Discovery of an orally active non-peptide fibrinogen receptor antagonist based on the hydantoin scaffold

1158

J . Med. Chem. 2001, 44, 1158-1176

Discover y of a n Or a lly Active Non -P ep tid e F ibr in ogen Recep tor An ta gon ist

Ba sed on th e Hyd a n toin Sca ffold

Hans Ulrich Stilz,*^,#Wolfgang Guba,^#Bernd J ablonka,^§Melitta J ust,^§Otmar Klingler,^#Wolfgang Ko¨nig,^#

Volkmar Wehner,^#and Gerhard Zoller^#

Chemistry and DG Cardiovascular Agents, Aventis Pharma AG, D-65926 Frankfurt am Main, Germany

Received August 31, 2000

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a

promising new class of antithrombotic agents. The binding of fibrinogen to the fibrinogen

receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural

modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP

IIb/IIIa antagonist 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible

manner, human and dog platelet aggregation as well as ¹²⁵I-fibrinogen binding to ADP-activated

human gel filtered platelets and isolated GP IIb/IIIa with K_ivalues of 9 nM and 0.17 nM,

respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying

the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural

features which are important for binding. Hydrophobic substitutions both at the hydantoin

nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The

crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which

is a promising drug candidate for the treatment of thrombotic diseases in humans.

In tr od u ction

surface protein. A normal platelet contains approxi-

mately 50 000 receptor complexes which bind like sev-

eral other integrins to an Arg-Gly-Asp-Ser (RGDS) tet-

rapeptide recognition sequence.⁸Despite the common

motif, integrins are quite specific in their interaction

with different adhesive proteins, such as fibrinogen,

vitronectin, fibronectin, laminin, collagen, or von Will-

ebrand factor (vWF).

Platelet GP IIb/IIIa antagonists described to date can

be divided into different classes. Monoclonal antibod-

ies,^9,10polypeptides from snake venoms¹¹and leeches,¹²

linear and cyclic peptides containing either the arginine-

glycine-aspartic acid (RGD) recognition sequence^13-17or

the carboxyl-terminal sequence from the fibrinogen

γ-chain,¹⁸and non-peptide antagonists.^7,19-28Several

series of non-peptide antagonists which mimic the RGD

sequence have been identified. Many of these com-

pounds have been shown to inhibit platelet aggregation

independently of the activating agonist, as well as to

prevent thrombus formation in animal models or in

humans.^{7,15b,19,20a,22,23,29,30}

Derived from the RGDS recognition sequence, we

have generated a novel series of potent, selective, and

orally active heterocyclic non-peptide GP IIb/IIIa an-

tagonists, incorporating a hydantoin substructure. In

this publication, we describe the design and synthesis

of the hydantoin series which finally resulted in the

ethylester prodrug 48 (HMR 1794) as a promising

candidate for further clinical profiling. A 3D-QSAR

model applying the GRID/GOLPE ^43,44methodology was

developed to rationalize the SAR data and to highlight

structural features important for binding to the fibrino-

gen receptor.

Vascular injury, thrombus formation, and their is-

chemic complications culminate in a variety of vasooc-

clusive disorders such as unstable angina, myocardial

infarction, stroke, or peripheral arterial disease.^1-3The

acute vascular occlusion is caused by excessive platelet

deposition and aggregation at the sites of atherosclerotic

plaques or damaged arterial surfaces leading to platelet-

rich thrombus formation.⁴Platelet aggregation may be

stimulated by collagen, thrombin, thromboxane A2,

serotonin, adenosine diphosphate (ADP), platelet acti-

vating factor (PAF), epinephrine, or most probably by

a combination of these factors. The primary mechanism

of platelet aggregation is initiated by activation of the

platelet glycoprotein GP IIb/IIIa and linking of platelets

by fibrinogen bound to the receptor GP IIb/IIIa.^5,6Many

of the present antiplatelet agents including aspirin,

ticlopidine, and clopidogrel only interfere with one single

agonistic pathway. However, the specific inhibition of

a single agonistic pathway leaves alternative routes to

platelet activation unaffected and is thus of limited

effectiveness in preventing thrombus formation. There-

fore blocking of GP IIb/IIIa offers a superior approach

in effectively preventing arterial thrombosis.⁷This has

been demonstrated with abciximab (ReoPro), the Fab

fragment of the chimeric anti-GP IIb/IIIa monoclonal

antibody. Abciximab has proven to be beneficial in

several clinical studies of percutaneous transluminal

coronary angioplasty (PTCA), unstable angina, and non-

Q-wave myocardial infarction. Efficacy has been dem-

onstrated after short- and long-term treatment.^9b

The glycoprotein GP IIb/IIIa belongs to the integrin

superfamily and is the most abundant platelet cell

Ch em istr y

* To whom correspondence should be addressed. Tel: (011-49-69)-

305-83294. Fax: (011-49-69)331399. E-mail: Ulrich.Stilz@aventis.com.

The hydantoin ring represents a common feature of

all GP IIb/IIIa inhibitors described in this paper.

#

Chemistry, Aventis Pharma AG.

^§DG Cardiovascular Agents, Aventis Pharma AG.

Published on Web 03/16/2001

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1159

Sch em e 1

The final steps in Scheme 2 were carried out by

standard peptide chemistry. Briefly, hydantoin 3 and

Boc protected dipeptide fragments were coupled using

DCC and HOBt in DMF. The arginine based inhibitors

4-9 were obtained after deprotection and purification

by chromatography. During all these different steps the

guanidino group of arginine was always protonated and

did not cause significant side reactions.Many of the

compounds listed in Table 2 were obtained from hydan-

toin building blocks prepared as shown in Scheme 3

(route B), Scheme 4 (route A), and Scheme 5 (route C).

The hydantoin building blocks 28 were synthesized

starting from the p-bromophenyl ketones 24 by treat-

ment with copper cyanide in DMF to furnish the

p-cyanophenyl ketones 25.³¹Reaction of 25 with potas-

sium cyanide and ammonium carbonate in water/

ethanol yielded the hydantoin derivatives 26.³²Treat-

ment of 26 with chloroacetic acid methylester in the

presence of potassium iodide and sodium methylate

using methanol as solvent provided intermediates 27,

which were converted to the hydantoin building blocks

28 using the Pinner reaction (Scheme 3).³³This ap-

proach was used to prepare the target compounds 16-

18 and 21-23 (Table 2). Compound 13 was synthesized

analogously using 4-nitroacetophenone instead of the

p-bromophenyl ketones for the hydantoin formation.

Ta ble 1. Arginine Based GP IIb/IIIa Inhibitors

Scheme 4 shows the synthesis of the hydantoin build-

ing block 34 starting from methyl 2-(p-nitrophenyl)-

acetic acid 29 which was treated with isoamylnitrite and

sodium methylate in methanol/diethyl ether to yield

oxime 30. Subsequent hydrogenolysis with Pd/C as

catalyst afforded the amino acid derivative 31. Com-

pound 31 was then reacted with ethoxycarbonylmeth-

ylisocyanate in DMF in the presence of N-ethylmorpho-

line to give the urea derivative 32 which was converted

with benzyloxycarbonyl-S-methylisothiourea to the cor-

responding protected guanidino derivative 33. Cycliza-

tion of 33 was conducted in HCl/water/acetic acid at 80

°C to yield hydantoin 34 and finally the target com-

pound 12. The hydantoin precursors of compounds 10

and 11 were prepared analogously starting from (R,S)-

amino-(3-nitro-phenyl)-acetic acid methylester and

2-(R,S)-amino-3-(4-nitro-phenyl)-propionic acid methyl-

ester, respectively. Route A was also used to prepare

15 from (S)-2-amino-5-benzyloxycarbonylamino-pen-

tanoic acid benzyl ester and 3-isocyanato-benzoic acid

ethyl ester.

The synthesis of hydantoin building block 20c is

shown in Scheme 5 (route C). The initial step was the

condensation of (2,5-dioxo-imidazolin-1-yl)acetic acid

methyl ester with 4-cyanobenzaldehyde followed by the

Pinner reaction and cleavage of the ester group to yield

the acetic acid derivative 20c.

The final step in the synthesis of compounds 10-23

and compounds 35-43 was carried out by coupling

(DCC, HOBt) of H-Asp(OBu^t)-Phg-OBu^thydrochloride

(Table 2) with the corresponding hydantoin building

blocks and by coupling of hydantoin 28a with selected

amino building blocks H-R₂(Table 3). The coupling of

building block 14c to the GPIIb/IIIa inhibitor 14 serves

as an illustrative example (Scheme 6). Precursor 14c

was prepared from 27a in a three-step synthesis.

a

Human platelets were isolated from platelet rich plasma

(PRP) by gel filtration on Sepharose 2B. The resulting suspension

of gel-filtered platelets was activated with 10 µM ADP in the

presence of 1 mg/mL fibrinogen. Aggregation was measured as

the maximal increase in light transmittance. Compounds were

added to GFP 2 min before activation with ADP. Inhibition of

aggregation was expressed as IC₅₀value, i.e., the mean concentra-

tion requiring 50% inhibition in GFP samples of two to six different

donors.

However, a variety of different synthetic routes were

used to build up the hydantoin scaffold, depending on

the availability of the corresponding starting materials

(Scheme 1). Amino acids (route A), ketones/aldehydes

(route B), and unsubstituted hydantoins (route C) were

used as precursors.

Compounds 4-9 in Table 1 were prepared as outlined

in Scheme 2 (route A). Arginine methyl ester dihydro-

chloride 1 was reacted with ethoxycarbonylmethyl iso-

cyanate in DMF in the presence of N-ethylmorpholine

to give the urea derivative 2, which was cyclized using

6 N HCl under reflux to obtain the hydantoin 3 in 99%

yield.

The four individual stereoisomers 44, 45, 46, and 47

(Table 4) of the diastereomeric mixture 39 were pre-

1160 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

Sch em e 2^a

a

(a) Ethoxycarbonylmethyl isocyanate, N-ethylmorpholine, DMF; (b) HCl, reflux; (c) DCC, HOBt, DMF; (d) TFA.

pared by a convergent synthesis using (S or R)-3-amino-

3-phenylpropionic acid ethyl ester hydrochloride (49a

or 49b) and (4-(R or S)-((4-amino-imino-methyl)-phenyl)-

4-methyl-2,5-dioxoimidazolidin-1-yl)-acetic acid (57a or

57b ) as building blocks as has been reported previ-

ously.^29c,dThe synthesis of the enantiomerically pure

hydantoin 57a (R) was carried out by separation of the

racemate 52 to the enantiomer 53a (R, > 99% ee) as

outlined in Scheme 7. The corresponding (S) enanti-

omers were prepared analogously.

indicates that the GPIIb/IIIa receptor exposes a hydro-

phobic pocket close to the carboxylic acid binding site

as has already been suggested by others.⁷

A variety of different arginine mimetics were evalu-

ated in order to further constrain the molecules (Table

2). Replacement of the flexible arginine side chain by a

meta substituted phenyl ring provided the diastereo-

meric mixture 10 which was less active than the parent

compound 9. Compounds 10 and 12 demonstrate the

influence of the guanidino position. The guanidino group

in the para position (12) enhances potency 30-fold in

comparison to the meta position (10) and nearly 5-fold

in comparison to 9. These results correlate quite well

with the data from the ¹²⁵I-fibrinogen binding assay^29b

(12, K_i) 0.03 µM, 4-fold more active than 9). This

dramatic increase is reversed on incorporation of a more

flexible phenylmethylene linker (11).

Resu lts a n d Discu ssion

To determine the potential of the compounds for the

treatment of thrombotic diseases, they were evaluated

in vitro for their ability to inhibit ADP-mediated plate-

let aggregation of human gel filtered platelets (IC₅₀,

Table 1). The RGDS tetrapeptide, was used as an ini-

Substitution of the hydrogen atom at the 4-position

of the hydantoin ring by a methyl group (13) enhances

potency and makes this stereocenter stable against

racemization, which is favorable for the isolation of the

individual stereoisomers.

Another approach to improve the potency of the

compounds involves replacement of the strong basic

guanidino center by the less basic amino group. Sur-

prisingly, compound 14, the amino analogue of 13,

exhibited similar activity. Interestingly, the phenyl ring

can be moved to the position of the glycine moiety as in

compound 15 without a dramatic loss of activity com-

pared to 14. However, we could not further improve the

activity within the amino series.

After intensive structure-activity relationship stud-

ies, the amidino derivative 16 turned out to be the most

active molecule inhibiting human platelet aggregation

with an IC₅₀of 40 nM. The diastereomeric mixture 16

was separated by chromatography, providing the two

stereoisomers 17 and 18. Stereoisomer 18 is the more

active fibrinogen receptor antagonist (IC₅₀) 25 nM).

Compound 18 is 36-fold more active than the parent

arginine derivative 9, suggesting that a benzamidine

is an excellent substitute for the arginine as has been

reported by others as well.^7a,b,15bLess active compounds

19 and 20 were obtained by replacing the phenyl linker

in 16 by a phenylmethylene (19) or a phenylmethine

tial lead compound with only moderate activity (IC₅₀

)

100 µM). Although our early compound 4 is closely

related to the RGDS structure, we were surprised to

find that the potency could be improved by a factor of

10 (IC₅₀) 10 µM). On the basis of that result we de-

cided to conserve the hydantoin scaffold which is

responsible for conformationally constraining the linear

peptide.

Further variations at the carboxy terminus were

made to optimize the potency. The serine residue in 4

was replaced by a variety of different amino acids. The

most active compounds were obtained by incorporating

amino acids having a lipophilic side chain (Table 1).

Replacing the serine residue by a leucine in compound

5 improved the activity by a factor of 3 (IC₅₀) 3 µM).

Even more potent molecules were obtained by replacing

serine by valine in compound 6 (IC₅₀) 1 µM), with

hexahydrophenylglycine in compound 7 (IC₅₀) 1.5 µM),

or with phenylglycine in compound 9 (IC₅₀) 0.9 µM).

In addition, 9 inhibited ¹²⁵I-fibrinogen binding to ADP-

activated human GFP in a concentration dependent

manner with a K_ivalue of 0.12 µM. Interestingly, the

serine derivative 4 and the phenylalanine derivative 8

showed the same activity (IC₅₀) 10 µM) in inhibition

of platelet aggregation. In conclusion, the activity of

compounds can be improved by replacing the carboxy

terminal serine with aliphatic or aromatic residues. This

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1161

Ta ble 2. Arginine Replaced GP IIb/IIIa Inhibitors

a

Diastereomers separated.

(20) linker, indicating that a specific three-dimensional

separation of the amidino group and of the carboxylic

acid group of the aspartic acid is particularly favorable

for receptor binding.

ability of compound 12 was determined to be 3% (dog,

3 mg/kg p.o.; duration of action 1.1 h). This behavior

called for further structural modification. One effort was

aimed at reducing the peptidic nature of the compounds

by replacement of the C-terminal amino acids. This

might lead to metabolically more stable compounds with

prolonged duration of action. Another effort was aimed

at improving the oral bioavailability. Attempts to im-

prove the oral activity of 16 by the synthesis of a variety

of prodrugs in order to mask the charged ionic residues

by suitable protective groups led to compounds with

increased bioavailability whose property profile, how-

ever, still did not meet our desire.

Replacement of the methyl group of the hydantoin in

16 by selected groups such as ethyl, cyclopropyl, and

benzyl should further elucidate the substituent effects

at the 4-position of the hydantoin. Compounds 21 (IC₅₀

) 25 nM) and 22 (IC₅₀) 20 nM) are slightly more active

than 16. Compound 23 (IC₅₀) 50 nM) with a larger

side chain as in 16 is slightly less active.

The compounds shown in Table 2 are potent platelet

aggregation inhibitors. However, the bioavailability and

pharmacokinetic profile of these first generation inhibi-

tors was not yet optimal. For example, the oral bioavail-

Variations of the C-terminal R-amino acids are given

in Table 3.

1162 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

Sch em e 3^a

a

(a) CuCN, DMF, reflux; (b) KCN, (NH₄)₂CO₃; (c) Cl-CH₂CO₂CH₃, KI, NaOCH₃; (d) HCl, ethanol; (e) NH₃, isopropanol; (f) HCl, reflux.

Sch em e 4^a

a

(a) Isoamylnitrite, methanol, NaOCH₃; (b) HCl, methanol, DMF, 10% Pd/C, H₂; (c) ethoxycarbonylmethyl isocyanate, N-ethylmorpholine,

DMF, -20 °C; (d) 1-benzyloxycarbonyl-2-methyl-isothiourea, CH₃CO₂H, methanol; (e) HCl, CH₃CO₂H, 80 °C.

Sch em e 5^a

a

(a) NaOAc, CH₃CO₂H, 135 °C, 3 h; (b) HCl, methanol; (c) NH₃, methanol; (d) 6 N HCl.

Again, it is worth noting that the presence of a free

carboxylic acid group of the aspartic acid unit is favor-

able for high activity. Blockade of this carboxylic acid

group by an ester as in compound 36 resulted in

molecules that were less active in the in vitro platelet

aggregation assay. In contrast, blockade of the second

carboxylic acid group had a very limited influence on

the activity as indicated by compound 35. We therefore

focused our attention on replacing the carboxyl terminal

dipeptide moiety with structural elements having only

one carboxylic acid group. Active compounds were

discovered within three different structural classes

including aspartic acid amides (37), diaminopropionic

acid derivatives (38), and â-amino acid derivatives (39).

A variety of different â-amino acids were synthesized

to study this structural class in more detail. Incorpora-

tion of smaller alkyl substituents such as a methyl

group or larger aliphatic residues such as an adamantyl

residue into the side chain of the â-amino acids resulted

in the less active compounds 40 and 41. The phenyl

residue proved to be a particularly favorable residue

within the â-amino acids series.

Compound 39 is a potent inhibitor of human platelet

aggregation with an IC₅₀value of 0.2 µM. In addition,

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1163

Ta ble 3. C-Terminal Variation of GPIIb/IIIa Inhibitors

change unfavorable for binding. Alkylation of the NH

group of the hydantoin ring in derivative 42 had only a

minor effect on receptor binding. Interestingly, this

alkylation influences the pharmacokinetics of the mol-

ecule. Oral dosing of respective ester prodrugs resulted

in higher drug plasma levels in the case of 39 than in

the case of 42. For this reason, further studies were

concentrated on compound 39.

Compound 39 is a mixture of the four possible stereo-

isomers. As has been reported elsewhere, the SS ste-

reoisomers 44 proved to be the most potent fibrinogen

receptor antagonist, inhibiting ADP-activated human

platelet aggregation with an IC₅₀of 20 nM.^29cThe other

three stereoisomers, 45, 46, and 47, are less active

(Table 4). Platelet aggregation activatation by other po-

tent agonists like collagen (IC₅₀) 65 nM), PAF (IC₅₀

)

30 nM), and thrombin (IC₅₀) 40 nM) was also effec-

tively inhibited by 44. The ¹²⁵I-binding to ADP-activated

human GFP and isolated GP IIb/IIIa was inhibited in

a concentration dependent manner with K_ivalues of 9

and 0.17 nM, respectively, suggesting that 44 is a re-

versible inhibitor of GP IIb/IIIa-mediated platelet ag-

gregation. Selectivity of 44 for GP IIb/IIIa was indicated

by testing the inhibition of ristocetin-induced von Will-

ebrand factor binding to GP Ib-IX⁴⁰(IC₅₀> 100 µM) or

39

vitronectin binding to integrin R_vâ₃(IC₅₀> 100 µM).

The drug 44 (S 1197) is a zwitterion containing a

positively charged amidino group and a negatively

charged carboxylic acid group. Probably due to its

zwitterionic nature, the compound is not very soluble

in water. We also assumed that the zwitterionic char-

acter of the molecule could be limiting for the oral

bioavailability. In addition, this compound is amor-

phous, making its large-scale production less feasable.

Several ester prodrugs were synthesized in order to

protect the ionizable carboxylic-acid group of the drug.

The crystalline maleic acid salt 48 (HMR 1794) of the

ethylester prodrug was selected for further studies.

The oral antiplatelet activity of the ethylester prodrug

48 was measured by ex vivo platelet aggregometry. The

prodrug was given orally at a dose of 1 mg/kg to a group

of eight conscious dogs. Between 1 and 8 h after

administration of 48, ADP-induced aggregation was

almost completely inhibited. Platelet function normal-

ized within 24 h, indicating that the fibrinogen receptor

blockade was fully reversible. A favorable terminal

elimination half-life of 9.9 h for the active drug was

observed. Primary hemostasis as determined by skin

bleeding time was not significantly influenced at any

time point during the experiment. These results confirm

that 48 is a promising drug substance for the chronic

treatment and prophylaxis of thrombotic diseases in

humans.

it was found that the compound is orally active in dogs.²⁹

Interestingly, orally active fibrinogen receptor antago-

nists where a â-amino acid derivative was incorporated

at the carboxy terminus but based on quite different

scaffolds have also been reported by other research

groups.^20c,23aSome limited structure/activity studies on

compound 39 were carried out. In particular, the

importance of the two NH groups for receptor binding

was explored. Alkylation of the NH group by a methyl

group at the carboxy-terminus resulted in the less active

diastereomeric mixture 43. Presumably, the respective

NH group has a convenient influence on receptor

binding or the N-alkylation results in a conformational

P h a r m a cop h or e Mod elin g a n d 3D-QSAR

An a lysis

From the conformational analysis of cyclic peptides

were derived various pharmacophoric models exhibiting

turn-extended-turn, cupped, Gly-Asp â-turn, and Arg-

Gly â-turn motifs for the RGD triad.³⁴These models

formed the basis for rational design and 3D database

searches and eventually led to potent nonpeptide inhibi-

tors with structurally diverse central scaffolds. The

common motif between these different structural classes

1164 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

Sch em e 6^a

which was originally proposed by Ku, Miller et al.³⁵The

validity and the predictivity of a 3D-QSAR analysis is

critically dependent on the assumption that all the

examined molecules have the same binding mode. Since

the enlarged binding site hypothesis implies that a

common binding mode can only be assumed within a

given structural class of GP IIb/IIIa inhibitors, separate

3D-QSAR models have to be developed for each scaffold

such as the hydantoins presented here.

Ta ble 4. Stereoisomers of 39

The development of a pharmacophore model involves

the following three main aspects. First, key features for

determining the biological activity have to be selected,

i.e., the pharmacophore hypothesis has to be formulated.

Second, a common conformation has to be defined which

can be adopted by all the active compounds in the data

set, assuming a single binding mode. Ideally this com-

mon conformation is identical to the bioactive conforma-

tion, but there may also be other common conformations

yielding stable, predictive models. The last aspect in

pharmacophore mapping deals with the superposition

of a series of compounds onto a reference conformation

which defines the so-called alignment rule.

In 3D-QSAR the aligned compounds are described by

thousands of molecular field variables which are derived

by calculating at each grid node the interaction energy

between the respective compound and a probe which

represents a chemical group. Each compound will have

different interaction energies around the surface, and

these differences in interaction energies between the

examined molecules are correlated with the observed

changes in biological activity by a partial least squares

(PLS) model. Thus, 3D-QSAR methods can be used to

establish statistical models for the quantitative correla-

tion between 3D molecular structures and biological

activities. Furthermore, 3D-QSAR models can also be

interpreted in terms of molecular structures by high-

lighting regions in 3D space which either increase or

diminish the activity.

Within this general context, the pharmacophore map-

ping procedure for the hydantoin-based fibrinogen

receptor antagonists applying QXP and the resulting

3D-QSAR model generated with GRID/GOLPE will be

described.^43,44

of fibrinogen receptor antagonists is a distance between

10 and 16 Å between the acidic and basic center

depending on the central scaffold. This structural

diversity strongly hints at an enlarged binding site

The structures of compounds 22, 35, 37, and 44 were

selected for defining the 3D pharmacophore (Figure 1)

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1165

Sch em e 7^a

a

(a) NaOH, 145 °C, 10 bar; (b) HCl, ethanol; (c) R-mandelic acid, isopropanol, diisopropyl ether; (d) ethoxycarbonylmethyl isocyanate,

DMF, N-ethylmorpholine; (e) 6 N HCl; (f) CuCN, DMF; (g) HCl, ethanol; (h) NH₃, ethanol.

fibrinogen receptor binding, the recently developed

variable selection methodology smart region definition

(SRD) was defined.⁴³This procedure discards noninfor-

mative interaction energies and is embedded in the

program GOLPE for building, validating, and interpret-

ing 3D-QSAR models.⁴⁴Thus, the original 16 588 X-

variables were finally reduced to 1623 X-variables.

The predictive ability of the models was evaluated by

cross-validation using five randomly assigned groups of

approximately the same size. Here, a two-component

model with a q²) 0.88 and a standard error of

prediction of 0.34 log units was obtained. The corre-

sponding r²value is 0.97 for the model generated with

the complete data set (Figure 2). This is an excellent

result and proves both the validity of the proposed

pharmacophore and the predictive quality of the 3D-

QSAR model for the hydantoin series of fibrinogen

receptor antagonists.

F igu r e 1. Pharmacophore derived by superposition of 44, 22,

and 37 with QXP. For the sake of clarity, 35 has been omitted.

because they represent the most diverse subset of active

compounds within the hydantoin series. Compound 22

is a mimic of the tetrapeptide template RGDF, 37 is an

analogue of the tripeptide RGD C-terminally capped

with an adamantyl moiety, and 44 is the most potent

member with a C-terminal â-amino acid. Compound 35

was added to enforce the superposition of the carboxylic

acid function following glycine, because this compound

proved that the second C-terminal acid moiety of the

tetrapeptide analogues was not essential for interacting

with the fibrinogen receptor. To define a common

pharmacophoric pattern in 3D space, the program QXP

was applied.³⁶QXP uses a superposition force field

which automatically assigns short-range attractive forces

to atoms with similar hydrogen bonding character or

hydrophobicity in different molecules. QXP allows the

molecules to adopt low-energy conformations which

maximize the overlap of similar atoms. The interaction

energies between the superimposed molecules and the

phenolic OH probe were calculated by GRID with a grid

spacing of 1 Å.³⁷Thus, for each molecule 16 588 descrip-

tors (interaction energies) were generated, the so-called

X-matrix. The corresponding IC₅₀values were divided

by the number of diastereomers (where applicable), and

the negative log-transform was applied to linearize the

range of activities. Since a lot of the X-variables do not

contribute to the correlation with the inhibition of

The 3D-QSAR model can also be readily interpreted

by the use of PLS coefficient plots. For each grid point,

the values of the coefficients are a measure for the

correlation of the probe-ligand interaction energies with

the biological activity. In this study the phenolic OH

probe was used; therefore, negative coefficients indicate

that favorable polar interactions increase the activity,

whereas unfavorable steric interactions, i.e., bulky

groups, decrease the activity. Conversely, positive coef-

ficients indicate that polar interactions decrease the

activity, whereas large, hydrophobic moieties increase

the biological activity. As it can be seen in Figure 3, the

contour maps of the positive PLS coefficients (yellow)

at the level 0.002 encompass a much greater volume

than the negative PLS coefficients (cyan) and, therefore,

have a greater impact on correlating structural features

with biological activity. The yellow regions indicate that

bulky, hydrophobic substitutions at the hydantoin

nucleus carrying the amidino moiety and at the C-

terminus increase the affinity toward the fibrinogen

receptor. This is in accordance with the qualitative SAR

derived above.

1166 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

F igu r e 2. Experimental vs calculated activities (pIC₅₀). The highly active compounds are positioned in the upper right corner of

the plot; the less active compounds are located in the lower left corner.

A pharmacophore mapping procedure and 3D-QSAR

analysis resulted in a predictive and stable statistical

model. The model can be readily interpreted in chemical

terms, and the main determinants of affinity toward the

fibrinogen receptor are hydrophobic substitutions at the

hydantoin nucleus and the C-terminus. This is con-

firmed by the qualitative SAR derived from the com-

parison of the different optimization series.

The ethyl ester prodrug 48 (HMR 1794) is an orally

active antithrombotic agent with a favorable terminal

half-life of 9.9 h in dogs. Further studies with respect

to the use of 48 for the chronic treatment and prophyl-

axis of thrombotic diseases in humans including un-

stable angina pectoris, myocardial infarction, stroke, or

peripheral arterial disease are under investigation.

F igu r e 3. Contour maps of the negative (cyan) and positive

(yellow) PLS regression coefficients contoured at 0.002 and

-0.002, respectively. Steric interactions in the yellow regions

will increase the activity. To aid the association of the contour

maps with molecular features, 44 has been displayed for better

orientation.

Exp er im en ta l Section

Con clu sion s

All starting materials not described below were purchased

from commercial sources. All reagents and solvents were used

as received from commercial sources without additional pu-

rification.

Structural modifications of an RGDS tetrapeptide

lead structure have led to the discovery of a non-peptide

fibrinogen receptor antagonist. The drug 44 is a highly

potent, competitive, and selective fibrinogen receptor

antagonist and a much more effective antithrombotic

agent than aspirin, which is currently used to prevent

arterial thrombosis.²⁹The design of 44 is based on the

hydantoin heterocycle onto which the important phar-

macophoric groups are assembled. The conformational

rigidity and well-defined stereochemistry at the two

chiral centers of 44 most likely account for the very high

affinity and specificity of 44 for the fibrinogen receptor.

In addition, the structural rigidity might contribute to

the enhanced metabolic stability and oral activity of 44

compared to the much more flexible tetrapeptide lead

structure.

Melting points were determined on a Bu¨chi capillary melt-

ing point apparatus and are uncorrected. ¹H NMR spectra were

recorded on a Varian Gemini 200 (200 MHz), a Bruker AM

400 (400 MHz), a Bruker Avance DRX 400 (400 MHz), or a

Bruker ARX 500 (500 MHz) spectrometer; δ values in ppm

relative to tetramethylsilane are given. Low-mass spectra were

recorded with fast atom bombardment positive ionization

(+FAB): VG ZAB SEQ; dissociation chemical ionization

(DCI): Kratos MS 80 or VG Trio 2000; positive electrospray

ionization (+ESI): VG BIO-Q. High-resolution spectra were

recorded on a VG ZAB SEQ (+FAB). Optical rotations were

determined on a Perkin-Elmer 241 polarimeter. The specific

rotation has not been corrected. Normal phase silica gel (EM

Science silica gel 60 (70-230 mesh) was used for chromatog-

raphy. TLC plates coated with silica gel 60 F254 (Merck) were

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1167

used; detection by UV light (254 nm) or ninhydrin solution or

iodine vapor was applied. The KHSO₄/K₂SO₄solution was

prepared by dissolving a mixture of KHSO₄(50 g) and K₂SO₄

(100 g) in water (1000 mL). The following solvent mixtures

were used: solvent A: n-butanol (60):water (24):pyridine (20):

glacial acetic acid (6); solvent B: CH₂Cl₂(9):methanol (1):

glacial acetic acid (0.1):water (0.1); solvent C: n-butanol (17):

glacial acetic acid (1):water (2).

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cetyl}-Asp (OBu ^t)-Va l-OBu ^t(6a ). ¹H NMR (DMSO-d₆)

δ 0.88 (d, J ) 7.5 Hz, 6H), 1.38 (s, 18H), 1.55 (m, 2H), 2.00

(m, 1H), 2.40 (dd, J ) 7.5, J ) 15 Hz, 1H), 2.65 (dd, J ) 5 Hz,

J ) 15 Hz, 1H), 3.10 (m, 2H), 3.95 (br s, 3H), 4.10 (br s, 1H),

4.70 (m, 1H), 7.65 (br s, NH), 7.90 (br s, NH), 8.60 (m, NH);

MS m/z (FAB) 584.4 (M + H)⁺.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cet yl}-Asp -Va l-OH Tr iflu or oa cet ic Acid Sa lt (6).

2-(S)-(3-Eth oxyca r bon ylm eth ylu r eid o)a r gin in e Eth yl

Ester (2). At 0 °C, N-ethylmorpholine (19.5 mL, 150 mmol)

and ethoxycarbonylmethylisocyanate (18.45 mL, 150 mmol)

were added to a solution of arginine methyl ester dihydrochlo-

ride 1 (39.18 g, 150 mmol) in DMF (450 mL). The mixture was

stirred at 0 °C for 1 h and at 22 °C for 4 h. The reaction was

monitored by TLC [solvent A]. After completion, the precipitate

was filtered off and the filtrate was evaporated under reduced

pressure to give 103.33 g of a DMF containing oil.

1

[R]²¹) -36.6° (c ) 1, water); H NMR (DMSO-d₆) δ 0.88 (d,

D

J ) 5 Hz, 6H), 1.55 (m, 2H), 2.05 (m, 1H), 2.45 (m, 1H), 2.70

(m, 1H), 3.10 (m, 2H), 3.95-4.10 (m, 4H), 4.63 (m, 1H), 7.15

(br s, 4H, NH), 7.75 (m, 2H, NH), 8.38 (s, 1H, NH), 8.50 (d,

1H, NH). Mass calcd for C₁₈H₃₀N₇O₈, 472.2156; found, 472.2159.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-1-yl]a cet yl}-

Asp (OBu ^t)-(S)-h exa h yd r op h en ylglycin e-OBu ^t(7a ). [R]²⁰

D

) -26.9° (c ) 1, methanol); ¹H NMR (DMSO-d₆) δ 0.95-1.16

(m, 7H), 1.38 (s, 18 H), 1.50-1.80 (m, 8H), 2.40 (m, 1H), 2.66

(m, 1H), 2.96 (m, 2H), 3.07 (m, 2H), 3.95 (m, 2H), 4.68 (m,

1H), 7.13 (br s, 4H, NH), 7.60 (br s, 1H, NH), 7.88 (m, 1H,

NH), 8.35 (m, 1H, NH), 8.50 (m, 1H, NH); MS m/z (FAB) 624.3

(M + H)⁺.

(4-(S)-(3-Gu a n id in op r op yl-2,5-d ioxo-im id a zolid in -1-

yl)a cetic Acid (3). Crude material of 2 (103.33 g) was refluxed

in HCl (6 N, 1000 mL) for 30 min. The solvent was removed

under reduced pressure, and the remaining residue was

adjusted to pH ) 7 with saturated NaHCO₃solution. The

product crystallized from the aqueous solution. The crystals

were sucked, washed with water, and dried under reduced

pressure to give 31.75 g (99%) of 3. ¹H NMR (DMSO-d₆) δ

1.40-1.80 (m, 4H), 2.13 (m, 2H), 3.05 (m, 2H), 3.43 (m, 2H),

4.00 (m, 1H), 7.60-7.90 (m, 5H, NH), 8.20 (m, 1H, NH) 9.40

(m, 1H, NH); MS m/z 272 (M + H)⁺.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cet yl}-Asp -(S)-H exa h yd r op h en ylglycin e-OH Tr i-

flu or oa cetic Acid Sa lt (7). [R]²⁰) -30.6° (c ) 1, water);

D

¹H NMR (DMSO-d₆) δ 0.9-1.16 (m, 7H), 1.26-1.80 (8H), 2.45

(m, 1H), 2.70 (m, 1H), 3.10 (m, 4H), 4.00 (m, 1H), 4.08 (m,

1H), 4.63 (m, 1H), 7.13 (br s, 4H, NH), 7.60 (t, 1H, NH), 7.75

(d, 1H, NH), 8.36 (s, 1H, NH), 8.50 (d, 1H, NH); Mass calcd

for C₂₁H₃₄N₇O₈, 512.2469; found, 512.2469.

Similar methods were employed for compounds 4-9.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cetyl}-Asp (OBu ^t)-Ser (Bu t)-OBu ^t(4a ). At 0 °C, DCC

(660 mg, 3 mmol) was added to a solution of 3 (770 mg, 3

mmol), H-Asp(OBu^t)-Ser(But)-OBu^thydrochloride (1.27 g, 3

mmol), and HOBt (410 mg, 3 mmol) in DMF (20 mL). The

mixture was stirred at 0 °C for 1 h and at 22 °C for 15 h. The

precipitate (urea) was filtered off, and the filtrate was evapo-

rated. After chromatography (silica gel, solvent B) the residue

was freeze-dried to give 745 mg (40%) of 4a . ¹H NMR (DMSO-

d₆) δ 1.13 (s, 9H), 1.40 (s, 18H), 1.50-1.83 (m, 4H), 2.40 (dd,

J ) 15 Hz, J ) 7.5 Hz, 1H), 2.68 (dd, J ) 15 Hz, J ) 7.5 Hz,-

1H), 3.13 (m, 2H), 3.43 (dd, J ) 10 Hz, J ) 5 Hz, 1H), 3.61

(dd, J ) 10 Hz, J ) 5 Hz, 1H), 3.98 (s, 2H), 4.11 (m, 1H), 4.25

(m, 1H), 4.68 (m, 1H), 7.14 (br s, 4H, NH), 7.64 (br t, 1H, NH),

7.91 (br d, 1H, NH), 8.38 (s, 1H, NH), 8.50 (br d, 1H, NH); MS

m/z (FAB) 628.6 (M + H)⁺.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in 1-

yl]a cetyl}-Asp (OBu ^t)-P h e-OBu ^t(8a ). [R]²²) -25.1° (c )

D

1

1, methanol); H NMR (DMSO-d₆) δ 1.40 (s, 18H), 1.48-1.93

(m, 4H), 2.50 (m, 1H), 3.08 (m, 1H), 4.13-4.58 (m, 7H), 7.39

(m, 5H); MS m/z (FAB) 632.6 (M + H)⁺.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cet yl}-Asp -P h e-OH Tr iflu or oa cet ic Acid Sa lt (8).

[R]²³) -18.2° (c ) 1, water); ¹H NMR (DMSO-d₆) δ 1.90 (m,

D

2H), 2.70 (m, 1H), 2.83 (m, 1H), 3.02 (m, 1H), 3.23 (m, 5H),

4.22 (m, 2H), 4.38 (m, 1H), 4.70 (m, 2H); Mass calcd for

C

₂₂H₃₀N₇O₈, 520.2156; found, 520.2156.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]acetyl}-Asp(OBu ^t)-(R,S)-(S)-ph en ylglycin e-OBu ^t(9a).

1

[R]¹⁹) -0.5° (c ) 1, methanol); H NMR (DMSO-d₆) δ 1.33

D

(s, 9H), 1.40 (s, 9H), 1.50-1.80 (m, 4H), 2.45 (m, 1H), 2.70 (m,

1H), 3.13 (m, 2H), 3.98 (s, 2H), 4.10 (br s, 1H), 4.73 (m, 1H),

5.23 (d, J ) 7.5 Hz, 1H), 7.13 (br s, 4H, NH), 7.35 (s, 5H), 7.65

(m, 1H, NH), 8.38 (s, 1H, NH), 8.50 (d, 1H, NH), 8.59 (d, 1H,

NH); MS m/z (FAB) 618.2 (M + H)⁺.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cet yl}-Asp -Ser -OH Tr iflu or oa cet ic Acid Sa lt (4).

Compound 4a (700 mg, 1.12 mmol) was treated with TFA (7

mL, 90%) for 1 h at 22 °C. The solvent was removed, and the

residue was distributed between water and diethyl ether. The

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cetyl}-Asp -(S)-p h en ylglycin e-OH Tr iflu or oa cetic

Acid Sa lt (9). [R]¹⁹_D) +20.7° (c ) 1, water); ¹H NMR (DMSO-

d₆) δ 1.60 (m, 4H), 2.43 (m, 1H), 2.75 (m, 1H), 3.10 (m, 2H),

4.00 (s, 2H), 4.15 (m, 1H), 4.65 (m, 1H), 5.13 (m, 1H), 7.18 (br

s, 4H, NH), 7.34 (m, 5H), 8.29-8.50 (m, 2H, NH), 8.55 (m, 1H,

NH). Mass calcd for C₂₁H₂₈N₇O₈, 506.1999; found, 506.2000.

Similar methods were employed for compounds 10 and 11.

(R,S)-(3-Eth oxycar bon ylm eth ylu r eido)-(3-n itr o-ph en yl)-

a cetic Acid Meth yl Ester (10a ). To a solution of (R,S)-amino-

(3-nitro-phenyl)-acetic acid methyl ester hydrochloride (4.6 g,

19 mmol) and isocyanato-acetic acid ethyl ester (2.4 g, 19

mmol) in DMF (25 mL) was added N-ethylmorpholin (2.5 mL,

20 mmol). The solution was stirred at room temperature for

12 h. The solution was evaporated and treated with CH₂Cl₂

and aqueous hydrochloric acid. The organic layer was sepa-

rated and evaporated to yield 4.55 g (70%) of 10a .

[4-(R,S)-(3-Nitr o-p h en yl)-2,5-d ioxo-im id a zolid in -1-yl]-

a cetic Acid (10b). Compound 10a (4.5 g, 13.2 mmol) was

heated in a mixture of water (25 mL), concentrated HCl (25

mL), and acetic acid (15 mL) at 100 °C for 30 min. The mixture

was cooled, and the product filtered to yield 3.1 g (84%) of 10b.

[4-(R,S)-(3-Am in o-p h en yl)-2,5-d ioxo-im id a zolid in -1-yl]-

a cetic Acid (10c). Compound 10b (300 mg, 1 mmol) was

hydrogenated in methanol (50 mL) and DMF (10 mL) with

aqueous solution was freeze-dried to give 579 mg (90%) of 4.

1

[R]²¹) -19.1° (c ) 1, water); H NMR (DMSO-d₆) δ 1.48-

D

1.83 (m, 4H), 2.45 (m, 1H), 2.69 (m, 1H), 3.10 (m, 2H), 3.65

(m, 2H), 4.00 (s, 2H), 4.13 (br s, 1H) 4.18 (m, 1H), 4.52 (m,

1H), 7.05-7.33 (m, 4H, NH), 7.69 (br s, 1H, NH), 7.93 (m, 1H,

NH), 8.35 (m, 1H, NH), 8.50 (m, 1H, NH). Mass calcd for

C

₁₆H₂₆N₇O₉, 460.1792; found, 460.1786.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cetyl}-Asp (OBu ^t)-Leu -OBu ^t(5a ). mp 79-81 °C; [R]²¹

D

1

) -37.9° (c ) 1, methanol); H NMR (DMSO-d₆) δ 0.84 (d, J

) 6 Hz, 3H), 0.90 (d, J ) 6 Hz, 3H), 1.38 (s, 18 H), 1.40-1.80

(m, 7H), 2.40 (dd, J ) 8 Hz, J ) 16 Hz, 1H), 2.65 (dd, J ) 5

Hz, J ) 16 Hz, 1H), 3.15 (m, 2H), 3.99 (m, 2H), 4.03-4.16 (m,

2H), 4.65 (m, 1H), 7.16 (br s, 4H, NH), 7.66 (t, 1H, NH), 8.07

(d, 1H, NH), 8.38 (s, 1H, NH), 8.47 (d, 1H, NH); MS m/z (FAB)

597.9 (M + H)⁺.

{2-[4-(S)-(3-Gu a n id in op r op yl)-2,5-d ioxo-im id a zolid in -

1-yl]a cet yl}-Asp -Leu -OH Tr iflu or oa cet ic Acid Sa lt (5).

[R]²⁰) -40.5° (c ) 1, water); ¹H NMR (D₂O) δ 0.87 (d, J ) 6

D

Hz, 3H), 0.94 (d, J ) 6 Hz, 3H), 1.53-2.03 (m, 7H), 2.82 (dd,

J ) 8 Hz, J ) 18 Hz, 1H), 2.96 (dd, J ) 6 Hz, J ) 18 Hz, 1H),

3.23 (m, 2H), 4.28 (m, 2H), 4.38 (m, 2H). Mass calcd for

C

₁₉H₃₂N₇O₈, 486.2312; found, 486.2308.

1168 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

Pd/C (100 mg, 10%). After 8 h the catalyst was filtered, and

the filtrate evaporated to yield 140 mg (56%) of 10c.

and the compound crystallized (ethyl acetate) to yield 32 (29.75

g, 69.4%). mp 105-110 °C.

(R,S)-(3-Eth oxyca r bon ylm eth yl-u r eid o)-(4-ben zyloxy-

ca r bon yl-gu a n id in o-p h en yl)a cetic Acid Meth yl Ester

(33). A solution of 32 (11.03 g, 35.7 mmol), 1-benzyloxycarbo-

nyl-2-methyl-isothiourea (8 g, 35.7 mmol) and acetic acid (7.5

g, 124.9 mmol) in methanol (72 mL) was stirred for 12 h. The

solid was filtered and dried to yield 33 (10.68 g, 62%). mp 173-

174 °C.

(R,S)-[4-(4-Ben zyloxyca r bon yl-gu a n id in o-p h en yl)-2,5-

d ioxo-im id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (34).

A mixture of 33 (5.6 g, 11.5 mmol) in water (42 mL),

concentrated HCl (28 mL), and acetic acid (112 mL) was heated

at 80 °C for 90 min. The mixture was evaporated and stirred

with water and methanol to give 4.25 g (80%) 34. mp 214-

216 °C.

[4-(R,S)-(3-Nitr o-gu a n id in o-p h en yl)-2,5-d ioxo-im id a zo-

lid in -1-yl]a cetic Acid (10d ). Compound 10c (700 mg, 2.8

mmol) and 1-nitro-2-methyl-isothiourea (570 mg, 4.2 mmol)

were stirred in NaOH (1 N, 3.5 mL) for 11 h at 80 °C. The

solution was filtered, the aqueous layer was acidified to pH 3

with HCl, and the product was extracted with ethyl acetate.

The compound was purified over Sephadex LH20 using solvent

C and freeze-dried to yield 48 mg (5%) of 10d .

{2-[4-(R,S)-(3-Nit r o-gu a n id in o-p h en yl)-2,5-d ioxo-im i-

d a zolid in -1-yl]a ce t yl}-Asp (OBu ^t)-(S )-p h e n ylglycin e -

OBu ^t(10e). To a solution of 10d (30 mg, 0.09 mmol) in DMF

(5 mL) were added at 0 °C DCC (21 mg, 0.1 mmol), HOBt (12.2

mg, 0.09 mmol), Asp(OBu^t)-(S)-phenylglycine-OBu^thydrochlo-

ride (38.6 mg, 0.09 mmol), and N-ethylmorpholin (10.4 mg,

0.09 mmol). The solution was stirred for 2 h at 0 °C and 12 h

at room temperature. The solvent was removed in vacuo and

the residue treated with ethyl acetate. The dicyclohexyl urea

was filtered, and the filtrate was extracted with aqueous

NaHCO₃solution and KHSO₄solution, washed with water,

dried, and evaporated to give 69 mg of crude material of 10e

which was used in the next step without further purification.

{2-[4-(R,S)-(3-Gu a n id in o-p h en yl)-2,5-d ioxo-im id a zoli-

d in -1-yl]a cetyl}-Asp -(S)-p h en ylglycin e-OH Tr iflu or oa ce-

tic Acid Sa lt (10). Compound 10e (65 mg, 0.09 mmol) was

stirred with TFA (5 mL, 90%) for 3 h and evaporated. The

residue was dissolved in methanol (50 mL) and hydrogenated

at room temperature with Pd/C (0.1 g, 10%) for 2 h. The

catalyst was removed by filtration, and the filtrate was

evaporated, purified over Sephadex LH20 using solvent C, and

freeze-dried to yield 19.5 mg (39%) of 10. mp 150 °C; ¹H NMR

(DMSO-d₆) δ 2.4-2.5 (m, 1H), 2.6-2.8 (m, 1H), 4.05-4.15 (m,

2H), 4.6-4.7 (m, 1H), 5.1-5.25 (m, 1H), 5.33 (s, 1H), 7.1-7.7

(m, 10H), 8.5-8.7 (b, NH), 8.8 (s, NH). Mass calcd for

{2-[4-(R ,S )-(4-Be n zyloxyca r b on yl-gu a n id in o-p h e n -

yl)-2,5-d ioxo-im id a zolid in -1-yl]a ce t yl}-Asp (OBu ^t)-(S)-

p h en ylglycin e-OBu ^t(12a ). The method for compound 4a was

employed to give 3.46 g (63%) of 12a starting from 34.

{2-[4-(R,S)-(4-Gu a n id in o-p h en yl)-2,5-d ioxo-im id a zoli-

d in -1-yl]a cetyl}-Asp -(S)-p h en ylglycin e-OH Tr iflu or oa ce-

tic Acid Sa lt (12). Compound 12a (1.6 g, 2 mmol) was stirred

with TFA (16 mL, 90%) for 1 h, evaporated, and lyophylized.

The residue was dissolved in methanol (100 mL) and hydro-

genated at room temperature with Pd/C (0.5 g, 10%) for 3 h.

The catalyst was removed by filtration, and the filtrate was

evaporated, purified over Sephadex LH20 using solvent C, and

1

freeze-dried to give 0.72 g (65.5%) of 12. H NMR (DMSO-d₆)

δ 2.4-2.55 (m, 1H), 2.65-2.75 (m, 1H), 3.95-4.05 (m, 2H),

4.6-4.75 (m, 1H), 5.2-5.3 (m, 1H), 5.32 (s, 1H), 7.20-7.70 (m,

11H), 8.4-8.5 (m, NH), 8.80 (s, NH), 9.90 (s, NH), 12.5 (b, OH).

Mass calcd for C₂₄H₂₆N₇O₈, 540.1843; found, 540.1835.

(R,S)-5-Meth yl-5-(4-n itr o-p h en yl)-im id a zolid in e-2,4-d i-

on e (13a ). To potassium cyanide (20.8 g, 0.32 mol) and

ammonium carbonate (96.1 g, 1 mol) in water (250 mL) was

added carefully 4-nitroacetophenone (49.5 g, 0.3 mol) in

ethanol (250 mL). The solution was stirred at 50 °C for 5 h.

After cooling, the product was filtered and washed with diethyl

ether to give 52.7 g (75%) of 13a . mp 237-240 °C.

(R,S)-[4-Met h yl-4-(4-n it r o-p h en yl)-2,5-d ioxo-im id a zo-

lid in -1-yl]a cetic Acid Meth yl Ester (13b). Under inert

atmosphere sodium (1.68 g, 73 mmol) was carefully dissolved

in methanol (200 mL). After completion, 13a (17.17 g, 73

mmol) was added and refluxed for 2 h. After addition of

chloroacetic acid methylester (7.92 g, 73 mmol) and KI (12.12

g, 73 mmol), the mixture was refluxed for 10 h and cooled,

and the precipitate was removed by filtration. Evaporation of

the filtrate afford more crude material. Recrystallization (ethyl

acetate) yielded 18.1 g (81%) of 13b.

[4-(R,S)-(4-Am in o-p h en yl)-4-m eth yl-2,5-d ioxo-im id a zo-

lid in -1-yl]a cetic Acid Meth yl Ester (13c). To a solution of

13b (22.2 g, 72.2 mmol) in ethanol (600 mL) and water (133

mL) were added calcium chloride (7.4 g) in water (11 mL),

acetic acid (7.4 mL), and zinc powder (37 g). The mixture was

heated under reflux for 2 h and filtered hot. The residue was

extracted with ethyl acetate, and the filtrates were combined

and evaporated to yield 10.2 g (51%) of 13c.

[4-(R,S)-(4-Ben zyloxyca r b on ylgu a n id in o-p h en yl)-4-

m eth yl-2,5-d ioxo-im id a zolid in -1-yl]a cetic Acid Meth yl

Ester (13d ). The method for compound 33 was employed to

give 2.85 g (58%) of 13d starting from 13c.

C

₂₄H₂₆N₇O₈, 540.1843; found, 540.1850.

{2-[4-(R,S)-(4-Gu a n id in o-b en zyl)-2,5-d ioxo-im id a zoli-

d in -1-yl]a cetyl}-Asp -(S)-p h en ylglycin e-OH Tr iflu or oa ce-

tic Acid Sa lt (11). mp 195-200 °C; ¹H NMR (DMSO-d₆) δ

2.4-2.5 (m, 1H), 2.6-2.75 (m, 1H), 2.85-2.95 (m,1H), 3.0-

3.15 (m,1H), 3.9 (s, 2H), 4.4-4.5 (m, 1H), 4.6-4.7 (m, 1H), 5.2-

5.3 (m, 1H), 7.1-7.2 (m, 2H), 7.4-7.7 (m, 11H), 8.2 (s, NH),

8.4 (d, NH), 8.5 (d, NH), 9.8-10 (b, NH), 12.1-12.8 (b, OH).

Mass calcd for C₂₅H₂₈N₇O₈, 554.1999; found, 554.1990.

Hyd r oxyim in o-(4-n itr o-p h en yl)-a cetic Acid Meth yl Es-

ter (30). To a solution of (4-nitro-phenyl)-acetic acid methyl

ester 29 (245 g, 1.255 mol) in diethyl ether (2500 mL) were

added a solution of isoamyl nitrite (323.5 g, 2.76 mol) in diethyl

ether (1400 mL) and then sodium methoxide (108.3 g, 2.0 mol)

in methanol (1600 mL). The mixture was stirred at room

temperature for 12 h. The solid was filtered, and the filtrate

was evaporated to 3000 mL and acidified to pH 2. The solid

was dissolved in water and acidified with HCl to pH 2. The

precipitations of both treatments were combined and recrys-

tallized from methanol to give 175.9 g (62.5%) of 30. mp 200-

201 °C.

(R,S)-Am in o-(4-a m in o-p h en yl)a cetic Acid Meth yl Es-

ter Hyd r och lor id e (31). A solution of 30 (30 g, 133.8 mmol)

in methanol (1000 mL) and DMF (250 mL) was hydrogenated

over Pd/C (2 g, 10%). The pH value was adjusted between 3

and 4 by addition of HCl in methanol (3.3 mol/L). After 8 h

the catalyst was filtered, the filtrate evaporated, and after

addition of ethyl acetate the product was precipitated. Recrys-

tallization from 2-propanol/ethyl acetate gave 24.5 g (84.5%)-

of 31. mp 155-158 °C.

[4-(R,S)-(4-Ben zyloxyca r b on ylgu a n id in o-p h en yl)-4-

m eth yl-2,5-d ioxo-im id a zolid in -1-yl]a cetic Acid (13e). The

method for compound 34 was employed to give 850 mg (31%)

of 13e.

{2-[4-(R,S)-(4-Ben zyloxyca r bon yl-gu a n id in o-p h en yl)-

4-m eth yl-2,5-d ioxo-im id a zolid in -1-yl]a cetyl-a m in o}-Asp -

(OBu ^t)-(S)-p h en ylglycin e-OBu ^t(13f). The method for com-

pound 4a was employed to give 370 mg (74%) of 13f starting

from of 13e.

(R,S)-(4-Am in o-p h en yl)-(3-eth oxyca r bon ylm eth yl-u r e-

id o)a cetic Acid Meth yl Ester (32). To a solution of 31 (30

g, 138.5 mmol) and N-ethylmorpholin (41.5 g, 360 mmol) in

DMF (200 mL) was slowly added isocyanato acetic acid ethyl

ester (18 g, 139.4 mmol) at -20 to -30 °C. After warming to

room temperature the mixture was evaporated, and water and

ethyl acetate were added. The organic layer was separated,

and the aqueous layer was adjusted to pH 8 and extracted with

ethyl acetate. The combined organic layers were evaporated,

{2-[4-(R,S)-(4-Gu a n id in o-p h en yl)-4-m et h yl-2,5-d ioxo-

im id a zolid in -1-yl]a cetyl}-Asp -(S)-p h en ylglycin e-OH (13).

The method for compound 10 was employed to give 120 mg

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1169

(47%) of 13 starting from 13f. mp > 180 °C;¹H NMR (DMSO-

d₆) δ 1.7 (s, 3H), 2.4-2.55 (m, 1), 2.6-2.75 (m, 1H), 3.95-4.05

(m, 2H), 4.6-4.7 (m, 1H), 5.15-5.25 (m, 1H), 7.2-7.67 (m,

14H), 8.45 (d, NH), 8.55 (d, NH), 9. 0 (m, NH), 9.9 (b, OH).

Mass calcd for C₂₅H₂₈N₇O₈, 554.1999; found, 554.1996.

2-[4-(R ,S )-(4-Am in om e t h yl-p h e n yl)-4-m e t h yl-2,5-d i-

oxoim id a zolid in -1-yl]a cetic Acid Meth yl Ester (14a ). To

a solution of 27a (1 g, 3.48 mmol) in ethanol (8 mL) and acetic

acid (2 mL, 50%) was added Pd/C (200 mg, 10%). The mixture

was hydrogenated at room temperature for 2 h at 3 bar. The

catalyst was filtered off (Celite), and the filtrate was evapo-

rated to give 800 mg (79%) after chromatography (silica gel,

CH₂Cl₂/MeOH, 8:2); MS m/z (FAB) 292.1 (M + H)⁺.

2-[4-(R ,S )-(4-Am in om e t h yl-p h e n yl)-4-m e t h yl-2,5-d i-

oxoim id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (14b). A

solution of 15a (750 mg, 2.57 mmol) in concentrated HCl (15

mL) was heated for 6 h at 100 °C. The solution was evaporated

and the residue freeze-dried to give 700 mg (87%). ¹H NMR

(DMSO-d₆) δ 1.70 (s, 3H), 4.00 (q, J ) 5 Hz, 2H), 4.08 (s, 2H),

7.53 (s, 4H), 8.48 (br s, 3H, NH), 9.10 (s, 1H, NH), MS m/z

(FAB) 278 (M + H)⁺.

2-[4-(R,S)-(4-ter t-Bu t oxyca r b on yla m in om et h yl-p h en -

yl)-4-m eth yl-2,5-d ioxoim id a zolid in -1-yl]a cetic Acid (14c).

A mixture of 14b (300 mg, 0.96 mmol) in dioxane (2 mL) and

water (1 mL) was adjusted to pH 8.0 with NaOH (1 N, 1 mL)

and subsequently cooled to 0 °C. Di-tert-butyl dicarbonate (230

mg, 1.05 mmol) was added while stirring. The reaction mixture

was stirred at room temperature for 3 h. NaOH (1 N, 1.2 mL)

was added continueously to maintain pH at 8.0. The reaction

mixture was evaporated. The residue was adjusted to pH 2.0

(KHSO₄, 0 °C) and distributed between water and ethyl

acetate. The organic extracts were washed with water and

dried, and the solvent was evaporated. The residue was freeze-

dried to give 340 mg (94%). ¹H NMR (DMSO-d₆) δ 1.40 (s, 9H),

1.70 (s, 3H), 4.00-4.19 (m, 4H), 7.25 (d, J ) 9 Hz, 2H), 7.40

(m, 1H, NH), 7.48 (d, J ) 9 Hz, 2H), 8.98 (s, 1H, NH), 13.00

(br s, 1H, COOH).

{2-[4-(R,S)-(4-ter t-Bu toxyca r bon yla m in om eth yl-p h en -

yl)-4-m eth yl-2,5-dioxoim idazolidin -1-yl]acetyl}-Asp(OBu ^t)-

(S)-p h en ylglycin e-OBu ^t(14d ). The method for compound 4a

was employed to give 320 mg (54%) of 14d starting from 14c.

¹H NMR (DMSO-d₆) δ 1.33 (s, 9H), 1.40 (s, 18H), 1.70 (s, 3H),

2.43 (m, 1H), 2.68 (m, 1H), 4.00 (br s, 2H), 4.09 (d, J ) 5 Hz,

2H), 4.70 (m, 1H), 5.20 (m, 1H), 7.24 (d, J ) 9 Hz, 2H), 7.35

(m, 5H), 7.45 (d, J ) 9 Hz, 2H), 7.95 (s, 1H, NH), 8.50 (m, 1H,

NH), 8.60 (d, J ) 7.5 Hz, 1H, NH), 8.86 (s, 1H, NH).

with CH₂Cl₂and potassium hydrogen sulfate solution. The

organic layer was separated and evaporated to yield 187 mg

(68%) of 15c. ¹H NMR (DMSO-d₆) δ 1.50 (s, 9H), 1.55-1.85

(m, 4H), 2.95 (m, 2H), 4.25 (m, 1H), 6.85 (m, 1H, NH), 7.60

(m, 2H), 7.90 (m, 2H), 8.60 (m, 1H, NH).

{3-[4-(S)-(3-ter t-Bu toxyca r bon yla m in o-p r op yl)-2,5-d i-

oxo-im id a zolid in -1-yl]ben zoyl}-Asp (OBu ^t)-(S)-p h en ylgly-

cin e-OBu ^t(15d ). The method for compound 4a was employed

to give 500 mg of crude material 15d starting from 15c which

was used for the next step without further purification.

{3-[4-(S)-(3-Am in o-p r op yl)-2,5-d ioxo-im id a zolid in -1-yl]-

ben zoyl}-Asp -(S)-p h en ylglycin e-OH (15). The method for

compound 10 was employed to give 244 mg (93%, based on

15c) of 15 starting from 15d . mp 201 °C; ¹H NMR (DMSO-d₆)

δ 1.5-1.95 (m, 4H), 2.65-2.95 (m, 4H), 4.00 (br s, 2H), 4.3 (t,

1H), 4.9 (m, 1H), 5.3 (d, 1H), 7.25-8.0 (m, 12H), 8.6 (d, NH),

8.65 (d, NH), 8.8 (d, NH), 12-13.2 (b, OH). Mass calcd for

C

₂₅H₂₈N₅O₈, 526.1938; found, 526.1949.

{2-[4-(R,S)-(4-Am in oim in om et h yl-p h en yl)-4-m et h yl-

2,5-dioxo-im idazolidin -1-yl]acetyl}-Asp-(S)-ph en ylglycin e-

OH Tr iflu or oa cetic Acid Sa lt (16). The method for com-

pound 4a was employed starting from 28a followed by

treatment with TFA to give 390 mg. [R]²⁵) +1.3 ° (c ) 1, in

D

1

methanol); H NMR (DMSO-d₆) δ 1.74 (s, 3H), 2.48 (m, 1H),

3.70 (m, 1H), 4.05 (s, 2H), 4.65 (m, 1H), 5.24 (m, 1H), 7.35 (m,

5H), 7.79 (m, 4H), 8.54 (m, 1H, NH), 8.55 (m, 1H, NH), 9.08

(s, 1H, NH), 9.30 (br s, 4H, NH). Mass calcd for C₂₅H₂₇N₆O₈,

539.1890; found, 539.1892.

{2-[4-(S or R)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cet yl}-Asp -(S)-p h en ylgly-

cin e Tr iflu or oa cetic Acid Sa lt (17). The diastereomeric

mixture 16 was resolved by chromatography on a LiChroprep-

RP-18 reversed-phase column (10 µm) using a water/acetoni-

trile mixture (880 mL of water; 120 mL of acetonitrile; 1 mL

of TFA). Fractions containing the first peak were concentrated

and freeze-dried. [R]³⁰) -14 ° (c ) 1, in water); ¹H NMR

D

(DMSO-d₆) δ 1.75 (s, 3H), 2.45 (m, 1H), 2.70 (m, 1H), 4.03 (s,

2H), 4.63 (m, 1H), 5.30 (d, J ) 7.5 Hz, 1H), 7.38 (m, 5H), 7.70-

7.90 (AA′BB′, 4H), 8.53 (m, 2H, NH), 9.05 (s, 1H,NH), 9.15

(br s, 2H, NH, 9.30 (br s, 2H, NH), 12.70 (br s, 2H, COOH);

MS m/z (FAB) 539 (M + H)⁺.

{2-[4-(R or S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-dioxo-im idazolidin -1-yl]acetyl}-Asp-(S)-ph en ylglycin e-

OH Tr iflu or oa cetic Acid Sa lt(18). Compound 18 was iso-

lated from the diastereomeric mixture 16 by chromatography

on a LiChroprep-RP-18 reversed-phase column (10 µm). Frac-

{2-[4-(R,S)-(4-Am in om et h yl-p h en yl)-4-m et h yl-2,5-d i-

oxoim id a zolid in -1-yl]a ce t yl}-Asp -(S )-p h e n ylglycin e -

OH Tr iflu or oa cetic Acid Sa lt (14). The method for com-

pound 10 was employed to give 160 mg (59%) of 14 starting

tions containing the second peak were concentrated and freeze-

1

dried; [R]³⁰) +20° (c ) 1, in water); H NMR (DMSO-d₆) δ

D

1.75 (s, 3H), 2.45 (m, 1H), 2.70 (m, 1H), 4.03 (s, 2H), 4.63 (m,

1H), 5.30 (d, J ) 7.5 Hz, 1H), 7.38 (m, 5H), 7.70-7.90 (AA′BB′,

4H), 8.53 (m, 2H, NH), 9.05 (s, 1H, NH), 9.15 (br s, 2H, NH),

9.30 (br s, 2H, NH), 12.70 (br s, 2H, COOH; MS m/z (FAB)

539 (M + H)⁺.

from 14d . [R]²³) +1.7° (c ) 1, methanol); ¹H NMR (DMSO-

D

d₆) δ 1.70 (s, 3H), 2.45 (m, 1H), 2.70 (m, 1H), 4.00 (br s, 2H),

4.00 (m, 4H), 4.63 (m, 1H), 5.20 (m, 1H), 7.20-7.60 (m, 9H),

8.38 (m, 1H, NH), 8.53 (d, 1H, J ) 7.5 Hz, NH), 8.95 (s, 1H,

NH). Mass calcd for C₂₅H₂₈N₅O₈, 526.1938; found, 526.1938.

3-[3-(1-Ben zyloxycar bon yl-4-ben zyloxycar bon ylam in o-

bu tyl)-u r eid o]ben zoic Acid Eth yl Ester (15a ). To a solu-

tion of (S)-2-amino-5-benzyloxycarbonylamino-pentanoic acid

benzyl ester hydrochloride (4.1 g, 10.5 mmol) and 3-isocyanato-

benzoic acid ethyl ester (2.0 g, 10.5 mmol) in DMF (20 mL)

was added 1.7 g of N-ethylmorpholin (14.7 mmol), and the

solution was stirred at room temperature for 12 h. The solution

was evaporated and treated with ethyl acetate and KHSO₄

solution. The organic layer was separated and evaporated to

yield 4.1 g (72%) of 15a .

{2-[4-(R,S)-(4-Am in oim in om eth yl-ben zyl)-2,5-d ioxo-im -

id a zolid in -1-yl]a cet yl}-Asp -(S)-p h en ylglycin e-OH Tr i-

flu or oa cetic Acid Sa lt (19). The method for compound 4 was

1

employed; [R]²⁸) +9.7 ° (c ) 1, in water); H NMR (DMSO-

D

d₆) δ 1.74 (s, 3H), 2.65-2.75 (m, 2H), 3.05 (m, 2H), 3.93 (m,

2H), 4.53 (m, 1H), 4.68 (m, 1H), 5.20 (m, 1H), 7.28-7.40 (m,

5H), 7.48 (m, 2H), 7.78 (m, 2H), 8.35-8.53 (m, 2H, NH), 8.90

(m, 3H, NH), 9.40 (m, 2H, NH). Mass calcd for C₂₅H₂₇N₆O₈,

539.1890; found, 539.1882.

[4-(4-Cya n o-ben zylid en e)-2,5-d ioxo-im id a zolid in -1-yl]-

a cetic Acid Meth yl Ester (20a ). A mixture of (2,5-dioxo-

imidazolidin-1-yl)acetic acid methyl ester (30 g, 174 mmol),

4-cyanobenzaldehyde (28.8 g, 220 mmol), and anhydrous

sodium acetate (36 g, 432 mmol) was heated to 135 °C. Acetic

acid (60 mL) was added, and the mixture was stirred for 3 h.

After cooling the mixture was added to 600 mL of water and

70 mL of methanol. The precipitation was filtered and dried

in a vacuum to yield 37.39 g (75%) of 20a . mp 265 °C.

[4-(4-Am in oim in om et h yl-b en zylid en e)-2,5-d ioxo-im i-

d a zolid in -1-yl]a cetic Acid Meth yl Ester Hyd r och lor id e

(20b). Dry HCl was bubbled through a solution of 20a (10 g,

3-[4-(S)-(3-Am in o-p r op yl)-2,5-d ioxo-im id a zolid in -1-yl]-

ben zoic Acid Hyd r och lor id e (15b). The method for com-

pound 12e was employed to give 230 mg (10%) of 15b starting

from 15a . mp 174 °C.

3-[4-(S)-(3-ter t-Bu toxycar bon ylam in o-pr opyl)-2,5-dioxo-

im id a zolid in -1-yl]ben zoic Acid (15c). A solution of 15b (230

mg, 0.73 mmol), di-tert-butyl dicarbonate (270 mg, 1.2 mmol),

and N-ethylmorpholin (200 mg, 1.7 mmol) in DMF (20 mL)

was stirred for 2 h. The solution was evaporated and treated

1170 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

35.1 mmol) in CH₂Cl₂(600 mL) and methanol (100 mL) at 0

°C for 7 h. The mixture was stirred overnight at room

temperature and the precipitate filtered. The precipitate was

dissoved in methanol (1400 mL), and NH₃in methanol (17 mL,

2.935 mol/L) was added. After the mixture was stirred

overnight, diethyl ether was added and the precipitate filtered

and dried to yield 9.3 g (78%) of 20b. mp 290 °C.

for 4 h under reflux. The suspension was allowed to cool to 70

°C, and poured into a solution of of iron(III) chloride (40 g),

concentrated HCl (10 mL), and water (60 mL). The mixture

was stirred at 70 °C for 20 min and extracted three times with

toluene (90 mL). The organic extracts were washed with HCl

(2 N, 250 mL) and with NaOH (2 N, 250 mL) and evaporated.

The solid residue was triturated with ligroine to give 14.57 g

(85%). ¹H NMR (DMSO-d₆) δ 1.10 (m, 4H), 2.93 (m, 1H), 8.00-

8.24 (AA′BB′, 4H); MS m/z (FAB) 172 (M + H)⁺.

4-Cyan ph en ylben zylm eth an on e (25d) starting from 4-bro-

mphenylbenzylmethanone. ¹H NMR (DMSO-d₆) δ 4.45 (s, 2H),

7.30 (m, 5H), 8.00-8.25 (AA′BB′, 4H); MS m/z (FAB) 222 (M

+ H)⁺.

[4-(4-Am in oim in om et h yl-b en zylid en e)-2,5-d ioxo-im i-

d a zolid in -1-yl]a cetic Acid Hyd r och lor id e (20c). Com-

pound 21b (1.61 g, 4.75 mmol) was heated in HCl (6 N, 32

mL) for 2 h under reflux. After cooling, the solid was filtered

1

and dried to give 1.42 g (92%) of 20c. mp > 300 °C; H NMR

(DMSO-d₆) δ 4.20 (s, 2H), 6.66 (s, 1H), 2.70 (m, 1H), 7.75-

7.95 (m, 4H), 9.15-9.50 (m, 4H), 11.23 (s, 1H, NH).

Similar methods were employed for compounds 26a , 26b,

26c, and 26d .

{2-[4-(4-Am in oim in om eth yl-ben zylid en e)-2,5-d ioxo-im -

id a zolid in -1-yl]a cetyl-Asp -(S)-p h en ylglycin e-OH Tr iflu o-

r oa cetic Acid Sa lt (20). The method for compound 4a was

employed starting from 20c followed by treatment with TFA

4-(R,S)-(4-Cya n op h en yl)-4-m eth yl-2,5-d ioxoim id a zoli-

d in e (26a ). 4-Acetyl benzonitrile (20 g, 138 mmol), ammonium

carbonate (115.6 g, 1.21 mol), and potassium cyanide (11.6 g,

178 mmol) were dissolved in ethanol/water (600 mL, 1:1). The

mixture was stirred for 5 h at 55 °C and for 12 h at room

temperature. The solution was adjusted to pH ) 6.3 with 6 N

HCl and subsequently stirred at room temperature for 2 h.

The precipitate was filtered off, washed with water, and dried

over phosphorus pentoxide under high vacuum to give 22.33

1

to give 0.21 g (13%) of 20. mp 255 °C; H NMR (DMSO-d₆) δ

2.4-2.55 (m, 1H), 2.65-2.8 (m, 1H), 4.15 (s, 2H), 4.65-4.75

(m, 1H), 5.05-5.2 (m, 1H), 6.65 (s, 1H), 7.20-7.45 (m, 8H),

7.7-7.95 (m, 7H), 8.2-8.4 (m, NH), 8.6-8.75 (m, NH), 9.05-

10.1 (b, NH), 10.5-11.0 (b,OH). Mass calcd for C₂₅H₂₅N₆O₈,

537.1734; found, 537.1740.

1

g (75%). H NMR (DMSO-d₆) δ 1.66 (s, 3H), 7.68 (d, J ) 7.5

{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cet yl}-Asp (OBu ^t)-(S)-p h en yl-

glycin e-OBu ^tHyd r och lor id e (21a ). The method for com-

pound 16a was employed starting from 28b. ¹H NMR (DMSO-

d₆) δ 1.85 (m, 3H), 1.30-1.40 (s, 18H), 1.90-2.20 (m, 2H), 2.43

(dd, J ) 9 Hz, J ) 15 Hz, 1H), 2.70 (dd, J ) 5 Hz, J ) 15 Hz,

1H), 4.00 (s, 2H), 4.70 (m, 1H), 5.20 (m, 1H), 7.38 (m, 5H),

7.70-7.90 (AA′BB′, 4H); MS m/z (FAB) 665.4 (M + H)⁺.

{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-eth yl-2,5-

dioxo-im idazolidin -1-yl]acetyl}-Asp-(S)-ph en ylglycin e Tr i-

flu or oa cetic Acid Sa lt (21). The method for compound 16

was employed starting from 21a . ¹H NMR (D₂O) δ 0.90 (m,

3H), 2.10-2.40 (m, 2H), 2.70-3.00 (m, 2H), 4.25 (m, 2H), 5.35

(d, J ) 10 Hz, 1H), 7.25-7.40 (m, 5H), 7.70-7.85 (m, 4H). Mass

calcd for C₂₆H₂₉N₆O₈, 553.2047; found, 553.2048.

{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-cyclop r o-

p yl-2,5-d ioxo-im id a zolid in -1-yl]a cet yl}-Asp (OBu ^t)-(S)-

p h en ylglycin e-OBu ^tHyd r och lor id e (22a ). The method for

compound 16a was employed starting from 28c. Compound

22a was used without further purification.

{2-[4-(R ,S )-(4-Am in oim in om e t h yl-p h e n yl)-4-cyclo-

p r o p y l-2,5-d io x o -im id a zo lid in -1-y l]a c e t y l}-As p -(S )-

p h en ylglycin e-OH Tr iflu or oa cetic Acid Sa lt (22). The

method for compound 16 was employed starting from 22a . ¹H

NMR (DMSO-d₆) δ 0.30-0.75 (m, 4H), 1.70 (m, 1H), 2.40 (m,

1H), 2.75 (m, 1H), 4.08 (m, 2H), 4.65 (m, 1H), 4.88 (m, 1H),

7.10-7.33 (m, 5H), 7.73-8.00 (m, 5H), 8.70-9.10 (m, 4H, NH),

11.20 br s, 1H, NH), 11.40 (br s, 1H, NH). Mass calcd for

Hz, 2H), 7.90 (d, J ) 7.5 Hz, 2H), 8.74 (s, 1H, NH), 10.9 (s,

1H, NH); MS m/z (FAB) 216 (M + H)⁺.

(R ,S )-(4-Cya n op h e n yl)-4-e t h yl-2,5-d ioxoim id a zoli-

d in e (26b). ¹H NMR (DMSO-d₆) δ 0.80 (t, J ) 7.5 Hz, 3H),

1.80-2.10 (m, 2H), 7.68-7.93 (AA′BB′, 4H), 8.75 (s, 1H, NH),

10.90 (s, 1H, NH); MS m/z (FAB) 230 (M + H)⁺.

(R,S)-(4-Cya n op h en yl)-4-cyclop r op yl-2,5-d ioxoim id a -

1

zolid in e (26c). H NMR (DMSO-d₆) δ 0.40 (m, 2H), 0.50 (m,

2H), 1.65 (m, 1H), 7.70-7.95 (AA′BB′, 4H), 8.50 (s, 1H, NH),

10.95 (s, 1H, NH); MS m/z (FAB) 242 (M + H)⁺.

(R,S)-(4-Cya n op h en yl)-4-b en zyl-2,5-d ioxoim id a zoli-

d in e (26d ). ¹H NMR (DMSO-d₆) δ 3.00 (d, J ) 15 Hz, 1H),

3.50 (d, J ) 15 Hz, 1H), 7.25 (m, 5H), 7.50 (m, 1H, NH), 7.83-

7.95 (AA′BB′, 4H), 8.78 (s, 1H, NH); MS m/z (FAB) 292 (M +

H)⁺.

Similar methods were employed for compounds 27a , 27b,

27c, and 27d .

[(R,S)-4-(4-Cya n op h en yl)-4-m et h yl-2,5-d ioxoim id a zo-

lid in -1-yl]a cetic Acid Meth yl Ester (27a ). Sodium (1.068

g, 46.47 mmol) was dissolved under nitrogen in dry methanol

(110 mL). Compound 26a (10 g, 46.47 mmol) was added to the

clear solution, and the mixture was boiled under reflux for 2

h. Potassium iodide (7.75 g, 46.48 mmol) was added, and a

solution of methyl chloroacetate (4.53 mL, 51.3 mmol) in

methanol (5 mL) was added dropwise within 1 h. The mixture

was heated to reflux for 6 h, left to stand at room temperature

overnight, and then concentrated. The oily residue was chro-

matographed on silica gel using CH₂Cl₂/ethyl acetate (9:1) to

C

₂₇H₂₉N₆O₈, 565.2047; found, 565.2038.

1

give 8.81 g (66%). H NMR (DMSO-d₆) δ 1.75 (s, 3H), 3.65 (s,

{2-[4-(R,S)-(4-Am in oim in om e t h yl-p h e n yl)-4-b e n zyl-

2,5-d io x o -im id a zo lid in -1-y l]a c e t y l}-As p (O B u ^t)-(S )-

p h en ylglycin e-OBu ^tHyd r och lor id e (23a ). The method for

compound 16a was employed starting from 28d . Compound

23a was used without further purification.

3H), 4.25 (s, 2H), 7.73 (d, J ) 9 Hz, 2H), 7.9 5 (d, J ) 9 Hz,

2H), 9.2 0 (s, 1H, NH); MS m/z (FAB) 288.1 (M + H)⁺.

[(4-(R,S)-(4-Cya n op h en yl)-4-eth yl-2,5-d ioxoim id a zoli-

d in -1-yl]a cetic Acid Meth yl Ester (27b). ¹H NMR (DMSO-

d₆) δ 0.88 (t, J ) 7.5 Hz, 3H), 1.90-2.30 (m, 2H), 3.65 (s, 3H),

4.24 (s, 2H), 7.70-7.98 (AA′BB′, 4H), 9.25 (s, 1H, NH); MS

m/z (FAB) 302 (M + H)⁺.

{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-ben zyl-2,5-

d ioxo-im id a zolid in -1-yl]a cet yl}-Asp -(S)-p h en ylglycin e-

OH Tr iflu or oa cetic Acid Sa lt (23). The method for com-

1

pound 16 was employed starting from 23a . H NMR (DMSO-

[(4-(R,S)-(4-Cya n op h en yl)-4-cyclop r op yl-2,5-d ioxoim i-

d a zolid in -1-yl]a cetic Acid Meth yl Ester (27c). ¹H NMR

(DMSO-d₆) δ 0.45-0.65 (m, 4H), 1.66 (M, 1H), 3.70 (s, 3H,

OCH₃), 4.24 (s, 2H), 7.75-7.98 (AA′BB′, 4H), 9.00 (s, 1H, NH);

MS m/z (FAB) 314 (M + H)⁺.

[(4-(R,S)-(4-Cya n op h en yl)-4-ben zyl-2,5-d ioxoim id a zo-

lid in -1-yl]a cetic Acid Meth yl Ester (27d ). MS m/z (FAB)

364 (M + H)⁺.

d₆) δ 2.38 (m, 1H), 2.70 (m, 1H), 3.50-3.80 (m, 4H), 4.55 (m,

1H), 4.88 (m, 1H), 7.25 (m, 1OH), 7.78-7.98 (AA′BB′, 4H); MS

m/z (FAB) 615 (M + H)⁺.

Similar methods were employed for compounds 25b, 25c,

and 25d .

4-Cya n p h en yleth ylm eth a n on e (25b) from 4-bromphen-

ylethylmethanone. ¹H NMR (DMSO-d₆) δ 1.10 (t, J 6.3 Hz, 3H),

3.10 (q, J ) 6.3 Hz, 2H), 7.95-8.15 (AA′BB′, 4H); MS m/z

(FAB) 160 (M + H)⁺.

Similar methods were employed for compounds 28a , 28b,

28c, and 28d .

4-Cya n p h en ylcyclop r op ylm eth a n on e (25c). 4-Bromo-

phenylcyclopropylmethanone (22.5 g, 100 mmol) and CuCN

(10.3 g, 100 mmol) were dissolved in DMF (15 mL) and stirred

[4-(R,S)-(4-Am in oim in om et h yl-p h en yl)-4-m et h yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (28a ).

A cooled (0 °C) solution of 27a (28.8 g, 100 mmol) in ethanol

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1171

(400 mL) was purged with dry HCl while maintaining the

temperature below 10 °C until the characteristic absorption

of the nitrile group had disappeared (IR). The solvent was

evaporated to give 38.4 g (99%) of (4-(R,S)-((4-ethoxyimino-

methyl)-phenyl)-4-methyl-2,5-dioxo-imidazolidin-1-yl) acetic

acid ethyl ester hydrochloride: MS m/z (FAB) 348 (M + H)⁺.

This intermediate was immediately suspended in 2-propanol

(380 mL) and treated with a solution of NH₃in 2-propanol (2

N, 115 mL). The reaction mixture was stirred for 2 h at 50 °C

and allowed to cool to room temperature. The product was

precipitated by addition of diethyl ether (2000 mL). The

material was filtered and dried in high vacuum to give 24.8 g

(71%) of (4-(S)-((4-amino-imino-methyl)- phenyl)-4-methyl-2,5-

dioxoimidazolidin-1-yl)-acetic acid ethylester hydrochloride:

MS m/z (FAB) 319 (M + H)⁺. The ester (24.7 g/mmol) was

dissolved in concentrated HCl (375 mL) and refluxed for 6 h.

The solvent was removed under reduced pressure. The residue

was freeze-dried to give 21.65 g (95%) of 28a . ¹H NMR (DMSO-

d₆) δ 1.74 (s, 3H), 4.09 (s, 2H), 7.7-7.9 (AA′BB′, 4H), 9.2 (s,

1H), 9.28 (s, 2H), 9.45 (s, 2H); MS m/z (FAB) 291.1 (M + H)⁺.

[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-eth yl-2,5-d i-

oxo-im id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (28b).

¹H NMR (DMSO-d₆) δ 0.88 (s, 3H), 2.10 (m, 2H), 4.10 (s, 2H),

7.70-7.90 (AA′BB′, 4H), 9.25 (m, 3H, NH), 9.45 (s, 2H, NH);

MS m/z 305 (FAB) (M + H)⁺.

9H), 2.80 (m, 2H), 3.90 (m, 1H), 5.05 (s, 2H), 7.35 (br s, 5H),

7.50 (m, 1H, NH); MS m/z (FAB) 295.1 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-2-S-ben zyloxy-

ca r bon yla m in o-p r op ion ic Acid Hyd r och lor id e (38). The

method for compound 4a was employed starting from 28a to

give crude material (6.32 g) which was treated with TFA (3.6

mL), water (0.4 mL), and ethanedithiole (0.4 mL) to give 350

1

mg. H NMR (DMSO-d₆) δ 1.75 (s, 3H), 3.50 (m, 2H), 4.00 (br

s, 3H), 5.03 6 (s, 2H), 7.33 (br s, 6H), 7.80 (AA′BB′, 4H), 8.25

(m, 1H, NH), 9.05 (s, 1H, NH), 9.25 (br s, 2H, NH), 9.50 (br s,

2H, NH). Mass calcd for C₂₄H₂₇N₆O₇, 511.1941; found, 511.1940.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-p h en yl-

p r op ion ic Acid Eth yl Ester Hyd r och lor id e (39a ). The

method for compound 4a was employed starting from 28a to

give 597 mg (61%). ¹H NMR (DMSO-d₆) δ 1.10 (q, 3H), 1.70

(s, 3H), 1. 80 (s, 3H), 2.75 (m, 2H), 3.90-4.10 (m, 4H), 5.15

(m, 1 H), 7.20-7.40 (m, 5H), 7.68-7.85 (AA′BB′), 8.80 (d, 1H,

NH), 9.60 (brs, 4H, NH); MS m/z 466 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-p h en yl-

p r op ion ic Acid Hyd r och lor id e (39). Compound 39a (580

mg, 1.19 mmol) was dissolved in concentrated HCl (55 mL)

and left at room temperature for 5.5 h. The solution was

evaporated, and the residue (540 mg) was chromatographed

on Sephadex LH20 (solvent C). The residue was freeze-dried

to give 477 mg (85%). ¹H NMR (DMSO-d₆) δ 1.75 (s, 3H), 2.60

(d, J ) 7.5 Hz, 2H), 4.03 (s, 2H), 5.10 (dd, J ) 7.5 Hz, J ) 15

Hz, 1H), 7.30 (m, 5H), 7.70-7.90 (AA′BB′, 4H), 8.75 (d, J )

7.5 Hz, 1H, NH), 9.07 (br s, 1H, NH), 9.60 (br s, 4H). Mass

calcd for C₂₂H₂₄N₅O₅, 438.1777; found, 438.1761.

[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-cyclop r op yl-

2,5-d ioxo-im id a zolid in -1-yl]a cet ic Acid H yd r och lor id e

1

(28c). H NMR (DMSO-d₆) δ 0.55 (m, 4H), 1.68 (m, 1H), 4.10

(s, 2H), 7.80-7.93 (AA′BB′, 4H), 9.00 (s, 1H, NH), 9.25 (br s,

2H, NH), 9.44 (br s, 2H, NH); MS m/z (FAB) 317 (M + H)⁺.

[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-ben zyl-2,5-d i-

oxo-im id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (28d ).

¹H NMR (DMSO-d₆) δ 3.20 (d, J ) 15 Hz, 1H), 3.55 (d, J ) 15

Hz, 1H), 3.78 (d, J ) 5 Hz, 2H), 7.25 (m, 5H), 7.90 (s, 4H),

9.20-9.50 (m, 5H, NH); MS m/z (FAB) 367.2 (M + H)⁺.

{2-[4-(R,S)-(4-Am in oim in om et h yl-p h en yl)-4-m et h yl-

2,5-dioxo-im idazolidin -1-yl]acetyl}-Asp-(S)-ph en ylglycin e-

OCH₃Hyd r och lor id e (35). The method for compound 4a was

employed. ¹H NMR (DMSO-d₆) δ 1.88 (s, 3H), 2.80 (dd, J )

7.5 Hz, J ) 17.5 Hz, 1H), 3.96 (dd, J ) 5 Hz, J ) 17.5 Hz,

1H), 3.65-3.74 (s, 3H), 4.30 (br s, 3H), 5.46 (m, 1H),

7.25-7.40 (m, 5H), 7.70-7.90 (AA′BB′, 4H). Mass calcd for

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-m eth yl-

p r op ion ic Acid Meth yl Ester Hyd r och lor id e (40a ). The

method for compound 4a was employed starting from 28a . ¹H

NMR (DMSO-d₆) δ 1.18 (t, J 7.5 Hz, 3H), 1.63 (m, 1H), 1.73

(s, 3H), 2.30 (m, 2H), 3.08 (m, 3H), 3.84 (m, 2H), 4.04 (q, J )

7.5 Hz, 2H), 7.70-7.88 (AA′BB′), 8.18 (m, 1H, NH); MS m/z

404 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-m eth yl-

p r op ion ic Acid Hyd r och lor id e (40). The method for com-

pound 39 was employed to give 477 mg (85%). ¹H NMR

(DMSO-d₆) δ 1.60 (m, 1H), 1.73 (s, 3H), 2.21 (m, 2H), 3.05 (m,

3H), 3.53 (m, 2H), 3.98 (s, 2H), 7.73-7.88 (AA′BB′), 8.15 (m,

1H, NH), 8.95 (m, 1H, NH), 9.03 (br s, 2H, NH), 9.33 (br s,

2H, NH), 12.08 (br s, 1H). Mass calcd for C₁₇H₂₂N₅O₅, 376.1621;

found, 376.1617.

C

₂₆H₂₉N₆O₈, 553.2047; found, 553.2048.

{2-[4-(R,S)-(4-Am in oim in om et h yl-p h en yl)-4-m et h yl-

2,5-d i o x o -i m i d a z o li d i n -1-y l]a c e t y l}-As p -O C H ₃-(S )-

p h en ylglycin e-OCH₃Hyd r och lor id e (36). The method for

compound 4a was employed starting from 28a to give 437 mg

1

(16%). H NMR (DMSO-d₆) δ 1.75 (s, 3H), 2.45 (m, 1H), 2.65

(m, 1H), 3.60 (OCH₃, 6H), 4.03 (br s, 2H), 4.70 (m, 1H), 5.40

(m, 1H), 7.35 (m, 5H), 7.75 (d, J ) 7.5 Hz, 2H), 7.85 (d, J )

7.5 Hz, 2H), 8.55 (m, 1H, NH), 8.80 (m, 1H, NH), 9.10 (br s,

3H), 9.40 (br s, 2H). Mass calcd for C₂₇H₃₁N₆O₈, 567.2203;

found, 567.2194.

1-Ad a m a n tyl ca r ba ld eh yd e (41a ). A solution of 1-(hy-

droxymethyl)adamantane (19.98 g, 120 mmol) in CH₂Cl₂(180

mL) was added to a suspension of pyridinium chlorochromate

(36.9 g, 171 mmol) in CH₂Cl₂(240 mL) at 12-18 °C during 15

min. After the mixture was stirred at 12-18 °C for 90 min,

heptane (900 mL) was added. The mixture was filtered, and

the filtrate was evaporated under reduced pressure. The

residue was dissolved in pentane, and the pentane phase was

washed with NaOH (0.2 N, 3 × 120 mL) and water (2 × 120

mL). The solvent was dried and evaporated to give 14.58 g of

crude material which was used for the next step without

further purification

{2-[4-(R,S)-(4-Am in oim in om et h yl-p h en yl)-4-m et h yl-

2,5-d ioxo-im id a zolid in -1-yl]a cet yl}-Asp -2-Ad a m a n t yl-

a m id e Tr iflu or oa cetic Acid Sa lt (37). The method for

compound 4a was employed starting from 28a followed by

1

treatment with TFA to give 615.8 mg. H NMR (DMSO-d₆) δ

1.35-1.90 (m, 17H), 2.35-2.65 (m, 2H), 3.78 (m, 1H), 4.58 (m,

1H), 7.70-7.90 (AA′BB′, 4H), 7.80 (1H, NH), 8.50 (m, 1H, NH),

9.08 (br s, 1H, NH). Mass calcd for C₂₇H₃₅N₆O₆, 539.2618;

found, 539.2621.

(R,S)-3-Am in o-3-(1-a d a m a n tyl)p r op ion ic Acid (41b).

Malonic acid (5.64 g 54.2 mmol), ammonium acetate (8.67 g,

112.5 mmol), and 41a (8.9 g, 54.2 mmol) in absolute ethanol

(14 mL) were stirred under reflux for 4 h. The precipitate was

filtered and washed with ethanol. The solvent was removed,

and the residue distributed between CH₂Cl₂and water. The

water phase was freeze-dried and the residue treated with a

small amount of water. lt was filtered and dried in vacuo over

phosphorus pentoxide to give 1.75 g (15%) of 41b as a white

3-Am in o-2-S-ben zyloxyca r bon yl-a m in op r op ion ic Acid

ter t-Bu tyl Ester (38a ). A cooled suspension of 3-amino-2-^L-

benzyloxycarbonylaminopropionic acid (5 g, 21 mmol) in di-

oxane (50 mL) was treated with concentrated H₂SO₄(5 mL).

The slightly yellowish solution was cooled with dry ice, and

condensed isobutylene (50 mL) was added. The mixture was

shaken at room temperature for 3 d under nitrogen at 20 atm.

Subsequently, excess isobutylene was removed by nitrogen.

The solution was adjusted to a pH of 10 with NaCO₃(2 M, 70

mL) and extracted three times with diethyl ether (200 mL).

The organic phase was washed with water, dried, and evapo-

rated to give 4.31 g of oil (70%). ¹H NMR (DMSO-d₆) δ 1.38 (s,

1

solid. H NMR (CD₃OD) δ 1.5-2.05 (m, 15H), 2.1 (dd, J ) 5.5

Hz, J ) 10.0 Hz, 1H), 2.5 (dd, J ) 1.5 Hz, J ) 10.0 Hz, 1H),

2.95 (dd, J ) 5.5 Hz, J ) 1.5 Hz, 1H).

1172 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

1

(R,S)-3-Am in o-3-(1-a d a m a n tyl)p r op ion ic Acid Meth yl

Ester (41c). A solution of 41b (1.75 g, 7.87 mmol) in metha-

nolic HCl (2 N) was stirred under reflux for 4 h. The solvent

was removed in vacuo and the residue distributed between

diethyl ether (50 mL) and water (50 mL). The phases were

separated, and the water phase was extracted with diethyl

ether (50 mL). The water phase was adjusted to pH 9.3 with

NaOH (2 N) and extracted with CH₂Cl₂(2 × 50 mL). After

drying over anhydrous Na₂SO₄, the solvent was evaporated

to give 1.45 g (78%), pale yellow oil. ¹H NMR (DMSO-d₆) δ

1.22-1.72 (m, 15H), 1.85-2.05 (m, 4H), 2.43-2.55 (m, 1H),

3.58 (s, 3H).

monium acetate). H NMR (DMSO-d₆) δ 2.3 (s, 3H), 2.2-2.4

(m, 2H), 3.85 (m, 1H), 7.2-7.4 (m, 5H); MS m/z (CI) 180 (M +

H)⁺.

(R,S)-3-N-Meth yla m in o-3-p h en ylp r op ion ic Acid Meth -

yl Ester Hyd r och lor id e (43b). Thionyl chloride (10.7 mL,

145 mmol) was added dropwise to absolute methanol (100 mL)

at - 15 °C. Compound 43a (13 g, 73.4 mmol) was added, and

the solution was allowed to stand overnight at room temper-

ature. The solvent was removed, and the residue was purified

by flash chromatography on silica gel (CH₂Cl₂/methanol 9:1)

to give 7.1 g (50%). ¹H NMR (DMSO-d₆) δ 2.32 (s, 3H), 3.1 (m,

2H), 3.52 (s, 3H), 4.48 (dd, J ) 4.5 Hz, J ) 2.5 Hz, 1H), 7.4-

7.6 (m, 5H), 8.6-9.8 (bs, 2H, NH₂⁺); MS m/z (ESI) 194 (M +

H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cet yl-N-m et h yla m in o}-3-

(R,S)-p h en ylp r op ion ic Acid Meth yl Ester Hyd r och lor id e

(43c). TOTU (O-(cyano(ethoxycarbonyl)-methyleneamino)-

1,1,3,3-tetramethyluronium-tetrafluoroborate (328 mg, 1 mmol)

was added to a solution of 28a (327 mg, 1 mmol), 43b (230

mg, 1 mmol), and N-ethylmorpholine (115 mg, 1 mmol) in DMF

(15 mL) at 0 °C. The solution was allowed to stir at room

temperature for 24 h. The solvent was evaporated and the

residue was chromatographed on Sephadex LH20 (solvent C)

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-(1-a d a -

m a n t yl)p r op ion ic Acid Met h yl E st er H yd r och lor id e

(41d ). The method for compound 4a was employed starting

from 28a to give 800 mg (45%). ¹H NMR (DMSO-d₆) δ 1.3-

2.0 (m, 18H), 2.1-2.3 (m,1H), 2.4-2.6 (m,1H), 3.5, 3.55 (2s,

3H), 3.73-3.87 (m, 1H), 3.85-4.05 (AB, 2H), 7.68-7.85 (m,

4H), 7.92 (d, J ) 5.0 Hz, 1H), 9.0-9.9 (bs, 5H); MS m/z (ESI)

510 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-(1-a d a -

m a n tyl)p r op ion ic Acid Hyd r och lor id e (41). The method

for compound 39 was employed to give 260 mg (89%). ¹H NMR

(DMSO-d₆) δ 1.3-2.1 (m, 19H), 2.3-2.45 (m, 1H), 2.4-2.6 (m,

1H), 3.73-3.9 (m, 1H), 3.87-4.03 (AB, 2H), 7.7-8.0 (m, 5H)

8.8-11.0 (2bs, 5H). Mass calcd for C₂₆H₃₄N₅O₅, 496.2660;

found, 496.2582.

1

and freeze-dried to give 160 mg (32%). H NMR (DMSO-d₆) δ

1.78 (s, 3H), 2.78, 3.23 (2s, 3H), 2.8-3.0 (m, 1H), 3.05-3.25

(m, 1H), 3.52, 3.58, 3.68, 3.7 (4s, 3H), 4.18-4.38, 4.55 (2AB,

2H), 5.47-5.55, 5.9-6.03 (2m, 1H), 7.2-7.5 (m, 5H), 7.73-

7.95 (m, 4H), 8.85-9.4 (bs, 5H); MS m/z (ESI) 466 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-

2,5-d ioxo-im id a zolid in -1-yl]a cet yl-N-m et h yla m in o}-3-

(R,S)-p h en ylp r op ion ic Acid Hyd r och lor id e (43). Com-

pound 43c (270 mg, 0.54 mmol) was dissolved in HCl (6 N, 15

mL) and stirred at room temperature for 7 h. The solution is

2-[4-(R ,S )-(4-Cya n op h e n yl)-3-e t h yl-4-m e t h yl-2,5-d i-

oxoim id a zolid in -1-yl]a cetic Acid Meth yl Ester (42a ). A

solution of 2-[4-(S,R)-(4-cyanophenyl)-4-methyl-2,5-dioxoimi-

dazolidin-1-yl]acetic acid methyl ester (4.5 g, 15.7 mmol) in

dried DMF (25 mL) was treated (Argon) with NaH (753.6 mg,

17.3 mmol, 50% oil dispersion). After the mixture was stirred

for 15 min, ethyl iodide (1.39 mL, 17.3 mmol) was added, and

stirring was continued for 4 h at room temperature and left

overnight. The solution was concentrated, and the residue was

chromatographed on silica CH₂Cl₂/ethyl acetate (9.5:0.5) to give

1

diluted with water and freeze-dried to give 240 mg (91%). H

NMR (DMSO-d₆) δ 1.78 (s, 3H), 2.52,2.75 (2s, 3H), 2.63-

2.83,3.0-3.2 (2m, 2H), 4.18-4.35,4.48-4.7 (2,AB, 2H), 5.45-

5.55,5.95-6.0 (2m, 1H), 7.13-7.65 (2s, 1H), 7.2-7.45 (m, 5H),

7.73-7.95 (m, 4H), 9.15 (s, 1H), 9.35-9.65 (2bs, 4H), 12.35-

12.65 (bs, 1H); MS m/z (ESI) 452 (M + H)⁺.

1

2.55 g of oil (51%). H NMR (DMSO-d₆) δ 0.98 (t, J ) 6.3 Hz,

(S)-3-Am in o-3-p h en ylp r op ion ic Acid Eth yl Ester Hy-

d r och lor id e (49a ). To a solution of (R,S)-3-amino-3-phenyl-

propionic acid ethyl ester (46.27 g, 239.4 mmol) in absolute

ethanol (492 mL) was slowly added Z-^L-Phe-OH (71.67 g, 239.4

mmol) under stirring. The solution was kept at 4 °C for 12 h.

The precipitate was filtered, washed with a small proportion

of cold absolute ethanol, and dried in vacuo over phosphorus

pentoxide to give 53.19 g of the S isomer as Z-^L-Phe-OH salt

containing 3.2% of the R isomer as determined by GC analysis.

It was recrystallized from absolute ethanol to give 47.49 g

which was confirmed by GC analysis to be optically pure

(>99% ee). The optically pure Z-^L-Phe-OH salt (47.49 g, 96.4

mmol) was suspended in a mixture of ethyl acetate (2400 mL)

and of water (2400 mL). Solid NaHCO₃was added until a pH

of 7.5 was reached. The organic phase was separated, and the

aqueous phase was washed twice with ethyl acetate (1000 mL).

The combined organic phases were washed with water (100

mL) and dried over anhydrous MgSO₄. The solvent was

evaporated under reduced pressure, and the residue was taken

up in absolute ethanol (1000 mL). A saturated solution of HCl

(10 mL) in ethanol was added. The mixture was stirred,

filtered, and the solvent was removed under reduced pressure.

The remaining residue was treated with diethyl ether. It was

filtered, washed with diethyl ether, and dried in vacuo over

phosphorus pentoxide to give 20 g (30%) of 49a . [R]²²_D) +5.8°

3H), 1.85 (s, 3H), 3.10 (m, 2H), 3.70 (s, 3H), 4.30 (s, 2H), 7.63

(d, J ) 9 Hz, 2H), 7.96 (d, J ) 9 Hz, 2H); MS m/z (FAB) 316

(M + H)⁺.

2-[4-(R,S)-(4-Am in oim in om eth yl-ph en yl)-3-eth yl-4-m eth -

yl-2,5-dioxoim idazolidin -1-yl]acetic Acid (42b). The method

1

for compound 28a was employed starting from 42a . H NMR

(DMSO-d₆) δ 1.00 (t, 3H), 1.88 (s, 3H), 3.05 (m, 2H), 4.18 (s,

2H), 7.65 (d, J ) 7.5 Hz, 2H), 7.90 (d, J ) 7.5 Hz, 2H), 9.30-

9.50 (m, 4H, NH); MS m/z (FAB) 319.2 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-3-eth yl-4-

m eth yl-2,5-d ioxoim id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-

p h en ylp r op ion ic Acid Eth yl Ester Hyd r och lor id e (42c).

The method for compound 4a was employed starting from 42b.

¹H NMR (DMSO-d₆) δ 0.95 (t, J ) 6.3 Hz, 3H), 1.10 (m, 3H),

1.73 (s, 3H, CH₃COOH), 1.84 (s, 3H), 2.75 (m, 2H), 2.90-3.40

(m, 2H), 4.00 (q, J ) 6.3 Hz, 2H), 4.10 (br s, 2H), 5.10 (m, 1H),

7.30 (m, 5H), 7.65-7.90 (AA′BB′, 4H), 8.80 (d, 1H, NH); MS

m/z (FAB) 494 (M + H)⁺.

3-{2-[4-(R,S)-(4-Am in oim in om eth yl-p h en yl)-3-eth yl-4-

m eth yl-2,5-d ioxoim id a zolid in -1-yl]a cetyla m in o}-3-(R,S)-

p h en ylp r op ion ic Acid Hyd r och lor id e (42). The method for

compound 39 was employed.

(R,S)-3-N-Meth yla m in o-3-p h en ylp r op ion ic a cid (43a ).

Methylammonium chloride (20.26 g, 300 mmol) and sodium

acetate (24.61 g, 300 mmol) in absolute ethanol (40 mL) were

stirred for 30 min. Malonic acid (10.4 g, 100 mmol) and

benzaldehyde (10.61 g, 100 mmol) were added, and the mixture

was stirred under reflux for 3.5 h. The precipitate was filtered

and washed with ethanol. The solvent was removed and the

residue distributed between ethyl acetate and water. The

water phase was concentrated, and the residue was purified

by flash chromatography on silica gel using methanol as eluent

to give 26.23 g of 43a (contains some amounts of methylam-

1

(c ) 1; methanol); H NMR (DMSO-d₆) δ 1.1 (t, J ) 5 Hz, 3

H), 1.96 (br s, 2H), 2.6 (m, 2H), 4.0 (q, J ) 5 Hz, 2H), 4.2 (t, J

) 5 Hz, 3H), 7.17-7.4 (m, 5H); MS m/z (FAB) 194 (M + H)⁺.

(R)-3-Am in o-3-p h en ylp r op ion ic Acid Eth yl Ester Hy-

d r och lor id e (49b). The method for compound 49a was

employed starting from (R,S)-3-amino-3-phenyl-propionic acid

ethyl ester hydrochloride by using Z-^D-Phe-OH to resolve the

racemate. [R]²²) -5.8° (c ) 1; methanol); ¹H NMR (DMSO-

D

d₆) δ 1.1 (t, J ) 5 Hz, 3H), 1.96 (br s, 2H), 2.6 (m, 2H), 4.0 (q,

Discovery of a Non-Peptide Fibrinogen Receptor Antagonist

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8 1173

J ) 5 Hz, 2H), 4.2 (t, J ) 5 Hz, 3H), 7.17-7.4 (m, 5H); MS

m/z (FAB) 194 (M + H)⁺.

(R,S)-4-(4-Br om op h en yl)-4-m eth yl-2,5-d ioxoim id a zoli-

d in e (50). The method for compound 26a was employed

starting from 4-bromoacetophenone to give 65.3 g (97%). ¹H

NMR (DMSO-d₆) δ 1.65 (s, 3 H), 7.4-7.65 (AA′BB′, 4 H), 8.63

(s, 1 H), 10.85 (br s, 1 H); MS m/z (FAB) 269 (M + H)⁺.

3H), 4.07 (s, 2 H), 7.68-7.95 (AA′BB′, 4H), 9.15 (s, 1H), 13.15

(br s, 1H); MS m/z (FAB) 274 (M + H)⁺.

[4-(S)-((4-Am in oim in om eth yl)-p h en yl)-4-m eth yl-2,5-d i-

oxo-im id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (57a ).

The method for compound 25a was employed starting from

56a to give 21.65 g (95%). [R]²²_D) +42.7° (c ) 1; 1 N HCl); ¹H

NMR (DMSO-d₆) δ 1.74 (s, 3H), 4.09 (s, 2H), 7.7-7.9 (AA′BB′,

4H), 9.2 (s, 1H), 9.28 (s, 2H), 9.45 (s, 2H); MS m/z (FAB) 291

(M + H)⁺.

(R,S)-2-Am in o-2-(4-br om op h en yl)-p r op ion ic Acid (51).

A solution of NaOH (3 N, 50 mL) was added to 50 (5.3 g, 20

mmol). The resulting suspension was heated in an autoclave

under nitrogen (10 bar) for 1 h at 145 °C. The reaction mixture

was allowed to cool to room temperature. It was diluted with

water (150 mL), and the pH was adjusted to 4 by addition of

acetic acid using an ice bath for cooling. The reaction mixture

was stirred for 2 h at 0 °C. The precipitate was isolated,

washed with water, and dried in vacuo over phosphorus

[4-(R)-((4-Am in oim in om eth yl)-p h en yl)-4-m eth yl-2,5-d i-

oxo-im id a zolid in -1-yl]a cetic Acid Hyd r och lor id e (57b).

The method for compound 57a was employed starting from

1

53b. [R]²²) - 42.7° (c ) 1; 1 N HCl); H NMR (DMSO-d₆) δ

D

1.74 (s, 3 H), 4.09 (s, 2 H), 7.7-7.9 (AA′BB′, 4 H), 9.2 (s, 1 H),

9.28 (s, 2 H), 9.45 (s, 2 H); MS m/z (FAB) 291 (M + H)⁺.

3-{2-[4-(S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(S)-p h en ylp r op i-

on ic Acid Eth yl Ester Aceta te (44a ). The method for

1

pentoxide to give 3.65 g (75%)of 51 as a white solid. H NMR

(DMSO-d₆) δ 1.6 (s, 3H), 7.4-7.6 (AA′BB′, 4H); MS m/z (FAB)

244 (M + H)⁺.

compound 4a was employed starting from 57a and 49a to give

1

35 g (83%). [R]²²) -56.7° (c ) 1; water); H NMR (DMSO-

D

(R,S)-2-Am in o-2-(4-br om op h en yl)-p r op ion ic Acid Eth -

yl Ester (52). A solution of HCl (9.8 N, 150 mL) in ethanol

was added to 51 (27.3 g, 112.3 mmol). The suspension was

refluxed for 18 h. The solvent was removed, and the remaining

residue was distributed between ethyl acetate and an satu-

rated aqueous solution of NaHCO₃. The organic layer was

washed with water and dried over anhydrous magnesium

sulfate. The solvent was removed and the crude product (23.22

g) was distilled in vacuo to give 20.7 g (68%) of 52 as a colorless

d₆) δ 1.12 (t, J ) 6 Hz, 3H), 1.73 (s, 6H), 2.75 (d, J ) 6 Hz,

2H), 3.95-4.1 (m, 4H), 5.18 (q, J ) 6 Hz, 1H), 7.2-7.38 (m,

5H), 7.64-7.85 (AA′BB′, 4H), 8.78 (d, J ) 8 Hz, 1H), 9.6 (br s,

5 H). Mass calcd for C₂₄H₂₈N₅O₅, 466.2090; found, 466.2093.

3-{2-[4-(S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(S)-p h en ylp r op i-

on ic Acid Hyd r och lor id e (44). The method for compound

12e was employed starting from 44a to give 10.55 g (94%).

1

[R]²²) -54.0° (c ) 1; water); H NMR (DMSO-d₆) δ 1.73 (s,

1

D

oil, bp 129-130 °C/2 mm. H NMR (DMSO-d₆) δ 1.14 (t, J )

3H), 2.7 (d, J ) 7 Hz, 2H), 4.03 (s, 2H), 5.13 (q, J ) 7 Hz, 1H),

7.2-7.38 (m, 5H), 7.7-7.9 (AA′BB′, 4H), 8.78 (d, J ) 8 Hz,

1H), 9.08 (s, 1H), 9.19 (s, 2H), 9.39 (s, 2H), 12.3 (s, 1H). Mass

calcd for C₂₂H₂₄N₅O₅, 438.1777; found, 438.1782.

8 Hz, 3H), 1.5 (s, 3H), 2.38 (br s, 2H), 4.08 (q, J ) 8 Hz, 2H),

7.38-7.58 (AA′BB′, 4H); MS m/z (FAB) 272 (M + H)⁺.

(S)-2-Am in o-2-(4-br om op h en yl)-p r op ion ic Acid Eth yl

Ester (53a ). To a solution of 52 (44.3 g, 163 mmol) and

R-mandelic acid (24.8 g, 163 mmol) in 2-propanol (138 mL)

was added diisopropyl ether (414 mL) at room temperature.

The suspension was kept at 0 °C for 12 h. The precipitated

salt was isolated and recrystallized twice in the same way to

3-{2-[4-(R)-(4-Am in oim in om eth yl-ph en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(S)-p h en ylp r op i-

on ic Acid Eth yl Ester Aceta te (45a ). The method for

compound 4a was employed starting from 57b and 49a . ¹H

NMR (DMSO-d₆) δ 1.10 (t, J ) 6 Hz, 3H), 1.75 (s, 6H), 2.78

(d, J ) 6 Hz, 2H), 3.95-4.1 (m, 4H), 5.18 (q, J ) 6 Hz, 1H),

7.2-7.38 (m, 5H), 7.64-7.85 (AA′BB′, 4H), 8.83 (d, J ) 8 Hz,

1H), 9.09 (s, 1H), 9.24 (br s, 2H), 9.45 (br s, 2H); MS m/z (FAB)

466 (M + H)⁺.

give 20 g of enantiopure 53a as mandelic acid salt, [R]²²

)

D

-14° (c ) 1, 2.15 N HCl in ethanol). The precipitate was

distributed between ethyl acetate and an aqueous solution of

NaHCO₃. The organic layer was washed with water and dried

over anhydrous magnesium sulfate. The solvent was removed

to give 12.5 g (28%) of 53a as a colorless oil which was

confirmed by means of the R(-)-R-methoxy-R-trifluor-meth-

3-{2-[4-(R)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(S)-p h en ylp r op i-

on ic Acid Hyd r och lor id e (45). The method for compound

ylphenyl acetic acid derivative (Mosher) to be optically pure

12e was employed starting from 45a . [R]²²) -110.0° (c ) 1;

1

D

(>99% ee). [R]²²) +52.7° (c ) 1; 2.15 N HCl in ethanol); H

D

1

water); H NMR (DMSO-d₆) δ 1.75 (s, 3H), 2.78 (d, J ) 7 Hz,

NMR (DMSO-d₆) δ 1.14 (t, J ) 8 Hz, 3H), 1.5 (s, 3H), 2.38 (br

s, 2H), 4.08 (q, J ) 8 Hz, 2H), 7.38-7.58 (AA′BB′, 4H); MS

m/z (FAB) 272 (M + H)⁺.

2H), 4.00 (s, 2H), 5.15 (q, J ) 7 Hz, 1H), 7.20-7.40 (m, 5H),

7.7-7.9 (AA′BB′, 4H), 8.73 (d, J ) 8 Hz, 1H), 9.09 (s, 1H), 9.24

(s, 2H), 9.45 (s, 2H), 12.3 (s, 1H). Mass calcd for C₂₂H₂₄N₅O₅,

438.1777; found, 438.1765.

(R)-2-Am in o-2-(4-br om op h en yl)-p r op ion ic Acid Eth yl

Ester (53b). The method for compound 53a was employed by

using S-mandelic acid to resolve the racemate. [R]²²_D) -52.7°

3-{2-[4-(S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R)-p h en ylp r op i-

on ic Acid Eth yl Ester Aceta te (46a ). The method for

compound 4a was employed starting from 57a and 49b. ¹H

NMR (DMSO-d₆) δ 1.12 (t, J ) 6 Hz, 3H), 1.73 (s, 6H), 2.75

(d, J ) 6 Hz, 2H), 3.95-4.1 (m, 4H), 5.18 (q, J ) 6 Hz, 1H),

7.2-7.38 (m, 5H), 7.64-7.85 (AA′BB′, 4H), 8.78 (d, J ) 8 Hz,

1H), 9.6 (br s, 5H); MS m/z (FAB) 466 (M + H)⁺.

1

(c ) 1; 2.15 N HCl in ethanol). H NMR (DMSO-d₆) δ 1.14 (t,

J ) 8 Hz, 3H), 1.5 (s, 3H), 2.38 (br s, 2H), 4.08 (q, J ) 8 Hz,

2H), 7.38-7.58 (AA′BB′, 4H); MS m/z (FAB) 272 (M + H)⁺.

2-(S)-(4-Br om op h en yl)-2-(3-et h oxyca r b on yl-m et h yl-

u r eid o)-p r op ion ic Acid Eth yl Ester (54a ). The method for

compound 2 was employed starting from 53a to give 18.1 g

(99%) of 54a as a white solid. [R]²²) +10.7° (c ) 1; 2.15 N

D

3-{2-[4-(S)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R)-p h en ylp r op i-

on ic Acid Hyd r och lor id e (46). The method for compound

HCl in ethanol); ¹H NMR (DMSO-d₆) δ 1.17 (t, J ) 7.5 Hz,

3H), 1.25 (t, J ) 7.5 Hz, 3H), 2.02 (s, 3H), 3.91 (d, J ) 6 Hz,

2H), 4.05-4.25 (m, 4H), 5.02 (t, J ) 6 Hz, 1H), 6.08 (s, 1H),

7.29-7.51 (AA′BB′, 4H); MS m/z (FAB) 401 (M + H)⁺.

1

12e was employed starting from 46a . H NMR (DMSO-d₆) δ

1.75 (s, 3H), 2.38 (m, 2H), 4.03 (m, 2H), 5.18 (m, 1H), 7.10 (m,

1H), 7.25-7.33 (m, 5H), 7.7-7.9 (AA′BB′, 4H), 8.70 (m, 1H),

9.20-9.80 (br s, 4H), 12.3 (s, 1H); MS m/z (FAB) 438 (M +

H)⁺.

[4-(S)-(4-Br om oph en yl)-4-m eth yl-2,5-dioxoim idazolidin -

1-yl)a cetic Acid (55a ). The method for compound 3 was

employed starting from 54a to give 11.4 g (78%) of 55a . [R]²²

D

) +32.8° (c ) 1; 2.15 N HCl in ethanol); ¹H NMR (DMSO-d₆)

δ 1.69 (s, 3H), 4.06 (s, 2H), 7.4-7.65 (AA′BB′, 4H), 9.05 (s,

1H), 13.1 (br s, 1H); MS m/z (FAB) 327 (M + H)⁺.

3-{2-[4-(R)-(4-Am in oim in om eth yl-p h en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R)-p h en ylp r op i-

on ic Acid Eth yl Ester Aceta te (47a ). The method for

[4-(S)-(4-Cyan oph en yl)-4-m eth yl-2,5-dioxoim idazolidin -

1-yl]a cetic Acid (56a ). The method for compound 22c was

employed starting from 55a to give 9.3 g (95%). [R]²²_D) +33.4°

(c ) 1; 2.15 N HCl in ethanol); ¹H NMR (DMSO-d₆) d 1.73 (s,

compound 4a was employed starting from 57b and 49b. [R]²²

D

1

) +56.7° (c ) 1; water); H NMR (DMSO-d₆) δ 1.12 (t, J ) 6

Hz, 3H), 1.73 (s, 6H), 2.75 (d, J ) 6 Hz, 2H), 3.95-4.1 (m,

4H), 5.18 (q, J ) 6 Hz, 1H), 7.2-7.38 (m, 5H), 7.64-7.85

1174 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 8

Stilz et al.

(AA′BB′, 4H), 8.78 (d, J ) 8 Hz, 1H), 9.6 (br s, 5H); MS m/z

(FAB) 466 (M + H)⁺.

The predictive ability of the models was evaluated by cross-

validation using five groups of approximately the same size

to which the objects were assigned randomly. Each of these

groups was omitted from the data set at a time, and a reduced

PLS model was built with the remaining compounds. The

whole procedure was repeated 20 times. This cross-validation

scheme yields a more realistic estimate of the predictivity of

the statistical model than the usual leave-one-out (LOO).

Blood Sa m p lin g a n d P la telet P r ep a r a tion . Blood was

collected from healthy adult volunteers who denied taking any

medication for the previous two weeks. A total of 8.4 mL of

blood was drawn without tourniquet from the antecubital vein

into 1.4 mL of acid citrate-dectrose (ACD, 0.8% citric acid, 2.2%

sodium citrate, 2.45% glucose). Platelet rich plasma (PRP) was

separated by centrifugation of blood for 15 min at 160g at room

temperature. Gel-filtered platelets (GFP) were prepared as

described. Briefly, PRP was acidified to pH 6.5 with ACD

(approximately 1 mL of ACD + 10 mL of PRP) and centrifuged

for 20 min at 400g. Platelets were resuspended with Tyrode’s

solution containing hirudin (1 IU/mL), albumin (0.35%), and

apyrase (500 µg/mL). The platelet suspension was applied to

a Sepharose CL 2B acrylic glass column, and platelets were

eluted at 2 mL/min using Tyrode’s solution without additives.

The resulting GFP was set to a platelet count of 4 × 10⁸cells/

mL.

3-{2-[4-(R)-(4-Am in oim in om eth yl-ph en yl)-4-m eth yl-2,5-

d ioxo-im id a zolid in -1-yl]a cetyla m in o}-3-(R)-p h en ylp r op i-

on ic Acid Hyd r och lor id e (47). The method for compound

12e was employed starting from 47a . [R]²²) -60.7 ° (c ) 1;

D

1

water); H NMR (DMSO-d₆) δ 1.75 (s, 3H), 2.70 (d, J ) 7 Hz,

2H), 4.03 (s, 2H), 5.13 (q, J ) 7 Hz, 1H), 7.23 (m, 1H), 7.26-

7.35 (m, 5H), 7.73-7.85 (AA′BB′, 4H), 8.78 (d, J ) 8 Hz, 1H),

9.06 (s, 1H), 9.15 (s, 2H), 9.36 (s, 2H), 12.3 (s, 1H); MS m/z

(FAB) 438 (M + H)⁺.

QXP P h a r m a cop h or e Mod elin g. The routine TFIT (as

implemented within the QXP program, version flo99.6S) with

2000 iterations was applied. As an additional constraint, the

superposition of the carbon atoms of the essential carboxylic

acid functions was enforced by adding zero-order bonds.³⁶The

lowest energy pharmacophore was chosen, since the remaining

49 solutions hardly differed. In contrast to the procedure

suggested in the manual, explicit hydrogen atoms were also

attached to carbon atoms. The final pharmacophore consisted

of an ensemble of the above-mentioned four molecules, and

the remaining molecules in the dataset with the exception of

compounds 16 (diasteromeric mixture of compound 17), 18

(wrong stereoisomer), 36 (double ester), and 39 (diastereomeric

mixture of 44) were separately superimposed by template

fitting with TFIT. Each superposition was visually inspected,

and with few exceptions the highest scoring solution was

selected. In the case of RGDS, 4 and 19 the second and both

the second and fifth solutions, respectively, was chosen. For

20 both the first and fourth superposition and for 11 and 40

both the first and second solutions were chosen, and during

the later QSAR analysis the conformers with the greatest

deviations in predicted activity were discarded.

P la telet Aggr ega tion Assa y. A suspension of gel filtered

platelets (GFP) containing 3 × 10⁸platelets/mL was activated

with 10 µM ADP in the presence of 1 mg/mL fibrinogen and

stirred at 1000 rpm at 37 °C in an aggregometer (PAP 4,

Biodata, Hatboro, PA). Aggregation was measured as the

maximal increase in light transmittance. Compounds were

added to GFP at 37 °C 2 min before the activation with ADP.

Inhibition of aggregation was expressed as IC₅₀value, i.e.,

mean concentration requiring 50% inhibition in GFP samples

of two to six different donors.

GRID Ca lcu la tion s. The interaction energies between the

superimposed molecules and the phenolic OH probe were

calculated by GRID (Molecular Discovery Ltd., version 17) with

a grid spacing of 1 Å.³⁷Thus, for each molecule, 16 588

descriptors (interaction energies) were generated.

F ibr in ogen Bin d in g to GF P . ¹²⁵I-Fibrinogen binding to

GFP was determined according to Marguerie et al.³⁸with slight

modifications from Kornecki et al.⁴¹and Bennett and Vilaire.⁴²

In displacement experiments, the inhibition of 40 nM ¹²⁵I-

fibrinogen by increasing concentrations of the competitor was

characterized in the presence of the platelet agonist ADP (10

µM). The incubation was started by the addition of 250 µL of

GFP (4 × 10⁸cells/mL), yielding a total sample volume of 500

µL. After 30 min incubation at room temperature, 100 µL

aliquots were layered onto a 20% sucrose solution, centrifuged

for 2 min at 10700g, and the radioactivity of the resulting

platelet pellet was measured in a γ counter.

GOLP E An a lysis. For building, validating, and interpret-

ing the 3D-QSAR model, the program GOLPE (version 4.5.1)

with the new methodology smart region definition (SRD) was

applied.^43,44After eliminating columns in the X-matrix with a

total sum of squares < 10^-7, interaction energies with a range

between -0.02 and 0.02 were zeroed and columns with a

standard deviation < 0.21 kcal/mol were removed. To avoid a

skewed distribution in the X-matrix unique (N - 1) variables

were also eliminated. This variable pretreatment reduced the

original number of 16 588 to 2556 variables. A principal

components analysis projects the high-dimensional informa-

tion into a few latent variables. The score plot of the first two

dimensions, t1 and t2 (data not shown), shows a clear separa-

tion between highly active and low active compounds. The only

exceptions are 41 and 43 which are close to 44 and which were

also detected as outliers in the PLS analysis correlating the

grid variables with the IC₅₀s. Since both compounds exhibit a

high internal strain energy after superposition onto the

pharmacophore, they were omitted from the further analysis.

To improve the interpretability and the predictivity of the

3D-QSAR model, the novel SRD algorithm (critical distance

) 1.0 Å, collapsing distance ) 2.0 Å) implemented in GOLPE

was applied. Regions around the molecules carrying the same

statistical information are comprised into groups, and those

regions which do not contribute to the predictivity of the model

are eliminated. As a consequence, the risk of chance correla-

tions is minimized and the grouping of variables in 3D space

facilitates the interpretation of the loading contours. To

evaluate the effect of the grouped X-variables on the predic-

tivity, a fractional factorial design (FFD) scheme for variable

selection (two dimensions, the remaining parameters were at

their default settings) was applied to check the effect of

temporarily removed groups on the predictivity of the model.

Those regions which did not increase the predictive power of

the model were eliminated. Thus, the original 16 588 X-

variables were finally reduced to 1623 X-variables.

F ibr in ogen Bin d in g Assa y. GP IIb/IIIa isolation and

immobilization, fibrinogen binding assay, and data analysis

were carried out as described.^29d

Ack n ow led gm en t. We thank Birgit Herzog, Dieter

Hill, Oda J ooss, Ulrich Nickel, Reinhold Reibert, Peter

Pokorny, Holger Gaul, Manuela Schnierer, Susanne

Stamm, Dirk Timme, Bianca Seitz, Claus Keleschovsky,

and Karin Zentgraf for skillful technical assistance.

Su p p or tin g In for m a tion Ava ila ble: Analytical data.

This material is available free of charge via the Internet at

http://pubs.acs.org.

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J M001068S

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Discovery of an orally active non-peptide fibrinogen receptor antagonist based on the hydantoin scaffold

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