Novel, potent and selective chimeric FXa inhibitors featuring hydrophobic P1-ketoamide moieties

Article abstract of DOI:10.1016/S0960-894X(00)00458-3

Judicious combination of P-region sequences of highly potent anticoagulant proteins including NAP5, NAP6, Ecotin, and Antistasin with SAR from small molecule FXa inhibitors led to a series of chimeric inhibitors of formula 1a-j. We report herein the desig

Full text of DOI:10.1016/S0960-894X(00)00458-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2305±2309
Novel, Potent and Selective Chimeric FXa Inhibitors Featuring
Hydrophobic P1-Ketoamide Moietiesy
J. Edward Semple,* Odile E. Levy, Nathaniel K. Minami, Timothy D. Owens
and Daniel V. Siev
Department of Medicinal Chemistry, Corvas International, Inc., 3030 Science Park Road, San Diego, CA 92121, USA
Received 23 June 2000; accepted 3 August 2000
Dedicated to the Memory of Joseph E. Semple
AbstractÐJudicious combination of P-region sequences of highly potent anticoagulant proteins including NAP5, NAP6, Ecotin,
and Antistasin with SAR from small molecule FXa inhibitors led to a series of chimeric inhibitors of formula 1a j. We report
herein the design, synthesis, and biological activity of this novel family of FXa inhibitors that express both high in vitro potency
and superb selectivity against related serine proteases. # 2000 Elsevier Science Ltd. All rights reserved.
Factor Xa (FXa) is a trypsin-like serine protease that
catalyzes the penultimate step of the coagulation cascade
following initiation. Assembly of FXa with cofactor FVa
on the surface of an anionic phospholipid membrane in
the presence of calcium forms the prothrombinase
(PTase) complex, which in turn proteolytically activates
the zymogen prothrombin to thrombin (FIIa). Thrombin
is the terminal serine protease of the cascade, converting
soluble ®brinogen to insoluble ®brin and activating plate-
lets that ultimately form a blood clot. Consequently, FXa,
PTase, and FIIa play key roles in the regulation of normal
hemostasis and abnormal intravascular thrombus
development (thrombosis).1 The development of eca-
cious small molecule inhibitors of thrombosis would
ful®ll a major unmet medical need, and continues to be
an area of intensive investigation in the pharmaceutical
industry.2 Selective FXa and PTase inhibitors may have
distinct therapeutic advantages over thrombin inhibi-
tors (i.e., less potential for bleeding complications) and
may ultimately provide greater safety and ecacy for
both venous and arterial antithrombotic indications.
potent and selective FXa and PTase inhibitors AcAP5
(NAP5) and AcAP6 (NAP6)5 that incorporate nontradi-
tional hydrophobic amino acids in their corresponding P1
recognition site led us to investigate new hybrid classes of
synthetic inhibitors. In this letter, we describe the design,
synthesis, and biological activity of potent chimeric FXa
inhibitors 1a±j, which feature novel P3-d-arginine-P1-
hydrophobic ketoamide pharmacophores (Fig. 1).
Inhibitor Design Strategy
Certain small proteins secreted by hematophagous
organisms, including NAP6 (AcAP6, Corvas, Ki FXa
996 pM, PTase 207 pM; P3±P1 putative reactive site
sequence: R-S-F)5 and Ecotin (UCSF, from Escherichia
coli, Ki FXa 54pM; P3±P1 sequence: S±T±M)1b,6 demon-
strate powerful anticoagulant properties in part due to
their potent inhibition of FXa and prothrombinase. In
contrast to related protein FXa inhibitors, including
NAP5 (AcAP5, Corvas, Ki FXa 43 pM, PTase 144 pM),5
Antistasin (Merck, ATS; Ki FXa 500 pM, PTase
114 pM),7 and small molecule inhibitors of FXa that
feature the P3-R-P1-R bis-cationic motif,1,2,4a NAP6
and ecotin utilize aromatic Phe and hydrophobic Met
residues at their P1-positions, respectively. Intrigued by the
presence of such rare, nonbasic residues and cognizant of
the structure±activity relationships (SAR) of synthetic
FXa inhibitors,2,4 we designed novel chimeric targets
1a±j that feature non-basic, aromatic or hydrophobic
P1-a-ketoamide units (Fig. 1).
A large variety of novel, potent, and selective small mol-
ecule thrombin3 and FXa4 inhibitor sca€olds have emerged
from our laboratories. Concurrently, the discovery of a
new family of anticoagulant proteins containing the
*Corresponding author. Tel.: +1-858-455-9800 ext. 1140; fax: +1-858-
455-5169; e-mail: ed_semple@corvas.com
yPortions of this work were presented to the Division of Medicinal
Chemistry, 219th American Chemical Society National Meeting, San
Francisco, CA, 26±30 March, 2000. MEDI.193.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00458-3

2306
J. E. Semple et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2305±2309
Figure 1. Design o0f P1-hydrophobic ketoamide FXa inhibitors 1a±j. X=H, OH, OR; dotted lines represent either aromatic or cyclohexyl rings. R
0
denotes suitable P1 ±Pn moieties.
Our new targets utilize a ketoamide moiety as the tran-
sition state analogue (TSA) functionality and feature a
P3-d-arginine±P1-cyclohexyl/aromatic sca€old. Based
on previous SAR trends, the optimal P4-benzyl sulfo-
namide and P2-sarcosine residues were maintained.4,8
Replacement of the highly basic P1-arginine side chain
(guanidinium cation pKa ꢀ12.5) with hydrophobic/aro-
matic residues was deemed an attractive strategy for
potentially improving the PK pro®les in this series of
inhibitors.4b Furthermore, the aromatic sidechains may
participate in edge-to-face and cation±p interactions at
which was converted in three steps to the acylguanidine
derivative 4 in 88% overall yield. Ring-opening of 4
with the appropriate sarcosine±aluminum amide reagent
followed by ester cleavage to 3 was very ecient and
proved serviceable for multigram preparations of this
key intermediate. To the best of our knowledge, such
ring-opening processes are unprecedented.
Our approach to the P1-Tyr ketoamides 1a,b proceeded
as outlined in Scheme 2. Our recently described TFA-
mediated Passerini strategy11,12 was employed for the
0
0
the S1±S1 pockets of FXa,1,2,9 potentially conferring
concise construction of all key P1±P1 precursors. Oxi-
enhanced binding anity and improved potency. We
report herein the synthesis and biological activity of this
novel family of FXa inhibitors that express high in vitro
potency and superb selectivity against related serine
proteases.
dation of protected tyrosinol to the corresponding
aldehyde13 and immediate three-component reaction
with ethyl isocyanoacetate and TFA in the presence of
pyridine bu€er directly provided multigram quantities
of adduct 5 in good overall yield as a ca. 1:1 mixture of
diastereomers at the newly created a-hydroxy center.
Chiral HPLC analysis (Chiracel AD; isopropanol, hex-
ane gradient) showed only two peaks, indicating reten-
tion of con®guration at the original chiral center.11a
Deprotection and coupling with acid fragment 3 deliv-
ered advanced intermediate 6, which was processed by
standard chemistry to deliver the P1-tyrosine ketoa-
mides 1a,b in satisfactory overall yields. P1-Phenylala-
nine target 1c, a direct analogue of 1a, was produced in
a similar fashion.
Chemistry
Two complementary routes to the P2±P4 fragment 3 are
outlined in Scheme 1.10 In the initial, somewhat capri-
cious peptide-coupling route, commercial Boc-d-
Arg(NO2)±OH was elaborated over two steps to 2,
which in turn was converted to the key intermediate 3.8a
The yields shown were oftentimes dicult to reproduce
especially on multigram scales, which led us to investi-
gate a more reliable alternative route to 3. A novel ring-
opening strategy proceeded from d-Arg(NO2)±OMe,
A convergent approach to the extended P1-Phe ketoamide
1d is outlined in Scheme 3. Boc-Phe±OH was elaborated
ꢁ
ꢁ
.
Scheme 1. Reagents and conditions: (a) HCl Sar±OMe, EDC, HOBt, NMM, CH3CN, 0 C to rt; (b) HCl, MeOH, 0 C to rt, 52±72% overall; (c)
BnSO2Cl, Et3N, CH3CN, 0 ꢁC to rt, 45±72%; (d) LiOH, MeOH, H2O, 0 ꢁC to rt, Dowex H+, ꢀquant; (e) BnSO2Cl, NMM, DMF, 88%; (f) EDC,
ꢁ
ꢁ
.
HOBt, NMM, MeCN, ꢀquant.; (g) Me3Al, HCl Sar-OtBu, THF, 0 C to rt, 75%-quant; (h) TFA, CH2Cl2, 0 C to rt, ꢀquant.

J. E. Semple et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2305±2309
2307
Scheme 2. Reagents and conditions: (a) EDC, Cl2CHCO2H, DMSO, toluene, ꢀ5 ꢁC to rt, ꢀquant; (b) CNCH2CO2Et, TFA, pyridine, CH2Cl2, 0 ꢁC
to rt, 69%; (c) Et2NH, CH2Cl2, 0 ꢁC to rt, ꢀquant; (d) 3, DIPC, HOBt, CH3CN, rt, 49%; (e) H2, Pd/C, EtOH, HOAc, H2O, ꢀquant; (f) RP-HPLC
puri®cation, 42%; (g) for R=H (1a): TFA, CH2Cl2, 0 ꢁC to rt, ꢀquant.
Scheme 3. Reagents and conditions: (a) MeNH(OMe) HCl, HOBt, EDC, DIEA, MeCN, quant; (b) LiAlH4, THF, 78 ꢁC; H+, 99%; (c) tert-
.
BuNC, TFA, 2,4,6-collidine, CH2Cl2, 0 ꢁC to rt, 78%, (Passerini rxn. on ꢀ70 g scale); (d) 6 N HCl, re¯ux; (e) Boc2O, Na2CO3, dioxane, H2O; H+
,
93% for two steps; (f) PhEtNH2, EDC, HOBt, NMM, MeCN, 0 ꢁC to rt, quant; (g) HCl, EtOAc, 0 ꢁC to rt, ꢀquant.; (h) EDC, HOBt, NMM,
MeCN, 0 ꢁC to rt, 95%; (i) 3, HATU, HOAt, NMM, MeCN, 0 ꢁC to rt, ꢀquant; (j) H2, Pd/C, EtOH, HOAc, H2O, ꢀquant; (k) EDC, Cl2CHCO2H,
DMSO, toluene, ꢀ5 ꢁC to rt; (l) RP-HPLC, 34±43% overall.
in ®ve steps to the norstatine derivative 7 on a ca. 20±30
g scale, again featuring a key Passerini reaction step. P1 ±
The synthesis of the P1-cyclohexylalanine ketoamides
1e±j is outlined in Scheme 4. Cyclohexylalanine was
converted in four steps to 11 which after hydrolysis and
protection a€orded the Chx-norstatine derivative 12.
Condensation of 12 with various amines and deprotec-
0
P2 fragment 8 was assembled and coupled with 7 to a€ord
0
9 which, in turn, was coupled with 3 to produce the
advanced intermediate 10. Elaboration of 10 over three
0
0
steps provided the P4±P2 target 1d. This convergent (12-
tion gave the P1±P1 fragments 13a±e. Standard coupling
linear steps longest sequence) methodology proved reli-
able and was scalable, a€ording multigram quantities of
the ®nal target 1d for further biological evaluations.
of acid 3 with amines 13a±e gave the corresponding
products 14a±e, which were converted as outlined to
targets 1e±j.

2308
J. E. Semple et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2305±2309
.
Scheme 4. Reagents and conditions: (a) Boc2O, 1 N NaOH, MeCN, 16 h, quant; (b) MeNH(OMe) HCl, HOBt, EDC, DIEA, MeCN, quant; (c)
LiAlH4, THF, 78 ꢁC; H+0, 93%; (d) tert-BuNC, TFA, pyridine, 0 ꢁC to rt, 46%; (e) 6 N HCl, re¯ux ; (f) Boc2O, K2CO3, dioxane, H2O; H+, 72%
for two steps; (g) Couple P1 -amine, EDC, HOBt, DMF, 75±90%; (h) 4 N HCl, dioxane, 0 ꢁC, ꢀquant; (i) 3, HATU or EDC, HOBt, DIEA, DMF,
62±80%; (j) H2, Pd/C, EtOH, H2O, AcOH, ꢀquant; (k) EDC, DMSO, DCA, toluene, 0 ꢁC to rt; (l) RP-HPLC, 45±63%.
Biological Activity
candidates from series 1 showed classical slow binding
kinetics of inhibition, the reported IC50 values are at
T=30 min. Limited available data indicates these new
targets are only modestly active as PTase inhibitors,
with IC50's=9±29 nM.
The in vitro activity of the P1-ketoamides 1a±j along
with the argininal standard CVS 23714b,8 is summarized
in Table 1. CVS 2371 demonstrated high in vitro
potency on both amidolytic FXa and PTase,4c but
lacked selectivity on plasmin and trypsin. Good to
excellent FXa inhibition was observed for the new tar-
The inhibitory potency of ketoamides 1a±j may result
from numerous energetically favorable interactions
0
0
gets, with IC50's=0.78±46 nM. Within a de®ned P4±P2
throughout the active site P4±P2 manifold, including
sca€old, variations at P1 produced the following SAR
results: ChxAla (1e, 2.1 nM) > Phe (1c, 6.6 nM) ꢂ Tyr
(1a, 7.6 nM) >> Tyr(t-Bu) (1b, 246 nM). Amongst the
four di€erent P1 groups prepared, the hydrophobic P1-
ChxAla target 1e expressed optimal activity. Further
exploration of 1e at the P1 ±P2 region led to superior
inhibitors, with 1h (IC50=0.78 nM) emerging as the top
candidate. Very high selectivity against thrombin, plas-
min and trypsin was noted for most targets. Leading
canonical b-sheet, hydrophobic, van der Waals and
aromatic edge-to-face interactions.4,9 Binding to FXa
probably occurs in a normal extended substrate-like
mode, with the P3-d-Arg residue participating in key
cation±p interactions at the S4 pocket.1b,2c,4 Docking of
0
0
0
our new P1 residues at either the S1 (most likely) or S1
speci®city pockets is tentative, but the biological results
suggest that such unconventional aromatic/hydrophobic
binding interactions are viable in the FXa active site.
Table 1. In vitro activity of P1-hydrophobic ketoamide FXa inhibitors 1a±j
IC50 Values (nM)a
Compound
P1-Sidechain
R
FXa
0.86
7.63
246
6.6
7.89
2.13
13.2
46.4b
0.78
3.26
1.87
PTase
3.4
Plasmin
1200
FIIa
Hu Trypsin
74.7
CVS 2371 Reference
>2500
1a
1b
1c
1d
1e
1f
1g
1h
1i
4-(OH)PhCH2
4-(t-BuO)PhCH2
PhCH2
CH2CO2Et
CH2CO2Et
CH2CO2Et
CH2CONHPhEt
CH2CO2Et
PhEt
(3,4-OCH2O)PhCH2
(3,4-OCH2O)PhCH2
PhCH2
29.2
ND
25.1
25.9
ND
ND
ND
9.0
Inact.
>2500
Inact.
Inact.
Inact.
Inact.
Inact.
>2500
>2500
>2500
>2500
Inact.
>2500
>2500
>2500
2210
>2500
1720
1960
Inact.
Inact.
>2500
Inact.
Inact.
>2500
Inact.
>2500
2500
PhCH2
c-C6H11CH2
c-C6H11CH2
c-C6H11CH2
c-C6H11CH2
c-C6H11CH2
c-C6H11CH2
ND
ND
1j
n-Pentyl
3830
163
aConcentration of compounds 1a±j necessary to inhibit FXa, PTase, plasmin, FIIa, and human trypsin cleavage of the chromogenic substrates
described in ref 3 by 50%. Reported value for each compound is from a single IC50 determination which con®rmed initial range values.
b1g is the P3-d-Arg(NO2) derivative (see Scheme 4).

J. E. Semple et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2305±2309
2309
Conclusion
J. Z.; Levy, O. E.; Gibson, T. S.; Nguyen, K.; Semple, J. E.
ibid. 1999, 9, 3459. (c) Tamura, S. Y.; Goldman, E. A.; Ber-
gum, P. W.; Semple, J. E. ibid. 1999, 9, 2573.
Judicious combination of structural information
gleaned from naturally occurring small protein anti-
coagulants5 7 with evolving SAR trends from small
molecule antithrombotic inhibitors2,4 led us to design
and synthesize a series of novel covalent FXa inhibitors
1a±j, which incorporate hydrophobic or aromatic P1-
ketoamide moieties. Application of our recently descri-
bed Passerini technology11 expedited the assembly of
these targets and facilitated rapid SAR development. In
vitro evaluation revealed potent inhibitors of FXa,
which demonstrated good to excellent selectivity pro®les
towards thrombin (FIIa), plasmin and trypsin. Deriva-
tive 1h (IC50 FXa=0.78 nM, PTase=9.0 nM) emerged
as the top candidate and constitutes a novel structural
paradigm. Numerous active site interactions coupled
with optimal P1-substitution and presentation geometry
are important for conferring good FXa inhibitory
potency and high selectivity into this class.
5. Corvas NAP5 (AcAP5) and NAP6 (AcAP6), novel anti-
coagulant hookworm proteins: Stanssens, P.; Bergum, P. W.;
Ganesmans, Y.; Jespers, L.; Laroche, Y.; Huang, S.; Maki, S.;
Messens, J.; Lauwereys, M.; Cappello, M.; Hotez, P. J.; Vla-
suk, G. P. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2149 and
refs cited therein.
6. Ecotin anticoagulant protein: (a) Seymour, J. L.; Lindquist,
R. N.; Dennis, M. S.; Mo€at, B.; Yansura, D.; Reilly, D.;
Wessinger, M. E.; Lazarus, R. A. Biochemistry 1994, 33, 3949.
(b) McGrath, M. E.; Erpel, T.; Bystro€, C.; Fletterick, R. J.
EMBO J. 1994, 13, 1502. (c) McGrath, M. E.; Hines, W. M.;
Sakanari, J. A.; Fletterick, R. J.; Craik, C. S. J. Biol. Chem.
1991, 266, 6620.
7. Antistasin (ATS): (a) Dunwiddie, C.; Thornberry, N. A.;
Bull, H. G.; Sardana, M.; Friedman, P. A.; Jacobs, J. W.;
Simpson, E. J. Biol. Chem. 1989, 264, 16694. (b) Dunwiddie,
C. T.; Smith, D. E.; Nutt, E. M.; Vlasuk, G. P. Thromb. Res.
1991, 64, 787.
8. (a) Abelman, M. M.; Miller, T. A.; Nutt, R. F. US Patent
5696231, 1997; Chem Abstr. 1996, 125, 196382. (b) Marlowe,
C. K.; Scarborough, R. M.; Laibelman, A. M.; Sinha, U.;
Zhu. B.-Y. US Patent 5721214, 1998; Chem Abstr. 1998, 128,
205143. (c) Marlowe, C. K.; Sinha, U.; Gunn, A. C.; Scarbor-
ough, R. M. Bioorg. Med. Chem. Lett. 2000, 10, 13.
Acknowledgements
We gratefully acknowledge S. M. Anderson, L. Truong,
and P. R. Bergum for all in vitro pharmacological
studies, K. Nguyen for synthesis support, T. G. Nolan
for analytical support, and G. P. Vlasuk, T. K. Brunck
and S. Y. Tamura for stimulating discussions regarding
antithrombotic inhibitor targets.
9. (a) Renatus, M.; Bode, W.; Huber, R.; Sturzebecher, J.;
Stubbs, M. T. J. Med. Chem. 1998, 41, 5445. (b) Krishnan, R.;
Zhang, E.; Hakansson, K.; Arni, R. K.; Tulinsky, A.; Lim-
Wilby, M. S. L.; Levy, O. L.; Semple, J. E.; Brunck, T. K.
Biochemistry 1998, 37, 12094.
10. All new compounds were characterized by full spectro-
scopic (NMR, IR, LR/HRMS) data. Yields refer to spectro-
scopically and chromatographically homogeneous (ꢂ95% by
1H NMR, HPLC, TLC) materials.
References and Notes
11. (a) Semple, J. E.; Owens, T. D.; Nguyen, K.; Levy, O. E.
Org. Lett. 2000, 2, 2769. (b) Semple, J. E. Division of Organic
Chemistry, 219th American Chemical Society National Meet-
ing, San Francisco, CA, 26±30 March, 2000. ORGN.667. (c)
Semple, J. E. Joint American Chemical Society 55th South-
west/15th Rocky Mountain Regional Meeting, El Paso, TX,
21±23 October, 1999. ORGN.202.
12. Passerini reaction: (a) Passerini, M. Gazz. Chim. Ital.
1921, 51, 126. (b) Passerini, M.; Ragni, G.; Gazz. Chim. Ital.
1931, 61, 964. (c) Authoritative review: Ugi, I. In Isonitrile
Chemistry; Ugi, I., Ed.; Academic: New York, 1971; Chapter
7. (d) TiCl4 activation: Caro®glio, T.; Cozzi, P. G.; Floriani,
C.; Chiesa-Villa, A.; Rizzoli, C. Organometallics 1993, 12,
2726. (e) Seebach, D.; Adam, G.; Gees, T.; Schiess, M.; Wei-
gand, W. Chem. Ber. 1988, 121, 507. (f) Seebach, D.; Schiess,
M. Helv. Chim. Acta 1983, 66, 1618. (g) With TFA and pyri-
dine: Lumma, W. J. Org. Chem. 1981, 46, 3668.
1. Vlasuk, G. P. In New Therapeutic Agents in Thrombosis and
Thrombolysis; Sasahara, A. A., Loscalzo, J., Eds.; Marcel
Dekker: New York, 1997; Chapter 15. (b) Ripka, W. C. In
Structure-Based Drug Design; Veerapandian, P., Ed.; Marcel
Dekker: New York, 1997; Chapter 11.
2. Recent reviews: (a) Fevig, J. M.; Wexler, R. R. Annu. Rep.
Med. Chem. 1999, 34, 81. (b) Vacca, J. P. Annu. Rep. Med.
Chem. 1998, 33, 81. (c) Ripka, W. C.; Vlasuk G. P. Annu. Rep.
Med. Chem. 1997, 32, 71.
3. Recent Corvas FIIa inhibitors: (a) Minami, N. K.; Reiner,
J. E.; Semple, J. E. Bioorg. Med. Chem. Lett. 1999, 9, 2625. (b)
Reiner, J. E.; Lim-Wilby, M. S.; Brunck, T. K. Uong, T. H.;
Goldman, E. A.; Abelman, M. A.; Nutt, R. F.; Semple. J. E.;
Tamura, S. Y. ibid. 1999, 9, 895. (c) Owens, T. D.; Semple, J.
E. ibid. 1998, 8, 3683. (d) Semple, J. E.; Rowley, D. C.; Owens,
T. D.; Minami, N. K.; Uong, T. H.; Brunck, T. K. ibid. 1998,
8, 3525. (e) Semple, J. E. Tetrahedron Lett. 1998, 39, 6645. (f)
Semple, J. E. Bioorg. Med. Chem. Lett. 1998, 8, 2501.
4. Recent Corvas FXa Inhibitors: (a) Tamura, S. Y.; Levy, O.
E.; Reiner, J. E.; Uong, T. H.; Goldman, E. A.; Brunck, T. K.
Semple, J. E. Bioorg. Med. Chem. Lett. 2000, 10, 745. (b) Ho,
13. (a) Jurczak, J.; Golebiowski, A. Chem. Rev. 1989, 89, 149.
(b) Ho, P. T.; Ngu, K. J. Org. Chem. 1993, 58, 2313. (c) Hyun,
S. I.; Kim, Y. G. Tetrahedron Lett. 1998, 39, 4299. (d) Myers,
A. G.; Zhong, B.; Movassaghi, M.; Kung, D. W.; Lanman, B.
A.; Kwon, S. Tetrahedron Lett. 2000, 41, 1359.