Scholarly article on C23H31N5O6S*ClH 179746-31-7 from Journal of Medicinal Chemistry p. 1984

1984

J. Med. Chem. 2005, 48, 1984-2008

In-Depth Study of Tripeptide-Based r-Ketoheterocycles as Inhibitors of

Thrombin. Effective Utilization of the S₁′ Subsite and Its Implications to

Structure-Based Drug Design¹

Michael J. Costanzo,* Harold R. Almond, Jr., Leonard R. Hecker, Mary R. Schott, Stephen C. Yabut,

Han-Cheng Zhang, Patricia Andrade-Gordon, Thomas W. Corcoran, Edward C. Giardino, Jack A. Kauffman,

Joan M. Lewis, Lawrence de Garavilla, Barbara J. Haertlein, and Bruce E. Maryanoff *

Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development,^†Spring House, Pennsylvania 19477-0776

Received August 14, 2003

Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrom-

botic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on

the ^D-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors.

The peptide-based R-ketoheterocycles were typically prepared by either an imidate or a Weinreb

amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds

were generally assayed for inhibition of human R-thrombin and bovine trypsin. From a

structure-based design standpoint, the heterocycle allows one to explore and adjust interactions

within the S1′ subsite of thrombin. The preferred R-ketoheterocycle is a π-rich 2-substituted

azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a

preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K_ivalue of 0.2 nM and ca.

15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin

inhibition (K_i) 0.000 65 nM; slow tight binding). Several R-ketoheterocycles had thrombin K_i

values in the range 0.1-400 nM. The “Arg” unit in the R-ketoheterocycles can be sensitive to

stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily

epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable

conditions had to be selected for the enzymatic assays. Lead ^D-Phe-Pro-Arg 2-benzothiazoles

3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g.,

factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for

thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in

vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation

induced by R-thrombin (IC₅₀) 30-40 nM). They also proved to be potent anticoagulant/

antithrombotic agents in vivo on intravenous administration, as determined in the canine

arteriovenous shunt (ED₅₀) 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED₅₀

)

0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs

caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were

also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other

than ^D-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished

cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect

to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-

hirugen and 4-thrombin-hirugen depict novel interactions in the S₁′ region, with the

benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction

with Trp-60D of thrombin’s insertion loop. The benzothiazole ring of 3 displaces the Lys-60F

side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4

forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti

conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in

binding energy resulting from this salt bridge is apparently offset by perturbations across the

enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors,

such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However,

energy contour calculations with the computer program GRID also indicate a favorable

interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of

thrombin.

The analysis of macromolecular structures by X-ray

crystallography, NMR studies, and computer-assisted

molecular modeling can provide well-defined, three-

dimensional structures for use in drug design and

optimization.²Structure-based drug design has been

especially well suited for agents that inactivate enzyme

targets. In this regard, particular attention has been

directed to the serine protease R-thrombin (EC 3.4.21.5)³

following the first X-ray structure, which was reported

in 1989.⁴Since thrombin has a critical position in the

* Address correspondence to these authors. For M.J.C.: phone, 215-

628-5648; fax, 215-628-4985; e-mail, mcostanz@prdus.jnj.com. For

B.E.M.: phone, 215-628-5530; fax, 215-628-4985; e-mail, bmaryano@

prdus.jnj.com.

^†Formerly The R. W. Johnson Pharmaceutical Research Institute.

Published on Web 10/09/2004

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1985

blood coagulation cascade and thus a central role in the

regulation of hemostasis and thrombosis,⁵inhibitors of

this enzyme could lead to useful drugs for treating

thrombotic disorders,⁶which constitute a serious source

of mortality and morbidity in patients worldwide.

mined the molecular structure of the CtA-thrombin-

hirugen complex.^12aIn addition to the expected features

of CtA complexation relative to its Pro-Arg motif and

its electrophilic R-ketoamide, we also found an aromatic

stacking interaction within the S₁′ region of the active

site between the hydroxyphenyl group of CtA and Trp-

60D. This observation supported the idea of finding new

worthwhile interactions in the S₁′ domain of thrombin,

and we pursued this avenue by means of peptide-based

R-ketoheterocycles. This “heterocycle-activated ketone”

approach was quite successful, as documented in our

preliminary publication on a series of potent thrombin

inhibitors.¹⁴Subsequently, we presented details on the

X-ray crystal structures of thrombin complexed with our

inhibitors RWJ-50353 (3)^15aand RWJ-51438 (4),^15bas

well as on the antithrombotic properties of 3.¹⁶

Bode et al.⁴described the molecular structure of

human R-thrombin complexed with D-Phe-Pro-Arg-CH₂-

Cl (1a, PPACK),⁷an irreversible active-site inhibitor (a

“transition-state analogue”).⁸This archetypal complex

reveals numerous key interactions at the atomic level:

(1) an antiparallel â-strand between the extended

backbone of the ligand and the backbone of Ser-214/

Trp-215/Gly-216; (2) the guanidine occupying the S₁

specificity pocket; (3) a tetrahedral adduct formed with

Ser-195; (4) alkylation of the ꢀ-nitrogen of His-57 via

the chloromethyl group; (5) the methylene groups of Pro

tucked into a hydrophobic S₂cleft partly defined by

residues of the 60A-I insertion loop; and (6) the phenyl

group of D-Phe in a hydrophobic S₃pocket with an

aromatic stacking interaction involving Trp-215.⁹Ad-

ditionally, the X-ray crystal structure of thrombin-

PPACK (viz. 1a) suggests an opportunity for specific

interactions with the unique 60A-I insertion loop of

thrombin, especially with the side chains of Tyr-60A,

Trp-60D, and Lys-60F. Shortly thereafter, Tulinsky,

Bode, and co-workers¹⁰determined the molecular struc-

ture of human R-thrombin complexed with r-hirudin, a

des-sulfate version of the famous anticoagulant protein

from the European medicinal leech, which depicts an

elaborate collection of hydrogen bonds and electrostatic

interactions. These achievements set the stage for

thrombin to become a preeminent target for structure-

based drug design.

In 1991, we developed an interest in thrombin as a

therapeutic target and embarked on a project to discover

novel inhibitors with the aid of published atomic coor-

dinates and computer-based molecular modeling. One

of our approaches involved the synthesis and biological

evaluation of peptide-based R-ketoheterocycles (also

termed “acylheterocycles”), adapted from the proven

thrombin recognition motif D-Phe-Pro-Arg-X, which is

exemplified by the chloromethyl ketone PPACK (1a)⁷

and the aldehyde efegatran (1b).¹¹Such tripeptide

species tend to operate as “transition-state analogues”

in the region of the thrombin catalytic triad, the

focal point of substrate proteolytic cleavage.⁸We envi-

sioned the substitution of “X” with heterocycles such as

2-oxazolyl or 2-pyridyl to obtain “heterocycle-activated

ketones” that would form a reversible hemiketal adduct

with the γ-oxygen of Ser-195, a key residue of the active-

site catalytic triad. This strategy seemed particularly

attractive because heterocycles, besides furnishing the

necessary electrophilicity to the arginine carbonyl, could

serve as a “C-terminal extensor” into the S₁′ region of

the thrombin active site to probe for additional useful

interactions. This group, along with its substituents,

would allow for modulation of electronic and steric

properties to adjust the interactions.

During the early phase of our studies, Edwards et al.

published pioneering work on peptide-based R-ketohet-

erocycles as serine protease inhibitors, in their case for

elastase.¹⁷A seminal paper in 1992 described the first

examples of such compounds and presented an X-ray

crystal structure of Ac-Val-Pro-Val-(2-benzoxazole) com-

plexed with porcine pancreatic elastase (PPE).^17aThis

structure revealed the key features of hemiketal forma-

tion with Ser-195 and hydrogen bonding between the

benzoxazole nitrogen and His-57.^17aSuch interactions

were subsequently identified in our work on peptide-

based R-ketoheterocycles and thrombin.^14,15Over the

ensuing years, there have been many other papers

dealing with peptide-based R-ketoheterocycles as inhibi-

tors of thrombin,¹⁸various serine proteases,¹⁹and

cysteine proteases.²⁰While this structural motif has

Concurrently, we had been investigating the sponge-

derived natural product cyclotheonamide A (2, CtA),¹²

a macrocyclic thrombin inhibitor, as well as its conge-

ners.¹³CtA intrigued us in several ways, but it was most

useful as a tool for elucidating new types of interactions

within the active site of thrombin. Therefore, we deter-

1986 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Table 1. Biological Data for P3 Analogues

Costanzo et al.

^aTrifluoroacetate salt. ^bThe 2S/2R epimeric ratio was determined by analytical HPLC except where otherwise noted. ^cAbbreviations:

thr, human R-thrombin; try, bovine trypsin; ND, not determined; SB, slow-binding kinetics. Standard errors are given for N experiments,

which are indicated in parentheses. ^dIC₅₀) 30 ( 5 nM (N ) 5). ^eThe L-Phe diastereomer of 5 (78; 2S/2R ) 99:1) had thrombin and

trypsin IC₅₀values of 17000 ( 1300 nM and 5.8 ( 0.6 nM (N ) 2), respectively. ^fSlow-binding K_i) 0.0055 ( 0.0043 nM (N ) 2).

^gDetermined by ¹H NMR via the ratio of the R-methine peak integrals. ^hSlow-binding K_i) 0.00065 ( 0.00009 nM (N ) 2). ⁱIC₅₀) 30

( 7 nM (N ) 4). ^jDetermined by ¹H NMR via the ratio of the N-methyl peak integrals. ^kIC₅₀) 32 ( 14 nM (N ) 4). ^lSlow-binding K_i

) 0.018 ( 0.011 nM (N ) 2).

generally afforded very potent enzyme inhibitors, the

verdict is still out as to whether such “transition-state

analogues” can also provide viable drug candidates for

clinical development.

In this article, we present a detailed report on our

studies with peptide-based R-ketoheterocycles as inhibi-

tors of thrombin. This effort has encompassed a diverse

set of molecular entities, which are represented by

structures 3-77. Overall, we address (1) the design and

synthesis of potent, selective thrombin inhibitors, gen-

erally based on D-Phe-Pro-Arg and related motifs; (2)

the epimerization potential at the stereogenic center R

to the heterocycle-activated ketone; (3) structure-

activity relationships (SAR); (4) the efficacy of selected

inhibitors in animal models of thrombosis; (5) structural

changes to circumvent problematic side effects; and (6)

noteworthy aspects of intermolecular interactions within

the active site of thrombin from X-ray structures of

complexes.

Results and Discussion

Initially, we set out to investigate tripeptide-based

R-ketoheterocycles based on the D-Phe-Pro-Arg sequence

(3-68) (Tables 1-5), a proven thrombin recognition

motif. Later, we attached the favored R-keto-2-ben-

zothiazole to other known thrombin recognition motifs

(69-77) (Table 6). An N-terminal N-methyl group was

installed in most of the D-Phe-Pro-Arg compounds and

R-Ketoheterocycles as Inhibitors of Thrombin

Table 2. Biological Data for P₂Analogues

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1987

^aTrifluoroacetate salt. ^bThe 2S/2R epimeric ratio was determined by analytical HPLC except where otherwise noted. ^cAbbreviations:

thr, human R-thrombin; try, bovine trypsin; NA, not active (less than 10% inhibition at the specified concentration); ND, not determined.

Standard errors are given for N experiments (indicated in parentheses). ^dCompound reported in ref 19j. ^eDetermined by ¹³C NMR via

the integrals for the peaks at δ 122.32 and 122.16 ppm in the presence of 4.3 mol equiv of (R)-(-)-2,2,2-trifluoro-1-(9-anthryl)ethanol in

CDCl₃at 40 °C. ^fDetermined by ¹H NMR via integration of the N-methyl peaks.

25

congeners to avoid possible imine formation between a

primary amino N-terminus and the activated ketone,

which could lead to self-condensation or decomposition.

Whereas the tripeptide aldehyde D-Phe-Pro-Arg-H (GYKI-

14166) self-condenses to an imidazo[1,2-a]pyrrolo[2,1-

c]pyrazine system,^11athe corresponding N-methyl ana-

logue (1b, efegatran) is not plagued by this problem.¹¹

We prepared numerous tripeptide R-ketoheterocycles

with 2-substituted π-rich heterocycles (3-49, 51-55,

57-77), as well as two with 2-substituted π-deficient

heterocycles (50 and 56) (Tables 1-6). Our emphasis

on the π-rich heterocycles was derived from the fact that

they generally exhibited much better potency as throm-

bin inhibitors (vide infra).

Synthetic Chemistry. Target compounds 3-77 were

synthesized by two main routes, which are illustrated

for the synthesis of 3 and 4 in Schemes 1 and 2,

respectively.²¹In the first route, the heterocycle was

formed by reacting a hydroxyimidate, such as 3b, with

an appropriate bifunctional amine (Scheme 1). The

hydroxyimidates were prepared by reacting either a

tripeptidyl aldehyde, such as 3a,^11aor a protected amino

acid aldehyde with acetone cyanohydrin^17aand treating

the resulting adduct with methanolic HCl. As an

example, reaction of 3b with 2-aminothiophenol fur-

nished benzothiazole 3c, which was oxidized with the

Dess-Martin periodinane,²³deprotected with HF, and

purified by HPLC to furnish benzothiazole 3.

In the second route, the heterocycle was introduced

by reacting a Weinreb amide, such as 4a,²⁴with an

appropriate lithioheterocycle at low temperature (Scheme

2). Excess lithium reagent was required to overcome

quenching caused by the exchangeable protons in the

substrate. We preferred arylsulfonyl protecting groups

for the Arg guanidine because benzyloxycarbonyl (Cbz)

and 9-fluorenylmethylcarbonyl (Fmoc) groups were

cleaved too readily under the reaction conditions. Hence,

treatment of Weinreb amide 4a with 5-8 mol equiv of

lithium 2-lithiobenzothiazole-6-carboxylate at -78 °C

provided the corresponding ketone, which was reduced

with NaBH₄

(to avoid side reactions during the

ensuing steps), esterified with trimethylsilyldiazo-

methane, and deprotected with trifluoroacetic acid

(TFA) to yield alcohols 4b. Dipeptide 4c was coupled to

4b with the agency of dicyclohexylcarbodiimide (DCC)

to give intermediate 4d, which was saponified and

converted to benzothiazolecarboylic acid 4 by the meth-

ods used for 3c (Scheme 1). In the synthesis of lysine-

based and analogous target compounds (37-42, 69-72),

the amino side chain was protected with a 4-methoxy-

2,3,6-trimethylbenzenesulfonyl (Mtr) group, which was

removed by using anhydrous HBr in the final depro-

tection step. The diastereomeric alcohols corresponding

to 3, 48, and 49, were prepared by HF deprotection of

3c followed by HPLC separation. The second route

(Scheme 2) is more convergent and proved to be more

versatile.

The crude target compounds from both routes usually

contained 10-20% of the epimeric D-amino ketone

diastereomer (e.g., 27, 33, 36, 38, 40, and 42),²⁶most of

which was removed during purification by reverse-phase

HPLC. The D-amino ketone diastereomer, which gener-

ally eluted before the corresponding L-diastereomer, had

greatly diminished thrombin and trypsin affinities

relative to the L-diastereomer (vide infra). The L/D-amino

ketone diastereomer ratios (2S/2R) were usually deter-

mined by either analytical HPLC or proton NMR

integration of the N-methyl or amino ketone R-methine

signals.

Compounds 28, 41/42, 43, 71/72 required the synthe-

sis of various unnatural amino acids (Schemes 3-6).

R-L-Methylarginine derivative 28 was prepared by the

route illustrated in Scheme 3. Orthogonal protection of

R-L-ornithine²⁷(28a) with Cbz and tert-butyloxycarbonyl

(Boc) groups gave 28b, which was guanylated to provide

protected Arg derivative 28c. After removal of the two

Cbz groups from 28c, tosylation and esterification

furnished ester 28d. Reaction of 28d with 2-lithioben-

zothiazole, followed by reduction of the ketone moiety

and removal of the Boc group, furnished 28e, which was

1988 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Table 3. Biological Data for P₁Analogues

Costanzo et al.

^aTrifluoroacetate salt. ^bThe 2S/2R epimeric ratio was determined by analytical HPLC except where otherwise noted. ^cAbbreviations:

thr, human R-thrombin; try, bovine trypsin; NA, not active (less than 10% inhibition at the specified concentration); ND, not determined;

SB, slow-binding kinetics. Standard errors are given for N experiments (indicated in parentheses). ^dDetermined by ¹H NMR by the ratio

of the N-methyl peak integrals. ^eSlow-binding K_i) 430 ( 110 nM (N ) 11). ^fIC₅₀) 630 (N ) 1). ^gIC₅₀) 540 ( 50 nM (N ) 3). ^hDetermined

by ¹H NMR by the ratio of the R-methine peak integrals.

converted to 28 in the usual manner. Ester 28d was

reacted directly with 2-lithiobenzothiazole because the

Weinreb amide could not be obtained from the corre-

sponding acid despite our attempts to employ various

coupling reagents (BOP, PyBOP, PyBrOP, or BOP-

Cl).²⁸

trans-4-Aminomethylcyclohexylglycine derivatives 41

and 42 were prepared by a Strecker reaction (Scheme

4).²⁹Esterfication of acid 41a followed by Mtr protection

of the amino group gave ester 41b, and reduction of 41b

to the corresponding alcohol followed by oxidation with

the Dess-Martin periodinane gave aldehyde 41c. Reac-

tion of 41c with sodium cyanide in the presence of

ammonium carbonate followed by hydrolysis with 4 M

sodium hydroxide provided the desired R-amino acid.

This material was protected with a Boc group and

coupled to N-methoxy-N-methylamine to yield Weinreb

amide 41d, which was converted to a mixture of 41 and

42 via the Weinreb amide route (Scheme 2).

L-Methoxypropylglycine derivative 43 was synthe-

sized via Evans chiral N-acyl-2-oxazolidinone chemistry

(Scheme 5).³⁰Alcohol 43a was oxidized to acid 43b,

which was converted to the oxazolidinone 43c.³⁰Lithia-

tion of 43c, followed by reaction with di-tert-butyl

azodicarboxylate, provided 43d, which was hydrolyzed

to give hydrazine 43e. Removal of the Boc groups,

reduction of the hydrazine, Boc protection of the amine,

and Weinreb amide formation gave 43f, which was

reacted with 2-lithiobenzothiazole. The resulting ketone

was reduced to provide alcohol 43g, which was con-

verted to 43 via the Weinreb amide route (Scheme 2).

2-(4-Aminocyclohexyl)-â-alanines 71 and 72 were

prepared by a modified Knoevenagel procedure (Scheme

6).³¹Knoevenagel condensation of aldehyde 69d in the

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1989

Table 4. Biological Data for Non-Ketone Analogues

^aIsolated as a trifluoroacetate salt. ^bRefers to the stereogenic carbon bearing the guanidinopropyl side chain. The 2S/2R epimeric

ratio was determined by analytical HPLC. ^cAbbreviations: thr, human R-thrombin; try, bovine trypsin; ND, not determined. Standard

errors are given for N experiments, which are indicated in parentheses. ^dIC₅₀) 32000 nM (N ) 1). ^eIC₅₀) 30000 nM (N ) 1). ^fEnriched

in one diastereomer; however, the absolute stereochemistry is unknown.

presence of ammonium acetate provided â-alanine 71a,

which was protected with a Boc group and converted to

the Weinreb amide 71b. Compound 71b provided 71

and 72 via the Weinreb amide route (Scheme 2).

Epimerization Studies. One can appreciate that the

proton on the stereogenic center adjacent to the acti-

vated ketone of peptide-based R-ketoheterocycles is

somewhat labile such that stereomutation might take

place during synthetic processes, biochemical assays, or

in vivo experiments. Therefore, it is important to

understand the potential for stereomutation, especially

under the conditions used for the bioassays. We evalu-

ated the epimerization of thiazole 59 (L-Arg/D-Arg ) 98:

2), an early lead compound, in the buffers that were

used for thrombin (pH 7.40 and 7.85) and trypsin (pH

7.40 and 8.40) inhibition assays (Table 7³²). After 35 min

at 37 °C (enzyme assay conditions), some epimerization

of the L-arginine to the D-arginine diastereomer was

apparent in both buffer systems by analytical HPLC.

The L-arginine content of 59, which was initially 98%,

decreased to 92% in the pH 7.85 thrombin buffer and

to 93% in the pH 7.4 thrombin buffer, whereas it

decreased to 73% in the pH 8.4 trypsin buffer. At pH

7.4, epimerization was slightly faster in the trypsin

buffer (87%) than in the thrombin buffer (93%). Similar

epimerization results were reported for efegatran (1b).^11b

A solution of 59 in distilled water (pH 6.5) did not

epimerize after 3 h at 23 °C but changed to a 73:27

mixture after 16 days. Although 59 epimerized under

mildly basic conditions, it is reasonably stable stereo-

chemically under mildly acidic conditions, such as pH

3-4.

L-Arg/D-Arg diastereomer ratio at equilibrium for 4 and

59 is essentially 1:1. On the basis of these epimerization

studies, we confidently performed the thrombin and

trypsin inhibition assays at pH 7.4 (normal pH of

human plasma) within a 30-min time frame.

To approximate in vivo epimerization for the keto-

benzothiazoles, we also monitored the rate of equilibra-

tion of 3 in rabbit and rat plasma over 120 min at pH

7.4 and 37 °C. In both milieus, a 53:47 mixture of L-Arg/

D-Arg epimers, 3:27, was obtained after 60 min, indicat-

ing that 3 would be almost completely epimerized within

1 h under physiological conditions.

Structure-Activity Relationships. A broad range

of compounds (3-77) was assessed for the inhibition

of human R-thrombin and bovine trypsin (Tables

1-6). Compounds 3-77 generally displayed competi-

tive Michaelis-Menten kinetics, although some exhib-

ited slow-binding (i.e., time-dependent) kinetics for

thrombin. Hence, the thrombin IC₅₀values are also

given because they are a better comparator for assessing

the relative potency of slow-binding inhibitors with

Michaelis-Menten inhibitors, since all IC₅₀values were

determined under the same experimental conditions.

We included trypsin inhibition as a primary selectivity

screen because of trypsin’s high structural homology

with thrombin. In the X-ray crystal structure of human

R-thrombin, 195 out of 259 R-carbons in the B chain

occupy topologically equivalent positions relative to

bovine trypsin with a root-mean-squared (rms) deviation

of 0.82 Å.^4bConsequently, good selectivity (i.e., >100-

fold) for thrombin over trypsin can be difficult to attain.

Trypsin inhibition might present a source of adverse

effects following oral administration.^33,34

In a separate study, benzothiazole 4 (L-Arg/D-Arg )

98:2) was dissolved in the pH 7.4 thrombin buffer at 37

°C and epimerization was monitored. The L-Arg/D-Arg

isomer ratio changed to 70:30 after 0.5 h, 60:40 after 1

h, and 50:50 after 6.5 h. Under these conditions, 59 (L-

Arg/D-Arg ) 98:2) gave a 70:30 mixture after 0.5 h and

a 50:50 mixture after 1 h (and after 6.5 h). Thus, the

Early in the project, we discovered that the 2-ben-

zothiazole group together with the L-amino acid ster-

eochemistry at P₁, as in 3, confer excellent thrombin

affinity (K_i) 0.20 nM) and moderate selectivity over

trypsin (16-fold). This finding was investigated further

with systematic changes to the P₃, P₂, P₁, and P₁′

1990 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Table 5. Biological Data for P₁′ Analogues

Costanzo et al.

^aTrifluoroacetate salt. ^bThe 2S/2R epimeric ratio was determined by analytical HPLC, except where otherwise noted. ^cAbbreviations:

thr, human R-thrombin; try, bovine trypsin; NA, not active (less than 10% inhibition at the specified concentration); ND, not determined.

Standard errors are given for N experiments (given in parentheses). ^dDetermined by ¹H NMR via integration of the N-methyl signals.

^eDetermined by ¹H NMR via integration of the R-methine signals. ^fSlow binding K_i) 0.0070 ( 0.0022 nM (2).

segments of the D-Phe-Pro-Arg-X pharmacophore (Fig-

ure 1). Modifications of the P₃residue were conducted

with cognizance of the SAR knowledge base for this

tripeptide motif.^7a,11Thus, the D-amino acid stereochem-

istry and a hydrophobic side chain were generally

maintained. The presence of a suitable P₃residue, such

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1991

Table 6. Biological Data for Other Thrombin Recognition Motifs

^aTrifluoroacetate salt. ^bThe 2S/2R epimeric ratio was determined by analytical HPLC except where otherwise noted. ^cAbbreviations:

thr, human R-thrombin; try, bovine trypsin; ND, not determined; SB, slow-binding kinetics. Standard errors are given for N experiments

(given in parentheses). ^dThe relative stereochemistry of the cyclohexyl ring is trans. ^eThe absolute stereochemistry of the carbon that

bears the cyclohexyl ring is unknown; its configuration is drawn arbitrarily. ^fCompound is essentially a single diastereomer (for 71,

71/72 ) 99:1; for 72, 71/72 ) 1:99). ^gIC₅₀) 58000 nM (N ) 1). ^hIC₅₀) 22000 nM (N ) 1). ⁱDetermined by ¹H NMR via the ratio of the

R-methine peak integrals.

Scheme 1. Imidate Route to 3

as the D-Phe unit of 3, is critical for thrombin affinity,

as reflected in the lack of potency for 20 (Table 1). Since

D-cyclohexylalanine, as in 8, did not diminish thrombin

affinity, the π stacking interaction between a P₃aryl

group and Trp-215 (Figure 1) can obviously be compen-

sated for by general hydrophobic interactions (i.e.,

“grease”). This point is also illustrated by results for the

D-phenylglycine (15) and D-cyclohexylglycine (16) ana-

1992 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Scheme 2. Weinreb Amide Route to 4

Costanzo et al.

Scheme 3. Synthesis of L-R-Methylarginine Derivative 28

Scheme 4. Synthesis of trans-4-Aminomethylcyclohexylglycine Derivatives 41 and 42

logues. Substitution of D-Phe with a 4-fluoro substituent

led to a minor improvement in thrombin affinity,

whereas perfluorination decreased thrombin affinity (cf.

3 with 6 and 7). Methyl substitution at the D-Phe

stereogenic center was tolerated because there was no

effect on thrombin affinity (cf. 5 with 11). A basic

nitrogen in P₃, which in D-Phe-Pro-Arg thrombin inhibi-

tors is typically hydrogen-bonded to Gly-216 (Figure 1),

conferred potent thrombin affinity (cf. 3 with 9 and 10,

as well as 13 with 14 and 19). Although des-amino

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1993

Scheme 5. Synthesis of L-(3-Methoxypropyl)glycine Derivative 43

Scheme 6. Synthesis of 2-(4-Aminocyclohexyl)-â-alanine Derivatives 71 and 72

compound 14 is less potent than the corresponding

amino derivative 13, it still had respectable thrombin

affinity (K_i) 1.1 nM). Alkylation of the N-terminal

nitrogen may eliminate slow-binding kinetics, as for 3

or 12 vs 5. D-Diphenylalanine^11cand D-cyclohexyl-

glycine^11dderivatives 13 and 16 are particularly note-

worthy because they displayed the greatest thrombin

affinity of the compounds studied, with thrombin K_i

values of 0.000 65 and 0.018 nM, respectively (slow-

binding kinetics). Compounds 13 and 16 were also

among the most potent inhibitors in terms of thrombin

IC₅₀values: 4.5 and 5.3 nM, respectively.

The presence of a suitable P₂residue, similar to Pro,

is important for high thrombin and trypsin affinity, as

illustrated by 25 and 26 vs the truncated analogues 21-

24 (Table 2). Substitution of L-Val for L-Pro led to

diminished thrombin affinity and equipotent trypsin

activity relative to 3, whereas substitution with L-

homoproline resulted in activity virtually identical to

that of 3. The attached hydrophobic P₃residue, such as

D-Phe, is very important (Table 2). The higher trypsin

inhibitory potencies for 22 and 23 relative to 21 may

be attributed to reestablishing the hydrogen bond

between the Arg R-NH of the inhibitor and the Ser-214

carbonyl of the â-sheet (Figure 1). Compound 23 is

intriguing as a trypsin inhibitor because of its relatively

high affinity (K_i) 30 nM) and low molecular weight

(387 Da).^19j,35

Modifications of the P₁residue, i.e., the L-Arg moiety

of 3, illustrate the importance of the L-amino acid

stereochemistry and the basic functionality for potent

thrombin and trypsin activity, especially in the case of

transition-state analogues (Table 3). Considering the

L-amino acid analogues with reasonable potency, that

is, with thrombin inhibition in the nanomolar range,

they had significantly higher thrombin and trypsin

affinities than did their D-epimer counterparts (cf. 3

with 27, 32 with 33, 35 with 36, and 37 with 38. Much

of the thrombin inhibition attributed to the D-epimers

is probably due to the presence of small amounts of the

more potent L-epimers. Because of this situation, we

methylated the L-arginine stereogenic center to prevent

epimerization and provide pure, stable D and L isomers

for study. However, this structural change resulted in

slow-binding kinetics and a significant decrease in

thrombin affinity presumably because of steric factors

(cf. 3 with 28). A similar outcome for R-methyl substitu-

tion was reported for the analogous aldehyde series, i.e.,

D-MePhe-Pro-Arg(R-Me)H.³⁶N-Methylation of the argi-

nine R-amino group severely affected the thrombin and

1994 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Costanzo et al.

Figure 1. Diagram representing the key interactions between 4 (RWJ-51438) and thrombin.^15b

trypsin potency, as would be expected from disruption

of a hydrogen bond between the Arg R-NH and the

carbonyl group of Ser-216 of the â-sheet (cf. 3 with 29;

Figure 1). Since basic P₁groups of thrombin inhibitors

typically form a strong salt bridge with Asp-189 in the

thrombin specificity pocket (Figure 1), it is not surpris-

ing that there was a strong correlation between de-

creasing thrombin affinity and decreasing basicity (cf.

3 with 32, 37, and 43-45). Additionally, extension of

the Arg side chain by just one methylene dramatically

decreased thrombin affinity (cf. 3 with 30). The sensitiv-

ity of positioning the basic group was also observed in

the significantly higher thrombin affinity for 3-amidi-

nophenylalanine 32 relative to 4-amidinophenylalanine

31 and for (S)-1-amidinopiperidine derivative 34 relative

to its corresponding (R)-isomer 35. Methylation of the

amino group of the P₁Lys side chain caused a dramatic

decrease in thrombin and trypsin affinities (cf. 37 with

39). The trans-4-aminomethylcyclohexylglycine deriva-

tive 41 displayed weak thrombin affinity (K_i) 920 nM)

and modest trypsin activity (K_i) 54 nM). In the neutral

P₁compounds 43-45, butyl analogue 44 was essentially

devoid of thrombin inhibition, whereas benzyl analogue

45 had very weak thrombin inhibition (K_i) 7100 nM).

The weak K_ivalue of 3900 nM for methoxypropylglycine

analogue 43 is in stark contrast with the potent K_iof 7

nM reported for a related methoxypropyl derivative in

a boronic acid series of thrombin inhibitors.³⁷

Some nonketone analogues, 46-49, were prepared to

ascertain the importance of an activated carbonyl

functionality for thrombin inhibition (Table 4). The

methylene analogues 46/47 and the alcohol derivatives

48/49 had extremely weak thrombin potency relative

to 3. The somewhat better thrombin affinity of alcohols

48/49 relative to 46/47 might be attributed to the

formation of a hydrogen bond with Gly-193 or Ser-195

(Figure 1). The results for non-ketones 46-49 attest to

the crucial role of the electrophilic ketone for strong

enzyme inhibition, consistent with a transition-state

analogue.

Modifications of the activating heterocycle at P₁′, i.e.,

the benzothiazole moiety of 3, had a dramatic impact

on thrombin affinity (cf. 50-68 and 4; Table 5). At least

two heteroatoms proximal to the carbon bearing the

carbonyl group are required for potent activity (cf. 3

with 50 and 51). Although pyridine derivative 50

contains a π-deficient heterocycle that can activate the

adjacent carbonyl, it showed modest potency relative to

3. Benzothiophene 51 was found to have 12000-fold less

thrombin affinity than benzothiazole 3, indicating the

key importance of the heterocyclic nitrogen atom. The

significant increase in potency for 3 relative to benzox-

azole 55 (30-fold) and N-methylbenzimidazoles 53/54

(40/50-fold) may be associated with the relative π

electron-withdrawing power of these heterocycles³⁸and/

or a hydrophobic patch for interaction of the sulfur atom

on the surface of thrombin (vide infra). Methyl substitu-

tion in the imidazole series did not improve thrombin

inhibition; however, it did enhance the selectivity for

thrombin over trypsin (entries 52-54). In the thiazole

series, substituted thiazoles always exhibited less throm-

bin inhibition relative to the unsubstituted thiazole (cf.

58-61 with 57). Moreover, the thiazoles usually exhib-

ited higher affinity for trypsin than for thrombin (entries

57-61). Benzo-fused thiazoles, oxazoles, and imidazoles

consistently had greater thrombin inhibition and more

selectivity over trypsin than their non-benzo counter-

parts. Benzothiazole 3 had 10-fold more thrombin

affinity than thiazole 57, and benzimidazole 54 had 25-

fold more thrombin affinity than imidazole 52. This

result may be due to the benzo-fused heterocycles

having increased electronic induction, thereby increas-

ing the electrophilicity of the keto group. However,

although the thrombin affinity of benzothiazole 3 rela-

tive to thiazole 57 increased, the trypsin affinity re-

mained essentially unchanged. We attribute this in-

crease in thrombin affinity and trypsin selectivity to an

edge-to-face aromatic-stacking interaction between the

benzene ring of the benzoheterocycle and the indole ring

of Trp-60D of the thrombin 60A-I insertion loop (Figure

1), which has been observed by X-ray crystallography

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1995

(vide infra).¹⁵Fusion of another phenyl ring to generate

a naphthothiazole, as with 62, did not enhance thrombin

affinity relative to 3. Reduction of the benzothiazole

benzene unit caused a 17-fold reduction in thrombin

affinity and did not affect trypsin affinity (cf. 3 with 61),

consistent with an aromatic-stacking interaction be-

tween the benzoheterocycle and the indole ring of Trp-

60D, as saturated hydrophobicity will not effectively

replace the planar benzene ring. Substitution at the

benzothiazole 6-position with hydroxy, hydroxymethyl,

methoxy, and fluoro groups did not significantly affect

thrombin affinity (cf. 63-66). 6-Carboxamido substitu-

tion (67) led to one of the most potent compounds in

terms of thrombin IC₅₀values, although slow-tight

binding kinetics were obtained. Relative to 3, 6-car-

bomethoxy (68) and 6-carboxy (4) substitution caused

a 6-fold and 10-fold reduction, respectively, of thrombin

affinity in terms of K_ivalues.

We tested the heterocycle-activated ketone approach

with other thrombin recognition motifs (viz. 69, 70, 73-

77 (Table 6)). Compounds 69 and 70, ketobenzothiazole

analogues of a N-methylketoamide inhibitor L-370,518,

exhibited only modest thrombin inhibition (K_ivalues of

110 and 53 nM, respectively) and also lacked selectivity

over trypsin, which is in sharp contrast to the results

reported for L-370,518 (thrombin K_i) 0.09 nM; trypsin

K_i) 5 µM).³⁹A molecular modeling study suggested

that steric interactions between the 4-aminocyclohexane

groups of 69 and 70 and the benzothiazole may be

responsible for the modest thrombin potency and that

insertion of a methylene spacer between the side chain

and the activated carbonyl group would help alleviate

the problem. Hence, 71 and 72 were prepared, but they

were essentially devoid of thrombin inhibition. Com-

pound 73, a ketobenzothiazole analogue of a selective

boroarginine derivative,⁴⁰had only weak thrombin

inhibition and no selectivity over trypsin. More encour-

aging results were obtained with 74-77, which had

thrombin K_ivalues in the range 1.4-16 nM. Compounds

74-76 are analogues of orally bioavailable arginals,⁴¹

and 77 is an analogue of an orally bioavailable, selective

agmatine⁴²derivative. Of these ketobenzothiazoles, only

75 exhibited selectivity over trypsin, albeit by just a

factor of 11.

The in vitro antithrombotic activity of selected com-

pounds from 3-68 was assessed by the inhibition of gel-

filtered platelet (GFP) aggregation induced by R-throm-

bin (Table 8). GFP preparations allow the determina-

tion of antiplatelet activity in the absence of plasma

coagulation factors and fibrinogen. The most potent

compound from this group was L-homoproline derivative

26, with an aggregation IC₅₀value of 10 nM (thrombin

K_i) 0.34 nM). In comparison, reference thrombin

inhibitor efegatran (1b) had an IC₅₀value of 83 nM.

Compounds with the P₃substituent modified to D-

cyclohexylalanine 12, D-phenylglycine 15, D-cyclohexyl-

glycine 16, and L-aspartate 75 also displayed potent

activity, with GFP aggregation IC₅₀values of less than

20 nM. Higher thrombin affinity did not necessarily

translate into more potent inhibition of aggregation (cf.

3 with 75 and 77). Lead compounds 3, 4, and 68

inhibited GFP aggregation with relatively potent IC₅₀

values of 32, 41, and 39 nM, respectively.

Table 8. Gel-Filtered Platelet Aggregation and Blood Pressure

Effect in Guinea Pigs^a

cmpd

thr K_i, nM^b

GFP aggr IC₅₀, nM^c∆BP% @ dose, mg/kg^d

1b

3

18 ( 4 (12)

0.18 ( 0.02 (18)

2.0 ( 0.9 (6)

0.18 ( 0.03 (4)

24 ( 4 (3)

0.36 ( 0.09 (4)

SB

3.1 ( 0.6 (6)

0.46 ( 0.05 (6)

ND

3.1 ( 1.2 (3)

21 ( 7 (6)

83 ( 28 (9)

32 ( 6 (10)

41 ( 19 (4)

28 ( 1 (2)

46 ( 9 (3)

16 ( 4 (4)

43 ( 14 (3)

29 ( 9 (4)

17 ( 6 (3)

17 ( 3 (2)

ND

-21 @ 10

-85 @ 3

-32 @ 10

-49 @ 3

-36 @ 10

-47 @ 10

-54 @ 3

-25 @ 3

-63 @ 3

-21 @ 3

-70 @ 10

-47 @ 3

-45 ( 15 @ 3 (2)

-45 @ 3

-41 @ 3

-16 @ 1

-68 ( 4 @ 10 (2)

-30 @ 3

-70 @ 10

-51 @ 10

-44 @ 3

-34 @ 3

ND

4

8

10

12

13

14

15

16

19

25

26

37

38

39

52

54

61

62

63

64

65

68

69

70

74

75

76

77

180 ( 18 (2)

10 ( 1 (2)

ND

0.34 ( 0.05 (6)

38 ( 5 (3)

210 ( 10 (3)

3700 ( 600 (3)

200 ( 70 (6)

8.1 ( 1.2 (6)

3.4 ( 1.0 (6)

0.58 ( 0.08 (6)

0.14 ( 0.01 (6)

0.23 ( 0.03 (5)

0.15 ( 0.05 (5)

1.3 ( 0.1 (3)

110 ( 30 (9)

53 ( 3 (6)

ND

5200 ( 1800 (2)

230 ( 40 (3)

59 ( 16 (2)

22 ( 5 (5)

ND

39 ( 13 (7)

76 ( 22 (4)

120 ( 28 (4)

47 ( 14 (3)

16 ( 2 (2)

37 ( 17 (2)

34 ( 13 (3)

-16 @ 10

-25 @ 3

ND

-39 @ 10

ND

-64 @ 10

-30 @ 20

16 ( 4 (3)

1.4 ( 0.2 (9)

1.5 ( 0.4 (6)

10 ( 2 (6)

^aAbbreviations: ND, not determined; SB, slow-binding kinetics;

thr, human R-thrombin; GFP aggr, gel-filtered platelet aggrega-

tion; BP, blood pressure. The number of experiments (N) is in

parentheses. ^bK_ivalues (mean ( standard error) for thrombin

inhibition. ^cIC₅₀values (mean ( standard error) for inhibition of

human platelet aggregation. ^dPercent change in blood pressure

in guinea pigs at the given dose (iv).

Our intent was to assess selected compounds in vivo

for their anticoagulant/antithrombotic activity. As a

prelude, we examined the general cardiovascular effects

of certain lead compounds on intravenous (iv) admin-

istration to anesthetized guinea pigs.⁴³Unfortunately,

3 and some of its analogues elicited pronounced hy-

potension and electrocardiogram (ECG) effects (Table

8).^43bHypotensive side effects have been reported in

other series of thrombin inhibitors,^40,44and 1b (efegat-

ran) in our hands caused hypotension at a 10-fold higher

dose than the dose of 3 (30 vs 3 mg/kg). Thus, we tested

representative analogues of 3 for their hypotensive

effects. The P₁Arg side chain was not the primary cause

of hypotension because the corresponding Lys and

4-aminocyclohexylglycine analogues, 37 and 69, also

caused a drop in blood pressure of 45% and 25% at 3

mg/kg, respectively. Many modifications, at the P₃(8,

12-16, and 19), P₂(25, 26), and P₁′ (52, 54, 61-65, 68)

positions, caused a substantial decrease in blood pres-

sure. Hypotension was also induced by thrombin rec-

ognition motifs other than D-Phe-Pro, such as in 74-

77. Fortunately, carboxylic acid and carboxylic ester

analogues 4 and 68 (methyl ester of 4) displayed

significantly diminished hypotension and ECG effects

relative to 3, allowing for their use in subsequent in vivo

studies (vide infra).⁴⁵

Several agents and pathways were evaluated as

possible causes of the hypotension and ECG effects of 3

and its analogues, including nitric oxide, bioactive

amines, cholinergic activation, and activation of the

complement cascade. We especially suspected the comple-

1996 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Table 9. Inhibition of Thrombin Relative to Other Serine Proteases

Costanzo et al.

selectivity ratio (K_iother/K_ithr)^b

cmpd

thr K_i, nM^a

trypsin

fXa

plasmin

prot Ca

SK

tPA

uPA

4

68

3

2.0 ( 0.9 (9)

1.3 ( 0.1 (3)

0.2 ( 0.02 (30)

18 ( 4 (12)

1.4

24

600

390

2400

1800

19000

72

210

760

6300

72

21

8500

1800

23000

130

380

110

45

16

2400

5300

47000

12000

28

39000

330

78

37000

1b (efegatran)

argatroban

0.5

470

9.9 ( 0.7 (7)

54000

>10⁵

10⁶

^aK_ivalues (mean ( standard error) for thrombin inhibition; the number of experiments (N) is in parentheses. ^bSelectivity is defined

as the ratio of the K_ivalue of the serine protease over the K_ivalue for thrombin. For the original K_idata, see Supporting Information.

Abbreviations: thr, thrombin; fXa, activated factor X; tPA, two-chain tissue-type plasminogen activator; prot Ca, activated protein C; SK

) streptokinase; uPA, two-chain urokinase-type plasminogen activator.

Table 10. In Vivo Efficacy in Dogs and Rabbits and Effect on Blood Pressure in Guinea Pigs

GP hypotension^a

dog A-V shunt^b

rabbit DVT^b,c

cmpd

N

ED_-25(mg/kg)

N

ED₅₀(mg/kg)

safety ratio^d(ED_-25:ED₅₀

)

N

ED₅₀(mg/kg, iv)

safety ratio^d(ED_-25:ED₅₀

)

3

5

4

2

5

0.9 ( 0.5

3

0.46 ( 0.10

0.14 ( 0.07

0.47 ( 0.05

0.66 ( 0.16

2

93

29

43

3

0.25 ( 0.03

0.43 ( 0.14

0.35 ( 0.12

0.21 ( 0.19

4

30

4

13.0 ( 6.8

13.4 ( 2.1

28.6 ( 1.6

68

1b

38

136

^aHypotensive response in anesthetized guinea pigs (GP). ED_-25is the dose (iv) for eliciting a 25% decrease in blood pressure (N ) 3).

^bDog arteriovenous shunt model. ED₅₀is the dose (iv) that reduces thrombus weight accumulation on a silk fiber by 50%. ^cRabbit deep

vein thrombosis model. ED₅₀is the dose (iv) that elicits a 50% reduction of thrombus formation (N ) 3). ^dCardiovascular safety estimation,

which is based on a comparison of the hypotensive response in anesthetized guinea pigs to the anticoagulant/antithrombotic response in

dogs or rabbits.

ment cascade because it was implicated as the source

of hypotension associated with DuP-714, a D-Phe-Pro-

based boroarginine thrombin inhibitor that turned out

to inhibit complement factor I, as well.⁴⁰Ultimately,

none of these mechanisms proved to be responsible, and

the cause of the cardiovascular side effects remains

unknown. For each possibility, a specific inhibitor or

receptor antagonist was given prior to intravenous

administration of 3 and blood pressure was monitored.

Nitric oxide was ruled out because L-NAME,⁴⁶an

inhibitor of nitric oxide synthesis, did not prevent the

hypotension. H1 and H2 histamine receptors were ruled

out because suitable antagonists did not prevent hy-

potension.⁴⁷Blockade of cholinergic activation with

atropine did not prevent hypotension.⁴⁸Inhibition of

complement with tranexamic acid⁴⁹and depletion of the

factors in the complement cascade with cobra venom

factor⁵⁰did not prevent hypotension. In addition, acute,

homologous desensitization did not occur.

Assessment of Advanced Leads. We evaluated 3,

4, and 68, as well as the reference standards efegatran

(1b) and argatroban, for selectivity vs seven important

enzymes: trypsin, factor Xa, plasmin, activated protein

C, streptokinase, tissue-type plasminogen activator

(tPA), and urokinase-type plasminogen activator (uPA)

(Table 9). Although 3, 4, and 68 are not as selective as

argatroban,^45bthey still show some worthwhile selectiv-

ity. Argatroban has a selectivity ratio of 470-fold in favor

of thrombin vs trypsin inhibition, whereas 3, 4, and 68

have modest selectivities at best: 16-, 1.4-, and 24-fold,

respectively. Generally speaking, poor selectivity for

thrombin over trypsin has been observed for inhibitors

with a D-Phe-Pro-Arg motif. Nevertheless, the selectivity

for 3, 4, and 68 is better than that for efegatran (try/

thr ) 0.5).^11a

generated in the canine model contain both platelets

and fibrin, whereas the thrombi generated in the rabbit

model are mainly comprised of fibrin. In the canine

arteriovenous shunt model (Figure 2), 4 was signifi-

cantly more potent than the other compounds, with an

ED₅₀value of 0.14 mg/kg (dose needed to elicit a 50%

inhibition of thrombus formation). Compounds 3, 68,

and efegatran (1b) had ED₅₀values of 0.46, 0.47, and

0.66 mg/kg, respectively. In addition, 3, 68, and efegat-

ran at a 1 mg/kg dose, as well as 4 at a 0.3-mg/kg dose,

had no effect on platelet counts, blood pressure, or heart

rate. In the rabbit deep vein thrombosis model (Figure

3), 3, 4, 68, and efegatran had ED₅₀values of 0.29, 0.30,

0.41, and 0.13 mg/kg, respectively.

We needed to estimate the cardiovascular side effect

liability of the lead compounds relative to their anti-

thrombotic efficacy. By way of a working therapeutic

index, we calculated a ratio that compares the ED₅₀

values of 3, 4, 68, and efegatran in each efficacy model

to the guinea pig hypotensive ED_-25values, which

reflect the dose needed to elicit a 25% reduction in blood

pressure (Table 10). The resulting “safety ratios” al-

lowed the lead compounds to be ranked. The safety

ratios using the dog AV shunt model for 4 and 68 were

93 and 29, respectively, which is a meaningful improve-

ment over 3 (safety ratio ) 3). These ratios compare

favorably with that for efegatran (safety ratio ) 43).

Similarly, safety ratios using the rabbit model for 4 and

68 were 30 and 38, whereas 3 had a safety ratio of only

4. Consequently, benzothiazole-6-carboxylic acid 4 was

deemed to have a favorable safety ratio, or therapeutic

index.

Blood samples taken after an iv dose of 1 mg/kg of 4

in anesthetized rats were used to determine percent

plasma thrombin inhibition and to extrapolate drug

concentration (µM). Compound 4 was found to have an

iv plasma t_1/2of 25 ( 7 min. Conscious rats were dosed

with 10 mg/kg of 4 by gavage, and an oral t_1/2of 60 (

16 min and oral bioavailability of only 3 ( 1% were

determined. As a consequence of the poor oral bioavail-

The in vivo anticoagulant/antithrombotic activity of

3, 4, and 68 was assessed in two different animal models

of thrombosis (iv administration): the canine arterio-

venous (AV) shunt model and the rabbit deep vein

thrombosis model (Table 10).¹⁶Blood clots (thrombi)

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1997

Figure 2. Effects of 4, 68, 3, efegatran (1b), argatroban, and heparin on thrombus weight in the canine arteriovenous shunt

model. Values are mean (( standard error) thrombus weight for N g 3 for each group. The thrombus weight was determined

following 15 min of exposure to shunt flow at each dose. Doses were administered as a bolus plus infusion over the 20 min shunt

flow period. The asterisk (/) indicates p < 0.05; i.e., the value is significantly different from the control thrombus weight.

Figure 3. Effects of 4, 68, 3, efegatran (1b), argatroban, and heparin on thrombus weight in the rabbit deep vein thrombosis

model. Vehicle and each dose of compound were tested in separate groups of animals. Values are mean (( standard error) of

percent inhibition of thrombus weight. The asterisk (/) indicates p < 0.05; i.e., the value is significantly different from the control

thrombus weight. The number of experiments (N) was 11 for the vehicle and as follows for the test compounds: 4, N ) 1 for 0.1

mg/kg, N ) 3 for 0.3 and 1 mg/kg; 68, N ) 3 for 0.1, 0.3, and 1.0 mg/kg; 3, N ) 7 for vehicle, N ) 3 for 0.1, 0.3, and 1 mg/kg; 1b,

N ) 3 for 0.1, 0.3, and 1 mg/kg; argatroban, N ) 3 for 0.3, 1, and 3 mg/kg; heparin, N ) 4 for 10, 30, and 100 U/kg bolus + 50%

dose/20-min infusion (iv).

ability of 4, there was a lack of interest in moving the

compound forward into preclinical development. In this

regard, it is interesting to note that a closely related

R-ketothiazole thrombin inhibitor exhibited reasonably

good oral bioavailability in rats (F ) 23%).^18l

X-ray Crystallography and Molecular Modeling.

We have reported on the X-ray crystal structures of the

ternary complexes of 3^15aand 4^15bwith thrombin-

hirugen (Figure 4).⁵¹Compounds 3 and 4 are structur-

ally analogous to PPACK (D-Phe-Pro-Arg-CH₂Cl), but

they have a methyl group on the N-terminus and a

2-benzothiazole instead of a chloromethyl group. The

overall interactions for the D-Phe-Pro-Arg segments in

the complexes of 3 and 4 are similar to those observed

in PPACK-thrombin (Figure 1).⁴Both inhibitors form

a hemiketal adduct between the Ser-195 hydroxyl and

the arginine carbonyl, and the tripeptide motif (P₃-P₂-

P₁) has the standard interactions previously observed

in thrombin-inhibitor complexes of this type. However,

the benzothiazole binds in an interesting manner at the

S₁′ subsite, which is mainly defined by His-57, Tyr-60A,

Trp-60D, and Lys-60F. The S₁′ subsite of thrombin,

dominated by Lys-60F,⁵²can accommodate smaller side

chains as observed from the P₁′ residues of several

natural substrates.⁵³These residues include Gly and

Thr and, in some cases, apolar residues, such as Val of

fibrinogen A, Leu-13 of protein C, and Ile-153 and Ile-

37 of factor VII and factor XI, respectively.^53d,eThe steric

interactions between substrates with Lys-60F in the S₁′

subsite create a preference for small P₁′ residues.^4,53The

benzothiazole rings of 3 and 4 hydrogen-bond within the

S₁′ subsite via their nitrogen atoms to His-57 and stack

in a face-to-edge manner with the indole ring of Trp-

60D (Figure 1). Consequently, the amino group of Lys-

1998 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Costanzo et al.

Figure 4. Stereoview of the 4-thrombin complex (white) overlapped with 3-thrombin complex (green). Hydrogens were omitted

for clarity. Steric interactions with the carboxylic acid moiety of 4 perturb the 60A-I insertion loop relative to 3-thrombin.

60F in both structures does not form a hydrogen bond

with the carbonyl oxygen of His-57. Usually, this

hydrogen bond is formed when the S₁′ subsite is either

unoccupied or occupied by a small residue such as Gly.⁴

Remarkably, 3 is a very potent thrombin inhibitor even

though the benzothiazole ring displaces the side chain

of Lys-60F from its normal, extended position such that

the aminobutyl group is folded into a U-shaped gauche

conformation (rmsd of side chain is 1.6 Å). In contrast,

the carboxylic acid substituent on the benzothiazole ring

of 4 forms a salt bridge with the NZ atom of Lys-60F,

thereby drawing it out into an extended anti conforma-

tion. It also hydrogen-bonds with a water molecule and

forms a salt bridge with the NZ atoms of the symmetry-

related residues Lys-236 and Lys-240. Steric interac-

tions with the carboxylic acid moiety of 4 perturb the

60A-I insertion loop relative to 3-thrombin (Figure 4).

Whereas the nonbonded distance between the ben-

zothiazole and the Trp-60D of 4-thrombin is shorter,

leading to stronger van der Waals interactions, the

remaining residues are more distant. Apparently, the

advantage in binding energy that would be conferred

by the salt bridge between Lys-60F NZ and the car-

boxylic acid of 4 is more than offset by perturbations in

the enzyme from steric interactions such that the

binding affinity of 4 (K_i) 2 nM) is reduced 10-fold

relative to that of 3 (K_i) 0.2 nM).

The SAR data for 3-77, along with the X-ray crystal-

lographic evidence with 3 and 4, suggest that the

improved affinity and selectivity relative to aldehyde

1b (efegatran) may be primarily due to two factors: (1)

the relative π-electron-withdrawing power of the ben-

zothiazole group and (2) the edge-to-face aromatic-

stacking interaction between the benzothiazole phenyl

group of 3 and the indole ring of Trp-60D. However, we

sought to determine if there might be some additional

factors that could contribute to the increased affinity/

selectivity of 3 relative to aldehyde 1b (efegatran).

Hence, we employed computer modeling to compare the

potential interactions between 3 and the active sites of

thrombin and trypsin with the aid of Goodford’s GRID

program.⁵⁴A three-dimensional grid of points (0.5 Å

cubes) was generated that encompassed the entire

active-site region of both enzymes. Probes possessing

the characteristics of various atom types (e.g., hydro-

phobic, polar, H-bonding, etc.) were placed at each grid

point, and the interaction energies with thrombin and

trypsin were calculated. These energy fields were then

contoured and compared to locate energetically favor-

able regions or “hot spots” that would favor a particular

atom type of an inhibitor in thrombin over trypsin. The

hydrophobic aromatic CH probe contoured at -2 kcal

proved to be particularly revealing (Figure 5). In

thrombin (panel A), the energy contour passes directly

through the thiazole ring of 3 and almost completely

envelops the sulfur atom, whereas in trypsin (panel B)

the thiazole ring and its sulfur atom are left completely

exposed, which is energetically less favorable. In con-

trast, a similar energy difference between these hydro-

phobic energy contours is not possible for aldehyde 1b

(efegatran). This viewpoint is supported by observa-

tions that 3 has better affinity and selectivity than

benzoxazole 55 and benzimidazole 53, and by the

increased affinity and selectivity of N-methylbenz-

imidazole 54 relative to benzimidazole 53 (Table 5).

Although the increased affinity and selectivity of 3 may

be mostly due to the aromatic-stacking interaction with

Trp-60D of the thrombin 60A-I insertion loop, it may

also be partly due to a hydrophobic interaction between

the thiazole ring of 3 and a hydrophobic patch on the

surface of thrombin. This binding mechanism for pep-

tidyl R-ketoheterocycle inhibitors and human thrombin

differs somewhat from the binding mechanisms reported

for human neutrophil elastase¹⁷and human mast cell

tryptase.^19jIn the case of neutrophil elastase, a deter-

mining factor for high affinity appears to be hydrogen

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1999

Figure 5. GRID contours for aromatic CH probe (-2 kcal) of 3 bound to thrombin (A) and trypsin (B). In thrombin (A), the

energy contour passes directly through the thiazole ring of 3 and almost completely envelops the sulfur atom, whereas in trypsin

(B) the thiazole ring and its sulfur atom are left completely exposed, which is energetically less favorable.

bonding between the sp²-hybridized nitrogen of the

heterocycle and the Nꢀ of His-57.¹⁷In the case of

tryptase, a determining factor for high affinity appears

to be the electrophilicity of the heterocycle-activated

ketone and/or a hydrophobic interaction with the Cys-

42/Cys-58 disulfide bond.^19j

selectivity for 3 may be due to interactions of the

benzothiazole sulfur atom with a hydrophobic patch on

the surface of thrombin. This binding mechanism for 3

and thrombin differs somewhat from those for peptide

R-ketoheterocycles and human neutrophil elastase¹⁷or

human mast cell tryptase.^19j

Conclusion

Experimental Section³²

We have applied structure-based drug design to the

discovery of reversible active-site-directed thrombin

inhibitors. Our efforts led to a series of potent R-keto-

heterocycle inhibitors primarily based on the D-Phe-Pro-

Arg motif (3-45, 50-72). Thrombin inhibition K_ivalues

as potent as 0.000 65 nM (13) were obtained, and

several compounds had subnanomolar affinities. We

extended the R-ketoheterocycle approach to other throm-

bin recognition motifs (73-77) to obtain thrombin K_i

values in the range 0.7-400 nM. Compound 3 and

several of its analogues caused hypotension and ECG

effects in guinea pigs, which were significantly dimin-

ished by introducing a carboxylic acid (4 and 12) or a

carboxylic ester (68) onto the benzothiazole.⁵⁵Ben-

zothiazolecarboxylic acid 4 was efficacious in two animal

models of thrombosis, and its therapeutic index com-

pared favorably with that of efegatran (1b). However,

since 4 has a low oral bioavailability in rats (F ) 3%),

its clinical use would be limited to parenteral admin-

istration. The crystallographic structure of a complex

between 4 and thrombin reveals novel interactions in

the S₁′ region, where the carboxylic acid moiety ap-

pended to the benzothiazole forms a salt bridge with

the side chain of Lys-60F. Surprisingly, formation of the

salt bridge did not result in enhanced thrombin affinity,

presumably because of perturbation of the enzyme to

accommodate the carboxylic acid group. The SAR data

for 3-77, along with the X-ray crystallographic evidence

for 3 and 4, suggest that their improved affinity/

selectivity relative to aldehyde congener 1b may be

primarily due to two factors: (1) the relative π-electron-

withdrawing power of the benzothiazole group and (2)

the edge-to-face aromatic stacking interaction between

the benzothiazole benzene ring of 3 and the indole of

Trp-60D. In addition, computational analysis with the

GRID program suggests that the improved affinity/

General Information and Procedures. Commercially

available reagents were used without purification unless

specifically necessary. All reactions were conducted under

argon or nitrogen in rigorously dried solvents with magnetic

stirring at room temperature unless noted otherwise. All

glassware used in the lithiation experiments was flame-dried

while assembled with an argon purge, and all butyllithium

reagents were assayed immediately before use by titration

with 1-pyreneacetic acid.⁵⁶A pH meter was used to monitor

pH adjustments. Multiple extractions are designated within

parentheses; for example “(3×)” indicates three extractions.

Solutions and residues were never heated above 40 °C while

concentrating in vacuo. Normal-phase preparative chroma-

tography was performed on an Isco Combiflash Separation

System Sg 100c equipped with a Biotage FLASH Si 40M silica

gel cartridge (KP-Sil silica, 32-63 µm, 60 Å; 4 cm × 15 cm),

eluting at 35 mL/min with detection at 254 nm. Reverse-phase

preparative chromatography was performed on a Waters

Delta-Prep 3000 HPLC equipped with three PrepPak reverse-

phase cartridges connected in series (Bondapak C-18; 40 mm

× 300 mm; 15-20 µm, 125 Å), eluting at 40 mL/min with

detection at 220 nm unless noted otherwise; gradient elutions

were generally conducted over a 60-min period. Reverse-phase

chromatography fractions were lyophilized on a FTS Systems

Flexi-Dry MP lyophilizer. Melting points were determined on

a Thomas-Hoover apparatus calibrated with a set of melting

point standards. Optical rotations were measured on a Perkin-

Elmer 241 polarimeter at the sodium D line (λ ) 589 nm) in

MeOH (c 1.00) unless noted otherwise. Infrared spectra were

recorded on a Nicolet SX-60 spectrometer in KBr pellets with

a resolution of 4 cm^-^{1 1}H NMR spectra were acquired at

.

300.14 MHz on a Bruker Avance-300 spectrometer in CD₃OD

unless indicated otherwise, using Me₄Si as an internal stan-

dard. NMR abbreviations used were the following: s, singlet;

d, doublet; dd, doublet of doublets; t, triplet; m, multiplet; br,

broad; ov, overlapping. ¹³C NMR spectra were acquired at

125.76 MHz at 40 °C on a Bruker Avance-500 spectrometer.

HPLC analysis was performed on a Hewlett-Packard series

1100 HPLC instrument, eluting with a gradient of water/

MeCN/TFA (from 10:90:0.2 to 90:10:0.2) over 4 min with a flow

rate of 0.75 mL/min on a Kromasil C18 column (50 mm × 2.0

2000 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Costanzo et al.

mm; 3.5 µm particle size) or on a Supelcosil ABZ+Plus column

(50 mm × 2.1 mm; 3.0 µm particle size) at 32 °C. Signals were

recorded simultaneously at 220, 254, and 305 nm with a diode

array detector; purity is reported for the 220 nm signal.

Electrospray (ES) mass spectra were obtained on a Micromass

Platform LC single quadrupole mass spectrometer in the

positive mode. Accurate mass determination (HRMS) was

performed on an Autospec E high-resolution magnetic sector

mass spectrometer tuned to a resolution of 6 K. The ions were

produced in a fast atom bombardment (FAB) source at an

accelerating voltage of 8 kV. Linear voltage scans were

collected to include the sample ion and two poly(ethylene

glycol) ions, which were used as internal reference standards.

Elemental analysis and Karl Fischer water (KF) analysis were

obtained from Robertson Microlit Laboratories, Inc., Madison,

NJ. Abbreviations used were the following: Boc ) tert-

butoxycarbonyl; BOP-Cl ) bis(2-oxo-3-oxazolidinyl)phosphinic

chloride; BOP reagent ) benzotriazol-1-yloxytris(dimethylami-

no)phosphonium hexafluorophosphate; Cbz ) benzyloxycar-

bonyl; DBU ) 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC )

dicyclohexylcarbodiimide; DIBAL ) diisobutylaluminum hy-

dride; DMAP ) 4-(dimethylamino)pyridine; DMF ) N,N-

dimethylformamide; DMSO ) dimethyl sulfoxide; EtOAc )

ethyl acetate; Fmoc ) 9-fluorenylmethylcarbonyl; HOAc )

glacial acetic acid; HOBT ) 1-hydroxybenzotriazole hydrate;

Mtr ) 4-methoxy-2,3,6-trimethylbenzenesufonyl; PyBrOP )

bromotris(pyrrolidino)phosphonium hexafluorophosphate; TFA

) trifluoroacetic acid; THF ) tetrahydrofuran.

(1S)- and (1R)-N-Methyl-^D-phenylalanyl-N-[4-[(ami-

noiminomethyl)amino]-1-(2-benzothiazolylcarbonyl)bu-

tyl]-^L-prolinamide (3 and 27). Imidate Route Starting

with a Tripeptidyl Aldehyde (Scheme 1). A mixture of

tripeptide aldehyde 3a^11a(9.92 g, 14.5 mmol), CH₂Cl₂(48 mL),

acetone cyanohydrin (4.0 mL, 43.4 mmol), and triethylamine

(1.2 mL, 8.7 mmol) was stirred for 3 h. An additional portion

of acetone cyanohydrin (1.3 mL, 14.2 mmol) was added

followed by another hour of stirring. The mixture was con-

centrated in vacuo and partitioned between water (125 mL)

and EtOAc (125 mL). The resulting organic layer was washed

with water (2×) and brine, dried (Na₂SO₄), and concentrated

in vacuo. The residue was triturated with with hexane (3×)

and the resulting solid was dried in vacuo to give the crude

cyanohydrin intermediate (9.73 g, 94%) as an off-white foam.

min. The resulting aqueous layer was isolated and extracted

with EtOAc (3×) and the combined organic extracts were

washed with successive portions of water and brine, dried (Na₂-

SO₄), and concentrated in vacuo to give the corresponding

protected ketone (2.90 g, 94%; CAS no. 179746-19-1) as a white

foam.

The protected ketone (2.97 g, 3.63 mmol) and anhydrous

anisole (10 mL) were placed in a Teflon reaction tube of an

HF apparatus and cooled to -78 °C. Anhydrous HF (15-20

mL) was condensed into the tube, and the temperature was

increased to 0 °C. The reaction mixture was stirred at 0 °C

for 2 h, concentrated in vacuo, and triturated with Et₂O (3×)

to give a yellow solid. This solid was purified by reverse-phase

HPLC, eluting with water/MeCN/TFA (70:30:0.2) to provide

diastereomers 3 and 27 as glasses. Each diastereomer was

dissolved in water and lyophilized to give 3 (1.11 g, 35%; slower

elution) and 27 (0.10 g; 3%; faster elution) as white foams.

Compound 3: [R]²⁰-72.5°; ¹H NMR δ 1.35-1.45 (m, 1H),

D

1.70-2.00 (m, 7H), 2.15-2.25 (m 1H), 2.40-2.50 (m, 1H), 2.70

(s, 3H), 3.00-3.10 (m, 1H), 3.25-3.35 (m, 2H), 3.40-3.50 (m,

1H), 4.35-4.45 (m, 2H), 5.60-5.70 (m, 1H), 7.25-7.37 (ov m,

5H), 7.55-7.65 (m, 2H), 8.10 (d, 1H, J ) 6.9 Hz), 8.20 (d, 1H,

J ) 6.9 Hz); MS (FAB) m/z 550.3 (MH)⁺. Anal. (C₂₈H₃₅N₇O₃S‚

2.3CF₃CO₂H‚0.8H₂O) C, H, N, F, H₂O. Compound 27: mp

110-120 °C; [R]²⁰-67.5° (c 0.67); IR ν_max1674, 1549, 1481,

D

1427, 1203 cm^-1; ¹H NMR δ 1.30-1.45 (m, 1H), 1.70-2.05 (m,

7H), 2.10-2.25 (m, 1H), 2.40-2.55 (m, 1H), 2.65 (s, 3H), 3.00-

3.10 (m, 1H), 3.20-3.50 (m, 3H), 4.30-4.45 (m, 2H), 5.68-

5.78 (m, 1H), 7.20-7.40 (ov m, 5H), 7.55-7.65 (m, 2H), 8.10

(d, 1H, J ) 6.9 Hz), 8.20 (d, 1H, J ) 6.9 Hz); MS (FAB) m/z

550.3 (MH)⁺. Anal. (C₂₈H₃₅N₇O₃S‚2.6CF₃CO₂H‚1.5H₂O) C, H,

N, H₂O.

N-Methyl-^D-phenylalanyl-N-[(1S)-4-[(aminoiminome-

thyl)amino]-1-(2-benzoxazolylcarbonyl)butyl]-^L-prolin-

amide (55). Imidate Route Starting with an Amino Acid

Aldehyde.^21cAnhydrous HCl gas (24 g) was bubbled into a

solution of 55a^12b(3.0 g, 6.3 mmol; CAS no. 186181-88-4) and

Gaseous HCl (51 g) was bubbled into a solution of the

cyanohydrin (9.3 g, 13.1 mmol) and anhydrous MeOH (200 mL)

at -50 to -70 °C under an atmosphere of argon over 1.5 h.

The reaction mixture was placed in a refrigerator at 0 °C for

2 days, transferred to a separatory funnel under argon, and

added dropwise at 0-6 °C to a stirred mixture of water (700

mL), EtOAc (250 mL), and NaHCO₃(159 g, 1.4 mol). The pH

of the quenching solution was not allowed to go below pH 6.8.

Additional portions of water and NaHCO₃were added (as

dictated by the measured pH) until the reaction mixture was

neutralized. The resulting mixture was filtered through a filter

agent, and the filtrate was extracted with EtOAc (3×). The

combined organic layers were washed with brine, dried (Na₂-

SO₄), and concentrated in vacuo to give the imidate 3b (7.51

g, 74%) as an off-white foam.

2-Aminothiophenol (2.32 g, 18.5 mmol) was added to a

degassed solution of 3b (7.23 g, 9.26 mmol) in absolute EtOH

(290 mL). The resulting mixture was heated at reflux for 4.75

h, cooled to room temperature, exposed to atmospheric oxygen

for 18 h, and concentrated in vacuo. The residue was purified

by chromatography on silica gel, eluting with CH₂Cl₂/MeOH

(9:1) to provide the alcohol intermediate 3c (3.33 g, 44%; CAS

no. 178926-05-1) as a white foam.

anhydrous MeOH (60 mL) at -78 °C at a rate such that the

temperature did not exceed -40 °C. After the addition, the

reaction mixture was stirred for 30 h at 0 °C. The reaction

mixture was added to vigorously stirred saturated aqueous

NaHCO₃while maintaining the pH above 6.8. The mixture

was extracted with EtOAc (100 mL), the layers were separated,

and the aqueous layer was extracted with EtOAc (3×). The

combined EtOAc extracts were washed with water (2×), dried

(Na₂SO₄), and concentrated in vacuo to give 55b (CAS no.

179746-31-7) as a white solid; MS (FAB) m/z 506.2 (MH)⁺. A

solution of 55b (1.00 g, 1.84 mmol), 2-aminophenol (0.22 g,

2.03 mmol), and absolute EtOH (40 mL) was heated at reflux

under N₂for 24 h and concentrated in vacuo. The residue was

purified by normal-phase chromatography, eluting with CH₂-

Cl₂/MeOH (95:5) to obtain 55c as a white foam (1.04 g, 100%;

The Dess-Martin periodinane (1.59 g, 3.76 mmol) was

added to a solution of 3c (3.08 g, 3.76 mmol) in CH₂Cl₂(125

mL). The resulting mixture was stirred for 45 min, and an

additional portion of periodinane (1.2 g, 2.83 mmol) was added,

followed by another 15 min of stirring. The excess periodinane

was consumed by the addition of 300 mL of quench solution

(75 g of Na₂S₂O₃in 300 mL of saturated aqueous NaHCO₃),

diluted with EtOAc (300 mL), and vigorously stirred for 15

CAS no. 179746-32-8); mp 81-91 °C; [R]²⁵-2.9° (c 0.68,

D

CHCl₃). Anal. (C₂₈H₃₁N₅O₆S‚0.6H₂O) C, H, N, H₂O.

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2001

A mixture of benzoxazole 55c (0.87 g, 1.53 mmol), 20% Pd-

(OH)₂on activated carbon (0.21 g), and absolute EtOH (19 mL)

was placed on an atmospheric hydrogenator and stirred under

hydrogen for 16 h. The resulting mixture was filtered through

filter agent and concentrated in vacuo to give 55d as pale-

yellow foam (0.47 g, 71%; CAS no. 179746-33-9). Compound

55d (0.47 g, 1.09 mmol) was converted to 55 according to the

methods described for 4 to furnish benzoxazole 55 (0.054 g)

dissolved in 140 mL of TFA/CH₂Cl₂(1:4), stirred for 2 h, and

concentrated in vacuo. The residue was diluted with CH₂Cl₂

and concentrated under high vacuum to give the trifluoro-

acetate salt of amino alcohols 4b (2.55 g, 91%) as a light-yellow

solid. A solution of 4b (2.55 g, 4.25 mmol) and HOBT (0.57 g,

4.25 mmol) in MeCN (196 mL) was neutralized by the addition

of triethylamine (ca. 2 mL) under N₂. DCC (2.63 g, 12.8 mmol)

and 4c (0.41 g, 4.25 mmol) were added, and the reaction

mixture was stirred over 18 h. The reaction mixture was

filtered through filter agent, and the filtrate was concentrated

in vacuo and dissolved in EtOAc. The EtOAc solution was

extracted with 10% aqueous citric acid (2×), saturated aqueous

NaHCO₃(2×), and brine (2×), dried (Na₂SO₄), and concen-

trated in vacuo. The residue was purified by normal-phase

chromatography, eluting with CH₂Cl₂/MeOH (19:1) to give the

coupled product 4d (3.05 g, 80%) as a solid.

as a white powder: mp 44-54 °C; [R]²⁵-63.3° (c 0.58); IR

D

ν_max3407, 1676, 1204, 1137 cm^-1; ¹H NMR [(CD₃)₂CO] δ 1.35-

1.45 (m, 1H), 1.07-2.00 (ov m, 8H), 2.15 (s, 2.9H), 2.25 (s,

0.1H), 2.40-2.50 (m, 1H), 3.20-3.65 (ov m, 4H), 4.40-4.50 (m,

1H), 4.50-4.60 (m, 1H), 5.45-5.55 (m, 1H), 7.30-7.40 (m, 5H),

7.55 (dd, 1H, J ) 7.3, 7.3 Hz), 7.64 (dd, 1H, J ) 7.3, 7.3 Hz),

7.79 (d, 1H, J ) 7.3 Hz), 7.93 (d, 1H, J ) 7.3 Hz); MS (FAB)

m/z 534.3 (MH)⁺. Anal. (C₂₈H₃₅N₇O₄‚0.4CF₃CO₂H‚0.25H₂O) C,

H, N, H₂O.

A solution of anhydrous LiOH (1.27 g, 52.9 mmol) in water

(10 mL) was added to a stirred solution of 4d (2.26 g, 2.52

mmol) in 1,4-dioxane (100 mL) and stirred for 4 h. The

resulting mixture was acidified to pH 3.0 with 10% aqueous

citric acid and extracted with EtOAc (3×). The combined

organic extracts were washed with brine (2×), dried (Na₂SO₄),

and concentrated in vacuo. The residue (2.09 g, 2.36 mmol)

was dissolved in CH₂Cl₂(40 mL) and treated with the Dess-

Martin periodinane (1.50 g, 3.53 mmol). After 2 h, the reaction

mixture was treated with 100 mL of 10% aqueous Na₂S₂O₃

and stirred for 15 min. The resulting aqueous layer was

isolated and extracted with CH₂Cl₂(3×). The combined organic

extracts were washed with water (2×), dried (Na₂SO₄), and

concentrated in vacuo to furnish a white foam. This foam was

combined with anhydrous anisole (12 mL) in a Teflon reaction

tube, placed on a HF apparatus, and cooled to -78 °C.

Anhydrous HF (ca. 24 mL) was condensed into the reaction

tube, the temperature of the mixture was raised to 5 °C, and

the mixture was stirred for 5.5 h. The reaction mixture was

concentrated in vacuo and triturated three times with Et₂O

(50 mL). The resulting tan solid was purified by reverse-phase

HPLC, eluting with water/MeCN/TFA (90:10:0.2) to afford 4

N-Methyl-^D-phenylalanyl-N-[(1S)-4-[(aminoiminome-

thyl)amino]-1-[(6-carboxy-2-benzothiazolyl)carbonyl]bu-

tyl]-^L-prolinamide (4). Weinreb Amide Route (Scheme

2). A solution of n-butyllithium (103 mL of 2.08 M in hexane,

214 mmol) was added dropwise to a stirred solution of

benzothiazole-6-carboxylic acid (19.7 g, 111 mmol) in THF

(1,386 mL) at -78 °C at a rate that kept the reaction

temperature below -70 °C. On completion of addition, the

dark-red reaction mixture was stirred for 34 min at -78 °C

and a solution of 4a²⁴(4.36 g, 9.25 mmol) in THF (185 mL)

was added dropwise over 15 min at a rate that maintained

the reaction temperature below 70 °C. After the addition, the

temperature was immediately raised to -20 °C and main-

tained at -24 to -20 °C for 1.5 h. The reaction was quenched

with saturated aqueous NH₄Cl (790 mL), the layers were

separated, and the aqueous layer was extracted with EtOAc

(3×). The combined organic extracts were washed with water

(2×) and brine (2×), dried (Na₂SO₄), filtered through a filter

agent, and concentrated in vacuo to give crude 4e as a solid

(5.50 g, 100% yield; CAS no. 179746-75-9).

(0.76 g, 37%) as an off-white solid: mp 110-125 °C; [R]²⁵

D

1

-79.6°; IR ν_max3374, 1674, 1203 cm^-1; H NMR δ 1.40-1.45

(m, 1H) 1.73-1.97 (m, 8H), 2.15-2.25 (m, 1H), 2.50-2.60 (m,

1H), 2.70 (s, 3H), 3.00-3.10 (m, 1H), 3.20-3.30 (m, 1H), 3.40-

3.50 (m, 1H), 4.35-4.45 (m, 2H), 5.60-5.70 (m, 1H), 7.25-

7.37 (ov m, 5H), 8.25 (ov s, 2H), 8.82 (s, 1H); MS (FAB) m/z

594.4 (MH)⁺. Anal. (C₂₉H₃₅N₇O₅S‚2.5CF₃CO₂H‚1.0H₂O) C, H,

N, F, H₂O.

D-Phenylalanyl-N-[(1S)-4-[(aminoiminomethyl)amino]-

1-(2-benzothiazolylcarbonyl)butyl]-^L-prolinamide (5). A

solution of butyllithium (360 mL of 0.58 M in hexane, 0.207

mol) was added dropwise over 30 min to a stirred solution of

freshly distilled benzothiazole (103.1 g, 0.763 mol) in THF (2.5

L) at -78 °C under argon at a rate that kept the reaction

temperature below -70 °C. Upon completion of addition, the

reaction mixture was stirred for 30 min at -70 °C and a

solution of 4a²⁴(18.0 g, 38.2 mmol; Scheme 2) in THF (1.1 L)

was added dropwise over 50 min at a rate that maintained

the reaction temperature below -70 °C. After the addition,

the reaction mixture was stirred at -70 °C for 2 h and poured

rapidly into saturated aqueous NH₄Cl (2.0 L) and vigorously

stirred for 30 min. Ethyl acetate (500 mL) was added, the

mixture was stirred for 15 min, and the layers were separated.

The aqueous layer was extracted with EtOAc (500 mL) and

the combined organic extracts were washed with water (3 ×

800 mL) and brine (3 × 800 mL), dried (Na₂SO₄), filtered

through filter agent, and concentrated in vacuo to afford a

brown oil. This oil was partially purified by normal-phase

chromatography, eluting with EtOAc/hexane (5:1) followed by

trituration with hexane (4 × 500 mL) to provide 5a as an

amber glass (23.0 g, 110%).

Sodium borohydride (2.12 g, 56.0 mmol) was added in one

portion to a solution of the above crude acid (5.50 g, 9.34 mmol)

in MeOH (140 mL) at -25 °C, and the reaction mixture was

stirred at -25 to -20 °C for 1 h. The reaction was quenched

by the addition of acetone (31 mL), and the mixture was stirred

for 15 min and concentrated in vacuo. The residue was

dissolved in water (100 mL), extracted with EtOAc (3×), and

acidified to pH 3.0 with 10% aqueous citric acid. The acidic

aqueous layer was extracted with EtOAc (3×) and the com-

bined organic extracts were washed with brine (2×), dried

(Na₂SO₄), and concentrated in vacuo to give crude 4f as a light-

yellow solid (4.00 g, 72%). Compound 4f (9.70 g, 16.4 mmol)

was dissolved in CH₂Cl₂/MeOH (17:3), cooled to 0 °C, and

treated dropwise with (trimethylsilyl)diazomethane (2 M in

hexane, 56 mL, 112 mmol) over 30 min. After 1 h, the yellow

reaction mixture was concentrated in vacuo and purified by

normal-phase chromatography, eluting with CH₂Cl₂/MeOH (9:

1) to give 4g as an off-white foam (2.93 g, 30%; CAS no.

186182-15-0). A solution of 4g (2.73 g, 4.51 mmol) was

Compound 5a (20.8 g, 38 mmol) was dissolved in MeOH (700

mL), cooled to -20 °C, treated with sodium borohydride (4.35

g, 115 mmol), and stirred at -20 °C for 30 min. The reaction

mixture was quenched by the addition of acetone (100 mL)

and warmed to room temperature over 30 min. The reaction

2002 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Costanzo et al.

saturated with NaCl. Ethyl acetate (100 mL) was added, and

the aqueous layer was adjusted to pH 4.0 with 25% aqueous

citric acid (CAUTION: evolution of CO₂) while stirring at 5

°C. The layers were separated, and the acidic aqueous layer

was extracted with EtOAc (5×). The combined EtOAc extracts

were dried (MgSO₄), filtered through filter agent, and concen-

trated in vacuo to give 28f (3.70 g, 56%; CAS no. 179746-65-

7) as a clear glass: FAB-MS m/z 381 (MH)⁺.

mixture was concentrated in vacuo, and the green residue was

partitioned between EtOAc (300 mL) and water (150 mL). The

aqueous layer was extracted with EtOAc (2 × 300 mL), and

the combined organic extracts were washed with brine (2 ×

50 mL), dried (Na₂SO₄), and decolorized with activated carbon.

The resulting solution was filtered through filter agent and

concentrated in vacuo to provide 5b as a light-yellow solid (23

g, 110%; CAS no. 186181-82-8). Compound 5b was added to a

mixture of CH₂Cl₂(300 mL) and TFA (75 mL). The resulting

solution was stirred for 2.5 h and concentrated in vacuo. The

residue was triturated with Et₂O (2 × 1 L) to furnish 5c as a

yellow solid (21.3 g, 82.6%; CAS no. 179746-24-8). A solution

containing 5c (0.796 g, 1.29 mmol), N-carbobenzyloxy-^D-

phenylalanyl-^L-proline (0.509 g, 1.29 mmol), and HOBT (0.265

g, 1.96 mmol) in MeCN (25 mL) was adjusted to pH 7-8 by

the addition of triethylamine (approximately 500 µL) under

argon. DCC (0.404 g, 1.96 mmol) was added, and the reaction

mixture was stirred over 18 h. The reaction mixture was

filtered through filter agent, and the filtrate was concentrated

in vacuo and dissolved in EtOAc. The EtOAc solution was

extracted with 10% aqueous citric acid (2×), saturated aqueous

NaHCO₃(2×), and brine (2×), dried (Na₂SO₄), and concen-

trated in vacuo. The residue was purified by normal-phase

chromatography, eluting with CH₂Cl₂/MeOH (19:1) to give 5d

(0.85 g, 80%) as a solid.

Compound 28f (6.86 g, 18 mmol) was dissolved in 180 mL

of MeOH and combined with 20% Pd(OH)₂on activated carbon

(1.37 g) and placed under hydrogen pressure (60 psig) on a

Parr hydrogenator for 18 h. The reaction mixture was filtered,

and the filter cake was extracted with four 150 mL portions

of hot MeOH (CAUTION). The combined MeOH extracts were

filtered through filter agent and concentrated in vacuo to give

28b (3.50 g, 79%) as a white solid: FAB-MS m/z 247 (MH)⁺.

Compound 28b (3.40 g, 13.8 mmol) was combined with

triethylamine (5.77 mL, 41.4 mmol) in DMF (75 mL), and to

the resulting suspension was added 1,3-bis(benzyloxycarbo-

nyl)-2-methyl-2-thiopseudourea (7.42 g, 20.7 mmol; CAS no.

25508-20-7). The resulting mixture was stirred for 1.7 days,

treated with triethylamine (2.0 mL, 14.4 mmol) and 1,3-bis-

(benzyloxycarbonyl)-2-methyl-2-thiopseudourea (0.78 g, 2.2

mmol), stirred for 2 days, treated again with triethylamine

(2.0 mL, 14.4 mmol) and 1,3-bis(benzyloxycarbonyl)-2-methyl-

2-thiopseudourea (1.48 g, 4.1 mmol), and stirred at 22 °C for

3 days. The crude reaction mixture was filtered through filter

agent, and the DMF was removed in vacuo. The oily residue

was partitioned between 0.1 N NaOH (200 mL) and Et₂O (200

mL). The basic aqueous layer was extracted with Et₂O (4×),

cooled to 5 °C, and adjusted to pH 3.5 with citric acid. The

acidic aqueous layer was extracted with EtOAc (5×), and the

combined EtOAc extracts were dried (Na₂SO₄), filtered through

filter agent, and concentrated in vacuo. The residue was

purified by normal-phase chromatography, eluting with CH₂-

Cl₂/MeOH (95:5) to give 28c (5.94 g, 77%; CAS no. 179746-

67-9) as a white foam: FAB-MS m/z 557 (MH)⁺.

A solution of 28c (5.86 g, 10.5 mmol) in 210 mL of MeOH

was treated with 20% Pd(OH)₂on activated carbon (1.47 g),

and the mixture was placed under 60 psig of hydrogen pressure

on a Parr hydrogenator. After 24 h, another portion of 20%

Pd(OH)₂on activated carbon (0.74 g) was added, and the

reaction was placed under 60 psig of hydrogen for 6 h. The

crude reaction mixture was filtered through filter agent and

concentrated in vacuo to afford 28g (3.03 g, 100%; CAS no.

179746-68-0) as a white foam: FAB-MS m/z 289 (MH)⁺.

Compound 28g (0.52 g, 1.80 mmol) was dissolved in 2.7 mL of

4 N KOH, diluted with 9.0 mL of acetone, and cooled to -18

°C. To this solution was added a solution of p-toluenesulfonyl

chloride (0.68 g, 3.60 mmol) in acetone (3.6 mL) over 15 min

while stirring vigorously. After 1 h at -18 to -15 °C, the

reaction was slowly warmed to 22 °C over 2.5 h. The acetone

was removed in vacuo at 20 °C, and the aqueous residue was

diluted with 25 mL of water and extracted with Et₂O (3×).

The aqueous layer was saturated with NaCl, layered with

EtOAc, cooled to 5 °C, and adjusted to pH 3.5 with 1 N HCl.

The acidic aqueous layer was extracted with EtOAc (4×), and

Compound 5d (0.75 g) was oxidized with the Dess-Martin

periodinane and deprotected with anhydrous HF as described

for compound 4. The resulting solid was purified by reverse-

phase HPLC, eluting with water/MeCN/TFA (70:30:0.2) to give

5 (0.24 g) as a white solid: [R]²⁵-79.0°; IR ν_max2923, 1328,

D

1153, 582 cm^-1; ¹H NMR δ 1.50 (m, 1H), 1.70-2.05 (ov m, 7H),

2.60-2.70 (m, 1H), 3.10-3.20 (m, 2H), 3.21-3.30 (m, 2H),

3.40-3.50 (m, 1H), 4.35-4.45 (m, 2H), 5.60-5.70 (m, 1H),

7.20-7.30 (m, 2H), 7.31-7.40 (m, 3H), 7.55-7.65 (m, 2H), 8.10

(d, 1H, J ) 6.9 Hz), 8.20 (d, 1H, J ) 6.9 Hz); MS (FAB) m/z

536 (MH)⁺. Anal. (C₂₇H₃₃N₇O₃S‚2.36CF₃CO₂H‚0.85H₂O) C, H,

N, F, H₂O.

N-Methyl-^D-phenylalanyl-N-[(1S)-4-[(aminoiminome-

thyl)amino]-1-(2-benzothiazolylcarbonyl)-1-methylbutyl]-

L-prolinamide (28, Scheme 3). A suspension of 28a²⁷(7.03

g, 35 mmol) in 60 mL of water was cooled to 5 °C, and the pH

was adjusted to 11.0 with 3 N NaOH. Benzyl chloroformate

(6.00 mL, 42 mmol) was added dropwise over 4 h while stirring

at 5 °C. The reaction mixture was slowly warmed to 22 °C

over 16 h and then extracted with Et₂O (3×). The aqueous

layer was cooled to 5 °C, adjusted to pH 2.0 with 3 N HCl,

and extracted with EtOAc (3×). The cold acidic aqueous layer

was readjusted to pH 11.0 with 3 N NaOH and diluted with

175 mL of 1,4-dioxane. Di-tert-butyl dicarbonate (38.2 g, 175

mmol) was added in one portion at 5 °C while stirring. The

reaction mixture was slowly warmed to 22 °C over 16 h, the

pH was adjusted to 11.0 with 3 N NaOH, and another portion

of di-tert-butyl dicarbonate (38.2 g, 175 mmol) was added and

stirred at 22 °C over 24 h. The 1,4-dioxane was removed in

vacuo, and the reaction mixture was adjusted to pH 11 with 3

N NaOH, extracted with Et₂O (3×), cooled to 5 °C, and

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2003

the combined EtOAc extracts were washed with saturated

aqueous NaCl (2×), dried (Na₂SO₄), and concentrated in vacuo

to give 28h (0.72 g, 90%; CAS no. 179746-69-1) as a white

foam; FAB-MS m/z 443 (MH)⁺. Compound 28h was dissolved

in 29 mL of CH₂Cl₂/MeOH (9:1), cooled to 5 °C, and treated

dropwise over 10 min with 2 M (trimethylsilyl)diazomethane

in hexane (4.3 mL, 8.63 mmol). After 10 min, the reaction

mixture was concentrated in vacuo at 22 °C and the residue

was purified by normal-phase chromatography, eluting with

EtOAc/hexane (3:2) to give 28d (1.06 g, 81%) as a white foam:

FAB-MS m/z 457 (MH)⁺.

MS (FAB) m/z 564.3 (MH)⁺. Anal. (C₂₉H₃₇N₇O₃S‚2.3CF₃CO₂H‚

1.9H₂O) C, H, N, F. H₂O: calcd 3.98; found 3.40.

(1S)- and (1R)-N-Methyl-^D-phenylalanyl-N-[1-[[4-trans-

(aminomethyl)cyclohexyl]methyl]-2-(2-benzothiazolyl)-

2-oxoethyl]-L-prolinamide (41 and 42, Scheme 4). A slurry

of 41a (5.00 g, 31.8 mmol; CAS no. 1197-18-8; Scheme 4) in

MeOH (50 mL) was treated with 4 M HCl in 1,4-dioxane, and

the resulting solution was stirred for 18 h. The reaction

mixture was concentrated in vacuo, dissolved in CH₂Cl₂(50

mL), adjusted to pH 7-8 with triethylamine, and treated with

4-methoxy-2,3,6-trimethylbenzenesulfonyl chloride (7.89 g,

31.8 mmol) while cooling with a 23 °C water bath. After 4 h,

the reaction mixture was filtered through filter agent and the

filtrate was washed with 1 M KHSO₄(3×), saturated aqueous

NaHCO₃(2×), and brine, dried (Na₂SO₄), filtered, and con-

centrated in vacuo to give 41b (11.4 g, 94%).

A solution of 41b (11.4 g, 29.7 mmol) in THF (150 mL) was

cooled to 0 °C and treated dropwise with 1 M LiAlH₄in THF

(60 mL, 60 mmol) over 45 min. After the addition, the reaction

mixture was slowly warmed to room temperature over 18 h.

The reaction mixture was quenched with excess 1 N NaOH

(CAUTION), filtered through filter agent, and concentrated

in vacuo. The residue was dissolved in EtOAc, extracted with

1 N HCl (2×) and brine, dried (Na₂SO₄), filtered, and concen-

trated in vacuo to afford 41e (8.09 g, 77%). A solution of 41e

A solution of benzothiazole (2.63 g, 19.5 mmol) in 20 mL of

THF was cooled to -75 °C with stirring under nitrogen.

Butyllithium (1.6 M in hexane, 9.46 mL, 15.1 mmol) was added

dropwise over 15 min at -75 to -65 °C and stirred for 15 min.

To this solution was added dropwise over 15 min a solution of

28d (0.46 g, 1.00 mmol) in 10 mL of THF at -75 to -70 °C.

The reaction mixture was stirred at -75 °C for 2 h, and the

reaction was quenched with 160 mL of saturated aqueous NH₄-

Cl, saturated with NaCl, and extracted with EtOAc (3×). The

combined EtOAc extracts were washed with saturated aqueous

NaCl (3×), dried (Na₂SO₄), and concentrated in vacuo. The

residue was triturated with hexane (3×), dissolved in 150 mL

of EtOAc/hexane (3:2), filtered through filter agent, and

purified by normal-phase chromatography, eluting with EtOAc/

hexane (3:2) to give 0.42 g (74%) of 28i (0.42 g, 74%; CAS no.

179746-71-5) as clear glass: FAB-MS m/z 560 (MH)⁺. A

solution of 28i (0.40 g, 0.72 mmol) in MeOH (14 mL) was cooled

to -20 °C while stirring and treated with NaBH₄(0.081 g, 2.14

mmol). After 2.5 h, acetone (2.6 mL) was added and the

reaction mixture was warmed to room temperature over 30

min. The reaction mixture was concentrated in vacuo and

partitioned between water (15 mL) and EtOAc (30 mL). The

aqueous layer was extracted with EtOAc (2×), and the

combined organic extracts were washed with brine (2×), dried

(Na₂SO₄), and concentrated in vacuo to give 28j (0.40 g, 99%)

as a tan foam. This material (0.39 g, 0.69 mmol) was dissolved

in 14 mL of TFA/CH₂Cl₂(1:4), stirred for 2 h, and concentrated

in vacuo at 10 °C. The residue was partitioned between 20

mL of 1 N NaOH previously saturated with NaCl and of CH₂-

Cl₂(20 mL). The basic aqueous layer was extracted with CH₂-

Cl₂(3×), and the combined CH₂Cl₂extracts were washed with

saturated aqueous NaCl, dried (Na₂SO₄), and concentrated in

vacuo to furnish 28e (0.32 g, 100%) as a brown amorphous

solid: FAB-MS m/z 462 (MH)⁺.

A solution of 4c (0.309 g, 0.753 mmol) and diisopropylethyl-

amine (132 µL, 0.758 mmol) in 10 mL of anhydrous CH₂Cl₂

was cooled to 5 °C while stirring under argon. Bis(2-oxo-3-

oxazolidinyl)phosphinic chloride (BOP-Cl; 0.193 g, 0.758 mmol)

was added, and the reaction mixture was stirred for 15 min.

A solution of 28e (0.316 g, 0.685 mmol) and diisopropylethyl-

amine (119 µL, 0.684 mmol) in 10 mL of anhydrous CH₂Cl₂

was added over 10 min at 5 °C, and the reaction mixture was

slowly warmed to 22 °C over 4.5 h. The reaction mixture was

concentrated in vacuo at 40 °C, and the residue was partitioned

between EtOAc and 1 M KHSO₄. The organic layer was

extracted two more times with 1 M KHSO₄, three times with

saturated aqueous NaHCO₃, and twice with saturated aqueous

NaCl, dried (Na₂SO₄), and concentrated in vacuo at 40 °C. The

residue was purified by normal-phase chromatography, eluting

with EtOAc/hexane (4:1) to give (1S)-N-methyl-N-[(phenyl-

methoxy)carbonyl]-^D-phenylalanyl-N-[1-(2-benzothiazolylhy-

droxymethyl)-4-[[imino[[(4-methylphenyl)sulfonyl]amino]methyl]-

amino]-1-methylbutyl]-^L-prolinamide (0.381 g, 65%; CAS no.

186182-14-9) as a white foam: FAB-MS m/z 855 (MH)⁺. This

substance was converted to 28 according to the methods

described for 5. The resulting crude material was purified by

reverse-phase HPLC, eluting with water/MeCN/TFA (60:40:

0.2) to afford 28 as a white solid (0.094 g): mp 122-130 °C;

[R]²⁵_D-85.3° (c 0.26); IR ν_max3375, 1679, 1203 cm^-1; ¹H NMR

δ 1.05-1.15 (m, 2H), 1.4-1.8 (ov m, 7H), 2.10-2.20 (m, 1H),

2.21-2.29 (m, 1H), 2.40-2.50 (m, 1H), 2.58 (s, 2.4H), 2.55 (s,

0.2H), 2.95-3.05 (m, 1H), 3.1-3.2 (m, 4H), 4.25-4.35 (m, 2H),

7.15-7.3 (ov m, 5H), 7.50-7.60 (m, 2H), 8.05-8.15 (m, 2H);

(8.09 g, 22.8 mmol) in CH₂Cl₂(100 mL) was treated with the

Dess-Martin periodinane (14.5 g, 34.1 mmol). After 2 h, 75

mL of the quench solution (25 g of Na₂S₂O₃in 100 mL of

saturated aqueous NaHCO₃) was added and the reaction

mixture was stirred for 18 h. The layers were separated, and

the aqueous layer was extracted with CH₂Cl₂. The combined

organic extracts were washed with brine, dried (MgSO₄), and

concentrated in vacuo. The residue was purified by normal-

phase chromatography, eluting with hexane/EtOAc (7:3) to

give 41c (7.52 g, 93%). A slurry of 41c (2.50 g, 7.07 mmol),

ammonium carbonate (2.65 g, 27.5 mmol), and sodium cyanide

(0.69 g, 10.6 mmol) in 14 mL of EtOH/water (1:1) was heated

at reflux for 4 h. The reaction mixture was cooled to room

temperature and acidified to pH 3 with 1 N HCl. A white

precipitate formed, which was isolated by filtration and

recrystallized from MeOH (1.35 g, 45%) to give 41f. A slurry

of 41f in 4 N NaOH (25 mL) was heated at reflux for 18 h,

cooled to 0 °C, and treated with di-tert-butyl dicarbonate (0.393

g, 1.80 mmol). The reaction mixture was slowly warmed to

room temperature over 18 h, cooled to 5 °C, acidified to pH 3

with citric acid, and extracted with EtOAc (3×). The combined

organic extracts were washed with brine, dried (Na₂SO₄),

filtered, and concentrated in vacuo to furnish 41g (0.605 g,

76%).

A solution of 41g (0.605 g, 1.22 mmol), N,O-dimethylhydroxyl-

amine‚HCl (0.170 g, 1.72 mmol), and triethylamine (323 µL,

2.32 mmol) in CH₂Cl₂(30 mL) was treated with BOP-Cl (0.443

g, 1.74 mmol). After 4 h, the reaction mixture was quenched

2004 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

Costanzo et al.

with water (15 mL). The layers were separated and the organic

layer was extracted with water, 10% aqueous citric acid (2×),

saturated aqueous NaHCO₃(2×), and brine, dried (MgSO₄),

filtered, and concentrated in vacuo to afford 41d (0.585, 93%).

This material was converted to a mixture of 41 and 42

according to the methods described for 37 and 38.³²The

resulting crude solid was purified by reverse-phase HPLC,

eluting with water/MeCN/TFA (from 80:20:0.2 to 70:30:0.2) to

afford 41 (0.041 g; slower elution) and 42 (0.046 g, faster

elution) as white solids. Compound 41: ¹H NMR δ 1.05-1.15

(m, 2H), 1.35-1.45 (m, 2H), 1.60-2.00 (ov m, 8H), 2.10-2.20

(m, 2H), 2.40-2.50 (m, 1H), 2.70 (s, 3H), 2.70-2.80 (m, 2H),

3.00-3.10 (m, 1H), 3.20-3.30 (m, 1H), 3.35-3.45 (m, 1H),

4.45-3.55 (m, 2H), 5.60-5.70 (m, 1H), 7.20-7.30 (m, 2H),

7.31-7.40 (m, 3H), 7.60-7.70 (m, 2H), 8.10 (d, 1H, J ) 7.1

Hz), 8.20 (d, 1H, J ) 7.1 Hz); MS (ES) m/z 562.6 (MH)⁺. Anal.

(C₃₁H₃₉N₅O₃S‚2.3CF₃CO₂H‚0.09HBr‚1.7H₂O) C, H, N, F. Br:

calcd 0.83; found 0.50. H₂O: calcd 3.55; found 2.46. Compound

42: ¹H NMR δ 1.00-1.10 (m, 2H), 1.20-1.40 (ov m, 3H), 1.50-

1.60 (m, 1H), 1.80-1.90 (m, 6H), 1.95-2.05 (m, 1H), 2.10-

2.20 (m, 1H), 2.45-2.55 (m, 1H), 2.65 (s, 3H), 2.70-2.80 (m,

2H), 3.00-3.10 (m, 1H), 3.20-3.30 (m, 1H), 3.35-3.45 (m, 1H),

4.35-4.45 (m, 1H), 4.40-4.50 (m, 1H), 5.75-5.80 (m, 1H),

7.20-7.25 (m, 2H), 7.30-7.35 (m, 3H), 7.60-7.70 (m, 2H), 8.15

(d, 1H, J ) 7.1 Hz), 8.20 (d, 1H, J ) 7.1 Hz); MS (ES) m/z

562.6 (MH)⁺. Anal. C₃₁H₃₉N₅O₃S‚2.27CF₃CO₂H‚0.05HBr‚

1.6H₂O) C, H, N, Br, F, H₂O.

N-Methyl-^D-phenylalanyl-N-[(1S)-1-(2-benzothiazolyl-

carbonyl)-4-methoxybutyl]-^L-prolinamide (43, Scheme

5). A solution of 5-methoxypentanoic acid 43b⁶²(6.80 g, 52

mmol) and triethylamine (6.80 g, 67 mmol) in 100 mL of

anhydrous THF was cooled to -78 °C while stirring under

argon. Pivaloyl chloride (6.80 g, 57 mmol) was added to the

reaction mixture dropwise over 15 min at -78 °C. After 15

min the reaction mixture was warmed to 0 °C, stirred for 1.5

h, and then recooled to -78 °C. Meanwhile, 1.6 M BuLi in

hexane (58 mL, 93 mmol) was added dropwise to a solution of

(4S,5R)-(-)-4-methyl-5-phenyl-2-oxazolidinone (16.4 g, 93 mmol)

in 100 mL of THF over 30 min at -78 °C. After 15 min, this

reaction mixture was added slowly to the above mixed-

anhydride reaction while stirring under argon at -78 °C. The

reaction mixture was slowly warmed to room temperature over

18 h, quenched with 1 N KHSO₄, and concentrated in vacuo.

The residue was partitioned between water and CH₂Cl₂, and

the aqueous layer was extracted with CH₂Cl₂(3×). The

combined organic extracts were washed with saturated aque-

ous NaCl (2×), dried (Na₂SO₄), and concentrated in vacuo. The

residue was purified by normal-phase chromatography, eluting

with CH₂Cl₂/EtOAc (95:5) to give 43c (8.2 g, 55%) as a white

solid: MS (FAB) m/z 292 (MH)⁺.

dissolved in absolute EtOH (100 mL), combined with PtO₂

(0.300 g), and placed on a Parr hydrogenator under hydrogen

pressure (55 psig) over 24 h. The reaction mixture was filtered

through filter agent and concentrated in vacuo. The residue

was dissolved in MeOH (50 mL), and the pH was adjusted to

pH 8 with triethylamine (ca. 2 mL). The solution was cooled

to 0 °C, treated with di-tert-butyl dicarbonate (2.0 g, 9.0 mmol),

and slowly warmed to room temperature over 20 h. The

reaction mixture was concentrated in vacuo and partitioned

between EtOAc (50 mL) and cold (5 °C) 1 N HCl (50 mL). The

organic extract was extracted with cold 1 N HCl (50 mL),

washed with saturated aqueous NaCl, dried (Na₂SO₄), and

concentrated in vacuo to provide N-[(1,1-dimethylethoxy)-

carbonyl]-5-methoxy-L-norvaline (2.15 g, 68%; CAS no. 167496-

30-2) as a white solid: MS (FAB) m/z 248 (MH)⁺. This material

(1.48 g, 6.0 mmol) was combined with N,O-dimethylhydroxy-

lamine hydrochloride (0.878 g, 9.0 mmol) and dissolved in 70

mL of CH₂Cl₂. The resulting mixture was adjusted to pH 8

with triethylamine and treated with BOP-Cl (2.29 g, 9.0

mmol). After 2 h, the reaction mixture was partitioned between

CH₂Cl₂and cold 1 N HCl. The organic layer was extracted with

saturated aqueous NaHCO₃and saturated aqueous NaCl,

dried (MgSO₄), and concentrated in vacuo. The residue was

partially dissolved in MeOH, and the insoluble material was

removed by filtration. The filtrate was concentrated in vacuo

to afford Weinreb amide 43f (1.00 g, 57%) as a white solid:

MS (FAB) m/z 291 (MH)⁺. Weinreb amide 43f (0.87 g, 3.0

mmol) was converted to 43 according to the methods described

for 5. The resulting crude product was purified by reverse-

phase HPLC, eluting with water/MeCN/TFA (50:50:0.2) to

afford 43 (0.120 g) as a white solid: [R]²⁵_D-18.0°; IR ν_max3430,

2929, 1677, 1204 cm^-1; ¹H NMR (CDCl₃) δ 1.35-1.45 (m, 1H),

1.70-2.00 (ov m, 6H), 2.15-2.25 (m, 2H), 2.40-2.50 (m, 1H),

2.65 (s, 3H), 2.95-3.05 (m, 1H), 3.30 (s, 3H), 3.40-3.50 (m,

3H), 4.30-4.40 (m, 2H), 5.60-5.65 (m, 0.8H), 5.65-5.70 (m,

0.2H), 7.20-7.29 (m, 2H), 7.30-7.35 (m, 3H), 7.55-7.65 (m,

2H), 8.10 (d, 1H, J ) 7.1 Hz), 8.20 (d, 1H, J ) 7.1 Hz); MS

(FAB) m/z 523.4 (MH)⁺. Anal. (C₂₈H₃₄N₄O₄S‚1.5CF₃CO₂H‚

1.1H₂O) C, H, N, F, H₂O.

N-Methyl-^D-phenylalanyl-N-[1-[trans-4-aminocyclo-

hexyl]-3-(2-benzothiazolyl)-3-oxopropyl]-^L-prolinamide

(71 and 72, Scheme 6). A slurry of 69d³²(4.41 g, 13.0 mmol),

ammonium acetate (1.50 g, 19.5 mmol), and malonic acid (1.35

g, 13.0 mmol) in absolute EtOH (25 mL) was heated at reflux

for 18 h. The reaction mixture was cooled to room temperature

and diluted with water (25 mL). The resulting precipitate was

collected and dried in vacuo to give 71a (2.95 g, 57%) as a white

solid. This material (2.95 g, 7.4 mmol) was dissolved in MeOH

(70 mL), basified with triethylamine (3.1 mL, 22 mmol), and

treated with di-tert-butyl dicarbonate (1.80 g, 8.30 mmol) while

stirring. After 18 h, the reaction mixture was dissolved in

EtOAc and extracted with 1 M KHSO₄(2×) and brine, dried

(Na₂SO₄), filtered, and concentrated in vacuo. The residue was

purified by normal-phase chromatography, eluting with CH₂-

Cl₂/MeOH (19:1) to give the corresponding Boc-protected

product (0.635 g, 17%). This material (0.24 g, 0.48 mmol) was

converted to Weinreb amide 71d as described for 41. Com-

pound 71b (0.10 g, 0.18 mmol) was converted to a 1:1 mixture

of 71 and 72 according to the methods used for 41. The

resulting crude solid was purified by reverse-phase HPLC,

eluting with water/MeCN/TFA (from 90:10:0.2 to 60:40:0.2) to

afford 71 (0.015 g, slower elution) and 72 (0.015 g, faster

elution) as white solids. Compound 71: HPLC purity ) 97%;

¹H NMR δ 1.15-2.30 (ov m, 16H), 2.80-3.20 (ov m, 6H), 3.55-

3.73 (ov m, 1H), 4.25-4.45 (ov m, 2H), 5.60-5.70 (ov m, 1H),

7.20-7.35 (m, 5H), 8.10-8.25 (ov m, 2H); MS (ES) m/z 562.3

(MH)⁺; HRMS m/z 562.2852 (562.2852 calcd for C₃₁H₃₉N₅O₃S

+ H⁺). Compound 72: HPLC purity ) 99%; ¹H NMR δ 1.27-

1.43 (ov m, 5H), 1.65-1.77 (ov m, 4H), 2.03-2.13 (ov m, 4H),

2.40-2.45 (m, 1H), 2.64 (s, 3H), 3.01-3.40 (m, 5H), 3.60-3.65

(m, 1H), 4.10-4.18 (m, 1H), 4.30-4.35 (m, 2H), 7.20-7.26 (m,

2H), 7.30-7.35 (m, 3H), 7.57-7.65 (m, 2H), 8.08 (d, 1H, J )

7.65 Hz), 8.18 (d, 1H, J ) 7.65 Hz); MS (ES) m/z 562.3 (MH)⁺;

HRMS m/z 562.2873 (562.2852 calcd for C₃₁H₃₉N₅O₃S + H⁺).

Compound 43c (3.81 g, 13 mmol) was reacted with di-tert-

butyl azodicarboxylate (0.48 g, 15 mmol) according to the

general method of Evans et al.⁶³The crude product was

purified by normal-phase chromatography, eluting with CH₂-

Cl₂/hexane/MeCN (70:30:7) to give 43d (2.33 g, 33%) as a white

solid: MS (FAB) m/z 522 (MH)⁺.

Compound 43d (2.33 g, 4.5 mmol) was dissolved in 18 mL

of THF, cooled to 0 °C, and treated with a solution of LiOH‚

H₂O (0.46 g, 10.7 mmol) in 9 mL of H₂O. Aqueous H₂O₂(1.1

mL, 30%, 10 mmol) was added, and the reaction mixture was

stirred at 0 °C for 3 h. The reaction mixture was partitioned

between 1 N HCl and CH₂Cl₂, and the acidic aqueous layer

was extracted with CH₂Cl₂(2×). The combined organic ex-

tracts were washed with saturated aqueous NaCl, dried

(MgSO₄), and concentrated in vacuo. The residue was purified

by normal-phase chromatography eluting with EtOAc/hexane/

HOAc to afford 43e (0.87 g, 56%) as a white solid: MS (FAB)

m/z 363 (MH)⁺.

Compound 43e (2.15 g, 5.93 mmol) was dissolved in 22 mL

of TFA/CH₂Cl₂(1:4), stirred for 1.5 h, and concentrated in

vacuo. The solvents were removed in vacuo at 20 °C to furnish

(S)-2-hydrazino-5-methoxypentanoic acid monotrifluoroacetate

(2.07 g, >100%): MS (FAB) m/z 163 (MH)⁺. This material was

R-Ketoheterocycles as Inhibitors of Thrombin

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2005

Thrombin Inhibition Assay.³²Thrombin-catalyzed hy-

drolysis rates were measured spectrophotometrically using

commercial human R-thrombin (American Diagnostica), a

chromogenic substrate (Spectozyme TH (H-^D-HHT-Ala-Arg-

pNA‚2AcOH), American Diagnostica) in aqueous buffer (10

mM Tris, 10 mM Hepes, 150 mM NaCl, 0.1% PEG; pH 7.4),

and a microplate reader (Molecular Devices). Changes in

absorbance at 405 nm were monitored (Softmax, Molecular

Devices) upon addition of enzyme both with and without

inhibitor present at 37 °C over 30 min. The IC₅₀values were

determined by fixing the enzyme and substrate concentrations

and varying the inhibitor concentration (1 nM thrombin, 50

µM Spectrozyme TH). Michaelis-Menton kinetics were applied

to the initial reaction slopes. Inhibition constants (K_i) were

determined by fixing the enzyme and inhibitor concentrations

and varying the substrate concentration (1 nM thrombin,

5-100 µM Spectrozyme TH). Michaelis-Menton kinetics were

applied to the initial reaction slopes using the program K‚Cat

(Bio Metallics Inc.).

(2) Reviews pertinent to structure-based drug design and discov-

ery: (a) Appelt, K. Bacquet, R. J.; Bartlett, C. A.; Booth, C. L.;

Freer, S. T.; Fuhry, M. A.; Gehring, M. R.; Herrmann, S. M.;

Howland, E. F.; Janson, C. A.; et al. Design of enzyme inhibitors

using iterative protein crystallographic analysis. J. Med. Chem.

1991, 34, 1925-1934. (b) Greer, J.; Erickson, J. W.; Baldwin, J.

J.; Varney, M. D. Application of the three-dimensional structures

of protein target molecules in structure-based drug design. J.

Med. Chem. 1994, 37, 1035-1054. (c) Reickson, J. W.; Fesik, S.

W. Macromolecular X-ray crystallography and NMR as tools for

structure-based drug design. Annu. Rep. Med. Chem. 1992, 27,

217-289. (d) McDowell, R. S.; Artis, D. R. Structure-based design

from flexible ligands. J. Med. Chem. 1995, 30, 265-274. (e)

Kuntz, I. D.; Meng, E. C.; Shoichet, B. K. Structure-based

molecular design. Acc. Chem. Res. 1994, 27, 117-123. (f) Bode,

W.; Huber, R. Structural basis of the endoproteinase-protein

inhibitor interaction. Biochim. Biophys. Acta 2000, 1477, 241-

252.

(3) (a) R-Thrombin is a member of the trypsin-like serine protease

family. The catalytic machinery in the active site of thrombin

and other serine proteases is comprised of the catalytic triad

His-57, Asp-104, and Ser-195 (chymotrypsinogen numbering

system^4a). (b) Throughout this paper, the term “thrombin” refers

to “R-thrombin”.

(4) (a) Bode, W.; Mayr, I.; Baumann, U.; Huber, R.; Stone, S. R.;

Hofsteenge, J. The refined 1.9 Å crystal structure of R-throm-

bin: interaction with D-Phe-Pro-Arg chloromethylketone and

significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO

J. 1989, 8, 3467-3475. (b) Bode, W.; Turk, D.; Karshikov, A.

The refined 1.9 Å X-ray crystal structure of D-Phe-Pro-Arg

chloromethylketone-inhibited human R-thrombin: structure

analysis, overall structure, electrostatic properties, detailed

active-site geometry, and structure-function relationships. Pro-

tein Sci. 1992, 1, 426-471.

(5) (a) Berliner, L. J., Ed. Thrombin: Structure and Function;

Plenum Press: New York, 1992. (b) Davie, E. W.; Fugikawa,

K.; Kisiel, W. The coagulation cascade: initiation, maintenance,

and regulation. Biochemistry 1991, 30, 10363-10370.

(6) For reviews on thrombin inhibitors, see the following: (a)

Maffrand, J. P. Direct thrombin inhibitors. Nouv. Rev. Fr.

Hematol. 1992, 34, 405-419. (b) Talbot, M. D.; Butler, K. D.

Potential clinical uses of thrombin inhibitors. Drug News Per-

spect. 1990, 3, 357-363. (c) Das, J.; Kimball, S. D. Thrombin

active site inhibitors. Bioorg. Med. Chem. 1995, 3, 999-1007.

(d) Scarborough, R. M. Anticoagulant strategies targeting throm-

bin and factor Xa. Annu. Rep. Med. Chem. 1995, 30, 71-80. (e)

Kimball, S. D. Thrombin active site inhibitors. Curr. Pharm. Des.

1995, 1, 441-468. (f) Fareed, J.; Callas, D. D. Pharmacological

aspects of thrombin inhibitors: a developmental perspective.

Vessels 1995, 1, 15-24. (g) Lefkovits, J.; Topol, E. J. Direct

thrombin inhibitors in cardiovascular medicine. Circulation

1994, 90, 1522-1536. (h) Betschmann, P.; Lerner, C.; Sahli, S.;

Obst, U.; Diederich, F. Molecular recognition with biological

receptors: structure-based design of thrombin inhibitors. Chimia

2000, 54, 633-639. (i) Menear, K. Direct thrombin inhibitors:

Current status and future prospects. Expert Opin. Invest. Drugs

1999, 8, 1373-1384. (j) Ripka, W. C.; Vlasuk, G. P. Antithrom-

botics/serine proteases. Annu. Rep. Med. Chem. 1997, 32, 71-

89. (k) Sanderson, P. E. J. Small, noncovalent serine protease

inhibitors. Med. Res. Rev. 1999, 19, 179-197. (l) Kimball, S. D.

Challenges in the development of orally bioavailable thrombin

active site inhibitors. Blood Coagulation Fibrinolysis 1995, 6,

511-519.

(7) (a) Kettner, C.; Shaw, E. D-Phe-Pro-ArgCH₂Clsa selective

affinity label for thrombin. Thromb. Res. 1979, 14, 969-973. (b)

Hauptmann, J.; Markwardt, F. Studies on the anticoagulant and

antithrombotic action of an irreversible thrombin inhibitor.

Thromb. Res. 1980, 20, 347-351.

(8) (a) Please note that the term “transition-state analogue” is used

somewhat loosely in this paper. “Reaction coordinate analogue”

has been proposed as a more precise term for a reversible enzyme

inhibitor involving an electrophilic center that covalently at-

taches to a group in the enzyme active site.^8b(b) Christianson,

D. W.; Lipscomb, W. N. Carboxypeptidase A. Acc. Chem. Res.

1989, 22, 62-69.

Trypsin Inhibition Assay.³²Trypsin-catalyzed hydrolysis

rates were measured using the same method as the thrombin

procedure. Bovine type 1 trypsin (Sigma) and Spectrozyme

TRY (Cbo-Gly-^D-Ala-Arg-pNA‚AcOH, American Diagnostics)

replaced their thrombin equivalents in a concentration range

of 3.2 U/mL trypsin and 0.1-0.3 mM Spectrozyme.

Gel-Filtered Platelet Aggregation. Gel-filtered platelet

preparations allow assessment of antiplatelet activity in the

absence of plasma coagulation factors and fibrinogen. R-Throm-

bin, which induces platelet aggregation and fibrin clot forma-

tion, can only be tested in such a preparation. Human platelet-

rich plasma (PRP) concentrate was gel-filtered on a Sepharose

2B column with Tyrode’s buffer (140 mM NaCl, 2.7 mM KCl,

12 mM NaHCO₃, 0.76 mM Na₂HPO₄, 5.5 mM glucose, 5.0 mM

Hepes, and 2 mg/mL BSA; pH 7.4). The gel-filtered platelets

were diluted with Tyrode’s buffer, compound solution in buffer,

and 2 mM CaCl₂to achieve ca. 142 900 platelets/µL final assay

concentration on the 96-well assay plates. Platelet aggregation

was initiated by the addition of a concentration of human

R-thrombin shown in preliminary experiments (on the day of

the test) to achieve 80% aggregation (0.005-0.25 NIH U/mL).

The assay plate was stirred and intermittently placed in a

microplate reader to read optical density (650 nM) at 0 and 7

min after the thrombin addition. Aggregation was calculated

to be the decrease in optical density between the time zero

and 7 min measurements. The assay was determined to be

valid when the percent aggregation of the control is >75%.

Each concentration of compounds, vehicles, and buffer controls

were run in duplicate. Inhibition was calculated by comparing

to wells containing no inhibitor (vehicle), and IC₅₀values were

determined using a four-parameter fit logistics model.

Acknowledgment. We thank Dr. Lisa Minor and

Yuanping Wang for several enzymatic studies, Michael

Addo for the platelet aggregation studies, George Greco

for some synthetic intermediates, and Michael Moyer

and Rekha Shah for epimerization studies and analyti-

cal HPLC assays. We are grateful to Dr. Michael Greco

and Dr. Enrico Di Cera (Washington University) for

helpful discussions and advice.

Supporting Information Available: Table 7, experimen-

tal procedures and characterization data for 6-20, 22, 24-

26, 29-40, 44-54, 56-70, and 73-77 (with associated refs

57-61 and 64-70), stereochemical stability profile of 3 in

rabbit and rat plasma, enzyme inhibition assay procedures,

K_ivalues for Table 9, in vivo assay procedures, and microana-

lytical data. This material is available free of charge via the

Internet at http://pubs.acs.org.

(9) For ease of discussion, Schechter and Berger’s nomenclature

system designating the amino acid residues flanking the scissile

bond is used throughout this paper. Schechter, I.; Berger, A. On

the size of the active site in proteases. I. Papain. Biochem.

Biophys. Res. Commun. 1967, 27, 157-162.

(10) Rydel, T. J.; Ravichandran, K. G.; Tulinsky, A.; Bode, W.; Huber,

R.; Roitsch, C.; Fenton, J. W., II. The structure of a complex of

recombinant hirudin and human R-thrombin. Science 1990, 249,

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Dioszegi, M.; Fittler, Z.; Szabo, G.; Juhasz, A.; Tomori, E.;

Szilagyi, G. Highly active and selective anticoagulants: D-Phe-

Pro-Arg-H, a free tripeptide aldehyde prone to spontaneous

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2006 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6

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JM0303857

Article Doi

In-depth study of tripeptide-based α-ketoheterocycles as inhibitors of thrombin. Effective utilization of the S1′ subsite and its implications to structure-based drug design

DOI: 10.1021/jm0303857

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