Sulfonamidopyrrolidinone factor Xa inhibitors: Potency and selectivity enhancements via P-1 and P-4 optimization

3572

J . Med. Chem. 1999, 42, 3572-3587

Su lfon a m id op yr r olid in on e F a ctor Xa In h ibitor s: P oten cy a n d Selectivity

En h a n cem en ts via P -1 a n d P -4 Op tim iza tion

Yong Mi Choi-Sledeski,* Daniel G. McGarry, Daniel M. Green,^†Helen J . Mason,^‡Michael R. Becker,

Roderick S. Davis, William R. Ewing, William P. Dankulich, Vincent E. Manetta, Robert L. Morris,

Alfred P. Spada, Daniel L. Cheney,^‡Karen D. Brown, Dennis J . Colussi, Valeria Chu, Christopher L. Heran,

Suzanne R. Morgan, Ross G. Bentley, Robert J . Leadley, Sebastien Maignan, J ean-Pierre Guilloteau,

Christopher T. Dunwiddie,^§and Henry W. Pauls*

Departments of Cardiovascular Drug Discovery and New Leads Generation, Rhoˆne-Poulenc Rorer, 500 Arcola Road,

Collegeville, Pennsylvania 19426-0107

Received J anuary 25, 1999

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa)

inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy

in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation

of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were

initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were

discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and

molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxyben-

zamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the

pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially

in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a

potent fXa inhibitor (K_i) 2 nM) with selectivity against structurally related serine proteinases

(>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor

in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in

vivo (e.g. rat FeCl₂-induced carotid artery thrombosis model).

In tr od u ction

Vascular injury stimulates a series of proteolytic

cleavage reactions culminating in the sequential activa-

tion of coagulation factor X and prothrombin.¹Factor

X is secreted by the liver as a zymogen which is

hydrolytically activated to factor Xa (fXa) by the factor

VIIa/tisssue factor complex (extrinsic pathway) or by the

factor IXa/factor VIIIa complex (intrinsic pathway). The

trypsin-like serine proteinase fXa combines with factor

Va and calcium on membrane surfaces to form the

prothrombinase complex. fXa is responsible for the

catalytic activity of prothrombinase, i.e. the conversion

of prothrombin (factor II) to thrombin (factor IIa), the

last enzyme in the coagulation cascade. Upon assembly,

one unit of prothrombinase can generate over 100

molecules of thrombin,²which in turn is responsible for

A common approach to the design of proteinase

fibrin formation, platelet activation, and other physi-

inhibitors is the modification of peptidyl substrates;⁷

inhibitors which result are often burdened with the

drawbacks associated with peptides in general.⁸Previ-

ously we disclosed novel non-peptidyl inhibitors⁹which

used the X-ray structure of the fXa active site¹⁰and

existing SAR as the departure points for design. This

resulted in the discovery of a 3-sulfonamidopyrrolidi-

none as a useful template for the presentation of ligands

to the S-1 and S-4 subsites of fXa. Further optimization

led to RPR120844, a reversible inhibitor of fXa (K_i)

6.9 nM) which displays moderate selectivity against

related serine proteinases and efficacy in vivo. Having

established optimal template characteristics (ring size,

chirality, and linkage type) in the previous work,⁹we

anticipated that potency and selectivity enhancements

ological events.³Although direct thrombin inhibitors

have been pursued⁴as therapies for thrombosis, the

inhibition of fXa in the prothrombinase complex has

emerged as an alternative antithrombotic approach.⁵

Indeed, the unique position of fXa at the convergence

of the intrinsic and extrinsic pathways and its role as

the singular enzyme for thrombin activation make fXa

an attractive intervention point for the development of

antithrombotic therapy.⁶

^†Current address: Wyeth-Ayerst Research, 145 King of Prussia Rd,

Radnor, PA 19087.

^‡Current address: Bristol Myers Squibb, Rte 206 and Province Line

Rd, Lawrenceville, NJ 08648.

^§Current address: Lilly Research Laboratory, Lilly Co. Center,

Indianapolis, IN 46285.

Published on Web 08/17/1999

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3573

F igu r e 1. Connolly surface of the fXa active site showing features which are potentially important for binding: S-1, S-4 (with

“cation hole”), â-sheet, and the catalytic triad region (CT). This stereoview was generated using fXa coordinates¹⁰available in the

Brookhaven database: 1HCG.

Sch em e 1. General Synthesis of Sulfonyl Chlorides^a

were unavailable at the time this work was initiated;

however, a truncated fXa structure¹⁰had been published

(Figure 1). The S-4 subsite is an open-ended box with

the floor and walls defined by two electron-rich aromatic

side chains (Trp215, Tyr99) and a phenylalanine residue

(Phe174). The terminus of the box is lined with H-bond

acceptors, and an acidic side chain is located at the

periphery (Glu97). This juxtaposition of functionality,

termed the “cation hole”, is not found in other trypsin-

like serine proteinases. It has been invoked to explain

the potency of inhibitors with positively charged and/

or basic moieties at P-4.¹³Of the substitution patterns

explored in our earlier work, biaryls were found to be

effective P-4 ligands.⁹We incorporated weakly basic sp²-

hybridized nitrogens into the distal ring of the biaryl

moiety in hopes of modifying physicochemical properties

while increasing anti-fXa potency.

Herein we report our findings on the use of para-

substituted benzamidines as effective P-1 ligands in the

pyrrolidinone series. In addition, the selectivity en-

hancements obtained by P-4 manipulation are de-

scribed. The synergies realized by the combination of

these ligands into hybrid molecules, as demonstrated

by in vitro potencies, and the in vivo effectiveness of a

potent inhibitor 20b (RPR130737) will be discussed.

a

(a) i. t-BuLi or n-BuLi, THF, -78 °C, 10 min, ii. ZnCl₂, -78

°C to rt, iii. 1-bromo-4-iodobenzene, Pd(Ph₃)₄; (b) i. t-BuLi or

n-BuLi, THF, -78 °C, ii. SO₂gas, -78 °C to rt, iii. SO₂Cl₂, hexane;

(c) i. Mg, Et₂O, reflux, ii. 3-bromopyridine or 4-bromopyridine,

NiCl₂dppp, Et₂O, rt.

could be realized by further manipulation of the P-1 and

P-4 ligands.

In this respect we were intrigued by the publications

of Sturzebecher and Walsmann in the field of trypsin-

like serine proteinases.¹¹In these papers the effect of

heteroatom substitution on the activity of benzamidine

“needles”¹²was described. Modest increases in activity

against several trypsin-like serine proteinases, including

fXa, trypsin, and thrombin (fIIa), were reported for

4-aminobenzamidine (2) versus benzamidine (1) (vide

infra). We envisioned that similar modifications could

be made for the P-1 groups of the pyrrolidinone inhibi-

tors. Our modeling efforts indicated that substituents

para to the P-1 benzamidine of a sulfonamidopyrroli-

dinone would be placed in an environment rich in

H-bond donors and acceptors. Therefore, we set about

to synthesize fXa inhibitors which incorporated p-

aminobenzamidines.

Ch em istr y

The pyrrolidinone benzamidine inhibitors 3 were

prepared using the methods of the companion paper;⁹

however, a number of novel P-4 groups are described

herein. The requisite sulfonyl chlorides were prepared

by lithiation of the biaryl intermediates 5 and 7, then

treatment with sulfur dioxide, and conversion of the

resulting sulfinic acid to the corresponding sulfonyl

chloride (Scheme 1). Palladium(0)-mediated cross-

coupling reactions were used to generate the biaryl

intermediates 5. Cross-coupling reaction of 2-thienyl-

magnesium bromide with bromopyridines utilizing NiCl₂-

(dppp) catalyst¹⁴gave better yields of the pyridylthio-

phene intermediates 7 than Suzuki coupling.¹⁵

In addition to potency enhancement, we were inter-

ested in modifications which could significantly alter the

physicochemical properties of our inhibitors. Modifica-

tions were incorporated into the aromatic P-4 group

because of the unique features of the aryl binding pocket

of fXa (S-4). X-ray structures of fXa/inhibitor complexes

The aminobenzamidine inhibitors were prepared as

shown in Scheme 2. (2-Oxopyrrolidin-3(S)-yl)carbamic

acid tert-butyl ester (10)⁹was alkylated with the ap-

propriate benzyl bromide (obtained in three steps start-

3574 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Sch em e 2. Synthesis of Aminobenzamidine Derivatives^a

Choi-Sledeski et al.

a

(a) SnCl₂, EtOH; (b) benzophenone, pTsOH, toluene; (c) NBS, (C₆H₅CO)₂O₂, CCl₄; (d) NaH, THF/DMF, 0 °C; (e) NaH, MeI, DMF, 0

°C; (f) HCl gas, EtOAc, 0 °C; (g) ArSO₂Cl, Et₃N, CH₃CN; (h) acetyl chloride, DMAP, Et₃N, 1,2-DCE; (i) HCl gas, EtOH, rt; (j) NH₃gas,

MeOH, reflux.

Sch em e 3. Synthesis of Hydroxybenzamidine Derivatives^a

a

(a) ICl, CH₂Cl₂; (b) i. NaH, MEMCl, DMPU, THF, ii. NaBH₄; (c) NBS, Ph₃P, THF; (d) NaH, THF, DMPU; (e) Zn(CN)₂, Pd(PPh₃)₄,

DMF; (f) HCl gas, EtOAc, 0 °C; (g) ArSO₂Cl, pyridine, rt; (h) HCl gas, EtOH; (i) NH₃gas, MeOH, reflux.

ing from 3-methyl-4-nitrobenzonitrile in the case of 9);

subsequent BOC deprotection and sulfonylation yielded

the penultimate intermediate. Depending on the reac-

tion time and temperature, deprotection of the BOC

group using HCl gas led to partial or complete removal

of the benzhydrylidene protecting group. Nevertheless,

the sulfonylation step proceeded smoothly on either

intermediate to give the desired sulfonamides (see

Experimental Section). Standard conditions⁹(i.e. Pinner

reaction and ammonolysis) were used to convert the

nitrile to the target amidine 4. The meta-isomer 15 was

prepared similarly using 3-methyl-5-nitrobenzonitrile as

the starting material. For the acetylated derivative 14

an alkylation step (11 to 12) was inserted prior to BOC

removal and sulfonylation. Nitrile 13 was acetylated

and converted to the amidine as usual.

The hydroxybenzamidine inhibitor 20 was prepared

as described in Scheme 3 by alkylation of 10 with the

appropriate bromide (16 in the case of 20). Cyanation

of the iodo intermediate 17 followed by deprotection

gave the 3-aminopyrrolidinone 18. Sulfonylation in

pyridine provided the intermediate nitrile 19; standard

conditions were used to convert the nitrile to the target

compound 20. Inhibitor 21 was prepared similarly using

p-hydroxybenzaldehyde as the starting material.

Compound 27 was prepared by alkylation of 10 with

benzyl bromide 24, deprotection, sulfonylation, and then

conversion to the amidine using mild conditions¹⁶

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3575

Sch em e 4. Synthesis of Acylating Inhibitor 27 and Acid 28^a

a

(a) t-BuLi, O₂, THF, -78 °C; (b) methyl bromoacetate, K₂CO₃, n-Bu₄NI, DMF, 80 °C; (c) NBS, (C₆H₅CO)₂O₂, CCl₄; (d) NaH, THF/

DMF, 0 °C; (e) HCl(g), EtOAc; (f) 7-methoxynaphthalene-2-sulfonyl chloride, Et₃N, CH₃CN, rt; (g) H₂S, Et₃N, pyridine; (h) MeI, acetone,

reflux; (i) NH₄OAc, MeOH, reflux; (j) 10 N NaOH, EtOH, rt.

Ta ble 1. Dependence of fXa Inhibition on Benzamidine and

Ta ble 2. SAR of Biaryl P-4 Ligands

Sulfonamide Substitution Patterns

(Scheme 4). The functionalized benzyl bromide 24 was

prepared in three steps from 4-bromo-3-methylbenzoni-

trile. Treatment of 4-bromo-3-methylbenzonitrile with

t-BuLi followed by oxygen gas gave the hydroxyben-

zonitrile 22. Alkylation with methyl bromoacetate fol-

lowed by benzylic bromination yielded the desired

benzyl bromide intermediate 24. The acid derivative 28

was obtained by base hydrolysis of the ester 27.

Resu lts a n d Discu ssion

Our initial efforts at P-1 optimization were fruitful

and led to the identification of inhibitor 4a as a

3576 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Choi-Sledeski et al.

F igu r e 2. Stereoview of the trypsin/20a crystal structure.

F igu r e 3. Stereoview of the binding model of 20a in the fXa active site.

significant step forward (Table 1). This compound was

8-fold more potent against fXa than its parent 3a ,⁹and

in this case, the activity against other serine proteinases

(thrombin, 5-fold; bovine trypsin, 3-fold) was also en-

hanced. Our earlier work⁹had demonstrated that N-

methylation of the sulfonamide was tolerated and in

some instances led to modest enhancements in potency

(3a vs 3b); this result was paralleled by the aminoben-

zamidine pair 4a and 4b.

serine proteinases also increased (thrombin, 7-fold;

bovine trypsin, 3-fold) although selectivity favored fXa.

The activities of 15 and 21 demonstrate the strict

positional requirements for heteroatom substitution.

Examination of the S-1 pocket suggests that substitu-

tion in the 3-position of the benzamidine ring results

in steric clashes upon binding; correspondingly, com-

pound 15 was weakly active. Substitution at the 2-posi-

tion would appear to be sterically tolerated; nonetheless

compound 21 was much less active than its 4-positional

isomer. How can the large potency increases seen with

the 4-heterobenzamidines and the 4-hydroxy com-

pounds, in particular, be rationalized? Benzamidines

substituted in the para-position generally exist in

solution as protonated cations;¹⁷similarly, unsubsti-

tuted benzamidines are protonated at physiological pH

The requirements of the P-1 ligand were explored in

more detail. Work on simple benzamidines¹¹had sug-

gested that 4-hydroxy substitution could also lead to

potency enhancements, albeit less than 4-amino sub-

stitution. Thus, we were gratified to discover that the

corresponding compound 20a was more potent than the

parent 3a (16-fold). Once again, activity against other

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3577

Ta ble 3. Heterobenzamidine Hybrids

Ta ble 4. In Vitro Comparisons of Selected fXa Inhibitors

F igu r e 4. Comparison of RPR120844 and RPR130737 in the

rat FeCl₂-induced carotid artery thrombosis model. Dose is

bolus plus 60 min of infusion. Vehicle time-to-occlusion: 18

min.

(pK_a) 11.4). Thus, invoking the increased basicity of

amino- and hydroxybenzamidines to explain the potency

increases observed is not a compelling argument.

The possibility of obtaining fXa/20a crystal structures

to help explain our SAR was considered. Unfortunately,

unlike the related serine proteinases, trypsin and

thrombin, X-ray structures of fXa with small molecule

inhibitors are rare. Bode et al. have obtained X-ray

structures of the bisamidine inhibitor DX-9065a in both

fXa¹³and trypsin.¹⁸Similar inhibitor binding modes

were observed for both complexes, reflecting the large

degree of homology between the two enzymes; these

results were the impetus for our own protein structure

work. A crystal structure of 20a (trypsin K_i) 206 nM)

with trypsin was solved to 1.9 Å resolution (Figure 2).

The salient features of this structure include a twin-

twin interaction between the P-1 amidine and the S-1

carboxylate (Asp189) which is part of an extensive

H-bonding network (Ser190 and Gly219). An H-bond

from the pyrrolidinone carbonyl to Gly219 is observed,

and the methoxynaphthyl group is in close contact with

the S-4 subsite. The role of the hydroxyl is difficult to

define since it hydrogen bonds to an artifactual sulfate

ion; this anion also forms H-bonds to the catalytic triad

residues Ser195 and His57.

pected to be extensively delocalized with participation

of the substituent; consequently the hydroxyl is an

excellent H-bond donor or may ionize readily. Several

H-bond acceptors (and donors) are found in this region

of fXa; our binding model indicates that the Ser195 or

His57 side chains and the Ser214 carbonyl are likely

candidates for direct or water-mediated interaction. This

additional interaction could account for the potency

enhancements seen in fXa, and similar arguments can

be made for the 4-aminobenzamidines. In contrast, the

hydroxyl group of the less active isomer 21 cannot access

this region of the enzyme.

A recent publication from Berlex Biosciences describes

a very similar para-substituted benzamidine effect on

an unrelated series of fXa inhibitors, namely 1,6-

diphenoxypyridines.¹⁹The potency enhancements for

4-amino substitution were comparable to our own

observations on pyrrolidinones. However, the 4-hydroxy

analogue was 67-fold more active than the unsubsti-

tuted parent of the Berlex series. This contrasts with

the more modest enhancements (up to 20-fold) observed

for the pyrrolidinones. It is by no means certain that

the rather small potency increases from para-substitu-

tion first described by Sturzebecher and Walsmann for

trypsin-like serine proteinases in general¹¹would apply

equally to all classes of fXa inhibitors, and it appears

that the magnitude and relative specificity of this effect

is class-dependent.

All the important interactions of 20a with the trypsin

active site are duplicated in the fXa/20a binding model

(Figure 3), and the conformations of the inhibitor in the

two active sites are very similar. The protonated ben-

zamidine engages in a hydrogen bond network to

Asp189, Ser190, and Gly218 of the S-1 subsite. An

H-bond from the pyrrolidinone carbonyl to Gly218 is

observed and the methoxynaphthyl inserts deep into the

S-4 subsite. The 4-hydroxybenzamidine cation is ex-

It occurred to us that the 4-hydroxy and/or 4-amino

functions could serve as handles to append moieties with

potential to interact covalently with the catalytic triad

(i.e. Ser195). Thus, compounds 14 and 27 were prepared

3578 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Choi-Sledeski et al.

as potential acylating probes, and in fact, the latter

compound was shown to be an effective inhibitor of fXa

(K_i) 8 nM). We propose that the ester is functioning

as an alternate substrate inhibitor of fXa, ultimately

hydrolyzed by water to yield acid 28, a relatively weak

inhibitor. In support of this mechanism we were able

to demonstrate, by HPLC analysis, that incubation of

ester 27 with fXa yielded acid 28 and that the inhibition

of fXa by 27 was time-dependent. Compound 27 also

showed significantly enhanced trypsin activity. Because

of the chemical lability and lack of selectivity of 27, we

decided to focus our efforts on nonacylating inhibitors.

Nonetheless, this was a useful result since it provided

further support for the pyrrolidinone binding model in

fXa.

has low activity against the fibrinolytic serine protein-

ase tissue plasminogen activator (t-PA). Kinetic analysis

showed that RPR130737 was a reversible, competitive

inhibitor of fXa.²¹This compound inhibited coagulation

of human plasma in vitro: i.e. RPR130737 doubled

activated partial thromboplastin time at a concentration

of 0.32 µM. This result indicates its effectiveness against

fXa in the prothrombinase complex which is the physi-

ologically relevant form of the proteinase. In this

respect, note also the improvement in activity versus

RPR120844; similar results were seen in rat and dog

plasma (data not shown).

The more polar nature of the pyridyl-containing

inhibitors is reflected in the calculated log P (Table 4).

The Clog P for RPR130737 (1.1) is significantly less than

for the naphthalene-containing inhibitors (Clog P g 2).

The fXa inhibitor RPR130737 was evaluated in vivo

in a rat model of ferrous chloride-induced arterial

thrombosis; this data is presented in Figure 4. A dose-

dependent reduction in thrombus mass and a concomi-

tant increase in the time to occlusion were observed.

Efficacy is comparable to that of RPR120844 at about

one-tenth the dose. Qualitatively similar results were

obtained in a canine model of electrolytic injury-induced

thrombosis in the jugular vein and carotid artery and

will be published separately. Clearly, the potency

enhancements measured in vitro are reflected in the

animal models of thrombosis. Inhibitors containing

positively charged amidino or guanidino functions are

often compromised by their nonspecific effects on car-

diovascular function.²²Blood pressure and heart rate

were monitored during the course of our in vivo experi-

ments with RPR130737; hemodynamic parameters were

unchanged.

Efforts to optimize the P-4 group are summarized in

Table 2. Initial attempts to incorporate nitrogen into

the distal ring yielded compounds 3d -3f, equipotent to

the simple biphenyl 3c. It was observed, as was typical

for the biaryl P-4s, that selectivities against trypsin and

thrombin were 20-fold or better. Attempts to incorporate

discrete positive charges in P-4, entries 3g and 3i, did

not increase potency. It is possible that the very nearly

linear presentation of these biphenyl systems does not

allow for productive interaction of the basic and/or

charged systems with the S-4 “cation hole”.

In contrast, similar substitutions with thiophene as

the proximal aromatic ring yielded two compounds (3k

and 3l) with 3-fold enhancements in fXa activity with

respect to the simple biphenyl. For this pair, relative

selectivity against fIIa and trypsin was high; the isox-

azole 3n was also potent and selective. In the two X-ray

structures of fXa with small molecule inhibitors,^13,20

a

bound water molecule at the terminus of the S-4 pocket

is observed. A weak interaction with this water may be

invoked to rationalize the modest enhancements in

activity observed for compounds 3k , 3l, and 3n . In the

less active isomer 3j, the nitrogen lone pair is directed

away from the bound water.

Con clu sion

The SAR of the pyrrolidinone class of fXa inhibitors

has been expanded to yield 20b (RPR130737) as a

potent member (K_i) 2 nM) with high selectivity (>1000

times) against other serine proteinases. RPR130737 is

a reversible inhibitor of fXa and inhibits coagulation of

human plasma at submicromolar levels. In vivo experi-

ments have shown that this compound is an effective

antithrombotic agent in animal models of thrombosis.

X-ray studies in trypsin support our model for the

inhibition of fXa by pyrrolidinone inhibitors with a

hydroxybenzamidine moiety in P-1. It suggests that the

twin-twin interaction between the amidine and Asp189

is augmented by hydrogen bonding between the p-

hydroxyl and the proximal residues of the enzyme. The

excellent selectivity achieved for RPR130737 is, in part,

due to the P-4 pyridylthiophene, which is complemen-

tary to the unique S-4 subsite of fXa.

To further exploit these observations, the pyridin-3-

yl and isoxazolylthienyl P-4s were grafted onto the

hydroxy- and aminobenzamidine-containing templates.

The hydroxybenzamidine hybrids in Table 3 (20b and

20c) were found to be potent inhibitors of fXa (K_i) 2

and 3 nM, respectively), comparable to the best of the

naphthalene-containing compounds (20a ) with the ad-

ditional benefit of improved selectivity against trypsin

and thrombin (3 orders of magnitude). The aminoben-

zamidines 4c and 4d were somewhat less effective

than the hydroxy counterparts and less selective. Note

that, in contrast, the fluoro analogue 29 (K_i) 130 nM)

was significantly less active than even the unsubsti-

tuted parent 3k (K_i) 41 nM). Although the amino

function conferred good activity, it appears that hy-

droxybenzamidine is generally a superior ligand for S-1

in this series. Compound 20b, in particular (designated

RPR130737), had an attractive profile and was chosen

for further study as an intravenous fXa inhibitor.

Exp er im en ta l Section

En zym e Assa ys. Human fXa, thrombin, and activated

protein C (aPC) were obtained from Enzyme Research Labo-

ratories, Inc. (South Bend, IN). Bovine trypsin was obtained

from Sigma Chemical Co. (St. Louis, MO). Plasmin was

purchased from Pharmacia Hepar (Franklin, OH). Tissue

plasminogen activator (t-PA) was obtained from Genentech

(San Fransisco, CA). The chromogenic substrates used were

Spectrozyme FXa (American Diagonostica Inc., Greenwich, CT)

for fXa; Pefachrome TH (Centerchem, Inc., Stamford, CT) for

thrombin; S-2765 (Diapharma Group Inc., Franklin, OH) for

trypsin; Pefachrome-tPA (Centerchem Inc.) for t-PA. The

Table 4 compares in vitro data for RPR130737 with

earlier pyrrolidinone prototypes. Optimal selectivity is

obtained by the combination of the pyridylthiophene P-4

and the hydroxybenzamidine. Most significantly, the

compound is selective against anticoagulant serine

proteinases such as aPC and plasmin. In addition, it

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3579

chromogenic substrate S-2366 (Diapharma Group Inc.) was

used for both plasmin and aPC assays.

placed on top of the vessel downstream from the flow probe

as described in the methods of Kurz et al., with minor

modifications.²⁵The filter paper was removed after 10 min.

The carotid artery was removed 60 min after the application

of the filter paper whether or not occlusion had occurred. Time-

to-occlusion was defined as the time from the application of

FeCl₂until blood flow decreased to zero. If the vessel did not

occlude by 60 min, the time to occlusion was assigned a 60-

min value for data analysis. The thrombus was removed, and

the wet weight was determined immediately on a Sartorius

BP160P balance (Sartorius AG, Gottingen, Germany). Blood

pressure and heart rate were continuously monitored on a

Gould TA6000 recorder (Gould Instruments, Valley View, OH)

and reported at control, after 15 min of infusion, and at 15,

30, 45, and 60 min after FeCl₂application.

Molecu la r Mod elin g. The inhibitor 20a was flexibly

docked into the fXa active site using an automated protocol

developed in-house.²⁶In this method, lower-energy conforma-

tions of the ligand are generated on the fly using a modifed

version of a rule-based method implemented in Chem-X

(January 1997 release, Chemical Design Ltd., Chipping Norton,

Oxfordshire OX7 5SR, U.K.). Using ChemDBS-3D with in-

house customization, an attempt is made to fit each conforma-

tion in turn onto a predefined, minimal pharmacophore model

using a steric shell of the active site as an added constraint.

Matching conformations are then passed to the program

DISCOVER (MSI, 9685 Scranton Rd, San Diego, CA 92121-

3752) for optimization in a partially relaxed active site model

using the CFF97 force field (April 1997 release, developed by

the Potential Energy Functions Consortium, MSI, 9685 Scran-

ton Rd, San Diego, CA 92121-3752).²⁷The resulting docked

complexes are scored on the basis of total force field energy.

fXa, thrombin, and plasmin were assayed in

a buffer

containing 0.05 M Tris, 0.15 M NaCl, and 0.1% PEG-8000 at

pH 7.5.²³Trypsin and aPC were assayed in the aforementioned

buffer with addition of 0.02 M CaCl_2.Tissue plasminogen

activator was assayed in the first buffer, but PEG-8000 was

replaced with 0.1% (w/v) BSA (bovine serum albumin; Sigma).

The final substrate concentrations in the reactions were 200,

50, 250, and 500 µM for Spectrozyme FXa, Pefachrome TH,

S-2765, and Pefachrome-tPA, respectively. The final concen-

trations of S-2366 for the plasmin and aPC assays were 300

and 500 µM, respectively. All enzyme assays were carried out

at room temperature in 96-well microtiter plates with a final

enzyme concentration of 1 nM. Compound dilutions were

added to the wells containing buffer and enzyme and prein-

cubated for 30 min. The enzyme reactions were initiated by

the addition of substrate, and the color developed from the

release of p-nitroanilide from each chromogenic substrate was

monitored continuously for 5 min at 405 nm on a Thermomax

microtiter plate reader (Molecular Devices, Sunnyvale, CA).

Under the experimental conditions, less than 10% of the

substrate was consumed in all assays. The initial velocities

measured were used to determine the amount of inhibitor

required to diminish 50% of the control velocity; this concen-

tration was defined as the IC₅₀of the inhibitor. Assuming the

kinetic mechanisms were all competitive, the apparent K_i

values were then calculated according to the Cheng-Prusoff

equation: K_i) IC₅₀/1 + [S]/K_m.²⁴

fXa -In d u ced Clea va ge of In h ibitor 27. Compound 27 and

fXa were combined in buffer at a final concentration of 62.5

and 125 nM, respectively. The ratio of 27 and fXa was high

(500:1) to enable HPLC analysis of the reaction mixture. The

mixture was incubated at 37 °C, and aliquots were removed

for analysis and anti-fXa activity estimation.

Clog P’s were calculated using ACD Lab software (Advanced

Chemistry Development, 133 Richmond St W, Suite 605,

Toronto, Ontario, Canada, M5A 2L3).

Tr yp sin /20a Cr ysta l Str u ctu r e Deter m in a tion . Cr ys-

tallization an d in h ibitor soakin g: Bovine pancreatic trypsin

(Sigma T8003) was solubilized in 1 mM CaCl₂, 60 mM

benzamidine, 50 mM Mes-NaOH (pH 6.0) buffer to a final

concentration of 30 mg/mL. The hanging drops crystallization

method was used. Single crystals were obtained with am-

monium sulfate (1.4-1.8 M), pH 6.0, in the “open” form

(P2₁2₁2₁, a ) 59.9 Å, b ) 64.2 Å, c ) 69.9 Å) for which the

binding site is free of any contact with crystallographic

symmetry-related molecules. The crystals were soaked in 50

µL of 3.3 M ammonium sulfate, 1 mM 20a , 6% DMF, 1 mM

CaCl₂, 50 mM Mes-NaOH (pH 6.0) for 5 h.

Da ta collection a n d p r ocessin g: For data collection a

crystal was placed in the previous soaking solution + 20%

glycerol for 5 min and then flash-cooled in a gas stream of N₂

at 90 K. Data collection was performed on a FR591 rotating

anode generator (Nonius, The Netherlands) equipped with a

DIP2020K image plate detector (Mac Science, J apan). Data

images were processed using DENZO and SCALEPACK,²⁸and

structure refinement was performed using X-PLOR.²⁹The

trypsin structure used as a starting point is the uncomplexed

1.55 Å form (2ptn Protein Data Bank entry).³⁰

Ch em istr y. All starting materials, reagents, and solvents

were used as received from commercial sources except for

N-bromosuccinimide which was recrystallized from water and

dried under vacuum. In general, reactions were performed

under an inert atmosphere such as nitrogen or argon. Workup

indicates drying over sodium or magnesium sulfate, filtering,

and concentrating in vacuo. Flash column chromatography of

intermediates was performed using 230-400 mesh silica gel

60 (E. Merck). Proton NMRs were recorded on a Bruker ARX

300 MHz or ACF 300 spectrometer, and mass spectra were

obtained from a Varian VG-70SE spectrometer. Elemental

analyses were performed by Quantitative Technologies of

Whitehorse, NJ .

At the early time points (5 and 30 min) fXa was completely

inhibited, and very little conversion had taken place. After 1-h

incubation with fXa, HPLC analysis revealed that 48% of

compound 27 (K_i) 8 nM) was converted to acid 28 (K_i) 1.3

µM). This corresponded to an anti-fXa activity of 98% for the

mixture. After 3-h incubation, the ester 27 was completely

converted to acid 28; the anti-fXa activity of the mixture was

14%.

Coa gu la tion Assa y. Activated partial thromboplastin time

(APTT) was measured with a MLA Electra 800 automatic

coagulation timer (Orthodiagnostics, NJ ). Citrated human

(George King Biomedical Inc: Overland Park, KS), dog

(mongrel; Covance Research Product, Alice, TX), and rat

(Sprague-Dawley; Charles River) plasma were used in the

assays. One hundred microliters of freshly thawed plasma was

mixed with 100 µL of compound dilutions followed by auto-

matic addition of 100 µL of actin-activated cephaloplastin

reagent (Dade, Miami, FL) and 100 µL of 0.035 M calcium

chloride to start the clot formation. Anticoagulant activity of

a compound was reported as the concentration required for

doubling the plasma clotting time.

Rat FeCl₂-In du ced Car otid Ar ter y Th r om bosis Model.²⁵

Experiments were performed on male Sprague-Dawley rats

anesthetized with inactin (100 mg/kg, ip). The trachea was

intubated via a tracheostomy with polyethylene tubing (PE

205) to ensure airway patency. Catheters (PE50) were inserted

into the right jugular vein for drug administration and the

right femoral artery for collection of blood samples and

recording of blood pressure. The right carotid artery was

isolated, and a piece of parafilm “M” (American Can Co.,

Greenwich, CT) was placed under the vessel to separate it from

the surrounding tissue. An electromagnetic flow probe (0.95

mm lumen) was placed on the carotid artery and attached to

a model 1401 flow meter (Skalar, Delft, The Netherlands).

Following baseline flow measurements of 10 min, compounds

were administered as an intravenous bolus plus a constant

infusion for 75 min. After 15 min of drug infusion, vascular

damage was produced by the local application of a piece of filter

paper (2 × 5 mm) saturated with a 50% solution of FeCl₂

Final products were generally purified by preparative

reverse-phase HPLC. Purification was performed on a Rainin

SD-1 Dynamax system with a 2-in. C-18 reverse-phase Dy-

namax 60 Å column using a gradient of 10-40% acetonitrile/

3580 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

0.1% TFA water to 80-100% acetonitrile/0.1% TFA water over

Choi-Sledeski et al.

described in part B except that 2 equiv of tert-butyllithium

were used to generate the starting anion. The crude solid

product was purified by washing with copious amounts of

hexane and ether and was used without further purification:

MS (EI) m/z 253, 255 (M⁺).

(1-Meth yl-1H-im id a zol-2-yl)ben zen e-4-su lfon yl Ch lo-

r id e (6d ). (1-Methyl-1H-imidazol-2-yl)benzene was treated as

described in part B but using 1.02 equiv of n-butyllithium: MS

(EI) m/z 256, 258 (M⁺).

5-P yr id in -3-ylth iop h en e-2-su lfon yl Ch lor id e (8a ). 3-

Thiophene-2-ylpyridine^14awas treated as described in part B

but using 1.02 equiv of n-butyllithium. The crude solid product

was diluted with EtOAc and water. The organic layer was

washed with saturated NaHCO₃and saturated NaCl and then

worked up. The crude solid was suspended in ether and the

insoluble black solid filtered off. The ethereal solution was

concentrated in vacuo to give a light yellow solid which was

used without further purification: ¹H NMR (CDCl₃) δ 8.95 (bs,

1H), 8.70 (bs, 1H), 7.95 (d, 1H), 7.90 (d, 1H), 7.45 (bs, 1H),

7.44 (d, 1H); MS (EI) m/z 259, 261 (M⁺).

5-P yr id in -4-ylth iop h en e-2-su lfon yl Ch lor id e (8b). 4-

Thiophene-2-ylpyridine^14bwas treated as described in part B

but using 1.02 equiv of n-butyllithium. The crude solid product

was diluted with EtOAc and water. The organic layer was

washed with saturated NaHCO₃and saturated NaCl and then

worked up to give the product as a yellow solid which was used

without further purification: ¹H NMR (CDCl₃) δ 8.75 (d, 2H),

8.60 (d, 1H), 7.90 (d, 1H), 7.51 (d, 2H); MS (EI) m/z 259, 261

(M⁺).

P r ep a r a tion of 4-Am in o-3-[3-(S)-(7-m eth oxyn a p h th a -

len -2-ylsu lfon yla m in o)-2-oxop yr r olid in -1-ylm eth yl]ben -

za m id in e Tr iflu or oa ceta te (4a ). Rep r esen ta tive P r oce-

d u r e for th e Syn th esis of Com p ou n d s 4c a n d 4d . P a r t

A: Tin(II) chloride (13.9 g, 61.7 mmol) was added to a solution

of 3-methyl-4-nitrobenzonitrile (2.0 g, 12.3 mmol) in EtOH (160

mL). The resulting mixture was refluxed for 2 h and then

cooled to ambient temperatures. The reaction mixture was

poured into ice water (100 mL), and the pH of the solution

was adjusted to >7 with saturated NaHCO₃. The solution was

diluted with EtOAc, and the resulting mixture was filtered

through Celite. The organic layer was separated and the

aqueous layer further extracted with EtOAc. The combined

organic layers were worked up to yield 4-amino-3-methylben-

zonitrile (1.57 g, 96%) as a white solid: ¹H NMR (CDCl₃) δ

7.30-7.35 (m, 2H), 6.63 (d, 1H), 4.10 (bs, 2H), 2.15 (m, 3H);

MS (EI) m/z 132 (M⁺).

P a r t B: Benzophenone (1.74 g, 9.53 mmol) and p-toluene-

sulfonic acid (0.43 g, 2.1 mmol) were added to a solution of

4-amino-3-methylbenzonitrile (1.2 g, 9.08 mmol) in toluene (75

mL). The reaction vessel was fitted with a Dean-Stark trap

and heated to reflux. After 24 h, the solution was cooled to

ambient temperature and concentrated in vacuo. The residue

was chromatographed on silica gel eluting with a gradient of

3% EtOAc/hexanes to 10% EtOAc/hexanes to give 4-(benzhy-

drylidenylamino)-3-methylbenzonitrile (2.43 g, 90%) as an

oil: ¹H NMR (CDCl₃) δ 7.80 (m, 2H), 7.23-7.48 (m, 7H), 7.15

(d, 1H), 7.05 (bs, 2H), 6.50 (d, 1H), 2.20 (s, 3H); MS (EI) m/z

296 (M⁺).

P a r t C: N-Bromosuccinimide (0.84 g, 4.7 mmol) and benzoyl

peroxide (0.22 g, 0.64 mmol) were added to a solution of

4-(benzhydrylidenylamino)-3-methylbenzonitrile (1.36 g, 4.27

mmol) in CCl₄(40 mL). The solution was refluxed for 16 h

and then cooled to ambient temperature. The mixture was

diluted with CH₂Cl₂and washed with 1 N NaOH and satu-

rated NaCl. The organic layer was worked up, and the residue

was chromatographed on silica gel eluting with a gradient of

5% EtOAc/hexanes to 10% EtOAc/hexanes to give 4-(benzhy-

drylidenylamino)-3-(bromomethyl)benzonitrile (9) (0.91 g, 57%)

as an oil: ¹H NMR (CDCl₃) δ 7.70-7.80 (m, 2H), 7.60 (d, 1H),

7.25-7.53 (m, 8H), 7.15 (dd, 1H), 6.35 (d, 1H), 4.55 (s, 2H);

MS (EI) m/z 374, 376 (M⁺).

a

30-50-min period and a flow rate of 40-50 mL/min.

Fractions containing the desired material were concentrated

and lyophilized to obtain the final products as white solids.

P r ep a r a tion of 4-P yr id in -2-ylben zen esu lfon yl Ch lo-

r id e (6a ). Rep r esen ta tive P r oced u r e for th e Syn th esis

of Bia r yl Su lfon yl Ch lor id es. P a r t A: A solution of 2-bro-

mopyridine (1.0 g, 6.3 mmol) in THF (25 mL) was treated with

tert-butyllithium (7.5 mL, 1.7 M solution in pentane) at -78

°C for 10 min followed by zinc chloride in ether (6.6 mL, 6.6

mmol). The reaction was warmed to room temperature over a

2-h period and then treated with palladium tetrakis(triph-

enylphosphine) (35.4 mg, 0.031 mmol) and 1-bromo-4-iodo-

benzene (1.78 g, 6.29 mmol) in THF (10 mL). The reaction

mixture was stirred for 72 h, then concentrated in vacuo, and

diluted with 10% NH₄OH solution and EtOAc. The organic

layer was washed with saturated NaCl and worked up. The

crude residue was purified by flash chromatography to give

4-(pyridin-2-yl)bromobenzene (5a ) (0.75 g, 51%): ¹H NMR

(CDCl₃) δ 8.71 (d, 1H), 7.88 (d, 2H), 7.66-7.79 (m, 2H), 7.56

(d, 2H), 7.22 (d, 1H); MS (EI) m/z 233, 235 (M⁺).

4-(P yr id in -3-yl)br om oben zen e (5b). 3-Bromopyridine (6

g, 38 mmol) was treated as described above with n-butyl-

lithium (28.5 mL, 1.6 M solution in THF) and 1-bromo-4-

iodobenzene (8.96 g, 31.7 mmol). The crude product was

purified by chromatography (30% EtOAc/hexanes) to obtain

5b (3.5 g, 47%): MS (EI) m/z 233, 235 (M⁺).

4-(P yr id in -4-yl)br om oben zen e (5c). 4-Bromopyridine hy-

drochloride was free based with 1 N NaOH solution (0.95

equiv) and extracted into methylene chloride. The organic

solution was concentrated at 0 °C and used immediately

without further purification. A portion of the solid obtained

(3.0 g, 19 mmol) was treated as described above with n-

butyllithium (14.25 mL, 1.6 M solution in THF) and 1-bromo-

4-iodobenzene (5.39 g, 19 mmol). The crude product was

purified by chromatography (30% EtOAc/hexanes to 60%

EtOAc/hexanes) to obtain 4-(pyridin-4-yl)bromobenzene (5c)

(2.59 g, 58%): MS (EI) m/z 233, 235 (M⁺).

(1-Meth yl-1H-im id a zol-2-yl)ben zen e (5d ). (1-Methyl-1H-

imidazol-2-yl)benzene was prepared using the method of Bell

et al.³¹with 1-bromo-4-iodobenzene and 1-methylimidazole as

starting materials: ¹H NMR (CDCl₃) δ 7.58 (d, 2H), 7.5 (d,

2H), 7.11 (d, 1H), 6.98 (d, 1H), 3.75 (s, 3H); MS (EI) m/z 236,

238 (M⁺).

P a r t B: tert-Butyllithium (3.72 mL, 1.7 M solution in

pentane) was added dropwise to a solution of 4-(pyridin-2-yl)-

bromobenzene (0.75 g, 3.16 mmol) in THF/ether (20 mL, 1:1

v/v) at -78 °C. In a separate flask, SO₂gas was condensed

(10 mL) at -78 °C and then diluted with ether (20 mL). The

lithium anion was added dropwise via cannula to the solution

of sulfur dioxide, and the reaction mixture was allowed to

warm to room temperature overnight. The suspension was

concentrated in vacuo, and the resulting solid washed with

ether. The collected solid was diluted with anhydrous hexane

(20 mL) and treated with SO₂Cl₂(3.32 mL, 3.32 mmol, 1 M

solution in CH₂Cl₂) at 0 °C. The suspension was allowed to

warm to room temperature over 4 h and then concentrated in

vacuo. The crude solid product was washed with hexane and

triturated with warm benzene followed by methylene chloride

to give 4-pyridin-2-ylbenzenesulfonyl chloride (6a ) (0.5 g,

63%): ¹H NMR (CD₃OD) δ 8.72 (d, 1H), 8.34 (AB, 2H), 8.19

(AB, 2H), 7.85-8.05 (m, 2H), 7.47 (t, 1H); MS (EI) m/z 253,

255 (M⁺).

4-P yr id in -3-ylben zen esu lfon yl Ch lor id e (6b). The title

compound was prepared from 4-(pyridin-3-yl)bromobenzene

(1.75 g, 7.5 mmol) as described in part B except that 2 equiv

of tert-butyllithium were used to generate the starting anion.

The crude solid product was purified by washing with copious

amounts of hexane followed by 100 mL of hot anhydrous CH₂-

Cl₂and was used without further purification (1.98 g, 100%):

¹H NMR (DMSO-d₆) δ 9.23 (bs, 1H), 8.80 (m, 2H), 8.04 (m,

1H), 7.65-7.83 (AB, 4H); MS (EI) m/z 253, 255 (M⁺).

4-P yr id in -4-ylben zen esu lfon yl Ch lor id e (6c). The title

compound was prepared from 4-(pyridin-4-yl)bromobenzene as

P a r t D: Sodium hydride (0.028 g, 0.70 mmol, 60% oil

dispersion) was added to a solution of (2-oxopyrrolidin-3-(S)-

yl)carbamic acid tert-butyl ester⁹(10) (0.134 g, 0.67 mmol) in

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3581

THF/DMF (10 mL, 9:1 v/v) at 0 °C. After 5 min, 4-(benzhy-

drylidenylamino)-3-(bromomethyl)benzonitrile (9) (0.252 g,

0.67 mmol) in THF (1 mL) was added dropwise via cannula to

the reaction mixture. The ice bath was removed and the

resulting solution stirred for an additional 4 h during which

time the solution turned cloudy. The reaction was quenched

with saturated NH₄Cl solution and diluted with EtOAc. The

organic layer was separated and the aqueous layer washed

further with EtOAc. The combined organic layers were worked

up, and the crude residue was chromatographed on silica gel

eluting with a gradient of 20% EtOAc/hexanes to 30% EtOAc/

hexanes to give {1-[2-(benzhydrylidenylamino)-5-cyanobenzyl]-

2-oxopyrrolidin-3-yl}carbamic acid tert-butyl ester (11) (0.284

g, 86%) as a foamy solid: ¹H NMR (CDCl₃) δ 7.60-7.80 (bs,

2H), 7.40 (s, 1H), 7.25-7.40 (bs, 6H), 7.22 (d, 1H), 7.00-7.15

(bs, 2H), 6.48 (d, 1H), 5.00 (d, 1H, 4.45 (AB, 2H), 4.15 (m, 1H),

3.30 (m, 2H), 2.61 (m, 1H), 1.90 (m, 1H), 1.45 (s, 9H); MS (FAB)

m/z 495 (M + H)⁺.

P a r t E: Hydrogen chloride gas was bubbled through a

solution of {1-[2-(benzhydrylidenylamino)-5-cyanobenzyl]-2-

oxopyrrolidin-3-yl}carbamic acid tert-butyl ester (11) (0.70 g,

1.42 mmol) in EtOAc (75 mL) at 0 °C for 5 min. After 1 h, the

solution was concentrated in vacuo. The resulting residue was

dissolved in CH₃CN (50 mL); then triethylamine (0.79 mL, 5.68

mmol) and 7-methoxynaphthalene-2-sulfonyl chloride (0.38 g,

1.49 mmol) were added. After 5 h, the reaction mixture was

concentrated in vacuo, diluted with EtOAc, and washed with

saturated NaHCO₃and saturated NaCl. The organic layer was

worked up, and the crude material was purified by chroma-

tography eluting with 5% CH₃OH/CH₂Cl₂to give 7-methoxy-

naphthalene-2-sulfonic acid [1-(2-amino-5-cyanobenzyl)-2-oxo-

pyrrolidin-3-(S)-yl]amide (0.60 g, 94%) as a yellow solid: ¹H

NMR (CDCl₃) δ 8.30 (s, 1H), 7.90 (d, 1H), 7.80 (d, 1H), 7.70

(d, 1H), 7.30-7.41 (m, 4H), 6.55 (d, 1H), 5.25 (d, 1H), 4.90 (s,

2H), 4.30 (AB, 2H), 3.95 (s, 3H), 3.75 (m, 1H), 3.20 (m, 2H),

2.55 (m, 1H), 2.00 (m, 1H); MS (FAB) m/z 451 (M + H)⁺.

5-P yr id in -3-ylth iop h en e-2-su lfon ic Acid [1-(2-Am in o-

5-cya n oben zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR

(CDCl₃) δ 8.85 (d, 1H), 8.61 (dd, 1H), 7.82 (m, 1H), 7.68 (d,

1H), 7.26-7.38 (m, 4H), 6.60 (d, 1H), 5.35 (d, 1H), 4.89 (s, 2H),

4.30 (AB, 2H), 3.97 (m, 1H), 3.30 (m, 2H), 2.65 (m, 1H), 2.09

(m, 1H); MS (FAB) m/z 454 (M + H)⁺.

5-Isoxa zol-3-ylth iop h en e-2-su lfon ic Acid [1-(2-Am in o-

5-cya n oben zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR

(CDCl₃) δ 8.35 (d, 1H), 7.81 (d, 1H), 7.69 (d, 1H), 7.50 (d, 2H),

7.39 (dd, 1H), 6.65 (d, 1H), 6.59 (d, 1H), 4.32 (AB, 2H), 4.02

(m, 1H), 3.30 (m, 2H), 2.60 (m, 1H), 2.08 (m, 1H).

P a r t F : Hydrogen chloride gas was bubbled through a

solution of 7-methoxynaphthalene-2-sulfonic acid [1-(2-amino-

5-cyanobenzyl)-2-oxopyrrolidin-3-yl]amide (0.60 g, 1.33 mmol)

in EtOH (60 mL) at 0 °C for 10 min. The reaction flask was

stoppered and allowed to warm to ambient temperature

overnight. The reaction mixture was concentrated in vacuo,

and the crude imidate was taken up in MeOH (50 mL) and

cooled to 0 °C. Ammonia gas was bubbled through the solution

for 10 min; then the reaction flask was fitted with a reflux

condenser and balloon. The reaction mixture was heated at

60 °C for 4.5 h and then concentrated in vacuo to yield the

crude product. Purification by reverse-phase HPLC and lyo-

philization of the aqueous fractions containing the desired

product yielded 4-amino-3-[3-(S)-(7-methoxynaphthalen-2-yl-

sulfonylamino)-2-oxopyrrolidin-1-ylmethyl]benzamidine tri-

fluoroacetate (4a ) (0.58 g, 75%) as a white solid: ¹H NMR

(DMSO-d₆) δ 8.80 (bs, 2H), 8.45 (bs, 2H), 8.35 (s, 1H), 8.10 (d,

1H), 8.00 (d, 1H), 7.90 (d, 1H), 7.70 (dd, 1H), 7.50 (m, 2H),

7.40 (d, 1H), 7.35 (dd, 1H), 6.70 (d, 1H), 6.20 (bs, 2H), 4.15

(AB, 2H), 4.10 (m, 1H), 3.90 (s, 3H), 3.12 (m, 2H), 1.98 (m,

1H), 1.55 (m, 1H); MS (FAB) m/z 468 (M + H)⁺. Anal.

(C₂₃H₂₅N₅O₄S‚1.5TFA‚0.5H₂O) C, H, N.

2H), 4.11-4.25 (m, 3H), 3.15 (m, 2H), 2.18 (m, 1H), 1.62 (m,

1H); MS (FAB) m/z 471 (M + H)⁺; HRMS (ESI) calcd for

C₂₁H₂₂N₆O₃S₂471.1273, found 471.1268 (M + H)⁺; HPLC

analysis (C18, 15-min linear gradient; elution 10-80% CH₃-

CN/0.1% TFA H₂O; flow rate 1.0 mL/min) indicated that the

product was 99A%.

4-Am in o-3-[3-(S)-(5-isoxa zol-3-ylt h iop h en e-2-ylsu lfo-

n yla m in o)-2-oxop yr r olid in -1-ylm eth yl]ben za m id in e Tr i-

flu or oa ceta te (4d ). ¹H NMR (DMSO-d₆) δ 8.80 (bs, 2H), 8.75

(d, 1H), 8.70 (d, 1H), 8.52 (bs, 2H), 7.75 (s, 2H), 7.52 (dd, 1H),

7.45 (d, 1H), 7.10 (d, 1H), 6.71 (d, 1H), 6.20 (bs, 1H), 4.28 (m,

1H), 4.21 (AB, 2H), 3.20 (m, 2H), 2.20 (m, 1H), 1.70 (m, 1H);

MS (ESI) m/z 461 (M + H)⁺. Anal. (C₁₉H₂₀N₆O₄S‚TFA‚1.07H₂O)

C, H, N.

P r ep a r a tion of 4-Am in o-3-[3-(S)-(7-m eth oxyn a p h th a -

len -2-ylsu lfon ylm eth yla m in o)-2-oxop yr r olid in -1-ylm eth -

yl]ben za m id in e Tr iflu or oa ceta te (4b). P a r t A: Sodium

hydride (0.35 g, 8.7 mmol, 60% mineral oil dispersion) was

added to a solution of {1-[2-(benzhydrylidenylamino)-5-cy-

anobenzyl]-2-oxopyrrolidin-3-yl}carbamic acid tert-butyl ester

(11) (3.94 g, 7.98 mmol) in DMF (8 mL) at 0 °C. After 20 min,

methyl iodide (0.99 mL, 15.9 mmol) was added. The reaction

mixture was stirred for 2 h, then treated with saturated NH₄-

Cl, and diluted with EtOAc. The organic layer was separated

and the aqueous layer washed with additional EtOAc. The

combined organic layers were washed with saturated NaCl and

worked up. The crude material was purified by column

chromatography eluting with a gradient of 30% EtOAc/hexanes

to 50% EtOAc/hexanes to give {1-[2-(benzhydrylidenylamino)-

5-cyanobenzyl]-2-oxopyrrolidin-3-yl}-N-methylcarbamic acid

tert-butyl ester (12) (3.72 g, 92%) as a yellow solid: ¹H NMR

(CDCl₃) δ 7.70 (bs, 2H), 7.45 (m, 8H), 7.10 (bs, 2H), 6.45 (dd,

1H), 4.70 (m, 1H), 4.49 (AB, 2H), 3.30 (m, 2H), 2.83 (s, 3H),

2.35 (m, 1H), 2.10 (m, 1H), 1.50 (s, 9H); MS (FAB) m/z 509 (M

+ H)⁺.

P a r t B: {1-[2-(Benzhydrylidenylamino)-5-cyanobenzyl]-2-

oxopyrrolidin-3-yl}-N-methylcarbamic acid tert-butyl ester (12)

was treated as described in example 4a , part E, to give

7-methoxynaphthalene-2-sulfonic acid [1-(2-amino-5-cyanoben-

zyl)-2-oxopyrrolidin-3-yl]methylamide (13) as a foamy yellow

solid: ¹H NMR (CDCl₃) δ 8.38 (d, 1H), 7.90 (d, 1H), 7.78 (d,

1H), 7.72 (dd, 1H), 7.32 (dd, 1H), 7.30 (dd, 1H), 7.28 (d, 1H),

7.23 (d, 1H), 6.55 (d, 1H), 4.98 (s, 2H), 4.95 (m, 1H), 4.25 (AB,

2H), 3.98 (s, 3H), 3.20 (m, 2H), 2.70 (s, 3H), 1.95 (m, 1H); MS

(FAB) m/z 465 (M + H)⁺.

P a r t C: 7-Methoxynaphthalene-2-sulfonic acid [1-(2-amino-

5-cyanobenzyl)-2-oxopyrrolidin-3-yl]methylamide (13) was

treated as described in example 4a , part F, to give 4-amino-

3-[3-(S)-(7-methoxynaphthalen-2-ylsulfonylmethylamino)-2-ox-

opyrrolidin-1-ylmethyl]benzamidine trifluoroacetate (4b) as a

white solid: ¹H NMR (DMSO-d₆) δ 8.90 (bs, 2H), 8.75 (bs, 2H),

8.40 (s, 1H), 8.050 (d, 1H), 7.95 (d, 1H), 7.70 (dd, 1H), 7.60 (d,

1H), 7.55 (dd, 1H), 7.48 (d, 1H), 7.39 (dd, 1H), 6.70 (d, 1H),

6.00 (bs, 1H), 4.98 (m, 1H), 4.20 (AB, 2H), 3.90 (s, 3H), 3.15

(m, 2H), 2.67 (s, 3H), 2.05 (m, 1H), 1.70 (m, 1H); MS (FAB)

m/z 482 (M + H)⁺. Anal. (C₂₃H₂₅N₅O₄S‚2.0TFA) C, H, N.

P r ep a r a tion of N-(4-Ca r ba m im id oyl-2-{3-[(7-m eth -

oxyn a p h th a len -2-ylsu lfon yl)m eth yla m in o]-2-oxop yr r o-

lid in -1-(S)-ylm eth yl}p h en yl)a ceta m id e Tr iflu r oa ceta te

(14). P a r t A: Triethylamine (0.25 mL, 1.81 mmol), N,N-

(dimethylamino)pyridine (0.01 g, 0.061 mmol) and acetyl

chloride (0.43 mL, 6.05 mmol), were added to a solution of

7-methoxynaphthalene-2-sulfonic acid [1-(2-amino-5-cyanoben-

zyl)-2-oxopyrrolidin-3-yl]methylamide (13) (0.28 g, 0.61 mmol)

in 1,2-dichloroethane (20 mL). The solution was heated to 60

°C for 16 h, then cooled to ambient temperature, and diluted

with EtOAc. The organic layer was washed with saturated

NaHCO₃and saturated NaCl and then worked up. The crude

residue was purified by column chromatography eluting with

20% EtOAc/CH₂Cl₂to give N-(4-cyano-2-{3-[(7-methoxynaph-

thalen-2-ylsulfonyl)methylamino]-2-oxopyrrolidin-1-ylmethyl}-

phenyl)acetamide (0.232 g, 76%) as a white solid: ¹H NMR

(CDCl₃) δ 9.50 (s, 1H), 8.50 (d, 1H), 8.30 (s, 1H), 7.89 (d, 1H),

7.80 (d, 1H), 7.76 (dd, 1H), 7.60 (d, 1H), 7.40 (d, 1H), 7.20 (m,

4-Am in o-3-[2-oxo-3-(S)-(5-p yr id in -3-ylt h iop h en e-2-yl-

su lfon yla m in o)p yr r olid in -1-ylm eth yl]ben za m id in e Tr i-

1

flu or oa ceta te (4c). H NMR (DMSO-d₆) δ 8.95 (d, 1H), 8.75

(s, 2H), 8.58 (d, 1H), 8.50 (d, 1H), 8.48 (s, 2H), 8.10 (d, 1H),

7.70 (s, 2H), 7.50 (m, 2H), 7.40 (d, 1H), 6.71 (d, 1H), 6.20 (bs,

3582 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Choi-Sledeski et al.

2H), 4.90 (m, 1H), 4.30 (AB, 2H), 3.90 (s, 3H), 3.30 (m, 2H),

2.75 (s, 3H), 2.35 (m, 1H), 2.05 (m, 1H), 1.90 (s, 3H); MS (FAB)

m/z 507 (M + H)⁺.

NMR (CDCl₃) δ 10.91 (s, 1H), 9.82 (s, 1H), 7.84 (d, 1H), 7.75

(dd, 1H), 6.75 (d, 1H); MS (EI) m/z 248 (M)⁺.

P a r t B: Sodium hydride (1.2 g, 52 mmol, 60% mineral oil

dispersion) was added to a solution of 2-hydroxy-5-iodoben-

zaldehyde (7.0 g, 28 mmol) in THF (25 mL) at 0 °C. After 5

min, 2-methoxyethoxymethoxy chloride (3.4 mL, 30 mmol) and

1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (4 mL)

were added to the reaction mixture. The solution was allowed

to warm to ambient temperature. After 45 min, the solution

was cooled to -15 °C and sodium borohydride (6 mL, 2 M

solution in THF) was added dropwise. The solution was stirred

for 10 min; then 2 M HCl (24 mL) was added. The resulting

solution was diluted with ether and washed with water and

saturated NaCl. The organic layer was worked up and the

crude material purified by column chromatography eluting

with 40% EtOAc/hexanes to give 5-iodo-2-(2-methoxyethoxy-

methoxy)benzyl alcohol (7.6 g, 80%) as a white solid: ¹H NMR

(CDCl₃) δ 7.64 (d, 1H), 7.52 (dd, 1H), 6.90 (d, 1H), 5.30 (s, 2H),

4.62 (d, 2H), 3.82 (m, 2H), 3.54 (m, 2H), 3.35 (s, 3H), 2.53 (t,

1H); MS (EI) m/z 338 (M)⁺.

P a r t C: Triphenylphosphine (30.6 g, 117 mmol) and N-

bromosuccinimide (20.8 g, 117 mmol) were added to a solution

of 5-iodo-2-(2-methoxyethoxymethoxy)benzyl alcohol (36.2 g,

107 mmol) in THF (290 mL) at 5 °C. The solution was stirred

for 10 min then allowed to warm to ambient temperature. After

20 min, the solution was concentrated in vacuo and the crude

material purified by column chromatography eluting with

EtOAc:CH₂Cl₂:hexane (3:1:6) to give 5-iodo-2-(2-methoxy-

ethoxymethoxy)benzyl bromide (16) (29.6 g, 69%) as an oil:

¹H NMR (CDCl₃) δ 7.60 (d, 1H), 7.50 (dd, 1H), 6.91 (s, 1H),

5.30 (s, 2H), 4.43 (dd, 1h), 3.84 (m, 2H), 3.53 (m, 2H), 3.35 (s,

3H); MS (EI) m/z 400 (M)⁺.

P a r t D: 3-(S)-(tert-Butoxycarbonylamino)-1-(5-iodo-2-(2-

methoxyethoxymethoxy)benzyl)pyrrolidin-2-one (17) was pre-

pared as described in example 4a , part D, substituting 5-iodo-

2-(2-methoxyethoxymethoxy)benzyl bromide (16) for 4-(benzhy-

drylidenylamino)-3-(bromomethyl)benzonitrile. The product

was purified by column chromatography eluting with EtOAc:

CH₂Cl₂:hexane (3:1:1): ¹H NMR (CDCl₃) δ 7.53 (dd, 1H), 7.45

(d, 1H), 6.93 (d, 1H),. 5.26 (s, 2H), 5.20 (bs, 1H), 4.50 (d, 1H),

4.45 (d, 1H), 4.19 (m, 1H), 3.80 (m, 2H), 3.53 (m, 2H), 3.37 (s,

3H), 3.22 (m, 2H), 2.62 (m, 1H), 1.84 (m, 1H), 1.46 (s, 9H); MS

(ESI) m/z 521 (M + H)⁺.

P a r t E: Zinc cyanide (16.6 g, 141 mmol) and tetrakis-

(triphenylphosphine)palladium(0) (3.93 g, 3.40 mmol) were

added to a solution of 3-(S)-(tert-butoxycarbonylamino)-1-(5-

iodo-2-(2-methoxyethoxymethoxy)benzyl)pyrrolidin-2-one (17)

(25.3 g, 48.6 mmol) in DMF (150 mL). The solution was heated

to 80 °C for 4 h, then cooled to room temperature, and diluted

with EtOAc. The resulting solution was washed with 1 N NH₄-

OH, water, and saturated NaCl. The organic layer was worked

up and the crude product purified by column chromatography

eluting with EtOAc:CH₂Cl₂:hexane (3:1:1) to give 3-(S)-(tert-

butoxycarbonylamino)-1-(5-cyano-2-(2-methoxyethoxymethoxy-

)benzyl)pyrrolidin-2-one (18.8 g, 92%) as a yellow oil: ¹H NMR

(CDCl₃) δ 7.54 (dd, 1H), 7.45 (d, 1H), 7.20 (d, 1H), 5.35 (s, 2H),

5.20 (bs, 1H), 4.50 (d, 1H),1.45 (s, 9H), 4.45 (d, 1H), 4.15 (m,

1H), 3.77 (m, 2H), 3.51 (m, 2H), 3.35 (s, 3H), 3.24 (m, 2H),

2.60 (m, 1H), 1.90 (m, 1H); MS (EI) m/z 420 (M)⁺.

P a r t B: N-(4-Cyano-2-{3-[(7-methoxynaphthalen-2-ylsul-

fon yl)m et h yla m in o]-2-oxopyr r olidin -1-ylm et h yl}ph en yl)-

acetamide was treated as described in example 4a , part F, to

yield the title compound 14: ¹H NMR (DMSO-d₆) δ 9.70 (s,

1H), 9.23 (bs, 2H), 9.00 (bs, 1H), 8.40 (s, 1H), 8.00 (d, 1H),

7.98 (d, 1H), 7.70 (m, 2H), 7.60 (m, 2H), 7.35 (dd, 1H), 4.97

(m, 1H), 4.40 (AB, 2H), 3.90 (s, 3H), 3.20 (m, 2H), 2.68 (s, 3H),

2.10 (m, 1H), 2.00 (s, 3H), 1.80 (m, 1H); MS (FAB) m/z 524 (M

+ H)⁺. Anal. (C₂₆H₂₉N₅O₅S‚TFA‚1.5H₂O) C, H, N.

P r ep a r a tion of 3-Am in o-5-[3-(S)-(7-m eth oxyn a p h th a -

len -2-ylsu lfon yla m in o)-2-oxop yr r olid in -1-ylm eth yl]ben -

za m id in e Bistr iflu or oa ceta te (15). P a r t A: 3-Methyl-5-

nitrobenzonitrile³²was treated in a similar manner as example

4a , part A, to give 3-amino-5-methylbenzonitrile: ¹H NMR

(CDCl₃) δ 6.83 (s, 1H), 6.70 (s, 1H), 6.68 (s, 1H), 3.70 (bs, 2H),

2.30 (s, 3H); MS (EI) m/z 132 (M⁺).

P a r t B: 3-Amino-5-methylbenzonitrile was treated as

described in example 4a , part B, to give 3-(benzhydrylidenyl-

amino)-5-methylbenzonitrile: ¹H NMR (CDCl₃) δ 7.73 (d, 2H),

7.45 (m, 2H), 7.30 (m, 4H), 7.05 (dd, 2H), 7.00 (s, 1H), 6.78 (s,

2H), 6.71 (s, 1H), 2.20 (s, 3H); MS (EI) m/z 296 (M)⁺.

P a r t C: 3-(Benzhydrylidenylamino)-5-methylbenzonitrile

was treated in the same manner as described in example 4a ,

part C, to yield 3-(benzhydrylidenylamino)-5-(bromomethyl)-

benzonitrile: ¹H NMR (CDCl₃) δ 7.75 (d, 2H), 7.50 (m, 1H),

7.40 (m, 2H), 7.30 (m, 4H), 7.05 (m, 2H), 6.95 (s, 1H), 6.89 (s,

1H), 4.30 (s, 2H); MS (EI) m/z 374 (M)⁺.

P a r t D: 3-(Benzhydrylidenylamino)-5-(bromomethyl)ben-

zonitrile was treated in the same manner as described in

example 4a , part D, to give {1-[3-(benzhydrylidenylamino)-

5-cyanobenzyl]-2-oxopyrrolidin-3-yl}carbamic acid tert-bu-

tyl ester: ¹H NMR (CDCl₃) δ 7.75 (d, 2H), 7.50 (m, 1H),

7.40 (m, 2H), 7.30 (m, 4H), 7.10 (m, 1H), 6.95 (s, 1H), 6.65

(s, 1H), 5.10 (bs, 1H), 4.30 (AB, 2H), 4.05 (m, 1H), 3.85 (m,

2H), 2.55 (m, 1H), 1.75 (m, 1H), 1.40 (s, 9H); MS (EI) m/z

495 (M)⁺.

P a r t E: {1-[3-(Benzhydrylidenylamino)-5-cyanobenzyl]-2-

oxopyrrolidin-3-yl}carbamic acid tert-butyl ester was treated

in the same manner as described in example 4a , part E, to

give 7-methoxynaphthalene-2-sulfonic acid [1-(3-benzhydrylide-

nylamino-5-cyanobenzyl)-2-oxopyrrolidin-3-yl]amide: ¹H NMR

(CDCl₃) δ 8.35 (s, 1H), 7.90 (d, 1H), 7.80 (d, 1H), 7.75 (dd, 1H),

7.70 (d, 2H), 7.50 (m, 1H), 7.40 (m, 2H), 7.25 (m, 5H), 7.00 (m,

4H), 6.55 (s, 1H), 5.25 (s, 1H), 4.25 (AB, 2H), 3.95 (s, 3H), 3.65

(m, 1H), 2.80 (m, 2H), 2.45 (m, 1H), 1.95 (m, 1H); MS (FAB)

m/z 615 (M + H)⁺.

P a r t F : 7-Methoxynaphthalene-2-sulfonic acid [1-(3-ben-

zhydrylidenylamino-5-cyanobenzyl)-2-oxopyrrolidin-3-yl]-

amide was treated in the same manner as described in

example 4a , part F, to give 3-amino-5-[3-(S)-(7-methoxynaph-

thalen-2-ylsulfonylamino)-2-oxopyrrolidin-1-ylmethyl]benzami-

dine bistrifluoroacetate (15): ¹H NMR (DMSO-d₆) δ 9.15 (s,

1H), 9.00 (bs, 2H), 8.35 (s, 1H), 8.20 (d, 1H), 8.05 (d, 1H), 7.95

(d, 1H), 7.70 (dd, 1H), 7.60 (d, 1H), 7.20 (dd, 1H), 6.70 (s, 1H),

6.65 (s, 1H), 6.60 (s, 1H), 5.80 (bs, 2H), 4.20 (AB, 2H), 4.10

(m, 1H), 3.90 (s, 3H), 3.00 (m, 2H), 2.00 (m, 1H), 1.50 (m, 1H);

MS (FAB) m/z 468 (M + H)⁺. Anal. (C₂₆H₂₉N₅O₅S‚2.5TFA‚

1.25H₂O) C, H, N.

P r ep a r a tion of 4-Hyd r oxy-3-[2-oxo-3-(S)-(5-p yr id in -3-

ylth iop h en e-2-ylsu lfon yla m in o)p yr r olid in -1-ylm eth yl]-

ben zam idin e Tr iflu or oacetate (20b). Repr esen tative P r o-

ced u r e for th e Syn th esis of Com p ou n d s 20a a n d 20c.

P a r t A: Iodine monochloride (250 mL, 1 M solution in CH₂-

Cl₂) was added to a solution of salicylaldehyde (30.6 g, 251

mmol) in CH₂Cl₂(150 mL). The reaction mixture was stirred

for 14 h; then saturated sodium sulfite (500 mL) was added

until the color disappeared. The mixture was diluted with CH₂-

Cl₂, and the layers were separated. The organic layer was

washed with water and saturated NaCl and then worked up.

The crude material was recrystallized from cyclohexane to give

2-hydroxy-5-iodobenzaldehyde (31.3 g, 50%) as white solid: ¹H

P a r t F : Hydrogen chloride gas was bubbled through EtOAc

(340 mL) at 0 °C for 10 min; then a solution of 3-(S)-(tert-

butoxycarbonylamino)-1-(5-cyano-2-(2-methoxyethoxymethoxy-

)benzyl)pyrrolidin-2-one (18.8 g, 44.9 mmol) in EtOAc (160 mL)

was added dropwise via cannula. The mixture was stirred for

30 min during which time a precipitate formed. The reaction

mixture was diluted with diethyl ether and then quickly

filtered to yield 3-(S)-[(3-amino-2-oxopyrrolidin-1-yl)methyl]-

4-hydroxybenzonitrile hydrochloride (18) (10.6 g, 88%) as a

white hygroscopic solid: ¹H NMR (DMSO-d₆) δ 8.50 (bs, 3H),

7.50 (dd, 1H), 7.40 (d, 1H), 7.00 (d, 1H), 4.26 (d, 2H), 4.00 (m,

1H), 3.25 (m, 2H), 2.30 (m, 1H), 1.90 (m, 1H); MS (EI) m/z

231 (M)⁺.

P a r t G: 5-Pyridin-3-ylthiophene-2-sulfonyl chloride (3.38

g, 13.0 mmol) and 3-(S)-[(3-amino-2-oxopyrrolidin-1-yl)methyl]-

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3583

4-hydroxybenzonitrile hydrochloride (18) (3.47 g, 13.0 mmol)

were stirred in pyridine (70 mL) at ambient temperature

overnight. The reaction mixture was concentrated in vacuo and

then purified by chromatography eluting with 10% MeOH/CH₂-

Cl₂to afford 5-pyridin-3-ylthiophene-2-sulfonic acid {1-[5-

cyano-2-hydroxybenzyl]-2-oxopyrrolidin-3-(S)-yl}amide (3.6 g,

61%) as a foamy white solid: ¹H NMR (CDCl₃) δ 8.82 (bs, 1H),

8.61 (bs, 1H), 7.85 (d, 1H), 7.65 (d, 1H), 7.63 (d, 1H), 7.55 (s,

1H), 7.53 (dd, 1H), 7.41 (d, 1H), 7.31 (m, 1H), 7.28 (d, 1H),

6.91 (d, 1H), 4.41 (s, 2H), 4.12 (m, 1H), 3.35 (m, 2H), 2.55 (m,

1H), 2.09 (m, 1H); MS (FAB) m/z 455 (M + H)⁺.

(CDCl₃) δ 7.62 (s, 1H), 7.14 (dd, 1H), 7.05 (d, 1H), 5.31 (s, 2H),

5.13 (s, 1H), 4.35 (AB, 2H), 4.18 (m, 1H), 3.85 (t, 2H), 3.55 (t,

2H), 3.35 (s, 3H), 3.18 (m, 2H), 2.60 (m, 1H), 1.85 (m, 1H),

1.45 (s, 9H); MS (EI) m/z 520 (M)⁺.

P a r t E : 3-(S)-(tert-Butoxycarbonylamino)-1-(3-iodo-4-(2-

methoxyethoxymethoxy)benzyl)pyrrolidin-2-one was treated in

the same manner as described in example 20b, part E, to give

3-(S)-(tert-butoxycarbonylamino)-1-(3-cyano-4-(2-methoxy-

ethoxymethoxy)benzyl)pyrrolidin-2-one: ¹H NMR (CDCl₃) δ

7.42 (d, 1H), 7.40 (dd, 1 H), 7.23 (s, 1H), 5.38 (s, 2H), 5.13 (,

1H), 4.40 (AB, 2H), 4.17 (m, 1 H), 3.88 (t, 2H), 3.55 (t, 2H),

3.38 (s, 3H), 3.20 (m, 2H), 2.60 (m, 1H), 1.91 (m, 1H) 1.45 (s,

9H); MS (FAB) m/z 420 (M + H)⁺.

P a r t F : 3-(S)-(tert-Butoxycarbonylamino)-1-(3-cyano-4-(2-

methoxy)ethoxymethoxy)benzyl)pyrrolidin-2-one was treated

in the same manner as described in example 20b, part F, to

give 5-[(3-(S)-amino-2-oxopyrrolidin-1-yl)methyl]-2-hydroxy-

benzonitrile hydrochloride: ¹H NMR (CD₃OD) δ 7.45 (d, 1H),

7.40 (dd, 1H), 4.40 (AB, 2H), 4.10 (m, 1 H), 3.35 (m, 2H), 2.55

(m, 1H), 1.95 (m, 1H).

P a r t G: 5-[(3-(S)-Amino-2-oxopyrrolidin-1-yl)methyl]-2-hy-

droxybenzonitrile hydrochloride was treated in the same

manner as described in example 20b, part G, to give 7-meth-

oxynaphthalene-2-sulfonic acid [1-(3-cyano-4-hydroxybenzyl)-

2-oxopyrrolidin-3-(S)-yl]amide: ¹H NMR (CD₃OD) δ 8.40 (s,

1H), 7.93 (d, 1H), 7.85 (d, 1H), 7.73 (dd, 1H), 7.40 (dd, 2H),

7.30 (m, 2H), 6.90 (d, 1H), 4.30 (AB, 2H), 4.10 (m, 1H), 3.91

(s, 3H), 3.10 (m, 2H), 2.15 (m, 1H), 1.65 (m, 1H); MS (FAB)

m/z 452 (M + H)⁺.

7-Meth oxyn a p h th a len e-2-su lfon ic Acid {1-[5-Cya n o-2-

h ydr oxyben zyl]-2-oxopyr r olidin -3-(S)-yl}am ide. MS (FAB)

m/z 452 (M + H)⁺.

5-Isoxa zol-3-ylth iop h en e-2-su lfon ic Acid [1-(5-Cya n o-

2-h ydr oxyben zyl)-2-oxopyr r olidin -3-(S)-yl]am ide. ¹H NMR

(CDCl₃) δ 9.37 (bs, 1H), 8.35 (d, 2H), 7.50 (m, 2H), 7.41 (d,

1H), 7.00 (d, 1H), 6.57 (d, 1H), 5.48 (bs, 1H), 4.35 (AB, 2H),

4.10 (m, 1H), 3.50 (m, 2H), 2.70 (m, 1H), 2.20 (m, 1H).

P a r t H: 5-Pyridin-3-ylthiophene-2-sulfonic acid {1-[5-cyano-

2-hydroxybenzyl]-2-oxopyrrolidin-3-(S)-yl}amide was treated

in the same manner as described in example 4a , part F, to

afford 4-hydroxy-3-[2-oxo-3-(S)-(5-pyridin-3-ylthiophene-2-yl-

sulfonylamino)pyrrolidin-1-ylmethyl]benzamidine trifluoro-

acetate (20b) as an amorphous white solid: ¹H NMR (DMSO-

d₆) δ 10.91 (s, 1H), 9.06 (s, 2H), 8.98 (s, 1H), 8.67 (s, 2H),

8.61 (d, 1H), 8.55 (d, 1H), 8.16 (d, 1H), 7.71 (m, 2H), 7.60 (d,

1H), 7.51 (m, 1H), 7.42 (s, 1H), 7.00 (d, 1H), 4.35 (AB, 2H),

4.21 (m, 1H), 3.21 (m, 2H), 2.20 (m, 1H), 1.71 (m, 1H); MS

(FAB) m/z 472 (M + H)⁺. Anal. (C₂₁H₂₁N₅O₄S‚2TFA‚1.4H₂O)

C, H, N.

4-H yd r oxy-3-[3-(S)-(7-m et h oxyn a p h t h a len -2-ylsu lfo-

n ylam in o)-2-oxopyr r olidin -1-ylm eth yl]ben zam idin e (20a).

7-Methoxynaphthalene-2-sulfonic acid {1-[5-cyano-2-hydroxy-

benzyl]-2-oxopyrrolidin-3-(S)-yl}amide was treated in the same

manner as described in example 4a , part F, except the crude

material was purified by chromatography eluting with 1%

Et₃N/15% MeOH in CH₂Cl₂to afford the title product as an

amorphous solid: ¹H NMR (DMSO-d₆) δ 8.36 (bs, 1H), 8.0 (d,

1H), 7.93 (d, 1H), 7.72 (m, 1H), 7.53 (bs, 1H), 7.40 (m, 1H),

7.32 (m, 1H), 7.19 (bs, 1H), 6.22 (d, 1H), 4.20 (d, 1H), 4.12 (d,

1H), 4.10 (t, 1H), 3.90 (s, 3H), 3.12 (m, 2H), 1.94 (m, 1H), 1.55

(m, 1H); MS (FAB) m/z 469 (M + H)⁺. Anal. (C₂₃H₂₄N₄O₅S‚

0.75H₂O) C, H, N.

4-Hyd r oxy-3-[3-(S)-(5-isoxa zol-3-ylth iop h en e-2-ylsu lfo-

n yla m in o)-2-oxop yr r olid in -1-ylm eth yl]ben za m id in e Tr i-

flu or oa ceta te (20c). ¹H NMR (DMSO-d₆) δ 9.04 (bs, 2H), 8.87

(bs, 2H), 8.73 (d, 1H), 8.67 (d, 1H), 7.74 (m, 2H), 7.61 (dd, 1H),

7.42 (d, 1H), 7.09 (d, 1H), 6.97 (d, 1H), 4.33 (AB, 2H), 4.23 (m,

1H), 3.21 (m, 2H), 2.21 (m, 1H), 1.75 (m, 1H); MS (ESI) m/z

462 (M + H)⁺. Anal. (C₁₉H₁₉N₅O₅S₂‚TFA‚0.875H₂O) C, H, N.

P r ep a r a tion of 2-Hyd r oxy-5-[3-(S)-(7-m eth oxyn a p h -

t h a len -2-ylsu lfon yla m in o)-2-oxop yr r olid in -1-ylm et h yl-

]ben za m id in e (21). P a r t A: p-Hydroxybenzaldehyde was

treated in the same manner as described in example 20b, part

A, to give 4-hydroxy-3-iodobenzaldehyde: ¹H NMR (CDCl₃) δ

9.82 (s, 1H), 8.24 (s, 1H), 7.80 (d, 1H), 7.12 (d, 1H), 5.95 (s,

1H); MS (EI) m/z 248 (M)⁺.

P a r t B: 4-Hydroxy-3-iodobenzaldehyde was treated in the

same manner as described in example 20b, part B, to give

3-iodo-4-(2-methoxyethoxymethoxy)benzyl alcohol: ¹H NMR

(CDCl₃) δ 7.78 (s, 1H),7.25 (dd, 1H), 7.10 (d, 1H), 5.31 (s, 2H),

4.59 (d, 2H), 3.88 (t, 2H), 3.55 (t, 2H), 3.36 (s, 3H); MS (EI)

m/z 338 (M)⁺.

P a r t H: 7-Methoxynaphthalene-2-sulfonic acid [1-(3-cyano-

4-hydroxybenzyl)-2-oxopyrrolidin-3-(S)-yl]amide was treated in

the same manner as described in example 20b, part H, to give

2-hydroxy-5-[3-(7-methoxynaphthalen-1-ylsulfonylamino)-2-oxo-

pyrrolidin-1-yl]benzamidine, trifluoroacetic acid (21): ¹H NMR

(CD₃OD) δ 8.4 (s, 1H), 7.95 (d, 1H), 7.88 (d, 1H), 7.75 (dd, 1H),

7.45 (d, 1H), 7.40 (m, 1H), 7.30 (dd, 1H), 6.98 (d, 1H), 4.35 (d,

2H), 4.15 (t, 1H), 3.95 (s, 3H), 3.20 (m, 2H), 2.20 (m, 1H), 1.70

(m, 1H); MS (FAB) m/z 469 (M + H)⁺; HRMS (ESI) calcd for

C

₂₃H₂₄N₄O₅S 469.1545, found 469.1531 (M + H)⁺; HPLC

analysis (C18, 20-min linear gradient; elution 10-100% CH₃-

CN/0.1% TFA H₂O; flow rate 1.0 mL/min) indicated that the

product was 99A%.

P r ep a r a t ion of {4-(Am in oim in om et h yl)-2-[3-(S)-(7-

m eth oxyn aph th alen -2-ylsu lfon ylam in o)-2-oxopyr r olidin -

1-ylm eth yl]p h en oxy}a cetic Acid Meth yl Ester Tr iflu o-

r oa ceta te (27). P a r t A: tert-Butyllithium (31.1 mL, 52.9

mmol, 1.7 M solution in pentane) was added dropwise to a

solution of 4-bromo-3-methylbenzonitrile (5.18 g, 26.4 mmol)

in THF (165 mL) at -78 °C. After 5 min, CuBr‚SMe₂(11.42 g,

55.5 mmol) was added. The resulting solution was stirred for

10 min; then O₂was slowly and ca r efu lly bubbled through

the reaction mixture for 30 min. After this time, the solution

was allowed to warm to ambient temperature and the solution

was stirred for 16 h. The reaction mixture was then poured

into cold H₂O (75 mL) and diluted with EtOAc. The organic

layer was washed with saturated NH₄SO₄(3 × 75 mL) and

then extracted with 10 N NaOH (2 × 30 mL). The basic

aqueous layers were acidified to pH 6 with 6 N HCl and then

extracted with EtOAc. The combined organic layers were

worked up to yield 4-hydroxy-3-methylbenzonitrile (22) (1.27

g, 36%) as a white solid which was used in the subsequent

step without further purification: ¹H NMR (CDCl₃) δ 9.00 (s,

1H), 7.45 (s, 1H), 7.40 (d, 1H), 6.80 (d, 1H), 2.26 (s, 3H); MS

(EI) m/z 133 (M)⁺.

P a r t C: 3-Iodo-4-(2-methoxyethoxymethoxy)benzyl alcohol

was treated in the same manner as described in example 20b,

part C, to give 3-iodo-4-(2-methoxyethoxymethoxy)benzyl bro-

mide: ¹H NMR (CDCl₃) δ 7.80 (s, 1H), 7.30 (d, 1H), 7.05 (d,

1H), 5.32 (s, 2H), 4.40 (s, 2H), 3.85 (t, 2H), 3.55 (t, 2H), 3.37

(s, 3H); MS (EI) m/z 400 (M)⁺.

P a r t D: 3-Iodo-4-(2-methoxyethoxymethoxy)benzyl bromide

was treated in the same manner as described in example 20b,

part D, to give 3-(S)-(tert-butoxycarbonylamino)-1-(3-iodo-4-

(2-methoxyethoxymethoxy)benzyl)pyrrolidin-2-one: ¹H NMR

P a r t B: Methyl bromoacetate (0.56 mL, 5.92 mmol) was

added to a solution of 4-hydroxy-3-methylbenzonitrile (22)

(0.70 g, 5.26 mmol), K₂CO₃(1.61 g, 11.6 mmol), and tetrabu-

tylammonium iodide (0.57 g, 1.53 mmol) in DMF (30 mL). The

resulting mixture was heated to 80 °C for 16 h and then cooled

to ambient temperature. The reaction mixture was diluted

with EtOAc and washed with H₂O and saturated NaCl. The

organic layer was worked up and the crude material purified

by column chromatography eluting with a gradient of 10%

EtOAc/hexanes to 50% EtOAc/hexanes to afford (4-cyano-2-

3584 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Choi-Sledeski et al.

methylphenoxy)acetic acid methyl ester (23) (0.82 g, 76%): ¹H

NMR (CDCl₃) δ 7.40-7.48 (m, 2H), 6.70 (d, 1H), 4.68 (s, 2H),

3.80 (s, 3H), 2.25 (s, 3H); MS (EI) m/z 205 (M)⁺.

1.95 (m, 1H), 1.55 (m, 1H); MS (FAB) m/z 527 (M + H)⁺. Anal.

(C₂₅H₂₆N₄O₇S‚2.0TFA) C, H, N.

P r ep a r a tion of Bip h en yl-4-su lfon ic Acid {1-[3-(Am i-

n oim in om eth yl)ben zyl]-2-oxop yr r olid in -3-(S)-yl}a m id e

Tr iflu or oa ceta te (3c). Rep r esen ta tive P r oced u r e for th e

Syn t h esis of Com p ou n d s 3d -3f, 3i-3l, 3n . P a r t A: Bi-

phenyl-4-sulfonyl chloride (0.28 g, 1.09 mmol) was added to a

solution of triethylamine (0.42 mL, 3.0 mml) and 3-(3-(S)-

amino-2-oxopyrrolidin-1-ylmethyl)benzonitrile hydrochloride⁹

(0.25 g, 1.0 mmol) in CH₃CN (10 mL). After 4 h, the reac-

tion mixture was concentrated in vacuo, diluted with EtOAc,

and then washed with saturated NaHCO₃and saturated NaCl.

The organic layer was worked up and purified by chromatog-

raphy eluting with 50% EtOAc/CH₂Cl₂to give biphenyl-4-

sulfonic acid [1-(3-cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide

(0.30 g, 70%): ¹H NMR (CDCl₃) δ 8.14 (s, 1H), 7.95 (d, 1H),

7.82 (d, 1H), 7.64 (m, 5H), 7.47 (m, 6H), 5.42 (bs, 1H), 4.42

(AB, 2H), 3.82 (m, 1H), 3.22 (m, 1H), 2.62 (m, 1H), 2.13 (m,

1H).

P a r t C: (4-Cyano-2-methylphenoxy)acetic acid methyl ester

(23) was treated in the same manner as described in example

4a , part C, to give (2-bromomethyl-4-cyanophenoxy)acetic acid

methyl ester (24) as a white solid: ¹H NMR (CDCl₃) δ 7.65 (d,

1H), 7.55 (dd, 1H), 6.80 (d, 1H), 4.80 (s, 2H), 4.55 (s, 2H), 3.80

(s, 3H); MS (EI) m/z 283, 285 (M)⁺.

P a r t D: (2-Bromomethyl-4-cyanophenoxy)acetic acid meth-

yl ester (24) was treated in the same manner as described in

example 4a , part D, to give [2-(3-tert-butoxycarbonylamino-

2-oxopyrrolidin-1-ylmethyl)-4-cyanophenoxy]acetic acid methyl

ester (25) as a white solid: ¹H NMR (CDCl₃) δ 7.50-7.58 (m,

2H), 6.78 (d, 1H), 5.10 (bs, 1H), 4.70 (s, 2H), 4.55 (AB, 2H),

4.15 (m, 1H), 3.80 (s, 3H), 3.20 (m, 2H), 2.60 (s, 2H), 1.90 (m,

1H), 1.58 (s, 9H).

P a r t E: [2-(3-tert-Butoxycarbonylamino-2-oxopyrrolidin-1-

ylmethyl)-4-cyanophenoxy]acetic acid methyl ester (25) was

treated in the same manner as described in example 4a , part

E, to give {4-cyano-2-[3-(7-methoxynaphthalen-2-ylsulfonyl-

amino)-2-oxopyrrolidin-1-ylmethyl]phenoxy}acetic acid methyl

ester (26) as a foamy white solid: ¹H NMR (CDCl₃) δ 8.35 (s,

1H), 7.90 (d, 1H), 7.75 (dd, 1H), 7.55 (dd, 1H), 7.42 (d, 1H),

7.30 (dd, 1H), 7.20 (m, 1H), 6.70 (d, 1H), 5.40 (d, 1H), 4.65 (s,

2H), 3.95 (s, 3H), 3.70 (m, 1H), 3.20 (m, 2H), 2.50 (m, 1H),

2.05 (m, 1H); MS (FAB) m/z 524 (M + H)⁺.

4-P yr id in -2-ylben zen esu lfon ic Acid [1-(3-Cya n oben -

zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR (CD₃OD) δ

8.73 (bs, 2H), 8.16 (d, 2H), 8.02 (d, 1H), 7.80 (m, 2H), 7.57 (d,

1H), 7.46 (m, 3H), 7.32 (m, 1H), 7.26 (s, 2H), 5.42 (bs, 2H),

4.55 (s, 1H), 4.23 (t, 1H), 3.20 (m, 2H), 2.33 (m, 1H), 1.87 (m,

1H); MS (FAB) m/z 433 (M + H)⁺.

4-P yr id in -3-ylben zen esu lfon ic Acid [1-(3-Cya n oben -

zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR (CD₃OD) δ

8.85 (bs, 2H), 8.57 (bs, 2H), 8.16 (d, 1H), 7.94 (AB, 4H), 7.46-

7.65 (m, 5H), 4.44 (AB, 2H), 4.23 (t, 1H), 3.20 (m, 2H), 2.33

(m, 1H), 1.87 (m, 1H); MS (FAB) m/z 433 (M + H)⁺.

4-P yr id in -4-ylben zen esu lfon ic Acid [1-(3-Cya n oben -

zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR (CDCl₃) δ

8.78 (m, 2H), 8.11 (d, 2H), 7.66 (m, 2H), 7.47-7.58 (m, 5H),

5.48 (s, 1H), 4.50 (AB, 2H), 3.88 (t, 1H), 3.29 (dd, 2H), 2.58

(m, 1H), 2.17 (m, 1H); MS (FAB) m/z 433 (M + H)⁺.

(1-Met h yl-1H -im id a zol-2-yl)b en zen e-4-su lfon ic Acid

[1-(3-Cya n ob en zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H

NMR (CDCl₃) δ 7.60 (m, 3H), 7.45 (m, 5H), 7.15 (s, 1H), 6.98

(s, 1H), 4.48 (AB, 2H), 3.95 (s, 3H), 3.75 (m, 1H), 3.20 (m, 2H),

2.60 (m, 1H), 2.00 (m, 1H); MS (FAB) m/z 436 (M + H)⁺.

5-P yr id -2-ylth iop h en e-2-su lfon ic Acid [1-(3-Cya n oben -

zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR (CDCl₃) δ

8.62 (m, 1H), 7.78 (m, 1H), 7.69 (m, 1H), 7.58 (m, 2H), 7.50 (d,

1H), 7.46 (m, 2H), 7.20 (m, 2H), 5.43 (bs, 1H), 4.42 (AB, 2H),

3.98 (m, 1H), 3.26 (m, 2H), 2.68 (m, 1H), 2.15 (m, 1H); MS

(FAB) m/z 439 (M + H)⁺.

P a r t F : Hydrogen sulfide gas was bubbled for 5 min

through a solution of {4-cyano-2-[3-(7-methoxynaphthalen-2-

ylsulfonylamino)-2-oxopyrrolidin-1-ylmethyl]phenoxy}acetic acid

methyl ester (26) (0.12 g, 0.23 mmol) in pyridine/TEA (5.4 mL,

10:1 v/v) at room temperature. After stirring the pale green

solution for a period of 18 h, the reaction mixture was

concentrated in vacuo. The residue was diluted with EtOAc

and washed with 0.5 N HCl and saturated NaCl. The organic

layer was worked up to give the crude thioamide. Methyl iodide

(0.56 mL, 9.00 mmol) was added to a solution of the thioamide

in acetone (9 mL) and then heated to reflux for 2 h. The

reaction mixture was cooled to room temperature and concen-

trated in vacuo to provide the crude thioimidate hydroiodide.

The thioimidate hydroiodide was dissolved in MeOH (6 mL),

and NH₄OAc (0.058 g, 0.75 mmol) was added. The resulting

mixture was heated at reflux for 4 h, allowed to cool to room

temperature, and stirred overnight. The reaction mixture was

concentrated in vacuo and the crude product purified by

reverse-phase HPLC. Lyophilization of the aqueous fractions

containing the desired product yielded {4-(aminoiminomethyl)-

2-[3-(S)-(7-methoxynaphthalen-2-ylsulfonylamino)-2-oxopyrro-

lidin-1-ylmethyl]phenoxy}acetic acid methyl ester trifluoroac-

etate (27) (0.030 g, 21%) as a white solid: ¹H NMR (DMSO-

d₆) δ 9.00 (bs, 4H), 8.30 (s, 1H), 7.97 (d, 1H), 7.90 (d, 1H), 7.65

(m, 2H), 7.50 (s, 1H), 7.37 (s, 1H), 7.25 (dd, 1H), 7.10 (d, 1H),

4.95 (AB, 2H), 4.30 (AB, 2H), 4.05 (m, 1H), 3.80 (s, 3H), 3.60

(s, 3H), 3.15 (m, 2H), 1.95 (m, 1H), 1.55 (m, 1H); MS (FAB)

m/z 541 (M + H)⁺. Anal. (C₂₆H₂₈N₄O₇S‚TFA‚3.40H₂O) C, H,

N.

5-P yr idin -3-ylth ioph en e-2-su lfon ic Acid [1-(3-Cyan oben -

zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR (CDCl₃) δ

8.90 (d, 1H), 8.63 (d, 1H), 7.85 (dd, 1H), 7.68 (d, 1H), 7.61 (m,

1H), 7.41-7.45 (m, 3H), 7.34 (dd, 1H), 7.31 (d, 1H), 5.45 (s,

1H), 4.50 (s, 2H), 3.95 (m, 1H), 3.25 (m, 2H), 2.68 (m, 1H),

2.15 (m, 1H); MS (FAB) m/z 439 (M + H)⁺.

5-P yr idin -4-ylth ioph en e-2-su lfon ic Acid [1-(3-Cyan oben -

zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. ¹H NMR (CDCl₃) δ

8.70 (d, 2H), 7.68 (d, 1H), 7.60 (m, 1H), 7.40-7.50 (m, 6H),

5.65 (bs, 1H), 4.48 (AB, 2H), 3.98 (m, 1H), 3.28 (m, 2H), 2.68

(m, 1H), 2.17 (m, 1H); MS (FAB) m/z 439 (M + H)⁺.

5-Isoxa zol-3-ylth iop h en e-2-su lfon ic Acid [1-(3-Cya n o-

P r ep a r a t ion of {4-(Am in oim in om et h yl)-2-[3-(S)-(7-

m eth oxyn aph th alen -2-ylsu lfon ylam in o)-2-oxopyr r olidin -

1-ylm eth yl]p h en oxy}a cetic Acid Tr iflu or oa ceta te (28).

10 N NaOH (0.05 mL) was added to a solution of {4-

(aminoiminomethyl)-2-[3-(S)-(7-methoxynaphthalen-2-ylsulfo-

nylamino)-2-oxopyrrolidin-1-ylmethyl]phenoxy}acetic acid meth-

yl ester trifluoroacetate (27) (0.1 g, 0.18 mmol) in EtOH (2

mL). The reaction mixture was stirred for 5 h and then

concentrated in vacuo. The residue was dissolved in H₂O (2

mL), and the pH was adjusted to 3 using 1 N HCl. The solid

which formed was collected by filtration and purified by

reverse-phase HPLC. The appropriate fractions were lyophi-

lized to afford the title compound 28 (0.05 g, 44%) as a white

solid: ¹H NMR (DMSO-d₆) δ 9.10 (bs, 2H), 8.70 (bs, 2H), 8.35

(s, 1H), 8.15 (d, 1H), 8.00 (d, 1H), 7.90 (d, 1H), 7.70 (m, 2H),

7.50 (s, 1H), 7.45 (s, 1H), 7.30 (m, 1H), 7.10 (m, 1H), 4.85 (s,

1H), 4.30 (AB, 2H), 4.05 (m, 1H), 3.80 (s, 3H), 3.10 (m, 2H),

1

ben zyl)-2-oxop yr r olid in -3-(S)-yl]a m id e. H NMR (CDCl₃)

δ 8.36 (d, 1H), 7.70 (d, 1H), 7.61 (m, 1H), 7.49 (m, 4H), 6.57

(d, 1H), 5.55 (bs, 1H), 4.51 (s, 2H), 3.97 (m, 1H), 3.29 (m, 2H),

2.69 (m, 1H), 2.17 (m, 1H).

P a r t B: Biphenyl-4-sulfonic acid [1-(3-cyanobenzyl)-2-oxo-

pyrrolidin-3-(S)-yl]amide was treated in the same manner as

described in example 4a , part F, to give biphenyl-4-sulfonic

acid {1-[3-(aminoiminomethyl)benzyl]-2-oxopyrrolidin-3-(S)-

yl}amide trifluoroacetate 3c as a white solid: ¹H NMR (DMSO-

d₆) δ 9.31 (bs, 2H), 9.14 (bs, 2H), 8.22 (d, 1H), 7.91 (m, 6H),

7.60 (m, 8H), 4.45 (AB, 2H), 4.16 (m, 1H), 3.12 (m, 1H), 2.07

(m, 1H), 1.65 (m, 1H); MS (FAB) m/z 449 (M + H)⁺. Anal.

(C₂₄H₂₄N₄O₃S‚TFA‚0.25H₂O) C, H, N.

4-P yr id in -2-ylben zen esu lfon ic Acid {1-[3-(Am in oim i-

n om eth yl)ben zyl]-2-oxop yr r olid in -3-(S)-yl}a m id e Bistr i-

flu or oa ceta te (3d ). ¹H NMR (CD₃OD) δ 8.72 (d, 2H), 8.15

Sulfonamidopyrrolidinone fXa Inhibitors

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3585

(d, 2H), 7.97-8.06 (m, 4H), 7.55-7.83 (m, 4H), 7.50 (m, 1H),

4.54 (AB, 2H), 4.21 (t, 1H), 3.28 (m, 2H), 2.30 (m, 1H), 1.80

(m, 1H); MS (FAB) m/z 450 (M + H)⁺. Anal. (C₂₃H₂₃N₅O₃S‚

2TFA‚H₂O) C, H, N.

4-P yr id in -3-ylben zen e Su lfon ic Acid {1-[3-(Am in oim i-

n om eth yl)ben zyl]-2-oxop yr r olid in -3-(S)-yl}a m id e Bistr i-

flu or oa ceta te (3e). ¹H NMR (DMSO-d₆) δ 9.27 (bs, 2H), 9.05

(bs, 2H), 8.23 (m, 2H), 7.93 (m, 4H), 7.62 (m, 2H), 7.51 (m,

3H), 4.40 (m, 2H), 4.15 (m, 1H), 3.10 (m, 2H), 2.05 (m, 1H),

1.60 (m, 1H); MS (FAB) m/z 450 (M + H)⁺; HRMS (ESI) calcd

for C₂₃H₂₃N₅O₃S 450.1600, found 450.1611 (M + H)⁺; HPLC

analysis (C18, 15-min linear gradient; elution 10-80% CH₃-

CN/0.1% TFA H₂O; flow rate 1.0 mL/min) indicated that the

product was >97A%.

3-Chloroperoxybenzoic acid (0.017 g, 0.966 mmol) was added

to a solution of 4-pyridin-4-ylbenzenesulfonic acid [1-(3-cy-

anobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide (0.038 g, 0.088 mmol)

in CHCl₃(2 mL) at room temperature. The mixture was stirred

for 18 h and then diluted with CH₂Cl₂. The organic layer was

washed with saturated NaHCO₃and saturated NaCl and then

worked up to yield 4-(1-oxidopyridin-4-yl)benzenesulfonic acid

[1-(3-cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide (0.039 g, 100%)

which was used in the subsequent step without further

purification: ¹H NMR (CDCl₃) δ 8.34 (d, 2H), 8.09 (d, 2H), 7.8

(d, 2H), 7.69 (m, 2H), 7.64 (m, 1H), 7.51 (d, 1H), 7.48 (s, 1H),

4.48 (ABq, 2H), 4.55 (s, 1H), 4.02 (t, 1H), 3.26 (m, 2H), 2.56

(m, 1H), 2.04 (m, 1H); MS (ESI) m/z 449 (M + H)⁺.

P a r t B: 4-(1-Oxidopyridin-4-yl)benzenesulfonic acid [1-(3-

cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide was treated as

described in example 4a , part F, to give the title compound

3h : ¹H NMR (DMSO-d₆) δ 9.28 (bs, 2H), 8.98 (bs, 2H), 8.30

(m, 3H), 7.75-8.02 (m, 6H), 7.44-7.78 (m, 4H), 4.35 (m, 2H),

4.12 (m, 1H), 3.10 (m, 2H), 2.05 (m, 1H), 1.64 (m, 1H); HRMS

(ESI) calcd for C₂₃H₂₃N₅O₄S 466.1549, found 466.1526 (M +

H)⁺; HPLC analysis (C18, 15-min linear gradient; elution 10-

80% CH₃CN/0.1% TFA H₂O; flow rate 1.0 mL/min) indicated

that the product was >90A%.

P r ep a r a tion of 3-{3-(S)-[5-(2-Meth oxyp yr im id in -4-

y l)t h iop h e n e -2-y lsu lfon yla m in o ]-2-o x op y r r o lid in -1-

ylm eth yl}ben za m id in e Tr iflu or oa ceta te (3m ). P a r t A:

[5-(2-Methylsulfanylpyrimidin-4-yl)thiophene-2-sulfonic acid

[1-(3-cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide was prepared

in a similar manner as described in example 3c, Part A. ¹H

NMR (CDCl₃, 300 MHz) δ 8.55 (d, 1H), 7.70 (d, 1H), 7.65 (d,

1H), 7.60 (m, 1H), 7.50 (s, 1H), 7.46 (m, 2H), 7.23 (d, 1H), 5.84

(s, 1H), 4.48 (s, 2H), 3.97 (m, 1H), 3.28 (m, 2H), 2.66 (m, 1H),

2.60 (s, 3H), 2.15 (m, 1H).

P a r t B: 3-Chloroperoxybenzoic acid (75%, 1.05 g, 4.58 mmol)

in CHCl₃(50 mL) was added dropwise to a solution of [5-(2-

methylsulfanylpyrimidin-4-yl)thiophene-2-sulfonic acid [1-(3-

cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide (1.48 g, 3.05 mmol)

in CHCl₃(30 mL) at 0 °C. The resulting mixture was allowed

to warm to room temperature and stirred for 16 h. The reaction

mixture was diluted with CH₂Cl₂and washed successively with

saturated Na₂SO₃, 10% Na₂CO₃, and water. The organic layer

was worked up to give a mixture of [5-(2-methylsulfinylpyri-

midin-4-yl)thiophene-2-sulfonic acid [1-(3-cyanobenzyl)-2-oxo-

pyrrolidin-3-(S)-yl]amide and [5-(2-methylsulfonylpyrimidin-

4-yl)thiophene-2-sulfonic acid [1-(3-cyanobenzyl)-2-oxopyrro-

lidin-3-(S)-yl]amide (1.46 g, 2.82 mmol) as a solid which was

used in the subsequent step without further purification: MS

(FAB) m/z 502, 518 (M + H)⁺.

P a r t C: A mixture of [5-(2-methylsulfinylpyrimidin-4-yl)-

thiophene-2-sulfonic acid [1-(3-cyanobenzyl)-2-oxopyrrolidin-

3-(S)-yl]amide and [5-(2-methylsulfonylpyrimidin-4-yl)thiophene-

2-sulfonic acid [1-(3-cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide

(0.19 g, 0.37 mmol) was dissolved in MeOH (10 mL) and CH₂-

Cl₂(1 mL). Ammonia gas was bubbled through the solution

for 5 min; then the reaction mixture was heated to reflux for

4 h. After this time, the solution was concentrated in vacuo to

give [5-(2-methoxypyrimidin-4-yl)thiophene-2-sulfonic acid [1-(3-

cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide (0.17 g, 97%) as

a solid: ¹H NMR (DMSO-d₆) δ 8.70 (d, 1H), 8.09 (d, 1H), 7.75

(m, 3H), 7.66 (s, 1H), 7.55 (m, 3H), 4.42 (AB, 2H), 4.27 (m,

1H), 3.95 (s, 3H), 3.15 (m, 2H), 2.17 (m, 1H), 1.68 (m, 1H).

P a r t D: [5-(2-Methoxypyrimidin-4-yl)thiophene-2-sulfonic

acid [1-(3-cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide was

treated in the same manner as described in example 4a , part

F, to give 3-{3-(S)-[5-(2-methoxypyrimidin-4-yl)thiophene-2-

ylsulfonylamino]-2-oxopyrrolidin-1-ylmethyl}benzamidine tri-

fluoroacetate (3m ): ¹H NMR (DMSO-d₆) δ 9.29 (bs, 2H), 9.11

(bs, 2H), 8.70 (d, 1H), 8.62 (d, 1H), 8.09 (d, 1H), 7.75 (m, 2H),

7.68 (m, 1H), 7.57 (m, 3H), 4.45 (AB, 2H), 4.25 (m, 1H), 3.95

(s, 3H), 3.16 (m, 2H), 2.18 (m, 1H), 1.70 (m, 1H); MS (FAB)

m/z 487 (M + H)⁺. Anal. (C₂₁H₂₂N₆O₄S₂‚1.5TFA‚0.975H₂O) C,

H, N.

4-P yr id in -4-ylben zen esu lfon ic Acid {1-[3-(Am in oim i-

n om eth yl)ben zyl]-2-oxop yr r olid in -3-(S)-yl}a m id e Bistr i-

1

flu or oa ceta te (3f). H NMR (DMSO-d₆) δ 9.28 bs, 2H), 9.13

(bs, 2H), 8.80(bs, 1H), 8.33 (m, 1H), 7.97 (m, 5H), 7.62 (m, 1H),

7.51 (m, 3H), 4.40 (m, 2H), 4.15 (m, 1H), 3.10 (m, 2H), 2.05

(m, 1H), 1.60 (m, 1H); MS (FAB) m/z 450 (M + H)⁺. Anal.

(C₂₃H₂₃N₅O₃S‚2TFA) C, H, N.

(1-Meth yl-1H-im idazol-2-yl)ben zen e-4-su lfon ic Acid {1-

[3-(Am in oim in om eth yl)ben zyl]-2-oxop yr r olid in -3-(S)-yl}-

a m id e Tr iflu or oa ceta te (3i). ¹H NMR (DMSO-d₆) δ 9.30 (bs,

2H), 8.89 (bs, 2H), 8.70 (d, 1H), 7.90 (m, 1H), 7.69 (m, 4H),

7.55 (m, 5H), 4.45 (s, 2H), 4.10 (m, 1H), 3.90 (s, 3H), 3.20 (m,

2H), 2.20 (m, 1H), 1.80 (m, 1H). MS (FAB) m/z 453 (M + H)⁺.

Anal. (C₂₂H₂₄N₆O₃S‚2TFA‚0.8H₂O) C, H, N.

5-P yr id -2-ylth iop h en e-2-su lfon ic Acid {1-[3-(Am in oim -

in om et h yl)b en zyl]-2-oxop yr r olid in -3-(S)-yl}a m id e t r i-

1

flu or oa ceta te (3j). H NMR (DMSO-d₆) δ 9.32 (bs, 2H), 9.13

(bs, 2H), 8.56 (d, 1H), 8.49 (d, 1H), 8.04 (d, 1H), 7.89 (m, 3H),

7.58 (m, 4H), 7.38 (m, 1H), 4.46 (AB, 2H), 4.23 (m, 1H), 3.16

(m, 2H), 2.16 (m, 1H), 1.70 (m, 1H); MS (FAB) m/z 456 (M +

H)⁺. Anal. (C₂₁H₂₁N₅O₃S₂‚2TFA) C, H, N.

3-[2-Oxo-3-(S)-(5-p yr id in -3-ylt h iop h en e-2-ylsu lfon yl-

a m in o)p yr r olid in -1-ylm et h yl]b en za m id in e Tr iflu or o-

a ceta te (3k ). ¹H NMR (DMSO-d₆) δ 9.30 (s, 2H), 9.05 (s, 2H),

8.95 (s, 1H), 8.63 (d, 1H), 8.55 (d, 1H), 8.16 (d, 1H), 7.65-7.71

(m, 3H), 7.48-7.60 (m, 4H), 4.48 (AB, 2H), 4.28 (m, 1H), 3.15

(m, 2H), 2.20 (m, 1H), 1.71 (m, 1H); MS (FAB) m/z 456 (M +

H)⁺. Anal. (C₂₁H₂₁N₅O₃S₂‚2TFA‚0.8H₂O) C, H, N.

3-[2-Oxo-3-(S)-(5-p yr id in -4-ylt h iop h en e-2-ylsu lfon yl-

a m in o)p yr r olid in -1-ylm et h yl]b en za m id in e Tr iflu or o-

1

a ceta te (3l). H NMR (DMSO-d₆) δ 9.30 (s, 2H), 9.05 (s, 2H),

8.69 (d, 2H), 8.67 (d, 1H), 7.89 (d, 1H), 7.85 (d, 2H), 7.77 (d,

1H), 7.68 (m, 2H), 7.51-7.60 (m, 3H), 4.42 (AB, 2H), 4.26 (m,

1H), 3.18 (m, 2H), 2.21 (m, 1H), 1.70 (m, 1H); MS (FAB) m/z

456 (M + H)⁺; HRMS (FAB) calcd for C₂₁H₂₁N₅O₃S₂456.1164,

found 456.1205 (M + H)⁺; HPLC analysis (C18, 15-min linear

gradient; elution 10-80% CH₃CN/0.1% TFA H₂O; flow rate

1.0 mL/min) indicated that the product was 99A%.

3-[3-(S)-(5-Isoxa zol-3-ylth iop h en e-2-ylsu lfon yla m in o)-

2-oxop yr r olid in -1-ylm eth yl]ben za m id in e Tr iflu or oa ce-

1

ta te (3n ). H NMR (DMSO-d₆) δ 9.31 (bs, 2H), 9.22 (bs, 2H),

8.74 (s, 1H), 8.70 (d, 1H), 7.75 (m, 3H), 7.57 (m, 3H), 7.10 (d,

1H), 4.44 (AB, 2H), 4.26 (m, 1H), 3.15 (m, 2H), 2.20 (m, 1H),

1.71 (m, 1H); MS (FAB) m/z 446 (M + H)⁺. Anal. (C₁₉H₁₉N₅O₄S₂‚

1.5TFA) C, H, N.

P r ep a r a tion of N-Meth ylp yr id -4-ylben zen e-4-su lfon ic

Acid {1-[3-(Am in oim in om eth yl)ben zyl]-2-oxop yr r olid in -

3(S)-yl}a m id e Tr iflu or oa cet a t e (3g). 4-Pyridin-4-ylben-

zenesulfonic acid [1-(3-cyanobenzyl)-2-oxopyrrolidin-3-(S)-yl]-

amide was treated as described in example 27, part F: ¹H

NMR (CD₃OD) δ 8.42,8.98 (AB, 4H), 8.16 (s, 4H) 7.56-7.73

(m, 3H), 7.59 (s, 1H), 4.50 (AB, 2H), 4.43 (s, 3H), 4.27 (t, 1H),

3.26 (m, 2H), 2.33 (m, 1H), 1.80 (m, 1H); MS (FAB) m/z 464

(M + H)⁺; HRMS (ESI) calcd for C₂₄H₂₆N₅O₃S 464.1756, found

464.1751 (M)⁺. HPLC analysis (C18, 15-min linear gradient;

elution 10-80% CH₃CN/0.1% TFA H₂O; flow rate 1.0 mL/min)

indicated that the product was >97A%.

P r ep a r a tion of 4-(1-Oxid op yr id in -4-yl)ben zen e-4-su l-

fon ic Acid {1-[3-(Am in oim in om eth yl)ben zyl]-2-oxop yr -

r olid in -3-(S)-yl}a m id e Tr iflu or oa cet a t e (3h ). P a r t A:

P r ep a r a tion of 4-F lu or o-3-[3-(S)-(5-p yr id in -3-ylth io-

p h en e-2-ylsu lfon yla m in o)-2-oxop yr r olid in -1-ylm et h yl-

3586 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18

Choi-Sledeski et al.

]ben za m id e Tr iflu or oa ceta te (29). P a r t A: Sodium boro-

hydride (5 mL, 2.0 M solution in triglyme, 10.0 mmol) was

added to a solution of 5-bromo-2-fluorobenzaldehyde (6.10 g,

30.0 mmol) in THF (30 mL) at 0 °C. The reaction mixture was

stirred at 0 °C for 25 min and then quenched by the addition

of 1 N HCl. The mixture was diluted with EtOAc, and the

layers were separated. The organic layer was washed with

water and saturated NaCl and then worked up. The crude

product was purified by column chromatography eluting with

15% EtOAc/hexanes to afford 5-bromo-2-fluorobenzyl alcohol

(6.00 g, 98%): ¹H NMR (CDCl₃) δ 7.55 (dd, 1H), 7.36 (m, 1H),

6.89 (t, 1H), 4.71 (d, 2H), 2.11 (bs, 1H); MS (EI) m/z 204, 206

(M)⁺.

CH₃CN/0.1% TFA H₂O; flow rate 1.0 mL/min) indicated that

the product was 99A%.

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P a r t B: Triphenylphosphine (4.10 g, 15.6 mmol) and

N-bromosuccinimide (2.67 g, 15.0 mmol) were added to a

solution of 5-bromo-2-fluorobenzyl alcohol (3.10 g, 15.1 mmol)

in THF (30 mL) at 10 °C. The ice bath was removed and the

resulting solution stirred for 20 min at room temperature. The

reaction mixture was concentrated in vacuo and the crude

material purified by column chromatography eluting with 5%

EtOAc/hexanes to give 5-bromo-2-fluorobenzyl bromide (3.90

g, 93%): ¹H NMR (CDCl₃) δ 7.50 (dd, 1H), 7.37 (m, 1H), 6.92

(t, 1H), 4.42 (s, 2H); MS (EI) m/z 266, 268, 270 (M)⁺.

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example 4a , part D, substituting 5-bromo-2-fluorobenzyl bro-

mide for 4-(benzhydrylidenylamino)-3-(bromomethyl)benzoni-

trile. The product was purified by column chromatography

eluting with EtOAc:CH₂Cl₂:hexane (3:1:1): ¹H NMR (CDCl₃)

δ 7.40 (m, 2H), 6.96 (t, 1H), 5.20 (bs, 1H), 4.50 (s, 2H), 4.18

(m, 1H), 3.28 (m, 2H), 2.64 (m, 1H), 1.90 (m, 1H), 1.45 (s, 9H);

MS (EI) m/z 287, 389 (M)⁺.

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P a r t D: 3-(S)-(tert-Butoxycarbonylamino)-1-(5-cyano-2-fluo-

robenzyl)pyrrolidin-2-one was prepared as described in ex-

ample 20b, part E, substituting 3-(S)-(tert-butoxycarbonylami-

no)-1-(5-bromo-2-fluorobenzyl)pyrrolidin-2-one for 3-(S)-(tert-

butoxycarbonylamino)-1-(5-iodo-2-(2-methoxyethoxymeth-

oxy)benzyl)pyrrolidin-2-one (17). The crude material was puri-

fied by column chromatography eluting with 3:1:1 EtOAc:

hexane:CH₂Cl₂: ¹H NMR (CDCl₃) δ 7.70 (bd, 1H), 7.61 (m, 1H),

7.19 (t, 1H), 5.18 (bs, 1H), 4.63 (d, 1H), 4.50 (d, 1H), 4.18 (m,

1H), 3.32 (m, 2H), 2.64 (m, 1H), 2.00 (m, 1H), 1.47 (s, 9H); MS

(EI) m/z 334 (M)⁺.

P a r t E: 3-(S)-[(3-Amino-2-oxopyrrolidin-1-yl)methyl]-4-fluo-

robenzonitrile hydrochloride was prepared as described in

example 20b, part F, substituting 3-(S)-(tert-butoxycarbonyl-

amino)-1-(5-cyano-2-fluorobenzyl)pyrrolidin-2-one for 3-(S)-

(ter t-bu t oxyca r bon yla m in o)-1-(5-cya n o-2-(2-m et h oxyet h -

oxymethoxy)benzyl)pyrrolidin-2-one: ¹H NMR (DMSO-d₆) δ

8.73 (bs, 3H), 7.92 (m, 1H), 7.86 (d, 1H), 7.50 (t, 1H), 4.54 (s,

2H), 4.10 (m, 1H), 3.38 (m, 2H), 2.42 (m, 1H), 2.07 (m, 1H);

MS (EI) m/z 233 (M)⁺.

P a r t F : 5-Pyridin-3-ylthiophene-2-sulfonic acid [1-(5-cyano-

2-fluorobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide was prepared as

described in example 20b, part G, using 3-(S)-[(3-amino-2-

oxopyrrolidin-1-yl)methyl]-4-fluorobenzonitrile hydrochloride

and 5-pyridin-3-ylthiophene-2-sulfonyl chloride. The crude

product was triturated with Et₂O to give the product as a white

solid: ¹H NMR (CDCl₃+ CD₃OD) δ 8.87 (s, 1H), 8.55 (d, 1H),

8.08 (d, 1H), 7.73 (m, 3H), 7.53 (m, 1H), 7.48 (d, 1H), 7.28 (t,

1H), 4.54 (AB, 2H), 4.20 (m, 1H), 3.34 (m, 2H), 2.49 (m, 1H),

1.98 (m, 1H); MS (FAB) m/z 457 (M + H)⁺.

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P a r t G: 5-Pyridin-3-ylthiophene-2-sulfonic acid [1-(5-cyano-

2-fluorobenzyl)-2-oxopyrrolidin-3-(S)-yl]amide was treated in

the same manner as described in example 4a , part F, to give

4-fluoro-3-[3-(S)-(5-pyridin-3-ylthiophene-2-ylsulfonylamino)-

2-oxopyrrolidin-1-ylmethyl]benzamide trifluoroacetate (29): ¹H

NMR (CD₃OD) δ 8.77 (s, 1H), 8.44 (d, 1H), 8.03 (d, 1H), 7.67

(m, 2H), 7.58 (d, 1H), 7.45 (d, 1H), 7.41 (m, 1H), 7.28 (t, 1H),

5.10 (s, 1H), 4.47 (s, 2H), 4.13 (m, 1H), 3.23 (m, 2H), 2.28 (m,

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J M990041+

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Sulfonamidopyrrolidinone factor Xa inhibitors: Potency and selectivity enhancements via P-1 and P-4 optimization

DOI: 10.1021/jm990041+

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