Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-dimethyl P4 moieties

Article abstract of DOI:10.1016/j.bmcl.2014.05.101

In an effort to identify a potential back-up to apixaban (Eliquis), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent

Full text of DOI:10.1016/j.bmcl.2014.05.101

Accepted Manuscript
Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-di-
methyl P4 moieties
Michael J. Orwat, Jennifer X. Qiao, Kan He, Alan R. Rendina, Joseph M.
Luettgen, Karen A. Rossi, Baomin Xin, Robert M. Knabb, Ruth R. Wexler,
Patrick Y.S. Lam, Donald J.P. Pinto
PII:
S0960-894X(14)00610-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.101
BMCL 21715
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 April 2014
27 May 2014
29 May 2014
Please cite this article as: Orwat, M.J., Qiao, J.X., He, K., Rendina, A.R., Luettgen, J.M., Rossi, K.A., Xin, B.,
Knabb, R.M., Wexler, R.R., Lam, P.Y.S., Pinto, D.J.P., Orally bioavailable factor Xa inhibitors containing alpha-
substituted gem-dimethyl P4 moieties, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/
10.1016/j.bmcl.2014.05.101
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Orally bioavailable factor Xa inhibitors containing alpha-
substituted gem-dimethyl P4 moieties
Michael J.Orwat*, Jennifer X. Qiao, Kan He, Alan R. Rendina, Joseph M. Luettgen,
Karen A. Rossi, Baomin Xin, Robert M. Knabb, Ruth R. Wexler, Patrick Y. S. Lam, and
Donald J. P. Pinto
Bristol-Myers Squibb Company, Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
Corresponding author: Michael Orwat. Tel: 609 818 5294. E-mail address: michael.orwat@bms.com
This is where the receipt/accepted dates will go; Received Month XX, 2014; Accepted Month XX, 2014 [BMCL RECEIPT]
Abstract—In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several
analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent
FXa binding affinity (FXa Ki), good human plasma anticoagulant activity (PT EC2x), cell permeability, and oral bioavailability. ©2000
Elsevier Science Ltd. All rights reserved.
Thromboembolic diseases remain the leading cause of
death and disability in developed countries.
Anticoagulants are key agents for the prophylaxis and
treatment of thromboembolic disorders. For many years,
the standard of care has been limited to warfarin
(Coumadin®)1 which has the limitations of a narrow
therapeutic index, dietary restrictions, and the need for
regular monitoring.2 For decades a replacement for
warfarin had been sought. The most prominent of these
approaches included targeting key enzymes of the
coagulation cascade, namely, factor Xa (FXa) and
thrombin. Recent regulatory approvals in order of
A deep back-up strategy was adopted early on at
Bristol-Myers Squibb to identify FXa inhibitors with
superior profiles. These efforts resulted in the
identification of apixaban (Eliquis) which has proven
to be safe and effective in patients with nonvalvular
atrial fibrillation (AF) and venous thromboembolism
(VTE).7 Bristol-Myers Squibb’s strategy for small-
molecule FXa inhibitors spanned over a decade and led
to multiple clinical candidates. Early clinical candidates
included DPC4238 and razaxaban,9 which, provided
clinical proof of concept for the FXa inhibitor class and
also helped with clinical dose selection for apixaban.
approval include
thrombin inhibitor, dabigatran
(Pradaxa®),3 and FXa inhibitors rivaroxaban
(Xarelto®),4 apixaban (Eliquis®),5 and edoxaban
(Lixiana®)6 (Figure 1), for venous thromboembolism
(VTE) prevention and stroke prevention in atrial
fibrillation have validated both mechanisms as novel
replacements for warfarin. Efficacy and bleeding results
in preclinical and clinical studies have suggested that
FXa inhibition may have a wider therapeutic index than
thrombin inhibition. The newer agents have the
potential to address several warfarin limitations, which
include eliminating the need for dietary restrictions,
regular monitoring, eliminating the concern around
multiple drug-drug interactions (DDIs) and long half-
life.
Figure 1: Recently approved oral anticoagulants
Given the promise that the mechanism held for
delivering superior medicines, our back-up strategy
focused on structural diversity with the goal of a

°C; d) BH3, THF; e) NH3, ethylene glycol, seal tube, 80 °C; f)
PCC, NaOAc; g) R'= amine, ZnCl2, NaBH3CN.
differentiated profile from previous candidates.
Considerable effort was directed at finding alternative
diverse P4 groups. This paper aims to highlight such an
approach which culminated in the discovery of geminal
methyl P4 groups which retained potent inhibition of
FXa, good overall protease selectivity, and oral
bioavailability. Recently, we described novel
cyclopropyl P4 entities that served as an alternative
group to the lactam P4 moiety of apixaban.10 We also
reasoned that while the cyclopropyl group incorporated
a certain degree of rigidity in the S4 region, there was
sufficient capacity to also include P4 groups with a
varied degree of conformational freedom. The focus of
this paper is to highlight additional strategies that would
mimic the cyclopropyl P4 group.
The syntheses of compounds listed in Table 1-2 are
shown in Schemes 1-3. In Scheme 1, compound 5a was
prepared in 40% yield through bis-alkylation of methyl
2-(4-iodophenyl) acetate with methyl iodide using
sodium hydride as the base. Ester hydrolysis (lithium
hydroxide) of 5a afforded carboxylic acid 5b in 47%
yield. Ullman coupling of 5b with ester 6a10 afforded
7a, which was reduced to alcohol 8 in a 70% yield.
Amidation of 8 (ammonia saturated ethylene glycol in a
sealed tube at 80 °C) provided 1 in 79% yield. Ullman
coupling of compound 5a with sulfone 6b10 afforded
ester 7b in 50% yield. Ester hydrolysis (lithium
hydroxide) followed by reduction and subsequent
oxidation with pyridinium chlorochromate and sodium
acetate afforded aldehyde 9a in 27% overall yield.
R
I
I
a
O
c
O
N
NH
+
N
OMe
OR'
O
Reductive
amination
of
9a
with
sodium
6a: R = CO2Et,
6b: R = SO2Me
5a: R' = Me, 40%
5b: R' = H, 47%
b
cyanoborohydride in the presence of zinc chloride and
the appropriate amine provided compounds 3a-c in 30-
53% yields. Compound 2 was obtained as a by-product
of the reductive amination of 9a in 20% yield.
Alternatively, aldehyde 9b was obtained by the
oxidation of 8 and converted to amines via reductive
amination, followed by amidation (as described
previously), to afford 4a-d in good yields.
MeO
R
EtO2C
N
d
N
N
N
O
N
N
O
OR'
O
OH
MeO
MeO
7a: R = CO2Et, R' = H, 45%
7b: R = SO2Me, R' = Me, 50%
8: 70%
e
In a two- and three-step sequence starting from 1-
(bromomethyl)-4-iodobenzene, intermediates 14a and
14b were prepared, respectively (Scheme 2). Ullman
coupling of compound 14a with pyrazole precursor 6a10
provided key pyrazole compound 15 in 45% yield.
Amidation of 15 provided carboxamide analog 10 in
88% yield. Hydrogenation of 10 provided compound 11
in 56% yield. Further lactamization via acylation with
4-bromovaleryl chloride and cyclization with potassium
t-butoxide afforded lactam analog 12.
f
b,d,f
R
H2NOC
N
N
N
N
O
N
O
O
N
H
OH
MeO
9a: R = SO2Me, 27%
9b: R = CO2Et, 50%
1: 79%
MeO
g,e
g
H2NOC
N
MeO2S
N
O
N
N
N
N
O
R'
R'
MeO
MeO
4a: R' = NMe2, 59%
2: R' = OH, 20%
4b: R' = NHcyclopropyl, 31%
4c: R' = pyrrolidine, 56%
4d: R' = morpholine, 54%
3a: R' = NMe2, 30%
3b: R' = pyrrolidine, 53%
3c: R' = morpholine, 38%
Scheme 1. Synthesis of Compounds 1, 2, 3a-c, 4a-d.
Reagents and Conditions: a) MeI, NaH, DMF; b) LiOH,
water, THF; c) K2CO3, CuI, 1,10-phenanthroline, DMSO, 130

Scheme 3. Synthesis of Compounds 16-19.
Reagents and Conditions: a) Me3Al, Me2N or pyrrolidine,
THF; b) TiCl4, THF; c) 2.5 eq. MeMgBr, THF;12 d) NaSMe,
DMF, quant.; e) K2CO3, CuI, 1,10-phenanthroline, DMSO,
130 °C; f) NH3, ethylene glycol, 80 °C; g) mCPBA, DCM;
h) MeI, NaH, DMF.
Scheme 2. Synthesis of Compounds 11 and 12.
Reagents and Conditions: a) KCN, MeOH, H2O b) TMSCl,
MeOH;11 c) NaH, MeI, THF; d) 6a, K2CO3, CuI, 1,10-
phenanthroline, DMSO, 130 °C; e) NH3, ethylene glycol, 80
°C; f) H2, 10% Pd/C, HCl, EtOH; g) 4-bromovaleryl chloride,
TEA, THF, then KOtBu, 0 °C to rt.
The strategy at BMS for follow-on compounds was
specifically designed for incorporation of structural
diversity with the goal of achieving significant
differentiation from previous clinical candidates such as
apixaban. Towards this end a concerted effort was
undertaken to look for diversity at the P4 position using
the general apixaban template. Table 1 lists examples of
several geminally substituted methyl P4 groups.
Compounds that contained a basic P4 group, such as
compounds 3a-c, 4a-d, 11, 17 and 18, were
significantly more potent (FXa Ki < 0.1 nM) and had
good in vitro clotting activity (PT EC2x < 4 µM). Neutral
compounds, such as compounds 1, 2, 10, 12, 16, and 19
were less active. Impressively, several analogs, 3c, 4d,
and 10, showed good permeability in the Caco-2 assay
(>10 x 10-6nm/s). Pyrazolo C3 carboxamide and
methanesulfonyl groups were evaluated. Consistent
with the SAR in discovery of apixaban, pyrazolo C3
carboxamide was the preferred group. It is interesting to
note that tertiary amino groups, such as 17 and 18 were
more potent (FXa Ki <0.2 nM) than sulfone analog 19
(FXa Ki = 0.48 nM).
In Scheme 3, compounds 20a and 20b were readily
obtained, although in low yields, by treatment of ethyl
4-iodobenzoate with dimethylamine or pyrrolidine with
trimethylaluminum, followed by the addition of
titanium tetrachloride and methylmagnesium bromide
(2.5 eq.).12 The utilization of excess methylmagnesium
bromide (2.5 eq.) on ethyl 4-iodobenzoate in THF
afforded the tertiary alcohol 21 in quantitative yield.
Compound 22 was obtained by the treatment of 4-
iodobenzyl bromide with sodium thiomethoxide
followed by Ullman coupling with 6a10 in 64% yield.
Oxidation of 22 with mCPBA afforded 23 in 70% yield.
Compounds 16-19 were then arrived at by the
procedures outlined in Scheme 3.
While intriguing, it is not surprising to see basic P4
moieties having potent FXa binding affinity and good
clotting activity, an observation seen with earlier
clinical candidates, such as razaxaban.9 Polar neutral P4
groups are also tolerated, as illustrated by compound 1,
10 and 19. These groups interact with the highly
charged residues in the S4 pocket, in a similar manner,
as razaxaban.9

19
CONH2 SO2Me
0.48
3.90
1.0
The excellent FXa inhibition and cell permeability
demonstrated by these analogs allowed us to further
evaluate several of these analogs in dogs via cassette
PK studies (Table 2).8,9 In general, compounds bearing
basic P4 moieties demonstrated moderate clearance (Cl
> 1 L/Kg/h), large volume of distribution (Vdss > 5
L/Kg), short to moderate half-life (< 5 h), and with
modest oral bioavailability (< 50%). Compound 3c had
good permeability (Caco-2 Papp 17 x10-6 nm/sec), but
low oral bioavailability in dogs (F% = 13) due to its
poor intrinsic PK profile (moderate Cl = 1.28 L/Kg/h
and high Vdss = 10.1 L/Kg). However, neutral P4
analogs such as compounds 1 and 19, showed improved
pharmacokinetics in dogs such as moderate to low Cl
(0.98 and 0.32 L/Kg/h), low Vdss (3.02 and 0.51 L/Kg)
and moderate t1/2 (4.95 and 1.70 h), and good oral
bioavailability (50 and 79%). Additionally, these
compounds have excellent selectivity profiles as shown
for compound 1 (Table 3).
aKi values are obtained from purified human FXa enzyme and are
averaged from two experiments (n=2). bPT values are measured according
to Refs 8 and 9. cCaco-2 permeability (A-to-B). d na = not available.
Table 2: In Vivo Dog Pharmacokinetic Profilesa
Compound
Cl
Vdss
t1/2
F%
(L/Kg/h)
(L/Kg)
(PO)(h)
apixaban
0.02
0.98
3.86
2.20
1.28
5.04
1.76
3.36
7.60
0.32
0.20
3.02
7.3
5.80
4.95
1.86
1.40
4.18
1.04
2.80
3.33
nab
58
50
34
10
13
41
10
44
nab
79
1
3a
3b
3c
4a
4d
11
18
19
5.50
10.1
6.57
5.01
11.8
12.0
0.51
Table 1: In Vitro Profiles
1.70
aDog phamacokinetics: compounds were dosed (iv/PO) as TFA salts in
different experiments in a cassette dosing N-in-one format at 0.5 mg/kg iv
and 0.2 mg/kg PO (n = 2) according to refs 8 and 9. bna = not available.
Table 3: Enzyme selectivity data for Compound 1
Human enzyme
Ki (nM)
Compound C3
R
-
FXa
Kia
PTb
EC2x
Caco-2
c
Papp
FXa
0.66
(nM)
(10-6nm/s)
(µM)
Trypsin
>15,000
2,578
apixaban
-
0.08
0.66
1.01
0.11
0.24
3.8
9
Thrombin
1
CONH2 CH2OH
3.8
5.8
3.6
4.1
4.3
Plasma Kallikrein
Chymotrypsin
Activated Protein C
Factor IXa
Factor VIIa
Factor XIa
Urokinase
tPA
>11,000
>40,000
>37,600
>15,000
>15,000
>15,000
>13,000
>43,000
2
SO2Me
SO2Me
SO2Me
CH2OH
3.9
3a
3b
CH2NMe2
0.67
0.92
CH2-N-
pyrrolidinyl
3c
SO2Me
CH2-N-
morpholinyl
0.32
3.7
17
4a
4b
CONH2 CH2NMe2
0.07
0.05
0.45
0.68
5.9
2.8
CONH2 CH2-N-
cyclopropyl
4c
4d
CONH2 CH2-N-
0.05
0.06
0.73
3.8
5.6
17
pyrrolidinyl
CONH2 CH2-N-
morpholinyl
10
11
12
CONH2 CN
1.22
0.15
1.5
3.4
18
CONH2 CH2NH2
CONH2 CH2-N-
0.69
7.4
0.7
8.3
piperidine-2-one
16
17
18
CONH2 OH
9.27
0.20
0.07
nad
nad
1.1
1.7
CONH2 NMe2
0.96
0.81
CONH2 N-pyrrolidinyl

Acknowledgements
We would like to acknowledge Jeff Bozarth and Tracy
Bozarth for performing the FXa Ki and clotting assays
and William Ewing and Joanne Smallheer for
reviewing this manuscript.
References and notes
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Figure 2: Overlay models of hydroxymethylene 1 (orange)
cyclopropylamino 4b (yellow), and apixaban (blue).
Figure 2 shows a CADD overlay of compound 1 (FXa
Ki = 0.66 nM), 4b (FXa Ki = 0.05 nM), and apixaban
(FXa Ki = 0.08 nM) in human FXa. In general, the
bound orientation for these analogs is similar to that
seen for apixaban. In the P4 region the
hydroxymethylene moiety of 1 adopts a perpendicular
configuration in the S4 pocket and does not have the
same stacking arrangement that the phenyl lactam P4
group of apixaban has with the S4 residues: Phe174,
Tyr99, and Trp215. This configuration, in part, may
explain the five-fold decrease in binding affinity. The
basic P4 substituents with ring appendages such as the
cyclopropyl group in 4b appear to elicit lipophilic
interactions with the backbone Glu97 residue. The
combination of a charge interaction coupled with the
lipophilic interactions of the substituents afford a
greater degree of FXa binding affinity. The hydroxyl
group of analog 1 interacts weakly with the carbonyl of
Lys96 and is not as strong as that observed with basic
P4 groups. In part, this could also explain some loss in
binding affinity of the less charged P4 group relative to
the charged basic P4 groups.
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In summary, structural diversity at the P4 position was
successfully incorporated using the pyrazole scaffold of
apixaban. Novel tertiary substituted P4 moieties were
identified which demonstrated potent inhibition of FXa
and with good translation into the clotting activity. In
general, basic P4 moieties provided enhanced FXa
binding affinities compared to neutral P4 analogs. Good
oral bioavailability was observed for several of these
analogs, however, their overall pharmacokinetic
properties were moderate to poor (moderate Cl > 1
L/Kg/h and high Vdss > 5 L/Kg). The neutral hydroxy
methyl analog 1 and methylsulfone analog 19 provided
the best balance of FXa affinity, clotting activity, and
good dog pharmacokinetics in terms of low clearance,
low volume of distribution, moderate half life and oral
bioavailability. However, these analogs did not
positively differentiate from apixaban and were not
considered for further evaluation.

Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-dimethyl P4 moieties
Michael J.Orwat, Jennifer X. Qiao, Kan He, Alan R. Rendina, Joseph M. Luettgen, Karen A. Rossi, Baomin Xin, Robert M.
Knabb, Ruth R. Wexler, Patrick Y. S. Lam, and Donald J. P. Pinto