) C, H, N.
(S)-4-[(3-Am id in oben zoyl)glycyl]-2-oxop ip er a zin e-1,3-
d ia cetic a cid (1g): amorphous powder (yield 29%); [R]23
D
+92.6° (c ) 0.30, H2O). Anal. (C19H23N5O7‚HCl‚H2O) C, H,
N.
(S)-4-[(3-Am id in oben zoyl)glycyl]-2-oxop ip er a zin e-1,3-
d ia cetic a cid (1h ): amorphous powder (yield 71%); [R]20
+73.7° (c ) 0.15, H2O). Anal. (C18H21N5O7‚HCl) C, H, N.
D
(S)-4-[[4-N,N-Dim eth ylgu a n id in o)ben zoyl]glycyl]-2-ox-
opiper azin e-1,3-diacetic acid (1i): amorphous powder (yield
58%); [R]23 +95.6° (c ) 0.15, H2O). Anal. (C20H26N6O7‚
D
HCl‚H2O) C, H, N.
ter t-Bu tyl (S)-3-[(Meth oxyca r bon yl)m eth yl]-2-oxop ip -
er a zin e-1-a ceta te (5b). A mixture of 3 (7.6 g, 34.7 mmol),
N-Cbz-Asp(O-t-Bu)-OH (8.8 g, 31.6 mmol), and EDC (7.87 g,
41 mmol) in CH2Cl2 (100 mL) was stirred at room temperature
for 2 h. The mixture was concentrated in vacuo. The residual
oil was dissolved in EtOAc, washed with aqueous 5% KHSO4
and then saturated NaHCO3, dried over anhydrous MgSO4,
and evaporated in vacuo. The residue and p-toluenesulfonic
acid (665 mg, 3.5 mmol) were dissolved in toluene (350 mL),
and the mixture was stirred at 70 °C for 1.5 h. After cooling,
the reaction mixture was washed with saturated NaHCO3,
dried over anhydrous MgSO4, and concentrated in vacuo. The
residual oil was dissolved in MeOH (500 mL), treated with
10% Pd/C (5 g), and hydrogenated under atmospheric pressure
at room temperature for 20 h. The reaction mixture was
filtered, and the filtrate was evaporated in vacuo to give 5b
(7.9 g, 72%) as a colorless oil, which was converted into an
oxalate and recrystallized from MeOH-EtOAc-hexane to give
EtOH gave colorless crystals: mp 278 °C dec; [R]20 +82.0° (c
D
) 1.0, H2O); IR (KBr) cm-1 3350, 3045, 1735, 1690, 1630, 1590,
1450, 1380, 1340; 1H NMR (D2O) 2.84-2.89 (2H, m), 3.05-
3.44 (2H, m), 3.60 (3H, s), 3.98-4.96 (6H, m), 5.08 (1H, t, J )
6.2 Hz), 7.20 (2H, d, J ) 8.4 Hz), 7.80 (2H, d, J ) 8.4 Hz).
Anal. (C19H23N5O7‚1/2H2O) C, H, N.
Compounds 10-14 and (R)-9 were prepared by the same
procedure as that described for the synthesis of 9.
4-[(4-Am id in ob en zoyl)glycyl]-2-oxop ip er a zin e-1-a ce-
tic a cid (10): recrystallization from H2O gave colorless prisms
(yield 69%); mp 255 °C dec. Anal. (C16H19N5O5‚3/2H2O) C, H,
N.
(S)-4-[(4-Am id in oben zoyl)glycyl]-3-(ca r boxym eth yl)-2-
oxop ip er a zin e-1-a cetic a cid (11): recrystallization from 50%
MeOH gave colorless crystals (yield 57%); mp 226-228 °C;
[R]20 +70.3° (c ) 0.4, H2O). Anal. (C18H22N6O6‚2H2O) C, H,
colorless crystals: mp 146-147 °C; [R]20 -26.0° (c ) 0.60,
D
D
MeOH); IR (KBr) cm-1 3450, 2975, 1730, 1660, 1490, 1445,
N.
1
1365, 1240, 1150; H NMR (DMSO-d6) 1.42 (9H, s), 2.71 (1H,
(S)-4-[(4-Am id in oben zoyl)glycyl]-3-ben zyl-2-oxop ip er -
a zin e-1-a cetic a cid (12): conversion into a hydrochloride and
recrystallization from H2O gave colorless crystals (yield 61%);
dd, J ) 16, 6.6 Hz), 2.84 (1H, dd, J ) 16, 5.6 Hz), 3.09-3.91
(7H, m), 3.63 (3H, s). Anal. (C13H22N2O5‚C2H2O4) C, H, N.

Novel Non-Peptide Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 499
mp 290-296 °C; [R]23 +96.9° (c ) 0.1, 1 N HCl). Anal.
100 °C for 24 h. After cooling, the mixture was poured into
ice-water and extracted with n-hexane. The extract was then
dried over anhydrous Na2SO4 and evaporated in vacuo. The
residual oil was subjected to column chromatography (Si2O,
D
(C23H25N5O3‚HCl) C, H, N.
(S)-4-[(4-Am id in oben zoyl)glycyl]-3-(2-ca r boxyeth yl)-2-
oxop ip er a zin e-1-a cetic a cid (13): recrystallization from
H2O-MeOH gave colorless prisms (yield 80%); mp 235-241
1:1 EtOAc/n-hexane) to give 15e (2.2 g, 65.3%) as a colorless
1
oil: [R]20 +10.0° (c ) 1.045, MeOH); H NMR 1.16 (3H, t, J
°C; [R]23 +66.6° (c ) 0.3, H2O). Anal. (C19H23N5O7‚1/2H2O)
D
D
) 6.0 Hz), 1.20 (3H, t, J ) 6.0 Hz), 2.59 (1H, dd, J ) 11.8, 5.0
Hz), 2.75 (1H, dd, J ) 11.8, 6.2 Hz), 2.89 (2H, dd, J ) 6.6, 3.4
Hz), 3.32-3.70 (5H, m), 7.13-7.38 (5H, m).
C, H, N.
(S)-4-[(4-Am id in ob e n zoyl)glycyl]-3-[2-(m e t h oxyca r -
bon yl)eth yl]-2-oxop ip er a zin e-1-a cetic a cid (14): amor-
phous powder (72%); [R]20 +57.8° (c ) 0.5, H2O). Anal.
ter t-Bu tyl (S)-3-[(Meth oxyca r bon yl)m eth yl]-2-oxop ip -
er a zin e-1-p r op ion a te (16a ). EDC (5.75 g, 30 mmol) was
added to a mixture of 15a (7.0 g, 27 mmol), N-Cbz-Asp(O-t-
Bu)-OH (9.2 g, 27 mmol), and CH2Cl2 (100 mL), and the
resulting solution was stirred at room temperature for 5 h.
The solution was evaporated in vacuo, and the residue was
dissolved in EtOAc, washed with aqueous 5% KHSO4, dried
over anhydrous MgSO4, and concentrated in vacuo. A mixture
of the residual oil and p-toluenesulfonic acid (500 mg, 2.6
mmol) in toluene (500 mL) was heated at 70 °C for 1 h. After
cooling, the solution was washed with saturated NaHCO3,
dried over anhydrous MgSO4, and evaporated in vacuo to give
a pale-yellow oil. The residual oil was dissolved in MeOH (200
mL), treated with 10% Pd/C, and hydrogenated under atmo-
spheric pressure at room temperature for 2 h. The reaction
mixture was filtered, and the filtrate was concentrated in
vacuo. The residue was subjected to column chromatography
(SiO2, 1:4 f 1:2 EtOAc/n-hexane) to give 16a (7.2 g, 78%) as
a colorless oil: IR (neat) cm-1 2932, 2742, 1726, 1646, 1366,
1251, 1153; 1H NMR 1.40 (9H, s), 2.27 (1H, b s), 2.53 (2H, t, J
) 7.0 Hz), 2.64 (1H, dd, J ) 17.2, 8.6 Hz), 2.91-3.32 (4H, m),
2.96 (1H, dd, J ) 17.2, 3.2 Hz), 3.58 (2H, t, J ) 7.0 Hz), 3.66
(1H, dd, J ) 8.6, 3.2 Hz).
D
(C20H25N5O7‚H2O) C, H, N.
(R)-4-[(4-Am id in oben zoyl)glycyl]-3-[(m eth oxyca r bon -
yl)m eth yl]-2-oxop ip er a zin e-1-a cetic a cid ((R)-9): recrys-
tallization from H2O-MeOH gave colorless crystals; mp 283
°C dec; [R]20D -81.0° (c ) 1.0, H2O). Anal. (C19H23N5O7‚1/2H2O)
C, H, N.
The optical purities of 9 and (R)-9 were elucidated by HPLC
analysis on a chiral column to be 100% and 98.3%, respectively;
column, SUMICHIRAL OA-5000 (150 × 4.6 mm); temperature,
room temperature; eluant, a methanol solution containing 4%
3 mM aqueous CuSO4; flow rate, 1.0 mL/min; detector, 237
nm; retention time, 33.07 min for 9 and 29.28 min for (R)-9.
ter t-Bu t yl 3-[N-(2-Diet h oxyet h yl)a m in o]p r op ion a t e
(15a ). A solution of 2,2-diethoxyethylamine (2.7 g, 20.3 mmol)
and tert-butyl acrylate (2.6 g, 20.3 mmol) in toluene (50 mL)
was heated under reflux for 3 h and evaporated in vacuo. The
residue was subjected to column chromatography (SiO2, 1:1
EtOAc/n-hexane) to give 15a (2.4 g, 45%) as a colorless oil:
1
IR (neat) cm-1 2974, 1728, 1367, 1157, 1129, 1062; H NMR
1.22 (6H, t, J ) 7.4 Hz), 1.45 (9H, s), 1.57 (1H, b s), 2.42 (2H,
t, J ) 6.6 Hz), 2.74 (2H, d, J ) 5.6 Hz), 2.87 (2H, t, J ) 6.6
Hz), 3.45-3.80 (4H, m), 4.59 (1H, t, J ) 5.6 Hz).
2-Oxopiperazine derivatives 16b-e were synthesized by the
same procedure as that described for the synthesis of 16a .
Meth yl (S)-3-[(ter t-bu toxyca r bon yl)m eth yl]-2-oxop ip -
er a zin e-1-a ceta te (16b): conversion into an oxalate and
recrystallization from MeOH-Et2O gave colorless crystals
Meth yl N-(2-Dim eth oxyeth yl)glycin a te (15b). Methyl
chloroacetate (15 g, 0.14 mol) was added to a solution of 2,2-
dimethoxyethylamine (15 g, 0.14 mol) and K2CO3 (20 g, 0.15
mol) in DMF (150 mL), and the resulting mixture was stirred
at room temperature for 7 h. The reaction mixture was poured
into ice-water and extracted with EtOAc. The organic layer
was washed with saturated NaCl, dried over anhydrous
MgSO4, and concentrated in vacuo. The residue was subjected
to column chromatography (SiO2, 1:1 EtOAc/n-hexane) to give
15b (15.2 g, 51.9%) as a colorless oil: IR (neat) cm-1 2975,
(yield 81%); mp 140-141 °C; [R]20 -29.0° (c ) 0.8, MeOH).
D
Anal. (C13H22N2O5‚C2H2O4) C, H, N.
ter t-Bu t yl (S)-1-(ca r b a m oylm et h yl)-2-oxop ip er a zin e-
3-a ceta te (16c): conversion into an oxalate and recrystalli-
zation from MeOH-EtOAc gave colorless crystals (yield 79%);
mp 160-163 °C dec; [R]20 -16.6° (c ) 0.53, MeOH). Anal.
1
2900, 1735, 1460, 1240, 1050, 855; H NMR 2.73 (2H, d, J )
D
(C12H21N3O4‚C2H2O4) C, H, N.
5.0 Hz), 3.39 (6H, s), 3.45 (2H, s), 3.73 (3H, s), 4.46 (1H, t, J
) 5.0 Hz).
ter t-Bu tyl (S)-2-[3-[(ter t-bu toxyca r bon yl)m eth yl]-2-ox-
op ip er a zin -1-yl]p r op ion a te (16d ): conversion into an ox-
alate and recrystallization from MeOH-EtOAc-n-hexane gave
colorless crystals (yield 50%); mp 175-177 °C dec; [R]20D -34.4°
(c ) 0.45, MeOH). Anal. (C17H30N2O5‚C2H2O4) C, H, N.
ter t-Bu tyl (S)-2-[3-[(ter t-bu toxyca r bon yl)m eth yl]-2-ox-
op ip er a zin -1-yl]p h en yla ceta te (16e): conversion into an
oxalate and recrystallization from MeOH-EtOAc gave color-
N-(2-Diet h oxyet h yl)glycin a m id e (15c). Chloroaceta-
mide (6.5 g, 0.14 mmol) was added to a solution of 2,2-
diethoxyethylamine (7.5 g, 70 mmol) and K2CO3 (10 g, 75
mmol) in DMF (50 mL), and the resulting solution was stirred
at room temperature for 5 h. The reaction solution was poured
into ice-water and extracted with EtOAc. The extract was
washed with saturated NaCl, dried over anhydrous MgSO4,
and evaporated in vacuo. The residue was subjected to column
chromatography (SiO2, EtOAc) to give 15c (7.0 g, 54%) as a
colorless oil: 1H NMR 1.22 (6H, t, J ) 7 Hz), 2.72 (2H, d, J )
5.2 Hz), 3.30 (2H, s), 3.45-3.78 (4H, m), 4.54 (1H, t, J ) 5.2
Hz), 5.75 (2H, b s).
less crystals (yield 71%); mp 180-181 °C dec; [R]20 -68.3° (c
D
) 0.21, MeOH). Anal. (C23H28N2O5‚C2H2O4) C, H, N.
(S)-4-(4-Am idin oben zoyl)-3-[(m eth oxycar bon yl)m eth yl]-
2-oxop ip er a zin e-1-p r op ion ic Acid (17). A mixture of 16a
(2.0 g, 5.85 mmol), N-Cbz-Gly-OH (1.2 g, 5.85 mmol), EDC (1.4
g, 7.0 mmol), and CH2Cl2 (20 mL) was stirred at room
temperature for 1 h. The mixture was evaporated in vacuo,
and the residual oil was dissolved in EtOAc, washed with
aqueous 5% KHSO4, dried over anhydrous MgSO4, and con-
centrated in vacuo. The residue was dissolved in MeOH (30
mL), treated with 10% Pd/C (100 mg), and hydrogenated under
atmospheric pressure at room temperature for 30 min. The
reaction mixture was filtered, and the filtrate was evaporated
in vacuo. To a mixture of the residue and NaHCO3 (420 mg,
5 mmol) in H2O (5 mL) and 1,4-dioxane (5 mL) was added
4-amidinobenzoyl chloride hydrochloride (283 mg, 1.3 mmol)
portionwise with stirring, and the resulting mixture was
stirred at room temperature for 2 h. The reaction mixture was
filtered, and the filtrate was concentrated in vacuo. The
residue was dissolved in TFA (20 mL) followed by stirring for
1 h. The reaction mixture was concentrated in vacuo. After
addition of 1 N HCl (5 mL), the residue was subjected to
ter t-Bu tyl N-(2-Dieth oxyeth yl)-L-a la n in a te (15d ).
A
suspension of H-Ala-O-t-Bu hydrochloride (5 g, 33.4 mmol),
K2CO3 (5.52 g, 40 mmol), sodium iodide (0.3 g), and 2-bromo-
1,1-diethoxyethane (7.9 g, 40 mmol) in DMF was heated at
100 °C for 24 h. After cooling, the mixture was poured into
ice-water and extracted with n-hexane. The extract was
washed with saturated NaCl, dried over anhydrous Na2SO4,
and evaporated in vacuo. The residue was subjected to column
chromatography (SiO2, 1:1 EtOAc/n-hexane) to give 15d (48%)
as a colorless oil: [R]20 -11.0° (c ) 0.5, MeOH); 1H NMR
D
1.17-1.46 (9H, m), 2.60 (1H, dd, J ) 11.6, 4.8 Hz), 2.76 (1H,
dd, J ) 11.6, 6.4 Hz), 3.25 (1H, dd, J ) 14, 7.0 Hz), 3.56-3.80
(2H, m), 4.60 (1H, t, J ) 4.8 Hz).
ter t-Bu tyl N-(2-Dieth oxyeth yl)-L-ph en ylalan in ate (15e).
A suspension of H-Phe-O-t-Bu hydrochloride (2.5 g, 10 mmol),
K2CO3 (2.76 g, 20 mmol), sodium iodide (0.1 g), and 2-bromo-
1,1-diethoxyethane (2.2 g, 11 mmol) in DMF was heated at

500 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4
Sugihara et al.
column chromatography (CHP-20P, 5:95 CH3CN/H2O) to give
amorphous powder (yield 69%); [R]20 +61.5° (c ) 0.24, H2O).
D
17 (110 mg, 23%) as a colorless powder: [R]20 +83.5° (c )
Anal. (C22H30N4O7‚HCl) C, H, N.
D
1.0, H2O); IR (KBr) cm-1 3390, 1639, 1549, 1448, 1302, 1201;
1H NMR (D2O) 2.44-2.54 (2H, m), 2.75-2.86 (2H, m), 3.20-
5.12 (10H, m), 7.90 (2H, d, J ) 8.0 Hz), 7.99 (2H, d, J ) 8.0
Hz). Anal. (C19H23N5O7‚HCl) C, H, N.
(S)-4-[[4-[2-(N,N-Dim eth ylam in o)eth yl]ben zoyl]glycyl]-
2-oxop ip er a zin e-1-a cetic a cid (30): isolated as a mixture
of free base and hydrochloride (1:1); amorphous powder (yield
75%); [R]20D +55.7° (c ) 0.2, H2O). Anal. (C23H32N4O7‚1/2HCl)
C, H, N.
Compounds 18-21 were prepared by the same procedure
as that described for the synthesis of 17.
Molecu la r Mod elin g Stu d ies. We chose four compounds
for molecular modeling: RGDF, 1a , 9, and 18. One hundred
stable conformers were generated for each model compound
by 500-ps molecular dynamics calculation at 900 K, subsequent
annealing to 300 K, and energy minimization using the CVFF
force field of Discover (Molecular Simulation Inc., San Diego,
CA). To avoid overestimation of the electrostatic effect, we
adopted a distance-dependent dielectric constant 4*R and
assumed that the Asp and Arg surrogates were not charged.
The distance between the central carbon atoms on the amidine
(or guanidine) and carboxylate groups was plotted versus
relative energy and conformer population. The SARs of
compounds 9-14 indicate that the carboxymethyl group at the
1-position on the 2-oxopiperazine ring is necessary for GP IIb-
IIIa antagonistic activity, while that at the 3-position is not.
We assumed that this relationship also holds true for 1a , which
has two carboxymethyl groups, and chose the carboxymethyl
group at the 1-position on the 2-oxopiperazine ring as the Asp
surrogate. All molecular modeling and calculations were
performed on a Silicon Graphics INDIGO2 computer using
InsightII/Discover (Molecular Simulation Inc.).
In Vitr o P la telet Aggr ega tion Stu d ies. Blood was
collected from healthy human volunteers by venipuncture.
Guinea pigs were anesthetized with sodium pentobarbital, and
blood was collected by aortic puncture. Blood was withdrawn
into a plastic syringe containing 3.8% (human) or 3.15%
(guinea pig) sodium citrate (1:10 citrate/blood, v/v). Platelet-
rich plasma (PRP) and platelet-poor plasma (PPP) were
obtained by centrifugation at 1000g for 3-5 s and at 1000g
for 20 min at room temperature, respectively. The platelet
count in PRP was adjusted to 3 × 105/mL (human) and 4 ×
105/mL (guinea pig) using an automatic blood cell counter
(Sysmex E2500, Touaiyoudenshi Co., Tokyo, J apan). Platelet
aggregation was measured using an 8-channel aggregometer
(Hematracer VI, Niko Bioscience, Tokyo, J apan). PRP (250
µL), in a cuvette stirred at 1000 rpm, was prewarmed for 2
min at 37 °C with various concentrations of test compounds
(25 µL). The change in light transmittance was measured
after the addition of aggregating reagents (25 µL). Submaxi-
mal concentrations of aggregating agents were used in each
experiment.
F ibr in ogen Bin d in g Stu d ies. The fibrinogen binding
assay was performed by the method of Yoshimura et al.41 In
brief, 100 µL of purified GP IIb-IIIa (1 µg/mL) in buffer A (20
mmol Tris-HCl, 150 mmol NaCl, 1 mmol CaCl2, and 0.02%
NaN3, pH 7.4) was added to the wells of a 96-well plate
(MaxiSorp, Nunc, Denmark) followed by incubation overnight
at 4 °C. The plate was blocked with buffer B (3.5% BSA, 50
mmol Tris-HCl, 100 mmol NaCl, 2 mmol CaCl2, and 0.02%
NaN3, pH 7.4) for 2 h at 30 °C. One hundred microliters of 1
nM biotinylated fibrinogen solutions containing various con-
centrations of test compounds was added to the wells followed
by incubation at 30 °C for 3 h. After the plate was washed
with buffer C (1 mg/mL BSA, 50 mM Tris-HCl, 100 mM NaCl,
2 mM CaCl2, pH 7.4), 100 µL of antibiotin-alkaline phos-
phatase conjugate (Sigma Chemical Co., St. Louis, MO; 1:2000
dilution) was added followed by incubation at 30 °C for 1 h.
One hundred microliters of p-nitrophenyl phosphate (Bio-Rad
Laboratories, Richmond, CA) was added to each well followed
by incubation at room temperature. When the absorbance of
the control wells at 405 nm reached 1.0-1.2 (ca. 2 h later),
100 µL of 0.4 N NaOH was added to stop the reaction, and
the absorbance at 405 nm was measured again. IC50 values
were determined from the dose-response curves.
(S)-4-[(4-Am id in oben zoyl)glycyl]-1-[(m eth oxyca r bon -
yl)m eth yl]-2-oxop ip er a zin e-3-a cetic a cid (18): amorphous
powder (yield 41%); [R]20 +91.4° (c ) 0.2, H2O). Anal.
D
(C19H23N5O7‚HCl‚1/2H2O) C, H, N.
(S)-4-[(4-Am idin oben zoyl)glycyl]-1-(car bam oylm eth yl)-
2-oxop ip er a zin e-3-a cetic a cid (19): amorphous powder
(yield 58%); [R]20D +115.9° (c ) 1.0, H2O). Anal. (C18H21N6O6-
Na‚2.3H2O) C, H, N.
(S)-2-[4-[(4-Am idin oben zoyl)glycyl]-3-(car boxym eth yl)-
2-oxop ip er a zin -1-yl]p r op ion ic a cid (20): amorphous pow-
der (yield 64%); [R]20 +52.7° (c ) 1.0, MeOH). Anal.
D
(C19H23N5O7‚HCl‚H2O) C, H, N.
(S)-2-[4-[(4-Am idin oben zoyl)glycyl]-3-(car boxym eth yl)-
2-oxop ip er a zin -1-yl]p h en yla cetic a cid (21): amorphous
powder (yield 39%); [R]20 -30.9° (c ) 0.3, H2O). Anal.
D
(C25H27N5O7‚HCl) C, H, N.
(S)-4-[(4-(Am in om et h yl)b en zoyl]glycyl]-2-oxop ip er a -
zin e-1,3-d ia cetic Acid (22). A solution of 5a (410 mg, 1
mmol), N-Cbz-Gly-OH (210 mg, 1 mmol), and EDC (285 mg,
1.5 mmol) in CH3CN (3 mL) was stirred at room temperature
for 2 h and then evaporated in vacuo. The residue was
dissolved in EtOAc, washed with aqueous 5% KHSO4, dried
over anhydrous MgSO4, and concentrated in vacuo. The
residue was dissolved in MeOH (10 mL), treated with 10%
Pd/C (50 mg), and hydrogenated under atmospheric pressure
at room temperature for 1 h. The reaction mixture was
filtered, and the filtrate was concentrated to dryness in vacuo.
To a solution of the residual oil in DMF (5 mL) were added
4-[(N-carbobenzoxyamino)methyl]benzoic acid (285 mg, 1 mmol),
DEPC (245 mg, 1.5 mmol), and triethylamine (0.3 mL) at room
temperature followed by stirring for 1 h. The reaction mixture
was diluted with EtOAc, and the resulting solution was
washed with 5% aqueous KHSO4, dried over anhydrous
MgSO4, and evaporated in vacuo. The residual oil was
dissolved in TFA (3 mL), and the solution was stirred for 1 h
at room temperature and then evaporated in vacuo. The
residue was dissolved in MeOH (10 mL), treated with 10%
Pd/C (100 mg), and hydrogenated under atmospheric pressure
at room temperature for 1 h. The reaction mixture was
filtered, and the filtrate was concentrated in vacuo. The
residue was subjected to column chromatography (CHP-20P,
10% CH3CN in H2O) to give 22 (520 mg, 65%) as a colorless
powder: [R]20 +90.4° (c ) 0.25, H2O); IR (KBr) cm-1 3400,
D
3050, 2925, 1730, 1645, 1540, 1505, 1450, 1340, 1200, 1185,
1135, 970, 835; 1H NMR (DMSO-d6) 2.68-2.95 (2H, m), 3.15-
3.50 (2H, m), 3.55-4.50 (10H, m), 4.91 (1H, t, J ) 6.0 Hz),
7.56 (2H, d, J ) 8.0 Hz), 7.93 (2H, d, J ) 8.0 Hz). Anal.
(C18H22N4O7‚CF3CO2H) C, H, N.
Compounds 23-30 were synthesized from 5a -h by the
same procedure as that described for the synthesis of 22.
(S)-4-[[4-(2-Am in oeth yl)ben zoyl]glycyl]-2-oxopiper azin e-
1,3-d ia cetic a cid (23): amorphous powder (49%); [R]20D +88.7°
(c ) 0.20, H2O). Anal. (C19H24N4O7‚CF3CO2H) C, H, N.
(S)-4-[[4-(3-Am in op r op yl)ben zoyl]glycyl]-2-oxop ip er a -
zin e-1,3-d ia cetic a cid (24): amorphous powder (yield 49%);
[R]20 +99.9° (c ) 0.20, H2O). Anal. (C20H26N4O7‚HCl) C, H,
D
N.
4-[[4-(2-Am in oeth yl)ben zoyl]glycyl]-2-oxop ip er a zin e-
1-a cetic a cid (25): amorphous powder (yield 67%). Anal.
(C17H22N4O5‚HCl) C, H, N.
(S)-4-[[4-(2-Am in oet h yl)b en zoyl]glycyl]-3-[(m et h oxy-
car bon yl)m eth yl]-2-oxopiper azin e-1-acetic acid (27): amor-
phous powder (yield 72%); [R]D20 +62.7° (c ) 0.26, H2O). Anal.
(C21H26N4O7‚HCl) C, H, N.
Ex Vivo Stu d ies. Male Hartley guinea pigs (300-400 g)
were used. At various times after iv or po administration of
test compounds, blood was collected, and PRP and PPP were
(S)-3-(2-Ca r b oxyet h yl)-4-[[4-[2-(N,N-d im et h yla m in o)-
eth yl]ben zoyl]glycyl]-2-oxop ip er a zin e-1-a cetic a cid (29):

Novel Non-Peptide Fibrinogen Receptor Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 4 501
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prepared as described for the in vitro study. ADP (20 µL) was
added to the cuvette containing the prewarmed PRP (220 µL).
To eliminate the possible influence of the sensitivity differences
of guinea pig platelets to ADP, which is dependent on the
animal lot and on experimental conditions including drug
administration protocol, two or three vehicle-treated animals
were used as a control at each measuring point. The percent-
age inhibition of platelet aggregation in drug-treated animals
was determined by comparison with aggregation in the
controls at each point.
Ack n ow led gm en t. The authors wish to thank Drs.
M. Fujino, K. Meguro, K. Nishikawa, and Y. Imura for
encouragement and helpful discussion throughout this
work.
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