Discovery of 3-amino-4-chlorophenyl P1 as a novel and potent benzamidine mimic via solid-phase synthesis of an isoxazoline library

Article abstract of DOI:10.1016/S0960-894X(03)00130-6

In an effort to identify orally bioavailable factor Xa inhibitors, two isoxazolines libraries were prepared to scan for novel P1 ligands. From this work, 4-chloro-3-aniline was identified as a novel and potent benzamidine mimic.

Full text of DOI:10.1016/S0960-894X(03)00130-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1795–1799
Discovery of 3-Amino-4-Chlorophenyl P1 as a Novel and Potent
Benzamidine Mimic Via Solid-Phase Synthesis of an Isoxazoline
Libraryy
Patrick Y. S. Lam,* Jessica J. Adams, Charles G. Clark, W. Jason Calhoun,
Joseph M. Luettgen, Robert M. Knabb and Ruth R. Wexler
Bristol-Myers Squibb Co., Experimental Station, PO Box 80500, Wilmington, DE 19880-0500, USA
Received 22 November 2002; revised 23 January 2003; accepted 3 February 2003
Abstract—In an effort to identify orally bioavailable factor Xa inhibitors, two isoxazolines libraries were prepared to scan for novel
P1 ligands. From this work, 4-chloro-3-aniline was identified as a novel and potent benzamidine mimic.
# 2003 Elsevier Science Ltd. All rights reserved.
Factor Xa (FXa) is the crucial enzyme at the con-
vergent point of the intrinsic and extrinsic coagulation
pathways.1À3 Together with FVa, calcium and phos-
pholipids, a prothrombinase complex is formed to
convert prothrombin to thrombin. Thrombin, in turn,
converts fibrinogen to fibrin and activates platelets,
eventually leading to the formation of thrombus or
blood clots. Inhibition of FXa has been demonstrated
to result in antithrombosis in both animal models4 and
clinical settings.5 Thus FXa has emerged as a promis-
ing target for the discovery of oral anticoagulants.
While several extremely potent FXa inhibitors con-
taining benzamidine as the P1 residue have been
identified, poor oral absorption and short duration of
action have precluded the development of these com-
pounds as oral agents. The poor pharmacokinetics and
low permeability are the result of the high basicity and
insufficient lipophilicity of the benzamidine (pKa 11.6).
In order to develop orally-bioavailable FXa inhibitors
with improved pharmacokinetics, many research
groups have focused on less basic, or neutral replace-
ments for the benzamidine moiety.6À8 We have
embarked on a comprehensive and rational design of
benzamidine mimics.9 To accompany the rational
approach, we have also undertaken a random library
approach to search for novel benzamidine mimics.
Herein, we would like to report on the discovery of
3-amino-4-chlorophenyl P1 as a novel and potent benz-
Figure 1. Nonbenzamidine P1 library of isoxazoline FXa inhibitors.
*Corresponding author. Tel.: +1-609-818-5720; fax: +1-609-818-3460; e-mail: patrick.lam@bms.com
yThis work was presented in part at The 220th ACS National Meeting, Washington, DC, Aug 19–24 (2000), MEDI-329. Lam, P. Y. S. ‘Structure-
based Design and Discovery of Orally-bioavailable Potent Nonbenzamidine Factor Xa Inhibitors’ in ‘New Advances in Cardiovascular Therapeutics.’
Symposium.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00130-6

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P. Y. S. Lam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1795–1799
Figure 2. Solid phase synthesis (SPS) of nonbenzamidine library.
Table 1. First nonbenzamidine P1 library
P1 FXa Ki, mM
P1 FXa Ki, mM
P1 FXa Ki, mM
P1 FXa Ki, mM

P. Y. S. Lam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1795–1799
Table 2. Second nonbenzamidine P1 library
1797
P1 FXa Ki, mM
P1 FXa Ki, mM
P1 FXa Ki, mM
P1 FXa Ki, mM
amidine mimic that resulted from our solid-phase
library synthesis approach.
was accomplished in 2% acetic acid in dimethylform-
amide at room temperature to yield isoxazoline 2.
We have recently reported the discovery of a potent
series of monobasic isoxazoline FXa inhibitors which
incorporate a benzamidine ligand in the P1 position10 as
represented by 1 (Fig. 1). The benzamidine ligand of 1
interacts via bidentate hydrogen bonding with Asp189
in the S1 pocket of FXa. To facilitate the search for
novel, less basic P1 ligands, a series of benzamidine
mimics was explored in an isoxazoline library, which
was synthesized using solid-phase synthesis (SPS).11
From the first diverse nonbenzamidine P1 library (22
members, Table 1), we found that a relatively short
alkyl chain (2d) has better FXa affinity than a long alkyl
chain (2m). 3-Carboxyphenyl 2s is inactive, probably
due to the electrostatic repulsion with Asp189 of FXa.
The 4-halophenylisoxazolines appear to be more active
than 3-halophenylisoxazolines (2a–c vs 2f, h, I, o) and 4-
chlorophenyl 2a is the most potent compound from the
first library. This result directed us to bias our second
library towards 4-substituted analogues.
The synthesis12 of the nonbenzamidine library (Fig. 2)
was initiated by condensing acrolyl chloride with Kaiser
oxime resin 313 to provide acryloyl oxime 4.14 A diverse
set of aldehydes 5 was condensed with hydroxylamine
followed by chlorination with N-chlorosuccinimide to
give hydroximoyl chloride 7. 1,3-Dipolar cycloaddition
of the nitrile oxide dipole, generated from 7 by the action
of base, and acryloyl dipolarophile 4 provided isoxazoline
8 regiospecifically.15 Cleavage of the isoxazoline from the
resin with concomitant amidation with biarylaniline 916
Methylenedioxyphenyl 11b was found to have better
FXa affinity than the corresponding ethylenedioxy-
phenyl derivative 11q (Table 2). Thiophene 11e is found
to be more potent than phenyl compound 2j (Table 1).
Styryl 11l is equivalent to the corresponding furan ana-
logue 11m. The flexible phenethyl 2q exhibits poor
binding affinity compared with the more con-
formationally rigid styryl 11l. It was interesting to note
that the large 4-biphenyl 11d can be accommodated as

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P. Y. S. Lam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1795–1799
the P1 moiety. In terms of para substituents, p-methoxy
11c has the best FXa Ki (97Â weaker than benzamidine
1). The p-methoxy derivative 11c is more potent than
difluoromethyl 11f, methyl 11k and hydroxy 11t. From
the library the most potent P1 ligand was found to be
3-amino-4-chlorophenyl 11a.17 Remarkably, compared
with benzamidine 1, it is only 18-fold weaker in FXa Ki.
This places 3-amino-4-chlorophenyl P1 as one of the
best benzamidine mimics reported.6,7,9
10. During the course of our investigation, two isoxazoline
library syntheses were reported. Solid phase: Cheng, J.-F.;
Mjalli, A. M. M. Tetrahedron Lett. 1998, 39, 939. Fluorous
phase: Studer, A.; Curran, D. P. Tetrahedron 1997, 53, 6681.
11. Representative procedure: 4-Chloro-3-nitrobenzaldehyde
(279 mg, 1.5 mmol) was placed in a 4 mL plastic vial with an
internal filter and a drain plug. Ethanol (2.5 mL) was added
followed by the addition of an aqueous hydroxylamine
hydrochloride solution (0.50 mL, 1.8 mmol, 3.74 M) and an
aqueous sodium acetate solution (0.79 mL, 1.2 mmol, 1.52 M).
The vial was rotated for 12 h on a Barnstead/Thermolyne
Labquake Rotator. The ethanol and water are removed under
a stream of nitrogen leaving a solid that was washed once with
water and dried under dynamic vacuum to give 4-chloro-3-
nitro-benzaldehyde oxime.
In summary, we have designed a versatile SPS route to
make libraries based on diverse nonbenzamidine P1
substituents and discovered a novel 3-amino-4-chloro-
phenyl P1 as one of the most potent benzamidine
mimics reported (loss of only 18Â in FXa Ki).8 The
optimization of this novel benzamidine mimic will be
described elsewhere.18,19
4-Chloro-3-nitro-benzaldehyde oxime was placed in a 4 mL
plastic vial with an internal filter and anhydrous chloroform (2
mL) and THF (1 mL) are added. N-chlorosuccinimide (200
mg, 1.5 mmol) was added followed by pyridine (15 mL, 0.1%
mol) and the solution was rotated for two h to give 4-chloro-3-
nitro-N-hydroxy-benzimoyl chloride.
References and Notes
The acryloyl oxime resin14 4 (200 mg, 0.15 mmol) was added
to the 4 mL plastic vial containing 4-chloro-3-nitro-N-
hydroxy-benzimoyl chloride followed by dropwise addition of
N,N-diisopropylethylamine (0.26 mL, 1.50 mmol) and the vial
was rotated for 2 h. The solution darkened in color over this
time. The vial was placed on a Baker-21 extraction system
SP24. Utilizing the drain plug, the resin was washed with
DMF (3Â), methanol (4Â), ethyl acetate (4Â), and lastly with
dichloromethane (4Â), and dried under a stream of air. IR: n
1772 cmÀ1 (C¼O), no 1400 cmÀ1 peak was detected.
1. (a) Rosenberg, J. S.; Beeler, D. L.; Rosenberg, R. D. Act. J.
Biol. Chem. 1995, 250, 1607. (b) Davre, E. W.; Fujikawa, K.;
Kisiel, W. T. Biochem. 1991, 30, 10363. (c) Rosenberg, R. D.;
Damus, P. S. J. Biol. Chem. 1973, 248, 6498.
2. Mann, K. G.; Nesheim, M. E.; Church, W. R.; Haley, P.;
Krishnaswamy, S. Blood 1990, 76, 1.
3. (a) Wong, P. C.; Quan, M. L.; Crain, E. J.; Watson, C. A.;
Wexler, R. R.; Knabb, R. M. J. Pharm. Exper. Therap. 2000,
292, 351. (b) Wong, P. C.; Crain, E. J.; Knabb, R. M.; Meade,
R. P.; Quan, M. L.; Watson, C. A.; Wexler, R. R.; Wright,
M. R.; Slee, A. M. ibid. 2000, 295, 212.
4. Murayama, N.; Tanaka, M.; Kunitada, S.; Yamada, H.;
Inoue, T.; Terada, Y.; Fujita, M.; Ikeda, Y. Clin. Pharm.
Therap. 2000, 66, 258.
5. (a) For reviews and references see: Vacca J. P. Thrombosis
and Coagulation. In Annual Reports in Med. Chem., Bristol, J.
A. Ed. Academic Press, 1998, 33, pp 81–90. (b) Fevig, J. M.;
Wexler, R. R. Annual Reports in Med. Chem., Doherty, A. M.
Ed. Academic Press, 1999, 34, pp 81–100. (c) Zhu, B.-Y.;
Scarborough, R. M. ibid 2000, 35, 83. (d) Sanderson, P. E. J.
ibid 2001, 36, 79.
6. (a) For reviews specifically on nonbenzamidines see: Me-
near, K. Curr. Med. Chem. 1998, 5, 457. (b) Rewinkel, J. B. M.;
Adang, A. E. P. Cur. Pharm. Design 1999, 5, 1043. (c) Peterlin-
Masic, L.; Kikelj, D. Tetrahedron 2001, 57, 7073.
7. Pinto, D. J.; Orwat, M. J.; Wang, S.; Fevig, J. M.; Quan,
M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.; Alexander,
R. S.; Wong, P. C.; Knabb, R. M.; Luettgen, J. M.; Aungst,
B. J.; Li, L.; Wright, M.; Jona, J. A.; Wexler, R. R.; Lam,
P. Y. S. J. Med. Chem. 2001, 44, 566.
8. Lam, P. Y. S.; Clark, C. G.; Li, R.; Pinto, D. J. P.; Orwat,
M. J.; Galemmo, R. J.; Fevig, J. M.; Alexander, R. S.; Small-
wood, A. M.; Rossi, K. A.; Wright, M. R.; Bai, S.; He, K.;
Luettgen, J. M.; Wong, P. C.; Knabb, R. M.; Wexler, R. R.
Submitted to J. Med. Chem.
40-Amino-biphenyl-2-sulfonic acid amide17 9 (74 mg, 0.30
mmol) and N,N-dimethylformamide (2 mL) were added to the
vial. The vial was capped, agitated for 60 s, and acetic acid (40
mL) was added and the solution was rotated for 14 h at room
temperature. The vial was placed onto the Baker-21 extraction
system, the solvent was collected into a test tube and the resin
was washed with DMF (2Â7 mL) and collected into the same
test tube. The collected solvent was placed in a separatory
funnel and partitioned with ethyl acetate and a 5% HCl solu-
tion. The organic phase was washed with 5% HCl solution
(5Â), saturated sodium bicarbonate solution (1Â), brine (1Â),
dried over sodium sulfate and the solvent was evaporated
under reduced pressure. The residue was gravity filtered
through a silica filled tube (Supelco SupelcleanTM LC-Si SPE
tube) using an ethyl acetate/hexane gradient to give 46 mg of 3
-(4-chloro-3-nitro-phenyl)-4,5-dihydro-isoxazole-5-(20-sulfamoyl-
biphenyl-4-yl)-carboxamide 11x (61% yield). The product is
judged to be >95% pure by NMR. 1H NMR (CDCl3) d 8.71 (s,
1H), 8.18 (d, 1H, J=1.8 Hz), 8.09 (d, 1H, J=8.1 Hz), 7.85–7.28
(m, 9H), 5.38 (dd, 1H, J=11.1, 4.8 Hz), 4.58 (bs, 2H), 3.87–3.78
(m, 2H). MS (CI)(M+NH4)+ 518/520 Cl pattern (20%). The
recovered resin showed no C¼O at 1772 cmÀ1 (IR).
12. Preparation of acryloyl oxime resin 4: To a 3-necked 2 L
round bottomed flask equipped with a mechanical stirrer,
septum capped inlet and a nitrogen bubbler was added p-
nitrobenzophenone oxime polystyrene (Kaiser) resin 3 (40 g,
0.76 mmol/g, 30.4 mmol). Dry methylene chloride (800 mL)
and N,N-diisopropylethylamine (20.9 mL, 120 mmol) were
added and the flask cooled in an ice bath to 0 ꢀC. To the stirred
suspension of the resin was added acryloyl chloride (7.4 mL, 90
mmol) dropwise via a syringe. The ice bath was removed and
the stirring continued for 12 h. The resin was isolated by fil-
tration and washed in the filter funnel successively with meth-
ylene chloride (2Â), methanol (2Â), ether (1Â), and methylene
chloride (2Â) and then dried under dynamic vacuum for 14 h.
9. (a) Quan, M. L.; Pruitt, J. R.; Ellis, C. D.; Liauw, A. Y.;
Galemmo, R. A.; Stouten, P. F. W.; Wityak, J.; Knabb, R. M.;
Thoolen, M. J.; Wong, P. C.; Wexler, R. R. Bioorg. Med.
Chem. Lett. 1997, 7, 2813. (b) Quan, M. J.; Liauw, A. Y.; Ellis,
C. D.; Pruitt, J. R.; Bostrom, L. L.; Carini, D. J.; Huang, P. P.;
Harrison, K.; Knabb, R. M.; Thoolen, M. J.; Wong, P. C.;
Wexler, R. R. J. Med. Chem. 1999, 42, 2752. (c) Quan, M. J.;
Ellis, C. D.; Liauw, A. Y.; Alexander, R.; Knabb, R. M.; Lam,
G. N.; Wong, P.C.; Wexler, R. R. J. Med. Chem. 1999, 42,
2760. (d) Quan, M. L.; Wexler, R. R. Curr. Top. Med. Chem.
2001, 1, 137.
IR: n 1756 (C¼O), 1632 (C¼C) cmÀ1
.
13. DeGrado, W. F.; Kaiser, E. T. J. Org. Chem. 1982, 47,
3258.

P. Y. S. Lam et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1795–1799
1799
14. IR: n 1772 (C¼O) cmÀ1
.
18. (a) Quan, M. L.; Ellis, C. D.; He, M. Y.; Liauw, A. Y.;
Woerner, F. J.; Alexander, R. S.; Knabb, R. M.; Lam, P. Y.
S.; Luettgen, J. M.; Wong, P. C.; Wright, M. R.; Wexler, R. R.
Bioorg. Med. Chem. Lett. 2003, 13, 369. (b) Quan, M. L.;
Ellis, C. D.; He, M. Y.; Liauw, A. Y.; Lam, P. Y. S.;
Rossi, K. A.; Knabb, R. M.; Luettgen, J. M.; Wright, M.
R.; Wong, P. C.; Wexler, R. R. Bioorg. Med. Chem. Lett
2003, 13, 1023.
19. The 3-amino-4-chlorophenyl P1 moiety was incorporated
into an analogous series and the pharmacokinetic profile
examined in dog. This compound was found to be highly
bioavailable (71%).
15. Quan, M. J.; Liauw, A. Y.; Ellis, C. D.; Pruitt, J. R.;
Bostrom, L. L.; Carini, D. J.; Huang, P. P.; Harrison, K.;
Knabb, R. M.; Thoolen, M. J.; Wong, P. C.; Wexler, R. R. J.
Med. Chem. 1999, 42, 2752.
106. Preparation of 40-amino-biphenyl-2-sulfonic acid amide 9:
4 -Amino-biphenyl-2-sulfonic acid tert-butylamide18 (5.00 g,
16.4 mmol) was heated to reflux in trifluoroacetic acid (80 mL)
for 7 h. The solvent was evaporated and the solid judged to be
of sufficient purity for further use.
ꢁ
17. 11a was obtained from 11x via the use of SnCl2 2H2O in
refluxing ethyl acetate.