Discovery of a High Affinity, Orally Bioavailable Macrocyclic FXIa Inhibitor with Antithrombotic Activity in Preclinical Species

Article abstract of DOI:10.1021/acs.jmedchem.0c00464

Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we report application of a previously disclosed cyclic carbamate P1 linker which provided improved oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency and good oral bioavailability but high in vivo clearance. Further structure-activity relationship (SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with selectivity against most of the relevant serine proteases. Compound 6f also demonstrated excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy model at doses which preserved hemostasis.

Full text of DOI:10.1021/acs.jmedchem.0c00464

Subscriber access provided by NATIONAL UNIV OF SINGAPORE
Article
Discovery of a High Affinity, Orally Bioavailable Macrocyclic
FXIa Inhibitor with Antithrombotic Activity in Preclinical Species
Wu Yang, Yufeng Wang, Amy Lai, Charles G. Clark, James R. Corte, Tianan Fang, Paul J. Gilligan,
Yoon Jeon, Kumar B Pabbisetty, Richard A. Rampulla, Arvind Mathur, Mahammed Kaspady, Premsai
Rai Neithnadka, Arunachalam Arumugam, Sivashankaran Raju, Karen A Rossi, Joseph E Myers, Steven
Sheriff, Zhen Lou, Joanna J. Zheng, Silvi A. Chacko, Jeffrey M. Bozarth, Yiming Wu, Earl J. Crain, Pancras
C Wong, Dietmar A Seiffert, Joseph M Luettgen, Patrick Y. S. Lam, Ruth R. Wexler, and William R. Eiwng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00464 • Publication Date (Web): 26 May 2020
Downloaded from pubs.acs.org on May 26, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Crain, Earl ; Bristol Myers Squibb Company, Research and Development
Wong, Pancras; Bristol Myers Squibb Company, Research and
Development
Seiffert, Dietmar; Bristol Myers Squibb Company, Research and
Development
Luettgen, Joseph; Bristol Myers Squibb Company, Research and
Development
Lam, Patrick; Bristol Myers Squibb Company, Research and Development
Wexler, Ruth; Bristol Myers Squibb Company, Research and
Development
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Eiwng, William; Bristol Myers Squibb Company, Research and
Development
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Discovery of a High Affinity, Orally Bioavailable
Macrocyclic FXIa Inhibitor with Antithrombotic
Activity in Preclinical Species
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a,
a
a
a
a
a
Wu Yang, * Yufeng Wang, Amy Lai, Charles G. Clark, James R. Corte, Tianan Fang, Paul
a
a
a
a
a
J. Gilligan, Yoon Jeon, Kumar B. Pabbisetty, Richard A. Rampulla, Arvind Mathur,
b
b
b
Mahammed Kaspady, Premsai Rai Neithnadka, Arunachalam Arumugam, Sivashankaran
b
a
a
a
a
a
Raju, Karen A. Rossi, Joseph E. Myers Jr., Steven Sheriff, Zhen Lou, Joanna J. Zheng, Silvi
a
a
a
a
a
A. Chacko, Jeffrey M. Bozarth, Yiming Wu, Earl J. Crain, Pancras C. Wong, Dietmar A.
a
a
a
a
a
Seiffert, Joseph M. Luettgen, Patrick Y. S. Lam, Ruth R. Wexler, William R. Ewing
aBristol Myers Squibb Company, Research and Development, P.O. Box 5400, Princeton, NJ
08540, USA
bBiocon Bristol Myers Squibb R&D Centre, Syngene International Ltd., Biocon Park, Plot No. 2
&
3, Bommasandra–Jigani Road, Bangalore 560100, India
*
Corresponding author e-mail: wu.yang@bms.com
KEYWORDS: Factor XIa inhibitors; FXIa; aPTT (Activated Partial Thromboplastin Time);
Macrocycles; Antithrombotic; Anticoagulant.
ABSTRACT:
Oral factor XIa (FXIa) inhibitors may provide a promising new antithrombotic therapy
with an improved benefit to bleeding risk profile over existing antithrombotic agents. Herein, we
report application of a previously disclosed cyclic carbamate P1 linker which provided improved
ACS Paragon Plus Environment
1

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
oral bioavailability in the imidazole-based 13-membered macrocycle to the 12-membered
macrocycle. This resulted in identification of compound 4 with desired FXIa inhibitory potency
and good oral bioavailability but high in vivo clearance. Further structure-activity relationship
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(SAR) studies of heterocyclic core modifications to replace the imidazole core as well as various
linkers to the P1 group led to the discovery of compound 6f, a potent FXIa inhibitor with
selectivity against most of the relevant serine proteases. Compound 6f also demonstrated
excellent pharmacokinetics (PK) profile (high oral bioavailability and low clearance) in multiple
preclinical species. Compound 6f achieved robust antithrombotic efficacy in a rabbit efficacy
model at doses which preserved hemostasis.
INTRODUCTION
The Global Burden of Disease Study for 2017 reports that cardiovascular diseases remain
the leading cause of death worldwide with total deaths from cardiovascular diseases increased by
2
1.1% from 2007 compared to 2017. Together, ischemic heart disease and stroke accounted for
1
over 8.9 million deaths in 2017, with thrombosis being the underlying cause for these deaths.
For over 50 years, vitamin K antagonists (warfarin) were the only available oral anticoagulant
option for the prevention of stroke in non-valvular atrial fibrillation (AF) and for the prevention
2
a
and treatment of venous thromboembolism (VTE). However, warfarin targets multiple factors
(or steps) within the coagulation cascade, has a recognized narrow therapeutic index and is prone
to multiple dietary and drug-drug interactions. These properties of warfarin result in an increased
potential for bleeding and a complex monitoring and dosing regimen. In the past 10 years, direct
acting oral anticoagulants directly targeting thrombin (dabigatran) or factor Xa (apixaban,
edoxaban, rivaroxaban) have been discovered and approved for prevention of stroke in AF, and
ACS Paragon Plus Environment
2

Journal of Medicinal Chemistry
Page 4 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for prevention and/or treatment of VTE. Although thrombin and FXa inhibitors address many of
the liabilities of warfarin, an unmet medical need remains as the risk of major bleeding with
currently approved drugs is as high as 2 to 3% per year in patients with AF. In addition, these
inhibitors are not widely approved for ischemic stroke or ACS (Acute Coronary Syndrome).
Thus, antithrombotic agents that can effectively reduce thrombosis without interrupting
hemostasis are needed. 2b, c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Factor XIa (FXIa) is a serine protease identified within the intrinsic blood coagulation
pathway. Since 1980, when thrombin was identified as a potential feedback activator of FXI, a
number of studies revealed that FXIa has a role in amplification of thrombin generation but a
3
limited role in hemostasis. This hypothesis is supported by genetic and epidemiologic evidence
as well as by preclinical and clinical proof-of-concept studies with pharmacologic agents
targeting FXI or FXIa. Factor XI gene disruption in mice is protective in thrombosis models and
does not lead to an increase in bleeding times. Human subjects with FXI deficiency4
(Hemophilia C; the most common of the rare bleeding disorders) do not suffer spontaneous
bleeding and the disorder often remains silent through adulthood. Some FXI-deficient subjects
have mild to moderate bleeding episodes following surgery or trauma in tissues with higher
levels of fibrinolytic activity, such as the oral cavity. Epidemiologic studies show that higher
levels of circulating concentrations of FXI are a thrombosis risk factor while low FXI levels may
5
confer some protection from thrombosis. Antibodies that bind FXI or FXIa and direct inhibitors
of FXIa that demonstrate antithrombotic efficacy have been reported with little or no effect on
6
hemostasis in animal models. In a Phase II clinical study in subjects undergoing elective total
knee arthroplasty, an antisense oligonucleotide targeting FXI (FXI ) reduced the incidence of
RX
VTE as compared to enoxaparin and appeared to be safe with respect to bleeding.7
ACS Paragon Plus Environment
3

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Our labs have reported on 12- and 13-membered macrocyclic FXIa inhibitors such as
8
compounds 1 and 2 discovered through structure-based design (Figure 1). Compounds 1 and 2,
having a chlorophenyltetrazole cinnamide as a P1 group, exhibited high affinity for FXIa (Ki < 1
nM) and potent anticoagulant activity as measured by prolongation of activated partial
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
thromboplastin time (aPTT, EC1.5x < 1 M). These compounds also possessed excellent
selectivity against other relevant blood coagulation enzymes. However, this series of
macrocyclic inhibitors lacked oral bioavailability potentially due to their high polar surface area
2
(
PSA, 169 Å for both 1 and 2) and the number of hydrogen bond donors (4 for both
compounds). To achieve good oral bioavailability, extensive efforts were made toward reducing
the PSA and the number of hydrogen bond donors while still maintaining high FXIa affinity.
Based on an X-ray crystal structure of 13-membered macrocycle 2 complexed with FXIa protein
and the unique s-trans conformation that the acrylamide adopted, conformationally constrained
analogs of the acrylamide P1 linker were prepared to pre-organize the chemotype into its
bioactive conformation.10 Cyclization also significantly improved binding affinity such that the
tetrazole group was no longer needed to achieve the desired FXIa affinity. This effort led to the
identification of six-membered cyclic P1 linkers as exemplified by cyclic carbamate 3a.
Compound 3a still retained reasonable affinity (FXIa Ki = 17 nM), with significant reduction in
2
PSA (from 169 to 126 Å ) and the number of hydrogen bond donors (from 4 to 3). As a result of
these changes, compound 3a achieved dramatically enhanced oral bioavailability in a rat PK
study, improving F% from less than 1% to 59%. Further optimization of substituents on the
terminal chlorophenyl ring identified 2,6-difluoro-substituted compound 3b, which gained a 3-
fold improvement in FXIa binding affinity and anticoagulant activity in the aPTT assay as
compared to compound 3a (Figure 1). However, 3b showed poor in vitro liver microsome
ACS Paragon Plus Environment
4

Journal of Medicinal Chemistry
Page 6 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
stability with t of 8 min (human) and 15 min (rat). Additionally, the FXIa affinity of 3b needed
1
/2
further improvement to achieve the desired range (Ki < 1 nM). In this manuscript, we report the
application of the same cyclization strategy to the 12-membered macrocycle series as represented
by compound 1 and further optimization of in vivo PK properties by carrying out SAR studies on
macrocycle cores as well as P1 linkers. This effort led to the discovery of compound 6f, with
desirable FXIa inhibitory activity (Ki < 1 nM) and good selectivity against many of the relevant
serine proteases. Compound 6f also demonstrated an excellent PK profile (oral bioavailability:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
9 - 94%, clearance: 1.8 - 17 mL/min/kg) in cyno, dog and rat PK studies. In a rabbit efficacy
model, compound 6f achieved robust antithrombotic efficacy. Also at the efficacious doses,
compound 6f did not prolong cuticle bleeding time when dosed alone or in combination with
aspirin in rabbit cuticle bleeding model.
1
3
O
N
N
13
N
N
O
NH
NH
O
1
2
N
O
H
N
N
HN
N
O
H
N
N
O
H
N
H
NH
O
H
N
N
R
O
R
N
O
O
O
N
H
N
N
H
N
O
O
N
linker
Cl
Cl
core
Cl
P1
1
2
3a (R = H)
FXIa Ki = 17 nM
3b (R = F)
FXIa Ki = 0.4 nM
FXIa Ki = 0.2 nM
FXIa Ki = 4.7 nM
aPTT EC1.5x = 0.4 M
aPTT EC1.5x = 0.3 M
aPTT EC1.5x = 5.9 M
aPTT EC1.5x = 2.0 M
PSA = 126 Å
PSA: 169 Å2
PSA: 169 Å
2
PSA: 126 Å
2
2
rat PK: F = 1.1% (des-Me analog)
rat PK: F = 0.8%
rat PK: F = 59%
Figure 1. Reported Macrocyclic FXIa Inhibitors 8,10
RESULTS AND DISCUSSION
With chlorophenyltetrazole cinnamide as the P1 group, the imidazole-based 12-
membered macrocycle 1 and 13-membered macrocycle 2 showed comparable FXIa binding
affinity (Figure 1, Ki = 0.4 nM for 1 and 0.2 nM for 2) and anticoagulant activity (aPTT EC1.5x
=
0
.4 M for 1, 0.2 M for 2 respectively). When the newly discovered 2,6-difluoro-3-
chlorophenyl substituted cyclic carbamate P1 was installed on to the 12-membered macrocycle
ACS Paragon Plus Environment
5

Page 7 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
, we were pleasantly surprised that the resulting compound 4 retained comparable FXIa activity
as compound 1 with chlorophenyltetrazole cinnamide P1 (Ki: 0.7 nM vs 0.4 nM, aPTT EC1.5x
:
0.6 M vs 0.4 M) (Figure 2). This is in contrast to the results obtained from the 13-membered
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
macrocycle series (Figure 1), where the same change from chlorophenyltetrazole cinnamide P1
(compound 2) to 2,6-difluoro-3-chlorophenyl substituted cyclic carbamate P1 (compound 3b)
resulted in a 24-fold loss in FXIa binding (Ki: 0.2 nM to 4.7 nM) and 7-fold loss in anticoagulant
activity (aPTT EC1.5x: 0.3 M to 2 M). However, consistent with the 13-membered macrocycle
with 2,6-difluoro-3-chlorophenyl substituted cyclic carbamate P1, compound 4, with reduced
2
2
PSA (169 Å to 126 Å ) and a decreased number of H-bond donors (4 to 3), showed good
permeability, with Caco-2 A to B of 38 nm/sec and B to A of 238 nm/sec.
O
O
1
2
HN
12
HN
N
N
O
O
H
comparable FXIa activities
H
H
H
N
N
N
N
O
N
O
N
N
F
O
F
N
H
N
O
N
O
4
1
Cl
Cl
FXIa Ki = 0.7 nM
FXIa Ki = 0.4 nM
aPTT EC1.5x = 0.6 M
aPTT EC1.5x = 0.4 M
2
PSA: 126
Å
PSA: 169 Å2
Caco: 38 nm/sec (A-B); 238 nm/sec (B-A)
Figure 2. Imidazole-based 12-Membered Macrocyclic FXIa Inhibitor with Cyclic Carbamate P1
With desired FXIa inhibitory activities (Ki < 1 nM, aPTT EC1.5x <1 M) and good Caco-
2 permeability, compound 4 was progressed into PK studies in multiple species (Table 1), where
it demonstrated good oral bioavailability, with F% of 29% in cyno, 50% in dog and 54% in rat,
respectively. However, compound 4 could not be progressed further due to its high in vivo
clearance (33 mL/min/kg in rat, 38 mL/min/kg in dog and 56 mL/min/kg in cyno) and short half-
life (1.4 - 1.9 h) across species.
Table 1. Pharmacokinetic Profile of Compound 4 in Pre-clinical Species
ACS Paragon Plus Environment
6

Journal of Medicinal Chemistry
Page 8 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Animal
species
LM T1/2
Clearance
(mL/min/kg)
Vdss
(L/kg)
AUC
(iv)(nM*h)
AUC
(po)(nM*h)
Dose iv/po
(mpk)
T
1/2 (h)
F (%)
54
(min)
Ratb
48
33 ± 0.7
1.4 ± 0.2
3.2 ± 0.4
520 ± 11
687 ± 78
0.6/1.2
Dogc
38
17
38
1.9
5.1
57
29
285
446 ± 4
0.4/1.1
0.5/1.5
Cynoc
56 ± 23
1.4 ± 0.1
9.2 ± 3.6
233 ± 94
263 ± 87
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
c
b
LM = Liver microsome stability. Vehicle for both iv and po: 10% ethanol; 35% PEG 400; 35% propylene glycol; 20% water, n = 3 animals.
Vehicle for both iv and po: 10% ethanol; 70% PEG 400; 20% water, n = 3 for each group except dog IV (n = 2, SD not calculated).
Our next goal was to reduce the in vivo clearance of compound 4 while maintaining high
FXIa affinity and good oral bioavailability. Based on our previous experience with FXIa
inhibitors in both the acyclic and macrocyclic series, the imidazole core in compound 4 was
1
1
replaced with various pyridine isomers (Table 2). The 2-, 3-, and 4-pyridine isomers
(compounds 5a-c) as well as the 2-fluoro-3-pyridine analog 5d all demonstrated comparable
FXIa inhibitory activities (both Ki and aPTT EC1.5x) as compound 4. The 3-pyridine compound
5
b showed lower liver microsomal stability compared to the imidazole core (4) and the 2- and 4-
pyridine isomers (5a and 5c). The 2-fluoro-3-pyridine analog 5d was prepared to test the
hypothesis that the 2-fluoro substitution would reduce the electron density of the 3-pyridine ring
and improve metabolic stability. However, compound 5d showed no improvement in metabolic
stability compared to the unsubstituted compound 5b. The 2- and 4-pyridine isomers 5a and 5c
exhibited moderate metabolic stability and were tested in rat PK studies. The 2-pyridine and 4-
pyridine isomers 5a and 5c showed similar oral bioavailability to 4, with the 2-pyridine analog
5
a having slightly lower clearance relative to compounds 4 and 5c. Based on these results and
PK studies of other related 2-pyridine analogs that consistently demonstrated lower clearance
relative to other cores, macrocycles with a 2-pyridine core were chosen as a template to explore
P1 linker modifications, with the goal of further improving in vivo clearance (Table 3).
Table 2. 12-Membered Macrocycles with Various Pyridine Cores
ACS Paragon Plus Environment
7

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
H
HN
N
O
O
O
F
O
F
N
core
Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
FXIa Kia
(nM)
b
LM T1/2
Clearanced
(mL/min/kg)
aPTT EC1.5x
Entry
4
Cores
%F
54
(M)
(h, d, r, c)(min.)
H
N
0.7
0.6
55, 38, 48, 17
33 ± 0.7
N
N
5
a
0.6
1.8
1.6
2.7
0.5
0.6
0.5
1.2
56, 27, 35, 12
17, 21, 9, 2
23 ± 4.7
37
NT
48
NTe
5
b
N
5c
36, 44, 29, 9
9, 15, 6, NT
33 ± 7.3
NT
N
5
d
NT
F
N
a
Ki values were obtained from purified human enzyme at 37 C and were averaged from multiple determinations (n≥2). baPTT (activated partial
thromboplastin time) in vitro clotting assay was performed in human plasma. EC1.5x refers to the plasma concentration, not the final concentration
after the addition of aPTT reagent and calcium. LM = liver microsome stability. rat PK vehicle for both iv and po: 10% ethanol; 35% PEG 400;
◦
c
d
e
3
5% propylene glycol; 20% water. n = 3 animals. NT = Not tested.
A metabolic ID study of 3a showed a major metabolite with molecular ion of M+H =
5
22, and metabolism likely occurred at the cyclic carbamate linker. This suggested that the
benzylic position attached to the terminal 2,6-difluoro-3-chlorophenyl might be the metabolic
soft spot, undergoing oxidation first, followed by the loss of CO. Based on this observation,
modifications around the P1 linker including the benzylic position were undertaken to try to
improve the liver microsomal stability and consequently lower in vivo clearance (Table 3).
Changing the heteroatom attached to the benzylic carbon from oxygen (cyclic carbamate 5a) to
nitrogen (cyclic urea 6a) or carbon (cyclic lactam 6b) maintained FXIa binding affinity.
However, the aPTT potency decreased by 15-fold for 6a (from 0.5 M to 7.5 M) along with
decreased metabolic stability in human liver microsomes. Similarly, the lactam analog 6b also
showed comparable FXIa binding affinity (0.9 nM vs 0.6 nM) as cyclic carbamate (5a) but
weaker anticoagulant activity (aPTT EC1.5x: 2.8 M vs 0.5 M) and lower liver microsomal
stability across species. When the benzylic carbon atom (cyclic carbamate 5a) was replaced with
ACS Paragon Plus Environment
8

Journal of Medicinal Chemistry
Page 10 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a nitrogen atom as in 2-ketopiperazine (6c), both FXIa inhibition and LM stability were
decreased. Addition of another carbonyl to 6c gave 2,3-diketopiperazine (6d) and 2,5-
diketopiperazine (6e), both exhibited increased LM stability but decreased FXIa affinity. Finally,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
the dihydropyridone linker (6f), which was designed to install a pre-oxidized sp carbon instead
3
of the sp carbon at benzylic positions in 5a, 6a or 6b, demonstrated much improved liver
microsomal stability across species while also maintaining excellent FXIa activity (Ki = 0.3 nM,
aPTT EC1.5x = 1.8 M).
Table 3. 2-Pyridine-Based 12-Membered Macrocycles with Various P1 linkers
O
H
HN
N
O
O
F
linker
N
F
Cl
c
b
LM T1/2
P1 with
Linkers
FXIa Kia
(nM)
aPTT EC1.5x
Entry
a
(M)
(
h, d, r, c)(min.)
O
5
a
O
N
N
0.6
0.5
0.9
4.5
14
0.5
7.5
2.8
4.6
4.3
56, 27, 35, 12
O
ad
HN
*
6
17, 27, 40, 2
8, 13, 15, 3
4, 6, 9, 1
O
O
O
O
6
be
N
N
N
N
6
c
N
O
6
d
76, 64, 60, 80
N
N
6
e
4.9
0.3
1.4
1.8
75, 72, 55, 27
103, 67, 60, 71
O
O
6
f
N
a
o
b
Ki values were obtained from purified human enzyme at 37 C and were averaged from multiple determinations (n≥ 2). aPTT (activated partial
thromboplastin time) in vitro clotting assay was performed in human plasma. EC1.5x refers to the plasma concentration, not the final concentration
c
d
e
after the addition of aPTT reagent and calcium. LM = liver microsome stability. Absolute stereochemistry not determined. 2:1 mixture of
diastereomers.
In addition to its excellent FXIa affinity and liver microsomal stability, compound 6f also
ACS Paragon Plus Environment
9

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
demonstrated good permeability (Caco-2 A to B: 55 nm/sec; B to A: 138 nm/sec). As a result of
this favorable profile, multiple PK studies with compound 6f were conducted showing desirable
PK properties as shown in Table 4. Across species, compound 6f showed very low to moderate
clearance (1.8 – 17 mL/min/kg) and good oral bioavailability (59 – 94%).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Pharmacokinetic Profile of Compound 6f in Different Species
Animal
species
Clearance
(mL/min/kg)
Vdss
(L/kg)
AUC
(iv)(nM*h)
AUC
(po)(nM*h)
Dose iv/po
(mpk)
T
1/2 (h)
F (%)
Rata
Dogb
10 ± 1.3
6.4 ± 1.1
17 ± 3.6
1.8 ± 0.4
1.7 ± 0.2
4.9 ± 0.9
1.4 ± 0.3
3.4 ± 2.1
1.1 ± 0.2
2.2 ± 0.8
2.0 ± 0.6
0.5 ± 0.2
1397 ± 196
2089 ± 319
765 ± 148
62
94
0.6/1.2
0.5/0.5
1735 ± 144
1895 ± 1404
817 ± 404
NTd
Cynob
Rabbitc
59
0.48/0.89
6681 ± 1365
NT
0.5/NT
a
b
Compound was dosed as TFA salt, vehicle for both iv and po: 10% ethanol; 70% PG; 20% water. Data were average of 3 animals. Compound
c
was dosed as HCl salt, vehicle for both iv and po: 10% ethanol; 70% PEG 400; 20% water. Data were average of 3 animals. Compound was
dosed as TFA salt, vehicle for iv: 76.5% Dextrose; 10% DMAc (dimethyl acetamide); 13.5% HP--CD. Data were average of 3 animals. NT =
not tested.
d
Compound 6f was further progressed to a rabbit electrically-induced carotid artery
thrombosis (ECAT) model to evaluate in vivo antithrombotic activity (Figure 3A, 3B).12a
Compound 6f (TFA salt, rabbit FXIa Ki = 1.2 nM, aPTT EC1.5x = 26 M) was dosed at 0.355 +
0.128, 1.42 + 0.52 and 3.55 + 1.28 mg/kg + mg/kg/h IV with 6 rabbits per dose. Figure 3A
shows the effects of vehicle and compound 6f treatment on carotid blood flow after injury-
induced thrombus formation. Integrated blood flow averaged 14 ± 2% of the baseline level in
vehicle treated animals following thrombus induction. By blocking the formation of thrombus,
compound 6f caused a dose-dependent increase in integrated blood flow compared with the
vehicle treated group. At the top dose (3.55 mg/kg + 1.28 mg/kg/h), integrated blood flow was
restored to 84 ± 4% of the baseline level. As shown in Figure 3B, compound 6f caused a dose-
dependent reduction of thrombus weight by inhibiting the formation of thrombus. At the top
dose, thrombus weight reduction was 69 ± 4% of the control level. Also at the top dose,
ACS Paragon Plus Environment
1
0

Journal of Medicinal Chemistry
Page 12 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 6f increased aPTT by 1.5-fold but did not increase PT (prothrombin time),13
consistent with the mechanism of FXIa inhibition. The potential for bleeding liability of
compound 6f was evaluated in a rabbit cuticle bleeding model (Figure 3C).12b, c At a dose
comparable to one that generated the highest antithrombotic efficacy (3.4 mg/kg + 5.4 mg/kg/h),
compound 6f did not increase cuticle bleeding time when compared with vehicle treated animals.
Since aspirin (ASA) is widely used in patients for the prevention and treatment of acute and
chronic arterial diseases, the potential exists that FXIa inhibitors would be dosed on top of ASA
in the clinical setting. Therefore, compound 6f was also tested in combination with ASA in the
cuticle bleeding model. Aspirin alone produced a 2.3-fold increase in bleeding time at its clinical
relevant dose (4 mg/kg/h).12c Compound 6f, when dosed together with ASA, increased bleeding
time by 2.1-fold, similar to that observed with ASA alone.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. A) Effects of compound 6f on carotid blood flow after thrombus induction in the rabbit ECAT model. B)
Effects of compound 6f on thrombus weight reduction (vehicle:10%DMA:15% HP-β-CD in D5W). C) Effects of
aspirin (ASA) and compound 6f on bleeding time in the rabbit cuticle bleeding time model.
The X-ray crystal structure of compound 6f bound to FXIa protein was determined and
found to be consistent with structures of our other macrocycles complexed with FXIa (Figure 4).
In the P1 region, the 6-fluoro of the terminal 2,6-diF-3-Cl-phenyl makes contact with Cys219
sulfur as well as orients the dihedral angle between phenyl/dihydropyridone closer to its
ACS Paragon Plus Environment
1
1

Page 13 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
bioactive conformation while the 2-fluoro fills a small hydrophobic pocket, making van der
Waals contact with Thr213 sidechain. The carbonyl of dihydropyridone points into the oxyanion
hole consisting of Gly193 and Ser195. The nitrogen of the 2-pyridine core forms a hydrogen
bond with a water molecule that extends into bulk solvent. In the P2 prime region, the methyl
carbamate forms two key hydrogen bonds: the NH interacts with the His40 carbonyl and the
other is an interaction with the Ile151 NH through the carbonyl of the carbamate via a water
molecule. Also consistent with structures of other macrocycles we had disclosed previously, the
amide NH in the macrocycle linker makes a crucial hydrogen bonding interaction to the carbonyl
of Leu41 that contributes significantly to the potency of the macrocycle series. The carbonyl of
the amide in the macrocycle makes a hydrogen bond with a water that extends to bulk solvent.
The R--methyl of the amide in the linker binds in a hydrophobic region near the S1' pocket.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. X-ray crystal structure of compound 6f bound to FXIa (1.95 Å resolution). The red solid spheres represent
water molecules and the dotted lines represent hydrogen bonds. PDB deposition number for 6f is 6W50.
ACS Paragon Plus Environment
1
2

Journal of Medicinal Chemistry
Page 14 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The single crystals of compound 6f were crystallized from a saturated solution of DMF at
room temperature and its X-ray structure is shown in Figure 5. The three dimensional packing
motif was built up with intermolecular interactions between the NH of the carbamate and the
carbonyl of the dihydropyridone lactam (shown in deep teal dashed lines) and between the
carbonyl of the macrocycle amide and the NH of a neighboring macrocycle amide (shown in
black). The extensive crystal packing resulted in the poor aqueous solubility of compound 6f (<
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.1 g/mL at pH 4.0 and above), which ultimately prevented it from advancing into clinical
development.
Figure 5. Single X-ray crystal structure of compound 6f. Cambridge Crystallographic Data Centre deposition
number for 6f is CCDC-1989806.
Compound 6f was tested against a panel of serine proteases and its selectivity is shown in
Table 5. Except for plasma kallikrein (Ki = 1.9 nM, 7-fold selective) and chymotrypsin (Ki = 20
nM, 77-fold selective), compound 6f generally exhibited good selectivity against other relevant
proteases.
ACS Paragon Plus Environment
1
3

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 5. Human Serine Protease Selectivity Profile for Compound 6f.
Human Enzyme
Ki (nM)
Factor XIa
Factor VIIa
Factor IXa
Factor Xaa
0.26
2400
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
> 27,000
> 9000
1300
Factor XIIa
Thrombina
> 13000
> 6000
1.9
Trypsina
Plasma Kallikrein
Activated Protein C
Plasmin
>21000
>15000
Tissue-type plasminogen
activator
4
100
Urokinase
>15000
20
Chymotrypsin
Tissue Kallikrein-1
>30,000
a
o
K
i
values in nM were obtained using human purified enzymes at 25 C within a standard compound screening panel. All others conducted at 37
o
C as part of a focused program panel.
CHEMISTRY
The synthesis of imidazole based 12-membered macrocycle with a chiral cyclic
carbamate P1 linker is shown in Scheme 1. Grignard addition of vinyl magnesium bromide to 3-
chloro-2,6-difluorobenzaldehyde followed by oxidation with Jones reagent gave the vinyl ketone
7. Treatment of 7 with trimethylsilyl chloride and sodium iodide afforded compound 8, which
.
was reduced to chiral R-alcohol 9 with (R)-methyl-CBS and BH THF. Alkylation of macrocycle
3
1
08b,11c with iodide 9 provided amino alcohol 11, which upon treatment with carbonyl
diimidazole gave cyclic carbamate 12. Finally, the SEM protecting group was removed by HCl
in dioxane at 70 ºC to afford compound 4.
Scheme 1. Synthesis of Cyclic Carbamate P1 Linker (compound 4) in 12-Membered Macrocycle
with Imidazole Core
ACS Paragon Plus Environment
1
4

Journal of Medicinal Chemistry
Page 16 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
F
F
O
F
F
O
F
OH
CHO
F
a, b
c
I
d
I
e
HN
+
H
N
F
8
F
9
N
O
O
H2N
SEM
Cl
Cl
Cl
Cl
N
O
7
10
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
HN
O
HN
H
N
H
F
OH
HN
f
g
H
N
H
N
N
O
F
O
F
N
O
N
F
O
N
N
O
N
N
O
N
O
H
N
O
SEM
F
SEM
F
Cl
Cl
Cl
11
12
4
Reagents and conditions: (a) Vinyl magnesium bromide, THF, 0 ºC, 2.5 h, 100%; (b) Jones reagent, acetone, 0 ºC, 30 min, 87%; (c) TMSCl, NaI,
.
water, CH
3 3 2 3 3
CN, 30 min, 94%; (d) (R)-Methyl-CBS, BH THF, THF, rt, 3 h, 65%; (e) K CO , CH CN, 75 ºC, 16 h, 64%; (f) CDI, rt, 16 h,
LiHMDS, 0 ºC, 1h, 82%; (g) 4M HCl in dioxane 75 ºC, 1 h, 70%.
The synthesis of intermediates 22b-d of 12-membered macrocycles containing various
pyridine cores is described in Scheme 2, following the same procedure as described in our recent
publication for the synthesis of 22a.11c Condensation of aldehydes a-d with either (S)-t-
butylsulfinamide (when X = N) or (R)-t-butylsulfinamide (when Y or Z = N) in the presence of
14
anhydrous copper sulfate or Ti(OEt) gave the chiral sulfinimines 13a-d. Grignard addition of
4
allyl magnesium bromide to sulfinimines 13a-d provided sulfinamides 14a-d, as a mixture of
1
5
diastereomers. The diastereoselectivities for the addition of allyl group to sulfinimines 13a-d
were significantly improved (de from 4-7:1 to >19:1) by employing allyl magnesium
16
bromide/indium (III) chloride for 13a and allyl bromide/In for 13c-d. Removal of chiral
auxiliaries in compounds 14a-d by hydrochloric acid followed by protection with a Boc group
gave compounds 15a-d. Suzuki-Miyaura coupling between chloropyridine 15a (or
bromopyridines 15b-d) and 2-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-5-nitroaniline 16 provided
1
7a-d. Reduction of the nitro group by zinc/ammonium chloride gave the bis-anilines, which at
low temperature selectively reacted with methyl chloroformate at the sterically less hindered
terminal aniline to give compounds 18a,18b and 18d. Compounds 18a and 18b were coupled
with (R)-2-methylbut-3-enoic acid using T3P and pyridine to give the amides 19a-b. It should be
noted that when diisopropylethylamine was used as the base instead of pyridine for the amide
ACS Paragon Plus Environment
1
5

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
coupling, significant epimerization was observed at the chiral center alpha- to the amide.
Intermediate 19c was prepared via a slightly different sequence; ie 17c was first coupled to
racemic 2-methylbut-3-enoic acid to give 18c, then reduction of nitro group followed by
carbamate formation afforded 19c. Following pretreatment with p-toluene sulfonic acid to form
the pyridinium ion, compounds 19a-d were cyclized via ring-closing metathesis (RCM) using
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
Grubbs (II) catalyst to give the pyridine-containing macrocycles 20a-d. Hydrogenation
followed by subsequent deprotection with TFA or hydrochloric acid provided the intermediate
amines 22a-d. The two diastereomers were separated after RCM for 20c or after hydrogenation
for 21d. Following the same sequence as described in Scheme 1, these intermediates 22a-d were
converted to their corresponding cyclic carbamates 5a-d.
Scheme 2. Synthesis of 12-Membered Macrocycles with Various Pyridine Cores and the Cyclic
Carbamate P1 linker
a
H2N
NO2
O
S
*
R
O
S
(
S)-t-Butylsulfinamide for a
O
R
b
c
d
O
B
N
R
Boc
R
X
Z
(R)-t-Butylsulfinamide forb-d
N
N
H
Y
X
Z
* H
O
Y
X
Z
X
Z
Y
Y
a: X = N, Y = Z = CH, R = Cl
b: Y = N, X = Z = CH, R = Br
c: Z = N, X = Y = CH, R = Br
d: Y = N, X = CF, Z = CH, R = Br
1
3a-d
14a-d
15a-d
16
O
*
O
H
N
HO
O
*
H2N
NO2
H2N
O
H
H
N
e
g
*
HN
O
h
HN
N
O
Boc
Boc
O
N
N
H
f
O
H
Boc
O
Boc
N
X
Z
X
N
H
Y
Y
H
X
Z
X
Z
Y
Y
17a-d
18a,b,d
19a,b: * chiral (R)
9c,d: *diast. mix.
20a-c: *chiral (R)
20d: *diast. mix.
1
e
O
HN
NO2
Boc
N
H
N
18c
O
H
O
O
HN
N
O
O
H
H
O
i
HN
N
O
j
HN
N
O
scheme 1
F
O
F
N
Boc
O
O
Z
N
H2N
X
Y
H
X
Z
X
Z
Y
Y
21a-d
22a-d
Cl
5a-d
4 2 3 4
Reagents and conditions: (a) (S)-t-butylsulfinamide CuSO , Cs CO , DCM, 94% for 13a or (R)-t-butylsulfinamide, Ti(OEt) , DCM, 85-93% for
1
3b-d; (b) Allyl magnesium bromide, InCl
3
2
, 106%, >19:1 de for 13a or Ally bromide, In, NaBr, 73-98% for 13b-d; (c) 1) HCl; 2) Boc O/TEA,
ACS Paragon Plus Environment
1
6

Journal of Medicinal Chemistry
Page 18 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
62-100% for two steps; (d) Pd(dppf)Cl
ºC to 0 ºC, 69-97%; (g) Ghosez′s reagent, TEA, 49%; (h) for 20a-c: 1) pTsOH, DCM; 2) Grubbs (II), 0.1 eq, DCM (0.012M), reflux, 53-63%; for
0d: Grubbs (II), 0.4 eq, DCE (0.016M), microwave 120 ºC, 25 min, 57%; i) H , PtO , MeOH/THF, 13-90%; j) 50% TFA/DCM, or HCl in
dioxane, 93-100%.
2 4 2 3
, DMSO, 81-100%; (e) 1) Zn/NH Cl; 2) ClCO Me, 74-86% for two steps; (f) T P, pyridine, EtOAc, -10
2
2
2
The synthesis of 12-membered macrocycles with a 2-pyridine core bearing different P1
linkers is shown in Scheme 3. Reductive amination of 22a with aldehyde 23 followed by
cyclization under microwave heating at 100 ºC afforded the cyclic urea, which was separated by
reverse phase preparative HPLC to give the two diastereomers, the more active one being 6a.
The keto-piperazine 6c was prepared by a two-step one-pot reaction of 22a with mesylate 24 at
120 ºC, albeit with low yield. The 2,3-diketopiperazine 6d was prepared by treatment of 22a
with aldehyde 25. The 2,5-diketopiperazine 6e was prepared by alkylation of 22a with -
chloroamide 26 to give 27, followed by acylation with 2-chloroacetyl chloride and cyclization
under basic condition. Michael addition of 22a to vinyl ketone 7 followed by acylation with
diethyl (2-chloro-2-oxoethyl)phosphonate, which upon cyclization with sodium methoxide
provided the dihydropyridone 6f. Hydrogenation of 6f afforded the lactam 6b as a 2:1 mixture of
diastereomers.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 3. Synthesis of Various Cyclic P1 Linkers with 2-Pyridine Core
ACS Paragon Plus Environment
1
7

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
N
F
N
F
6
c
CO Me
2
F
Cl
N
OMs
b
O
F
Ph
O
F
24
H
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
HN
O
N
Cl
Cl
O
O
O
O
1)
O
F
F
HN
F
a
23
26
H
N
F
HN
*
N
H
Cl
F
N
HN
N
O
2
) ClCOCH2Cl
Cl
N
F
O
O
d
3) Base
F
H N
F
2
Cl
6a
N
22a
27
Cl
6e
Cl
O
F
O
O
F
OEt
CHO
O
P
O
N
e
1)
EtO
EtO
2) NaOMe
c
Cl
F
25
7
F
Cl
N
Cl
O
O
O
N
O
F
N
F
F
H2
N
f
F
F
F
6d
6f
6b
Cl
CN, MeOH, rt, 64%; 2) NEt
DCE, 4Å MS, 23%; (d) 1) TEA, DMF, 70 ºC, 21%; 2) DIEA, THF, 0 ºC, 94%; 3) NaOMe/MeOH, 81%; (e) 1) DIEA, DCM, then diethyl 2-
chloro-2-oxoethylphosphonate, 70-93%; 2) NaOMe, MeOH, 88%; (f) H , PtO , EtOAc, 25% (2:1 mixture of diastereomers).
Cl
Cl
(
a) 1) NaBH
3
3 3 3
, CH CN, microwave 100 ºC, 10%. (b) Hunig’s base, DMA, 120 ºC, 4%; (c) NaBH(OAc) , HOAc,
2
2
The preparation of various precursors to the synthesis of different P1 linkers used in
Scheme 3 is described in Scheme 4. Condensation of 2,6-difluoro-5-chlorobenzaldehyde with t-
butylsulfinamide followed by Grignard addition of ally magnesium bromide gave compound 28.
Deprotection followed by reprotection with phenyl chloroformate provided phenyl carbamate
compound 29, which was oxidized by sodium periodate to afford compound 23. Alkylation of 3-
chloro-2,6-difluoroaniline with methyl 2-bromoacetate gave bis-alkylated compound 30.
Hydrolysis and reduction of the monoacid 31 provided alcohol 32, which was converted to the
mesylate 24. 3-Chloro-2,6-difluoroaniline could also be acylated with 2-chloroacetyl chloride to
give compound 26 or treated with ethyl 2-chloro-2-oxoacetate to give compound 33. Alkylation
of ketoamide 33 with allyl bromide followed by oxidation with sodium periodate afforded
aldehyde 25.
Scheme 4. Synthesis of Precursors to the Various P1 Linkers
ACS Paragon Plus Environment
1
8

Journal of Medicinal Chemistry
Page 20 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
S
O
O
O
S
F
Ph
Ph
S
F
HN
F
O
F
HN
F
O
O
CHO H N
F
N
F
F
HN
F
O
2
MgCl
c
g
k
d
F
a
b
Cl
Cl
Cl
F
Cl
Cl
28
29
CO2Me
23
CO2Me
CO2Me
CO2Me
CO2H
CO2Me
O
F
CO2Me
F
F
F
O
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
N
N
N
N
Br
OH
f
h
O
F
e
F
F
F
F
Cl
Cl
Cl
Cl
Cl
30
31
32
24
l
i
O
O
O
F
H
O
O
O
F
OEt
F
OEt
F
OEt
N
j
Cl
NH
N
N
CHO
O
F
F
F
F
Cl
26
Cl
33
Cl
34
Cl
25
Reagents and conditions: (a) t-Butylsulfinamide, Cs
MeOH; 2) Phenyl chloroformate, DIEA, CH
f) NaOH, MeOH, rt, 37%; (g) BH ·DMS, THF, rt, 68%; (h) Mesyl chloride, pyridine, DCM, 0 ºC, 54%; (i) Ethyl 2-chloro-2-oxoacetate, NEt
THF, rt, 72%; (j) Allyl bromide, NaH, THF, 0 ºC - rt, 68%; (k) NaIO , OsO , 2,6-lutidine, dioxane/H O, 83%; (l) 2-Chloroacetyl chloride, K CO
ºC - rt, 89%.
2
CO
3
, DCM, rt, 87%; (b) Allyl magnesium chloride, THF, -78 ºC, 100%; (c) 1) HCl/dioxane,
3
CN, rt, 91% for 2 steps; (d) NaIO
4
, OsO , MeOH/H O, 89%; (e) K HPO , KI, CH CN, 100 ºC, 45%;
4
2
2
4
3
(
3
3
,
4
4
2
2
3
,
0
CONCLUSION
We have applied a previously disclosed orally bioavailable cyclic carbamate P1 linker
into the 12-membered macrocycle series, resulting in identification of compound 4 that
possessed desired FXIa inhibitory potency and good oral bioavailability. To reduce in vivo
clearance of compound 4, SAR studies were carried out to optimize the macrocycle core, as well
as P1 linkers. We discovered that the 2-pyridine core gave lower clearance than the imidazole
core and that dihydropyridone P1 linker greatly improved liver microsomal stability over the
cyclic carbamate linker. Combining the two findings led to the discovery of compound 6f, with
good FXIa inhibitory activity and selectivity against many of the relevant serine proteases.
Compound 6f also demonstrated excellent PK profile (good oral bioavailability and low
clearance) in multiple preclinical species. Furthermore, compound 6f achieved robust
antithrombotic efficacy in a rabbit efficacy model and did not prolong cuticle bleeding time at
efficacious doses in a rabbit bleeding model either dosed alone or in combination with aspirin.
ACS Paragon Plus Environment
1
9

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compound 6f met our program criteria of high affinity and serine protease selectivity, excellent
oral PK properties, however, it has very low solubility which may require further development.
EXPERIMENTAL
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General method. Commercially available solvents and reagents were used to carry out
reactions without further purification. NMR chemical shifts were reported in ppm relative to
internal standard (TMS, CDCl or CD OD). Purification of intermediates and final products was
3
3
carried out via either normal or reverse phase chromatography. Normal phase chromatography
was carried out using prepacked SiO cartridges eluting with either gradients of hexanes and
2
ethyl acetate or DCM and MeOH. Reverse phase preparative HPLC was carried out using C18
columns eluting with gradients of Solvent A (90% water, 10% MeOH, 0.1% TFA) and Solvent B
(10% water, 90% MeOH, 0.1% TFA, UV 220 nm) or with gradients of Solvent A (90% water,
1
0% CH CN, 0.1% TFA) and Solvent B (10% water, 90% CH CN, 0.1% TFA, UV 220 nm).
3
3
Purity of final compounds was determined by using either orthogonal HPLC conditions: Sunfire
C18 column (3.5 m, 4.6 x 150 mm) eluted at 1.0 mL/min with gradient 10–100% B/A (solvent
A: 95% H O/5% CH CN /0.05% TFA; solvent B: 5% H O/95% CH CN /0.05% TFA) over 10
2
3
2
3
min and then 100% of B over 5 min (HPLC Method A) and Xbridge Phenyl column (3.5 m, 4.6
x 150 mm) eluted at 1.0 mL/min gradient 10–100% B/A (solvent A: 95% H O/5% CH CN
2
3
/
0.05% TFA; solvent B: 5% H O/95% CH CN /0.05% TFA) over 10 min and then 100% of B
2 3
over 5 min (HPLC Method B). The synthesis of intermediates 13a-22a and 10 were described in
our previous publications.10,11c Marvin Sketch 16.12.19 was used to name all macrocycles.
Methyl N‐[(10R,14S)‐14‐[(6R)‐6‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxo‐1,3‐oxazinan‐3‐
2
,7
yl]‐10‐methyl‐9‐oxo‐8,16,18‐triazatricyclo[13.2.1.0 ]octadeca‐1(17),2,4,6,15‐pentaen‐5‐yl]c
arbamate (4). HCl in Dioxane (6.7 mL, 4 M, 26.9 mmol) was added to a round bottom flask
ACS Paragon Plus Environment
2
0

Journal of Medicinal Chemistry
Page 22 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
containing compound 12 (215 mg, 0.30 mmol). The flask was sealed and heated at 75 ºC for 1 h
before cooling down to rt. The reaction mixture was concentrated to give a white solid mixture,
which was dissolved in DCM and washed with sat. NaHCO . The aqueous layer was extracted
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with DCM one more time. The combined organic layers were washed with brine, dried over
MgSO , filtered and concentrated. Purification by silica gel chromatography yielded 124 mg
4
white solid which was dissolved in DCM (50 mL). HCl in MeOH (0.3 mL, 1.25 M) was added.
The mixture was shaken before concentrating to give 4, HCl salt (135 mg, 71%) as a white solid.
+
MS (ESI) m/z: 588.3 (M+H) . Orthogonal HPLC purity: 99.7%, retention time = 6.26 min
1
(
HPLC Method A); 98.2%, retention time = 7.01 min (HPLC Method B). H NMR (500MHz,
CD OD)  7.66 - 7.50 (m, 4H), 7.48 - 7.39 (m, 1H), 7.17 - 7.07 (m, 1H), 5.92 - 5.82 (m, 1H),
3
5
1
.34 - 5.24 (m, 1H), 3.79 (s, 3H), 2.82 - 2.61 (m, 2H), 2.46 - 2.32 (m, 2H), 2.28 - 2.13 (m, 1H),
.87 - 1.75 (m, 1H), 1.69 - 1.58 (m, 1H), 1.56 - 1.45 (m, 1H), 1.42 - 1.27 (m, 2H), 1.13 - 1.02 (m,
1
9
3H), 0.89 - 0.73 (m, 1H). F NMR (471 MHz, CD OD) δ -115.89, -116.44.
3
Methyl N‐[(10R,14S)‐14‐[(6R)‐6‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxo‐1,3‐oxazinan‐3‐
2
,7
yl]‐10‐methyl‐9‐oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]ca
rbamate (5a). Following the procedure used for the preparation of 12, 22a (200 mg, 0.34 mmol)
was reacted with 9 (178 mg, 0.54 mmol) followed by cyclization to give 5a (100 mg, 47% for
+
two steps) as a yellow solid. MS (ESI) m/z: 599.2 (M+H) . Orthogonal HPLC purity: 100%,
retention time = 6.64 min (HPLC Method A); 100%, retention time = 7.17 min (HPLC Method
1
B). H NMR (500 MHz, CD OD) δ 8.77 (d, J=5.8 Hz, 1H), 8.01 (s, 1H), 7.84 (br d, J=5.2 Hz,
3
1
H), 7.69 - 7.63 (m, 1H), 7.62 - 7.54 (m, 3H), 7.09 (td, J=9.5, 1.7 Hz, 1H), 5.84 (dd, J=11.6, 2.8
Hz, 1H), 5.26 (dd, J=12.7, 4.7 Hz, 1H), 3.86 - 3.81 (m, 1H), 3.79 (s, 3H), 3.76 - 3.68 (m, 1H),
ACS Paragon Plus Environment
2
1

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
1
.69 - 2.52 (m, 2H), 2.37 - 2.27 (m, 2H), 2.20 - 2.09 (m, 1H), 1.98 - 1.89 (m, 1H), 1.66 - 1.56 (m,
H), 1.38 - 1.26 (m, 1H), 1.07 (d, J=6.9 Hz, 3H), 1.04 - 0.91 (m, 1H).
Methyl N‐[(10R,14S)‐14‐[(6R)‐6‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxo‐1,3‐oxazinan‐3‐
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2,7
yl]‐10‐methyl‐9‐oxo‐8,17‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]ca
rbamate (5b). Following the procedure used for the preparation of 12, 22b (HCl salt) (38 mg,
0
.069 mmol) was reacted with 9 (30 mg, 0.090 mmol) followed by cyclization to give 5b (2.1
+
mg, 4% for two steps) as a white solid. MS (ESI) m/z: 600.3 (M+H) . Orthogonal HPLC purity:
9
7.4%, retention time = 6.13 min (HPLC Method A); 99.8%, retention time = 6.87 min (HPLC
1
Method B). H NMR (400 MHz, CD OD)  8.87 (s, 2H), 8.59 (s, 1H), 7.70 - 7.46 (m, 4H), 7.06
3
(td, J=9.6, 1.6 Hz, 1H), 5.79 (dd, J=11.0, 2.7 Hz, 1H), 5.35 (dd, J=12.6, 3.8 Hz, 1H), 3.77 (s,
3
1
H), 3.67 (td, J=11.5, 4.4 Hz, 1H), 3.53 - 3.39 (m, 1H), 2.55 - 2.33 (m, 2H), 2.33 - 2.09 (m, 3H),
.82 (dd, J=9.6, 6.3 Hz, 1H), 1.67 - 1.43 (m, 2H), 1.14 (d, J=6.6 Hz, 3H), 0.96 (m, 1H).
Methyl N‐[(10R,14S)‐14‐[(6R)‐6‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxo‐1,3‐oxazinan‐3‐
2,7
yl]‐10‐methyl‐9‐oxo‐8,18‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]ca
rbamate (5c). Following the procedure used for the preparation of 12, 22c, HCl salt (62 mg,
0.14 mmol) was reacted with compound 9 (70.1 mg, 0.21 mmol) followed by cyclization to give
+
5
c (37.4 mg, 37% for two steps) as a white solid. MS (ESI) m/z: 598.9 (M+H) . Orthogonal
HPLC purity: 100%, retention time = 6.13 min (HPLC Method A); > 95%, retention time = 6.98
1
min (HPLC Method B). H NMR (500 MHz, CD CN) δ 8.66 (d, J=6.1 Hz, 1H), 8.15 (s, 1H),
3
7
7
.96 (s, 1H), 7.81 (s, 1H), 7.62 (d, J=8.5 Hz, 1H), 7.55 (d, J=5.8 Hz, 1H), 7.48 - 7.35 (m, 3H),
.00 - 6.90 (m, 1H), 5.61 (dd, J=11.4, 2.6 Hz, 1H), 5.19 - 5.10 (m, 1H), 3.65 (s, 3H), 3.47 (td,
J=11.8, 4.3 Hz, 1H), 3.36 - 3.29 (m, 1H), 2.38 - 2.24 (m, 2H), 2.16 - 2.08 (m, 1H), 2.02 - 1.95
ACS Paragon Plus Environment
2
2

Journal of Medicinal Chemistry
Page 24 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
m, 2H), 1.73 - 1.64 (m, 1H), 1.44 - 1.32 (m, 1H), 1.21 - 1.05 (m, 2H), 0.93 (d, J=6.9 Hz, 3H).
1
9
F NMR (471 MHz, CD CN) δ -76.45, -115.61, -116.04.
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Methyl N‐[(10R,14S)‐14‐[(6R)‐6‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxo‐1,3‐oxazinan‐3‐yl]‐16‐f
2
,7
luoro‐10‐methyl‐9‐oxo‐8,17‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl
]carbamate (5d). Following the procedure used for the preparation of 12, 22d, TFA salt (30 mg,
0
.049 mmol) was reacted with 9 (21.1 mg, 0.063 mmol) followed by cyclization to give 5d (6.3
+
mg, 18% for two steps) as an off-white solid. MS (ESI) m/z: 617.3 (M+H) . Orthogonal HPLC
purity: > 96.4%, retention time = 8.77 min (HPLC Method A); 96.6%, retention time = 8.48 min
1
(HPLC Method B). H NMR (500 MHz, CD OD) δ 8.25 (d, J=1.4 Hz, 1H), 8.11 (dd, J=9.5, 2.1
3
Hz, 1H), 7.53 - 7.65 (m, 3H), 7.51 (s, 1H), 7.00 - 7.15 (m, 1H), 5.69 (dd, J=11.0, 3.0 Hz, 1H),
5
.38 (dd, J=12.9, 3.3 Hz, 1H), 3.78 (s, 3H), 3.36 - 3.41 (m, 1H), 3.02 - 3.13 (m, 1H), 2.35 - 2.58
(m, 2H), 2.16 - 2.34 (m, 2H), 1.99 - 2.14 (m, 1H), 1.69 - 1.88 (m, 2H), 1.55 - 1.69 (m, 1H), 1.21
d, J=6.9 Hz, 3H), 0.92 (d, J=11.6 Hz, 1H).
(
Methyl N‐[(10R,14S)‐14‐[4‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxo‐1,3‐diazinan‐1‐yl]‐10‐methy
2
,7
l‐9‐oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]carbamate
(6a). To a solution of 23 (14.5 mg, 0.043 mmol) and 22a, 2 TFA (22 mg, 0.037 mmol) in MeOH
(0.5 mL) in a 2 dram vial was added NaCNBH (3 mg, 0.048 mmol). The reaction was stirred at
3
rt overnight. The reaction mixture was purified by preparative HPLC. The desired fractions were
neutralized with conc. Na CO , followed by extraction with 10% IPA in CHCl until no more
2
3
3
product detected in aqueous layer. The combined organic phases were dried over MgSO4,
filtered off solid, concentrated to yield a white solid product (19 mg, 64%) as a mixture of 2
ACS Paragon Plus Environment
2
3

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
isomers which was used directly in next step without separating the two isomers. MS (ESI) m/z:
+
6
92.3 (M+H) . The above diastereomeric mixture (19 mg, 0.027 mmol), NEt (7.65 µl, 0.055
3
mmol) in CH CN (2 mL) was sealed and microwaved at 100 °C for 10 min before cooling down
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to rt. Reverse phase preparative HPLC purification afforded two diastereomers, with later eluting
+
isomer 6a (2.0 mg, 10% yield) as a pale yellow solid. MS (ESI) m/z: 598.3 (M+H) . Orthogonal
HPLC purity: > 97.5%, retention time = 4.98 min (HPLC Method A); > 97.9%, retention time =
1
6
1
.25 min (HPLC Method B). H NMR (500 MHz, CD OD) δ 9.70 (s, 1H), 8.82 (d, J=6.1 Hz,
3
H), 8.14 (d, J=1.4 Hz, 1H), 7.95 (dd, J=6.2, 1.8 Hz, 1H), 7.68 (d, J=8.5 Hz, 1H), 7.60 (d,
J=1.9 Hz, 1H), 7.56 (dd, J=8.5, 2.2 Hz, 1H), 7.51 (td, J=8.7, 5.5 Hz, 1H), 7.08 - 7.02 (m, 1H),
5.13 - 5.02 (m, 2H), 3.80 (s, 3H), 3.66 - 3.52 (m, 2H), 2.71 (dt, J=6.7, 3.4 Hz, 1H), 2.54 - 2.45
(m, 1H), 2.32 - 2.23 (m, 2H), 2.12 - 2.03 (m, 1H), 1.93 (dtd, J=14.1, 7.1, 4.0 Hz, 1H), 1.69 - 1.59
1
9
(
m, 1H), 1.46 - 1.36 (m, 1H), 1.03 (d, J=6.9 Hz, 3H), 0.81 - 0.65 (m, 1H). F NMR (471 MHz,
CD OD) δ -77.34, -116.93, -117.42.
3
Methyl N‐[(10R,14S)‐14‐[4‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxopiperidin‐1‐yl]‐10‐methyl‐9‐
2
,7
oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]carbamate, TFA
salt (6b). To a solution of 6f (6.7 mg, 9.45 µmol) in EtOAc (1 mL) was added platinum(IV)
oxide (2.2 mg, 9.45 µmol). The reaction was purged with H (from a balloon) for several minutes
2
and then stirred at rt under H overnight. It was filtered and rinsed with EtOAc and DCM,
2
concentrated. Reverse phase preparative HPLC purification provided 6f, TFA salt (1.8 mg, 25%
+
yield) as an off-white solid. MS (ESI) m/z: 597.1 (M+H) . Orthogonal HPLC purity: 97.7%,
retention time = 7.17 min (HPLC Method A); 100%, retention time = 9.31 min (HPLC Method
1
B). H NMR (500 MHz, CD OD): a 2:1 mixture of diastereomers. δ 10.85 (br s, 1H), 10.02 -
3
ACS Paragon Plus Environment
2
4

Journal of Medicinal Chemistry
Page 26 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
.90 (m, 1H), 9.25 (br s, 1H), 9.05 (br s, 1H), 8.88 - 8.82 (m, 1H), 8.77 (br s, 2H), 8.62 (br d,
J=5.0 Hz, 1H), 8.26 - 8.13 (m, 1H), 6.64 - 6.56 (m, 1H), 4.97 (s, 3H), 4.91 - 4.73 (m, 3H), 4.07 -
3.95 (m, 1H), 3.97 - 3.76 (m, 2H), 3.60 - 3.44 (m, 2H), 3.35 - 3.20 (m, 2H), 3.16 - 3.03 (m, 1H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.82 (br d, J=5.5 Hz, 1H), 2.55 - 2.40 (m, 1H), 2.32 - 2.11 (m, 4H).
Methyl N‐[(10R,14S)‐14‐[4‐(3‐chloro‐2,6‐difluorophenyl)‐2‐oxopiperazin‐1‐yl]‐10‐m
2
,7
ethyl‐9‐oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]carbamate
,
TFA salt (6c). A clear, colorless solution of 22a (44 mg, 0.12 mmol), 24 (43 mg, 0.12 mmol)
and Hunig's base (0.10 mL, 0.60 mmol) in DMA (1.2 mL) was heated at 120 ºC overnight. The
reaction was diluted with methanol and purified by reverse phase chromatography (2x) to give 6c
+
+
(3.9 mg, 4%) as an off-white solid. MS (ESI) m/z: 598.2 (M+H) and 600.1 (M+2+H) . Orthogonal
HPLC purity: 96.7%, retention time = 5.69 min (HPLC Method A); 96.3%, retention time = 6.50
1
min (HPLC Method B). H NMR (500MHz, CD OD)  9.64 (br. s, 1H), 8.74 (d, J=5.8 Hz, 1H),
3
8.02 (d, J=1.4 Hz, 1H), 7.82 (dd, J=5.9, 1.8 Hz, 1H), 7.65 - 7.60 (m, 1H), 7.58 - 7.52 (m, 2H),
7
1
.22 (ddd, J=9.1, 8.0, 5.2 Hz, 1H), 6.98 (ddd, J=11.0, 9.1, 1.9 Hz, 1H), 5.39 (dd, J=12.7, 4.7 Hz,
H), 3.92 (s, 2H), 3.77 (s, 3H), 3.64 - 3.56 (m, 2H), 3.54 - 3.48 (m, 2H), 2.65 - 2.57 (m, 1H), 2.39
- 2.27 (m, 1H), 2.10 - 2.00 (m, 1H), 1.95 - 1.85 (m, 1H), 1.66 - 1.55 (m, 1H), 1.35 - 1.24 (m, 1H),
19
1
.05 (d, J=6.9 Hz, 3H), 1.02 - 0.90 (m, 1H). F NMR (471MHz, CD OD)  -77.44, -122.34 (d,
3
J=7.2 Hz), -122.74 (d, J=7.2 Hz).
Methyl N‐[(10R,14S)‐14‐[4‐(3‐chloro‐2,6‐difluorophenyl)‐2,3‐dioxopiperazin‐1‐yl]‐1
2,7
0
‐methyl‐9‐oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]carba
mate, TFA salt (6d). To a solution of 22a (7.0 mg, 0.019 mmol) in DCE (2 mL) was added
NaBH(OAc) (6.0 mg, 0.028 mmol), one drop of AcOH (1.1 µL, 0.019 mmol) and 4 Å
3
molecular sieve (10 mg, 0.019 mmol). The reaction was cooled down to 0 ºC, 25 (7.0 mg, 0.023
ACS Paragon Plus Environment
2
5

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mmol) in DCE (0.5 mL) was added. After 5 min, the reaction was warmed to rt. After 3 h, the
reaction mixture was filtered off solid and concentrated. Reverse phase preparative HPLC
purification afforded 6c, TFA salt (3.3 mg, 22% yield) as a pale yellow solid. MS (ESI) m/z:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
6
12.1 (M+H) . Orthogonal HPLC purity: > 95.2%, retention time = 6.02 min (HPLC Method A);
1
>
95.8%, retention time = 6.16 min (HPLC Method B). H NMR (500MHz, CD OD)  8.68 (dd,
3
J=5.4, 2.6 Hz, 1H), 7.83 (s, 1H), 7.64 - 7.55 (m, 3H), 7.54 - 7.48 (m, 2H), 7.22 - 7.14 (m, 1H),
5
3
.56 (dd, J=12.5, 4.5 Hz, 1H), 4.13 (br. s., 1H), 4.06 - 3.96 (m, 2H), 3.95 - 3.88 (m, 1H), 3.77 (s,
H), 2.63 (dd, J=6.6, 5.5 Hz, 1H), 2.24 - 2.14 (m, 1H), 2.03 (dt, J=12.0, 5.9 Hz, 1H), 1.93 (td,
J=8.7, 3.2 Hz, 1H), 1.56 (td, J=14.7, 8.0 Hz, 1H), 1.34 (br. s., 1H), 1.03 (d, J=6.9 Hz, 3H), 0.91
dd, J=13.1, 7.0 Hz, 1H).
Methyl N‐[(10R,14S)‐14‐[4‐(3‐chloro‐2,6‐difluorophenyl)‐2,5‐dioxopiperazin‐1‐yl]‐1
(
2
,7
0
‐methyl‐9‐oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]carba
mate, HCl salt (6e). To a solution of 27, 2 TFA (29 mg, 0.036 mmol) in THF (1 mL) was added
DIEA (0.03 ml, 0.18 mmol). The reaction mixture was sonicated and cooled down to 0 ºC. Two
drops of 2-chloroacetyl chloride (6.1 mg, 0.054 mmol) were added. After 2 min at 0 ºC, the
reaction mixture was diluted with EtOAc, washed with water, brine, dried over MgSO , filtered
4
and concentrated. Purification by silica gel chromatography yielded acylated intermediate (22
+
mg, 94% yield) as a white solid. MS (ESI) m/z: 648.1 (M+H) . To a solution of the acylated
o
intermediate (22 mg, 0.034 mmol) in MeOH (1 mL) at 0 C was added NaOMe (25 wt. % in
MeOH) (36.7 mg, 0.17 mmol) diluted in 1 mL of MeOH. The ice water bath was removed after
addition and the reaction mixture was stirred at rt overnight. The reaction was quenched with
HCl (1.25M in MeOH) (0.11 mL, 0.14 mmol), diluted with DCM, washed with water and brine,
dried over MgSO , filtered off solid and concentrated. Purification by silica gel chromatography
4
ACS Paragon Plus Environment
2
6

Journal of Medicinal Chemistry
Page 28 of 56
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
yielded 6e (20 mg, 81% yield) as a white solid. 6e was converted to its HCl salt by treatment
+
with HCl in Dioxane (4M). MS (ESI) m/z: 612.2 (M+H) . Orthogonal HPLC purity: 97.3%,
retention time = 5.95 min (HPLC Method A); 96.1%, retention time = 6.20 min (HPLC Method
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
B). H NMR (500MHz, CD OD)  8.82 (dd, J=6.2, 1.8 Hz, 1H), 8.28 (d, J=1.4 Hz, 1H), 8.04 (d,
3
J=6.3 Hz, 1H), 7.69 (d, J=8.3 Hz, 1H), 7.64 - 7.54 (m, 3H), 7.18 (td, J=9.2, 1.1 Hz, 1H), 5.36 -
5.27 (m, 1H), 4.54 - 4.32 (m, 4H), 3.77 (s, 3H), 2.68 (dd, J=6.9, 4.7 Hz, 1H), 2.42 - 2.30 (m,
1
H), 2.26 - 2.13 (m, 1H), 1.92 (td, J=9.2, 3.9 Hz, 1H), 1.63 (td, J=14.8, 7.6 Hz, 1H), 1.45 - 1.26
(m, 1H), 1.04 (d, J=6.9 Hz, 3H), 0.97 - 0.80 (m, 1H).
Methyl N‐[(10R,14S)‐14‐[4‐(3‐chloro‐2,6‐difluorophenyl)‐6‐oxo‐1,2,3,6‐tetrahydropyridin‐1
2
,7
‐
yl]‐10‐methyl‐9‐oxo‐8,16‐diazatricyclo[13.3.1.0 ]nonadeca‐1(19),2,4,6,15,17‐hexaen‐5‐yl]c
arbamate, Trifluoroacetic acid salt and Hydrochloric acid salt (6f). To a solution of 22a,
2TFA (300 mg, 0.46 mmol) in DCM (6.17 mL) was added DIEA (0.49 mL, 2.78 mmol). The
reaction was stirred for 45 min before compound 7 (94 mg, 0.46 mmol) was added in one
portion. The reaction was stirred for additional 5 h. Diethyl (2-chloro-2-oxoethyl)phosphonate
(248 mg, 1.16 mmol) in DCM (1.5 mL) was added dropwise. After 2 min, the reaction was
quenched by addition of NH Cl. It was extracted with DCM. The organic layer was washed 2x
4
with sat. NaHCO , brine, dried over MgSO , filtered and concentrated. Purification by silica gel
3
4
chromatography yielded intermediate phosphonate (260 mg, 75%) as a white solid. MS (ESI)
+
m/z: 749.0 (M+H) . To a clear solution of the intermediate phosphonate (260 mg, 0.35 mmol) in
MeOH (6.9 mL) was added NaOMe (25% in MeOH) (225 mg, 1.04 mmol) and the reaction was
stirred at rt for 5 min. HCl (0.55 mL, 1.25 N in MeOH) was added and MeOH was removed. The
residue was diluted with DCM and washed with H O. The aqueous layer was extracted with
2
ACS Paragon Plus Environment
2
7

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
DCM. The combined organic layers were washed with brine, dried over MgSO , filtered and
4
concentrated. Purification by silica gel chromatography yielded 148 mg of 6f as a white solid. To
a suspension of 6f in DCM (10 mL) at 0 ºC was added TFA (114 mg, 1 mmol). After removal of
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
solvents, 6f, TFA salt (169 mg, 68%) was obtained as a pale yellow solid. MS (ESI) m/z: 594.9
+
(
M+H) . Orthogonal HPLC purity: 100%, retention time = 6.94 min (HPLC Method A); 100%,
1
retention time = 6.68 min (HPLC Method B). H NMR (500 MHz, CD OD) δ 9.70 (s, 1H), 8.78
3
(d, J=6.1 Hz, 1H), 8.15 (s, 1H), 7.93 (dd, J=5.9, 1.5 Hz, 1H), 7.68 (d, J=8.3 Hz, 1H), 7.64 - 7.52
(m, 3H), 7.12 (td, J=9.2, 1.7 Hz, 1H), 6.13 (s, 1H), 5.40 (br dd, J=12.4, 4.7 Hz, 1H), 3.86 - 3.70
(m, 5H), 2.95 - 2.84 (m, 1H), 2.82 - 2.73 (m, 1H), 2.67 (br dd, J=6.9, 5.0 Hz, 1H), 2.38 - 2.27
(m, 1H), 2.18 - 2.06 (m, 1H), 2.00 - 1.90 (m, 1H), 1.71 - 1.59 (m, 1H), 1.42 - 1.27 (m, 1H), 1.08
d, J=6.9 Hz, 3H), 1.05 - 0.94 (m, 1H). A suspension of 6f (285 mg, 0.479 mmol) in MeOH (10
(
mL) was added HCl in MeOH (1.15 mL, 1.44 mmol) at 0 ºC. The suspension was taken out to rt
and was filtered through a Whatman autovial filter. The filtrate was concentrated to give a
yellowish solid. Small amount of MeOH was added to dissolve followed by addition of EtOAc.
White solid crashed out. The solid was filtered and rinsed with EtOAc, dried and lyopholized to
1
give 6f, HCl salt (232 mg, 77%) as a pale yellow solid. H NMR (500 MHz, CD OD) δ 10.06 (s,
3
1
H), 9.66 (s, 1H), 8.77 (d, J=6.10 Hz, 1H), 8.21 (s, 1H), 7.94 (d, J=5.57 Hz, 1H), 7.65 (d, J=8.17
Hz, 1H), 7.56 (s, 1H), 7.56 (m, 1H), 7.53 (m, 1H), 7.10 (td, J=9.33, 1.36 Hz, 1H), 6.09 (s, 1H),
.34 (dd, J=12.42, 4.68 Hz, 1H), 3.82 (m, 1H), 3.76 (s, 3H), 3.75 (m, 1H), 2.92 (m, 1H), 2.76 (dt,
5
J=17.72, 5.84 Hz, 1H), 2.69 (m, 1H), 2.35 (m, 1H), 2.10 (m, 1H), 1.94 (m, 1H), 1.62 (m, 1H),
13
1
1
.36 (m, 1H), 1.03 (d, J=6.75 Hz, 3H), 0.89 (m, 1H). C NMR (126 MHz, CD OD) δ 177.8,
3
67.6, 159.6 (dd, J=244.2, 5.4 Hz), 157.9, 156.3 (dd, J=250.8, 6.6 Hz), 156.1, 154.7, 144.4,
144.4, 144.0, 137.6, 132.5 (d, J=10.0 Hz), 131.7, 128.6, 127.4, 127.4, 125.5, 118.8, 118.5, 118.5
ACS Paragon Plus Environment
2
8