Potent and selective thrombin inhibitors incorporating the constrained arginine mimic L-3-piperidyl(N-guanidino)alanine at P1

Full text of DOI:10.1021/jm960607j

© Copyright 1996 by the American Chemical Society
Volume 39, Number 23
November 8, 1996
Communications to the Editor
P oten t a n d Selective Th r om bin
In h ibitor s In cor p or a tin g th e Con str a in ed
Ar gin in e Mim ic
L-3-P ip er id yl(N-gu a n id in o)a la n in e a t P 1
Odile E. Levy,* J . Edward Semple, Michael L. Lim,
J ohn Reiner, William E. Rote, Erin Dempsey,
Brigitte M. Richard, Erli Zhang,† Al Tulinsky,†
William C. Ripka, and Ruth F. Nutt
Corvas International, Inc., 3030 Science Park Road,
San Diego, California 92121-1102, and
Department of Chemistry, Michigan State University,
East Lansing, Michigan 48824-1322
Received August 23, 1996
Thrombin, a serine protease with trypsin-like speci-
ficity, plays a central role in the coagulation cascade by
mediating both the final conversion of fibrinogen to
fibrin and the activation of platelets.1 The prominent
anticoagulant therapeutic agents currently available,
heparin and its low molecular weight derivatives, and
the indirectly acting orally bioavailable coumarins suffer
from many side effects and limited efficacy.2 Consider-
able efforts to develop safer and novel thrombin inhibi-
tors are presently underway.3
Recently, in our laboratories, compound 1 (CVS 1123)
was identified as a potent transition-state thrombin
inhibitor which demonstrated good oral bioavailability
and selectivity profiles (see Figure 1).4 Molecular-
modeling considerations and structure-activity rela-
tionship (SAR) studies on 1 and related serine protease
inhibitors led to the design of compounds 2 (CVS 1578)
and 3 (CVS 1778),5 which incorporated a six- and a
seven-membered lactam sulfonamide moiety at P3-P4,6
respectively. The lactam template was based upon the
pioneering work of Freidinger.7 Both molecules dis-
played a high degree of selectivity for the inhibition of
thrombin over trypsin (Table 1).
F igu r e 1.
Ta ble 1. In Vitro IC50 Values (nM) and Selectivity Ratios of
Compounds 1-6 against Thrombin (FIIa), FXa, and Trypsina
compd FIIab trypsinc
FXab
trypsin/FIIa FXa/FIIa
1
2
3
1.2
1.2
1550
329
301
1
125
463.4
250.8
>201.6
29
12.4
>2500
0.71
20.6
4a
4b
5a
5b
6a
6b
1.7
1.5
113
69.7
60700
>2500
>2500
66.5
46.5
2107.6
102090
8388.9
80351
>1470.6
>1666.7
>868
>37313.4
>1388.9
>43859.6
28.8
>25000
>25000
>2500
0.67 68400
1.8 15100
0.57 45800
>25000
* Please send all correspondence to: Dr. Odile E. Levy, Corvas
International, Inc., 3030 Science Park Rd, San Diego, CA 92121-1102.
Tel: (619) 455-9800 (x127). Fax: (619) 455-7895. E-mail: odile_levy@
corvas.com.
a
Concentration (nM) of 1-6 required to inhibit enzymatic
cleavage of the chromogenic substrates described in reference 13
by 50%. Human thrombin and FXa were used. c Bovine trypsin
b
† Michigan State University.
was used.
S0022-2623(96)00607-3 CCC: $12.00 © 1996 American Chemical Society

4528 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Communications to the Editor
Sch em e 1a
Sch em e 2a
a
(a) BH3, THF, 84%; (b) H2 (40 psi), 10% PtO2, MeOH, HOAc,
quant; (c) bis-CBz-S-methylisothiourea, NMM, THF, 82%; (d) HCl,
MeOH, quant.
In our search for novel oral thrombin inhibitors with
selectivity against other enzymes including trypsin, we
investigated conformationally constrained arginine mim-
ics, in particular the novel L-3-piperidyl(N-guanidino)-
alaninal for P1.8 This communication describes the
syntheses, biological activities, and crystal structures
of several thrombin inhibitors which contain this novel
residue at P1 (compounds 4-6, Figure 1). Since the bis-
CBz protected L-3-piperidyl(N-guanidino)alaninols (11a
and 11b) were prepared nonstereoselectively at the Cγ
center, two diastereomers were obtained from each P1
substitution. In Figure 1 each isomer is labeled as “a”
and “b” according to the order of elution of the diaster-
a
(a) 11a , b, EDC, HOBt, NMM, CH3CN, 81%; (b) H2 (40 psi),
10% Pd/C, MeOH, quant; (c) EDC, DMSO, CH(Cl)2CO2H, toluene,
68%; (d) HPLC, separation of diastereomers.12
trypsin, and factor Xa (FXa) amidolytic activity.13
Selectivity ratios of trypsin/FIIa and FXa/FIIa are also
tabulated. Other proteases such as FVIIa, FXIa, FXIIa,
plasmin, urokinase, and activated protein C (aPC) were
also screened; however, all new targets were inactive
(IC50 > 2.5 µM) on these enzymes.
1
eomeric pair on a reverse phase C18 HPLC column. H
As exemplified by compounds 2 and 3, incorporation
of a six- or a seven-membered lactam ring at P3 with a
natural arginine side chain at P1 increased the trypsin
selectivity by ∼125- and 463.4-fold, respectively, com-
pared to 1. As shown with compounds 4a and 4b (Table
1), replacement of the arginine side chain in 1 (selectiv-
ity ratio ) 1) with that of the more rigid 3-piperidyl-
(N-guanidino)alaninal led to an increase in the desired
thrombin versus trypsin selectivity ratio (66.5 and 46.5,
respectively). Thus, L-3-piperidyl(N-guanidino)alaninal
in P1 represents an additional structure modification to
the lactams in P3 which confers selectivity for thrombin
over trypsin. The discovery of two independent struc-
tural features which enhance selectivity led us to
combine these features to give compounds 5 and 6, in
the hope that further increases in selectivity would be
obtained. As shown in Table 1, this goal was in fact
realized in that analogs 5 and 6 exhibited greatly
enhanced thrombin inhibitory potencies over trypsin.
Compound 5b, which incorporates both the novel P1
residue and the six-membered lactam at P3, has a
selectivity ratio of 102 090 and represents the most
selective transition-state inhibitor of thrombin described
to date. A similar gain in selectivity was observed for
compounds 5a , 6a , and 6b. It is evident from this data
that the introduction of the L-3-piperidyl(N-guanidino)-
alaninal moiety at P1 in conjunction with a lactam ring
at P3 resulted in a cooperative effect on selectivity. This
selectivity was achieved by abolishing the trypsin activ-
ity while retaining the thrombin potency in these
compounds.
NMR studies have shown that each of the two diaster-
eomeric aldehydes exists predominantly in the hydrate
form. Furthermore, the HPLC chromatograms show
single peaks. Unlike argininals 1-3, the conformation-
ally constrained mimics are incapable of undergoing
cyclic aminal formation.9
The preparation of the 3-piperidyl(N-guanidino)ala-
nine moiety is illustrated in Scheme 1. Commercially
available Boc-L-3-pyridylalanine 7 (purchased from Syn-
thetech Inc.) was reduced to Boc-L-3-pyridylalaninol 8
with BH3‚THF in high yields. Catalytic hydrogenation
with PtO2 in methanol generated Boc-L-3-piperidinyl-
alaninols 9a , b quantitatively, as an equal mixture of
diastereomers. Guanylation of the resulting piperidine
ring nitrogen atom with bis-CBz-protected S-methyl-
isothiourea,10 (compounds 10a ,b) and deprotection of
the Boc group afforded the corresponding hydrochloride
salts 11a ,b, which were then coupled to the desired
P2-P4 peptide segments.
The syntheses of the P2-P4 fragments for 4a ,b4a,8c
5a ,b, and 6a ,b5a,8d are reported elsewhere. A represen-
tative synthesis for the diastereomeric pair 4a ,b is
shown in Scheme 2. Segment condensation of the
P2-P4 fragment 12, with 11a ,b, furnished the fully
protected peptide alcohols 13a ,b in 81% yield. Hydro-
genation of the bis-CBz protecting groups in the pres-
ence of palladium on carbon in methanol resulted in
compounds 14a ,b in high yields. A modified Moffat
oxidation11 utilizing the water soluble carbodiimide
(EDC), DMSO, and dichloroacetic acid in toluene con-
verted the alcohols 14a ,b to the aldehydes 4a ,b. The
diastereomers were separated during final purification
by HPLC.12 The diastereomeric pairs 5a ,b and 6a ,b
were prepared analogously to 4a ,b, by condensation of
11a ,b with the corresponding P2-P4 fragments.
The compounds prepared are listed in Table 1 with
the IC50 values for the inhibition of thrombin (FIIa),
Compounds 4a ,b and 5a ,b were evaluated for anti-
thrombotic efficacy in a rat model of acute arterial
thrombosis.14 Three of the compounds 4b, 5a and 5b
showed a dose dependent reduction in the incidence of
thrombotic occlusion in this model, consistent with their
potent in vitro inhibition of thrombin. Additionally,
compound 5b was also demonstrated to be adsorbed

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23 4529
Further biological and pharmacological evaluation of
these compounds and additional details of structural
requirements for their unusual selectivity will be de-
scribed in a future paper.
Ack n ow led gm en t. We thank Terence K. Brunck,
Ph.D., and Marguerita Lim-Wilby, Ph.D., for useful
discussions. Many thanks to Suzanne M. Anderson,
Peter W. Bergum, and George P. Vlasuk, Ph.D., for all
pharmacological studies and Thomas G. Nolan, Ph.D.,
Steven A. Dixon, Malissa M. Lods, Michael G. Ma, and
Huan S. Tran for all analytical support.
Refer en ces
(1) (a) Colman, R. W.; Marder, V. J .; Salzman, E. W.; Hirsch, J .
Overview of hemostatis. In Hemostasis and Thrombosis: Basic
Principles and Clinical Practice, 3rd ed.; Colman, R. W., Hirsch,
J ., Marder, V. J ., Salzman, E. W., Eds.; J . B. Lippincott:
Philadelphia, 1994; pp 3-18. (b) Mann, K. G. Prothrombin and
Thrombin. Ibid. pp 184-199. (c) Colman, R. W.; Cook, J . J .;
Niewiarowski, S. Mechanisms of Platelet Aggregation. Ibid. pp
508-523. (d) Edit, J . F.; Allison, P.; Noble, S.; Ashton, J .; Golino,
P.; McNard, J .; Buja, L. M.; Willerson, J . T. Thrombin is an
important mediator of platelet aggregation in stenosed canine
coronary arteries with endothelial injury. J . Clin. Invest. 1989,
84, 18.
(2) (a) Tapparelli, C.; Metternich, R.; Ehrhardt, C.; Cook, N. S.
Synthetic low-molecular weight thrombin inhibitors: Molecular
design and pharmacological profile. Trends Pharm. Sci. 1993,
14, 366-376. (b) Hirsh, J .; Fuster, V. Guide to anticoagulant
therapy, part I: heparin. Circulation 1994, 89, 1449-1468. (c)
Hirsch, J . Oral anticoagulants. N. Engl. J . Med. 1991, 324,
1865-1875. Hirsch, J . Low molecular weight heparin. Thromb.
Haemost. 1993, 70, 204-207.
(3) (a) Lefkovits, J .; Topol, E. J . Direct thrombin inhibitors in
cardiovascular medicine. Circulation 1994, 90, 1523-1536. (b)
Stone, S. R.; Maraganore, J . M. Hirudin interactions with
thrombin. In Thrombin: Structure and Function; Berliner, L.
J ., Ed.; Plenum Press: New York, 1992; pp 21-256. (c) Topol,
E. J .; Bonan, R.; J ewitt, D.; Sigwart, U.; Kakkar, V. V.; Rothman,
M.; de Bono, D.; Fegurson, J .; Willerson, J . T.; Strony, J .; Ganz,
P.; Cohen, M. D.; Raymond, R.; Fox, I.; Maranganore, J .;
Adelman, B. Use of a direct antithrombin, hirulog, in place of
heparin during coronary angioplasty. Circulation 1993, 87,
1622-1629. (d) Harker, L. A.; Hanson, S. R.; Kelly, A. B.
Antithrombotic benefits and hemorrhagic risks of direct throm-
bin antagonists. Thromb. Haemost. 1995, 74, 464-472.
(4) (a) Vlasuk, G. P.; Webb, T. R.; Pearson, D. A. Inhibitors of
thrombosis. PCT Patent Application WO 93/15756, 1993. (b)
Richard, B. M.; Nolan, T. G.; Tran, H. S.; Miller, M. M.; Nutt,
R. F.; Vlasuk, G. P. Bioavailability of a novel direct thrombin
inhibitor in non-human primates following oral administration.
Circulation 1994, 90, I-180. (c) Rote, W. E.; Dempsey, E. M.;
Oldeschulte, G.; Ardecky, R. J .; Tamura, S. Y.; Nutt, R. F.;
Bergum, P. W.; Tran, H. S.; Anderson, S. M.; Vlasuk, G. P.
Evaluation of a novel orally active direct inhibitor of thrombin
in animal models of thrombosis. Circulation 1994, 90, I-344. (d)
Cousins, G. R.; Friedrichs, G. S.; Sudo, Y.; Rote, W. E.; Vlasuk,
G. P.; Nolan, T. G.; Mendoza, C.; Lucchesi, B. R. Orally effective
CVS-1123 prevents coronary artery thrombosis in the conscious
canine. Circulation 1995, 92, I-303. (e) Vlasuk, G.; Webb, T.;
Pearson, D.; Abelman, M. Inhibitors of thrombosis. U.S. Patent
5492895, 1996. (f) Tamura, S. Y.; Semple, J . E.; Ardecky, R. J .;
Leon, P.; Carpenter, S. H.; Ge, Y.; Shamblin, B. M.; Weinhouse,
M. I.; Ripka, W. C.; Nutt, R. F. Novel and general method for
the preparation of peptidyl argininals. Tetrahedron Lett. 1996,
37, 4109-4112.
(5) (a) Semple, J . E.; Ardecky, B.; Nutt, R. F.; Ripka, W. C.; Rowley,
D. C.; Lim-Wilby, R.; Brunck, T. K. 3-Amino-2-oxo-1-pipe-
ridineacetic acid derivatives as enzyme inhibitors. PCT patent
application, WO 95/35311, 1995. (b) Semple J . E.; Rowley, D.
C.; Brunck, T. K.; Tamura, S. Y.; Ardecky, R. J .; Carpenter, S.
H.; Ge, Y.; Goldman, E.; Minami, N. K.; Owens, T. D.; Siev, D.
V.; Uong, T. H.; Tulinsky, A.; Nutt, R. F.; Ripka, W. C. Design,
Synthesis and evolution of a novel, selective, and orally bio-
available class of thrombin inhibitors: P1-argininal derivatives
incorporating P3-P4 lactam sulfonamide moieties. J . Med. Chem.,
in press.
(6) Substrate and inhibitor nomenclature based on the following:
Schechter, I.; Berger, A. On the size of the active site in
proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967, 27,
157-162.
(7) (a) Freidinger, R. M.; Perlow, D. S.; Veber, D. F. Protected
lactam-bridged dipeptides for use as conformational constraints
in peptides. J . Org. Chem. 1982, 47, 104-109. (b) Bock, M. G.;
Evans, B. E.; Freidinger, R. M.; Gilbert, K.; Hobbs, D. W.;
F igu r e 2. Superimposed structures of inhibitors 5a ,b bound
in the thrombin active site.
following oral administration in conscious dogs to a level
of approximately 15%.15
The preliminary crystal structures of human R-throm-
bin complexed with 5a and 5b have been determined,
and the absolute configuration of each isomer at
P1-Cγ has been assigned.16 Thus, 5a and 5b have the
(R)- and the (S)-configuration at Cγ, respectively. X-ray
crystallographic analysis further revealed that both
inhibitors bind to thrombin in a substrate-like man-
ner, as expected, with the L-3-piperidyl(N-guanidino)-
alaninal occupying the S1 site and the carbonyl and
amine NH of the P3 residue hydrogen bonding with the
thrombin Gly 216 N,O atoms. Both structures were
conformationally very similar at the catalytic site and
at the S2 and S3 subsites (see Figure 2). The positioning
of the 5a and 5b lactam rings in the enzyme site was
also very similar to the structure of the lactam in the
argininal analog 2.5b The trypsin/thrombin selectivity
imparted by the L-3-piperidyl(N-guanidino)alaninal resi-
due does not appear to be due to a differences in
hydrophobicity between the S1 sites of trypsin and
thrombin since the hydrophobic -(CH2)3- loop of the
rigid arginine mimic is oriented out of the S1 site into
the solvent.
In summary, we have discovered novel, highly potent
and selective transition-state thrombin inhibitors which
incorporate the constrained arginine mimic, L-3-pip-
eridyl(N-guanidino)alaninal, at P1. While the inhibitory
potencies of these compounds were comparable to the
potencies achieved by their arginine-containing coun-
terparts, selectivity for thrombin over other serine
proteases such as trypsin and FXa was greatly in-
creased. Compound 5b with a trypsin/thrombin potency
ratio of 5 orders of magnitude is the most selective
transition-state thrombin inhibitor reported to date.

4530 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Communications to the Editor
Lundell, G. F.; Pettibone, D. J .; Rittle, K. E. Piperidinylcam-
phorsulfonyl oxytocin antagonists. PCT Patent Application WO
95/14025, 1995. (c) Freidinger, R. M. Computer graphics and
chemical synthesis in the study of conformation of biologically
active peptides. In Peptides: Synthesis, Structure, and Function;
Proc. Am. Pept. Symp., 7th; Rich, D. H.; Gross, E., Eds.; Pierce
Chem. Co.: Rockford, IL, 1981; pp 673-683.
enzymatic reactions were monitored in the wells of a microtitre
plate (Dynatech) by measuring the increase in absorbance at
405 nm, using a Thermomax microplate reader; this change in
absorbance directly reflected thrombin’s cleavage of Pefachrome
tPA and the release of pNA. A stock solution of inhibitors (1
mM) in ultrapure water was diluted to the desired range of 12
concentrations with Hepes-buffered saline containing BSA
(HBSA), 10 mM Hepes, 150 mM NaCl, and bovine serum
albumin, 0.1%, w/v, pH 7.5. All reactions were performed in
triplicate in a final volume of 200 µL of HBSA, containing at
final concentration 0.5 nM thrombin and 250 µM Pefachrome
tPA. To individual wells was added 50 µL of inhibitor or, in the
case of the control, 50 µL of HBSA, followed by 50 µL of HBSA
and 50 µL of human thrombin. After a 30 min incubation, the
reaction was initiated by the addition of 50 µL of Pefachrome
tPA. All reactions were under steady-state conditions, where less
than 3% of the substrate was consumed. The increasing absor-
bance was measured at 10 s intervals over 5 min, and the values
were stored by a dedicated computer, using Softmax software.
From the data, the software allowed for the calculation of the
velocity (change in absorbance per min); the averaged velocity
for a triplicate sample was plotted against the inhibitor concen-
tration. The data were then fit to a curve described by the four-
parameter equation: Y ) (A - D)/(1 + (X/C)B + D where the
IC50 is represented by term C in the equation. The selectivity of
the inhibitors was examined against 11 additional serine pro-
teinases, including trypsin. The IC50’s were calculated as out-
lined above for thrombin. The concentration of trypsin and
substrate S-2222 (phenacylisoleucylglutamyl(γ-O-methyl)glycyl-
arginine-p-nitroanilide‚HCl, 250 µM, chromogenix) employed
were 0.5 nM and 250 µM, respectively.
(8) (a) St Laurent, D. R.; Balasubramanian, N.; Han, W. T.; Trehan,
A.; Federici, M. E.; Meanwell, N. A.; Wright, J . J .; Seiler, S. M.
Active site directed thrombin inhibitors-II. Studies related to
arginine/guanidine bioisosteres. Biorg. Med. Chem. 1995, 3,
1145-1156. (b) Hilpert, K.; Ackermann, J .; Banner, D. W.; Gast,
A.; Gubernator, K.; Hadva´ry, P.; Labler, L.; Mu¨ller, K.; Schmid,
G.; Tschopp, T. B.; van de Waterbeemd, H. Design and synthesis
of potent and highly selective thrombin inhibitors. J . Med. Chem.
1994, 37, 3889-3901. (c) Levy, O. E.; Tamura, S. Y.; Nutt, R.
F.; Ripka, W. C. Arginine mimic derivatives as enzyme inhibi-
tors. PCT patent application, WO 95/35312, 1995. (d) Semple,
J . E.; Levy, O. E.; Nutt, R. F.; Ripka, W. C. 3-Amino-2-oxo-1-pi-
peridineacetic derivatives containing an arginine mimic as
enzyme inhibitors. PCT patent application, WO 95/35313, 1995.
(9) Bajusz, S. In The design of synthetic inhibitors of thrombin:
Chemistry and biology of the peptide anticoagulant D-MePhe-
Pro-Arg-H (GYKI-14766); Claeson, G., Ed.; Plenum Press: New
York, 1993; pp 91-108.
(10) Verdini, A. S.; Lucietto, P.; Fossati, G.; Giordani, C. A facile
preparation of Fmoc-Arg(Boc)2-OH and Z-Arg(Boc)2-OH, new
arginine derivatives for peptide synthesis. Tetrahedron Lett.
1992, 33, 6541-6542.
(11) (a) Pfitzer, K. E.; Moffatt, J . G. The synthesis of nucleoside-5′
aldehydes. J . Am. Chem. Soc. 1963, 85, 3027-3028. (b) Pfitzer,
K. E.; Moffatt, J . G. Sulfoxide-carbodiimide reactions. I. A facile
oxidation of alcohols. J . Am. Chem. Soc. 1965, 87, 5661-5670.
(12) Purification and separation of the diastereomers were performed
on a reverse phase HPLC column. Analytical runs were per-
formed with a 0.46 × 25 cm VYDAC column containing a C-18
resin comprised of 5 µm gel particles with a 300 Å pore size.
The analytical column was eluted with a water (containing 0.1%
trifluoroacetic acid)/acetonitrile gradient. The gradient was run
from 15% to 30% acetonitrile for 4a and 4b (retention times 15.6
and 16.7 min, respectively); 10% to 20% for 5a and 5b (retention
times 15.7 and 18 min, respectively); 5% to 25% for 6a and 6b
(retention times 20 and 21 min, respectively). Diastereomeric
ratios: 4a /4b ) 1.6/1; 5a /5b ) 1/1.5; 6a / 6b ) 1/1.7. All final
compounds were characterized by mass and 1H NMR spectros-
copy. Results of the high-resolution mass spectra for the purified
peptides are as follows: 4a and 4b (MH+ expected 551.3557,
obtained 551.3563 and 551.3572, respectively); 5a and 5b (MH+
expected 507.2390, obtained 507.2398 and 507.2393, respec-
tively); 6a and 6b (MH+ expected 521.2546, obtained 521.2551
and 521.2560).
(13) En zym e a ssa ys: IC50 Deter m in a tion s. Human thrombin was
purchased from Enzyme Research Laboratories, Inc. (South
Bend, IN); its concentration was predetermined by the supplier
from the absorbance at 280 nM and the extinction coefficient.
The activity of this material was 3806 NIH units/mg. The
potency of inhibitors (IC50) was determined from the inhibition
of the enzymatic (amidolytic) activity of thrombin at 22 °C, using
the chromogenic substrate Pefachrome tPA (CH3SO2-D-hexahy-
drotyrosylglycylarginine-p-nitroanilide‚HOAC, Pentapharm, Ltd,
Basel, Switzerland), obtained from the American distributor
Centerchem, Inc. (Tarrytown, NY). The substrate was reconsti-
tuted as a 4.0 mM stock with ultrapure water (18 mΩ-cm). The
(14) Compounds 4a ,b and 5a ,b were evaluated for antithrombotic
efficacy using a modification of the rat FeCl3-induced model of
acute arterial thrombosis originally described by Kurz et al.
(Kurz, K. D.; Main, B. W.; Sandusky, G. E. Thromb. Res. 1990,
60, 269-280). Each compound was administered to anesthetized
rats as a single intravenous bolus 5 min prior to the application
of the thrombogenic stimulus (FeCl3). The incidence of total
vascular occlusion as monitored by ultrasonic flowtometry was
used as the primary end point. Details of this model will be
published elsewhere.
(15) The absolute systemic bioavailability (% F) for compound 5b was
determined in fasted, conscious, purpose-bred beagle dogs (two
males and two females), following separate intravenous (5 mg/
kg) and oral (20 mg/kg) administration and collection of plasma
samples over a defined time course covering 6 h. The determi-
nation of plasma levels was accomplished using HPLC following
postcolumn fluorogenic derivitization using methodologies that
will be published elsewhere. The area under the plasma con-
centration versus time curves (AUC[0-∞]) for the oral (AUC-
[oral]) versus the intravenous (AUC[iv]) dosing regimens were
calculated by linear trapezoidal estimation using a noncompart-
mental model and were used to calculate %F (AUC[oral]/AUC-
[iv] × 100). Further details on the pharmacokinetic and phar-
macodynamic profile of these compounds will be published
elsewhere.
(16) Zhang, E.; Arni, R. K.; Levy, O. E.; Tulinsky, A. Stereochemical
considerations in drug design: The crystal structures of human
R-thrombin complexed with two tripeptidyl aldehyde inhibitors
at 2.1 A resolution. Abstract submitted to IUCR (International
Union of Crystallographic Research), August 1996, WA.
J M960607J