4536 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 23
Communications to the Editor
of benzo-fused, 7,5- and 7,6-fused azepinones as conformationally
restricted dipeptide mimetics. Tetrahedron Lett. 1995, 36, 1593-
1596. (c) Bernstein, P. R.; Gomes, B. C.; Kosmider, B. J .; Vacek,
E. P.; Williams, J . C. Nonpeptidic inhibitors of human leukocyte
elastase. 6. Design of a potent, intratracheally active, pyridone-
based trifluoromethyl ketone. J . Med. Chem. 1995, 38, 212-215.
(d) Skiles, J . W.; Sorcek, R.; J acober, S.; Miao, C.; Mui, P. W.;
Mc Nell, D.; Rosenthal, A. S. Elastase inhibitors containing
conformationally restricted lactams as P3-P2 dipeptide replace-
ments. Bioorg. Med. Chem. Lett. 1993, 3, 773-778. (e) Fairlie,
D. P.; Abbenante, G.; March, D. R. Macrocyclic peptidomimetics-
forcing peptides into bioactive conformations. Curr. Med. Chem.
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S.; Brooks, J . R.; Saperstein, R. Bioactive conformations of
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formationally constrained analog. Science 1980, 210, 656-658.
(14) (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.;
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.
Proceedings of the 7th American Peptide Symposium; Rich, D.
H., Gross, E., Eds.; Pierce Chemical Co.: Rockford, IL, 1981; pp
673-683.
(15) (a) Aube, J .; Wolfe, M. S. A divergent route toward lactam-based
dipeptidyl building blocks. Bioorg. Med. Chem. Lett. 1992, 2,
925-928. (b) Ede, N. J .; Lim, N.; Rae, I. D.; Ng, F. M.; Hearn,
M. T. W. Synthesis and evaluation of constrained peptide
analogs related to the N-terminal region of human growth
hormone. Pept. Res. 1991, 4, 171-176. (c) Williams, P. D.;
Perlow, D. S.; Payne, L. S.; Holloway, M. K.; Siegl, P. K. S.;
Schorn, T. W.; Lynch, R. J .; Doyle, J . J .; Strouse, J . F.; Vlasuk,
G. P.; Hoogsteen, K.; Springer, J . P.; Bush, B. L.; Halgren, T.
A.; Richards, A. D.; Kay, J .; Veber, D. F. Renin inhibitors
containing conformationally restricted P1-P1' dipeptide mimics.
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G.; Srivastava, L. K.; Mishra, R. K.; J ohnson, R. L. Dopamine
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including 8, 11, 12, 15, and 18. Typical reverse phase HPLC
analysis (Vydac 5 µm C-18, 0.1% TFA in acetonitrile, water
gradients) of each final target 3a -p showed three peaks,
indicative of the presence of a hydrate form and two diastere-
omeric cyclol forms. Although possessing similar retention times,
racemic argininal impurities are readily detectable via HPLC
analysis. Careful NMR analysis can also reveal racemized forms.
Either of the protocols utilized herein typically afforded crude
targets containing e5% racemized argininal byproduct. How-
ever, molecules incorporating argininal residues are regarded
as sensitive and reactive species and considerable care needs to
be taken when purifying and lyophilizing these substances. They
are stable for prolonged periods when stored in the lyophilized
solid form at 4 °C. Also, see refs 20a and 20d. (d) J urczak, J .;
Golebiowski, A. Optically active N-protected R-amino aldehydes
in organic synthesis. Chem. Rev. 1989, 89, 149-164.
(21) In our hands, modification of the reported one-pot, four-step
sequence by performing each step individually afforded more
consistent yields of intermediate 11 (Scheme 3, route I). As
outlined in Scheme 3, route II, we found an alternate four-step
method to 11 which was operationally superior to that described
above.
(22) Available from Sigma Chemical Co.
(23) Håkanson, K.; Tulinsky, A.; Brunck, T. K.; Levy, O. E.; Semple,
J . E.; et al. manuscript in preparation.
Deter m in a tion s. Human thrombin was
(24) En zym e Assa ys: IC50
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-
drotyrosyl-glycyl-arginine-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
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 the inhibitor (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 new inhibitor was also examined against FXa and trypsin.
The IC50 values were calculated as outlined above for thrombin.
Other chromogenic substrates were purchased from Chromoge-
nix. The concentration of enzyme and substrate employed
follows: FXa [0.25 nM], S-2765 [250 µM], trypsin [0.5 nM], and
S-2222 [250 µM].
(16) Semple, J . E.; Ardecky, R. J .; Nutt, R. F.; Ripka, W. C.; Rowley,
D. C.; Lim-Wilby, M. S.; Brunck, T. K. 3-Amino-2-oxo-1-pip-
eridineacetic acid derivatives as enzyme inhibitors. PCT Patent
Application WO 95/35311, 1995.
(17) (a) Stone, S. R. Thrombin inhibitors. A new generation of
antithrombotics. Trends Cardiovasc. Med. 1995, 5, 134-140. (b)
Banner, D. W.; Hadvary, P. Crystallographic analysis at 3.0-Å
resolution of the binding to human thrombin of four active site-
directed inhibitors. J . Biol. Chem. 1991, 266, 20085-20093. (c)
Bode, W.; Mayr, I.; Baumann, U.; Huber, R.; Stone, S. R.;
Hofsteenge, J . The refined 1.9-Å crystal structure of human
R-thrombin: interaction with D-Phe-Pro-Arg chloromethyl-
ketone and significance of the Tyr-Pro-Pro-Trp insertion seg-
ment. EMBO J . 1989, 8, 3467-3475. (d) Håkansson, K.;
Tulinsky, A.; Abelman, M. M.; Miller, T. A.; Vlasuk, G. P.;
Bergum, P. W.; Lim-Wilby, M. S. L.; Brunck, T. K. Crystal-
lographic structure of a peptidyl keto acid inhibitor and human
R-thrombin. Bioorg. Med. Chem. 1995, 3, 1009-1017.
(18) Iwanowicz, E. J .; Lau, W. F.; Lin, J .; Roberts, D. G. M.; Seiler,
S. M. Retro-binding thrombin active-site inhibitors: discovery,
synthesis, and molecular modeling. J . Med. Chem. 1994, 37,
2122-2124.
(19) Morgan, B. A.; Gainor, J . A. Approaches to the discovery of non-
peptide ligands for peptide receptors and peptidases. Annu. Rep.
Med. Chem. 1989, 24, 243-252.
(25) Subsequent synthetic investigations and in vitro biological
evaluation of related P3-lactam derivatives possessing the R-(R)
absolute stereochemistry corroborated the observation of sig-
nificantly decreased thrombin inhibitory potency.
(26) The absolute systemic bioavailability (%F) for compounds 3j, 3m ,
and 3n 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
determination of plasma levels was accomplished using HPLC
following postcolumn fluorogenic derivatization using method-
ologies that will be published elsewhere. The area under the
plasma concentration 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 non-
compartmental model, and were used to calculate %F (AUC-
[oral]/AUC[iv] × 100). Further details on the pharmacokinetic
and pharmacodynamic profile of these compounds will be
published elsewhere.
(20) (a) 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. (b) Webb, T. R.; Reiner, J . E.; Tamura, S. Y.; Ripka,
W. C.; Dagnino, R.; Nutt, R. F. Methods of synthesis of peptidyl
aldehydes. PCT Patent Application WO 95/35280, 1995. (c)
Based on the suggestions and concerns of a reviewer, the
following supporting comments regarding the chiral integrity
of the P1-argininal moiety may be appropriate. Due to their
ability to form 6-membered intramolecular hemiaminal rings
and to exist as hydrate forms, purified argininals produced via
either oxidation or hydrolysis protocols are configurationally
stable. The chiral integrity of precursors 5 and 6 was confirmed
through chiral HPLC analysis (Chiralpak AS; 95% hexane, 5%
ethanol) and by subsequent NMR and normal reverse phase
HPLC analysis of the derived diastereomeric intermediates
J M960572N