Orally bioavailable pyridine and pyrimidine-based Factor XIa inhibitors: Discovery of the methyl N-phenyl carbamate P2 prime group

Article abstract of DOI:10.1016/j.bmc.2016.03.062

Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H

Full text of DOI:10.1016/j.bmc.2016.03.062

Accepted Manuscript
Orally Bioavailable Pyridine and Pyrimidine-Based Factor XIa Inhibitors: Dis-
covery of the Methyl N-Phenyl Carbamate P2 Prime Group
James R. Corte, Tianan Fang, Donald J.P. Pinto, Michael J. Orwat, Alan R.
Rendina, Joseph M. Luettgen, Karen A. Rossi, Anzhi Wei, Vidhyashankar
Ramamurthy, Joseph E. Myers Jr., Steven Sheriff, Rangaraj Narayanan,
Timothy W. Harper, Joanna J. Zheng, Yi-Xin Li, Dietmar A. Seiffert, Ruth R.
Wexler, Mimi L. Quan
PII:
S0968-0896(16)30218-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.062
BMC 12911
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 January 2016
23 March 2016
30 March 2016
Please cite this article as: Corte, J.R., Fang, T., Pinto, D.J.P., Orwat, M.J., Rendina, A.R., Luettgen, J.M., Rossi,
K.A., Wei, A., Ramamurthy, V., Myers, J.E. Jr., Sheriff, S., Narayanan, R., Harper, T.W., Zheng, J.J., Li, Y-X.,
Seiffert, D.A., Wexler, R.R., Quan, M.L., Orally Bioavailable Pyridine and Pyrimidine-Based Factor XIa Inhibitors:
Discovery of the Methyl N-Phenyl Carbamate P2 Prime Group, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.03.062
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Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Orally Bioavailable Pyridine and Pyrimidine-Based Factor XIa Inhibitors: Discovery
of the Methyl N-Phenyl Carbamate P2 Prime Group
James R. Corte∗, Tianan Fang, Donald J. P. Pinto, Michael J. Orwat, Alan R. Rendina, Joseph M. Luettgen,
Karen A. Rossi, Anzhi Wei, Vidhyashankar Ramamurthy, Joseph E. Myers Jr., Steven Sheriff, Rangaraj
Narayanan, Timothy W. Harper, Joanna J. Zheng, Yi-Xin Li, Dietmar A. Seiffert, Ruth R. Wexler, and
Mimi L. Quan
Bristol-Myers Squibb Company, Research and Development, P.O. Box 5400, Princeton, New Jersey 08543, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1,
and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2
prime group which maintained FXIa activity, reduced the number of H-bond donors, and
improved the physicochemical properties compared to the amino indazole P2 prime moiety.
Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally
bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-
chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant
improvement in oral bioavailability compared to the previously reported imidazole (S)-23.
Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was
achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine
ring system.
Available online
Keywords:
Factor XIa
FXIa
Thrombosis
Activated Partial Thromboplastin Time
aPTT
2016 Elsevier Ltd. All rights reserved.
———
∗ James R. Corte. Tel.: +1-609-466-5030; fax: +1-609-818-3331; e-mail: james.corte@bms.com

1. Introduction
achieved.17 In an effort to discover structurally diverse FXIa
inhibitors, replacements for the imidazole scaffold were
investigated. It was found that the imidazole scaffold could be
replaced with a variety of 6-membered heterocyclic rings and this
work resulted in the discovery of pyridine (S)-2 (Figure 1), a
single-digit nanomolar FXIa inhibitor which exhibited excellent
potency in the in vitro clotting assay.18 The pyridine scaffold, as
well as some of the other 6-membered ring heterocycles, may
offer improved physicochemical properties compared to the
imidazole ring and help address bioavailability issues. Herein we
report on the optimization of the pyridine-based chemotype,
focusing on modifications to the P2 prime, P1, and scaffold
regions, which resulted in the discovery of compound (S)-24
which showed a significant improvement in oral bioavailability
compared to the previously reported imidazole analogs.
Factor XIa (FXIa), the activated form of the zymogen FXI, is
a trypsin-like serine protease and a key component in the intrinsic
pathway of the blood coagulation cascade. A growing body of
research indicates that FXIa plays a pivotal role in pathologic
thrombosis (blood clots) but only a minor role in hemostasis.1
Genetic,2-5 preclinical,6-7,15 and recently published clinical data8
support the hypothesis that FXI and FXIa are targets for
anticoagulant therapy.
In contrast to the severe bleeding episodes observed in
individuals with either a Factor VIII deficiency (Hemophilia A)
or Factor IX deficiency (Hemophilia B), individuals with a FXI
deficiency (Hemophilia C) rarely experience abnormal or
spontaneous bleeding.2 In fact, bleeding episodes which arise
following an injury or surgery are generally characterized as mild
or moderate for the majority of these FXI deficient individuals.
Moreover, analyses have shown that individuals with a severe
FXI deficiency have a lower risk for ischemic stroke and deep-
vein thrombosis (DVT).3 Similarly, individuals with elevated
levels of FXI experience a higher incidence of acute myocardial
infarction and DVT.4 The phenotype observed in FXI deficient
individuals was recapitulated in FXI deficient mice. FXI
deficient mice show reduced thrombus formation in venous and
arterial thrombosis models without an increase in injury-related
bleeding compared to wild-type mice.5
2. Results and discussion
Pyridine (S)-2 is a single-digit nanomolar FXIa inhibitor which
exhibited good potency in the in vitro clotting assay (aPTT
EC1.5x) but is not orally bioavailable. The lack of oral
bioavailability was attributed to the overall number of H-bond
donors present in the molecule, of which both the amino indazole
P2 prime and the basic cyclohexyl methyl amine P1 moieties
were major contributors.
The strategy to improve oral
bioavailability for the pyridine series was two-fold; 1)
optimization of the amino indazole P2 prime moiety and 2)
replacement of the basic cyclohexyl methyl amine P1 with a
neutral p-chlorophenyltetrazole P1.
Inhibition of the zymogen FXI with either neutralizing
antibodies or antisense oligonucleotides (ASO) was shown to be
effective in a number of animal thrombosis models, including
primates, with minimal effects on bleeding time.6,7 An antisense
oligonucleotide (ISIS 416858) which blocks the expression of
FXI is the most advanced in clinical trials.8 In a phase II study in
total knee replacement, ISIS-416858 reduced the incidence of
deep vein thrombosis and showed fewer bleeding events as
compared to subcutaneously administered enoxaparin.
In addition to inhibiting FXI production, a number of research
groups have targeted FXIa with either active site or allosteric
inhibitors.9-18 The active site irreversible FXIa inhibitors are
represented by β-lactam (BMS-262084), α-ketothiazole
peptidomimetics, boronic acid peptidomimetics, and natural
product clavatadine A.9-13 In 2013, Bristol-Myers Squibb (BMS)
NH2
NH2
N
O
O
N
N
N
N
N
H
N
H
H
HN
H2N
H2N
N
H
Cl
S
2
)-
(
scaffold
P1
P2'
FXIa Ki = 8.4 nM
aPTT EC1.5x = 0.5
1
S
(
)-
FXIa Ki = 0.31 nM
aPTT EC1.5x = 0.32
M
M
Figure 1. Imidazole and Pyridine-Based FXIa Inhibitors.
disclosed the first reversible FXIa inhibitor, based on
a
tetrahydroquinoline chemotype, which exhibited potent
antithrombotic efficacy in a rabbit thrombosis model with
minimal effects on cuticle bleeding.15 Recently BMS reported on
the discovery of imidazole (S)-1 (Figure 1), a picomolar FXIa
inhibitor with excellent activity in the in vitro clotting assay
(aPTT EC1.5x).16 Imidazole (S)-1, which contains a basic
cyclohexyl methyl amine P1 and amino indazole P2 prime, is a
potent, selective, and reversible FXIa inhibitor, but it is not orally
bioavailable. An intense effort was undertaken to address the
lack of oral bioavailability and we recently disclosed that a
modest improvement in oral bioavailability (%Fdog = 10) could be
Figure 2. 2A. The S2 prime pocket of FXIa from the X-ray crystal structure
of (S)-2 in FXIa. Key H-bonding interactions (dotted lines) between the
amino indazole moiety and the FXIa residues His40, Tyr143, and Ile151 are
shown. The red spheres depict water molecules. 2B. Regioisomeric 6,5-
fused amino indazole.

2.1. Optimization of the Amino Indazole P2 Prime Moiety:
Bicyclic P2 Prime Groups.
His40. Based on the X-ray crystal structure, the indazole N(1)H
serves as an H-bond donor with Tyr143. Replacing the H-bond
donor in ( )-2 with an H-bond acceptor as found in 3-amino
benzisoxazole ( )-5 (FXIa = 9.2 nM), led to a modest
improvement in FXIa activity. The corresponding chiral,
nonracemic derivative (S)-520 (FXIa Ki = 3.8 nM) was 2-fold
more active than the indazole (S)-2 in FXIa binding but it was
equipotent in the in vitro clotting assay (aPTT EC1.5x). Based on
the potency of 3-amino benzisoxazole (S)-5, it appears that an H-
bond acceptor for Tyr143 is preferred. Switching from the 6,5-
fused bicycle to a 6,6-fused bicycle, while maintaining the H-
bond donors for His40 on top and an H-bond acceptor for Tyr143
The amino indazole P2 prime group is a key binding element
providing a 30-fold increase in FXIa activity in the pyridine
series.18 X-ray crystallography of pyridine (S)-2 in the FXIa
active site revealed that all three N-H bonds of the amino
indazole were engaged in hydrogen bonding interactions (Figure
2A). Specifically, one of the NH bonds from the C3-amino
group forms an H-bond with the backbone carbonyl of His40 (3.0
Å) and the other NH forms an H-bond with the NH of His40 via
a network of water molecules. The indazole N(1)H serves as an
H-bond donor to Tyr143 (3.0 Å) and the N(2) forms an H-bond
through a conserved water to Ile151 (3.1 Å and 3.3 Å). We
probed these hydrogen bonds to determine which interactions
were critical for FXIa activity and if any of the NH bond donors
could be removed while maintaining FXIa activity and improving
oral bioavailability of the pyridine series. The results of this
exploration are described in Table 1.
on the bottom, led to 4-aminoquinazoline ( )-6.
The 4-
aminoquinazoline (FXIa Ki = 10 nM) maintained FXIa activity
but led to a loss in potency in the in vitro clotting assay. The
importance of the H-bond acceptor for binding to Tyr143 is
highlighted in 1-amino isoquinoline ( )-7. Removal of the
nitrogen led to a >10-fold loss in FXIa activity. Based on the
analogs described in Table 1, only the 3-amino benzisoxazole
derivative (S)-5 allowed for the removal of one H-bond donor
while maintaining both FXIa activity and potency in the in vitro
clotting assay.
Table 1. Compounds Containing Bicyclic P2 Prime Groups
O
R
N
H
Table 2. Compounds Containing Regioisomeric 6,5-Fused
Bicyclic P2 Prime Groups
H2N
N
Entry
R
FXIa Kia
(nM)
aPTTb EC1.5x
(µM)
O
R
N
H
H2N
N
8.4
13
87
97
0.5
(S)-2
( )-2
Entry
R
FXIa Kia
(nM)
aPTTb EC1.5x
(µM)
NTc
>40
NTc
53
61
26
NTc
0.5
( )-8
( )-3
( )-9
( )-4
( )-5
7.5
( )-10
9.2
3.8
10
NTc
0.6
(S)-10
3.7
87
0.4
>40
NTc
(S)-5
( )-6
( )-7
( )-11
( )-12
2.8
110
144
NTc
aKi values were obtained from purified human enzyme and were averaged
from multiple determinations (n=2), as described in Ref. 15b. baPTT
(activated partial thromboplastin time) in vitro clotting assay was performed
in human plasma as described in Ref. 15b. cNT = not tested.
aKi values were obtained from purified human enzyme and were averaged
from multiple determinations (n=2), as described in Ref. 15b. baPTT
(activated partial thromboplastin time) in vitro clotting assay was performed
in human plasma as described in Ref. 15b. cNT = Not tested.
In order to quickly survey the P2 prime region of FXIa,
racemic derivatives were initially synthesized. When potent
inhibitors were identified, the corresponding chiral, nonracemic
derivatives were prepared prior to profiling. Both racemic ( )-2
and the active enantiomer (S)-2 are included in Table 1 for
comparison. Removal of the 3-amino group gave indazole ( )-3
(FXIa = 87 nM) which led to a 6.5-fold loss in FXIa activity. A
similar loss in FXIa activity was observed when the 3-amino
moiety was replaced with a 3-hydroxy group which afforded the
3-hydroxy indazole analog ( )-4 (FXIa = 97 nM). The loss in
FXIa activity in ( )-4 could be attributed to the electrostatic
repulsion between the 3-hydroxy moiety and either His40 or the
water molecules.19 Compounds ( )-3 and ( )-4 demonstrated the
importance of maintaining an H-bond donor interaction with
2.2. Regioisomer Bicyclic P2 Prime Groups.
To confirm the optimal orientation of the P2 prime group, we
prepared the regioisomeric 6,5-fused heterocycles (Table 2). We
reasoned that if the 3-amino indazole in (S)-2 were flipped 180o,
the indazole N(1)H could form an H-bond to the carbonyl of
His40 and the 3-amino moiety could form an H-bond to Tyr143
(Figure 2B). However, in this orientation the 3-amino indazole
( )-8 (FXIa Ki = 53 nM) was 4-fold less active than ( )-2. In
fact, the 3-amino group did not influence FXIa activity as
indazole ( )-9 was equipotent to ( )-8. FXIa activity was
improved by replacing the 3-amino moiety with a hydroxyl,
which gave 3-hydroxy indazole ( )-10. The chiral, nonracemic
derivative (S)-10 (FXIa Ki = 3.7 nM) was approximately 2-fold

more potent than amino indazole (S)-2. Since the 3-hydroxy
indazole can exist as the 3-oxo tautomer, it was unclear which
functionality, the 3-hydroxy or the 3-oxo, was important for FXIa
activity as each could serve as an H-bond acceptor for Tyr143.19
In an attempt to answer this question, the 3-OMe indazole ( )-11
and the 3-oxo derivative ( )-12 were prepared. However, both
modifications led to a >10-fold loss in FXIa activity. With the
regioisomeric 6,5-fused heterocycles, only the 3-hydroxy
indazole derivative (S)-10 allowed for the removal of one H-bond
donor while maintaining FXIa activity and potency in the in vitro
clotting assay.
exploration of monocyclic P2 prime groups (Table 3). Removal
of either the amino group in compound ( )-14 or the carboxylic
acid group in compound ( )-15 led to a loss in FXIa activity.
Table 3. Compounds Containing Monocyclic P2 Prime
Groups
O
R
N
H
H2N
N
Entry
R
FXIa Kia
(nM)
aPTTb EC1.5x
(µM)
5.3
0.8
( )-13
( )-14
( )-15
205
56
NTc
35
24
12
NTc
3.7
( )-16
( )-17
(S)-17
6.3
2.6
Figure 3. X-ray crystal structure of (S)-10 in Factor XIa. PDB deposition
number is 5EXL. The red spheres depict water molecules and the dotted lines
depict hydrogen bonds. EDO stands for ethylene diol.
aKi values were obtained from purified human enzyme and were averaged
from multiple determinations (n=2), as described in Ref. 15b. baPTT
(activated partial thromboplastin time) in vitro clotting assay was performed
in human plasma as described in Ref. 15b. cNT = not tested.
An X-ray structure of 3-hydroxy indazole (S)-10 bound to
FXIa (2.3 Å resolution, R-work was 0.194 and R-free was 0.238,
Figure 3) was obtained. The binding mode was similar to that
described previously for the pyridine (S)-2, with the exception of
the P2 prime region.18 Compound (S)-10 occupies the S1, S1
prime, and the S2 prime binding pockets and participates in a
large number of hydrogen bonding interactions. The cyclohexyl
methyl amine P1 occupies the S1 pocket where the primary
amine is within bonding distance to either a conserved water
molecule (2.7 Å), Asp189 (2.9 Å), Ala190 (3.0 Å), or Gly218
(3.0 Å). The NH of the carboxamide forms an H-bond to Ser214
via a network of conserved water molecules. The carbonyl of the
amide interacts with the oxyanion hole which consists of the
backbone NH’s of Ser195 (3.6 Å), Asp194 and Gly193 (3.3 Å).
The inhibitor’s benzyl moiety occupies the S1 prime pocket with
an edge-on hydrophobic interaction between the phenyl ring and
the disulfide bridge Cys58-Cys42. The pyridine nitrogen forms
an H-bond to a conserved water molecule (2.9 Å). The 3-hydroxy
indazole occupies the S2 prime pocket and engages in a number
of H-bonds. The hydroxy group forms an H-bond with Tyr143
(2.6 Å), the indazole N(1)H forms an H-bond to the backbone
carbonyl of His40 (2.9 Å), and the indazole N(2) forms an H-
bond to the Ile151 via ethylene diol (EDO) and a water
molecule.21
Between these two compounds, the p-amino group was more
important for FXIa binding demonstrating the importance of H-
bond donation to His40. In order to improve the H-bond
donating ability of the aniline, amino pyridine ( )-16 and methyl
N-phenyl carbamate ( )-17 were prepared. Amino pyridine ( )-
16 provided a 2-fold increase in FXIa binding. Conversion of the
p-amino group in ( )-15 to methyl carbamate ( )-17 (FXIa = 12
nM) provided a 4 to 5-fold improvement in FXIa activity. The
corresponding chiral, nonracemic derivative (S)-17 (FXIa Ki =
6.3 nM) was found to be a single-digit nanomolar FXIa inhibitor
and showed good activity in the in vitro clotting assay. Analog
(S)-17 showed similar FXIa inhibition as amino indazole (S)-2
and was approximately 2-fold less active than either 3-amino
benzisoxazole (S)-5 or 3-hydroxy indazole (S)-10. Importantly,
the methyl N-phenyl carbamate P2 prime moiety allowed for
removal of two NH bond donors while maintaining FXIa activity
when compared to amino indazole (S)-2.
An X-ray structure of methyl N-phenyl carbamate (S)-17
bound to FXIa (2.1 Å resolution, R-work was 0.194 and R-free
was 0.226, Figure 4A) was obtained. The overall binding mode
is similar to that described for 3-hydroxy indazole (S)-10, with
the exception of the P2 prime region. As a result, only P2 prime
interactions will be described here. The methyl N-phenyl
carbamate of (S)-17 occupies the S2 prime pocket with the NH of
the carbamate forming an H-bond with the carbonyl of His40
(2.8 Å). The carbonyl of the carbamate forms an H-bond through
a conserved water to Ile151 (2.7 Å). Figure 4B shows an overlay
of the X-ray structures of amino indazole (S)-2 and methyl N-
2.3. Monocyclic P2 Prime Groups.
The 3-hydroxy indazole (S)-10 was one of the most potent P2
prime groups discovered in this series. In order to maintain the
interactions with His40 and Tyr143, we envisioned that an
anthranilic acid moiety could mimic the 3-hydroxy indazole.22
Indeed, anthranilic acid ( )-13 (FXIa = 5.3 nM) was found to be
equipotent to hydroxy indazole (S)-10 leading to further

phenyl carbamate (S)-17 in the S2 prime pocket of FXIa. The
methyl N-phenyl carbamate (S)-17 maintains two of the key H-
bonding interactions seen with the amino indazole (S)-2.
pyridine (S)-17.24 Importantly, a variety of 6-membered ring
scaffolds, specifically regioisomeric pyridines, pyrimidine, and
pyridinone, were identified which improved the potency in the
clotting assay.
Table 4. Compounds Containing Phenyl, Regioisomeric
Pyridines, Pyrimidine, and Pyridinone Scaffolds With Basic
P1.
Entry
X
FXIa Kia
(nM)
aPTTb EC1.5x
(µM)
6.3
17
2.6
13
(S)-17
(S)-18
(S)-19
8.4
1.3
7.9
9.1
0.9
1.2
(S)-20
(S)-21
(S)-22
2.4
0.4
aKi values were obtained from purified human enzyme and were averaged
from multiple determinations (n=2), as described in Ref. 15b. baPTT
(activated partial thromboplastin time) in vitro clotting assay was performed
in human plasma as described in Ref. 15b.
2.5. Replacement of the Basic Cyclohexyl Methyl Amine P1.
Recently we reported that when the basic cyclohexyl methyl
amine P1 was replaced with a neutral p-chlorophenyltetrazole P1,
as exemplified in imidazole (S)-23, a modest improvement in oral
bioavailability was achieved (Table 5 and Table 6).17 As a result,
the basic P1 in (S)-17 was replaced with the neutral p-
chlorophenyltetrazole P1 to give (S)-24. This modification
resulted in only a 3-fold loss in FXIa activity (FXIa Ki = 23 nM)
Table 5. Compounds Containing Imidazole, Regioisomeric
Pyridines, Pyrimidine, and Pyridinone Scaffolds With Neutral
P1.
Figure 4. 4A. X-ray crystal structure of (S)-17 in Factor XIa. PDB
deposition number is 5EXM. 4B. Overlay of X-ray crystal structures of (S)-
17 (magenta) and (S)-2 (cyan) in the S2 prime pocket of Factor XIa. The red
spheres depict water molecules and the dotted lines depict hydrogen bonds.
2.4. Scaffold Optimization.
The methyl N-phenyl carbamate (S)-17 exhibited good potency
in the in vitro clotting assay but it remains 5-fold less potent than
amino indazole (S)-2. The reason (S)-17 was less active in the
clotting assay was postulated to be due to higher protein
binding.23 To address the need for additional potency in the
clotting assay we sought to lower the protein binding by
replacing the pyridine scaffold with other 6-membered rings.18
The methyl N-phenyl carbamate P2 prime group was combined
with phenyl, regioisomeric pyridines, pyrimidine, and pyridinone
scaffolds (Table 4). The phenyl derivative (S)-18 was 2 to 3-fold
less active than pyridine (S)-17 and showed a 5-fold loss in the
clotting assay. The regioisomeric pyridines, (S)-19 and (S)-20,
and the pyrimidine (S)-21 were essentially equipotent to (S)-17 in
the FXIa binding assay and they showed a 2 to 3-fold
improvement in potency in the clotting assay. The pyridinone
(S)-22 (FXIa Ki = 2.4 nM; aPTT EC1.5x = 0.4 µM) was the only
scaffold which showed improvements in both FXIa affinity (2.5-
fold) and potency in the clotting assay (6.5-fold) compared to
FXIa Kia
(nM)
aPTTb EC1.5x
(µM)
Entry
X
(S)-23
6.7
8.6
(S)-24
(S)-25
23
>40
26
11
(S)-26
(S)-27
6.7
2.4
21
2.8
aKi values were obtained from purified human enzyme and were averaged
from multiple determinations (n=2), as described in Ref. 15b. baPTT
(activated partial thromboplastin time) in vitro clotting assay was performed
in human plasma as described in Ref. 15b.

however, a more significant loss (>15-fold) in potency in the
clotting assay was observed. The basic P1 was also replaced
with a p-chlorophenyltetrazole in the regioisomeric pyridine,
pyrimidine, and pyridinone analogs. As shown in Table 5, the
regioisomeric pyridine (S)-25, pyrimidine (S)-26, and pyridinone
(S)-27 improved both FXIa binding activity and potency in the
clotting assay compared to pyridine (S)-24. However, these
compounds were less potent in the clotting assay than the
corresponding analogs in Table 4 when the P1 moiety was the
basic cyclohexyl methyl amine. The loss of potency in the
clotting assay was a common feature on going from the basic
cyclohexyl methyl amine P1 to the neutral p-
chlorophenyltetrazole P1 and it was postulated that the loss was
due to an increase in protein binding.23
achieved with imidazole (S)-23, by replacing the basic
cyclohexyl methyl amine P1 with neutral p-
a
chlorophenyltetrazole P1, we next evaluated pyridine (S)-24,
regioisomeric pyridine (S)-25, pyrimidine (S)-26, and pyridinone
(S)-27 which possessed the neutral p-chlorophenyltetrazole P1.
Pyridine (S)-24 exhibited a low clearance, a long half-life, a high
volume of distribution, and was orally bioavailable in dog [%F =
34 (cassette); 53 (discrete)]. In comparing (S)-24 to (S)-17,
replacing the basic P1 with a neutral P1 led to improvements in
both clearance and half-life while maintaining oral exposure. In
comparing pyridine (S)-24 to imidazole (S)-23, the pyridine
scaffold showed a lower clearance and longer half-life which
resulted in
a significant improvement in oral exposure.
Regioisomeric pyridine (S)-25 and pyrimidine (S)-26 had a
similar PK profile to (S)-24, with low clearance, long half-life,
and oral bioavailability (%F = 28-34). The low clearance and
long half-life of (S)-24, (S)-25, and (S)-26 could be due to higher
protein binding which would be expected based on the potency in
the clotting assay (aPTT EC1.5x). Replacing the basic P1 with the
neutral P1 did not provide a general solution for improving the
PK profile, as pyridinone (S)-27 showed high clearance, a short
half-live, and a significant loss in oral exposure.
Table 6. Pharmacokinetic Profile For Selected Compounds.
Cla
(mL/h/kg) (h)
t1/2
Vdss
(L/kg) (%)
F
AUC
(nM*h)
Dose
iv/po (mpk)
Entry
(S)-2
50
54
27
17
21
6.1
1.2
2.7
3.2
1.1
2.4
8.0
3.4
10
0
63
66
<1
10
34
0
194
195
10
0.27/0.43
0.28/0.62
0.20/0.29
0.40/0.80
0.20/0.20
0.25/0.20
(S)-17
(S)-21
(S)-22
(S)-23
(S)-24
5.4
0.8
3.4
3.9
Figure 5. X-ray crystal structure of (S)-24 in Factor XIa. PDB deposition
number is 5EXN. The red spheres depict water molecules and the dotted
lines depict hydrogen bonds.
30
An X-ray structure of (S)-24 bound to FXIa (1.49 Å resolution,
R-work was 0.170 and R-free was 0.196, Figure 5) was obtained.
The overall binding mode is similar to that described for (S)-17,
with the exception of the P1 region. As a result, only the
chlorophenyltetrazole P1 interactions will be described here. The
chlorophenyl portion fills the S1 pocket with the chlorine atom
forming a π-Cl (3.7 Å) interaction with Tyr228. The tetrazole
portion extends out of the S1 pocket and interacts with the
Cys219-Cys191 disulfide bridge.25 In addition, the tetrazole N4
nitrogen forms an H-bond, via a series of water molecules, to
Gly218.
267
(53)b (1,878)b
(S)-25
(S)-26
(S)-27
5.6
7.3
30
3.7
6.8
1.5
1.6
4.0
1.6
34
28
0
280
230
0
0.19/0.19
0.20/0.20
0.20/0.20
aCompounds were dosed in dog in an N-in-1 format. Vehicle for both iv and
po: 70% water; 10% ethanol; 10% dimethylacetamide: 10% polypropylene
glycol. bCompound dosed in dog in a discrete format. PO dose: 1 mpk. PO
vehicle: 70% PEG400; 20%TPGS; 10% ethanol.
2.6. Pharmacokinetic Profiles.
2.7. Serine Protease Selectivity.
Several compounds were evaluated in dog, using cassette
dosing, in order to determine the pharmacokinetic (PK) profiles
(Table 6).26 Amino indazole (S)-2 was found to be a high
clearance compound and showed no oral exposure. However,
replacing the P2 prime of amino indazole (S)-2 with a methyl N-
phenyl carbamate, (S)-17, improved oral exposure in dog (%F =
63), albeit with both high clearance and high volume of
distribution. Similarly, pyrimidine analog (S)-21 was shown to
be orally bioavailable in dog (%F = 66) but was also a high
clearance and high volume of distribution compound. Pyridinone
(S)-22 exhibited both a lower clearance and lower volume of
distribution relative to either (S)-17 or (S)-21 but it was not orally
bioavailable. Even though the PK profiles for (S)-17 and (S)-21
were less than ideal with the high clearance and high volume of
distribution, it was encouraging to see oral exposure with analogs
containing the basic cyclohexyl methyl amine P1. Coupled with
the fact that a modest improvement in oral bioavailability was
The serine protease selectivity profiles for the amino indazole
(S)-2, hydroxy indazole (S)-10, and the methyl N-phenyl
carbamate (S)-17 containing the basic cyclohexyl methyl amine
P1 are listed in Table 7. Compound (S)-24, possessing methyl N-
phenyl carbamate P2 prime and p-chlorophenyltetrazole P1, was
also included. Compounds (S)-2 and (S)-17, possessing a basic
P1 group, showed >1,000-fold selectivity against many of the
relevant serine proteases, except for plasma kallikrein (< 7-fold)
and trypsin (<16-fold). Interestingly, the hydroxy indazole (S)-
10 showed improved selectivity for both plasma kallikrein (>140-
fold) and trypsin (>250-fold).27 Replacing the cyclohexyl methyl
amine P1 in (S)-17 with the p-chlorophenyltetrazole P1 in (S)-24
also led to an improvement in selectivity for both plasma
kallikrein (80-fold) and trypsin (>270-fold).28

Scheme 1. Synthesis of 4-chloropyridine 1c/(S)-1c and boronate 1g.a
O
a, b
1b
for : c
R
R
R
H2N
O
N
H
1e
for : f
H
N
N
N
N
Boc
1a
R = Cl
1c
R = Cl
1b
1e
R = Cl
R = Br
e
d
1d
R = Br
1c
(S)-
R = Cl
O
g
B
1f
1g
R = Br
R =
O
aReagents and conditions: (a) hydroxylamine hydrochloride, MeOH; (b), Zn, TFA, 0 °C, 74-98% over two steps; (c) Boc-tranexamic acid, EDC, HOBt, Hunig’s
base, DMF, rt, 66%; (d) chiral prep HPLC; (e) TMSBr, propionitrile, microwave 150°C, 64%; (f) Boc-tranexamic acid, BOP reagent, Et3N, DMF, rt, 84%; (g)
Pd(dppf)Cl2⋅CH2Cl2, bis(pinacolato)diboron, KOAc, dioxane, 80 °C, 4h, 60%.
Scheme 2. Synthesis of compounds with bicyclic P2 prime groups from 4-chloropyridine 1c or (S)-1c.a
aReagents and conditions: (a) Aryl boronic acid/ester, Pd2(dba)3, tBu3PHBF4, Cs2CO3, dioxane, 90 °C; or Pd(dppf)Cl2⋅CH2Cl2, K3PO4, DMSO, 90 °C; (b) 30-
50% TFA (v/v), CH2Cl2, rt, 11-73% over two steps; (c) hydrazine monohydrate, nBuOH, microwave, 160 °C, 16-71%; (d) acetohydroxamic acid, KOtBu, DMF,
43%; (e) formamidine acetate, DMA, 120 °C, 11%; (f) NaOH, MeOH, rt, 75%.
Scheme 3. Synthesis of compounds with bicyclic P2 prime groups from boronate 1g.a
aReagents and conditions: (a) Aryl bromide, Pd2(dba)3, tBu3PHBF4, Cs2CO3, dioxane, 90 °C; (b) 30% TFA (v/v), CH2Cl2, rt, 29-34% over two steps; (c)
Pd(OAc)2, BINAP, Cs2CO3, benzophenone imine, DMSO, 85 °C; (d) 30% TFA (v/v), CH2Cl2, rt; (e) 4M HCl in dioxane, water, 9% over three step

Hartwig amination of 3e with benzophenone imine, Boc-
deprotection and then hydrolysis of the imine.
The synthesis of the monocyclic P2 prime analogues 13-17 are
described in Scheme 4. Suzuki-Miyaura coupling of 1c or (S)-1c
with a variety of aryl- and heteroarylboronic acids gave
Table 7. Human Serine Protease Selectivity Profile for (S)-2,
(S)-10, (S)-17, and (S)-24.
Human Enzyme Ki
(nM)a
(S)-2
(S)-10
(S)-17
(S)-24
compounds 4a, 4c-4e, and (S)-4f.
Methyl ester 4a was
hydrolyzed to the anthranilic acid 4b with NaOH in MeOH at 60
°C and then deprotection with TFA in DCM provided 13.
Compounds 4c, 4d, 4e, and (S)-4f were deprotected with TFA in
DCM to give 4g, 15, 4h, and (S)-17, respectively. Saponification
of methyl ester 4g yielded carboxylic acid 14. Nucleophilic
aromatic substitution of fluoro pyridine 4h with ammonium
hydroxide at 140°C in a microwave gave amino pyridine 16.
Scheme 5 describes the syntheses of the phenyl and
Factor XIa
Factor VIIa
Factor IXa
Factor Xa
7.2
3.7
6.3
23
>10,890
>34,860
>9,000
>12,610
64
>15,180
>34,860
>9,000
>12,610
999
>12,170
>34,860
>9,000
>12,610
101
>13,390
NT
1,128
Thrombin
>13,340
>6218
1,850
Trypsin
regioisomeric pyridine scaffolds.
Condensation of 3-
Plasma Kallikrein
Activated Protein C
Plasmin
24
550
42
bromobenzaldehdye with lithium bis(trimethylsilyl)amide
generates the N-trimethylsilylaldimine, followed by the addition
of benzyl magnesium bromide according to the procedure of
Hart, gave after aqueous work up, the primary amine 5a.33 The
enantiomers were separated by chiral preparatory HPLC which
provided (S)-5a.30 Amide coupling of amine (S)-5a with Boc-
tranexamic acid, followed by Suzuki-Miyaura coupling with 4-
>21,400
>22,100
>21,900
>14,060
>20,780
>30,220
>22,100
>21,900
>14,060
>20,780
>30,220
>22,100
>21,900
>14,060
>20,780
>30,220
12,540
10,500
>29,730
29,540
TPA
Urokinase
Chymotrypsin
aAll Ki values in nM were obtained using human purified enzymes.
(methoxycarbonylamino)phenylboronic
acid
and
Boc-
deprotection afforded (S)-18.
Analogs containing the
regioisomeric pyridine scaffolds were prepared via a different
sequence. Negishi coupling of acid chlorides 5c and 5g with
benzyl zinc chloride in the presence of Pd(PPh3)4 afforded
ketones 5d and 5h. The ketones 5d and 5h were converted to
their corresponding amides (S)-5f and (S)-5j according to the
sequence previously described in Scheme 1. Suzuki-Miyaura
2.8. Chemistry
The synthesis of 4-chloropyridine 1c, (S)-1c, and boronate 1g,
key intermediates needed for the exploration of the S2 prime
pocket, are described in Scheme 1. Condensation of ketone 1a
with hydroxylamine hydrochloride generated the oxime and
subsequent reduction with zinc dust and TFA, according to the
procedure of Negi, provided amine 1b.29 Amide coupling of
amine 1b with Boc-tranexamic acid gave 1c. The enantiomers of
1c were separated by chiral preparatory HPLC to give (S)-1c.30
Efforts to convert 4-chloropyridine 1c to boronate 1g were not
successful, so 4-bromopyridine derivative 1f was prepared.
Using a modification of the procedure described by Schlosser,
ketone 1a was treated with trimethylsilyl bromide in propionitrile
at elevated temperature in a microwave to give the 4-
bromopyridine 1d.31 Compound 1d was then converted to 1f
according to the sequence described for 1c. Miyaura borylation
on 1f afforded boronate 1g. With the key intermediates in hand,
exploration of the S2 prime pocket was initiated.
The synthesis of analogues 2, 4-6, and 8-11 are described in
Scheme 2. Suzuki-Miyaura coupling of 1c or (S)-1c with a
variety of aryl boronic acids or esters, followed by Boc-
deprotection with TFA in DCM gave compounds 2b-2d, 2f, and
2h. Heating 2b and 2d with hydrazine monohydrate in n-butanol
at 160°C in a microwave provided amino indazole derivatives 2
and 8. Similarly, heating 2c and 2f with hydrazine monohydrate
in n-butanol at elevated temperatures afforded hydroxy indazole
derivatives 4 and 10. Amino benzisoxazole 5 was prepared by
reacting 2b with the potassium salt of N-hydroxyacetamide
according to the procedure described by Palermo.32 Heating 2a
with formamidine acetate in DMAC at 120°C afforded, after
coupling of amides (S)-5f
and (S)-5j with 4-
(methoxycarbonylamino)phenylboronic acid, followed by Boc-
deprotection, afforded (S)-19 and (S)-20.
Scheme 6 describes the synthesis of the pyrimidine scaffold.
Condensation of the β-ketoester 6a, prepared according to a
modified procedure of Maibaum, with formamidine acetate in the
presence of NaOMe gave pyrimidinone 6b.34,35
Heating
pyrimidinone 6b and POCl3 leads to the formation of the chloro
pyrimidine as well as the deprotection of the Boc group. The
amine was reprotected with Boc-anhydride to yield 6c. Suzuki-
Miyaura coupling with 4-(methoxycarbonylamino)phenylboronic
acid and Boc-deprotection afforded amine 6d. The enantiomers
were separated by chiral preparatory HPLC which provided (S)-
6d. Amide coupling with Boc-tranexamic acid and then Boc-
deprotection gave (S)-21.
Scheme 7 describes the synthesis of (S)-24 to (S)-26 analogs
which contain the neutral p-chlorophenyltetrazole P1. Suzuki-
Miyaura
coupling
of
(S)-7a
with
4-
(methoxycarbonylamino)phenylboronic acid followed by Boc-
deprotection afforded amine (S)-7b. Amide coupling of amine
(S)-7b with the activated cinnamic ester 7d yielded (S)-24. The
regioisomeric pyridine scaffold was prepared via a different
method than previously described in Scheme 5. Condensation of
aldehyde 7e, with (R)-2-methylpropane-2-sulfinamide in the
presence of Ti(OEt)4 gave the corresponding sulfinimine which
was reacted with benzylmagnesium chloride to give
diastereomeric sulfinamides (R,S)-7f and (R,S)-7f, which are
separable by normal phase chromatography.36 Suzuki-Miyaura
Boc-deprotection, 4-aminoquinazoline 6.
Indazole 9 was
prepared in a two-step sequence by heating 2e with hydrazine
monohydrate at elevated temperatures following Boc-
deprotection. Hydrolysis of the ethyl carbamate on 2h with
NaOH at room temperature yielded 3-OMe indazole 11.
Analogues 3, 7, and 12 were prepared according to Scheme 3.
Suzuki-Miyaura coupling of boronate 1g with appropriately
substituted arylbromides, 3a-3c, provided 3d-3f. Compounds 3d
and 3f were deprotected with TFA in DCM to give 3 and 12,
coupling
of
(R,S)-7f
with
4-
(methoxycarbonylamino)phenylboronic acid and removal of the
chiral auxiliary afforded amine (S)-7g. Amide coupling of amine
(S)-7g with cinnamic acid 7c provided (S)-25. The pyrimidine
analog (S)-26 was prepared by coupling amine (S)-6d with the
activated cinnamic ester 7d.
The synthesis of the pyridinone derivatives (S)-22 and (S)-27
respectively.
Compound 3e was converted to 1-amino
is described in Scheme 8.
Horner-Wadsworth-Emmons
isoquinoline 7 in a three step sequence, specifically Buchwald-

Scheme 4. Synthesis of compounds with monocyclic P2 prime groups from 4-chloropyridine 1c or (S)-1c.a
X
R1
R2
R1
R2
X
R1
R2
(HO)2B
X
O
O
c
a
1c
(S)-
or
1c
N
N
H
H
H
N
N
H2N
N
Boc
X = CH, R1 = NH2, R2 = CO2H
X = CH, R1 = H, R2 = CO2Me
X = CH, R1 = H, R2 = CO2H
X = CH, R1 = NH2, R2 = H
X = CH, R1 = NH2, R2 = CO2Me
X = CH, R1 = NH2, R2 = CO2H
X = CH, R1 = H, R2 = CO2Me
X = CH, R1 = NHBoc, R2 = H
X = N, R1 = F, R2 = H
13
4g
14
15
4h
16
17
4a
4b
4c
4d
4e
4f
b
b
d
X = N, R1 = F, R2 = H
X = N, R1 = NH2, R2 = H
X = CH, R1 = NHCO2Me, R2 = H
(S)-
X = CH, R1 = NHCO2Me, R2 = H
(S)-
aReagents and conditions: (a) Aryl- or heteroarylboronic acid, Pd2(dba)3, tBu3PHBF4, Cs2CO3, dioxane, 90 °C, 78-88%; (b) NaOH, MeOH, 60 °C; (c) 30% TFA
(v/v), CH2Cl2, rt, 6-87% over two steps; (d) ammonium hydroxide, 140 °C, microwave, 41%.
Scheme 5. Synthesis of compounds containing phenyl (S)-18 and regioisomeric pyridine (S)-19 and (S)-20 scaffolds.a
aReagents and conditions: (a) LiHMDS, THF, 0 °C, then BnMgCl, 80%, then chiral prep HPLC; (b) Boc-tranexamic acid, EDC, HOBt, Hunig’s base, DMF, rt,
47-84%, for 5j chiral prep HPLC; (c) Pd(Ph3P)4, BnZnCl, THF, -30 °C to 0 °C, 45-64%; (d) hydroxylamine hydrochloride, MeOH; (e), Zn, TFA, 0 °C, 35-50%
over two steps, for 5e chiral prep HPLC; (f) 4-(methoxycarbonylamino)phenylboronic acid, Pd(Ph3P)4, DME, water, 60 °C; (g) 30% TFA (v/v), CH2Cl2, rt, 34-
71% over two steps; (h) 4-(methoxycarbonylamino)phenylboronic acid, Pd(dppf)Cl2⋅CH2Cl2, K3PO4, DMSO, 90 °C; (i) 4-(methoxycarbonylamino)phenyl
boronic acid, Pd2(dba)3, tBu3PHBF4, Cs2CO3, dioxane, 90 °C.

reaction of β-ketophosphonate (S)-8a37 and 4-nitrobenzaldehyde
gave the corresponding α,β-unsaturated ketone which was
condensed with 1-(ethoxycarbonylmethyl)-pyridinium chloride in
the presence of ammonium acetate in EtOH at elevated
temperature to give scalemic pyridinone 8b.38 The nitro group
was reduced to the aniline and then reacted with methyl
chloroformate to give, following mild hydrolysis with sodium
hydroxide, carbamate 8c. After removing the Boc group, the
scalemic amine was purified by chiral preparatory hplc to give
(S)-8d. Amine (S)-8d was coupled with Boc-tranexamic acid and
then deprotection of the Boc-group gave (S)-22. Amine (S)-8d
was coupled with cinnamic acid 7c which provided (S)-27.
using EM Science silica gel 60 (230-400 mesh). Preparative
reverse-phase HPLC purifications were performed using the
following conditions: a binary solvent system where solvent A =
10% methanol, 90% water, 0.1% trifluoroacetic acid, and solvent
B = 90% methanol, 10% water, 0.1% trifluoroacetic acid,
detector 220 nm. The enantiomers were separated by chiral
preparatory HPLC and the conditions indicated in the
corresponding experimental procedure. Optical rotations were
performed on a Jasco P-1010 polarimeter.
4.1.1. 1‐(4‐Chloropyridin‐2‐yl)‐2‐phenylethan‐1‐one (1a).
To
a cooled (-40 °C) solution of methyl 4‐chloro-
pyridine‐2‐carboxylate (14.5g, 84.5 mmol) in THF (192 mL) was
added rapidly via cannula a cooled (-40 °C), pale brown solution
of 0.6 M benzylmagnesium chloride in THF (142 mL, 84.5
mmol). The resulting, clear orange solution was stirred at -40 °C.
After 1 h the reaction was quenched with glacial acetic acid (5.4
mL, 93 mmol) and the reaction was warmed to rt. The reaction
was partitioned between EtOAc and sat. NaHCO3 and the layers
were separated. The aqueous layer was extracted with EtOAc
(3x). The organic layers were combined and washed with brine,
dried over Na2SO4, filtered, and concentrated to give a red-brown
liquid weighing 21.6 g. Column chromatography on silica gel
(1.5:1 DCM:Hex) gave 10.1 g (52% yield) of 1a as an orange
liquid. LCMS: 232.1 (M+H)+ and 234.0 (M+2+H)+. 1H NMR
(500 MHz, CDCl3) δ: 8.63 (d, J = 5.5 Hz, 1H), 8.03 (d, J = 2.2
Hz, 1H), 7.46 (dd, J = 5.0, 2.2 Hz, 1H), 7.34-7.28 (m, 4H), 7.27-
7.22 (m, 1H), 4.52 (s, 2H). 13C NMR (125 MHz, CDCl3) δ:
197.9, 154.2, 149.8, 145.5, 134.2, 129.9, 128.5, 127.2, 126.8,
122.8, 44.1.
3. Conclusions
Pyridine-based FXIa inhibitor (S)-2 was optimized by
modifying the P2 prime, P1 and scaffold regions. Exploration of
the P2 prime region identified 3-amino benzisoxazole, 3-hydroxy
indazole, anthranilic acid, and methyl N-phenyl carbamate groups
as potent replacements for 3-amino indazole. The methyl N-
phenyl carbamate, found in (S)-17, became the preferred P2
prime moiety since it not only maintained FXIa activity but
improved oral exposure in dog even with the basic cyclohexyl
methyl amine P1. Moreover, replacing the basic cyclohexyl
methyl amine P1 with neutral p-chlorophenyltetrazole P1
resulted in the discovery of (S)-24, which showed a significant
improvement in oral bioavailability in dog compared to the
previously reported imidazole analog (S)-23.
Additional
improvements in FXIa binding affinity, while maintaining oral
bioavailability, was achieved with regioisomeric pyridine (S)-25
and pyrimidine (S)-26. Further efforts to improve both FXIa
potency and pharmacokinetics will be reported in due course.
4.1.2. 1‐(4‐Chloropyridin‐2‐yl)‐2‐phenylethan‐1‐amine (1b).
Compound 1b was prepared following a modified procedure of
Negi.29 To a clear, yellow solution of 1a (3.96 g, 17.1 mmol) in
MeOH (34 mL) was added hydroxylamine hydrochloride (3.56 g,
51.3 mmol). The resulting suspension was stirred at rt. After 14
h the reaction was concentrated to give a yellow solid. The solid
was dissolved in EtOAc and washed with sat. NaHCO3. The
layers were separated and the aqueous layer was extracted with
EtOAc. The organic layers were combined and washed with
brine, dried over Na2SO4, filtered, and concentrated to give 4.13
g of the corresponding oxime as a pink solid. LCMS: 247.1
(M+H)+ and 249.1 (M+2+H)+.
To a cooled (5 °C), clear, yellow solution of oxime (4.13 g) in
TFA (39.5 mL) was added in portions zinc dust (11.18 g, 171
mmol) so as to keep the temperature below 25 °C. After 1.5 h,
the reaction was filtered through a cotton plug, rinsing with TFA
(50 mL). The filtrate was poured slowly into a cold (0 °C),
vigorously stirred suspension of 2 M NaOH (700 mL) and DCM
(500 mL). The layers were separated and the aqueous layer was
extracted with DCM. The combined organic layers were washed
with brine, dried over MgSO4, filtered and concentrated to give
3.64 g as a clear, orange-brown liquid. Column chromatography
on silica gel (5% MeOH in DCM with 0.5% ammonium
hydroxide) gave 2.93 g (74% yield over two steps) of 1b as a
clear, yellow oil. LCMS: 233.1 (M+H)+ and 235.1 (M+2+H)+.
1H NMR (500 MHz, CDCl3) δ: 8.47 (d, J = 5.0 Hz, 1H), 7.31-
7.25 (m, 3H), 7.23-7.20 (m, 1H), 7.17-7.14 (m, 3H), 4.21 (dd, J =
8.8, 5.5 Hz, 1H), 3.15 (dd, J = 13.8, 5.0 Hz, 1H), 2.82 (dd, J =
13.8, 8.8 Hz, 1H), 1.60 (bs, 2H). 13C NMR (125 MHz, CDCl3) δ:
165.9, 150.1, 144.5, 138.4, 129.3, 128.5, 126.6, 122.4, 121.6,
58.5, 45.1.
4. Experimental section
4.1 Chemistry
All reactions were run under and atmosphere of dry nitrogen or
argon unless otherwise noted. Solvents and reagents were
obtained from commercial vendors in the appropriate grade and
used without further purification unless otherwise indicated.
Proton, carbon, and fluorine magnetic resonance (1H, 13C, and 19
F
NMR) spectra were recorded either on a Bruker Avance 400,
JEOL Eclipse 400, or a JEOL Eclipse 500 spectrometer.
Chemical shifts are given in parts per million (ppm) relative to
either the reference solvent of the sample in which they were run
or tetramethylsilane (TMS).
HPLC analyses of all final
compounds were performed using the following conditions:
Zorbax SB C18 4.6mm x 75 mm column with a binary solvent
system where solvent A = 10% methanol, 90% water, 0.2%
phosphoric acid, and solvent B = 90% methanol, 10% water,
0.2% phosphoric acid, flow rate = 2.5 mL/min, linear gradient
time = 8 min, start %B =0, final %B = 100, detection 220 nm.
All final compounds had an HPLC purity of ≥ 95% unless
specifically mentioned.
High resolution mass spectra were
measured on an Agilent TOF 6210 LC/MS system with
electrospray ionization (ESI). LCMS analyses were recorded on
a Shimadzu LC-10AT equipped with a SIL-10A injector, a SPD-
10AV detector normally operating at 220 nm, and interfaced with
a Micromass ZMD mass spectrometer. LCMS analyses used the
following conditions: Phenomenex Luna 5 µm C18 4.6 mm x 50
mm column with a binary solvent system where solvent A = 10%
methanol, 90% water, 0.1% trifluoroacetic acid, and solvent B =
90% methanol, 10% water, 0.1% trifluoroacetic acid, flow rate =
4 mL/min, linear gradient time = 4min, start %B =0, final %B =
100. Elemental analyses were performed by Robertson Microlit
Laboratories, Inc., Ledgewood, NJ 07852 and were within 0.4%
of the theoretical values. Flash chromatography was performed

Scheme 6. Synthesis of pyrimidine (S)-21 scaffold.a
aReagents and conditions: (a) formamidine acetate, NaOMe, MeOH, 58%; (b) POCl3, 50°C; (c) Boc2O, Et3N, DCM, 28% over two steps; (d) 4-
(methoxycarbonylamino)phenylboronic acid, Pd(dppf)Cl2⋅CH2Cl2, K3PO4, DMSO, 85 °C; (e) 30% TFA (v/v), CH2Cl2, rt, 37-46% over two steps; (f) chiral prep
HPLC; (g) Boc-tranexamic acid, EDC, HOBt, Hunig’s base, DMF.
Scheme 7. Synthesis of compounds (S)-24 to (S)-26 containing the neutral p-chlorophenyl tetrazole P1.a
aReagents and conditions: (a) 4-(methoxycarbonylamino)phenylboronic acid, Pd2(dba)3, tBu3PHBF4, Cs2CO3, dioxane, 90 °C, 86-99%; (b) 15-30% TFA (v/v),
CH2Cl2, rt, 87%; (c) 7c, EDC, HOBt, Et3N, DMF, 64-71%; (d) 7d, Hunig’s base, DMF, 58-75%; (e) (R)-2-methylpropane-2-sulfinamide, Ti(OEt)4, DCM, 59-
90%; (f) 1.0 M benzylmagnesium chloride or bromide in Et2O, DCM; for 7f: 71% yield as a 1.4:1 mixture of diastereromers which were separated by column
chromatography (g) 4M HCl in dioxane, MeOH, 44-81%.
Scheme 8. Synthesis of compounds (S)-22 and (S)-27 containing the pyridinone scaffold.a
aReagents and conditions: (a) 4-nitrobenzaldehyde, K2CO3, EtOH, rt; (b) 1-(ethoxycarbonylmethyl)-pyridinium chloride, NH4OAc, EtOH, 80 °C, 65% over two
steps; (c) zinc dust, NH4Cl, MeOH, rt to 60 °C; d) methyl chloroformate, pyridine, CH2Cl2; e) NaOH, MeOH, 0 °C; f) 50% TFA (v/v), CH2Cl2, rt, 35% over
four steps, then chiral prep HPLC; g) Boc-tranexamic acid, BOP reagent, Et3N, DMF, rt; h) 50% TFA (v/v), CH2Cl2, rt, 70% over two steps; i) 7c, EDC, HOBt,
Hunig’s base, DMF, 62%

4.1.5. General Procedure for Boc-Deprotection.
4.1.3. 2-Methyl-2-propanyl[(trans-4-{[(1S)-1-(4-chloro-2-
pyridinyl)-2-phenylethyl]carbamoyl}cyclohexyl)methyl]-
carbamate [(1c) and (S)-1c].
The compound was dissolved in a solution of 25-50% TFA in
DCM (v:v) to give a clear solution. After 1 h, the reaction was
concentrated to give a residue. The residue was dissolved in
DCM and concentrated. This was repeated twice to give a
residue which was either carried onto the next step without
purification or it was purified by reverse phase chromatography.
To a cooled (0 °C), clear, yellow solution of 1b (2.93 g, 12.6
mmol) in DMF (42 mL) was added sequentially Boc-tranexamic
acid (3.56 g, 13.8 mmol), HOBt (2.55 g, 18.8 mmol), and EDC
(3.62 g, 18.8 mmol). After 15 min. at 0 °C, the reaction was
warmed to rt. After 4 h, the reaction was poured into vigorously
stirred, cold (0 °C) water (200 mL) to give a white suspension.
Buchner funnel filtration gave a white solid which was washed
with water. The white solid was dissolved in dichloromethane
and washed with water, 0.5 M HCl, sat. NaHCO3, brine, dried
over MgSO4, filtered and concentrated to give a white solid
weighing 11 g. Column chromatography on silica gel (3:1
DCM:EtOAc) gave 3.91 g (66% yield) of 1c as a white solid.
The enantiomers were separated via chiral preparatory HPLC
[Daicel Chiralcel OD column; 30% IPA/Hex; 50 mL/min; 254
nm] which gave (R)-1c (>99%ee) as enantiomer A and (S)-1c
(>99%ee) as enantiomer B. HRMS m/z calc’d. for C26H35N3O3Cl
(M+H)+: 472.2367. Found 472.2357. 1H NMR (500 MHz,
CDCl3) δ: 8.41 (d, J = 5.4 Hz, 1H), 7.23-7.16 (m, 4H), 6.93-
6.92 (m, 2H), 6.89 (d, J = 1.3 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H),
5.22 (dd, J = 13.8, 7.7 Hz, 1H), 4.58 (bs, 1H), 3.16 (dd, J = 12.8,
6.0 Hz, 1H), 3.03 (dd, J = 13.4, 7.4 Hz, 1H), 2.97-2.95 (m, 2H),
2.07-2.02 (m, 1H), 1.89-1.79 (m, 4H), 1.50-1.37 (m, 12H), 0.97-
0.88 (m,2H). 13C NMR (125 MHz, CDCl3) δ: 175.0, 160.9,
156.1, 149.9, 144.3, 136.8, 129.4, 128.3, 126.7, 122.9, 122.8,
79.1, 54.7, 46.6, 45.4, 42.2, 37.7, 29.8, 29.7, 29.1, 28.8, 28.4.
Anal. calc’d for (C26H34N3O3Cl): C, 66.15; H, 7.26; N, 8.90; Cl,
4.1.6. trans-4-(Aminomethyl)-N-{(1S)-1-[4-(4-cyano-3-
fluorophenyl)-2-pyridinyl]-2-phenylethyl}cyclohexanecarbox-
amide, bis-trifluoroacetic acid salt [(S)-2b].
Compound (S)-1c was converted to (S)-2a using Suzuki-
Miyaura
coupling
method
B
with
(4‐cyano‐3‐
fluorophenyl)boronic acid to give (S)-2a. Boc-deprotection gave
0.234 g (54% yield over two steps) of (S)-2b as a white solid.
HRMS m/z calc’d for C28H30N4OF (M+H)+: 457.2404. Found
457.2420. 1H NMR (500 MHz, CD3OD) δ: 8.70 (d, J = 5.0 Hz,
1H), 7.93-7.90 (m, 1H), 7.77 (dd, J = 5.5, 1.6 Hz, 1H), 7.74 (dd,
J = 10.4, 1.6 Hz, 1H), 7.71-7.68 (m, 2H), 7.25-7.23 (m, 2H),
7.20-7.17 (m, 3H), 5.33 (dd, J = 8.8, 7.2 Hz, 1H), 3.24 (dd, J =
13.5, 6.9 Hz, 1H), 3.16 (dd, J = 13.5, 8.5 Hz, 1H), 2.76 (d, J =
7.2 Hz, 2H), 2.28-2.23 (m, 1H), 1.85-1.72 (m, 4H), 1.58-1.54 (m,
1H), 1.41-1.34 (m, 2H), 1.08-1.00 (m, 2H). 19F NMR (470 MHz,
CD3OD) δ: -77.27, -108.45.
4.1.7. tert‐Butyl N‐[1‐(6‐oxo‐1,6‐dihydropyrimidin‐4‐yl)‐2
‐phenylethyl]carbamate (6b).
To a solution of 0.5 M sodium methoxide in methanol (58.4
mL, 29.2 mmol) was added formamidine acetate (1.52 g, 14.6
mmol) to give a clear, colorless solution. Ethyl 4‐{[(tert‐
butoxy)carbonyl]amino}‐3‐oxo‐5‐phenylpentanoate (6a)34 (3.5 g,
10.4 mmol) in MeOH (20.9 mL) was added. The resulting clear
colorless solution was stirred at rt. After 8 h, the clear, yellow
solution was quenched with acetic acid (1.67 mL, 29.2 mmol)
and the reaction was concentrated to give a solid. The solid was
partitioned between water and CHCl3 (750 mL). The layers were
separated and the aqueous layer was extracted with CHCl3 (250
mL). The organic layers were combined and washed with sat.
NaHCO3, brine, dried over MgSO4, filtered, and concentrated to
give an off-white solid weighing 3.45 g. Recrystallization from
EtOAc gave a white solid weighing 1.49 g. The filtrate was
concentrated and then purified by column chromatography on
silica gel (gradient elution 0-8% MeOH/CHCl3) which gave
0.463 g. Combination of 1.49 g and 0.463 g gave 1.95 g (58%
yield) of 6b, as an off-white solid. LCMS: m/z 316.2 (M+H)+.
1H NMR (400MHz, DMSO-d6) δ: (rotamers) 12.47 (br. s., 1H),
8.18 (s, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.31 - 7.16 (m, 5H), 6.17
(s, 1H), 4.49 - 4.40 (m, 1H), 3.08 (dd, J = 13.8, 4.2 Hz, 1H), 2.72
(dd, J = 13.6, 10.5 Hz, 1H), 1.29 (s, 8H), 1.16 (br. s., 1H). 13C
NMR (100 MHz, DMSO-d6) δ: 168.5, 161.9, 155.5, 150.2,
138.9, 129.5, 128.6, 126.6, 110.8, 78.5, 57.1, 39.4 overlaps with
DMSO-d6, 28.6.
25
7.51. Found C, 65.97; H, 7.29; N, 8.66; Cl, 7.54. [α]D = -1.79
(c = 1.1; CHCl3).
4.1.4. General Procedures for the Suzuki-Miyaura coupling.
Method A:
To a flame-dried thick-walled vial was added the heteroaryl
halide or aryl halide (1.0 eq), Pd2(dba)3 (0.05 eq), tri-tert-
butylphosphonium tetrafluoroborate (0.12 eq), cesium carbonate
(2.0 eq) and the boronic acid or ester (1.3 eq). The vial was
sealed with a septum and then the vessel was purged with argon
for several minutes. Next, dioxane (0.2 M, degassed by bubbling
either nitrogen or argon) was added. Under a blanket of argon,
the septum was replaced with a Teflon-coated screw cap. The
reaction was stirred at rt for 1 h and then the reaction was
warmed to 90 °C for 12 to 24 h as determined by HPLC analysis
of the reaction mixture. The crude product was either used
without further purification or it was purified by either column
chromatography on silica gel or by reverse phase
chromatography.
Method B:
To a flame-dried flask was placed heteroaryl halide or aryl
halide (1.0 eq.), boronic acid or ester (2.5 eq),
Pd(dppf)Cl2⋅CH2Cl2 (0.10 eq) and K3PO4 (2.5 eq). The flask was
equipped with a reflux condenser and the reaction vessel was
purged with argon for several minutes. Next, degassed DMSO
(0.15 M) was added and the resulting suspension was heated to
90 °C. After 15 h, the reaction was cooled to rt, diluted with
DCM, washed with water, 2.0 M Na2CO3, brine, dried over
MgSO4, filtered and concentrated. The crude product was either
used without further purification or it was purified by either
column chromatography on silica gel or by reverse phase
chromatography.
4.1.8. tert‐Butyl N‐[1‐(6‐chloropyrimidin‐4‐yl)‐2‐phenyl-
ethyl]carbamate (6c).
An orange suspension of 6b (4.0 g, 12.68 mmol) in
phosphorus oxychloride (59.1mL, 634 mmol) was warmed to 50
°C to give a dark red solution. After 2 h, the reaction was cooled
to rt and concentrated to give an orange-brown residue. The
residue was dissolved in CH2Cl2 and then concentrated (2x). The
residue was dissolved in CH2Cl2 and then poured into cold (0 °C)
sat. NaHCO3. The layers were separated and the aqueous layer
was extracted with CH2Cl2 (1x). The organic layers were
combined and washed with brine, dried over MgSO4, filtered and
concentrated to give 2.75 g of 1-(6-chloropyrimidin-4-yl)-2-

phenylethan-1-amine as a red foam. LCMS: 234.0 (M+H)+; 236
(M+2+H)+.
To a flame-dried RBF was added (S)-7a (1.0 g, 3.0 mmol), 4-
(methoxycarbonylamino) phenylboronic acid (0.879 g, 4.51
mmol), Cs2CO3 (1.47 g, 4.51 mmol), Pd2dba3 (0.138 g, 0.15
mmol), and tri-tert-butylphosphine tetrafluoroborate (0.105 g,
0.361 mmol). The flask was purged with argon for several
minutes and then degassed 1,4-dioxane (15 mL) was added. The
reaction mixture was stirred at rt for 1 h and then warmed to 90
°C. After 4 h the reaction was stopped and cooled to rt. The
resulting gray/black suspension was filtered through a 0.45
micron glass membrane filter to give an orange filtrate. The
filtrate was concentrated to give a yellow foam. Column
chromatography on silica gel (gradient elution 0-50%
EtOAc:Hex) gave, after lyophilization, 1.16 g (86% yield) of
tert‐butyl N‐[(1S)‐1‐ (4‐{4‐[(methoxycarbonyl)amino]phenyl}-
pyridin‐2‐yl)‐2‐phenylethyl]carbamate, as a white solid. HRMS
m/z calc’d for C26H30N3O4 (M + H)+: 448.2240. Found
448.2231. 1H NMR (500 MHz, CDCl3) δ: 8.56 (d, J = 4.9 Hz,
1H), 7.50 - 7.29 (m, 5H), 7.25 - 7.14 (m, 3H), 7.05 - 6.97 (m,
3H), 6.92 (br. s., 1H), 5.88 (d, J = 7.7 Hz, 1H), 5.05 - 4.96 (m,
1H), 3.79 (s, 3H), 3.28 (dd, J = 13.2, 5.5 Hz, 1H), 3.02 (dd, J =
13.2, 8.2 Hz, 1H), 1.48 - 1.33 (m, 9H). 13C NMR (100 MHz,
CDCl3) δ: 159.8, 155.3, 153.8, 149.5, 147.7, 138.9, 137.6, 132.7,
129.5, 128.2, 127.6, 126.4, 119.8, 118.8, 79.3, 57.0, 52.4, 43.0,
28.4. [α]D23.4 = -47.43 (c =1.30, MeOH).
To a dark red solution of 1-(6-chloropyrimidin-4-yl)-2-
phenylethan-1-amine (2.73 g, 11.7 mmol) in CH2Cl2 (38.9 mL)
was added Boc2O (2.68 g, 12.3 mmol) and triethylamine (3.26
mL, 23.4 mmol). The reaction was stirred at rt for 1 h. Then, the
reaction was partitioned between CH2Cl2/sat. NaHCO3 and the
layers were separated. The aqueous layer was extracted with
CH2Cl2 (1x). The organic layers were combined and washed
with brine, dried over Na2SO4, filtered, and concentrated to give
an orange-brown oil. Purification by column chromatography on
silica gel (gradient elution 0-20% EtOAc/Hex) provided 1.17 g
(28% yield over two steps) of 6c as a pale, yellow solid. HRMS
m/z calc’d for C17H21ClN3O2 (M+H)+: 334.1322. Found
334.1325. 1H NMR (400 MHz, CDCl3) δ: 8.95 (s, 1H), 7.30-
7.18 (m, 3H), 7.05-6.96 (m, 3H), 5.41-5.37 (m, 1H), 4.99-4.92
(m, 1H), 3.17 (dd, J = 13.6, 6.6 Hz, 1H), 3.08 (dd, J = 13.6, 7.3
Hz, 1H), 1.41 (bs, 9H). 13C NMR (100 MHz, CDCl3) δ: 170.6,
161.4, 158.7, 155.0, 136.1, 129.2, 128.6, 127.0, 119.6, 80.1, 56.2,
41.5, 28.3.
4.1.9. Methyl N‐(4‐{6‐[(1S)‐1‐amino‐2‐phenylethyl]-
pyrimidin‐4‐yl}phenyl)carbamate, bis-trifluoroacetic acid salt
[(S)-6d].
Compound 6c was converted to 6d according to the Suzuki-
A
clear, yellow solution of tert‐butyl N‐[(1S)‐1‐(4‐{4‐
Miyaura
coupling
method
B
with
4-(methoxy-
[(methoxycarbonyl)amino]-phenyl}pyridin‐2‐yl)‐2‐phenylethyl]-
carbamate (0.850 g, 1.90 mmol) in 15% TFA/DCM (38.0 mL)
was stirred at rt. After 1.5 h, the reaction was concentrated to
give a clear, yellow residue. The residue was dissolved in DCM
and concentrated. This was repeated three times to give a yellow
residue. The residue was dissolved in DCM and then washed
with sat. NaHCO3, brine, dried over MgSO4, filtered and
concentrated to give a white foam. Column chromatography on
silica gel (gradient elution 0-10% MeOH/DCM) gave 0.575 g
(87% yield) of (S)-7b as a white foam. LCMS: 348.4 (M+H)+.
1H NMR (400 MHz, CD3OD) δ: 8.50 (d, J = 5.5 Hz, 1H), 7.61 -
7.53 (m, 4H), 7.51 (dd, J = 5.5, 1.6 Hz, 1H), 7.44 (d, J = 1.6 Hz,
1H), 7.25 - 7.19 (m, 2H), 7.19 - 7.13 (m, 1H), 7.11 - 7.06 (m,
2H), 4.24 (t, J = 7.1 Hz, 1H), 3.75 (s, 3H), 3.10 (dd, J = 13.2, 7.1
Hz, 1H), 3.02 (dd, J = 13.2, 7.1 Hz, 1H). 13C NMR (125 MHz,
CD3OD) δ: 164.8, 156.4, 150.3, 141.8, 139.7, 133.3, 130.6,
129.6, 128.7, 127.6, 121.0, 120.4, 120.1, 59.8, 52.8, 46.1.
[α]D23.7 = +12.63 (c =1.21, MeOH).
carbonylamino)phenylboronic acid, followed by Boc-
deprotection which gave 0.143 g (46% yield over two steps) of
6d as a white solid. The enantiomers were separated by chiral
prep HPLC [Chiralpak AS 5 x 50 cm; isocratic elution 30%
MeOH/EtOH(1:1):Heptane; 50 mL/min.; 220 nm] which gave
(R)-6d, enantiomer A (>97% ee), and (S)-6d, enantiomer B
(>98% ee).
HRMS m/z calc’d for C20H21N4O2 (M+H)+:
349.1665. Found 349.1663. 1H NMR (500 MHz, CD3OD) δ:
9.07 (s, 1 H), 7.99 (d, J = 8.8 Hz, 2 H), 7.66 (s, 1 H), 7.58 (d, J =
8.8 Hz, 2 H), 7.21 - 7.28 (m, 2 H), 7.15 - 7.20 (m, 1 H), 7.11 (d, J
= 7.2 Hz, 2 H), 4.21 (t, J = 7.2 Hz, 1 H), 3.76 (s, 3 H), 3.11 (dd, J
= 13.2, 7.2 Hz, 1 H), 3.04 (dd, J = 13.8, 7.7 Hz, 1 H). For (S)-6d:
[α]D22.4 = +1.05 (c = 1.0, MeOH). For (R)-6d: [α]D22.8 = -0.93 (c
= 1.1, MeOH).
4.1.10. tert‐Butyl N‐[(1S)‐1‐(4‐chloropyridin‐2‐yl)‐2‐phenyl-
ethyl]carbamate [(S)-7a].
To a cooled (0 °C) solution of 1b (15 g, 0.064 mol) in CH2Cl2
(150 mL) was added DMAP (0.78 g, 0.0064 mol) followed by
the portion wise addition of Boc2O (16.9 g, 0.0775 mol). The
reaction mixture was allowed to warm to rt and stir overnight.
The reaction mixture was concentrated. Column chromatography
on silica gel (EtOAc/Hex) gave 9.0 g (43 % yield) of 7a as a
white solid. The enantiomers were separated by chiral prep. SFC
[Chiralcel OD-H column: 3 x 25 cm, 5µm; Pressure 100 bars;
Temperature 35 ºC; Mobile phase: 88:12 CO2:MeOH; Flow rate:
120 mL/min; UV detection: 220 nm] which gave (S)-7a,
enantiomer A (>99% ee) and (R)-7a, enantiomer B (>98% ee).
HRMS m/z calc’d for C18H22ClN2O2 (M+H)+: 333.1364. Found
333.1366. LCMS: 333.0 (M+H)+ and 335.0 (M+2+H)+. 1H
NMR (400 MHz, CDCl3) δ: 8.46 (d, J = 5.5 Hz, 1H), 7.26 - 7.15
(m, 4H), 7.04 - 6.89 (m, 3H), 5.63 - 5.53 (m, 1H), 5.00 - 4.91 (m,
1H), 3.22 - 3.12 (m, 1H), 3.08 (dd, J = 13.2, 7.7 Hz, 1H), 1.42
(bs, 9H). 13C NMR (125 MHz, CDCl3) δ: 161.52, 155.09,
150.02, 144.11, 136.96, 129.37, 128.28, 126.56, 122.58, 122.52,
4.1.12. (2E)‐3‐[5‐Chloro‐2‐(1H‐1,2,3,4‐tetrazol‐1‐yl)phenyl]-
prop‐2‐enoic acid (7c).
To a cooled (0 °C) suspension of NaH (0.262 g, 6.56 mmol) in
THF (27.3 mL) was added drop wise methyl
2‐(dimethoxyphosphoryl)acetate (1.15 mL, 7.10 mmol). The
resulting thick, white suspension was diluted with additional
THF (15 mL) to facilitate mixing, and then the reaction was
allowed to warm to rt. After 45 min, a slightly cloudy blue
solution of 5‐chloro‐2‐(1H‐1,2,3,4‐tetrazol‐1‐yl)benzaldehyde39
(1.14 g, 5.46 mmol) in THF (8 mL) was added. The yellow-
green suspension was stirred vigorously. After 30 min, the
reaction was poured into cold sat. NH4Cl and extracted with
EtOAc. The organic layers were combined and washed with
brine, dried over Na2SO4, filtered and concentrated to give a
green-blue solid (1.76 g). The solid was dissolved in EtOAc and
filtered through a plug of silica gel and eluted with EtOAc. The
filtrate was concentrated to give a greenish solid (1.36 g).
Recrystallization from EtOAc gave an off-white solid (0.476 g).
Additional product was obtained by concentrating the filtrate,
adding methanol, sonicating, and collecting the resulting solid by
79.51, 56.58, 42.42, 28.31. [α]D25 = -5.73 (c = 1.0; CHCl3).
4.1.11. Methyl N‐(4‐{2‐[(1S)‐1‐amino‐2‐phenylethyl]pyridin‐
4‐yl}phenyl)carbamate [(S)-7b].
filtration.
A total of 0.829 g (57% yield) of methyl
(2E)‐3‐[5‐chloro‐2‐(1H‐1,2,3,4‐tetrazol‐1‐yl)phenyl]prop‐2‐

enoate was obtained. LCMS: 265.1 (M+H)+; 287.2 (M+Na)+.
1H NMR (500 MHz, CDCl3) δ: 8.80 (s, 1H), 7.78 (d, J = 2.2 Hz,
1H), 7.58 (dd, J = 8.8, 2.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.25
(d, J = 16.0 Hz, 1H), 6.45 (d, J = 16.0 Hz, 1H), 3.78 (s, 3H).
1H), 3.12 (dd, J = 13.7, 6.6 Hz, 1H), 3.00 (dd, J = 13.7, 8.2 Hz,
1H), 1.17 (s, 9H).
4.1.15. Methyl N‐(4‐{4‐[(1S)‐1‐amino‐2‐phenylethyl]pyridin‐
2‐yl}phenyl)carbamate, 2HCl [(S)-7g].
To
a white suspension of methyl (2E)‐3‐[5‐chloro‐2-
To a flame-dried RBF was added (R,S)-7f (0.060 g, 0.157
mmol), 4-(methoxy-carbonylamino)phenylboronic acid (0.046 g,
0.236 mmol), Cs2CO3 (0.077 g, 0.236 mmol), Pd2dba3 (0.0072 g,
0.0079 mmol), and tri-tert-butylphosphine tetrafluoroborate
(0.0055 g, 0.019 mmol). The flask was purged with argon for
several minutes and then degassed 1,4-dioxane (2 mL) was
added. The reaction mixture was stirred at rt for 1 h and then
warmed to 90 °C. After 16 h the reaction was stopped and
cooled to rt. The suspension was filtered and the filtrate was
concentrated. Column chromatography on silica gel (gradient
elution 0-50% EtOAc:Hex) gave, 0.040 g (56% yield) of methyl
N‐(4‐{4‐[(1S)‐1‐{[(R)‐2‐methylpropane‐2‐sulfinyl]amino}‐2‐phe
(1H‐1,2,3,4‐tetrazol‐1‐yl)phenyl]prop‐2‐enoate (0.140 g, 0.53
mmol) in MeOH (3.0 mL) was added 1.0 M NaOH (1.59 ml,
1.59 mmol). The resulting suspension was stirred vigorously at rt
for 2.5 h. The yellow suspension was neutralized with 1.0 N HCl
(1.60 mL), and concentrated to give a beige solid. The solid was
partitioned between 1.0 N HCl and EtOAc, and then the layers
were separated. The organic layer was washed with brine, dried
over Na2SO4, filtered and concentrated to give 0.137 g (100%
yield) of 7c as a white solid. LCMS: 251.1 (M+H)+. 1H NMR
(500 MHz, DMSO-d ) δ: 12.72 (s, 1H), 9.87 (s, 1H), 8.24 (d, J =
6
2.2 Hz, 1H), 7.77 (dd, J = 8.8, 2.2 Hz, 1H), 7.73 (d, J = 8.2 Hz,
1H), 6.98 (d, J = 16.0 Hz, 1H), 6.70 (d, J = 16.0 Hz, 1H).
nylethyl]pyridin‐2‐yl}phenyl)carbamate, as
a brown solid.
LCMS: 452.1 (M+H)+. 1H NMR (400 MHz, CDCl3) δ: 8.60 (d,
J=4.9 Hz, 1H), 7.92 - 7.87 (m, 2H), 7.55 (s, 1H), 7.50 (d, J=8.8
Hz, 2H), 7.33 - 7.22 (m, 3H), 7.13 - 7.08 (m, 3H), 6.89 (s, 1H),
4.74 - 4.68 (m, 1H), 3.79 (s, 3H), 3.61 (d, J=2.2 Hz, 1H), 3.14
(dd, J=13.7, 6.0 Hz, 1H), 3.05 (dd, J=13.7, 8.2 Hz, 1H), 1.18 (s,
9H).
4.1.13. 2,5‐Dioxopyrrolidin‐1‐yl (2E)‐3‐[5‐chloro‐2‐(1H‐
1,2,3,4‐tetrazol‐1‐yl)phenyl]prop‐ 2‐enoate (7d).
To a solution of 7c (5.00 g, 19.95 mmol) in THF (100 mL) and
DMF (10 mL) was added 1‐hydroxypyrrolidine‐2,5‐dione (2.53
g, 21.94 mmol) and DIC (3.42 mL, 21.94 mmol). The reaction
was stirred overnight. The white precipitate that formed was
collected by filtration, washed with methanol and water, and then
dried under vacuum to give 7.0 g (100% yield) of 7d as a white
solid. LCMS: 348.0 (M+H)+. 1H NMR (400 MHz, acetone-d6)
δ: 2.80 (s, 4H), 6.94 (d, J = 15.82 Hz, 1H), 7.45 (d, J = 15.82 Hz,
1H), 7.69-7.76 (m, J = 8.85, 2.2 Hz, 2H), 8.23 (d, J = 2.2 Hz,
1H), 9.52 (s, 1 H).
To a solution of methyl N‐(4‐{4‐[(1S)‐1‐{[(R)‐2‐methyl-
propane‐2‐sulfinyl]amino}‐2‐phenylethyl]pyridin‐2‐
yl}phenyl)carbamate (0.040 g, 0.089 mmol) in MeOH (1 mL)
was added 4M HCl in dioxane (1 mL). The reaction mixture was
stirred at rt. After 30 min, the reaction was concentrated to near
dryness. Diethyl ether was added to give a yellow suspension.
The solid was collected by filtration. The solid was rinsed with
diethyl ether and dried to give 0.029 g (78% yield) of (S)-7g as a
yellow solid. LCMS: 348.1 (M+H)+. 1H NMR (400 MHz,
CD3OD) δ: 8.78 (d, J = 6.0 Hz, 1H), 8.34 (d, J = 1.1 Hz, 1H),
7.93 - 7.86 (m, 3H), 7.77 (d, J = 8.8 Hz, 2H), 7.36 - 7.23 (m, 5H),
5.02 (t, J = 7.7 Hz, 1H), 3.78 (s, 3H), 3.50 (dd, J = 13.7, 6.6 Hz,
1H), 3.37 - 3.32 (m, 1H) partial overlap with CD3OD.
4.1.14. (R)‐N‐[(1S)‐1‐(2‐Bromopyridin‐4‐yl)‐2‐phenylethyl]‐
2‐methylpropane‐2‐sulfinamide [(R,S)-7f].
To a solution of 2‐bromopyridine‐4‐carbaldehyde (7e, 1 g,
5.38 mmol) and Ti(OEt)4 (5.64 mL, 26.9 mmol) in CH2Cl2 (10.75
mL) was added (R)-2-methylpropane-2-sulfinamide (0.717 g,
5.91 mmol). The reaction was stirred overnight at rt. The
reaction mixture was poured into a rapidly stirred mixture of
brine. The resulting suspension was filtered through a plug of
Celite® and the filter cake was washed with CH2Cl2 to give a
biphasic filtrate. The layers were separated. The organic layer
was washed with brine, dried over MgSO4, filtered and
concentrated. Column chromatography on silica gel (gradient
elution 0-30% EtOAc/Hex) gave 0.92 g (59% yield) of
(R)‐N‐[(1E)‐(2‐bromopyridin‐4‐yl)methylidene]‐2‐methylpropan
e‐2‐sulfinamide as a yellow oil. LCMS: 288.9 (M+H)+ and 290.9
(M+2+H)+. 1H NMR (400 MHz, CDCl3) δ: 8.56 - 8.52 (m, 2H),
7.89 (s, 1H), 7.64 (dd, J = 4.9, 1.6 Hz, 1H), 1.29 (s, 9H).
4.1.16. trans-N-{(1S)-1-[4-(3-Amino-1H-indazol-6-yl)-2-
pyridinyl]-2-phenylethyl}-4-(aminomethyl)cyclohexane-
carboxamide, bis-trifluoroacetic acid salt [(S)-2].
A suspension of (S)-2b (0.095 g, 0.138 mmol) in n-butanol
(3.0 mL) and hydrazine monohydrate (1.0 mL) was microwaved
at 150 °C for 10 min. The resulting clear, bright yellow solution
was concentrated. Purification by reverse phase chromatography
gave 0.069 g (71% yield) of (S)-2, as a yellow solid. Analytical
HPLC Purity, 100%. HRMS m/z calc’d for C28H33N6O (M+H)+:
469.2716. Found 469.2705. 1H NMR (500 MHz, CD3OD) δ:
8.69 (d, J = 5.5 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.92 (dd, J =
5.8, 1.9 Hz, 1H), 7.85 (bs, 1H), 7.75 (bs, 1H), 7.49 (dd, J = 8.5,
1.4 Hz, 1H), 7.28-7.25 (m, 2H), 7.22-7.19 (m, 3H), 5.35 (t, J =
7.7 Hz, 1H), 3.25 (d, J = 8.2 Hz, 2H), 2.77 (d, J = 6.6 Hz, 2H),
2.30-2.25 (m, 1H), 1.86-1.74 (m, 4H), 1.59-1.54 (m, 1H), 1.43-
1.33 (m, 2H), 1.10-1.01 (m, 2H). 13C NMR (100 MHz, CD3OD)
δ: 178.6, 162.3 (q, J = 34.9 Hz), 160.3, 155.8, 148.7, 145.8,
144.2, 139.9, 137.9, 130.4, 129.7, 128.2, 123.84, 123.75, 123.4,
To
a
cooled
(-78
ºC)
solution
of
(R)‐N‐[(1E)‐(2‐bromopyridin‐4‐yl)methylidene]‐2‐methyl-
propane‐2‐sulfinamide (0.686 g, 2.37 mmol) in 1:1 Et2O:CH2Cl2
(20 mL) was added 1.0 M benzylmagnesium bromide in THF
(2.61 mL, 2.61 mmol). The reaction was allowed to slowly warm
to rt and the reaction was stirred overnight. The reaction was
cooled to 0 ºC and additional 1.0 M benzylmagnesium bromide
in THF (0.52 mL) was added. After 2 h, the reaction was
quenched with sat. NH4Cl and diluted with EtOAc. The layers
were separated. The organic layer was washed with brine, dried
over MgSO4, filtered and concentrated. Column chromatography
on silica gel (gradient elution 0-50% EtOAc/Hex) gave 0.370 g
(41% yield) of (R,S)-7f as a brown oil and 0.273 g (30% yield) of
(R,R)-7f as an off-white solid. LCMS: 380.9 (M+H)+ and 382.9
(M+2+H)+. 1H NMR (400 MHz, CDCl3) δ: 8.30 (d, J = 4.9 Hz,
1H), 7.40 (s, 1H), 7.33 - 7.23 (m, 3H), 7.12 (dd, J = 4.9, 1.6 Hz,
1H), 7.09 - 7.05 (m, 2H), 4.66 - 4.60 (m, 1H), 3.58 (d, J = 2.2 Hz,
121.2, 117.8 (q, J = 289.6 Hz), 115.1, 111.6, 56.2, 46.2, 45.3,
25.2
41.6, 36.8, 30.3, 30.2, 29.8, 29.4. [α]D
= +5.52 (c = 0.80;
MeOH).
4.1.17. trans-4-(Aminomethyl)-N-{(1S)-1-[4-(3-hydroxy-1H-
indazol-5-yl)-2-pyridinyl]-2-phenylethyl}cyclohexane-
carboxamide, bis-trifluoroacetic acid salt [(S)-10].
Compound (S)-1c was converted to (S)-2f using Suzuki-
Miyaura coupling method
B with 5‐(dihydroxyboranyl)‐2‐
fluorobenzoic acid followed by Boc-deprotection. Compound

(S)-2f was converted to (S)-10 (0.150 g, 72% yield over three
steps, yellow solid) according to the procedure described in (S)-2,
with the exception that the reaction was microwaved at 160 °C
for 1 h. Analytical HPLC Purity, 100%. HRMS m/z calc’d for
C28H32N5O2 (M + H)+: 470.2556. Found 470.2544. 1H NMR
(500 MHz, CD3OD) δ: 8.58 (d, J = 5.5 Hz, 1H), 8.27 (s, 1H),
8.08 - 8.04 (m, 2H), 7.92 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.8 Hz,
1H), 7.31 - 7.25 (m, 2H), 7.25 - 7.19 (m, 3H), 5.34 (t, J = 8.0 Hz,
1H), 3.31 - 3.25 (m, 2H) overlap with CD3OD, 2.77 (d, J = 7.1
Hz, 2H), 2.34 - 2.24 (m, 1H), 1.90 - 1.80 (m, 3H), 1.80 - 1.72 (m,
1H), 1.63 - 1.51 (m, 1H), 1.45 - 1.31 (m, 2H), 1.13 - 0.99 (m,
2H). 13C NMR (125 MHz, CD3OD) δ: 179.0, 160.7, 159.2,
158.4, 146.1, 142.4, 137.3, 130.5, 129.9, 129.7, 128.5, 127.2,
124.0, 123.2, 122.6, 115.7, 113.4, 55.4, 46.3, 45.3, 41.2, 36.8,
30.4, 30.3, 29.8, 29.4. [α]D24.8 = + 1.79 (c = 1.41, MeOH).
DMF/MeOH mixture.
Purification by reverse phase
chromatography gave, after concentration and lyophilization,
0.141 g (71% yield) of (S)-24 as a fluffy, white solid. Analytical
HPLC Purity, 99.4%. HRMS m/z calc’d for C31H27ClN7O3 (M +
H)+: 580.1858. Found 580.1876. LCMS m/z 580.3 (M+H)+ and
582 (M+2+H)+. 1H NMR (500 MHz, DMSO-d6) δ: 9.97 (s, 1H),
9.84 (s, 1H), 8.86 (d, J = 8.2 Hz, 1H), 8.63 (d, J = 5.5 Hz, 1H),
7.97 (d, J = 2.2 Hz, 1H), 7.82 - 7.76 (m, 4H), 7.75 (dd, J = 8.8,
2.2 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H),
7.28 - 7.23 (m, 2H), 7.22 - 7.15 (m, 3H), 6.86 (d, J = 15.4 Hz,
1H), 6.81 (d, J = 15.4 Hz, 1H), 5.34 - 5.28 (m, 1H), 3.70 (s, 3H),
3.20 (dd, J = 13.7, 6.0 Hz, 1H), 3.12 (dd, J = 13.7, 8.8 Hz, 1H).
13C NMR (125 MHz, DMSO-d6) δ: 163.7, 159.4, 153.9, 150.4
(bs), 146.2 (bs), 145.2, 141.5, 137.5, 135.8, 132.9, 130.95,
130.92, 130.2, 129.4, 129.2, 129.0, 128.2, 128.0, 127.4, 127.0,
23.6
126.5, 120.1, 119.1, 118.4, 54.8, 51.8, 40.4. [α]D = 9.82 (c
=1.16, DMSO). Anal. calcd for C31H26ClN7O3 ⋅ 0.94 H2O ⋅ 0.92
C2HF3O2: C, 56.20; H, 4.14; N, 13.97; F, 7.47; Cl, 5.05. Found:
C, 56.50; H, 4.25; N, 13.78; F, 7.63; Cl, 4.96.
4.1.18. Methyl (4-{2-[(1S)-1-({[trans-4-(aminomethyl)-
cyclohexyl]0carbonyl}amino)-2-phenylethyl]-4-pyridinyl}-
phenyl)carbamate, bis-trifluoroacetic acid salt [(S)-17].
Compound (S)-1c was converted to (S)-4f using Suzuki-
Miyaura coupling method A with 4-(methoxy-carbonylamino)-
phenylboronic acid. Compound (S)-4f was converted to (S)-17
according to the Boc-deprotection procedure which gave 0.125 g
(87% yield over two steps) of (S)-17 as a white solid. Analytical
HPLC Purity, 100%. HRMS m/z calc’d for C29H35N4O3
487.2709 (M+H)+: Found 487.2706. 1H NMR (500 MHz,
CD3OD) δ: 8.57 (d, J = 6.0 Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H),
7.98 (dd, J = 6.0, 1.6 Hz, 1H), 7.82 - 7.77 (m, 2H), 7.69 - 7.64
(m, 2H), 7.29 - 7.24 (m, 2H), 7.24 - 7.19 (m, 3H), 5.35 (t, J = 8.0
Hz, 1H), 3.77 (s, 3H), 3.26 (d, J = 8.2 Hz, 2H), 2.77 (d, J = 7.1
Hz, 2H), 2.35 - 2.25 (m, 1H), 1.91 - 1.80 (m, 3H), 1.79 - 1.71 (m,
1H), 1.63 - 1.51 (m, 1H), 1.44 - 1.31 (m, 2H), 1.13 - 0.99 (m,
2H). 13C NMR (125 MHz, CD3OD) δ: 178.5, 159.8, 156.1,
155.5 (bs), 145.0 (bs), 143.7, 137.9, 130.5, 130.4, 129.6, 129.5,
128.0, 122.2, 121.4, 120.0, 56.0, 52.7, 46.1, 45.3, 41.5, 36.7,
30.3, 30.2, 29.7, 29.4. [α]D22.3 = -4.98 (c = 1.02, MeOH).
4.1.21. Methyl N‐(4‐{4‐[(1S)‐1‐[(2E)‐3‐[5‐chloro‐2‐(1H‐
1,2,3,4‐tetrazol‐1‐yl)phenyl]prop‐2‐enamido]‐2‐phenylethyl]-
pyridin‐2‐yl}phenyl)carbamate, trifluoroacetic acid salt [(S)-
25].
To a solution of (S)-7g (0.029 g, 0.069 mmol) in DMF (1 mL)
was added (2E)‐3‐[5‐chloro‐2‐(1H‐1,2,3,4‐tetrazol‐1‐yl)phenyl]-
prop‐2‐enoic acid (7c, 0.017 g, 0.069 mmol), EDC (0.026 g, 0.14
mmol), HOBt (0.021 g, 0.14 mmol) and TEA (0.048 mL, 0.34
mmol). The reaction mixture was stirred at rt. After 18 h, the
reaction was diluted with MeOH (3 mL). Purification by reverse
phase chromatography gave, after concentration and
lyophilization, 0.036 g (75 % yield) of (S)-25 as an off-white
solid. Analytical HPLC Purity, 99.5%. LCMS: 580.0 (M+H)+.
1H NMR (400 MHz, CD3OD) δ: 9.75 (s, 1H), 9.48 (s, 1H), 9.09 -
9.05 (m, 1H), 8.62 (d, J = 6.6 Hz, 1H), 8.02 (s, 1H), 7.97 (d, J =
2.2 Hz, 1H), 7.80 - 7.71 (m, 5H), 7.66 (dd, J = 8.8, 2.2 Hz, 1H),
7.56 (d, J = 8.2 Hz, 1H), 7.32 - 7.21 (m, 5H), 7.07 (d, J = 15.4
Hz, 1H), 6.73 (d, J = 15.9 Hz, 1H), 5.44 - 5.37 (m, 1H), 3.78 (s,
3H), 3.30 - 3.24 (m, 1H) partial overlap with CD3OD, 3.19 (dd, J
= 13.7, 7.1 Hz, 1H).
4.1.19. Methyl (4-{6-[(1S)-1-({[trans-4-(aminomethyl)-
cyclohexyl]carbonyl}amino)-2-phenylethyl]-4-pyrimidinyl}-
phenyl)carbamate, bis-trifluoroacetic acid salt [(S)-21].
Compound (S)-6d was converted to (S)-21 according to the
procedure (S)-1c, followed by Boc-deprotection which gave
0.034 g (37% yield over two steps) of (S)-21 as a pale, yellow
solid. Analytical HPLC Purity, 99.7%. HRMS m/z calc’d for
C28H34N5O3 (M+H)+: 488.2662. Found 488.2668. 1H NMR
4.1.22. Methyl N‐(4‐{6‐[(1S)‐1‐[(2E)‐3‐[5‐chloro‐2‐(1H‐
1,2,3,4‐tetrazol‐1‐yl)phenyl]prop‐ 2‐enamido]‐2‐phenylethyl]-
pyrimidin‐4‐yl}phenyl)carbamate, trifluoroacetic acid salt
[(S)-26].
To a suspension of (S)-6d (0.035 g, 0.076 mmol) and
2,5‐dioxopyrrolidin‐1‐yl (2E)‐3‐[5‐chloro‐2‐(1H‐1,2,3,4‐tetrazol‐
1‐yl)phenyl]prop‐2‐enoate (7d, 0.026 g, 0.076 mmol) in DMF
(0.757 mL) was added Hunig's base (0.066 mL, 0.378 mmol).
The resulting clear, slightly yellow solution was stirred at rt.
After 3 h, the reaction was partitioned between EtOAc/water and
the layers were separated. The aqueous layer was extracted with
EtOAc (1x). The organic layers were combined and washed with
sat. NaHCO3, brine, dried over Na2SO4, filtered and concentrated
(400MHz, CD3OD) δ: 9.55 (s, 1H), 9.09 (d, J = 1.1 Hz, 1H),
8.42 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.68 (s, 1H),
7.60 (d, J = 8.8 Hz, 2H), 7.28 - 7.16 (m, 5H), 5.29 - 5.21 (m, 1H),
3.76 (s, 3H), 3.26 (dd, J = 13.7, 6.0 Hz, 1H), 3.08 (dd, J = 13.7,
8.8 Hz, 1H), 2.77 (d, J = 7.1 Hz, 2H), 2.31 - 2.21 (m, 1H), 1.89 -
1.80 (m, 3H), 1.75 - 1.67 (m, 1H), 1.63 - 1.49 (m, 1H), 1.48 -
1.30 (m, 2H), 1.12 - 0.99 (m, 2H). 13C NMR (125 MHz,
CD3OD) δ: 178.3, 171.2, 165.4, 159.7, 156.3, 143.7, 138.9,
131.8, 130.5, 129.6, 129.3, 127.9, 119.7, 115.3, 57.1, 52.8, 46.3,
45.6, 41.7, 36.9, 30.5, 30.4, 29.9, 29.6. [α]D24.4 = -17.8 (c = 0.87,
MeOH).
to give a greenish solid.
Purification by reverse phase
chromatography gave, after concentration and lyophilization,
0.0281 g (64 % yield) of (S)-26 as a pale, yellow solid.
Analytical HPLC Purity, 99.5%. LCMS: 581.1 (M+H)+. 1H
NMR (400 MHz, DMSO-d6) δ: 10.02 (s, 1H), 9.85 (s, 1H), 9.14
(s, 1H), 8.84 (d, J = 8.3 Hz, 1H), 8.11 (d, J = 8.8 Hz, 2H), 7.98
(d, J = 2.2 Hz, 1H), 7.91 (s, 1H), 7.75 (dd, J = 8.3, 1.8 Hz, 1H),
7.71 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.29 - 7.15 (m,
5H), 6.87 (d, J = 15.8 Hz, 1H), 6.81 (d, J = 15.8 Hz, 1H), 5.26 -
5.17 (m, 1H), 3.69 (s, 3H), 3.18 (dd, J = 13.6, 5.7 Hz, 1H), 3.05
(dd, J = 13.6, 9.2 Hz, 1H).
4.1.20. Methyl N‐(4‐{2‐[(1S)‐1‐[(2E)‐3‐[5‐chloro‐2‐(1H‐
1,2,3,4‐tetrazol‐1‐yl)phenyl]prop‐ 2‐enamido]‐2‐phenylethyl]-
pyridin‐4‐yl}phenyl)carbamate, trifluoroacetic acid [(S)-24].
To a flask containing (S)-7b (0.100 g, 0.29 mmol) and
2,5‐dioxopyrrolidin‐1‐yl (2E)‐3‐[5‐chloro‐2‐(1H‐1,2,3,4‐tetrazol‐
1‐yl)phenyl]prop‐2‐enoate (7d, 0.100 g, 0.29 mmol) was added
DMF (1.9 mL) and Hunig's base (0.151 mL, 0.86 mmol). The
resulting clear, yellow solution was stirred at rt for 1 h. The
reaction was diluted with MeOH (1 mL) to give a 70%

4.2 X-Ray Crystallography
17-ID at the Advanced Photon Source was supported by the
companies of the Industrial Macromolecular Crystallography
Association through a contract with the Illinois Institute of
Technology. Use of the Advanced Photon Source was supported
by the U. S. Department of Energy, Office of Science, Office of
Basic Energy Sciences, under Contract No. DE-AC02-
06CH11357.
X-ray crystal structure data collection and structure
refinement (see Supplemental Information Tabulated
Parameters). Protein for Factor XIa crystals in complex with
inhibitors (S)-10 and (S)-17 was prepared as previously
described.16 Data for these crystals were collected in the
laboratory.
Raw data were processed with the program
HKL2000.40 The atomic coordinates of human Factor XIa (PDB
ID 4NA8) were used as a search model for rigid body only
refinement in AmoRe.41-43 Original refinement for compounds
was carried out with CNX (Accelrys) and inhibitor restraint
dictionaries were built with QUANTA (Accelrys), which was
also used for modeling. Later the structures were re-refined
using BUSTER/TNT44 (GlobalPhasing, Ltd.), MakeTNT
(GlobalPhasing, Ltd., Cambridge, UK) for inhibitor restraint
dictionaries, and COOT44 for modeling. Protein for Factor XIa in
complex with inhibitor (S)-24 was purchased from Proteros
Supplementary Material
Analytical data and experimental procedures for intermediates
1d-1g, 2g, 2h, 3c, 3e, 4a, 4g, 4h, (S)-5a, (S)-5b, 5d, (S)-5e, (S)-
5f, 5h, 5i, (S)-5j, 8b, and (S)-8d; Analytical data and
experimental procedures for final compounds 3-4, (S)-5, 6-9, 11-
16, (S)-18 to (S)-20, (S)-22, and (S)-27; and Crystallographic
data and refinement statistics for X-ray structures of the FXIa
complexes with compounds (S)-10, (S)-17, and (S)-24.
biostructures GmbH (Martinsried, Germany).
Data were
References and notes
collected at the Advanced Photon Source (APS) beamline 17-ID
and processed with XDS46,47 and AIMLESS48 through the use of
autoPROC (GlobalPhasing, Ltd., Cambridge, UK). The atomic
coordinates of human Factor XIa derived from PDB ID 3SOR
were used as search model for rigid body only refinement in
AmoRe.41-43 The structure was refined with BUSTER/TNT44
(GlobalPhasing, Ltd.), GRADE (GlobalPhasing, Ltd.,
Cambridge, UK) for inhibitor restraint dictionaries, and COOT45
for modeling. The PDB deposition numbers for compounds (S)-
10, (S)-17, and (S)-24 complexed to FXIa are 5EXL, 5EXM, and
5EXN, respectively.
1. Bane, C.E.; Gailani, D. Drug Discovery Today 2014, 19 (9), 1454-
1458.
2. Bane, C.E.; Neff, A.T.; Gailani, D. In Hemostasis and
Thrombosis: Practical Guidelines in Clinical Management; Saba,
H.I., Roberts, H.R., Eds.; John Wiley & Sons, Ltd.: Oxford, 2014;
pp 71-81.
3. (a) Salomon, O.; Steinberg, D.M.; Koren-Morag, N.; Tanne, D.;
Seligsohn, U. Blood 2008, 111 (8), 4113-4117. (b) Salomon, O.;
Steinberg, D.M.; Dardik, R.; Rosenberg, N.; Zivelin, A.; Tamarin,
I.; Ravid, B.; Berliner, S.; Seligsohn, U. J. Thromb. Haemost.
2003, 1, 658-661. (c) Salomon, O.; Steinberg, D.M.; Zucker, M.;
Varon, D.; Zivelin, A.; Seligsohn, U. Thromb. Haemost. 2011,
105, 269-273.
4. (a) Doggen, C.J.M; Rosendaal, F.R.; Meijers, J.C.M. Blood 2006,
108 (13), 4045-4051. (b) Meijers, J.C.M.; Tekelenburg, W.L.H.;
Bouma, B.N.; Bertina, R.M.; Rosendaal, F.R. N. Eng. J. Med.
2000, 342(10), 696-701.
5. (a) Wang, X.; Smith, P.L.; Hsu, M-Y.; Gailani, D.; Schumacher,
W.A.; Ogletree, M.L.; Seiffert, D.A. J. Thromb. Haemost. 2006, 4,
1982-1988. (b) Wang, X.; Cheng, Q.; Xu. L.; Feuerstein, G.Z.;
Hsu, M-Y.; Smith, P.L.; Seiffert, D.A.; Schumacher, W.A.;
Ogletree, M.L.; Gailani, D. J. Thromb. Haemost. 2005, 3, 695-
702. (c) Rosen, E.D.; Gailani, D.; Castellino, F.J. Thromb.
Haemost. 2002, 87, 774-776.
6. (a)Yamashita, A.; Nishihira, K.; Kitazawa, T.; Yoshihashi, K.;
Soeda, T.; Esaki, K.; Imamura, T.; Hattori, K.; Asada, Y. J.
Thromb. Haemost. 2006, 4, 1496-1501. (b) Tucker, E.I.; Marzec,
U.M.; White, T.C.; Hurst, S.; Rugonyi, S.; McCarty, O.J.T.;
Gailani, D.; Gruber, A.; Hanson, S.R. Blood 2009, 113 (4), 936-
944. (c) Gruber, A.; Hanson, S.R. Blood 2003, 102 (3), 953-955.
7. (a) Crosby, J.R.; Marzec, U.; Revenko, A.S.; Zhao, C.; Gao, D.;
Matafonov, A.; Gailani, D.; MacLeod, A.R.; Tucker, E.I.; Gruber,
A.; Hanson, S.; Monia, B.P. Arterioscler. Thromb. Vasc. Biol.
2013, 33, 1670-1678. (b) Zhang, H.; Lowenberg, E.C.; Crosby,
J.R.; MacLeod, A.R.; Zhao, C.; Gao, D.; Black, C.; Revenko,
A.S.; Meijers, J.C.M., Stroes, E.S.; Levi, M.; Monia, B.P. Blood
2010, 116, 4684-4692.
4.3 Enzyme and Coagulation Assays
The affinity of compounds for various enzymes was
determined using purified enzymes and synthetic peptide
substrates from commercial sources. The IC50 values were
−11 inhibitor concentrations assayed in duplicate
obtained over 4
at a fixed substrate concentration with a standard error of <20%.
Ki values were calculated using the relationship Ki equals IC50
divided by (1+S/Km), assuming competitive inhibition, where S
is the concentration of substrate, and Km is the
−Menten constant for the substrate.
Michaelis
The aPTT (activated partial thromboplastin time) in vitro
clotting assay was performed in an automated coagulation
analyzer using pooled normal human plasma and reagents from
commercial sources. The reported EC1.5x values are the FXIa
inhibitor plasma concentrations which produce a 50% increase in
the clotting time relative to the clotting time in the absence of the
inhibitor.
Please see reference 15b for a complete description of the in
vitro enzyme and plasma coagulation assays.
8. Buller, H.R.; Bethune, C.; Bhanot, S.; Gailani, D.; Monia, B.P.;
Raskob, G.E.; Segers, A.; Verhamme, P.; Weitz, J.I. N. Engl. J.
Med. 2015, 372, 232-240. The FXI ASO work published
subsequent to the work described herein.
9.
(a)Schumacher, W.A.; Seiler, S.E.; Steinbacher, T.E.; Stewart,
A.B.; Bostwick, J.S.; Hartl, K.S.; Liu, E.C.; Ogletree, M.L. Eur. J.
Pharmacol. 2007, 570, 167-174. (b) Wong, P.C.; Crain, E.J.;
Watson, C.A.; Schumacher, W.A. J. Thromb. Thrombolysis 2011,
32, 129-137.
Acknowledgments
The authors would like to thank Dauh-Rurng Wu, Leslie W.
Leith, Peng Li, Atsu Apedo, and Douglas B. Moore for chiral
separations of intermediates; the Department of Discovery
Synthesis and BMS Biocon Research Center for the preparation
of synthetic intermediates; the Department of Lead Discovery &
Optimization. Jeffrey M. Bozarth and Frank A. Barbera for in
vitro work; Mei-Mann Hsueh, Gang Lou, Zhen Lou, Qinling Qu,
and Baomin Xin for PK studies and analyses; William Ewing for
proof reading the manuscript. Use of the IMCA-CAT beamline
10. (a) Deng, H.; Bannister, T.D.; Jin, L.; Babine, R.E.; Quinn, J.;
Nagafuji, P.; Celatka, C.A.; Lin, J.; Lazarova, T.I.; Rynkiewicz,
M.J.; Bibbins, F.; Pandey, P.; Gorga, J.; Meyers, H.V.; Abdel-
Meguid, S.S.; Strickler, J.E. Bioorg. Med. Chem. Lett. 2006, 16,
3049-3054. (b) Lin, J.; Deng, H.; Jin, L.; Pandey, P.; Quinn, J.;
Cantin, S.; Rynkiewicz, M.J.; Gorga, J.C.; Bibbins, F.; Celatka,
C.A.; Nagafuji, P.; Bannister, T.D.; Meyers, H.V.; Babine, R.E.;
Hayward, N.J.; Weaver, D.; Benjamin, H.; Stassen, F.; Abdel-
Meguid, S.S.; Strickler, J.E. J. Med. Chem. 2006, 49, 7781-7791.

11. Lazarova, T.I.; Jin, L.; Rynkiewicz, M.; Gorga, J.C.; Bibbins, F.;
Meyers, H.V.; Babine, R.; Strickler, J. Bioorg. Med. Chem. Lett.
2006, 16, 5022-5027.
12. Buchanan, M.S.; Carroll, A.R.; Wessling, D.; Jobling, M.; Avery,
V.M.; Davis, R.A.; Feng, Y.; Xue, Y.; Oster, L.; Fex, T.; Deinum,
J.; Hooper, J.N.A.; Quinn, R.J. J. Med. Chem. 2008, 51, 3583-
3587.
13. It is not clear whether the macrocyclic indole from Hannesian is
an irreversible inhibitor, however, it was only weakly active
against FXIa (FXIa Ki = 51.6 µM), please see. Hanessian, S.;
Larsson, A.; Fex, T.; Knecht, W.; Blomberg, N. Bioorg. Med.
Chem. Lett. 2010, 20, 6925-6928.
14. Al-Horani, R.A.; Desai, U.R. J. Med. Chem. 2014, 57(11), 4805-
4818. (g) Sinha, D.; Badellino, K.O.; Marcinkiewicz, M.; Walsh,
P.N. Biochemistry 2004, 43, 7593-7600.
D.R.; Miller-Stein, C.; Pietrak, B.L.; Wallace, A.A.; White, R.B.;
Wong, B.; Yan, Y.; Nantermet, P.G. J. Med. Chem. 2004, 47,
2995-3008. The chlorophenyltetrazole moiety was first identified
in thrombin inhibitors and the authors propose that the tetrazole
ring interacts with the disulfide bridge via a donor atom-π
interaction.
26. Pinto, D. J. P.; Orwat, M. J.; Koch, S.; Rossi, K. A.; Alexander, R.
S.; Smallwood, A.; Wong, P. C.; Rendina, A. R.; Luettgen, J. M.;
Knabb, R. M.; He, K.; Xin, B.; Wexler, R. R.; Lam, P. Y. S. J.
Med. Chem. 2007, 50(22), 5339-5356.
27. The improvement in plasma kallikrein selectivity can be attributed
to the difference in an amino acid at position 143 in the S2 prime
pocket. Plasma kallikrein possesses a Phe143 whereas Factor XIa
contains Tyr143. The 3-hydroxyl indazole P2 prime moiety is not
as active in plasma kallikrein since it cannot form an H-bond to
Phe143.
15. (a) Wong, P. C.; Quan, M. L.; Watson, C.; Crain, E.; Wexler, R.
R.; Seiffert, D. Presented at the American Heart Association
Scientific Sessions, Dallas, TX, Nov. 2013; APS.709.03, Poster
#7050. (b) Quan, M.L.; Wong, P.C.; Wang, C.; Woerner, F.;
Smallheer, J.M.; Barbera, F.A.; Bozarth, J.M.; Brown, R.L.;
Harpel, M.R.; Luettgen, J.M.; Morin, P.E.; Peterson, T.;
Ramamurthy, V.; Rendina, A.R.; Rossi, K.A.; Watson, C.A.; Wei,
A.; Zhang, G.; Seiffert, D.; Wexler, R.R. J. Med. Chem. 2014,
57(3), 955-969. (c) Wong, P.C.; Quan, M.L.; Watson, C.A.;
Crain, E.J.; Harpel, M.R.; Rendina, A.R.; Luettgen, J.M.; Wexler,
R.R.; Schumacher, W.A.; Seiffert, D.A. J. Thromb.
Thrombolysis 2015, 40(4), 416-423.
16. Hangeland, J.J.; Friends, T.J.; Rossi, K.A.; Smallheer, J.M.; Wang,
C.; Sun, Z.; Corte, J.R.; Fang, T.; Wong, P.C.; Rendina, A.R.;
Barbera, F.A.; Bozarth, J.M.; Luettgen, J.M.; Watson, C.A.;
Zhang, G.; Wei, A.; Ramamurthy, V.; Morin, P.E.; Bisacchi, G.S.;
Subramaniam, S.; Arunachalam, P.; Mathur, A.; Seiffert, D.A.;
Wexler, R.R.; Quan, M.L. J. Med. Chem. 2014, 57, 9915-9932.
17. Pinto, D.J.P; Smallheer, J.M.; Corte, J.R.; Austin, E.J.D.; Wang,
C.; Fang, T., Smith, L.M.; Rossi, K.A.; Rendina, A.R.; Bozarth,
J.M.; Zhang, G.; Wei, A.; Ramamurthy, V.; Sheriff, S.; Myers,
J.E.; Morin, P.E.; Luettgen, J.M.; Seiffert, D.A.; Quan, M.L.;
Wexler, R.R. Bioorg. Med. Chem. Lett. 2015, 25, 1635-1642. It
should be noted that less potent FXIa inhibitors (FXIa Ki = 65-200
nM) were also shown to be orally bioavailable (%F = 26-49%),
however, imidazole (S)-23 provided the best combination of FXIa
Ki and %F.
18. Corte, J.R.; Fang, T.; Hangeland, J.J.; Friends, T.J.; Rendina, A.
R.; Luettgen, J.M.; Bozarth, J.M.; Barbera, F.A.; Rossi, K.A.;
Wei, A.; Ramamurthy, V.; Morin, P.E.; Seiffert, D.A.; Wexler,
R.R.; Quan, M.L. Bioorg. Med. Chem. Lett. 2015, 25, 925-930.
19. Baiocchi, L.; Corsi, G.; Palazzo, G. Synthesis 1978, 633-648. The
3-hydroxy indazole can also exist as the 3-oxo tautomer. If the 3-
oxo tautomer predominates, electrostatic repulsion exists between
the 3-oxo and the carbonyl of His 40 and/or the conserved water.
Similarly if the 3-hydroxy tautomer predominates, the hydroxyl
could maintain one H-bond, however, the oxygen lone pair would
undergo electrostatic repulsion with either the carbonyl of His40
or the water.
28. The improvement in trypsin selectivity can be attributed to the
difference in the size of the S1 pocket between trypsin and Factor
XIa. The S1 pocket of FXIa is slightly larger and more lipophilic
than trypsin
so it
can accommodate the larger
chlorophenyltetrazole P1. Please see, Colman, R. W.; Hirsch, J.;
Marder, V. J. Hemostasis and Thrombosis: Basic Principles and
Clinical Practice; Lippincott: Philadelphia, 1994.
29. Negi, S.; Matsukura, M.; Mizuno, M.; Miyake, K.; Minami, N.
Synthesis 1996, 991-996.
30. Each enantiomer was reacted with (S)-(+)-α-methoxyphenyl
acetic acid according to Trost (Trost, B.M. et al., J. Org. Chem.
1994, 59, 4202.) for determination of the absolute stereochemistry.
The absolute stereochemistry was confirmed by X-ray
crystallography of (S)-10, (S)-17, and (S)-24 bound to FXIa.
31. Schlosser, M.; Cottet, F. Eur. J. Org. Chem. 2002, 24, 4181-4184.
32. Palermo, M.G. Tetrahedron Lett. 1996, 37(17), 2885-2886.
33. Hart, D.J.; Kanai, K.; Thomas, D.G.; Yang, T-K. J. Org. Chem.
1983, 48(3), 289-294.
34. Maibaum, J.; Rich, D.H. J. Org. Chem. 1988, 53, 869-873.
35. Butters, M. J. Heterocycl. Chem. 1992, 29, 1369-1370.
36. Kuduk, S.D.; DiPardo, R.M.; Chang, R.K.; Ng, C.; Bock, M.G.
Tetrahedron Lett. 2004, 45, 6641-6643.
37. Rodriguez, A.C.; Ramos, A.P.; Hawkes, G.E.; Berti, F.; Resmini,
M. Tetrahedron: Asymmetry 2004, 15(12), 1847-1855.
38. Racemization of either the starting material or the intermediates to
the pyridinone occurred under the reaction conditions.
39. Howard, N.; Abell, C.; Blakemore, W.; Chessari, G.; Congreve,
M.; Howard, S.; Jhoti, H.; Murray, C.W.; Seavers, L.C.A.; van
Montfort, R.L.M. J. Med. Chem. 2006, 49, 1346-1355.
40. Otwinowski, Z.; Minor, W. Macromolecular Crystallography. Part
A; Carter, C.W., Sweet, R.M., Eds.; Methods in Enzymol. Vol.
276; Academic Press, Inc., San Diego, CA, 1997; pp 307-326.
41. Navaza, J. Acta Crystallogr. Sect. A 1994, 50, 157-163.
42. Navaza, J.; Vernoslova, E. Acta Crystallogr. Sect. A 1995, 51,
445-449.
43. Collaboration Computational Project, Number 4. The CCP4
Suite: programs for protein crystallography. Acta Crystallogr
Sect. D 1994, 50, 760-763.
20. The enantiomer (R)-5 had a FXIa Ki = 3,197 nM.
21. The presence of ethylene diol (EDO) is an artifact of the
crystallization process.
44. Blanc, E.; Roversi, P.; Vonrhein, C.; Flensburg, C.; Lea, S. M.;
Bricogne, G. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
60, 2210-2221.
22. Subsequent to our work, 1-acylindazol-3-ols were used as a
bioisostere for an anthranilic acid. See: Stiff, C.; Graber, D.R.;
Thorarensen, A.; Wakefield, B.D.; Marotti, K.R.; Melchior, E.P.;
Sweeney, M.T.; Han, F.; Rohrer, D.C.; Zurenko, G.E.; Romero,
D.L. Bioorg. Med. Chem. Lett. 2008, 18, 6293-6297.
23. The protein binding for (S)-2, (S)-5, (S)-10, and (S)-17 was not
determined. The protein binding for compounds (S)-24 to (S)-27
was not determined. However, based on unpublished data from
our Factor Xa and Factor XIa programs, protein binding does
impact the translation of Ki into the clotting assay. Specifically
poor translation into the clotting assay is at least in part due to
high protein binding.
45. Emsley, P.; Lokhamp, B.; Scott, W.G.; Cowtan, K. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486-501.
46. Kabsch, W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66,
125-132.
47. Kabsch, W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2010, 66,
133-144.
48. Evans, P.R.; Murshudov, G.N. Acta Crystallogr. Sect. D Biol.
Crystallogr. 2013, 69, 1204-1214.
24. An X-ray structure of (S)-22 bound to Factor XIa, unpublished
result, indicated that the carbonyl of the pyridinone scaffold was
picking up H-bonds with both Lys192 and Tyr143 through water
molecules and may explain the 2.5-fold increase in FXIa binding
relative to the pyridine (S)-17 scaffold. In addition, absolute
stereochemistry was confirmed to be (S).
25. Young, M.B.; Barrow, J.C.; Glass, K.L.; Lundell, G.F.; Newton,
C.L.; Pellicore, J.M.; Rittle, K.E.; Selnick, H.G.; Stauffer, K.J.;
Vacca, J.P.; Williams, P.D.; Bohn, D.; Clayton, F.C.; Cook, J.J.;
Kruger, J.A.; Kuo, L.C.; Lewis, S.D.; Lucas, B.J.; McMasters,

Graphical Abstract
Orally Bioavailable Pyridine and
Pyrimidine-Based Factor XIa Inhibitors:
Discovery of the Methyl N-Phenyl
Carbamate P2 Prime Group
James R. Corte∗, Tianan Fang, Donald J. P. Pinto, Michael J. Orwat, Alan R. Rendina, Joseph M. Luettgen,
Karen A. Rossi, Anzhi Wei, Vidhyashankar Ramamurthy, Joseph E. Myers Jr., Steven Sheriff, Rangaraj
Narayanan, Timothy W. Harper, Joanna J. Zheng, Yi-Xin Li, Dietmar A. Seiffert, Ruth R. Wexler, and Mimi L.
Quan
Bristol-Myers Squibb Company, Research and Development, P.O. Box 5400, Princeton, New Jersey 08543,
United States