Noncovalent thrombin inhibitors incorporating an imidazolylethynyl p1

Article abstract of DOI:10.1016/S0960-894X(00)00579-5

A series of noncovalent tripeptidic thrombin inhibitors incorporating a unidazolylethynyl moiety at P1 was investigated. A number of compounds of this series were highly potent and selective versus trypsin, and several compounds demonstrated good oral absorption in rats (F=58% for compound 19). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00579-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2775±2778
Noncovalent Thrombin Inhibitors Incorporating an
Imidazolylethynyl P1
Koo Lee,* Won-Hyuk Jung, Myunggyun Kang and Sung-Hack Lee
Life Science R&D, LG Chemical Ltd/Research Park, PO Box 61, Yu-Sung, Science Town, Taejon 305-380, South Korea
Received 14 August 2000; accepted 2 October 2000
Dedicated to Professor David F. Wiemer on the Occasion of His 50th Birthday
AbstractÐA series of noncovalent tripeptidic thrombin inhibitors incorporating a unidazolylethynyl moiety at P1 was investigated.
A number of compounds of this series were highly potent and selective versus trypsin, and several compounds demonstrated good
oral absorption in rats (F=58% for compound 19). # 2000 Elsevier Science Ltd. All rights reserved.
The development of an orally active thrombin inhibitor
represents a still elusive goal. Such a drug would be
e€ective and safe for the prophylaxis of venous and
arterial thrombosis. While numerous small molecule
thrombin inhibitors have been discovered,1 much recent
attention has been devoted to noncovalent tripeptides
that lack an electrophilic serine trap.2 4 Owing to their
fast-binding nature, the noncovalent inhibitors are more
ecacious and thus have a better therapeutic index than
covalent inhibitors.1b,4c,5 However, only a few compounds
of this class are reported to have oral bioavailability.
Unless their log P values are improved by incorporation of
highly lipophilic groups,6 thrombin inhibitors with
strongly basic P1 elements are likely to have poor oral
bioavailability. Compounds in this class are typi®ed by
the Merck products 1±3; the cyclohexylamine com-
pound 13b and its derivatives lack oral bioavailability
while the less basic aminopyridine analogue 33c is highly
bioavailable in dogs.
novel ethynyl P1 into the d-Phe-Pro sca€old and found
a new class of noncovalent thrombin inhibitors. In this
paper, we describe the results of this study.
We recently investigated a series of arylsulfonyl-pro-
pargylglycinamide thrombin inhibitors possessing a
variety of weakly basic P1 probes at the ethynyl termi-
nus, and identi®ed a number of potent and orally bio-
available thrombin inhibitors (e.g. 4, Ki=5nM).7 The
conformationally rigid ethynyl tether was best tolerated by
the S1 speci®city pocket of thrombin when it was sub-
stituted by an N-methylaniline moiety whereas the corre-
sponding alkyl and vinyl analogues8 were poorly tolerated.
As an extension of this work, we have incorporated this
With a readily available N-Boc-d-Phe-Pro-NH-CH2-
CꢀCH template, we explored several heterocyclic sub-
stituents at the acetylenic terminus including imidazole,
pyrazole, pyridine and 2-aminopyridine, chosen on the
basis of size, basicity, and potential capability of inter-
acting with Asp189 in the S1 active site of thrombin
(Table 1). The imidazole 5, as its acid salt, appeared to
be the most soluble compound in this series, and to
*Corresponding author. Tel.: +82-42-866-2258; fax: +82-42-861-
2566; e-mail: kleec@lgchem.co.kr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00579-5

2776
K. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2775±2778
Table 1. Thrombin inhibitory activity for compounds 5±9
Compounds
R
Thrombin
Ki (mM)
5
6
7
8
9
Imidazol-4-yl
3(N)-Me-imidazol-4-yl
Pyrazol-4-yl
Pyrid-4-yl
2-Aminopyrid-5-yl
1.2
5.5
5.4
>20
>20
exhibit the greatest thrombin anity. The aminopyridine
P1 substituent (compound 9) appeared to be too large to
be tolerated by the S1 pocket, while introduction of a
methyl group on the imidazole nitrogen of 5 closer to the
ethynyl substituent (compound 6) was detrimental.
In view of reports that replacement of d-Phe at P3 with
the unique amino acid d-diphenylalanine resulted in a
dramatic potency increase in the peptidic thrombin
inhibitors,3b,9 we anticipated that the potency of com-
pound 5 might be signi®cantly improved by utilizing
this amino acid. Indeed, the diphenylalanine compound
10 exhibited a 60-fold potency increase with a Ki of
24 nM and good selectivity against trypsin as well.
Remarkably, this compound was well absorbed when
administered orally in rats (Cmax=14.6 mM, Tmax=60
min, AUC=29 mMꢁ h, 30 mg/kg, n=3) whereas the des-
Boc analogue 1110 was signi®cantly less absorbed.
Figure 1. X-ray structure of compound 13 bound to thrombin.
be in close contact with the imidazole ring, it did not
show well-de®ned electron density, indicating a very
weak contribution of the phenyl ring to the enzyme
anity.
Compound 10 however exhibited only moderate anti-
coagulant activity when tested for its ability to prolong
activated partial thromboplastin time (aPTT) in human
plasma, suggesting that higher thrombin inhibitory
potency would be necessary for in vivo e€ectiveness.
Incorporation of highly lipophilic amino acid d-dicyclo-
hexylalanine6 gave analogue 12 that maintained com-
parable potency but exhibited a signi®cant loss of
anticoagulant activity. In view of a recent report that
the benzylsulfonyl group of 2 occupied a new binding
pocket in thrombin, thereby leading to greatly increased
anity,3a we prepared compound 13.10 It displayed a
10-fold enhanced potency and excellent anticoagulant
activity in the clotting assay, but the benzylsulfonyl
group proved detrimental to oral absorption.
Consistent with the X-ray crystallographic analysis, the
methanesulfonyl compound 14 retained potency with a
Ki of 2.6 nM. Interestingly, the related sulfamoyl com-
pound 15 exhibited even better potency with a sub-
nanomolar Ki. The potency of these sulfonyl compounds
translated well into good anticoagulant activity (see
Table 2) that compares favorably with that of the e-
cacious oral thrombin inhibitor LB30057,11 previously
discovered in our laboratories. However, both com-
pounds 14 and 15, like compound 13, were poorly absor-
bed. The acetyl and the carboxymethyl compounds 16±18
were to some extent orally bioavailable in rats (Cmax, 1.6±
5 mM, 30 mg/kg, po), but displayed limited potency.
Molecular modeling of the carbamate 10 revealed that
the Boc t-butyl moiety was exposed to solvent and did
not interact with the thrombin active site. We therefore
prepared the methyl carbamate 19, which indeed was
found to be equipotent to 10. In particular, the plasma
level of 19 was signi®cantly high after oral administra-
tion in rats (Cmax=23.6 mM, Tmax=60 min, AUC=
46 mMꢁ h, 30 mg/kg, po, n=3; Cl=12.8 mL/min/kg t1/
2= 40 min, AUC=26 mMꢁ h, 10 mg/kg, iv, n=3;
F=58%). Compound 19 also exhibited good antic-
oagulant activity, although still less than desired. By
contrast, and similar to the results obtained with 12, the
equipotent dicyclohexyl analogue 20 was relatively
inactive in the clotting assay. This result is in accord
with Merck's ®ndings.6
X-ray crystallographic studies on the complex of 13
with thrombin showed that the inhibitor was bound in
the active site in a fashion very similar to that of the
reported compound 2 (Fig. 1). The diphenyl and proline
rings spanned the distal and proximal hydrophobic
pockets, respectively, while the N H of the imidazolyl-
ethynyl side chain hydrogen-bonded to the carboxylic
acid of Asp 189 of the speci®city pocket (2.77 A). There
was also evidence for a hydrogen bond with a water
molecule involving the N H of the alternate imidazole
tautomer. One of the sulfonyl oxygens was hydrogen-
bonded with the N H of Gly 219 (3.27 A). Although
the phenyl ring of the benzylsulfonyl group appeared to

K. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2775±2778
2777
Table 2. Thrombin and trypsin inhibitory activities and coagulation parameter for compounds 10±20
Compds
R1
R2
Thrombina
Ki (nM)
Trypsinb
Ki (nM)
2ÂaPTTc
(mg/mL)
10
11
Boc
H
Diphenyl
Diphenyl
24
12
50
25
6.0
3.4
12
13
14
15
16
17
18
19
Boc
Dicyclohexyl
Diphenyl
Diphenyl
Diphenyl
Diphenyl
Diphenyl
Cyclohexyl
Diphenyl
Dicyclohexyl
29
30
24
14
18
ND
47
ND
24
>20
1.0
1.2
0.55
ND
ND
ND
1.6
BnS(O)2
MeSO2
NH2S(O)2
Ac
HO2CCH2
HO2CCH2
MeO(CO)
MeO(CO)
3.0
2.6
0.8
208
44
480
14
15
20
LB30057
30
33
>10
0.89
0.4
aHuman thrombin.
bBovine trypsin.
cConcentration of inhibitor in human plasma required to double the activated partial thromboplastin time (aPTT).
The synthesis of the imidazole thrombin inhibitors in
Table 1 and 2 is outlined in Scheme 1. The propargyl
intermediates 22, prepared by amide coupling of dipep-
tides of the general structure 216,12 with propargyl-
amine, underwent Pd-catalyzed acetylenic coupling with
N-trityl-4-iodoimidazole13 to give 23 in good yields. The
trityl group was eciently removed by treatment with
TFA and triisopropylsilane. Compounds 7±9 were also
prepared in this manner from 22 and the corresponding
heterocyclic bromides. Compound 6 could be prepared
by N-methylation (Me3OBF4) of 23 (R2=phenyl), fol-
lowed by trityl deprotection.
In summary, we have investigated a series of non-
covalent tripeptidic thrombin inhibitors that incorpo-
rate an imidazolylethynyl P1. With bene®t from the
weakly basic P1 motif, a number of compounds were
prepared that demonstrated good oral absorption in
rats. The most interesting compound in this series was
the carbamate 19, that showed both good oral bio-
availability and good anticoagulant activity. However,
utility of this compound was limited by its moderate
antithrombotic activity when evaluated in the rat
venous thrombosis model. Future communications will
report details of our continuing e€orts to develop more
ecacious oral thrombin inhibitors.
Acknowledgements
We wish to thank Mikyung Yun for obtaining the X-ray
crystal structure for the thrombin±13 complex and
Kyung Hee Yoon for performing aPTT determination.
References and Notes
1. For recent reviews see: (a) Hauptmann, J.; Sturzebecher, J.
Thromb. Res. 1999, 93, 203. (b) Sanderson, P. E. J. Med. Res.
Rev. 1999, 19, 179.
2. Lilly: (a) Wiley, M. R.; Chirgadze, N. Y.; Clawson, D.;
Craft, T. J.; Gi€ord-Moor, D. S.; Jones, N. D.; Olkowski, J.
L.; Schacht, A. L.; Weir, L. C.; Smith, G. F. Bioorg. Med.
Chem. Lett. 1995, 5, 2835. (b) Wiley, M. R.; Chirgadze, N. Y.;
Clawson, D.; Craft, T. J.; Gi€ord-Moor, D. S.; Jones, N. D.;
Olkowski, J. L.; Weir, L. C.; Smith, G. F. Bioorg. Med. Chem.
Lett. 1996, 6, 2387. (c) Wiley, M. R.; Weir, L. C.; Bliggs, S. L.;
Chirgadze, N. Y.; Clawson, D.; Gi€ord-Moor, D. S.; Smith,
G. F.; Vasudevan, V.; Zornes, L. L.; Klimkowski, V. J.
Bioorg. Med. Chem. Lett. 1999, 9, 2767.
Scheme 1. (a) Propargylamine, NMM, EDC, HOBT, quant.; (b)
Pd(Ph3P)4, CuI, Et3N, N-trityl-4-iodoimidazole, CH3CN, rt, 15 h, 60±
90%; (c) TFA, i-Pr3SiH, CH2Cl2, 0 ꢂC, 2 h; (d) TFA, neat, rt; (e) R1-Cl,
Et3N, CH2Cl2, 52±88%; (f) t-BuO2CCH2Br, Et3N, CH3CN, rt, 24 h, 70%.
3. Merck: (a) Lyle, T. A.; Chen, Z.; Appleby, S. D.; Frei-
dinger, R. M.; Gardell, S. J.; Lewis, S. D.; Li, Y.; Lyle, E. A.;
Lynch, J. J., Jr.; Mulichak, A. M.; Ng, A. S.; Naylor-Olsen, A.

2778
K. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2775±2778
M.; Sanders, W. M. Bioorg. Med. Chem. Lett. 1997, 7, 67. (b)
Tucker, T. J.; Lumma, W. C.; Mulichak, A. M.; Chen, Z.;
Naylor-Olsen, A. M.; Lewis, S. D.; Lucas, R.; Freidinger, R.
M.; Kuo, L. C. J. Med. Chem. 1997, 40, 830. (c) Feng, D.-M.;
Gardell, S. J.; Lewis, S. D.; Bock, M. G.; Chen, Z.; Freidinger,
R. M.; Naylor-Olsen, A. M.; Ramjit, H. G.; Woltmann, R.;
Baskin, E. P.; Lynch, J. J.; Lucas, R.; Shafer, J. A.; Chen, I-
W.; Dancheck, K. B.; Mao, S.-S.; Krueger, J. A.; Hare, T. R.;
Mulichak, A. M.; Vacca, J. P. J. Med. Chem. 1997, 40, 3726.
(d) Tucker, T. J.; Lumma, W. C.; Lewis, S. D.; Gardel, S. J.;
Lucas, B. J.; Sisko, J. T.; Lynch, J. J.; Lyle, E. A.; Baskin, E.
P.; Woltmann, R.; Appleby, S. D.; Chen, I-W.; Dancheck, K.
B.; Naylor-Olsen, A. M.; Krueger, J. A.; Cooper, C. M.;
Vacca, J. P. J. Med. Chem. 1997, 40, 3687.
4. Astra: (a) Gustafson, D.; Elg, M.; Lenfors, S.; Borjesson,
I.; Teger-Nilsson, A. C. Thromb. Haemost. 1995, 73, 1319. (b)
Gustafson, D.; Elg, M.; Lenfors, S.; Borjesson, I.; Teger-Nils-
son, A. C. Blood Coagul. Fibrinolys. 1996, 7, 69. (c) Elg, M.;
Gustafson, D.; Deinum, J. Thromb. Haemost. 1997, 78, 1286.
(d) Eriksson, B. I.; Carisson, S.; Halvarsson, M.; Risberg, B.;
Mattsson, C. Thromb. Haemost. 1997, 78, 1404. (e) Gustafson,
D.; Antonsson, T.; Bylund, R.; Eriksson, B. I.; Gyzander, E.;
Nilsson, I.; Elg, M.; Mattsson, C.; Deinum, J.; Pehrsson, S.;
Carlsson, S.; Nilsson, A.; Sorensen, H. Thromb. Haemost.
1998, 79, 110.
5. Stone, S. R.; Tapparelli, C. J. Enzyme Inhibition 1995, 9, 3.
6. Tucker, T. J.; Lumma, W. C.; Lewis, S. D.; Gardel, S. J.;
Lucas, R.; Baskin, E. P.; Woltmann, R.; Lynch, J. J., Jr.; Lyle,
E. A.; Appleby, S. D.; Chen, I-W.; Dancheck, K. B.; Vacca, J.
P. J. Med. Chem. 1997, 40, 1565.
7. Lee, K.; Hwang, S. Y.; Park, C. W. Bioorg. Med. Chem.
Lett. 1999, 9, 1013.
8. Lee, K. Unpublished results.
9. Chery, L.; Goodwin, C. A.; Schully, M. F.; Kakkar, V. V.;
Claeson, G. J. Med. Chem. 1992, 35, 3365.
10. Isaacs, R. C.; Naylor-Olsen, A. M.; Dorsey, B. D.; New-
ton, C. L. Patent Application WO 98/42342.
11. (a) Kim, I.-C.; Oh, Y. S.; Yun, M.; Hwang, S. Y.; Hong, S.;
Lee, Y. H.; Lee, K.; Kim, S.; Yoo, Y. J.; Yoon, K. H.; Kim, D.
S.; Lee, C. H. Circulation 1997, 96, I-41. (b) Oh, Y. S.; Yun, M.;
Hwang, S. Y.; Hong, S.; Shin, Y.; Lee, K.; Yoon, K. H.; Yoo, Y.
J.; Kim, D. S.; Lee, S. H.; Lee, Y. H.; Park, H. D.; Lee, C. H.;
Lee, S. K.; Kim, S. Bioorg. Med. Chem. Lett. 1998, 8, 631.
12. The starting material, N-Boc-d-diphenylalanine, was pre-
pared using Sibi's synthesis (Sibi, M. P.; Deshpande, P. K.; La
Loggia, A. J.; Christensen, J. W. Tetrahedron. Lett. 1995, 36,
8961) and also purchased from Synthetech Inc., Albany, OR,
USA. N-Boc-d-dicyclohexylalanine was prepared by catalytic
hydrogenation of N-Boc-d-diphenylalanine.3c
13. Kirk, K. I. J. Heterocycl. Chem. 1985, 22, 57.