Preparation of 1-(3-aminobenzo[d]isoxazol-5-yl)-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-ones as potent, selective, and efficacious inhibitors of coagulation factor Xa

Article abstract of DOI:10.1016/j.bmcl.2006.07.002

Previously, potent factor Xa inhibitors were described based on the 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one bicyclic core and a 4-methoxyphenyl P1 moiety. This manuscript describes 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and related bicyclic cores with the 3-

Full text of DOI:10.1016/j.bmcl.2006.07.002

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5176–5182
Preparation of 1-(3-aminobenzo[d]isoxazol-5-yl)-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-ones as potent, selective,
and efficacious inhibitors of coagulation factor Xa
Yun-Long Li,*,ꢀ John M. Fevig,* Joseph Cacciola,ꢁ Joseph Buriak, Jr.,§
Karen A. Rossi, Janan Jona,– Robert M. Knabb, Joseph M. Luettgen,
Pancras C. Wong, Stephen A. Bai,k Ruth R. Wexler and Patrick Y. S. Lam
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543-5400, USA
Received 6 June 2006; revised 26 June 2006; accepted 5 July 2006
Available online 25 July 2006
Abstract—Previously, potent factor Xa inhibitors were described based on the 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one bicyclic core
and a 4-methoxyphenyl P1 moiety. This manuscript describes 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and related bicyclic cores
with the 3-aminobenzisoxazole P1 moiety. Many of these compounds are potent, selective, and efficacious inhibitors of coagulation
factor Xa.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Thromboembolic diseases remain the leading cause of
death and disability in developed countries. This reality,
combined with the limitations of current therapies, has
led to extensive efforts to develop novel antithrombotic
agents.1 Factor Xa has become a major focus of phar-
maceutical intervention in the past decade because of
its central role in the blood coagulation cascade.2 Exten-
sive preclinical and clinical evidence has demonstrated
that inhibition of factor Xa is efficacious in both venous
and arterial thrombosis.3,4
F3C
N
F3C
N
F
F
H
H
NMe2 . HCl
N
N
N
N
N
SO2Me
O
O
N
NH2
2
1, razaxaban
fXa Ki = 0.19 nM
MeO
O
N
fXa Ki = 3.6 nM
R3
H2NOC
N
W
N
Y
Z
X
N
N
N
N
N
O
O
Previously, it was demonstrated that a series of non-ben-
zamidine N-arylpyrazole carboxamides, represented by
the 3-aminobenzisoxazole P1 analog razaxaban (1),5a
were highly potent, selective, and orally bioavailable
small molecule fXa inhibitors (Fig. 1). Razaxaban has
been shown to be efficacious in phase II deep vein
thrombosis (DVT) clinical trials.5b Furthermore, it was
Aryl
3
NH2
MeO
O
4
N
fXa Ki = 1.1 nM
Figure 1.
demonstrated that the 4-methoxyphenyl residue could
be an effective P1 group when combined with an opti-
mized pyrazole-P4 subunit, as in compound 2.6 Recent-
ly, bicyclic core variants of 2, represented by the
1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one 3,7 have been
shown to retain potent binding affinity for fXa. These
bicyclic variants were also expected to be less susceptible
to in vivo amide hydrolysis, which in the case of N-aryl-
pyrazole carboxamides such as 1 and 2 would liberate
a biarylaniline fragment. However, compound 3 was
Keywords: Factor Xa inhibitors; Anticoagulants; Antithrombotic
agents; Pyrazole; Bicyclic core; Pyrazolo[4,3-d]pyrimidin-7(6H)-ones.
*
Corresponding authors. Tel.: +1 609 818 5285; fax: +1 609 818
3450; e-mail: john.fevig@bms.com
ꢀ
ꢁ
§
Present address: Incyte Corporation, Wilmington, DE, USA.
Present address: AstraZeneca, Wilmington, DE, USA.
Present address: DuPont Ag Products, Newark, DE, USA.
Present address: Amgen, Thousand Oaks, CA, USA.
Present address: Centocor, Malvern, PA, USA.
–
k
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.002

Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5176–5182
5177
found to have only modest efficacy in a rabbit arterio-
venous (A-V) shunt thrombosis model8 relative to razax-
aban. This was rationalized on the basis of 3 being
5-fold less potent than razaxaban.7 This manuscript de-
scribes bicyclic variants in the 3-aminobenzisoxazole P1
series,9 represented by 4, which were prepared with the
expectation that this P1 series would display greater
potency and in vivo efficacy relative to analogs with
the 4-methoxyphenyl P1, such as 3.10
bicyclic core by refluxing in 95% formic acid. This treat-
ment also resulted in loss of the TBS group and subse-
quent conversion of the alcohol to a formate ester, to
give 10. Treatment of 10 with acetohydroxamic acid
and K2CO3 to form the 3-aminobenzisoxazole P1 moie-
ty12 was followed by hydrolysis of the formate ester, giv-
ing 11. Bromination of the alcohol and displacement
with pyrrolidine afforded the 1H-pyrazolo[4,3-d]pyrimi-
din-7(6H)-one example 12.
The initial bicyclic core example in the 3-aminobenzis-
oxazole P1 series, the 1H-pyrazolo[4,3-d]pyrimidin-
7(6H)-one 12, was prepared as described in Scheme 1,
using the synthesis route used for preparing compounds
such as 3.7 Catalytic hydrogenation of the nitro func-
tionality of 5 afforded an aniline, which upon diazotiza-
tion and treatment with ethyl cyanoacetate gave the
expected hydrazone intermediate. Treatment of this
hydrazone with methyl bromoacetate and K2CO3 at
90 ꢁC gave the 4-aminopyrazole diester 6 by N-alkyl-
ation and in situ ring closure. However, this sequence
proceeded in very low and irreproducible yield due to
the poor nucleophilicity of the nitrogen in the N-alkyl-
ation step. Nevertheless, aminopyrazole 6 was diazo-
tized and treated with sodium azide to form a
4-azidopyrazole. Selective hydrolysis of the methyl ester
was accomplished with lithium hydroxide to give 7.
Conversion of 7 to an acid chloride and subsequent
treatment with readily available biphenylaniline 811
afforded amide 9. Reduction of the azide afforded a
4-aminopyrazole-5-carboxamide, which was cleanly
cyclized to the 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one
Due to the poor yield of 6 discussed above, additional
examples in this bicyclic series were prepared via an
alternate route, described in Scheme 2, wherein the pyr-
azole R3 substituent was initially a methyl group.9b
Readily available pyrazole ester 139a was efficiently
and selectively nitrated at the pyrazole 4-position using
excess ammonium nitrate and trifluoroacetic anhydride
in TFA.13 Subsequent ester hydrolysis afforded acid
14, which was coupled with 4-bromoaniline to give 15.
Nitro reduction was unexpectedly difficult, with several
reagents (SnCl2, Zn/HCl, and Fe/HCl) resulting in sig-
nificant decomposition. However, potassium borohy-
dride and CuCl in ethanol afforded in moderate yield
the clean 4-aminopyrazole, which was efficiently cyclized
to the bicyclic core 16 by refluxing in either N,N-DMF
dimethyl acetal (R5 = H) or N,N-dimethylacetamide
dimethyl acetal (R5 = Me). Suzuki coupling with boron-
ic acid 17 readily gave the biphenyl aldehyde 18. The
completion of the synthesis of compounds 19a–h was
best accomplished by a four-step sequence. Thus, sodi-
um borohydride reduction of the aldehyde to the
alcohol was followed by the introduction of the
Me
N
Me
N
Me
N
NO2
H
N
NO2
EtO2C
N
H2N
OTBS
EtO2C
N
a,b
c
NO2
N3
NH2
d,e
a-c
N
N
N
CO2Et
CO2H
N
N
CO2H
O
CO2Me
8
Br
CN
F
f
CN
CN
CN
F
F
F
CN
EtO2C
CN
7
5
6
F
F
13
Me
14
15
Me
EtO2C
CHO
N
R5
N
O
R5
N3
N
(HO)2B
d,e
N
N
g-j
H
N
g,h
N
N
N
N
OTBS
N
OCHO
N
N
CHO
N
N
17
O
O
O
Br
f
CN
N
CN
16 R5 = H, Me
CN
CN
18 R5 = H, Me
F
F
F
F
10
9
19a R5 = H; X = -NMe2
Me
i,j
R5
19b R5 = H; X = N-pyrrolidine
EtO2C
N
EtO2C
N
N
19c R5 = H; X = 3-(S)-OH-N-pyrrolidine
19d R5 = H; X = -NHCH2-cyc-Pr
19e R5 = H; X = -NH-i-Pr
N
O
N
O
N
X
k,l
N
N
N
N
OH
O
N
N
19f
R
5 = Me; X = -NMe2
NH2
O
19g R5 = Me; X = 3-(R)-OH-N-pyrrolidine
19h R5 = Me; X = -NMe-i-Pr
N
19a-h
NH2
NH2
O
O
N
12
11
N
Scheme 1. Reagents and conditions: (a) H2 (1 atm), 10% Pd/C, EtOH
(95%); (b) NaNO2, HCl/H2O, 0 ꢁC; then ethyl cyanoacetate, NaOAc,
MeOH/H2O, 0 ꢁC (55%); (c) BrCH2CO2Me, K2CO3, DMF, 90 ꢁC (10–
20%); (d) NaNO2, TFA, 0 ꢁC; then NaN3 (80%); (e) LiOH, THF/H2O
(90%); (f) (COCl)2, CH2Cl2; then 8, DMAP, CH2Cl2 (70%); (g)
SnCl2ÆH2O, MeOH, 65 ꢁC; (h) 95% HCO2H, reflux (70% for two
steps); (i) AcNHOH, K2CO3, DMF; (j) K2CO3, EtOH, 65 ꢁC (45% for
two steps); (k) PBr3, CH2Cl2; (l) pyrrolidine, CH3CN, (50% for two
steps).
Scheme 2. Reagents and conditions: (a) NH4NO3 (2 equiv), TFAA
(7 equiv), TFA (90%); (b) LiOH, MeOH/H2O (80%); (c) (COCl)2,
CH2Cl2; then 4-bromoaniline, DMAP, CH2Cl2 (80%); (d) KBH4,
CuCl, EtOH, (50%); (e) N,N-DMF dimethyl acetal, reflux (R = H,
85%), or N,N-dimethylacetamide dimethyl acetal, reflux (R = Me,
75%); (f) 17, Pd(PPh3)4, Na2CO3, benzene, H2O, 80 ꢁC (70–80%); (g)
NaBH4, MeOH (50–65%); (h) HO-NHAc, K2CO3, DMF (60–70%); (i)
MnO2, CH2Cl2 (75-85%); (j) HNRR0, NaBH(OAc)3, HOAc, THF,
(50–70%).

5178
Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5176–5182
F3C
Scheme
2
F3C
N
F3C
N
F3C
N
F3C
N
F3C
N
H
N
NH2
H
N
NO2
NH2
H
N
NO2
O
c
a
b,c
a,b
N
26b-26c
20
+
N
N
N
N
N
N
N
CO2H
CO2H
CO2H
O
O
O
Br
Br
Br
CN
CN
CN
CN
CN
CN
F
F
F
F
F
F
21
22
~1 : 2
20
25
21
22
F3C
F3C
H
d
N
O
R5
N
d,
N
N
F3C
N
H
N
F3C
N
F3C
N
N
X
N
X
NO2
N
N
e,f
(27a)
NH2
O
then see
Scheme 2
steps f-j
g
X
O
N
N
N
O
O
NH2
NH2
Br
Br
O
Br
O
24a-b
23a-d
N
N
CN
CN
CN
F
29
Scheme
2
F
F
Scheme 3. Reagents and conditions: (a) NH4NO3 (2 equiv), TFAA
(7 equiv), TFA (60%); (b) (COCl)2, CH2Cl2; then 4-bromoaniline,
DMAP, CH2Cl2 (90%); (c) KBH4, CuCl, EtOH, (70%); (d) N,N-DMF
dimethyl acetal, (R = H, 90%), or N,N-dimethylacetamide dimethyl
acetal, reflux; then HOAc, reflux (R = Me, 75%).
27a (X=CO2Me)
27b (X=CN)
28
i
26d
h
(27b)
F3C
F3C
H
H
N
N
NH2
N
Scheme
2
Scheme
2
N
N
N
N
3-aminobenzisoxazole P1 moiety using acetohydroxa-
mic acid. The alcohol was then re-oxidized to the alde-
hyde with MnO2 and treated under standard reductive
amination conditions with an appropriate cyclic or acy-
clic amine, affording compounds 19a–h. Alternatively,
the alcohol could be brominated with PBr3 followed
by displacement with the appropriate amine as previous-
ly described.
26e
26f
O
O
Br
Br
CN
CN
30
31
F
F
Scheme 4. Reagents and conditions: (a) (COCl)2, CH2Cl2; then
4-bromoaniline, DMAP, CH2Cl2 (90%); (b) SnCl2, MeOH, reflux
(70%); (c) ClCOCl/PhMe, sealed tube, 110 ꢁC (85%); (d) (COCl)2,
DMF, CH2Cl2, then methyl p-bromophenylacetate, LDA, THF,
ꢀ78 ꢁC (X = CO2Me, 60%), or (COCl)2, DMF, CH2Cl2, then p-
bromophenylacetonitrile, LDA, THF, ꢀ78 ꢁC (X = CN, 75%); (e)
1.5 N HCl, dioxane, reflux; (f) Fe, 1N HCl, EtOH, reflux (65% for two
steps); (g) HC(OEt)3, 120 ꢁC (85%); (h) SnCl2, EtOH, reflux (50%); (i)
NaNO2,H2SO4, (80%).
Slight modification of this sequence, as shown in Scheme
3, afforded compounds 23a–d, where the pyrazole R3
group is trifluoromethyl.9b In this case, the pyrazole
nitration was best performed on the carboxylic acid
20.5a However, even after using an excess of reagents
and prolonged reaction times, it generally afforded only
an approximately 2:1 mixture of nitropyrazole 21 along
with recovered starting material 20. These materials
could be efficiently separated by prolonged stirring of
the solid in water, wherein the desired nitro compound
21 slowly went almost completely into solution. Filtra-
tion and extraction afforded clean product in about
60% yield. The starting material 20 could then be recy-
cled. Coupling with 4-bromoaniline and nitro reduction
as before gave 22. Where R5 = H, formation of the bicy-
clic core was accomplished as in Scheme 2 by refluxing
in N,N-DMF dimethyl acetal. However, where
R5 = Me, the cyclization was best accomplished by a
two-step sequence, wherein 22 was first refluxed in
N,N-dimethylacetamide dimethyl acetal and the result-
ing acetamidine intermediate was refluxed in glacial ace-
tic acid to effect the final cyclization.7 Once the CF3
bicyclic core was established, the synthetic sequence fol-
lowed that described in Scheme 2, to afford compounds
23a–d. The 5,6-dihydro-1H-pyrazolo[4,3-d]pyrimidin-
7(4H)-one examples 24a,b were prepared from a core
reduction side product isolated from the sodium boro-
hydride reduction of the corresponding biphenyl alde-
hyde, as described in Scheme 2.
Methods described in Scheme 2 allowed for the prepara-
tion of compounds 26b,c. Compound 26a was prepared
in a similar fashion, except that the fully elaborated P4
aniline was used in coupling with the carboxylic acid
21. For bicyclic cores where the amide nitrogen is re-
placed by carbon, conversion of 21 to the acid chloride
followed by treatment at ꢀ78 ꢁC with the lithium anion
of methyl p-bromophenylacetate and p-bromophenyl-
acetonitrile gave good yields of a-ketoester 27a and
a-cyanoketone 27b, respectively. Acid-catalyzed decar-
boxylation of 27a followed by nitro reduction afforded
28. Heating 28 in neat triethyl orthoformate induced
ring closure to the 1H-pyrazolo[4,3-b]pyridin-7(4H)-
one core 29. The a-cyanoketone 27b was treated with
SnCl2 in refluxing ethanol to effect nitro reduction and
subsequent ring closure to give the 5-amino substituted
1H-pyrazolo[4,3-b]pyridin-7(4H)-one bicyclic core 30.
Diazotization of 28 was followed by in situ trapping
by the adjacent enol to afford the 1H-pyrazolo[4,3-
c]pyridazin-7(4H)-one bicyclic core 31. Compounds
29–31 were converted by the methods described previ-
ously into 26d–f, respectively.
The SAR for the 1-(3-aminobenzo[d]isoxazol-5-yl)-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one core examples are
shown in Table 1. The initial bicyclic example, 12, was
an order of magnitude less potent than razaxaban 1 in
the in vitro fXa Ki assay, but was 4-fold more potent
and equipotent to 1 in the in vitro aPTT and PT clotting
Additional bicyclic core analogs were prepared as de-
scribed in Scheme 4. Aminopyrazole 22 was cyclized
by heating with phosgene in a sealed tube to afford the
1H-pyrazolo[4,3-d]pyrimidin-5,7(4H,6H)-dione core 25.

Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5176–5182
5179
Table 1. 1H-Pyrazolo[4,3-d]pyrimidin-7(6H)-one core
R3
R3
N
H
N
N
R5
N
N
X
N
X
N
N
O
O
NH2
NH2
O
O
N
N
12, 19a-h, 23a-d
24a-b
Compounda
R3
R5
X
fXa Kib (nM)
aPTT IC2xc (lM)
PT IC2xc (lM)
1
12
—
—
H
—
N-Pyrrolidine
NMe2
0.19
1.6
6.1
1.6
2.1
1.6
4.3
8.1
3.0
1.7
5.8
2.0
3.7
9.2
18.6
28
2.1
2.3
1.3
1.1
2.1
3.1
1.9
1.4
2.5
4.0
2.6
4.1
10.9
6.6
1.4
1.6
CO2Et
Me
Me
19a
19b
19c
19d
19e
19f
19g
19h
23a
23b
23c
23d
24a
24b
H
0.53
1.3
H
N-Pyrrolidine
3-(S)-OH-N-Pyrrolidine
–NHCH2-c-Pr
–NH-i-Pr
Me
Me
H
0.50
1.7
H
Me
Me
H
1.5
Me
Me
Me
H
NMe2
0.27
0.37
0.35
0.35
0.17
0.50
0.18
0.25
0.21
Me
Me
3-(R)-OH-N-Pyrrolidine
–NMe-i-Pr
NMe2
CF3
CF3
CF3
CF3
CF3
CF3
Me
Me
Me
—
—
NMe2
–NHCH2-c-Pr
3-(R)-OH-N-Pyrrolidine
NMe2
1.7
2.9
3-(R)-OH-N-Pyrrolidine
a All final compounds were purified by prep HPLC and gave satisfactory spectral data.
b Ki values were obtained from purified human enzyme and were averaged from multiple determinations (n = 2), as described in Ref. 5a.
c The aPTT (activated partial thromboplastin time) and PT (prothrombin time) in vitro clotting assays were performed in human plasma as described
in Ref. 5a.
assays, respectively. The 3-methylpyrazole series 19a–h
afforded compounds approaching the binding potency
of razaxaban, especially when R5 was also methyl, as
in compounds 19f–h. The 3-methyl series also exhibited
generally favorable potency in the clotting assays rela-
tive to 1. The 3-trifluoromethylpyrazole series 23a–d
provided even more potent compounds, but generally
at the expense of lower potency in the in vitro clotting
assays, presumably due to the higher protein binding
imparted by the CF3 group. For example, compounds
23b and 23d are essentially equipotent with razaxaban
in binding potency, but are less potent in the clotting as-
says. The reduced analogs of this core, the 5,6-dihydro-
1H-pyrazolo[4,3-d]pyrimidin-7(4H)-ones 24a,b, retain
excellent potency and now have very favorable clotting
assay activity relative to compounds 23a–d and to
razaxaban.
The SAR for additional bicyclic core analogs are shown
in Table 2. Compounds 26a–c contain the 1H-pyrazolo-
[4,3-d]pyrimidin-5,7(4H,6H)-dione bicyclic core.7 These
examples are characterized by excellent in vitro binding
Table 2. Examples of other bicyclic cores
F3C
H
F3C
H
N
N
Y
Z
F
Y
N
N
X
N
Z
X
N
N
O
O
N
NH2
NH2
O
26a
O
26b-f
N
N
Compounda
Y
Z
X
fXa Kib (nM)
apt IC2xc (lM)
PT IC2xc (lM)
1
—
—
N
N
N
C
—
NMe2
0.19
0.35
0.11
0.22
3.2
6.1
37.8
74.5
352
2.1
14.9
24.1
92.6
ND
230
26a
26b
26c
26d
26e
26f
C@O
C@O
C@O
CH
3-(R)-OH-N-Pyrrolidine
4-OH-N-Piperidine
NMe2
ND
638
C–NH2
N
C
4-OH-N-Piperidine
NMe2
0.77
5.5
C
ND
ND
a All final compounds were purified by prep HPLC and gave satisfactory spectral data.
b Ki values were obtained from purified human enzyme and were averaged from multiple determinations (n = 2), as described in Ref. 5a.
c The aPTT (activated partial thromboplastin time) and PT (prothrombin time) in vitro clotting assays were performed in human plasma as described
in Ref. 5a.

5180
Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5176–5182
potency, especially 26b, but also by uniformly poor
activity in the in vitro clotting assays, suggesting that
this core contributes to high protein binding. Examples
26d–f were prepared to determine if it was possible to re-
place the carboxamide nitrogen with carbon, thus negat-
ing the potential for generating an aniline degradant
in vivo. Only the 5-amino-1H-pyrazolo[4,3-b]pyridin-
7(4H)-one example 26e retains subnanomolar fXa bind-
ing potency. Unfortunately, this compound has poor
activity in the clotting assays, again suggesting that it
is highly protein bound.
>500-fold selectivity in all cases. The overall selectivity
profile for these compounds in most cases is comparable
to that of razaxaban 1.
The pharmacokinetic profiles of several compounds
were studied in dogs via a cassette dosing format, with
dosing at 0.5 mg/kg intravenously and 0.2 mg/kg orally
(Table 4). The 3-methylpyrazole 19a has high clearance
and only 2% bioavailability. Given its good Caco-2 per-
meability, this low bioavailability is more likely to be
related to high first pass metabolism, rather than poor
absorption. Addition of the 5-methyl substituent im-
proves the pharmacokinetic profile of this series, with
19f and 19g exhibiting lower clearance and higher bio-
availability relative to 19a and a half-life comparable
to razaxaban. The PK profile is further improved by
the 3-trifluoromethylpyrazole substituent, as shown by
23b and 23d. These compounds have lower clearance
and longer half-life than razaxaban, together with better
bioavailability than the 3-methylpyrazole examples.
Compound 24b has a profile similar to 19f and 19g.
Compounds with the 3-aminobenzisoxazole P1 residue
have previously been reported to show high selectivity
for fXa versus thrombin, trypsin and related serine pro-
teases.5a,9,11 Additional selectivity data are shown for
selected compounds in Table 3. Compounds 19f, 19g,
23b, and 24b all showed >1000-fold selectivity relative
to trypsin, aPC, factor IXa, factor VIIa, plasmin, tPA,
plasma kallikrein, urokinase, and chymotrypsin. They
are less selective relative to thrombin, but still show
Three of these compounds were also studied in the rab-
bit arterio-venous (A-V) shunt thrombosis model.8
Upon intravenous dosing, compounds 19f,19g, and
23b inhibited thrombus formation with an ID50 of 2.0,
2.4, and 1.9 lmol/kg/h, respectively (Table 5). Com-
pound 23b had an IC50 in this same assay of 290 nM.
This activity compares well to that of razaxaban in this
model.
Table 3. Human enzyme selectivity profile
Enzyme Ki (nM) 19f
19g
23b
24b
1
fXa
0.27
305
0.37
245
0.17
130
0.21
130
0.19
540
Thrombin
Trypsin
aPC
>4200 >4200 >4200 >4200 >10,000
19,000 >76,000 >37,000 >20,000 19,700
>41,000 >41,000 37,000 >41,000 9000
>15,000 >15,000 >25,000 >15,000 >15,000
23,000 >33,000 >40,000 >33,000 >33,000
>13,000 >13,000 >40,000 >13,000 >13,000
fIXa
Plasmin
tPA
Several examples from the 1H-pyrazolo[4,3-d]pyrimidin-
7(6H)-one core series were examined for their stability
under conditions of varying pH in order to assess their
potential for liberation of the P4 aniline via hydrolytic
cleavage of the bicyclic core. Data are shown for com-
pounds 19a, 19f, and 23a in Table 6. The results indicate
that the pyrimidinone ring of the bicyclic core decom-
poses at various rates depending on the pH and on the
substitution pattern of the bicyclic core. In most cases
two major degradants were observed, the identities of
which were determined by LC/MS. Compound II results
from hydrolysis of the C5-N6 bond to afford the N-for-
myl (R5 = H) or N-acetyl (R5 = Me) derivative. Further
hydrolysis affords the free amine III. For compound
19a, II was the predominant degradant observed at
pH 4.0, while the free amine III was the predominant
degradant at pH 1.0. Mechanistically, the N-formyl II
was thought to be an intermediate in the formation of
III. Evidence for this was gathered experimentally by
taking one half of the pH 4.0 samples and lowering them
to pH 1.0. After one week, the pH 1.0 samples showed
Urokinase
Chymotrypsin
960
580
>11,000 3240
8500
All Ki’s were obtained from purified human enzymes and are averaged
from multiple determinations (n = 2). See Ref. 5a for more details.
Table 4. Dog pharmacokinetic profiles
Compound Cl
Vdss
t1/2
F
Caco-2
(%) (Papp · 10ꢀ6 cm/s)
(L/h/kg) (L/kg) (h)
19aa
19fa
19ga
23ba
23da
24ba
3.3
1.7
1.2
0.7
0.6
0.98
9.5
8.9
6.6
2.5
2
11
6.1
4.7
19
4.2 23
3.8 48
13
14
63
8.4 58
4.3 27
3.4 84
6.6
4.2
5.3
ND
3.9
5.6
Razaxabanb 1.1
a Compounds were dosed as the TFA salts in an N-in-1 format at
0.5 mg/kg iv and 0.2 mg/kg po (n = 2).
b Ref. 5a. ND = not determined.
Table 5. Anticoagulant activity in rabbits
Compound
fXa Ki
(rabbit) (nM)
PT IC2x
(rabbit) (lM)
Rabbit A-V shunt8
IC50 (nM)
Rabbit A-V shunt8 ID50
Protein binding
(rabbit) (% bound)
(lmol/kg/h)
19f
19g
0.30
0.59
0.20
0.19
1.8
3.4
2.7
1.9
ND
ND
290
340
2.0
2.4
1.9
1.6
ND
91
23b
Razaxabana
97
93
a Ref. 5a. ND = not determined.

Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5176–5182
Table 6. Stability of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-ones
5181
with activity comparable to that of the clinical candi-
date, razaxaban. However, in aqueous solution stability
studies, these compounds were found to undergo vari-
able degrees of hydrolytic cleavage of the pyrimidinone
ring to generate pyrazole-5-carboxamide degradants.
While this core series is still promising, a major empha-
sis has been geared toward finding bicyclic cores with
greater aqueous stability.9b,14 Further efforts along these
lines will be described in due course.
R3
R3
R3
NHCOR5
H
NH2
H
N
N
O
R5
Ar
N
N
N
N
N
N
N
N
Ar
Ar
pH=4.0
(0.1M acetate)
O
O
pH=1.0
(0.1N HCl)
NH2
NH2
NH2
O
O
O
N
N
N
I
II
III
pH=1.0 (0.1N HCl)
Compounda Conditionsa I remainingb (%) IIb (%) IIIb (%)
Acknowledgments
19a
19f
23a
pH 1.0
pH 4.0
97.2
90.1
0.6
8.5
2.4
0.12
The authors thank Bruce Aungst, Frank Barbera, Tracy
Bozarth, Earl Crain, Andrew Leamy, Dale McCall, and
Carol Watson for technical assistance.
pH 1.0
pH 4.0
89.7
94.7
5.5
3.4
1.4
—
pH 1.0
pH 4.0
83.2
72.3
1.3
23.4
5.6
—
a pH 1.0 = 0.1 N HCl buffer; pH 4.0 = 0.1 M acetate buffer. Stability
studies were conducted at 40 ꢁC with an initial concentration of 5 lg/
mL.
b Values of % I–III were determined by HPLC after 2 days. The
identities of II and III were determined by LC/MS.
References and notes
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complete conversion to the amine III, while samples
remaining at pH 4.0 still showed a preponderance of
II. Thus, it appears that at both pH 1.0 and 4.0, initial
formation of II occurs, but only at pH 1.0 is II further
hydrolyzed appreciably to III. The degradation of 19f
is similar to that of 19a, with II (R5 = Me) being the ma-
jor component at pH 4.0. At pH 1.0 an appreciable
amount of II remains, while conversion to III is slower.
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hydrolysis of an N-acetyl group relative to an N-formyl
group. The 3-trifluoromethylpyrazole analog 23a under-
goes a more rapid decomposition than the 3-methylpy-
razole analogs, with 23% conversion to II at pH 4.0
after 2 days. Apparently, the CF3 group is activating
the pyrimidinone ring toward the initial hydrolysis,
thereby accelerating the decomposition. The observed
formation of II and III in these studies of 19a, 19f,
and 23a leads to the possibility that the free P4 aniline
could still be generated in vivo, although this risk is
slight, since it would require further hydrolysis of an
amide such as III, which can be viewed as a vinylogous
urea. The free P4 aniline was not observed in these
stability studies. Still, advancement of this series would
require use of an Ames negative P4 aniline.
In summary, several examples of pyrazole-fused bicyclic
core fXa inhibitors in the 3-aminobenzisoxazole P1 ser-
ies have been described. The 1H-pyrazolo[4,3-d]pyrimi-
din-7(6H)-one core is especially promising as a means
to maintain potent fXa inhibition while also reducing
the probability that in vivo amide hydrolysis will liberate
a biarylaniline fragment. Within this series, analogs 19f,
19g, and 23b are not only potent fXa inhibitors, but they
are also highly selective versus relevant serine proteases.
Compound 23b has a favorable pharmacokinetic profile
in dogs, with lower clearance and longer half-life relative
to razaxaban. Furthermore, 19f,19g, and 23b are highly
efficacious in the rabbit A-V shunt thrombosis model,

5182
Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5176–5182
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J.; Galemmo, R. A., Jr.; Wells, B.; Li, R.; Rossi, K. A.;
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Wexler, R. R.; Lam, P. Y. S. Abstracts of Papers, 230th
National Meeting of the American Chemical Society,
Washington, DC; American Chemical Society: Washing-
ton, DC, 2005; MEDI 279; (b) Li, Y.-L.; Fevig, J. M.; Han,
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14. Pinto, D. J. P. Presented at the 230th National Meeting of
the American Chemical Society, Washington, DC, August
2005; MEDI 249.