Design and synthesis of novel meta-linked phenylglycine macrocyclic FVIIa inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00375

Two novel series of meta-linked phenylglycine-based macrocyclic FVIIa inhibitors have been designed to improve the rodent metabolic stability and PK observed with the precursor para-linked phenylglycine macrocycles. Through iterative structure-based design and optimization, the TF/ FVIIa K_i was improved to subnanomolar levels with good clotting activity, metabolic stability, and permeability.

Full text of DOI:10.1021/acsmedchemlett.6b00375

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Letter
Design and Synthesis of Novel meta-Linked
Phenylglycine Macrocyclic FVIIa Inhibitors.
Jeremy M. Richter, Daniel L. Cheney, J. Alex Bates, Anzhi Wei, Joseph M
Luettgen, Alan R. Rendina, Timothy M. Harper, Rangaraj Narayanan, Pancras
C Wong, Dietmar A Seiffert, Ruth R. Wexler, and Eldon Scott Priestley
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00375 • Publication Date (Web): 01 Nov 2016
Downloaded from http://pubs.acs.org on November 6, 2016
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Design and Synthesis of Novel meta-Linked Phenylglycine Macrocy-
clic FVIIa Inhibitors.
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Jeremy M. Richter*, Daniel L. Cheney, J. Alex Bates, Anzhi Wei, Joseph M. Luettgen, Alan R. Rendi-
na, Timothy M. Harper, Rangaraj Narayanan, Pancras C. Wong, Dietmar Seiffert, Ruth R. Wexler, E.
Scott Priestley.
Bristol-Myers Squibb Research & Development, Hopewell, New Jersey, 08534, United States
KEYWORDS FVIIa, Factor VIIa, macrocycle, thrombosis, antithrombotic, anticoagulant.
ABSTRACT: Two novel series of meta-linked phenylglycine-based macrocyclic FVIIa inhibitors have been designed to improve
the rodent metabolic stability and PK observed with the precursor para-linked phenylglycine macrocycles. Through iterative struc-
ture-based design and optimization, the TF/FVIIa Ki was improved to sub-nanomolar levels with good clotting activity, metabolic
stability, and permeability.
Cardiovascular diseases remain the leading cause of death
worldwide in both developed and developing nations. Many of
these deaths can be attributed to arterial or venous thrombosis
(e.g. myocardial infarction, ischemic stroke, pulmonary embo-
lism, and deep vein thrombosis).1 In light of this high level of
medical need, the discovery of novel and selective antithrom-
botic therapies with high efficacy and low bleeding liability
would be beneficial.
have previously reported structures such as 1 that contain
weakly-basic benzamidine replacements that confer improve-
ments in permeability and oral exposure while maintaining
good of potency.24-26 This series of compounds exhibited many
favorable properties; however, low rodent oral bioavailability
was a key limitation. Biotransformation studies traced this
poor exposure to the carbamate upper linker in these macrocy-
cles, which was highly metabolized in rodent liver micro-
somes. Therefore, in an effort to design inhibitors that retained
favorable potency and selectivity, and incorporated improved
metabolic stability and oral bioavailability, a program was
initiated to modify the macrocyclic upper linker.27
Factor VIIa (FVIIa) resides at the beginning of the extrinsic
coagulation cascade.2 When blood vessel walls are compro-
mised, tissue factor (TF) is exposed to circulating FVIIa, lead-
ing to the conversion of FIX to FIXa. Both FIXa and FVIIa (in
conjunction with their cofactors FVIIIa and tissue factor, re-
spectively) cause the conversion of FX to FXa, which in turn
converts prothrombin to thrombin. Thrombin then promotes
clot formation by converting fibrinogen to fibrin and activat-
ing platelets, generating an extended fibrin/platelet network.3
Various blood coagulation factors have been the target of
pharmaceutical small molecule inhibitor research; including,
most notably FXa (e.g. apixaban and rivaroxaban) and throm-
bin (e.g. dabigatran).4 FVIIa inhibitors are appealing as antico-
agulant therapies for several reasons. First, the efficacy and
safety of FVIIa disruption has been demonstrated clinically
with recombinant nematode anticoagulant protein c2.5-7 Sec-
ondly, it has been demonstrated preclinically that FVIIa inhi-
bition has excellent efficacy and limited bleeding liability.8-15
This profile has made FVIIa a tantalizing target for anticoagu-
lant therapy for many years16-20 and has provided the impetus
for the investigations detailed herein.
Figure 1. General structures of FVIIa inhibitors.
The previously reported para-linked macrocycles (e.g. 1)
make several critical contacts with the FVIIa active site,24-26
leading to the exquisite potency observed. When designing
novel macrocyclic inhibitors, we were cognizant of the fact
that these interactions would need to be maintained or even
improved in order to retain the desired potency. One such in-
teraction that would need to be considered was the hydropho-
bic contact with the S2 binding pocket, which is primarily
made by the P2-methyl group labeled as “A” in structure 1. It
was reasoned that macrocycles linked at the meta position,
instead of the para position, of the P2-aryl ring could maintain
this favorable hydrophobic contact while significantly altering
the linker constitution and modulating the metabolic profile.
Of particular interest while considering the design of novel
FVIIa inhibitors are the two polar residues (Asp189 and
Ser190) that reside at the bottom of the primary specificity
pocket (S1). Historically, benzamidines are typically required
to bind to these residues and confer the required level of po-
tency; however, this comes at a cost of poor permeability and
oral bioavailability, necessitating a prodrug strategy.20-23 We
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ACS Medicinal Chemistry Letters
This publication details the design and synthesis of structures
Page 2 of 6
1
of type 2, which provided a novel scaffold for further elabora-
2
tion.
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7
8
9
Initial efforts surrounding the new chemotype 2 required
identification of the appropriate upper linker for the macrocy-
cle. The P’ N-H makes a key hydrogen bonding contact to a
conserved water (Figure 2A); therefore linkers were designed
to maintain this critical interaction. Molecular modeling also
indicated that 14 and 15 membered macrocyclic rings were
optimal, which focused our efforts on linkers that would main-
tain this ring size. As such, a series of linkers of different
lengths and constitutions were prepared and evaluated in the
TF/FVIIa activity assay (Table 1).15 Linkers of 4-atoms (3, 6,
7) and 5-atoms in length (4, 5, 8) were prepared, as they were
expected to be optimal based on molecular modeling overlays
with 1. Within the 4-atom linkers, the position of the oxygen
was varied (e.g. 3, 6) until it was discovered that removal of
the oxygen altogether (amide linker, 7) resulted in a significant
improvement in binding affinity. The lower activity of 4 may
originate from strain of the bound conformation in which the
ester portion of the carbamate is twisted out of plane, as ob-
served in the crystal structure of compound 4 bound to FVIIa
(Figure 2).28-32 Removal of the oxygen, as in the amide system
7, eliminates this penalty and possibly accounts for the im-
proved potency (See Figure 3 depicting the crystal structure of
7 bound to FVIIa). Furthermore, heteroatom substitution was
evaluated in the 5-atom linker series, which revealed that the
urea linker (8) was significantly more potent. In contrast to
the carbamates, the urea does not pay a similar thermodynamic
penalty to adopt this strained conformation, which can account
for the favorable impact on potency. From these results, the
urea (8) and amide (7) linkers were selected for further opti-
mization due to their promising potency. We elected to opti-
mize the urea linker first due to its superior potency relative to
the amide linker.
Figure 2. A. Crystallographic structure of compound 4 bound to
FVIIa determined at 1.9 Å resolution. The Fo-Fc omit electron
density map is depicted at 3 RMSD.33-34 B. Side view of the
strained carbamate substructure. Graphics were generated using
the program PyMol.35
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Figure 3. Crystallographic structure of compound 7 bound to
FVIIa determined at 1.85 Å resolution. The Fo-Fc omit electron
density map is depicted at 3 RMSD.33-34 Graphics were generated
using the program PyMol.35
Molecular modeling revealed the presence of a hydrophobic
binding pocket near the C-4 position of the P2-phenyl ring,
which the unsubstituted analogues were not able to contact
(vide supra) as shown in Figure 4. It was hypothesized that
substitution on the urea nitrogen could potentially improve the
binding affinity by contacting the hydrophobic binding pocket
above the P2 aryl ring. Additionally, the 4-
fluoroaminoisoquinoline P1 moiety was incorporated due to
its favorable impact on permeability.26 Disappointingly, a va-
riety of substitutions at the urea nitrogen actually led to a de-
crease in potency (10-13). Unable to improve the potency via
interactions with the P2 hydrophobic pocket from the urea
nitrogen, further inspiration was drawn from the binding mod-
el and substitution at the P2 benzylic position. Modeling sug-
gested that para-methoxy and benzylic substitution might
better fill the S2 pocket, so the para-methoxy derivatives with
both enantiomers of the corresponding benzylic methyl groups
were prepared. Gratifyingly, compound 14 exhibited greatly
improved potency when compared to the unsubstituted com-
pound. However the metabolic stability was poor across the
series of urea-based macrocycles and rat oral exposure of
compound 10 was extremely low (PAMPA pH 7.4 = 220
nm/s, rat po exposure below the limits of quantitation), which
led to the decision to discontinue the work on the urea series
and turn attention to the amide upper linker for further optimi-
zation.
Table 1. Upper Linker Optimization
O
X
NH2
NH
S
N
Me
N
N
H
O
O
O
TF/FVIIa Ki 25 TF/FVIIa Ki 37 oC
cmpd
X
oC (nM)a
(nM)a,c
3
4
5
6
7
8
-CH2O-
260
-CH2CH2O-
-CH2OCH2-b
-OCH2-
190
23
710
-CH2CH2-
<5
58
-CH2CH2N(CH3)-
6.3
aTF/FVIIa Ki values were obtained from recombinant human
enzyme and were averaged from two experiments.15 bContains 4-
OMe on the P2 aromatic ring. cDuring the course of optimiza-
tions, the assay was modified to 37 oC to better approximate phys-
iological conditions.
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ACS Medicinal Chemistry Letters
Figure 4. Overlay of crystal structure of the des-fluoro analogues
17
18
19
20
21
22
23
24
H
H
F
OMe
210
18
26
11
14
26
15
29
14
41
-,-
-,-
540,350
290,15
-,-
of 125 and model of 16 bound in FVIIa. Graphics were generated
1
2
3
4
5
6
7
8
using the program PyMol.
14,16
15,46
26,5
32,64
23,23
-,-
Table 2. Urea Optimization
OMe
H
H
290
77
R
X
N
cPr
Y
O
NH2
NH
S
Z
Me
N
H
OCH2CHF2
OCH2cPr
OH
27
-,88
-,-
N
N
H
9
H
26
F
O
O
O
10
11
12
13
14
15
16
17
18
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H
92
-,-
TF/FVIIa Ki TF/FVIIa Ki H,RLM t1/2
cmpd
X
Y
Z
R
25 oC (nM) 37 oC (nM)
(min)
F
OMe
410
14,8
-,-
aSee reference 15 for a detailed description of the FVII-
deficient prothrombin time assay.
9
10
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
17
14
-
-,-
5,6
-,-
200
11
Bn
80
-
-
12a
13a
14
CH2cPr
CH2cBu
Me
59
5,12
-,-
150
<5
47
-
S
OMe Me
OMe
3.8
-
4,4
6,6
Figure 5. Overlay of crystallographic structures of the des-fluoro
analogue of compound 125 (green) and 26 (yellow) bound in FVI-
Ia. Graphics were generated using the program PyMol.
15a,b
H
Me
Me
b
aRacemic at the phenylglycine center. des-Fluoro analogue of
Additional modeling suggested that the amide upper linker
may not optimally fill the S2 binding pocket, and we hypothe-
sized that a methyl substituent at the benzylic position in the
proper stereochemical orientation should improve this binding
interaction, analogous to the urea series (vide supra). Both
enantiomers of the macrocycle containing the benzylic methyl
group were prepared (Table 4), and it was discovered that the
(R)-enantiomer (26, assigned by X-ray co-crystal structure,
Figure 5) was significantly more potent than the (S)-
enantiomer (25), resulting in a 100-fold increase in TF/FVIIa
potency over the unsubstituted compound (21) (see Figure 5),
with concomitant improvement in the FVIIa deficient pro-
thrombin time assay. Selected compounds from this series
were studied in rat oral PK once promising potency, permea-
bility, and metabolic stability was achieved. Compounds 16
and 18 demonstrated very low (3.7 F%) or no rat oral expo-
sure, respectively, despite reasonable permeability (PAMPA
assay). While excellent potency against FVIIa and improved
metabolic stability were achieved with compound 26, off-
target liabilities, namely poor tissue kallikrein selectivity and
poor rat PK within this series limited its ability to progress
further.
the aminoisoquinoline.
In a manner analogous to the urea upper linkers, it was rea-
soned that substitution of the P2-aryl ring could improve the
potency by interactions with the hydrophobic binding pocket.
As such, amide-linked macrocycles containing substituted P2-
aryl rings were prepared and evaluated, the results of which
are delineated in Table 3. It was discovered that substitution at
the C-4 position led to approximately a ten-fold improvement
in TF/FVIIa potency (e.g. 18, 21, 22, vs 16). Conversely, sub-
stitution at the C-5 position had little to no effect on potency
(19), presumably because this position is solvent exposed and
substituents are not directed into the binding pocket. Mono-
substitution at the C-4 position provided relatively flat potency
SAR in both the TF/FVIIa and FVIIa deficient prothrombin
time assays (anti-clotting assay in human plasma, expressed as
the concentration required to prolong clotting time by two-
fold), but it modulated rat metabolic stability (as judged by
half-life in rat liver microsomes). Providing an appropriate
balance between potency and metabolic stability, the difluoro-
ethoxy substitution (21) was selected for further optimization.
Table 3. Amide optimization: P2 aryl
Table 4. Amide optimization: benzylic substitution
R
O
X
NH2
NH
S
F
O
O
X
Y
N
Me
N
F
NH2
NH
S
N
H
N
Me
N
F
O
O
O
N
H
F
O
O
FVII def.
PT EC2X
(µM)a
TF/FVIIa
Ki 37 oC
(nM)
O
H,RLM t1/2 PAMPA pH
cmpd
X
R
(min)
7.4,5.5 (nM/s)
FVII def.
PT EC2X
(µM)
TF/FVIIa TK1
aPC Ki
(nM)
H,RLM t1/2 PAMPA pH
(min) 7.4,5.5 (nM/s)
cmpd
X
Y
Ki 37 oC
(nM)
Ki
(nM)
16
H
H
260
10
13,33
120,67
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two steps. The lower yield of this two-step sequence is pri-
21
25
26
H
Me
H
H
H
27
67
140 1200
15
23
32,64
14,74
31,39
-,88
-,-
1
2
3
4
5
6
marily attributed to the material loss upon chiral separation
(50%) and final HPLC purification of the unprotected ami-
dine.
23
5700
39
Me
0.24
9.4
3.5
-,-
Scheme 2. Representative synthesis of compound 10
Scheme 1. Representative synthesis of compound 18
7
8
9
10
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a. p-NO2-PhOTeoc (1 equiv.), DIPEA (1.2 equiv.), MeOH, 2
h.; b. NaH (1.5 equiv.), DMF, 15 min.; MeI, 16 h.; c. KOAc (2.5
equiv.), bis(neopentyl glycolato)diboron (1.1 equiv.), PdCl2(dppf)
(0.05 equiv.), DMSO, 1,4-dioxane, 90 oC,
4
h.; d.
a. (COCl)2 (1.1 equiv.), DMF (0.1 equiv.), DCM, 30 min.;
DMAP (0.1 equiv.), TEA (1 equiv.), 28 (1.5 equiv.), 16 h.; b.
KOAc (2.5 equiv.), bis(neopentyl glycolato)diboron (1.4 equiv.),
PdCl2(dppf) (0.1 equiv.), DMSO, 1,4-dioxane, 90 oC, 3 h.; c.
CHOCO2H•H2O (1 equiv.), 31 (1 equiv.), DMF, MeCN, 60 oC, 16
h.; d. TBAF (10 equiv.), THF, 3 h.; e. BOP (2 equiv.), DMAP (4
equiv.), DIPEA (10 equiv.), DMF, DCM, 10 hour inverse addi-
tion.; f. Chiral HPLC; g. TFA.
CHOCO2H•H2O (1 equiv.), 31 (1 equiv.), DMF, MeCN, 60 oC, 16
h.; 37 (1.3 equiv.), DIPEA (5 equiv.), BOP (1 equiv.), 23 oC, 3 h.;
e. TBAF (10 equiv.), THF, 3 h.; f. Pyr (10 equiv.), p-
NO2PhOCOCl (5 equiv.), DCM, 0 to 23 to 50 oC, 16 h.; g. Chiral
HPLC; h. TFA.
Efficient access to the urea containing macrocycles was
more difficult and required extensive optimization of the syn-
thetic route to access the final compounds in good yield.
Scheme 2 delineates this optimized route. Beginning with the
amine 35, installation of a Teoc protecting group and alkyla-
tion of the resulting amine generated intermediate 38. Boronic
acid installation (39), Petasis reaction and subsequent amine
formation proceeded smoothly to generate intermediate 40.
Removal of the Teoc protecting group provided 41 and set the
stage for the key macrocyclization reaction. Extensive optimi-
zation eventually revealed that formation of the activated car-
bamate under carefully controlled conditions (5 equivalents of
p-NO2Ph-OCOCl and 10 equivalents of pyridine, 0 to 23 to 50
oC) could effect smooth urea formation in good yield to give
42. Chiral separation and deprotection provided compound
10.
Synthesis of the macrocyclic analogues involved two key
steps: 1) Petasis reaction of a functionalized P2 boronic acid
to form the phenyl glycine core, and 2) macrocyclization of
either the lower amide or upper linker amide, carbamate, or
urea. Synthesis of the amide macrocycles was typically ac-
complished via late stage lower amide macrocyclization
(Scheme 1). As such, the sequence began with preparation of
the appropriately substituted P2 acids according to modifica-
tions of literature protocols and known chemistry (27). For-
mation of the upper linker amide bond was achieved via con-
version of the acid 27 to the corresponding acid chloride fol-
lowed by base-mediated coupling with aniline 28 to afford
intermediate 29. The requisite boronic acid 30 was prepared
using Suzuki-Miyaura borylation conditions, which set the
stage for the key Petasis reaction. Condensation of the protect-
ed P1 aniline 31 with glyoxylic acid and boronic acid 30
smoothly provided the desired phenyl glycine core (32) in
good yield. Deprotection of 32 to provide amine 33 provided
the necessary intermediate for the final macrocycle formation.
Successful macrocyclization required slow inverse addition of
33 to a solution of the coupling reagents to provide good
yields of the desired macrocycle 34. Final chiral separation
and deprotection provided compound 18 in 14% yield over
In conclusion, a series of factor VIIa inhibitors were de-
signed and prepared in order to address the poor rodent meta-
bolic stability and oral exposure of compound 1. The meta-
linked macrocyclic series of factor VIIa inhibitors was de-
signed based on careful modeling of the VIIa active site. Us-
ing the information garnered through this analysis, the potency
of these novel inhibitors was improved from sub-micromolar
to sub-nanomolar with concurrent improvement in rodent met-
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Kotian, P.; Bantia, S.; Babu, Y. S. The Antithrombotic and Anti-
Inflammatory Effects of BCX-3607, a Small Molecule Tissue Fac-
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(15) Wong, P. C.; Luettgen, J. M.; Rendina, A. R.; Kettner, C. A.;
Xin, B.; Knabb, R. M.; Wexler, R. R.; Priestley, E. S. BMS-593214,
an Active Site-Directed Factor FVIIa Inhibitor: Enzyme Kinetics,
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2010, 104, 261 – 269.
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solve the larger issues of tissue kallikrein selectivity and poor
rodent PK, which precluded further advancement of the series.
ASSOCIATED CONTENT
Supporting Information
Synthesis and characterization data of key compounds. The Sup-
porting Information is available free of charge on the ACS Publi-
cations website.
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AUTHOR INFORMATION
Corresponding Author
* E-mail: jeremy.richter@bms.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Jeff Bozarth and Mojgan Abousleiman for help in ob-
taining the enzyme inhibition data referenced herein. We thank
Frank Barbera and Yiming Wu for help in obtaining the clotting
assay data reference herein. We thank colleagues at the BMS-
Biocon Research Center for synthesis of intermediates 31 and 37.
We thank Mei-Mann Hsueh and colleagues at the Biocon-BMS
Research Center for performing the PK studies referenced herein.
We thank Steven Sheriff both for preparing the coordinates and
data of compounds 4, 7, and 26 complexed to factor VIIa and
depositing them in the PDB.
ABBREVIATIONS
FVIIa, Factor VIIa; TF, Tissue Factor; H,RLM, human, rat liver
microsome metabolic stability; PAMPA, parallel artificial mem-
brane permeability assay; TK1, tissue kallikrein-1; aPC, activated
protein C; PK, pharmacokinetic; FIX, Factor IX; FIXa, Factor
Ixa; FVIIIa, Factor VIIIa; FX, Factor X; Fxa, Factor Xa; DMF,
N,N-dimethylformamide; DCM, dichloromethane; DMAP, dime-
thylamino pyridine; DMSO, dimethylsulfoxide; TBAF, tetra-N-
butylammonium fluoride; THF, tetrahydrofuran; BOP, (Benzotri-
azol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophos-
phate; DIPEA, diisopropylethylamine; HPLC, high performance
liquid chromatography; TFA, trifluoroacetic acid; HATU, 1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium
3-oxidhexafluorophosphate;
NMM,
N-
methylmorpholine.
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