Nonbenzamidine acylsulfonamide tissue factor-factor VIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.06.027

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P′ binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.

Full text of DOI:10.1016/j.bmcl.2013.06.027

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Nonbenzamidine acylsulfonamide tissue factor–factor VIIa inhibitors
a
a
a
a
a
Peter W. Glunz a, , Xiaojun Zhang , Yan Zou , Indawati Delucca , Alexandra H. Nirschl , Xuhong Cheng ,
Carolyn A. Weigelt a, Daniel L. Cheney a, Anzhi Wei a, Rushith Anumula b, Joseph M. Luettgen a,
Alan R. Rendina a, Mark Harpel a, Gang Luo a, Robert Knabb a, Pancras C. Wong a, Ruth R. Wexler a,
E. Scott Priestley a
⇑
a Bristol-Myers Squibb R&D, 311 Pennington-Rocky Hill Rd, Pennington, NJ 08534, United States
b Bristol-Myers Squibb Biocon Research Center, Hosur Road, Electronics City, Bangalore 560 100, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online xxxx
Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine
group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification
of additional hydrophobic and hydrophilic P0 binding interactions. The molecular details of these interac-
tions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing
the basicity of the P1 group results in improved oral bioavailability in this chemotype.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Factor VIIa
Tissue factor
TF-FVIIa inhibitor
Serine protease inhibitors
Structure-based design
Arterial thrombosis remains a leading cause of morbidity and
mortality worldwide.1 Patients on the current standard of care,
clopidogrel and aspirin, still experience cardiovascular events and
have an elevated bleeding risk.2 Recent acute coronary syndrome
trials with novel agents have shown limited efficacy and significant
bleeding.3 A major industry focus has been identifying efficacious
oral anticoagulants with wide therapeutic margin. Comparisons
of antithrombotic mechanisms have identified the tissue factor–
factor VIIa proteolytic complex (TF-FVIIa) as a biological target
which offers strong antithrombotic efficacy and low bleeding
liability in preclinical models.4 This serine protease initiates the
coagulation cascade by activating Factors IX and X to IXa and Xa,
respectively, leading to thrombin activation. Thrombin cleaves
fibrinogen to fibrin and activates platelets, thus triggering clot
formation.
One major challenge in the development of drugs inhibiting
TF-FVIIa is attaining oral bioavailability.5 In addition to limited
reports of nonbenzamidine FVIIa inhibitors lacking either potency
or permeability,6,7 the majority of reported FVIIa inhibitors bear a
benzamidine P1 moiety4f,8 as a mimic of the guanidine side-chain
of Arg in FVIIa’s natural substrates, factors IX and X. This highly-
basic P1 moiety (pKa ꢀ12) forms a strong salt-bridge interaction
with Asp189, resulting in inhibitors that are potent, but poorly
orally bioavailable due to low permeability under physiological
conditions. Approaches to overcome this issue include a prodrug
P2
P1
O
O
O
O
NH
R
H2N
N
H
N
O O
H
N
P'
N
H
S
N
H
S
O O
O
, R = NH2
3, R = H
O
1
2
Figure 1.
strategy,9 in which the basic group is transiently masked to allow
oral absorption, and an amidine replacement strategy.10 Replace-
ment of the amidine moiety constitutes a significant challenge as
this group is a major contributor to ligand binding affinity and
necessitates introduction of compensatory enzyme interactions
to maintain potency. Neither strategy has yet been successfully
demonstrated in the FVIIa field to yield an orally bioavailable,
clinical compound. We undertook an effort following this second
approach—replacement of the benzamidine moiety—starting with
the reported benzamidine-based acylsulfonamide inhibitor 14a,11
(Fig. 1). Herein, we detail the successful replacement of a benzam-
idine P1 group with aminoisoquinoline (2) and isoquinoline (3)
moieties and efforts to improve potency by optimizing substitution
of the P0 phenylsulfonyl group.
Aminoisoquinoline analogs 2 and 17–24 were prepared from
benzaldehyde 4 as outlined in Scheme 1. Cyanohydrin formation,
⇑
Corresponding author.
E-mail address: peter.glunz@bms.com (P.W. Glunz).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.027
Please cite this article in press as: Glunz, P. W.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.06.027

2
P. W. Glunz et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
A series of analogs of compound 1 with benzamidine replace-
Oi-Pr
OEt
Oi-Pr
OEt
ments was prepared and evaluated for inhibition of FVIIa (Table 1).
1-Aminoisoquinoline 2, an amidine replacement employed in the
context of serine protease inhibitors,17–19 was found to be the most
potent replacement, (FVIIa Ki = 16 nM). Isoquinoline 3 was five-
fold less potent. Additional P1 groups were found to be 300- to
5000-fold less potent then benzamidine, and thus are inadequate
as P1 replacements in this chemotype. P1 groups 15 and 16, which
were effectively incorporated into FXa inhibitors,20 afforded only
weakly potent inhibitors when incorporated into this chemotype.
Interestingly, 4-aminoquinazoline 13 is 30-fold less potent than 2.
Based upon the good potency observed for 2, we obtained an X-
ray co-crystal structure with FVIIa22 (Fig. 2a) and undertook a
structure-based design effort focusing on substitution of the phen-
ylsulfonyl P0 group to improve inhibitory activity via additional
interactions with the enzyme. The aminoisoquinoline P1 group
forms a bidenetate salt bridge with Asp189 and a hydrogen bond
with a water above Tyr228, mimicking the canonical binding inter-
actions of the Arg substrate and typical benzamidine P1 groups.
The acylsulfonamide carbonyl forms polar interactions with
Ser195 and His57. The phenyl group forms close contacts with
the His57 and Asp60 sidechains. In contrast to the X-ray crystal
structure for a benzamidine acylsulfonamide related to compound
1,4a there was no evidence for the ‘flipped’ conformation of the
Lys192–Gly193 peptide bond. Adjacent to the P0 phenyl group,
we identified a potential interaction with the hydrophilic binding
pocket comprised of the Asp60 and Tyr94 side chains and the
Gly97 and Thr98 backbone and a conserved water molecule
(Fig. 2b) and set out to target this interaction through addition of
polar substituent groups.
a,b
c,d,e
CHO
HO CO2Me
4
5
Oi-Pr
Oi-Pr
OEt
NPhth
OEt
PhthN
N
g
N
NH2
h,i,j
7
Cl CO2Bn
N
H
CO2Bn
f
6
8
Oi-Pr
Oi-Pr
OEt
OEt
PhthN
NH2
N
N
H
N
N
H
9
CO2H
N
H
S
O O
O
(R)-2
Scheme 1. Reagents and conditions: (a) NaHSO3, NaCN, EtOAc, 88%; (b) HCl, MeOH,
0 °C, 2 d; H2O, 1 h, 77%; (c) aq LiOH, THF, rt, 100%; (d) BnBr, Cs2CO3, DMF, rt, 95%; (e)
MsCl (1.1 equiv), TEA (1.5 equiv), CH2Cl2, 0 °C to rt, 92%; (f) DIEA, DMF, 90 °C, 17 h,
55%; (g) H2 (60 psi), 10% Pd-C, THF, 94%; (h) N-(trimethylsilyl)benzenesulfonamide
(10) (2 equiv), BOP (1.2 equiv), TEA (5 equiv), CH2Cl2/DMF (10:1), 68%; (i) ChiralPak
OD, 50% IPA/heptane + 0.1% TFA, (R)-enantiomer, followed by (S)-enantiomer; (j)
H2NNH2ꢁH2O, EtOH, 50 °C, 53%.
followed by conversion to the acetimidate and subsequent hydro-
lysis, afforded the
a-hydroxymethyl ester. The methyl ester was
converted to the benzyl ester 6 and the hydroxyl group was trans-
formed to the chloride, which could be displaced with protected
aminoisoquinoline 7.12 Hydrogenolysis of the benzylester gener-
ated acid 9, which was coupled13 with N-(trimethylsilyl)benzene-
sulfonamide (10)14 affording the acylsulfonamide derivative.
Separation of enantiomers and deprotection with hydrazine affor-
ded the aminoisoquinoline. Analogs 17–24 were synthesized by
substituting the appropriate N-(trimethylsilyl)arylsulfonamide,
prepared by silylation of the corresponding substituted arylsulf-
onamide. Analogs 13–16 were prepared via similar strategy as
has been described.15
Table 1
P1 SAR
Oi-Pr
OEt
H
R
N
N
H
S
O O
O
Isoquinoline analogs 3 and 25–33 were prepared by treatment
of benzaldehyde 4 with trimethylsilylcyanide and ammonia to
afford the 2-aminoacetonitrile derivative, which was converted
to the methyl ester via the acetimidate (Scheme 2). Pd-catalyzed
N-arylation16 of 6-bromoisoquinoline with aminoester 11 afforded,
after saponification, acid 12. Coupling with the corresponding
N-(trimethylsilyl)benzenesulfonamide afforded the acylsulfona-
mide derivative.
Compound
R
FVIIa Ki (nM)
pKa
NH
1
1.3
13.1a
H2N
NH2
2
16
9.3a, 8.9b
7.2a, 7.8b
7.0a
N
N
3
70
Oi-Pr
OEt
Oi-Pr
OEt
NH2
N
a, b
c, d
13
390
N
CHO
4
H2N CO2Me
N
11
H2N
14
15
670
10.9a
4.9a
N
H
Oi-Pr
OEt
Oi-Pr
OEt
H2N
N
e, f
1940
N
N
H
N
O O
O
N
CO2H
N
H
(R)-3
S
H2N
H
O
·HCl
16
2900
5.6a
12
N
N
H
Scheme 2. Reagents and conditions: (a) 7 N NH3/MeOH, TMSCN, 0 °C to rt, 67%; (b)
HCl, MeOH; H2O, 79%; (c) 6-bromoisoquinoline, Cs2CO3, BINAP, Pd2(dba)3, toluene,
100 °C, 72%; (d) aq LiOH, THF, rt, 57%; (e) N-(trimethylsilyl)benzenesulfonamide
(10), BOP, TEA, CH2Cl2/DMF (10:1), 88%; (e) ChiralPak AD, 50% IPA/heptane + 0.1%
TFA, (R)-enantiomer, followed by (S)-enantiomer.
Ki Determination, n P 2. See Ref. 21 for enzyme assay protocols.
a
pKa calculated using pKa dB module of ACD/Labs, version 12.0, Advanced
Chemistry Development, Inc., Toronto, ON.
b
pKa Determined experimentally.
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P. W. Glunz et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
tion of a methyl sulfone at the ortho position (28) and could be en-
hanced an additional two-fold with a larger ethyl sulfone (31).
Installing the methyl sulfone group at the meta (29) or para posi-
tion (30) resulted in 12-fold and 700-fold less potent compounds,
respectively. Thus, increased binding affinity was attained with
the introduction of either hydrophilic or hydrophobic P0 substitu-
ents. As with the 1-aminoisoquinoline FVIIa inhibitors, isoquino-
line-containing inhibitors were highly selective versus factor Xa,
thrombin and trypsin.
A crystal structure of sulfonamide 26 in FVIIa was obtained
(Fig. 3a),22 showing the primary sulfonamide engaged in the
hydrophilic interactions that we targeted. The nitrogen displaces
a crystallographic water molecule observed in the X-ray co-crystal
structure of 2 with FVIIa, forming an intermolecular hydrogen
bond to a structural water molecule bound to Asp60 sidechain
and Thr98 backbone, and an intramolecular hydrogen bond to
the 3-ethoxy group of P2. These binding interactions are consistent
with the SAR requirements for a primary sulfonamide (26 vs 32,
33). In addition, the isoquinoline P1 group is flipped 180° relative
to the observed binding mode of 1-aminoisoquinoline. Interactions
of the acylsulfonamide were similar to those shown in Figure 2a,
and again the peptide bond between Lys192 and Gly193 was not
‘flipped’. A binding model of ortho-sulfone 31 in FVIIa (Fig. 3b)
suggests that the alkylsulfone group is positioned in a hydrophobic
region on the prime side of the catalytic triad formed by Cys42–
Cys58 disulfide bridge, and Leu41 and His57 sidechains. This com-
pound appears to be interacting with a previously unreported
hydrophobic binding pocket of FVIIa. Efforts successfully combin-
ing this lipophilic interaction with the meta polar group (e.g., 26)
to engage both the hydrophobic and hydrophilic binding sites of
the S10 pocket of FVIIa will be described in due time.
b
hydrophilic
Asp60, Tyr94, Gly97, Thr98
S2 water
O
O
NH2
N
H
N
O O
N
H
S
O
Figure 2. (a) X-ray co-crystal structure of 2 with TF-FVIIa, electron density shown
as purple mesh. (b) Potential interactions of P0 phenyl substituents.
A series of substituted phenylsulfonyl analogs bearing the
1-aminoisoquinoline P1 group are listed in Table 2. A primary sul-
fonamide group at the meta position improves inhibition by three-
fold relative to the parent compound 2 ((R)-2 vs (R)-21), whereas
other substituents at the meta position are equipotent to or less
potent than 2. Substitution at the para position with either an hy-
droxyl or amino group slightly increased potency, whereas substi-
tution with methyl decreased potency, consistent with the para
position of inhibitor 2 being directed towards the Asp60 side chain
of FVIIa. All 1-aminoisoquinoline FVIIa inhibitors are highly selec-
tive versus factor Xa, thrombin and trypsin.
After success with the aminoisoquinoline replacement, we
switched to the less basic, but less potent isoquinoline P1 for P0
SAR exploration (Table 3). Incorporation of the meta primary sul-
fonamide (26) results in a similar improvement, as was observed
in the 1-aminoisoquinoline series. The meta-orientation of the pri-
mary sulfonamide appears optimal relative to either the ortho- or
para-substitution patterns, compounds 25 and 27, respectively.
Secondary and tertiary sulfonamides are 8- and 23-fold less potent,
respectively. Next, the effect of hydrophobic sulfone substituents
was examined. Potency was improved six-fold by the incorpora-
Additional in vitro studies were conducted on representative
compounds in these series. As expected for reversible, active site
inhibitors, compounds 1–3 were competitive versus a tripeptide
substrate, but exhibited noncompetitive inhibition versus the
physiological substrate, factor X, which interacts with TF-FVIIa pri-
marily at exosites. These findings are consistent with those de-
scribed for
a
benzamidine acylsulfonamide4a and in similar
fashion compounds 1–3 exhibited slow binding kinetic behavior
with the tripeptide substrate from which association rates were
derived.23 In vitro clotting times for the most potent compounds
((R)-21, IC2x = 14
lM; and 28; IC2x = 76 lM in a modified pro-
thrombin time assay24) were elevated compared to the benzami-
dine 1 (IC2x = 4
lM in the same assay).
Table 2
P0 SAR with 1-aminoisoquinoline P1
Oi-Pr
OEt
NH2
N
H
R
N
S
N
H
O O
O
R
FVIIa Ki (nM)
FXa Ki (nM)
Thrombin Ki (nM)
Trypsin Ki (nM)
2
H
H
H
3-OH
3-CONH2
3-CH2OH
3-NH2
3-SO2NH2
3-SO2NH2
4-OH
16
9.7
260
26
18
28
48
10
2.8
7
3500
7300
8200
5500
>8000
>8000
>8000
5900
5600
>8000
5800
3050
4300
6900
4700
(R)-2
(S)-2
17
18
19
20
21
(R)-21
22
23
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
3040
>13,000
11,000
>13,000
11,000
>13,000
>13,000
9400
>13,000
7400
2900
4-NH2
4-Me
9
197
24
Ki Determination, n P 2. See Ref. 21 for enzyme assay protocols.
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P. W. Glunz et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 3
P0 SAR with isoquinoline P1
Oi-Pr
OEt
N
H
N
O O
R
N
H
S
O
R
FVIIa Ki (nM)
FXa Ki (nM)
IIa Ki (nM)
Trypsin Ki (nM)
3
H
H
70
44
73
32
17
320
12
145
8100
6
250
780
5400
>13,000
>13,000
>13,000
>13,000
>13,000
>13,000
10,300
>13,000
>13,000
9400
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
(R)-3
25
26
(R)-26
27
28
29
30
31
32
>8000
>8000
>8000
>8000
>8000
>8000
>8000
>8000
>8000
7000
2-SO2NH2
3-SO2NH2
3-SO2NH2
4-SO2NH2
2-SO2Me
3-SO2Me
4-SO2Me
2-SO2Et
3-SO2HNMe
3-SO2NMe2
>13,000
>13,000
33
7200
Ki Determination, n P 2. See Ref. 21 for enzyme assay protocols.
Figure 3. (a) X-ray co-crystal structure of sulfonamide 26 in FVIIa. (b) Model of sulfone 31 in FVIIa with prime-side hydrophobic enzyme surface in green.
Table 4
group can result in improved oral bioavailability. In addition,
Dog pharmacokinetic profile for (R)-2 and (R)-3
substituting the isoquinoline for the aminoisoquinoline P1 group
removes two H-bond donors and may contribute to the observed
increase in bioavailability.
Oi-Pr
OEt
R
In conclusion, we have successfully replaced the highly basic
benzamidine P1 group of a FVIIa inhibitor with less basic 1-amino-
isoquinoline and isoquinoline groups. Importantly, we have identi-
fied both hydrophobic and hydrophilic P0 binding interactions to
compensate for this P1 change. These interactions have been
elucidated by X-ray crystallography and molecular modeling. We
have also shown that decreasing the basicity of the P1 group and
reducing the number of H-bond donors results in improved oral
bioavailability in this chemotype.
N
H
N
O O
N
H
S
O
Compound
(R)-2
(R)-3
R
NH2
10
36
H
44
>80
FVIIa Ki (nM)
Modified-PT, IC2x
pKa
MW, cLogP (calcd)
Dose (mg/kg)
iv CL (mL/min/kg)
iv t1/2 (h)
(lM)
3.1, 8.9
2.8, 7.8
519.6, 2.47
0.2 (iv) 0.5 (po)
9.3
0.9
0.6
534.6, 1.69
0.2 (iv) 0.5 (po)
24
1.5
1.5
4
Acknowledgments
iv Vss (L/kg)
Dog F%
The authors thank Frank Barbera, Jeffrey Bozarth, Randi Brown,
Karen Hartl, Diane Normandin, Tara Peterson and Ge Zhang for car-
rying out biological assays. We would also like to thank Steven
Sheriff for helping to prepare the coordinates and data for PDB
deposition.
36 (9,15,83)
PK data are an average of three experiments.
The pharmacokinetic parameters upon iv and oral dosing of
representative compounds (R)-2 and (R)-3 were determined
(Table 4). Aminoisoquinoline (R)-2 shows high clearance and little
oral bioavailability, whereas isoquinoline (R)-3 shows moderate
clearance and appreciable, but highly variable, oral bioavailability.
These results correlate to the decreased pKa of the P1 subunit of
isoquinoline (R)-3 (pKa = 7.8) as compared to aminoisoquinoline
(R)-2 (pKa = 8.9), suggesting that decreasing the basicity of the P1
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22. Compounds
2 and 26 were diffused into pre-formed FVIIa/benzamidine
crystals by soaking. The X-ray diffraction data were collected from these
FVIIa inhibitor complex crystals to 2.4 and 2.2 Å resolution with Rsym = 0.088
and 0.096, and completeness = 99.9% and 98.9%, respectively. The complex
structures were refined to R-work values of 0.18 and 0.20, respectively. The
structures have been deposited in the Protein Data Bank as 4JYU (2) and 4JYV
(26).
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Please cite this article in press as: Glunz, P. W.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.06.027