Potent thrombin inhibitors that probe the S1' subsite: Tripeptide transition state analogues based on a heterocycle-activated carbonyl group

Full text of DOI:10.1021/jm9603274

J . Med. Chem. 1996, 39, 3039-3043
3039
the benzoxazole nitrogen and His-57 of the catalytic
triad.12 More recently, Edwards et al.13 published two
additional papers on analogous elastase inhibitors,
which appear quite effective, and Tsutsumi et al.14
described R-keto heterocyclic inhibitors of prolyl en-
dopeptidase. We now wish to report on the synthesis
and biological activity of peptidoyl heterocycles with the
structural motif Me-(D-Phe)-Pro-Arg-Het, some of which
are potent inhibitors of thrombin and trypsin (Table 1).
Moreover, via X-ray crystallography, we determined the
molecular structure of a complex between the subnano-
molar inhibitor 2 and human R-thrombin, which depicts
some key interactions in the S1′ region of the active
site: The benzothiazole ring forms a hydrogen bond with
His-57 and an aromatic stacking interaction with Trp-
60D of thrombin’s unique insertion loop.15
P oten t Th r om bin In h ibitor s Th a t P r obe
th e S1′ Su bsite: Tr ip ep tid e Tr a n sition
Sta te An a logu es Ba sed on a
Heter ocycle-Activa ted Ca r bon yl Gr ou p 1
Michael J . Costanzo,† Bruce E. Maryanoff,*,†
Leonard R. Hecker,† Mary R. Schott,†
Stephen C. Yabut,† Han-Cheng Zhang,†
Patricia Andrade-Gordon,† J ack A. Kauffman,†
J oan M. Lewis,† Raman Krishnan,‡ and
Alexander Tulinsky‡
Drug Discovery, The R. W. J ohnson Pharmaceutical
Research Institute, Spring House, Pennsylvania 19477, and
Department of Chemistry, Michigan State University,
East Lansing, Michigan 48824
Received May 2, 1996
The pathology of thrombosis entails inappropriate
activity of the hemostatic mechanism, which results in
the interruption of normal blood flow and associated
damage to cells, tissues, and organs. Because this
condition is a serious source of mortality and morbidity
in patients worldwide, new types of antithrombotic
therapeutic agents are needed. Inhibitors of the serine
protease R-thrombin (EC 3.4.21.5), an enzyme that is
central to the regulation of hemostasis and thrombosis,
could represent potentially useful drugs in this area.2-9
In the area of thrombin inhibitors, we have been
investigating transition state analogues10 with a cyclic
structure, such as cyclotheonamide A (1) and its con-
geners,11 and with an acyclic structure based on the
classical tripeptide motif D-Phe-Pro-Arg.2,5-7 Besides
having the appropriate elements for molecular recogni-
tion in the active site of thrombin, particularly for the
S1 specificity pocket and the apolar S2 and S3 subsites,
these molecules also have an electrophilic arginine
carbonyl that can form a hemiacetal adduct with Ser-
195 of the catalytic triad as part of a complex hydrogen-
bonding network. At the outset of our work in 1991,
we sought to identify tripeptide transition state ana-
logues of the general form D-Phe-Pro-Arg-X, with new
types of carbonyl-activating groups, “X”. It occurred to
us that the use of certain heterocyclic entities for “X”,
such as 2-oxazolyl or 2-pyridyl (“Het”), would likely
afford the necessary electrophilicity to the arginine
carbonyl and thus provide an effective solution. We
favored this previously untested approach to enzyme
inhibitors because (1) the use of different, suitable
heterocycles, or substitution of a suitable heterocycle,
would modulate carbonyl electrophilicity and (2) the
heterocycle would provide a template for probing novel
molecular recognition within the underutilized S1′ do-
main of the thrombin active site.
Syn th etic Ch em istr y. The tripeptidoyl heterocycles
(Table 1) were generally synthesized by two routes,
which are illustrated for the synthesis of 2 in Schemes
1 and 2.17 In the first route, the heterocycle was formed
from keto imidate A (prepared by reacting aldehyde B19
with acetone cyanohydrin12 and treating the adduct
with methanolic HCl) and an appropriate bifunctional
amine (Scheme 1). Reaction of A with 2-aminothiophe-
nol furnished benzothiazole C, which was oxidized with
the Dess-Martin reagent,20 deprotected with HF, and
purified to furnish 2.
In the second route, the heterocycle was introduced
by reacting Weinreb amide D21 with an appropriate
lithio heterocycle at low temperature (Scheme 2). Ex-
cess lithium reagent is required to overcome quenching
caused by the exchangeable protons. Arylsulfonyl pro-
tecting groups are preferred for the Arg guanidine since
Cbz and Fmoc groups are cleaved under these reaction
conditions. Hence, treatment of D with 5-8 mol equiv
of 2-lithiobenzothiazole at -78 °C furnished the corre-
22
sponding ketone, which was reduced with NaBH4 (to
avoid side reactions during the ensuing steps) and
deprotected with TFA to give alcohols E. Coupling of
E with dipeptide F was effected by DCC, and interme-
diate G was converted to 2 by the methods used for C
(Scheme 1). Crude 2 from both routes contained 10-
20% of the epimeric D-Arg tripeptide,23 which was
removed during reverse-phase HPLC purification. The
second route (Scheme 2) is more convergent and more
generally applicable. The diastereomeric alcohols cor-
responding to 2, 4a ,b, were prepared by the HF depro-
tection of G and then separated by HPLC.
While we were pursuing this project, Edwards et al.
published a seminal paper in 1992 describing the first
examples of heterocycle activation in the design of novel
inhibitors of the serine protease elastase.12 In fact, an
X-ray crystal structure of Ac-Val-Pro-Val-(2-benzox-
azole) complexed with porcine pancreatic elastase (PPE)
suggested a new hydrogen-bonding interaction between
* Address correspondence to this author. Telefax: 215-628-4985.
E-mail: maryanoff@prius.jnj.com.
Biologica l Testin g. The compounds were tested for
inhibition of human R-thrombin and bovine trypsin. The
† R. W. J ohnson Pharmaceutical Research Institute.
‡ Michigan State University.
S0022-2623(96)00327-5 CCC: $12.00 © 1996 American Chemical Society

3040 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Communications to the Editor
Ta ble 1. Chemical Properties and Biological Data for Peptidoyl Heterocyclesa
Ki, nMd
compd
Het
mp, °Cb
93-104
[R]D,c deg
-71.2 (c 0.76)f
thr
try
try/thre
2
2-benzothiazole
2-thiazole
2-benzothiophene
2-benzoxazole
0.19 ( 0.07 (7)
3.6 ( 0.3 (3)
2400 ( 900 (6)
3.8 ( 1.4 (6)
3.4 ( 2.1 (6)
12.0 ( 1.0 (6)
8.1 ( 2.7 (6)
50 ( 13 (3)
15 ( 6 (6)
85 ( 44 (6)
5300 ( 2500 (6)
1600 ( 200 (3)
10.0 ( 5.0 (6)
10.0 ( 2.0 (6)
3.1 ( 1.7 (3)
1.6 ( 0.7 (2)
1340 ( 840 (6)
2.9 ( 1.9 (5)
4.4 ( 2.4 (5)
38 ( 4 (6)
290 ( 90 (6)
4400 ( 800 (3)
8.2 ( 1.4 (6)
73 ( 32 (6)
k
16
3a
3b
3c
3d
3e
3f
3g
3h
3ii
0.4
0.6
0.8
1.3
3.2
36
88
0.6
0.8
-78.3 (c 1.00)
-63.3 (c 0.58)
-71.7 (c 0.69)
-63.1 (c 1.00)h
-57.8 (c 1.00)h
-63.7 (c 1.00)
44-54
2-H4-benzothiazoleg
2-benzimidazole
N-Me-2-benzimidazole
N-Me-2-imidazole
2-quinazolin-4-one
2-pyridine
50-60
70-80
70-90
70-83
122-138
40-68
4a j
77-180 dec
65-175 dec
-61.8 (c 1.21)f
-69.7 (c 1.05)f
4bj
k
efegatranl
argatrobanl
3.9 ( 1.3 (6)
2900 ( 1200 (6)
0.39
290
a
All new compounds were isolated and purified by reverse-phase HPLC (MeCN:water:TFA, 30:70:0.1) and lyophilized as hydrated
trifluoroacetate (TFA) adducts. They are represented by standard molecular formulas with the following solvates: 2 (2.6 TFA, 1.5 water),
3a (2.0 TFA, 1.2 water), 3b (2.4 TFA, 1.5 water), 3c (4.0 TFA, 2.2 water), 3d (2.6 TFA, 1.4 water), 3e (3.8 TFA, 1.0 water), 3f (3.2 TFA,
1.1 water), 3g (3.9 TFA, 1.1 water), 3h (2.6 TFA, 3.0 water), 3i (2.8 TFA, 2.2 water), 4a (4.0 TFA, 1.5 water), and 4b (4.2 TFA, 1.8 water).
Microanalytical data (C, H, N, and water) were within the accepted range, unless noted otherwise. The new target compounds were
characterized by high-field NMR and mass spectrometry. Information on the enzymatic assays is contained in the Supporting Information.
b Melting point values are corrected. The products were amorphous powders obtained by lyophilization. c Optical rotations were determined
d
in MeOH at 25 °C, unless noted otherwise. The standard error is given for N measurements, indicated in parentheses. For methodology,
see the Supporting Information. e Ratio of the Ki value for trypsin to the Ki value for thrombin. f Determined at 20 °C. g 4,5,6,7-Tetrahydro-
h
i
j
2-benzothiazole. Determined in water. Mixture of L- and D-epimers at Arg CR in a 85:15 ratio. Single diastereomer of unestablished
configuration. Water analysis was out of range; calcd/found: 2.61/0.92 for 4a , 2.95/1.40 for 4b. k Greater than 150 000 nM; N ) 3. l Reference
thrombin inhibitor. Efegatran has been called GYKI-14766. Argatroban has been called MD-805 and argipidine.
Sch em e 1
data in Table 1 illustrate the importance of an aromatic
stacking interaction with Trp-60D for high affinity and
selectivity, as reflected in the X-ray crystallographic
results (vide infra): cf. 2 with 3a ,d and 3f with 3g.
Although methyl substitution in the imidazole series did
not improve thrombin inhibition, it did increase the
selectivity for thrombin over trypsin (entries 3e-g). Two
proximal heteroatoms are necessary, but not sufficient,
for potent activity, perhaps on account of a requirement
for effective activation of the electrophilic ketone (cf. 2
with 3b,i). Although pyridine derivative 3i has a
π-deficient heterocycle that can activate the adjacent
carbonyl, it showed fairly modest potency. The signifi-
cant increase in potency for benzothiazole 2 compared
to benzoxazole 3c (20-fold) and N-methylbenzimidazole
3f (40-fold) may be connected with the relative π
electron-withdrawing power of these heterocycles.24 The
results for alcohols 4a ,b further support the importance
of ketone activation for strong enzyme inhibition, con-
sistent with a transition state analogue.
The excellent thrombin inhibitor 2, as well as the
reference standards efegatran and argatroban, was
evaluated for selectivity versus other, important coagu-
lation enzymes: plasmin, tPA, activated protein C, and
streptokinase (Table 2). Although 2 is not as selective
as argatroban with these four enzymes,25 it still shows
noteworthy selectivity for potential development, and
2 is considerably more selective than efegatran.11a,26 In
the case of trypsin, 2 is a fairly potent inhibitor,
affording a selectivity ratio of just 16 in favor of
thrombin inhibition. It should be noted that a lack of
selectivity for thrombin over trypsin appears to be a

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3041
Sch em e 2
Ta ble 2. Inhibition of Thrombin Relative to Other Serine Proteasesa
selectivity (Ki other/Ki thr)b
compd
Ki, nM, thrombin
trypsin
plasmin
tPA
prot Ca
SK
6300
170
>50 000
2
0.19 ( 0.07 (7)
10.0 ( 5.0 (6)
10.0 ( 2.0 (6)
16
0.39
290
12 000
33
40 000
3300
130
37 000
19 000
72
>50 000
efegatran
argatroban
a
b
Ki values (nM, mean ( standard error) are given for thrombin inhibition; the number of experiments (N) is in parentheses. Selectivity
is defined as the ratio of the Ki value of the serine protease over the Ki value for thrombin. For the original Ki data, see the Supporting
Information. tPA, tissue-type plasminogen activator; prot Ca, activated protein C; SK, streptokinase.
general property of inhibitors with a D-Phe-Pro-Arg
motif;7c nevertheless, the selectivity exhibited by 2 for
thrombin over trypsin is significantly better than that
by efegatran (try/thr ) 0.39), which is currently involved
in clinical trials.
the active site of 2-thrombin-hirugen are depicted in
Figure 1.
Compound 2 is structurally analogous to PPACK (D-
Phe-Pro-Arg-CH2Cl), but it has a methyl group added
to the N-terminus and a 2-benzothiazole in place of the
chloromethyl group. The overall interactions for 2-
thrombin are similar to those for PPACK-thrombin
(Figure 2).16 The inhibitor forms a hemiketal adduct
between the Ser-195 hydroxyl and the arginine carbo-
nyl, and the tripeptide motif (P3-P2-P1) has the
standard interactions previously observed in thrombin-
inhibitor complexes of this type. However, the ben-
zothiazole group binds in an interesting manner at the
S1′ subsite, which is mainly defined by His-57, Tyr-60A,
Trp-60D, and Lys-60F. The benzothiazole ring (P1′)
hydrogen bonds via its nitrogen atom to His-57 (N-N
distance of 2.7 Å) and stacks in a face-to-edge manner
with the indole ring of Trp-60D. Edwards et al.12
inferred (as the benzoxazole O and N positions could
not be crystallographically distinguished) a similar
hydrogen bond in the crystal structure of their PPE
complex. Remarkably, despite the potent inhibition of
thrombin by 2, the benzothiazole ring is found to
displace the side chain of Lys-60F from its normal
position in other inhibitor-thrombin complexes, such
that the aminobutyl group has folded into a U-shaped,
unextended conformation.
We also examined 2 for functional activity with gel-
filtered human platelets, in comparison with efegatran
and argatroban. Compound 2 inhibited thrombin-
induced platelet aggregation with an IC50 value of 23
( 2 nM (N ) 5), which compares favorably with the
values obtained for efegatran (23 ( 12 nM; N ) 3) and
argatroban (52 ( 9 nM; N ) 3). Also, 2 exhibited potent
antithrombotic activity in several animal models on
intravenous administration, but the results of this work
will be reported separately.
X-r a y Cr ysta llogr a p h ic Str u ctu r e.27 A ternary
complex was prepared by adding 2 in 10-fold excess to
the hirugen-thrombin complex, and crystals were
grown by the hanging-drop method.28 Diffraction data
were collected by using a Siemens multiwire X-1000
detector (Rmerge ) 0.055). The monoclinic crystals are
isomorphous with crystals of other hirugen-thrombin
constructs28,29 and diffracted X-rays to about 2.3 Å.
After ca. 15 cycles of PROLSQ refinement (R-factor )
0.20), the electron density was compatible with two
possible orientations of the benzothiazole ring, with
interchanged nitrogen and sulfur atoms. The correct
position was established by occupancy refinement of the
sulfur atom in each position, also revealing a hydrogen
bond between N1 of the benzothiazole and NE2 of His-
57 of thrombin. Final refinement converged at R )
0.168 with 125 water molecules. Structural features of
Con clu sion . We have studied a series of peptidoyl
heterocycles with the structure Me-(D-Phe)-Pro-Arg-Het
and identified some potent inhibitors of human R-throm-
bin. The best thrombin inhibitor, 2 (RWJ -50353),
possesses a 2-benzothiazole group and has a Ki value
of 0.19 nM. This agent has excellent selectivity for

3042 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Communications to the Editor
F igu r e 1. Stereoview of 2 bound in the active-site cleft of thrombin. Color code: blue ) thrombin, red ) ligand 2. (The ligand
is also shown in bolder typeface.) Hydrogen bonds are indicated by broken red lines. Residues in thrombin are numbered with
the larger font, and water molecules are numbered with the smaller font.
F igu r e 2. Diagram representing the key interactions between 2 and thrombin.
thrombin vs four other enzymes important to blood
coagulation and is a powerful inhibitor of platelet
aggregation. The crystallographic structure of a com-
plex between 2 and thrombin reveals novel interactions
in the S1′ region, where the benzothiazole forms a
hydrogen bond with His-57 and an aromatic stacking
interaction with Trp-60D of the insertion loop of throm-
bin. Subnanomolar inhibition occurs with 2 despite the
Lys-60F side chain moving from its normal position to
a folded conformation because of the bulky benzothia-
zole group. Our work extends to thrombin inhibitors
the heterocycle-activated carbonyl approach pioneered
by Edwards and co-workers for elastase inhibitors12,13
and further supports the proposed mechanism of bind-
ing.
Ack n ow led gm en t. We thank Dr. Harold R. Al-
mond, J r., for molecular modeling and George E. Greco
for technical assistance. This work was partially sup-
ported by NIH Grant HL43229 (to A.T.).
Su p p or tin g In for m a tion Ava ila ble: Details for the
enzyme assays and Ki values for Table 2 (2 pages). See any
current masthead page for ordering information.
Refer en ces
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American Chemical Society, Chicago, IL, Aug. 20-24, 1994;
ORGN-25. (b) Abbreviations: Cbz, carbobenzoxy; Fmoc, 9-fluo-
renylmethylcarbonyl; TFA, trifluoroacetic acid; DCC, dicyclo-
hexylcarbodiimide.
(2) Berliner, L. J ., Ed. Thrombin: Structure and Function; Plenum
Press: New York, 1992.

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3043
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