Neutral macrocyclic factor VIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.04.008

Factor VIIa (FVIIa) inhibitors have shown strong antithrombotic efficacy in preclinical thrombosis models with limited bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful due to the requirement of a basi

Full text of DOI:10.1016/j.bmcl.2017.04.008

Accepted Manuscript
Neutral macrocyclic Factor VIIa inhibitors
Nicholas R. Wurtz, Brandon L. Parkhurst, Indawati DeLucca, Peter W. Glunz,
Wen Jiang, Xiaojun Zhang, Daniel L. Cheney, Jeffrey M. Bozarth, Alan R.
Rendina, Anzhi Wei, Tim Harper, Joseph M. Luettgen, Yiming Wu, Pancras C.
Wong, Dietmar A. Seiffert, Ruth R. Wexler, E. Scott Priestley
PII:
S0960-894X(17)30372-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.008
BMCL 24861
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 December 2016
28 March 2017
1 April 2017
Please cite this article as: Wurtz, N.R., Parkhurst, B.L., DeLucca, I., Glunz, P.W., Jiang, W., Zhang, X., Cheney,
D.L., Bozarth, J.M., Rendina, A.R., Wei, A., Harper, T., Luettgen, J.M., Wu, Y., Wong, P.C., Seiffert, D.A., Wexler,
R.R., Priestley, E.S., Neutral macrocyclic Factor VIIa inhibitors, Bioorganic & Medicinal Chemistry Letters (2017),
doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.008
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Neutral Macrocyclic Factor VIIa Inhibitors
Leave this area blank for abstract info.
Nicholas R. Wurtz, Brandon L. Parkhurst, Indawati DeLucca, Peter W. Glunz, Wen Jiang, Xiaojun Zhang,
Daniel L. Cheney, Jeffrey M. Bozarth, Alan R. Rendina, Anzhi Wei, Tim Harper, Joseph M. Luettgen, Yiming
Wu, Pancras C. Wong, Dietmar A. Seiffert, Ruth R. Wexler and E. Scott Priestley

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Neutral macrocyclic Factor VIIa inhibitors
Nicholas R. Wurtz*, Brandon L. Parkhurst, Indawati DeLucca, Peter W. Glunz, Wen Jiang, Xiaojun Zhang,
Daniel L. Cheney, Jeffrey M. Bozarth, Alan R. Rendina, Anzhi Wei, Tim Harper, Joseph M. Luettgen,
Yiming Wu, Pancras C. Wong, Dietmar A. Seiffert, Ruth R. Wexler and E. Scott Priestley
Bristol-Myers Squibb Research and Development, Princeton, NJ 08534, United States
ARTICLE INFO
ABSTRACT
Factor VIIa (FVIIa) inhibitors have shown strong antithrombotic efficacy in preclinical
thrombosis models with limited bleeding liabilities. Discovery of potent, orally active FVIIa
inhibitors has been largely unsuccessful due to the requirement of a basic P1 group to interact
with Asp189 in the S1 binding pocket, limiting their membrane permeability. We have
combined recently reported neutral P1 binding substituents with a highly optimized macrocyclic
chemotype to produce FVIIa inhibitors with low nanomolar potency and enhanced permeability.
Article history:
Received
Revised
Accepted
Available online
Keywords:
TF-FVIIa
2016 Elsevier Ltd. All rights reserved
.
macrocycles
anticoagulant
neutral P1
Vitamin K antagonists and heparins have been part of the
standard of care for thromboembolic disorders for many years,
but these antithrombotic therapies possess significant limitations.
Vitamin K antagonists, such as warfarin, have significant drug
and food interactions, which create a challenge for maintenance
of the desired therapeutic effect without excessive bleeding.1
Heparins are parenterally administered, which prevents their
widespread use in long-term therapy. Several decades of research
have produced anticoagulants targeting thrombin or FXa that
demonstrate improved efficacy with decreased bleeding in
clinical studies.2,3 Even though these new therapies are replacing
the older antithrombotics, research to find safe, effective
anticoagulants to complement existing therapies continues.
The extrinsic pathway in the clotting cascade is initiated by the
tissue factor-Factor VIIa complex (TF-FVIIa) that activates
Factors IX and X.4 This protein complex has attracted significant
interest because small molecule FVIIa inhibitors have strong
efficacy in preclinical models of thrombosis with minimal effect
on provoked bleeding at antithrombotic doses.5 Inhibition of the
TF-FVIIa complex may also have therapeutic potential in other
areas such as cancer or inflammation due both to its
antithrombotic effect as well as impact on other signaling
pathways.6-8 Despite years of study, the discovery of a potent,
orally bioavailable FVIIa inhibitor clinical candidate remains
elusive.9
O
P1
O
O
NH2
HN
O
P1
O
N
N
H
H2N
HN
N
O
O
O
S
O
2
3
1
FVIIa K, nMa
i
FVII def PT, µMa
1.8
3.5
2
190
8.6
13
7.3
87
24
PAMPA, nm/s, pH 7.4
tissue kallikrein Ki, nM
110
>10000
>10000
a FVIIa enzyme assays and the FVIIa-deficient prothrombin time assay
were performed according to established protocols.4
Figure 1. Comparison of phenylpyrrolidine FVIIa inhibitors with
different P1 groups
substituents (Figure 1).11 Compound 1 with a m-aminobenzamide
P1 group is moderately potent with good selectivity and served as
a starting point for optimization. A FVIIa fragment screening
effort lead to the identification of a series of bicyclic amide P1
binding groups, such as the isoquinolinone, which were
incorporated into the phenylpyrrolidine scaffold to afford
compounds such as 2, which resulted in a significant increase in
potency and permeability.11 These neutral P1 groups have
potential advantages over the more commonly reported basic P1
groups such as amidines which typically lack permeability since
they exist as charged species at physiological pH. Additionally,
Recently we have reported the discovery of potent
phenylpyrrolidine FVIIa inhibitors with a series of neutral P110
___
∗ Corresponding author. Tel.: +1-609-466-5099; e-mail:
nicholas.wurtz@bms.com (N. Wurtz)

neutral P1 substituents often have improved selectivity because
they lack the dominant salt bridge formed between cationic P1
groups, such as amidines, and Asp189, which is located at the
base of the S1 pocket of many serine proteases. Weakly basic P1
groups, such as the aminoisoquinoline shown in compound 3, are
a significant advance,12-15 but the discovery of orally active FXa
inhibitors demonstrates the potential advantages of utilizing
neutral groups that bind in the S1 pocket.2 While the installation
of neutral P1 groups in the phenylpyrrolidine chemotype
demonstrated improved permeability and selectivity over the
weakly basic aminoisoquinoline, the molecules still do not reach
sufficient potency and clotting activity for the desired
antithrombotic effect.
organolithium or Grignard species, but efforts to find a suitable
electrophile that did not require a multistep homologation were
unsuccessful. Palladium coupling with a zinc enolate occurred
selectively at the less hindered bromide, providing an undesired
product. After trying several vinylation protocols, we discovered
that coupling trimethyl(vinyl)silane in the presence of KF, n-
Bu4NCl and Pd(dba)219 was a robust, high yielding solution that
worked for both electron rich and electron poor substituents
(Scheme 1). After the Pd-mediated vinylation, the resulting
stilbenes could be converted to the desired phenethyl alcohol by
hydroborylation with 9-BBN. This sequence was routinely
carried out on multigram scale.
Figure 2. Introduction of the m-aminobenzamide P1 into the macrocyclic
FVIIa chemotype.
Macrocyclic inhibitors of FVIIa16-18 (e.g., compound 4, Figure
2) provide an alternative to the phenylpyrrolidines as a way to
restrict the bioactive conformation of phenylglycine-based
inhibitors. The rigid macrocyclic scaffold efficiently displays P1
groups as well as provides access to key hydrophobic S2 and S1’
binding pockets necessary for high affinity binding. Initially
reported with the weakly basic aminoisoquinoline, the
macrocycles are very potent compounds with little permeability
and poor selectivity vs. tissue kallikrein. Incorporation of the
neutral P1 groups into the macrocycle chemotype was undertaken
to determine if the resulting FVIIa inhibitors would be permeable
Scheme 2. Example of synthetic route for macrocycles. (a) NaH (2.5 eq),
THF, rt, 92%; (b) Bis(neopentyl glycolato)diboron, PdCl2(dppf), KOAc, 80
°C, 4 h; prep HPLC (0.1% TFA, MeOH/water), 59%; (c) 3-aminobenzamide,
glyoxylic acid, acetonitrile/DMF, µwave, 100 °C, 600 s, 86%; (d) HCl; (e)
PyBOP, DMAP, DIEA, 26% over 2 steps.
The synthesis of macrocycle 14 is shown as an example of
assembly of the key fragments (Scheme 2). p-Bromophenethanol
9a was coupled with the phenyl carbamate 10 in the presence of
sodium hydride in good yield. As previously reported,16 bromide
11 was converted into boronic acid 12 via Suzuki-Miyaura
conditions and the hydrolysis of the resulting boronic ester
during purification by preparative HPLC. A mixture of the P1
group, in this case m-aminobenzamide, glyoxylic acid and the
boronic acid was heated in the microwave to yield the desired
phenylglycine via a Petasis reaction.12,20 Deprotection of the Boc-
group followed by PyBOP or BOP mediated cyclization using
slow addition of the starting material to the coupling reagents
over several hours provided the desired macrocycle. A mixture of
atropisomers (2:1 inactive:active) was observed after cyclization.
Atropisomers could be separated by preparative HPLC and the
enantiomers could be separated by chiral HPLC either pre- or
post- cyclization. When the atropisomers did not readily
interconvert (e.g., compound 14), the inactive isomer could be
recycled by heating in DMSO at 100 °C for 2 days to again reach
the thermal dynamic equilibrium (2:1 mixture) and repurified.
Multiple inhibitor-FVIIa co-crystal structures have established
with improved potency and selectivity.
OH
I
X
Y
X
Y
X
Y
a
b
Br
Br
Br
8a, X = H, Y = Et (>95%)
8b, X = Et, Y = Et (>95%)
8c, X = Me, Y = Me (>95%)
9a, X = H, Y = Et (81%)
9b, X = Et, Y = Et (63%)
9c, X = Me, Y = Me (72%)
7a-c
Scheme 1. Synthesis of substituted p-bromophenethylalcohols. (a)
trimethyl(vinyl)silane (4 eq), KF (3 eq), n-Bu4NCl (2 eq), Pd(dba)2 (0.1 eq),
toluene (0.5 M), molecular sieves, 170 °C, 45 min; (b) 9-BBN (10 eq), THF,
120 °C; NaOH, H2O2.
The macrocycle chemotype can be assembled from three key
fragments containing the P1, P2 and P1’ binding units. We set
out to make a variety of substituents on the internal phenyl ethyl
alcohol to access the hydrophobic P2 binding pocket. p-
Bromophenylethanols, the key intermediates for this route,
proved difficult to synthesize when symmetrically disubstituted.
Using 1,3-substituted 5-bromo-2-iodobenzenes 7a-c as starting
materials, the aryliodide could be selectively exchanged in
preference to the arylbromide to form the corresponding
that the
R stereochemistry is the active enantiomer for
phenylglycine inhibitors of FVIIa.16-18
Table 1. Benzamide macrocycle SAR.

rotomer 19, which had similar potency to unsubstituted 18. The
crystal structure of compound 14 (Figure 3) shows the ethyl
efficiently filling the S2 binding pocket of FVIIa, whereas in the
less active conformer, the Et group must be projected into
solution, where it does not participate in favorable binding
interactions.22 The crystal structure also shows the benzamide
binding in the narrow P1 pocket and making key hydrogen bonds
with Asp189 and Gly219, and the isopropyl sulfone contacting
the C42-C58 disulfide bridge.
Compd #
FVIIa Ki
(nM)
FVIIa
Def PT
(µM)a
15
>10,000
16
9200
17
18
2100
940
78
14
23
2.9
(atropiso 1)
19
610
(atropiso 2)
20
91
42
a FVIIa enzyme assays and the FVIIa-deficient prothrombin assay were
performed according to established protocols.5
Initial incorporation of the m-aminobenzamide into the
macrocycle scaffold produced compound 5 with FVIIa Ki
>10,000 nM, which was not surprising considering the 100-fold
loss of potency observed in the phenylpyrrolidine series relative
to an aminoisoquinoline P1 (Cf. Figure 1 and Table 1). Despite
the poor activity of the initial neutral macrocycle 15, we decided
to build in functionality that had been shown to increase potency
in related FVIIa inhibitors. Ethyl substitution at the P2 position
produced compound 16 with FVIIa Ki = 9200 nM, which existed
as a mixture of atropisomers that readily isomerized at room
temperature after isolation. In order to avoid rotamers, we
installed a symmetrical P2 group which locks one ethyl group
into the bioactive conformation in compound 17, resulting in a 4-
fold increase in potency. Separately, an isopropyl sulfone that
reaches the C42-C58 disulfide bridge in the S1’ pocket was
added to produce 18, with comparable potency to the diethyl
analogue 17. Adding one ethyl group to this compound produced
atropisomers 14 and 19. Each isomer was isolated by preparatory
HPLC and was stable at room temperature in both organic and
aqueous solvents for days. Interestingly, the presence of the
internal methyl group on the amide apparently locks the ethyl
group on one side of the ring and prevents interconversion.
Atropisomer 14 was 30-fold more potent than the less active
Figure 3. Crystal structure of macrocycle 14 bound to FVIIa at 2.30 Å
resolution. The X-ray structure (5U6J) has been deposited in the PDB
(www.pdb.org). (a) Crystal structure depicted with surface rendering of the
FVIIa binding site. (b) Electron density for 14 is depicted with the omit Fc-Fo
map at 3 rmsd. Graphics were generated with the software program
PyMOL.23
Compound 14 demonstrated good potency and clotting
activity, but had only moderate permeability (PAMPA = 68
nm/sec).21 The sulfone was replaced with a sulfide to provide
compound 20 with higher permeability (PAMPA = 145 nm/sec)
albeit with slightly lower potency and the potential for
bioconversion to the more active species in vivo. Oral dosing in
rat of 20 showed 5.5% oral bioavailability but only traces of
conversion into the sulfone. Although the sulfide had only
moderate potency, the observed oral bioavailability shows a
possible path forward toward potent, orally active macrocycles.
Table 2. Macrocycles with isoquinolinone P1 groups.

hydrogen bond donor and moderate permeability. A comparison
of compounds 14 and 26 (Table 3) shows an increase in potency
with maintenance of good selectivity over other serine proteases.
Compd #
FVIIa Ki
(nM)
FVIIa
Def PT
(µM)a
Table 3. In vitro potency and selectivity of Compounds 14 and 26. Serine
protease enzyme assays and the FVIIa-deficient prothrombin assay were
performed according to established protocols.5
21
22
1500
840
Compound
14
23
26
4.2
FVIIa Ki, nM
FVIIa def PT, µM
PAMPA, nm/s, pH 5.5
Tissue Kallikrein Ki, nM
Factor Xa, Ki, nM
2.9
3.1
68
100
11.0
2800
4700
5800
O
Trypsin, FIXa, thrombin,
plasmin, plasma kallikrein,
TPA, Urokinas, aPC Ki, nM
O
NH
O
All > 10,000
All > 10,000
23
24
360 (R = Et)
9.6 (R = H)
H
N
HN
N
71
H
Initial replacement of the aminoisoquinoline P1 substituent in
a macrocyclic FVIIa inhibitor with the m-aminobenzamide
produced weakly active compounds. Addition of optimal P2 and
P1’ binding substituents produced permeable compounds that
inhibit FVIIa at low nanomolar concentrations with excellent
selectivity over other serine proteases. The benzamide P1 series
produced a moderately active compound with measurable oral
availability when the P1’ sulfone substituent was changed to the
corresponding sulfide. Potency in the series was further enhanced
with the isoquinolinone P1 in combination with the symmetrical
dimethyl P2, to afford 26, with excellent potency and clotting
activity. Despite the increased permeability, we believe that
active transport limits oral bioavailability. These results serve as
useful leads in the discovery of potent, orally active FVIIa
inhibitors.
O
O
R
O
25
26
61
17
4.3
3.1
a FVIIa enzyme assays and the FVIIa-deficient prothrombin assay were
performed according to established protocols.5
Acknowledgments
We would like to thank Atsu Apedo for carrying out separations of
final molecules and the BBRC DDS team for scaleup of
intermediates.
Benzamide 14 has promising activity, but more potency is
likely necessary for the desired antithrombotic efficacy. A
bicyclic series of more potent neutral P1 groups were discovered
while we were optimizing the neutral benzamide macrocycles.
The isoquinolinone P1 was chosen for incorporation into the
macrocycle scaffold since it had a good balance of permeability
and potency. In the phenylpyrrolidine chemotype, the analog
containing the isoquinolinone P1 was 10-fold more potent with
increased permeability relative to the corresponding benzamide.11
We chose the dimethyl P2 group to avoid atropisomer issues and
to mitigate the high protein binding of diethyl substitution (data
not shown). Combining these substituents produced compound
21 with a FVIIa Ki of 1500 nM. In order to determine if the
phenylpyrrolidine in combination with the macrocycle would
further rigidify the bioactive confirmation, we synthesized
compound 22. Since it is equipotent, it likely adopts a similar
conformation and presumably does not further stabilize the
bioactive confirmation. Addition of an acid adjacent to the amide
afforded compound 24, which adds a handle for a prodrug, and
provided an 8-fold increase in potency over compoound 21 to 9.6
nM but had weak clotting activity, indicative of high protein
binding. Sulfone 25, devoid of P2 substitution, had 15-fold
higher binding potency than the corresponding benzamide. These
compounds have ethyl and isopropyl sulfone P1’ binding groups
that have comparable potency, as previously reported.16
Combining the symmetrical dimethyl P2 binding group with a
cyclopropylsulfone P1’ group provided compound 26 with
excellent potency and clotting activity, but unfortunately the
compound had no oral bioavailability in mouse despite one less
References and Notes
1. Ansell, J.; Hirsh, J.; Hylek, E.; Jacobson, A.; Crowther, M.;
Palareti, G.; American College of Chest, P., Pharmacology and
management of the vitamin K antagonists: American College of
Chest Physicians Evidence-Based Clinical Practice Guidelines
(8th Edition). Chest 2008, 133 (6 Suppl), 160S.
2. Pinto, D. J.; Smallheer, J. M.; Cheney, D. L.; Knabb, R. M.;
Wexler, R. R. J. Med. Chem., 2010, 53, 6243.
3. Yeh, C. H.; Hogg, K.; Weitz, J. I. Atertio. Thromb. Vasc. Biol.,
2015, 35, 1056.
4. Girard, T. J.; Nicholson, N. S. Curr. Opin. Pharmacol., 2001, 1,
159.
5. Wong, P. C.; Luettgen, J. M.; Rendina, A. R.; Kettner, C. A.; Xin,
B.; Knabb, R. M.; Wexler, R. R.; Priestley, E. S. Thromb.
Haemost., 2010, 104, 261.
6. D. Loury, B. J, e. al., PCI-27483, a small molecule inhibitor of
Factor VIIa, inhibits growth of BxPC3 pancreatic adenocarcinoma
xenograft tumors, Proceedings of the Annual Meeting of the
American Association for Cancer Research San Diego, CA, 2008.
7. Rao, L. V.; Pendurthi, U. R. Arteriosclerosis, thrombosis, and
vascular biology 2005, 25 (1), 47.
8. Young, W. B.; Mordenti, J.; Torkelson, S.; Shrader, W. D.;
Kolesnikov, A.; Rai, R.; Liu, L.; Hu, H.; Leahy, E. M.; Green, M.
J.; Sprengeler, P. A.; Katz, B. A.; Yu, C.; Janc, J. W.; Elrod, K.
C.; Marzec, U. M.; Hanson, S. R. Bioorg. Med. Chem. Lett. 2006,
16 (7), 2037.
9. E.S. Priestley, Tissue factor-fVIIa inhibition: update on an
unfinished quest for a novel oral antithrombotic, Drug discovery
today, 19 (2014) 1440.

10. Schechter, I.; Berger, A. Biochem Biophys Res Commun 1967, 27
(2), 157-62.
11. Wurtz, N. R.; Parkhurst, B. L.; Jiang, W.; DeLucca, I.; Zhang, X.;
Ladziata, V.; Cheney, D. L.; Bozarth, J. R.; Rendina, A. R.; Wei,
A.; Luettgen, J. M.; Wu, Y.; Wong, P. C.; Seiffert, D. A.; Wexler,
R. R.; Priestley, E. S. ACS Med. Chem Lett, 2016, 7 (12), 1077.
12. Zhang, X.; Jiang, W.; Jacutin-Porte, S.; Glunz, P. W.; Zou, Y.;
Cheng, X.; Nirschl, A. H.; Wurtz, N. R.; Luettgen, J. M.; Rendina,
A. R.; Luo, G.; Harper, T. M.; Wei, A.; Anumula, R.; Cheney, D.
L.; Knabb, R. M.; Wong, P. C.; Wexler, R. R.; Priestley, E. S.
ACS Med. Chem. Lett. 2014, 5 (2), 188.
13. Miura, M.; Seki, N.; Koike, T.; Ishihara, T.; Niimi, T.; Hirayama,
F.; Shigenaga, T.; Sakai-Moritani, Y.; Tagawa, A.; Kawasaki, T.;
Sakamoto, S.; Okada, M.; Ohta, M.; Tsukamoto, S. Bioorg. Med.
Chem. 2007, 15 (1), 160.
14. Riggs, J. R.; Hu, H.; Kolesnikov, A.; Leahy, E. M.; Wesson, K.
E.; Shrader, W. D.; Vijaykumar, D.; Wahl, T. A.; Tong, Z.;
Sprengeler, P. A.; Green, M. J.; Yu, C.; Katz, B. A.; Sanford, E.;
Nguyen, M.; Cabuslay, R.; Young, W. B. Bioorg. Med. Chem.
Lett. 2006, 16 (12), 3197.
15. Trujillo John, I.; Huang, H.-C.; Neumann William, L.; Mahoney
Matthew, W.; Long, S.; Huang, W.; Garland Danny, J.; Kusturin,
C.; Abbas, Z.; South Michael, S.; Reitz David, B. Bioorg. Med.
Chem. Lett. 2007, 17 (16), 4568.
16. Priestley, E. S.; Cheney, D. L.; DeLucca, I.; Wei, A.; Luettgen, J.
M.; Rendina, A. R.; Wong, P. C.;Wexler, R. R. J. Med. Chem.,
2015, 58, 6225.
17. Glunz, P. W.; Mueller, L.; Cheney, D. L.; Ladziata, V.; Zou, Y.;
Wurtz, N. R.; Wei, A.; Wong, P. C.; Wexler, R. R.; Priestley, E. S.
J. Med. Chem., 2016, 59, 4007.
18. Zhang, X. G.; Glunz, P. W.; Johnson, J. A.; Jiang, W.; Jacutin-
Porte, S.; Ladziata, V.; Phillips, M. S.; Wurtz, N. R.; Parkhurst,
B.; Rendina, A. R.; Harper, T. M.; Cheney, D. L.; Luettgen, J. M.;
Wong, P. C.; Seiffert, D.; Wexler, R. R.; Priestley, E. S. J. Med.
Chem., 2016, 59, 7125.
19. Tuyet, J. Tet. Lett., 1999, 40, 1673.
20. Petasis, N. A.; Zavialov, I. A. J. Am. Chem. Soc. 1997, 199 (2),
445.
21. Avdeef,A.; Nielsen, P. E.; Tsinman, O. Eur J Pharm Sci, 2004,
22, 365.
22. Relative atomic coordinates for the crystallographic structure of
14 complexed with factor VIIa has been deposited in the PDB; the
deposition number is 5U6J. For procedures for crystallization, data
collection, and structure refinement, please see Ref 16.
23. The PyMOL Molecular Graphics System, Version 1.7
Schrödinger, LLC