Structure based design of macrocyclic factor XIa inhibitors: Discovery of cyclic P1 linker moieties with improved oral bioavailability

Article abstract of DOI:10.1016/j.bmcl.2019.08.008

This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability. Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1 moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmacokinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10, that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic P1 linkers demonstrated enhanced bioavailability and improved potency.

Full text of DOI:10.1016/j.bmcl.2019.08.008

Bioorganic&MedicinalChemistryLetters29(2019)126604
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure based design of macrocyclic factor XIa inhibitors: Discovery of
cyclic P1 linker moieties with improved oral bioavailability
Charles G. Clark⁎, Karen A. Rossi, James R. Corte, Tianan Fang, Joanne M. Smallheer,
Indawati De Lucca, David S. Nirschl, Michael J. Orwat, Donald J.P. Pinto, Zilun Hu,
Yufeng Wang, Wu Yang, Yoon Jeon, William R. Ewing, Joseph E. Myers Jr., Steven Sheriff,
Zhen Lou, Jeffrey M. Bozarth, Yiming Wu, Alan Rendina, Timothy Harper, Joanna Zheng,
Baomin Xin, Qian Xiang, Joseph M. Luettgen, Dietmar A. Seiffert, Ruth R. Wexler,
Patrick Y.S. Lam
Bristol-Myers Squibb Company, P.O. Box 4000, Princeton, NJ 08543, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
This manuscript describes the discovery of a series of macrocyclic inhibitors of FXIa with oral bioavailability.
Assisted by structure based drug design and ligand bound X-ray crystal structures, the group linking the P1
moiety to the macrocyclic core was modified with the goal of reducing H-bond donors to improve pharmaco-
kinetic performance versus 9. This effort resulted in the discovery of several cyclic P1 linkers, exemplified by 10,
that are constrained mimics of the bioactive conformation displayed by the acrylamide linker of 9. These cyclic
P1 linkers demonstrated enhanced bioavailability and improved potency.
Factor XIa inhibitors
FXIa
Activated partial thromboplastin time
aPTT
Thrombosis
Anticoagulant
Bioavailability
Cardiovascular disease (CVD) and stroke cause extensive economic
strain on society and are the leading cause of death worldwide.1 As of
2013, within the US alone, one out of every three deaths could be at-
tributed to some form of CVD despite the numerous strategies that exist
to reduce morbidity and mortality by improving or preserving good
cardiovascular health.2 Antithrombotic agents offer a well proven op-
tion for treatment and prevention.3 Warfarin, a decades old agent for
thromboembolic disease management, has a narrow therapeutic index
that requires frequent monitoring, and its inhibition is non-specific.
These drawbacks focused attention on the need for the discovery and
development of safer and more easily managed medicines.4 Within the
past decade novel oral anticoagulants (NOACS), which act as inhibitors
of thrombin or the coagulation factor Xa (FXa), have provided im-
proved benefits in the treatment or prevention of DVT (deep vein
thrombosis), PE (pulmonary embolism) and stroke arising from non-
valvular atrial fibrillation (NVAF).5
animal thrombosis models without a significant effect on bleeding
time.7 Moreover, clinical evidence from a FXI antisense oligonucleotide
showed a decreased occurrence of DVT during knee replacement sur-
gery, without increasing the number of bleeding events versus en-
oxaparin.8
As part of our program to discover FXIa inhibitors amenable to oral
dosing, we previously disclosed potent phenylimidazole compounds,9
exemplified by 1,10,11 which while potent and selective, exhibit low
oral bioavailability.
Coagulation factor XIa (FXIa) offers a newer target in antith-
rombotic therapy.6 In the classic waterfall model, FXIa lies upstream in
the intrinsic pathway, and inhibition may provide protection from
thrombosis while limiting bleeding risk. This is supported by the de-
monstrated efficacy of small molecule FXIa inhibitors in multiple
In this communication we describe the use of structure-based drug
design and ligand bound X-ray crystal structures that led to the
⁎ Corresponding author.
E-mail address: charles.clark@bms.com (C.G. Clark).
https://doi.org/10.1016/j.bmcl.2019.08.008
Received 23 May 2019; Received in revised form 30 July 2019; Accepted 4 August 2019
Availableonline16August2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.

C.G. Clark, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126604
Fig. 1. Amide hydrogen remval strategies for 1. Ki values shown were obtained at 25 °C.
O
H
O
N
H
N
H
β
H
s-trans
fXIa crystallographic
conformation
s-cis
expected
conformation
G = -1.8 kcal/mol
Fig. 2. X-ray crystal of 1 bound to FXIa with red arrow indicating potential point of conformational constraint. The red spheres represent water molecules.
discovery of a series of FXIa inhibitors with novel P1 linkers that have
greatly enhanced bioavailability and improved potency. To improve the
permeability and oral absorption of compounds represented by 1, the
number of hydrogen bond donors/acceptors and the polar surface area
(PSA) were reduced.12 The amide hydrogen and the tetrazole moiety,
which were responsible for 31% of the total PSA, were eliminated.13
Since removal of the tetrazole resulted in a significant loss in FXIa ac-
tivity,10 the aim was to lower the conformational energy by modifying
the acrylamide linker in a fashion that would pre-organize the system
towards the bioactive conformation. Moreover, rigidifying a molecule
through removal of rotatable bonds has been shown to have a positive
effect on permeability.14,15
Evaluation of the FXIa protein bound crystal structure of 116 in-
dicated an eclipsed nature of the amide proton with the β-proton of the
acrylamide (Fig. 2). This bound conformation exists in a slightly
strained s-trans orientation (Fig. 2 inset),17 and suggested the potential
for constraint via cyclization as indicated by the red arrow. The s-trans
conformation was unexpected as the vast majority of acyclic aliphatic
acrylamides found in the Cambridge Structural Database (109 out of
111) exist in the s-cis conformation.
Based on this observation a variety of cyclic linker motifs were
designed. These motifs were prioritized with the aid of molecular
modelling and synthesized on the chemotype of 1 removing the tetra-
zole moiety to improve permeability. The 6-membered cyclic urea 7
and cyclic carbamate 818 were found to maintain or improve fXIa af-
finity versus the acyclic urea and amide analogs 5 and 6 (Fig. 3).
Incorporation of the cyclic carbamate into our previously described
13-membered macrocyclic FXIa inhibitors19 provided a 78-fold gain in
FXIa affinity in 1020 as compared to the linear analog 8 (Fig. 4). A 33-
fold gain in FXIa affinity was also observed when the acrylamide moiety
of 9 was replaced in 10 by the cyclic carbamate, as compared to the
Previous attempts towards removing the amide hydrogen of 1 via N-
alkylation, in the case of 2, or by tying back to either the benzylic
methylene (cyclization route A to give 3) or to the imidazole (cycliza-
tion route B to give 4) were not successful with respect to maintaining
FXIa activity (Fig. 1). Therefore an alternate cyclization strategy (cy-
clization route C), which incorporates a conformational constraint be-
tween the amide nitrogen and the acrylamide linker, was designed.
2

C.G. Clark, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126604
Table 1
Cyclic linker modifications in the 13-membered macrocycles.
Compd #
L
R1
FXIa Ki
(nM)
aPTT EC1.5x
(μM)
LMa
t1/2 (min)
H, R
Fig. 3. Most active P1 cyclic linkers from initial screen. Ki values shown were
obtained at 25 °C.
10
12
13
14
15
16
17
A
B
C
A
B
C
D
H
H
H
F
17
5.9
3.7
3.0
2.0
1.8
1.4
> 40
NT
4.8
5.3
4.7
1.8
2.2
333
35, 18
6, 5
more moderate gain obtained with the linear imidazole compounds
shown in Fig. 3. The epimer 11 was less active with respect to both FXIa
affinity and in vitro anticoagulant activity (aPTT). Macrocyclic amide
10 met our targeted molecular properties goal of PSA < 130 Å2, while
minimizing the total number of H-bond donors and rotatable bonds.21
The urea and lactam linkers when incorporated in the macrocylic
series (1222 and 13,23 Table 1), also displayed both enhanced FXIa
affinity and aPTT activity. Fluoro substitution on the phenyl ring,
previously shown to further enhance activity,19,24 resulted in a 2–3 fold
improvement in both FXIa affinity and aPTT activity in compounds
14–16. Liver microsome (LM) stability for carbamate 14 and lactams
13 and 16 was determined to be low as compared to the ureas 12 and
15 and acrylamide 9.25 In an attempt to remedy the low metabolic
stability, the linker carbon atom was replaced with a nitrogen as in
piperazinone 17, which resulted in significantly reduced FXIa affinity
versus 13.
8, 15
41, 26
9, 7
F
F
H
15, NT
a
LM = liver microsome stability half-life in minutes for human (H) & rat (R).
To further explore the cyclic linker series, substituents on the P1
phenyl and cyclic carbamate ring were varied (Table 2). Transitioning
into the more potent chloro imidazole series (X = Cl) yielded a gain in
FXIa affinity and aPTT potency similar to earlier disclosed com-
pounds.10,19 Addition of a 6-fluoro substitution on the P1 phenyl in 20
showed a modest improvement in FXIa affinity. Compound 21 with a 3-
chloro-2-fluoro-6-trifluoromethyl phenyl P1 group had similar FXIa Ki
and aPTT to compound 20. The 2,6-di-fluoro analog 2226 gained FXIa
affinity and aPTT activity versus the mono-F analog 20; however, LM
stability remained poor. Since the benzylic position (R3) could serve as
a potential metabolic soft spot, an attempt to improve LM stability in
this series was explored. The deutero analog 23 was prepared in an
attempt to improve the microsomal stability. While it showed a 3-fold
gain in Ki and 2-fold gain in aPTT, no significant change in liver mi-
crosome stability was observed.
A comparison of the FXIa-bound crystal structures of cyclic carba-
mate 10 and the corresponding acrylamide 1819 show that they bind in
nearly identical conformations (Fig. 5), such that the P1 phenyl moiety
of each nearly superimposes. Interpretation of the crystal structure of
10 suggests that maintaining a hydrogen on the atom alpha to the
carbonyl (i.e., methylene in the case of the lactam and NH in the case of
the urea) could be beneficial in order to interact with the carbonyl of
Cys191 (distance 3.2 Å) and could account for the slightly greater ac-
tivity observed for the lactam and urea chemotypes versus the (R)-
carbamate.
The PK profiles of two of the three types of P1 linkers, cyclic car-
bamate 10 and lactam 13, plus the acrylamide, represented by com-
pounds 9 and 18, were evaluated in a rat PK study, the results of which
are summarized in Table 3. Compound 9 exhibited high clearance, a
Fig. 4. Introduction of cyclic linker into macrocyclic amide series leading to discovery of 10. Comparison of cyclic carbamate epimers 10 and 11.
3

C.G. Clark, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126604
O
O
HN
H
N
N
O
N
O
O
HN
O
O
10
Cl
FXIa Ki = 17 nM
O
N N
O
HN
H
N
N
N
N
N
H
HN
18
FXIa Ki = 0.16 nM
Cl
Fig. 5. Overlay of X-ray crystal structures of cyclic carbamate 10 (cyan) and chlorophenyl tetrazole acrylamide 18 (gold) bound to FXIa. The dotted line indicates a
potential H-bond from the position alpha to the carbonyl to the oxygen of Cys191.
Table 2
Table 4
Modification to P1 phenyl and cyclic carbamate in 13-membered macrocycle.
Human serine protease selectivity profile for 10.
Human Enzyme Ki (nM)a
10
13
Factor XIa
Factor VIIab
Factor IXa
Factor Xab
Factor XIIa
Thrombinb
Trypsinb
17
5.3
> 13,300
> 27,100
3660
4850
> 27,100
> 9,000
> 3050
> 13,300
> 6,260
> 7,160
> 15,200
> 6,150
> 15,100
> 3,050
> 13,300
6,260
Activated Protein C
Plasmin
> 21,500
> 15,200
> 6,150
> 15,100
TPA
Compd #
R1
R2
R3
X
FXIa Ki
(nM)
aPTT EC1.5x
(μM)
LMa
Urokinase
t1/2 (min)
H, R
a
b
Ki values in nM were obtained using human purified enzymes at 37 °C.
Ki values in nM were obtained using human purified enzymes at 25 °C.
10
19
20
21
22
23
H
H
F
H
H
H
F
H
H
H
H
H
D
H
17
5.9
NT
Cl
Cl
Cl
Cl
Cl
4.4
2.6
11, 14
5, 18
18, 49
9, 22
16, 31
2.1
1.9
well above hepatic blood flow and a shortened half-life with modest
oral bioavailability. Compound 18 displayed medium clearance, a re-
duced Vdss, a shorter half-life, and oral bioavailability of 0.8%.
Compound 10 was tested across a number of coagulation related
human serine proteases and trypsin showing at least several hundred
fold selectivity (Table 4).
CF3
F
1.8
2.4
F
0.95
0.27
0.91
0.45
F
F
a
LM = liver microsome stability half-life in minutes for human (H) & rat (R).
half-life of 1.4 hr, and oral bioavailability of 2%. Cyclic carbamate 1027
also displayed high clearance, but had a reduced steady state volume of
distribution (Vdss), a shorter half-life, and significant improvement in
oral bioavailability (F% = 59). Lactam 13 was shown to have clearance
Cyclic carbamate compounds 10 and 11 were synthesized as shown
in Scheme
1
starting from commercially available 3-chlor-
obenzaldehyde 24. Addition of vinyl magnesium bromide to the alde-
hyde at −78 °C provided vinyl alcohol 25, which was then oxidized
Table 3
Pharmacokinetic profile of selected compounds in rat studies.
Compd #
FXIa Ki
aPTT EC1.5x
LM
Caco-2
AB/BA
(nm/s)
AUC (nM*h)
Dose IV/PO
(mpk)
Cla
Vdss
T1/2
(h)
F
PSA
(Å2)
(nM)
(μM)
t1/2 (min)
H, R
(mL/min/kg)
(L/kg)
(%)
9
560
17
> 40
5.9
32, 45
NTd
< 15/324
NT
27
0.52/1.04b
0.68/1.36b
0.63/1.27c
0.79/1.58b
44
3.2
1.3
4.6
0.7
1.4
0.8
0.5
0.7
2
125
126
116
169
10
13
18
523
31
40
59
15
0.8
5.3
2.9
6, 5
46/309
< 15/58
188
28
0.16
0.27
41, 39
11
a
b
c
Vehicle for iv and po: 70% PG; 20% water; 10% ethanol.
Compound was dosed in a cassette format.
Compound was dosed in a discrete format.
See footnote 27.
d
4

C.G. Clark, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126604
Scheme 1. Reagents and conditions: (a) vinylmagnesium bromide, THF, −78 °C to rt, > 90%; (b) CrO3, H2SO4 (aq.), acetone, 0 °C to rt, > 90%; (c) TMSCl, NaI, H2O,
MeCN, > 90%; (d) NaBH4, H2O, THF; (e) 29a, K2CO3, MeCN, 80 °C, 41%; (f) CDI, TEA, dioxane, 110 °C, 56%; (g) 4 M HCl, dioxane, 75 °C, 54%; (h) prep chiral SFC to
separate isomers.
Scheme 2. Reagents and conditions: (a) vinylmagnesium bromide, THF, −78 °C to rt, 73% (23% de); (b) 4 M HCl in dioxane then Boc2O, TEA, MeCN, 40% over two
steps; (c) 9-BBN, THF, 100 °C, then H2O2, NaOH, EtOH, 45 °C, 73%; (d) MsCl, DIPEA, DCM, then NaI, acetone, reflux, 81%; (e) 29a, K2CO3, MeCN, 75 °C; (f) 20%
TFA, DCM, 37% over 2 steps; (g) CDI, THF, 67%; (h) 4 M HCl, dioxane, 75 °C, then preparatory reverse phase HPLC to separate isomers, 22%.
Scheme 3. Reagents and conditions: (a) methyl
acetoacetate, piperidine, MeOH; (b) NaOMe, re-
flux; (c) HCl, MeOH, reflux, 52% over three steps;
(d) NaOH, MeOH (e) BH3·DMS, 64% over two
steps, then preparatory chiral SFC to separate en-
antiomers; (f) MsCl, TEA, DCM, 0 °C, 89%; (g) 29a,
DIPEA, PhMe, 150 °C, 18%; (h) 4 M HCl, dioxane,
65 °C, 35%.
with Jones reagent to the corresponding vinyl ketone 26. Addition of
HI, generated in situ from NaI and TMSCl, by the method of Irifune
et al.,28 to the double bond gave the iodoketone 27, which was then
reduced with NaBH4 to the racemic iodoalcohol 28. Alternatively, io-
doketone 27 can be reduced using (S)-CBS reagent29 to yield the (R)-
iodo-alcohol 28 in > 90% yield and 60–70% ee. Alkylation of macro-
cyclic amine 29a with iodide 28 in the presence of K2CO3 in acetonitrile
at 80 °C provided the aminoalcohol intermediate 30. Cyclization by
treatment with CDI and TEA in refluxing dioxane provided cyclic car-
bamate 31. Removal of the SEM protecting group from the imidazole
and chiral separation afforded 10 and 11.
Compounds 14, and 19–22 were similarly synthesized by sub-
stituting the appropriately substituted benzaldehyde starting material
for 24 and the applicable macrocyclic amine 29a-b to generate the
desired compounds. Compound 23 was synthesized by use of NaBD4 to
reduce the appropriately substituted ketone 27 to a deutero-analog of
5

C.G. Clark, et al.
Bioorganic&MedicinalChemistryLetters29(2019)126604
Scheme 4. Reagents and conditions: (a) methyl 2-bromoacetate, K2HPO4, KI, MeCN, reflux, 48%; (b) NaOH, MeOH (c) BH3·DMS, 34% over two steps; (d) MsCl,
pyridine, DCM, 0 °C, 100%; (e) 29a, DIPEA, PhMe, 120 °C, 20%; (f) 4 M HCl, dioxane, 75 °C, 28%.
31, which was then used in the same fashion as shown in Scheme 1.
The cyclic urea compound 12 was synthesized as shown in Scheme
2 starting from commercially available (S)-N-(3-chlorobenzylidene)-2-
methylpropane-2-sulfinamide 32. Addition of vinyl magnesium bro-
mide to the sulfinimine at −78 °C provided the 1-allyl sulfinamide 33
in 23% de. Removal of the chiral auxillary with HCl and protection of
the amine with Boc2O yielded N-allyl Boc-amine 34, which was hy-
droborated using 9-BBN, and the intermediate oxidized with H2O2 to
give the Boc-protected amino alcohol 35. Treatment of 35 with mesyl
chloride followed by NaI in refluxing acetone provided the protected
amino iodide 36. This intermediate iodide 36 was used to alkylate
macrocyclic amine 29a in the presence of K2CO3 in acetonitrile at 75 °C,
followed by treatment with TFA to furnish the diamine 37. Cyclization
by treatment with CDI in THF at ambient temperature provided cyclic
urea 38. Subsequent removal of the SEM protecting group from the
imidazole and reverse phase HPLC separation of the diastereomeric
isomers afforded 12. Compound 15 was similarly synthesized by sub-
stituting (S)-N-(3-chloro-2,6-difluorobenzylidene)-2-methylpropane-2-
sulfinamide for 32 and the macrocyclic amine 29a to generate the
desired compound.
cyclic carbamate 10 was evaluated in a rat pharmacokinetic model and
found to impart a significant increase in oral bioavailability.
Acknowledgements
The authors thank Atsu Apedo and Douglas B. Moore for chiral se-
parations. 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. Use of the IMCA-
CAT beamline 17-ID at the Advanced Photon Source was supported by
the companies of the Industrial Macromolecular Crystallography
Association through a contract with Hauptman-Woodward Medical
Research Institute.
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11.. FXIa Ki values were obtained from purified human enzyme at 37 °C unless otherwise
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7