7-Azabicycloheptane Carboxylic Acid: A Proline Replacement in a Boroarginine Thrombin Inhibitor

Article abstract of DOI:10.1021/ol990294x

(Matrix Presented) The synthesis of thrombin inhibitor 3, which incorporates conformationally constrained 7-azabicycloheptane carboxylic acid (1) as a proline replacement, is described. The inhibition constant (K_i(thrombin) = 2.9 nM) indicates

Full text of DOI:10.1021/ol990294x

ORGANIC
LETTERS
1999
Vol. 1, No. 12
1875-1877
7-Azabicycloheptane Carboxylic Acid:
A Proline Replacement in a
Boroarginine Thrombin Inhibitor
Wei Han,* Jeffrey C. Pelletier,† Lawrence J. Mersinger, Charles A. Kettner, and
,‡
C. Nicholas Hodge*
Department of Chemical and Physical Sciences, DuPont Pharmaceuticals Company,
Experimental Station, P.O. Box 80500, Wilmington, Delaware 19880
wei.han@dupontpharma.com
Received September 20, 1999
ABSTRACT
The synthesis of thrombin inhibitor 3, which incorporates conformationally constrained 7-azabicycloheptane carboxylic acid (1) as a proline
replacement, is described. The inhibition constant (Ki(thrombin) ) 2.9 nM) indicates that 1 is a reasonable replacement of proline in the formation
of a â-turn tripeptide mimetic.
Peptidomimetic research owes its impetus to the desire to
discover nonpeptides that bind to peptide receptors but have
improved bioavailability, biostability, and selectivity over
endogenous or synthetic peptide ligands.1 One of several
ways to reach this goal is the use of conformationally
constrained analogues that mimic the receptor-bound con-
formation of the bioactive peptides. Conformationally locked
amino acids with spatially well-defined substitution patterns
are also of great value as building blocks for lead discovery
using combinatorial libraries.2 7-Azabicycloheptanecarboxy-
lic acid (1) is particularly attractive as a rigid proline analogue
that can project numerous substituents into defined regions
of space.3 This rigidity is illustrated in Figure 1: quenched
dynamics conformational searching4 of the bis-amide deriva-
† Present address: Wyeth Ayerst Research, 401 North Middletown Road,
Pearl River, NY 10965.
‡ Present address: Caliper Technologies Corporation, 605 Fairchild Drive,
Mountain View, CA 94043. E-mail: nick.hodge@calipertech.com.
(1) (a) Hirschmann, R. Angew. Chem., Int. Ed. Engl. 1991, 30, 1278.
(b) Rudinger, J. In Drug Design; Ariens, E. J., Ed.; Academic: New York,
1971; Vol. 2, p 319. (c) Evans, B. E.; Bock, M. G.; Rittle, K. E.; Dipardo,
R. M.; Whitter, W. L.; Veber, D. F.; Anderson, P. S.; Freidinger, R. M.
Proc. Natl. Acad. Sci. U.S.A.1986, 83, 4918.
Figure 1. Low-energy conformations of 2.4 Internal strain energies,
kcal: A, 11.1; B, 11.3; C, 12.3; D, 12.6.
(2) Ostresh, J. M.; Blondelle, S. E.; Dorner, B.; Houghten, R. A. Methods
Enzymol. 1996 (267: Combinatorial Libraries), 220-234.
10.1021/ol990294x CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/23/1999

tive 2 calculates only four significantly different conformers
within 10 kcal of 2A, the lowest energy conformer. These
conformations correspond to only two allowed φ angles each
for the trans (2A, 2B) and the cis (2C, 2D) amide. Given
these small differences in internal strain energies, one would
expect all of the isomers to exist in equilibrium at room
temperature. The similar energies of the cis and trans
conformations are consistent with the conformational prefer-
ences observed in short peptides containing proline, which
exist as a mixture of isomers.5 One of the trans conformations
(2B) incorporates an approximate â-turn. Our interest in these
compounds as proline mimetics led to the development of a
stereoselective synthesis of 1 and substituted analogues from
inexpensive starting materials.6
of fit.11 Conformer 2B, 0.2 kcal higher than the global
minimum, matched the relevant dihedral angles of DuP714
quite closely (Figure 2). In addition, inspection of the
Next we needed to evaluate these compounds as proline
mimetics in known proline-containing enzyme inhibitors.
N-Acetyl-D-Phe-Pro-boro-Arg (DuP714)7 is a prototypical
inhibitor of the serine protease thrombin, a clinical target of
Figure 2. Conformer 2B superimposed onto DuP714.
thrombin-DuP714 complex crystal structure showed suf-
ficient empty space to accommodate the ethano bridge of 1
(not shown). Taken together, these results suggest that 1
should substitute for proline in DuP714 without major
energetic penalties.
The potent and more readily accessible N-(3-phenylpro-
pionyl)boroarginine inhibitor 9, with a Ki against thrombin
of 0.10 nM,12 was selected as a reference compound for
assessing the effect of replacing proline with ethanoproline
1. The synthesis of target molecule 3 started from 7-(ben-
zyloxycarbonyl)-1-carboxy-7-azabicyclo[2.2.1]heptane tert-
butyl ester (4, Scheme 1).6 Hydrogenation of 4 followed by
great interest for treatment of thrombotic diseases.8 The
conformation of enzyme-bound DuP714 is known from
X-ray structures of its thrombin complex,9 and NMR studies
have demonstrated that free DuP714 is significantly preor-
ganized into its bound form in aqueous solution.10 We
superimposed the calculated low-energy conformers of
truncated tripeptide analogue 2 (Figure 1) onto the enzyme-
bound conformation of DuP714 to determine the goodness
Scheme 1
(3) Conformationally constrained 7-azabicycloheptane amino acids as
proline mimetics. Han, W.; Pelletier, J.; Sahli, A.; Mersinger, L. J.; Kettner,
C. A.; Hodge, C. N. Book of Abstracts, 213th National Meeting of the
American Chemical Society, San Francisco, April 13-17, 1997; American
Chemical Society: Washington, DC, 1997: MEDI-011.
(4) (a) Taber, D.; Christos, T. E.; Hodge, C. N. J. Org. Chem. 1996, 61,
2081. (b) Hodge, C. N.; Straatsma, T. P.; McCammon, J. A. In Structural
Biology of Viruses; Chiu, W., Ed.; Oxford, 1997; pp 451-473. (c) Hodge,
C. N.; Lam, P. Y.; Eyermann, C. J.; Jadhav, P. K.; Ru, Y.; Fernandez, C.
H.; DeLucca, G. V.; Chang, C. H.; Kaltenbach, R. F., III; Holler, E. E.;
Woerner, F. J.; Daneker, W. K.; Emmett, G. C.; Calabrese, J. C.; Aldrich,
P. E. J. Am. Chem. Soc. 1998, 120, 4570.
(5) Grathwohl, C.; Wuethrich, K. Biopolymers 1981, 20, 2623.
(6) Campbell, J. A.; Rapoport, H. J. Org. Chem. 1996, 61, 6313.
(7) (a) Kettner, C.; Mersinger, L.; Knabb, R. J. J. Biol. Chem. 1990,
265, 18289. (b) Wityak, J.; Earl, R. A.; Abelman, M. M.; Bethel, Y. B.;
Fisher, B. N.; Kauffman, G. S.; Kettner, C. A.; Ma, P.; McMillan, J. L.;
Mersinger, L. J.; Pesti, J.; Pierce, M. E. J. Org. Chem. 1995, 60, 3717.
(8) (a) Bazan, J. F. Nature 1996, 380, 21. (b) Tapparelli, C.; Meternich,
R.; Ehrhardt, C.; Cook, N. S. Trends Pharmacol. Sci. 1993, 14, 366. (c)
Lefkovits, J.; Topol, E. J. Circulation 1994, 90, 1523.
(9) Weber, P. C.; Lee, S.-L.; Lewandowski, F. A.; Schadt, M. C.; Chang,
C.-H.; Kettner, C. A. Biochemistry 1995, 34, 3750.
(10) Lim, M. S. L.; Johnston, E. R.; Kettner, C. J. J. Med. Chem. 1993,
36, 1831.
treatment with phenylpropionyl chloride yielded the corre-
sponding N-phenylpropionyl amide, which on treatment with
trifluoroacetic acid provided 1-carboxy-7-(phenylpropionyl)-
7-azabicyclo[2.2.1]heptane 5.13
(11) All heavy atoms of 2B and the corresponding heavy atoms of
DUP714 were used in the superposition calculation. The heavy atom root-
mean-square deviation was less than 0.5 Å.
(12) Fevig, J. M.; Abelman, M. M.; Amparo, E. C.; Cacciola, J.; Kettner,
C. A.; Pacovsky, G. J. U.S. Patent 5,462,964 (October 31, 1995).
1876
Org. Lett., Vol. 1, No. 12, 1999

Coupling of amino acid 5 with bromo amine 67a was
achieved by treatment with BOPCl and Et3N in DMF
(Scheme 2). Bromide 7 was converted to the corresponding
(pH 7.5), containing 0.20 M sodium chloride and 0.5% poly-
(ethylene glycol), MW 6000, and gave a value of 2.9 nM.
Under identical conditions, the parent proline compound had
a Ki of 0.10 nM. It is known that the boronic acid pinanediol
ester is rapidly hydrolyzed to boronic acid under these
conditions,15 so 2.9 nM is an accurate Ki for 3 as well.
If the assumptions made above were correct, the affinity
of constrained analogue 3 for thrombin would be expected
to increase somewhat due to an increase in buried hydro-
phobic surface area, and due to a slight entropic advantage
gained by freezing the proline ring into a single conforma-
tion.16 In the event, a 30-fold decrease in activity is observed
on substituting proline with 1, which suggests that the
ethanoproline derivative cannot attain the exact bound
conformation of Dup714 without an increase in internal strain
energy or steric contacts in the active site, despite the
qualitative overlay of the models. Nonetheless, the potency
of 3 and the ease of incorporation of 1 into peptide analogues
does suggest that 7-azabicycloheptanecarboxylic acid will
be useful as a proline mimetic in lead discovery and in
exploring possible bound conformations of bioactive proline-
containing peptides. We will report on the effect of replacing
the proline in other protease inhibitors with more elaborate
derivatives of 1 shortly.
Scheme 2
Acknowledgment. We wish to thank Professor Henry
Rapoport and Dr. Jeffrey A. Campbell for developing the
synthetic route to compound 4 through our postdoctoral
support program.
amine by displacement with azide followed by hydrogena-
tion. Finally, boroarginine 8 was obtained from the amine
by reaction with iminoaminomethanesulfonic acid in etha-
nol.9
Supporting Information Available: Experimental pro-
cedures and compound characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
The inhibition constant (Ki) of 8 against thrombin was
determined14 at 25 °C in 0.10 mM sodium phosphate buffer
(13) Data for compound 5: 1H NMR (CDCl3) δ 7.30-7.13 (m, 5H),
4.16 (t, 1H, J ) 4.4 Hz), 2.95 (t, 2H, J ) 9.3 Hz), 2.58 (t, 2H, J ) 9.3 Hz),
2.13 (m, 2H), 1.58 (m, 6H), 1.55 (s, 9H); 13CNMR (CDCl3) δ 172.87,
169.71, 141.31, 128.49, 128.40, 126.10, 80.98, 67.99, 59.07, 36.25, 32.47,
31.28, 30.31, 27.92. IR (Neat); 3026, 1730, 1664 cm-1; HRMS (ESI) calcd
for C20H27NO3 330.206919, found 330.206617.
OL990294X
(15) See ref 6a and Berry, S. C.; Fink, A. L.; Shenvi, A. B.; Kettner, C.
A. Proteins: Struct., Funct., Genet. 1988, 205.
(16) Lam, P.; Ru, Y.; Jadhav, P. K.; Aldrich, P. E.; DeLucca, G. V.;
Eyermann, C. J.; Chang, C. H.; Emmett, G.; Holler, E. R.; Daneker, W. F.;
Li, L.; Confalone, P. N.; McHugh, R. J.; Han, Q.; Li, R.; Markwalder, J.
A.; Seitz, S. P.; Sharpe, T. R.; Bacheler, L. T.; Rayner, M. R.; Klabe, R.
M.; Shum, L.; Winslow, D. L.; Kornhauser, D. M.; Jackson, D. A.; Erickson-
Viitanen, S.; Hodge, C. N. J. Med. Chem. 1996, 39, 3514.
(14) Data for compound 8: 1H NMR (CD3OD) δ 7.30-7.11 (m, 5H),
4.36 (bs, 1H), 4.18 (d, 1H, J ) 9.2 Hz), 4.04 (q, 1H, J ) 7.0 Hz), 2.88 (t,
2H, J ) 7.0 Hz), 2.67 (t, 2H, J ) 7.0 Hz), 2.55 (m, 1H), 2.33 (m, 1H),
2.18-1.45 (m, 16H), 1.37 (s, 3H), 1.29 (s, 3H), 0.85 (bs, 5H); HRMS (ESI)
calcd for C31H46BN5O4 564.371241, found 564.372111.
Org. Lett., Vol. 1, No. 12, 1999
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