Synthesis and SAR of benzamidine factor Xa inhibitors containing a vicinally-substituted heterocyclic core

Article abstract of DOI:10.1016/S0960-894X(01)00029-4

The selective inhibition of coagulation factor Xa has emerged as an attractive strategy for the discovery of novel antithrombotic agents. Here we describe highly potent benzamidine factor Xa inhibitors based on a vicinally-substituted, heterocyclic core.

Full text of DOI:10.1016/S0960-894X(01)00029-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 641±645
Synthesis and SAR of Benzamidine Factor Xa Inhibitors
Containing a Vicinally-Substituted Heterocyclic Core
John M. Fevig,* Donald J. Pinto,* Qi Han, Mimi L. Quan, James R. Pruitt,
Irina C. Jacobson, Robert A. Galemmo, Jr., Shuaige Wang, Michael J. Orwat,
Lori L. Bostrom, Robert M. Knabb, Pancras C. Wong, Patrick Y. S. Lam
and Ruth R. Wexler
DuPont Pharmaceuticals Company, PO Box 80500, Wilmington, DE 19880-0500, USA
Received 20 October 2000; accepted 22 December 2000
AbstractÐThe selective inhibition of coagulation factor Xa has emerged as an attractive strategy for the discovery of novel
antithrombotic agents. Here we describe highly potent benzamidine factor Xa inhibitors based on a vicinally-substituted hetero-
cyclic core. # 2001 DuPont Pharmaceuticals Company. Published by Elsevier Science Ltd. All rights reserved.
The serine protease factor Xa (FXa) is central to the
regulation of the coagulation cascade because it is situ-
ated at the con¯uence of the intrinsic and extrinsic
for comparative purposes. This manuscript will describe
the synthesis and SAR of these analogues in an attempt
to further optimize this series of FXa inhibitors.
pathways. FXa combines with factor Va in a Ca2+
-
dependent manner on phospholipid surfaces to form the
prothrombinase complex, which generates thrombin by
the limited proteolysis of prothrombin. The inhibition
of thrombin generation by selective FXa inhibition is
emerging as a promising strategy for the development of
e€ective antithrombotic agents, which may o€er advan-
tages relative to existing therapies.2 Previous reports
from our laboratories (Fig. 1) have described the isox-
azole benzamidine 13 and the benzamidines 2±4 as
potent inhibitors of human FXa.4 These inhibitors di€er
in the point of attachment of the benzamidine P1 resi-
due to the heterocyclic core, with 1 being `C-substituted'
on the core and 2±4 being `N-substituted' on the core.
The greater than an order of magnitude range of
potency between these inhibitors suggested that the
nature of the heterocyclic core is critical to binding
potency, and led us to prepare seven additional `C-
substituted' core analogues 5a±g and ®ve additional `N-
substituted' core analogues 6j±n (for structures, see
Table 1). Although we were primarily interested in pre-
paring ®ve-membered heterocycles, two examples con-
taining a six-membered core, 5h and 5i, were prepared
The general synthesis scheme for the heterocyclic ana-
logues 5a±h and 6j±n is shown in Scheme 1. These ana-
logues were prepared from intermediates 7, in which the
core heterocycle is substituted with an ester or acid
functionality, and a nitrile residue serves as the amidine
precursor. Direct coupling of the esters 7 (R=Et or Me)
with the aluminum reagent5 derived from 40-amino-N-
(tert-butyl)[1,10-biphenyl]-2-sulfonamide6 produced the
*Corresponding authors. E-mail: john.m.fevig@dupontpharma.com,
donald.j.pinto@dupontpharma.com
Figure 1.
0960-894X/01/$ - see front matter # 2001 DuPont Pharmaceuticals Company. Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00029-4

642
J. M. Fevig et al. / Bioorg. Med. Chem. Lett. 11 (2001) 641±645
amides 8 in good yields. In the case of the triazole acid
7m, amide coupling was accomplished via the acid
chloride to a€ord amide 8m. Standard Pinner reaction
on the nitriles 8 gave the corresponding amidines and
TFA-catalyzed deprotection a€orded the ®nal com-
pounds 5a±h and 6j±n.
in re¯uxing ethanol a€orded the methylthiazole 7f. The
unsubstituted thiazole was prepared by treating 12 with
thiourea in re¯uxing ethanol to furnish the amino-
thiazole 13. Treatment of 13 with tert-butyl nitrite
and copper(II) bromide in re¯uxing acetonitrile gave a
bromothiazole, which was reduced with zinc in re¯uxing
acetic acid to give thiazole 7g.
The phenyl core analogue 5i was prepared as shown in
Scheme 2. Amidation of 2-bromobenzoyl chloride 9 with
40-amino-N-(tert-butyl)[1,10-biphenyl]-2-sulfonamide in
the presence of DMAP was followed by Suzuki cou-
pling with 3-cyanophenyl boronic acid to a€ord 8i. This
material was subjected to the Pinner reaction and
TFA-catalyzed deprotection as described in Scheme 1 to
give the ®nal compound 5i.
The pyridine core intermediate 7h was prepared as
shown in Scheme 4. Oxidation of 2-bromo-3-methyl-
pyridine 14 with potassium permanganate gave a car-
boxylic acid, which was esteri®ed with DEAD,
triphenylphosphine, and methanol to a€ord the methyl
ester 15. Suzuki coupling of 15 with 3-cyanophenyl
boronic acid furnished the pyridine intermediate 7h.
The ®ve-membered heterocyclic C-linked compounds
7a±g were prepared as shown in Scheme 3. Treatment of
acid chloride 10b with ethyl isocyanoacetate in the pre-
sence of triethylamine a€orded the oxazole 7a. Treat-
ment of methyl ketone 10a with diethyl carbonate in the
presence of sodium hydride gave ketoester 11. Sequen-
tial treatment of 11 with N,N-dimethylformamide
dimethyl acetal and then with methyl hydrazine led to
the N-methylpyrazole 7b as the predominant regio-
isomer. Bromination of 11 with NBS gave 12, which
was a useful intermediate for preparing several of the
required heterocyclic intermediates 7. Heating 12 with
urea in DMF a€orded the imidazolone 7c. Sequential
treatment of 12 with potassium acetate in DMF and
then with ammonium acetate in re¯uxing acetic acid
gave an approximately 1:3 mixture of imidazole 7d and
oxazole 7e. Finally, treatment of 12 with thioacetamide
Scheme 3. Reagents: (a) ethyl isocyanoacetate, Et3N, CH2Cl2;
(b) NaH, diethyl carbonate, THF, 60 ꢀC; (c) Me2NCH(OMe)2, 100 ꢀC;
(d) methylhydrazine, EtOH, 80 ꢀC; (e) N-bromosuccinimide, CCl4;
(f) urea, DMF, 100 ꢀC; (g) KOAc, DMF; (h) NH4OAc, AcOH,
100 ꢀC; (i) thioacetamide, EtOH, 80 ꢀC; (j) thiourea, EtOH, 80 ꢀC; (k)
tert-butyl nitrite, CuBr2, CH3CN, 80 ꢀC; (l) Zn powder, HOAc,
100 ꢀC.
Scheme 1. Reagents: (a) AlMe3, 40-amino-N-(tert-butyl)[1,10-biphe-
nyl]-2-sulfonamide, CH2Cl2, 40 ꢀC; (b) (i) (COCl)2, CH2Cl2, DMF, (ii)
40-amino-N-(tert-butyl)[1,10-biphenyl]-2-sulfonamide, Et3N, CH2Cl2;
(c) (i) HCl (gas), MeOH, 0 ꢀC, (ii) (NH4)2CO3, MeOH; (d) TFA,
re¯ux.
Scheme 2. Reagents: (a) 40-amino-N-(tert-butyl)[1,10-biphenyl]-2-sul-
fonamide, DMAP, CHCl3, 0±25 ꢀC; (b) 3-cyanophenyl boronic acid,
(PPh3)4Pd, TBAB, 2 M Na2CO3, toluene, 80 ꢀC.
Scheme 4. Reagents: (a) KMnO4, H2O; (b) DEAD, PPh3, MeOH; (c)
3-cyanophenyl boronic acid, Pd(PPh3)2Cl2, TBAB, 2 M Na2CO3,
benzene, 80 ꢀC.

J. M. Fevig et al. / Bioorg. Med. Chem. Lett. 11 (2001) 641±645
643
The imidazole core intermediates 7j and 7k were pre-
pared as shown in Scheme 5. Reaction of 3-¯uoro-
benzonitrile 16 with imidazole or 4-methylimidazole
produced 17a and 17b, respectively. Regiospeci®c
deprotonation of 17a and 17b at C-2 of the imidazole
ring was accomplished with n-BuLi, and the resulting
anions were quenched with methyl chloroformate to
a€ord the required imidazole intermediates 7j and 7k,
respectively.
iminoyl chloride, which upon treatment with sodium
azide gave the tetrazole core intermediate 7n.
The binding results for the inhibitors 5a±i and 6j±n are
shown in Table 1, along with data for compounds 1±4.
Several general trends can be observed within this
somewhat limited set of 18 compounds. First, the `N-
substituted' compounds are generally more potent than
the `C-substituted' compounds. For example, the pyra-
zole 2 is 4- to 30-fold more potent than the structurally
similar `C-linked' compounds 5b, 5e, and 5f. Also, the
three most potent compounds (2, 6m, and 6n) are all
The remaining core intermediates 7l±n were prepared
from 3-aminobenzonitrile 18 as shown in Scheme 6.
Condensation of 18 with butyl glyoxalate in re¯uxing
ethanol gave an imine which, when treated with tosyl-
methyl isocyanate (TosMIC) and potassium carbonate
in methanol, underwent cyclization and transesteri®ca-
tion to give the imidazole methyl ester 7l. To prepare
the triazole 7m, 18 was diazotized and treated with
sodium azide to a€ord 3-azidobenzonitrile. This azide
underwent [3+2] cyclization with 3,3-diethoxy-1-pro-
pyne to produce a mixture of the triazoles 19a and 19b
in an approximately 2:1 ratio. The desired triazole 19b
was hydrolyzed to the aldehyde and subsequently oxi-
dized to the carboxylic acid 7m with silver nitrate.
Finally, the tetrazole 7n was prepared from 18 by a
three step sequence involving initial N-acylation with
ethyl oxalyl chloride. Treatment of the resulting amide
with triphenylphosphine/carbon tetrachloride gave an
Table 1. FXa, thrombin and trypsin inhibition results for 1±4 and
core analogues 5a±i and 6j±na
Scheme 5. Reagents: (a) imidazole or 4-methylimidazole, K2CO3,
DMF, 110 ꢀC; (b) (i) n-BuLi, THF, À78 ꢀC, (ii) ClCO2Me, THF,
À78 ꢀC.
Compounds FXa Ki (nM)b Thrombin Ki (nM)b Trypsin Ki (nM)b
1
2
3
4
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.15
0.013
0.16
0.80
2.5
0.20
2.3
6.0
0.40
0.06
0.82
1.0
2000
300
900
900
21
16
15
56
261
220
390
1100
190
60
3300
2640
6000
>21,000
3600
1250
>6300
15,400
>6300
800
97
260
780
26
27
58
8.2
6j
6k
6l
6m
6n
0.33
0.30
1.1
0.023
0.037
600
3200
300
3.4
6.4
300
aAll compounds were puri®ed by reverse phase HPLC (water/acetoni-
trile gradient+0.5% TFA) and isolated as TFA salts following lyo-
philization. All compounds gave satisfactory spectral and analytical
data.
bHuman puri®ed enzymes were used. Values are averages from multi-
ple determinations (nꢁ2) and the standard deviations were <30% of
the mean. Ki values were measured as described in ref 7.
Scheme 6. Reagents: (a) butyl glyoxalate, ethanol, 80 ꢀC; (b) TosMIC,
K2CO3, MeOH; (c) NaNO2, TFA; NaN3, H2O; (d) 3,3-diethoxy-1-
propyne, re¯ux; (e) 1 N HCl, THF/H2O; (f) AgNO3, NaOH, H2O; (g)
ethyl oxalyl chloride, Et3N, CH2Cl2; (h) PPh3, CCl4, re¯ux; (i) NaN3,
CH3CN.

644
J. M. Fevig et al. / Bioorg. Med. Chem. Lett. 11 (2001) 641±645
Table 2. Antithrombotic ecacy of 1, 2, 5a, 5f, and 6n in a rabbit
arterio-venous (A-V) shunt model8
actions with Gly218 and Cys220, which might explain
the preference for substitution at this position. The
preference for `N-linked' over `C-linked' heterocycles is
unclear without further structural work, but may
involve di€erences in the dihedral angle between the
heterocycle and the P1 benzamidine residue, which for 2
is about 70ꢀ.
Compound
Human FXa
Ki (nM)
Rabbit FXa
Ki (nM)
ID50
(mmol/kg/h)
1
2
5a
5f
6n
0.15
0.013
2.5
0.06
0.037
0.40
0.03
nda
0.08
nda
0.31
0.023
0.54
0.50
0.13
Several of these analogues (5a, 5f, and 6n) were tested
for in vivo antithrombotic ecacy in a rabbit arterio-
venous (A-V) shunt model,8 and the results are shown in
Table 2 along with comparative results for 13 and 2.4 All
of these compounds are potent in this assay, with each
and=not determined.
`N-linked.' Second, anity is increased by having a ter-
tiary nitrogen atom at the core position adjacent to the
P1 residue. Thus, the pyrazole 3 is 5-fold more potent
than the pyrrole 4 and the pyridine 5h is 8-fold more
potent than the phenyl analogue 5i. Also, this structural
feature is shared by the most potent inhibitors. For
example, in the `C-linked' series 5a±g, only those ana-
logues with this tertiary nitrogen display subnanomolar
binding potency. Third, anity is increased by sub-
stitution on the core distal to the P1 and P4 residues, as
evidenced by the greater potencies of the 3-methyl-
pyrazole 2 and the 2-methylthiazole 5f relative to the
pyrazole 3 and the thiazole 5g, respectively. This sub-
stitution also results in greater trypsin selectivity, again
seen by comparing 2 and 5f with 3 and 5g, respectively.
Also, a nitrogen atom at this position in the core
heterocycle enhances binding anity. Thus, the triazole
6m and the tetrazole 6n are considerably more potent
than the pyrazole 3. However, the nitrogen atom does
not lead to greater trypsin selectivity, as the trypsin
selectivity of 6m and 6n more closely resembles that
of pyrazole 3 than that of pyrazole 2. Finally, ®ve-
membered cores are more potent than six-membered
cores, as shown by the lower binding anities of the
pyridine 5h and the phenyl analogue 5i.
having a submicromolar ID50. The most potent
compounds are the pyrazole 2 and the tetrazole 6n.
In conclusion, we have described the SAR of a series of
FXa inhibitors in which various heterocyclic core tem-
plates are vicinally-substituted with a P1 benzamidine
residue and a P4 biphenylsulfonamide residue. This
structural motif has resulted in several highly potent
FXa inhibitors, especially pyrazole 2, thiazole 5f, tria-
zole 6m and tetrazole 6n, all of which have a FXa
Ki<0.1 nM. These potent templates provide the oppor-
tunity to replace the benzamidine with less basic P1
moieties, thereby sacri®cing some potency in exchange
for improved oral absorption. E€orts along these lines
are ongoing and will be reported in due course.1c,4b,9
Acknowledgements
The authors wish to thank Dale E. McCall, Joseph
M. Luettgen and Andrew W. Leamy for obtaining
compound binding data and Earl J. Crain for in vivo
studies.
The imidazoles 6j and 6k are interesting in that they do
not follow the general trends observed for the other
compounds. For example, both are quite potent without
the tertiary nitrogen adjacent to the P1 residue (com-
pare with 6l) and the substituted analogue 6k is
equipotent with the unsubstituted analogue 6j.
References
1. For preliminary accounts of this work, see: Pinto, D. J.;
Wang, S.; Orwat, M. J.; Quan, M. L.; Han, Q.; Jacobson, I.;
Lam, P. Y. S.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.
217th National Meeting of the American Chemical Society,
Anaheim, CA, March 21±25, 1999; American Chemical
Society: Washington, DC, 1999; MEDI 085. (b) Fevig, J. M.;
Buriak, J., Jr.; Pinto, D. J.; Han, Q.; Wang, S.; Alexander,
R. S.; Rossi, K. A.; Knabb, R. M.; Wong, P. C.; Wright, M. R.;
Wexler, R. R. 217th National Meeting of the American
Chemical Society, Anaheim, CA, March 21±25, 1999;
American Chemical Society: Washington, DC, 1999; MEDI
087. (c) Wexler, R. R.; Pinto, D. J.; Orwat, M. J.; Lam, P. Y. S.;
Fevig, J. M.; Quan, M. L.; Pruitt, J. R.; Alexander, R. S.;
Rossi, K. A.; Wright, M. R.; Wong, P. C.; Knabb, R. M.
27th National Medicinal Chemistry Symposium, Kansas City,
MO, June 13±17, 2000.
2. (a) Fevig, J. M.; Wexler, R. R. Annu. Rep. Med. Chem.
1999, 34, 81. (b) Ewing, W. R.; Pauls, H. W.; Spada, A. P.
Drugs Future 1999, 24, 771. (c) Zhu, B.-Y.; Scarborough,
R. M. Curr. Opin. Cardiovasc. Pulm. Renal Invest. Drugs 1999,
1, 63. (d) Vlasuk, G. P. In New Therapeutic Agents in Throm-
bosis and Thrombolysis; Sasahara, A. A., Loscalzo, J., Eds;
Marcel Dekker: New York, 1997; p 261.
The X-ray crystal structure of pyrazole 2 complexed to
the structurally similar serine protease trypsin4 reveals
features that might explain some of the FXa binding
results of these analogues, although due to space con-
siderations it is not shown here. As expected, the benz-
amidine is engaged in a bidentate interaction with
Asp189 in the S1 speci®city pocket and the biphenyl-
sulfonamide residue resides in the S4 aryl-binding
pocket. The pyrazole ring is oriented such that the N2
nitrogen interacts with the backbone N-H of Gln192
Ê
(3.2 A) and forms a van der Waals interaction with
Ê
Cys220 (3.3 A). Compounds 5c and 5d, each of which
have a tautomeric form resulting in an N-H in this
position, and 5i and 6l, which have a C-H projecting
toward Gln192 and Cys220, might have lower anity
due to the disruption of these interactions. In the crystal
structure, the methyl group of 2 projects toward solvent
and appears to have favorable van der Waals inter-
3. Pruitt, J. R.; Pinto, D. J.; Estrella, M. J.; Bostrom, L. L.;

J. M. Fevig et al. / Bioorg. Med. Chem. Lett. 11 (2001) 641±645
645
Knabb, R. M.; Wong, P. C.; Wright, M. R.; Wexler, R. R.
Bioorg. Med. Chem. Lett. 2000, 10, 865.
7. Kettner, C.; Mersinger, L.; Knabb, R. J. Biol. Chem. 1990,
265, 18289.
4. (a) Pinto, D. J.; Orwat, M. J.; Wang, S. Amparo, E. C.;
Pruitt, J. R.; Rossi, K. A.; Alexander, R. S.; Fevig, J. M.;
Cacciola, J.; Lam, P. Y. S.; Knabb, R. M.; Wong, P. C.;
Wexler, R. R. 217th National Meeting of the American Che-
mical Society, Anaheim, CA, March 21±25, 1999; American
Chemical Society: Washington, DC, 1999; MEDI 006. (b)
Pinto, D. J.; Orwat, M. J.; Wang, S.; Fevig, J. M.; Quan,
M. L.; Amparo, E. C.; Cacciola, J.; Rossi, K. A.; Alexander,
R. S.; Smallwood, A. S.; Luettgen, J. M.; Liang, L.; Aungst,
B. J.; Wright, M. R.; Knabb, R. M.; Wong, P. C.; Wexler,
R. R.; Lam, P. Y. S. J. Med. Chem. 2001, 44, 566.
5. Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett.
1977, 48, 4171.
6. Quan, M. L.; Liauw, A. Y.; Ellis, C. D.; Pruitt, J. R.; Car-
ini, D. J.; Bostrom, L. L.; Huang, P. P.; Harrison, K.; Knabb,
R. M.; Thoolen, M. J.; Wong, P. C.; Wexler, R. R. J. Med.
Chem. 1999, 42, 2752.
8. Wong, P. C.; Quan, M. L.; Crain, E. J.; Watson, C. A.;
Wexler, R. R.; Knabb, R. M. J. Pharmacol. Exp. Ther. 2000,
292, 351.
9. (a) For preliminary results, see Lam, P. Y. S.; Li, R.; Clark,
C. G.; Adams, J. J.; Galemmo, R. A., Jr.; Fevig, J. M.; Pinto,
D. J.; Quan, M. L.; He, M.; Han, Q.; Alexander, R. S.; Rossi,
K. A.; Wong, P. C.; Luettgen, J. M.; Aungst, B. J.; Wright, M.
R.; Bai, S. A.; Knabb, R. M.; Wexler, R. R. 220th National
Meeting of the American Chemical Society, Washington, DC,
August 20±24, 2000; American Chemical Society: Washington,
DC, 2000; MEDI 329. (b) Galemmo, R. A., Jr.; Fevig, J. M.;
Lam, P. Y. S.; Pinto, D. J.; Dominguez, C.; Alexander, R. S.;
Rossi, K. A.; Wells, B. L.; Drummond, S.; Clark, C. G.; Li,
R.; Wong, P. C.; Wright, M. R.; Knabb, R. M.; Wexler, R. R.
220th National Meeting of the American Chemical Society,
Washington, DC, August 20±24, 2000; American Chemical
Society: Washington, DC, 2000; MEDI 290.