Synthesis and P1′ SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.08.088

Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1′-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1′ group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1′ substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

Full text of DOI:10.1016/j.bmcl.2016.08.088

Bioorganic & Medicinal Chemistry Letters 26 (2016) 5051–5057
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and P10 SAR exploration of potent macrocyclic tissue
factor-factor VIIa inhibitors
y
⇑
Vladimir (Uladzimir) Ladziata , Peter W. Glunz, Yan Zou , Xiaojun Zhang, Wen Jiang,
Swanee Jacutin-Porte, Daniel L. Cheney, Anzhi Wei, Joseph M. Luettgen, Timothy M. Harper,
Pancras C. Wong, Dietmar Seiffert, Ruth R. Wexler, E. Scott Priestley
Bristol-Myers Squibb Research & Development, Princeton, NJ 08543, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for
treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S10-
binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P10 group. Over
the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P10 substituent emerged as
an improved alternative, offering increased metabolic stability and lower clearance, while maintaining
excellent potency and selectivity.
Received 3 August 2016
Revised 24 August 2016
Accepted 26 August 2016
Available online 28 August 2016
Keywords:
Thrombosis
Ó 2016 Elsevier Ltd. All rights reserved.
Anticoagulant
Factor VIIa-TF inhibitor
Macrocycle
Thrombosis-related disorders, such as myocardial infarction
(MI), thromboembolic stroke, deep vein thrombosis (DVT) and pul-
monary embolism (PE), are the leading cause of death in the indus-
trialized world.1 Traditional therapies, which include low
molecular weight heparin (LMWH) and warfarin, suffer from a
number of drawbacks which limit their utility for long-term use.
Namely, LMWH is administered only via parenteral route, while
use of warfarin is complicated by a slow onset of action, food
effects, and requires close monitoring due to a substantial bleeding
risk.2 Therefore, in the past several decades much research has
focused on discovery and development of thrombin and factor Xa
small molecule inhibitors with improved pharmacokinetic and
pharmacodynamic parameters.3 Among approved novel oral anti-
coagulants (NOACs), which target these two specific blood coagu-
lation cascade enzymes, are the thrombin inhibitor dabigatran
etexilate (PradaxaÒ),4a–d and the factor Xa inhibitors rivaroxaban
(XareltoÒ),4e–h apixaban (EliquisÒ)4i–k and edoxaban (SavaysaÒ).4l,m
The introduction of these novel antithrombotics into the clinical
practice enables replacement of the traditional therapies in many
patients.5
attractive class of anticoagulants, and much pharmaceutical
research efforts over the past two decades have been devoted to
finding orally bioavailable TF-FVIIa small molecule inhibitors.6
The TF-VIIa complex plays a key role in initiation of the extrinsic
coagulation pathway: circulating zymogen FVII interacts with
membrane-anchored TF, which is exposed upon pathophysiological
vascular injury or atherosclerotic plaque rupture, thereby forming
the proteolytically active TF-FVIIa complex.7a The initial step is then
amplified/propagated via activation of factors IX and X, leading to
direct generation of additional FVIIa in the former case, and throm-
bin formation in the latter; thrombin in turn cleaves fibrinogen to
fibrin and mobilizes platelets through protease-activated receptors,
which ultimately results in stable clot formation.7b Moreover, FVIIa
target validation was accomplished in numerous in vivo models
utilizing biological agents,8a–c as well as multiple small molecule
chemotypes,8d–l where effective anticoagulation coupled with low
risk of bleeding was consistently observed. Furthermore, apart
from their apparent utility as antithrombotics, TF-FVIIa inhibitors
may also possess clinical potential in tumor metastasis via
inhibition of protease-activated receptor 2 (PAR2) signaling,9 and
in inflammation.10
Given the fact that the tissue factor-factor VIIa complex
(TF-FVIIa) is situated at a critical initiation point of the coagulation
cascade, factor VIIa inhibitors are regarded as a pharmacologically
A number of diverse series of small molecule TF-FVIIa inhibitors
have been reported to date.11 Based on the interaction with the
polar primary specificity pocket, the reported FVIIa inhibitors could
be nominally divided into two categories. The first group is com-
prised of compounds bearing strongly basic benzamidine P1 moi-
eties,12a which are characterized by high TF-FVIIa inhibitory
⇑
Corresponding author. Tel.: +1 609 466 5055; fax: +1 609 818 3550.
E-mail address: vladimir.ladziata@bms.com (V. Ladziata).
Co-author deceased. Date of death: 03/21/2014.
y
http://dx.doi.org/10.1016/j.bmcl.2016.08.088
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

5052
V. Ladziata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5051–5057
potency, but exhibit low permeability and poor oral absorbance.
Conversely, the second group includes compounds with less basic
non-benzamidine P1 groups, which ordinarily possess improved
permeability, albeit at the expense of TF-FVIIa binding affinity.
Some representative examples of benzamidine (1–4)12b–e,8l and
non-benzamidine (5–8)12f–i small molecule TF-FVIIa inhibitors
are shown in Figure 1. Very recently our research team reported
novel 16-membered macrocyclic FVIIa inhibitors, bearing an
aminoisoquinoline P1 group.12i Macrocycle 8 is a potent FVIIa
inhibitor with excellent selectivity (>500 fold) against primary
serine protease anti-targets FIXa, FXa, thrombin and trypsin, and
attached to the macrocyclic scaffold (Fig. 2). We were delighted
to learn that upon p.o. and i.v. administration of compound 10 in
dogs oral bioavailability and clearance were much improved. It
should be noted that whereas analog 10, in comparison to sulfone
counterpart 9, possessed improved passive Caco-2 permeability
and maintained good metabolic stability, TF-FVIIa Ki was reduced
(ꢀ12 fold), along with selectivity against a number of serine pro-
teases (data not shown). Therefore, we decided to modify macrocy-
cle 10 at the cyclopropyl P10 region in order to improve TF-FVIIa
inhibitory activity and selectivity, while at the same time main-
taining the ADME profile. Equipped with structural information
for the S10 binding pocket we obtained previously,12e,i,18 we
aimed at expanding the cyclopropyl ring in order to enhance
hydrophobic interaction with the Cys42-Cys58 disulfide bridge,
as well as engaging Lys60A side chain via incorporating acidic
groups as shown in Figure 3. Since we previously demonstrated
that the 4-fluoroaminoisoquinoline P1 group imparts better
permeability due to reduced basicity,15 we decided to use 4-fluo-
roaminoisoquinoline bearing macrocyclic scaffolds for the P10
SAR examination described below.
excellent FVII deficient prothrombin time13 EC2x of 1.2
lM. Fur-
thermore, the macrocycle provided a rigid scaffold allowing for
direct optimization of P1, P2 and P10 groups.
Considering TF-FVIIa inhibition is likely to provide an apparent
therapeutic benefit for various thrombosis-related diseases, our
research laboratories aimed at developing potent and selective
TF-FVIIa inhibitors with improved ADME properties. Building on
the initial success of the macrocyclization strategy applied to FVIIa
inhibitors, Glunz et al. further enhanced this approach by utilizing
rational structure-guided atropisomer control via incorporation of
a P2 benzylic methyl group, and thereby locking macrocyclic scaf-
fold in the bioactive conformation.14 From the resulting conforma-
tionally stable macrocyclic template, Zhang et al. subsequently
undertook detailed structure–activity relationship (SAR) studies
in the P1 and P10 regions of the macrocyclic core.15 After surveying
a number of P10 moieties, cyclopropyl sulfone was identified as an
optimal group. Namely, compound 9 demonstrated outstanding
TF-FVIIa binding affinity and anti-clotting activity (Ki = 0.16 nM,
Synthesis of cyclopropyl P10 precursor 18 is depicted in Scheme 1.
Commercially available 2-bromobenzaldehyde 11 was nitrated, and
the obtained nitrobenzaldehyde 12 was treated sequentially with
MeNH2 and NaBH4 in MeOH to afford N-methyl benzylamine 13 in
good yield. Compound 13 was subsequently converted to the
corresponding Boc- and Cbz-protected derivatives 14 and 15 under
standard conditions. Synthesis of 4-cyclopropylaniline 18 was
achieved in three steps starting with Boc-protected bromobenzy-
lamine 14 via sequential Pd-catalyzed cyclopropanation,19 followed
by Zn-mediated reduction of the nitro group, and Boc removal. It
should be noted that reduction of 16 to 17 under hydrogenative
conditions (H2 (1 atm), Pd-C) resulted in a partial cyclopropyl ring
opening giving rise to the respective n-propyl contaminant.
In order to obtain advanced intermediates to explore the
Lys60A binding vector as shown in Figure 3, we carried out a six
step sequence starting with Cbz-protected bromoarene 15. As
shown in Scheme 2, CuI/(S)-Pro-catalyzed C-arylation of malo-
nates, disclosed by Ma and co-workers,20 afforded corresponding
malonate 20 and cyanoacetate 21 in excellent yields. Intermediates
20 and 21 were decarboxylated in a facile manner under Krapcho
conditions,21 and the subsequent succession of reduction and
EC2x = 1.1 lM) coupled with an excellent selectivity profile (Fig. 2).
Despite having good metabolic stability (dog liver micro-
somes16 t1/2 = 106 min), inhibitor 9 showed elevated dog clearance
at 29 mL/min/kg, indicating rapid elimination of the compound. In
addition, FVIIa inhibitor 9 demonstrated very low dog oral expo-
sure, presumably due to limited permeability.15 Indeed, passive
Caco-2 permeability17 for macrocycle
47 nm/s.
9 measured lower at
We envisioned that polar sulfone functionality in inhibitor 9
could be partially responsible for the poor dog pharmacokinetic
(PK) profile. To that end, we synthesized analog 10, where the
-SO2- fragment is excised, and the cyclopropyl group is directly
Figure 1. Selected structures of disclosed TF-FVIIa inhibitors 1–8.

V. Ladziata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5051–5057
5053
Figure 2. Comparison of sulfone P10 9 and des-sulfone P10 10.
Scheme 2. Synthesis of substituted cyclopropyl P10 building blocks 30 and 31.
Reagents and conditions: (a) CuI (0.2 equiv), (S)-Proline (0.4 equiv), Cs2CO3, DMSO,
50 °C, 3 d; (b) LiCl, DMSO–H2O, 130 °C, 2 h; (c) i. Zn, NH4Cl, MeOH–THF, rt, 2 h; (ii)
Boc2O, 85 °C; (iii) H2, Pd–C, Boc2O, MeOH, rt; (d) (CH2O)n, TDA-1, PhMe, 85 °C; (e)
[Me3SO]I (3.0 equiv), NaH, DMSO, rt to 60 °C, 3 h; (f) HCl, dioxane, 2 h.
Figure 3. Design strategy for novel macrocyclic FVIIa inhibitors.
Scheme 3. Preparation of carboxyethyl cycloalkyl P10 intermediates 42–45.
Reagents and conditions: (a) Zn, NH4Cl, MeOH–THF, rt, 2 h; (b) [NO][BF4], NaI, I2,
MeCN, ꢁ40 °C to 0 °C, 4 h; (c) Br-(L)-Br, NaH, DMF, 0 °C to rt; (d) acetimidamide, CuI
(0.15 equiv), (S)-Proline (0.3 equiv), Cs2CO3, DMF, 100 °C, 2 d; (e) H2 (1 atm), Pd–C,
EtOH, rt, 1.5 h.
Scheme 1. Synthesis of 1-cyclopropyl P10 building block 18. Reagents and
conditions: (a) HNO3, H2SO4, 0 °C, 2 h; (b) (i) MeNH2, MeOH, rt, 1 h; (ii) NaBH4,
MeOH, 0 °C to rt, 5 h; (c) Boc2O, TEA, THF, 40 °C, 2 h; (d) Cbz-Cl, TEA, THF, 0 °C to rt,
3 h; (e) cPrB(OH)2, Pd(OAc)2, PCy3, K3PO4, PhMe–H2O, 100 °C, 12 h; (f) Zn, NH4Cl,
MeOH–THF, rt then 40 °C, 3 h; (g) HCl, dioxane, 30 min.
moderate to good yields. Compounds 34–37 were then subjected
to Cu-catalyzed amination conditions23 to afford anilines 38–41,
which were conveniently deprotected under H2 (1 atm) in the
presence of Pd-C to furnish intended intermediates 42–45.
Having synthesized the necessary P10 intermediates 18, 30–31,
and 42–45, we were then equipped to assemble all the target
macrocyclic TF-FVIIa inhibitors. Macrocycles 10 (Fig. 2) and 46
(Table 1) were accessed starting from the precursor 18 utilizing a
general synthetic sequence we have disclosed earlier.14,15 Addi-
tional macrocyclic structures 52–55 were constructed as outlined
in Scheme 4 from previously described phenylglycine 47.15 BOP
coupling of the respective precursors afforded the required amino-
phenyl alcohols 48–52 in good yields. Our previously reported
two-step carbamoylation protocol14 involving phosgene function-
alization and subsequent slow addition to a dilute MeCN solution
of an amine base afforded the corresponding macrocyclic carba-
mates, which after saponification and Boc removal yielded the
desired macrocycles 53–57.24
protecting group swap resulted in di-Boc protected intermediates
24 and 25. Ensuing condensation with paraformaldehyde under
basic conditions provided styrenes 26 and 27. Gratifyingly, the
double bonds of 26 and 27 were efficiently cyclopropanated under
Corey-Chaykovsky conditions,22 and subsequent Boc removal pro-
vided building blocks 30 and 31 (Scheme 2).
In agreement with the design strategy, we set out to synthesize
an array of cycloalkyl P10 groups bearing carboxylate at the 1-posi-
tion. However, initial attempts to C-alkylate arylacetate 22 were
unsuccessful, presumably because of the poor nucleophilicity of
the corresponding anion due to extensive delocalization on the
nitroarene system. Thus, the synthetic sequence was modified to
sidestep the arylnitro precursor, which resulted in effectual deliv-
ery of substituted cycloalkyl carboxylates 42–45 (Scheme 3).
Namely, nitroarene 22 was reduced to the respective aniline 32,
which was then reacted with nitrosonium tetrafluoroborate, and
the intermediate diazonium species were treated with I2/NaI to
give rise to the required iodoarene precursor 33. In this instance,
treatment with NaH and the respective dibromo electrophiles
resulted in formation of substituted aryl cycloalkanes 34–37 in
To further probe charged interactions with Lys60A, an acid sur-
rogate was introduced at the P10 moiety. Cyclopropyl-bearing
macrocycle 53 was selected as a parent structure, and the tetrazole
moiety was chosen as an acid bioisostere (Scheme 5). Nitrile-
containing macrocycle 58 was prepared using a synthetic route
analogous to that outlined in Scheme 4. Subsequent treatment
with tributyltin azide in PhMe at 100 °C affected conversion of

5054
V. Ladziata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5051–5057
Table 1
Structure–activity relationship of substituted cycloalkyl P10 macrocycles
Compd
46
53
54
55
56
57
59
TF-FVIIa Ki 37 °Ca (nM)
FXa Ki 25 °Cb (nM)
18
0.55
6650
>13,300
2750
1100
70
4.7
0.14
850
>13,300
3400
1150
725
0.22
1800
>13,300
2200
1600
340
0.06
900
>13,300
505
1200
155
18
4.4
0.72
1050
>13,300
4900
960
3000
18,000
>6200
15
220
32
1400
>13,300
>6200
5300
1600
11
Thrombin Ki 25 °Cb (nM)
Trypsin Ki 25 °Cb (nM)
HK-1c Ki 37 °Cb (nM)
aPCd Ki 37 °Cb (nM)
175
10
e
FVII def. PT EC2X
(lM)
7.9
6.6
Human PPB (% free)
—
4.4
3.3
2.8
1.7
5.8
1.8
PAMPA pH 5.5, 7.4 (nm/s)
HLM t1/2 (min)
Clearance (mL/min/kg)
—, —
23
—
337, 117
100% rem. @10 min
—
209, 34
>120
56 (d)
211, 40
-
54 (d)
805, 80
94
—
26, 21
>120
—
119, 48
114
7 (d)
a
b
c
TF-FVIIa assays were performed with recombinant human enzyme with FX as a substrate (n P 2).
Ki’s for the indicated enzymes were determined by chromogenic substrate assays (n = 2).
Human tissue kallikrein-1.
Activated protein C.
See Ref. 13 for a detailed description.
d
e
Scheme 5. Preparation of macrocycle 59. Reagents and conditions: (a) Bu3SnN3,
PhMe, 105 °C, 2 d; (b) TFA, 15 min.
Scheme 4. Synthesis of the target macrocycles 52–55. Reagents and conditions: (a)
BOP, TEA, DMF–MeCN, rt, 30 min; (b) (i) COCl2, MeCN–DMPU, 0 °C; (ii) Et3N, MeCN
(ꢀ1 mM), 70 °C, 3 h; (c) chiral HPLC; (d) KO–TMS, THF, 60 °C, 18 h; (e) TFA, rt,
15 min.
differential steric requirements to bind to this anti-target. 4-THP
derivative 57 was prepared to introduce a mild degree of polarity
to the P10 ring system. As a result, 57 suffered ꢀ70-fold loss in
potency compared to the respective cyclohexyl analog 56, indicat-
ing that polar substituents may not be tolerated. Lastly, cyclo-
propyl tetrazole 59 showed an outstanding level of TF-FVIIa
inhibitory potency and selectivity, illustrating that this carboxylic
acid surrogate represents a competent pharmacophore for the
series.
Intrigued by the substantial increase in potency associated with
the introduction of cycloalkyl acid P10 groups we decided to gener-
ate X-ray crystal structures of macrocycles 54 and 56 bound to
FVIIa.25,26 The structures of acids 54 and 56 are presented in
Figure 4. The 4-F aminoisoquinoline P1 moiety as well as the
methylphenylene P2 moiety bound in a manner consistent with
X-ray structures described previously.12h,14 In contrast, the S10
binding pattern was quite different from the alkyl sulfone
moieties.18a As anticipated, 54 carboxylate appeared to interact
with the Lys60A side chain although via an intermediary water
molecule. Unexpectedly, the carboxylate of 56 forms a water
mediated interaction with Lys192 side chain. Both cyclobutyl
the nitrile moiety to the required tetrazole, and the sequence was
concluded with Boc removal to yield macrocyclic tetrazole 59.24
The series of prepared macrocycles was evaluated for TF-FVIIa
inhibition and compared to the corresponding parent macrocycle
46 bearing an unsubstituted cyclopropyl group at P10 region
(Table 1). Gratifyingly, introduction of the carboxylic acid moiety
in compound 53 resulted in an ꢀ30 fold boost in inhibitory
potency, and at the same time dramatically improved overall selec-
tivity for the compound. For example, human tissue kallikrein
changed from being essentially equipotent for cyclopropane 46
(HK-1 Ki/TF-FVIIa Ki = 0.8) to being fully selective for acid analog
53 (HK-1 Ki/TF-FVIIa Ki = 2000). Progressive expansion of the ring
size in homologs 53–56 produced a gradual increase in FVIIa inhi-
bitory activity, culminating in picomolar inhibitor 56 (TF-FVIIa
Ki = 60 pM). It should be noted that selectivity against protease
anti-targets was generally preserved in the homologous series
53–56. Of note, cyclobutyl analog 54 exhibited an optimal selectiv-
ity against aPC in the homologous 53–56 series, presumably due to

V. Ladziata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5051–5057
5055
Figure 5. X-ray crystal structure of the des-F aminoisoquinoline analog of 59
complexed with FVIIa (PDB ID: 5L30), solved at 1.73 Å. The omit Fo–Fc electron
density is displayed at 3.5 rmsd. Graphics were generated with the program
PyMOL.27
abovementioned structural data could also explain for 4-THP
analog 57 potency loss, where oxygen atom is presumably placed
inside a highly hydrophobic pocket, and is thus disfavored. The
interesting interactions of the carboxylic acids described above
prompted us to obtain an additional crystal structure of
a
tetrazole acid surrogate, the des-F aminoisoquinoline analog of
59 (Fig. 5). In this case, the tetrazole moiety is positioned in
much closer proximity to the Lys60A side chain making a direct
contact (2.9 Å), presumably due to the more spatially delocalized
nature of the tetrazolide anion vs the carboxylate.28 As observed
for analogs 54 and 56, the cyclopropyl group efficiently interacts
with the Cys42-Cys58 disulfide bridge. Overall, P10 residues in
54, 56 and 59 bind to FVIIa in a highly productive manner, provid-
ing the optimal level of ligand efficiency with almost every atom
engaged in a beneficial interaction with the enzyme.
Further profiling of macrocycles 53–57 and 59 revealed that the
compounds maintained good PAMPA permeability across the ser-
ies, with the exception of analog 57 (Table 1). Lower PAMPA values
in this case could be explained by increased polarity of the 4-THP
moiety. In addition, all macrocyclic inhibitors exhibited excellent
metabolic stability, and represented a substantial improvement
over parent analog 46. Measured FVII deficient prothrombin time
EC2x values13 showed a stronger correlation with human plasma
free fraction values rather than with the in vitro TF-FVIIa inhibition
constants. For instance, the most potent analog in vitro (56;
TF-FVIIa Ki = 60 pM) appeared to have the weakest anti-clotting
activity due to a low free fraction. On the other hand, ꢀ70-fold
weaker inhibitor 57 showed a more robust FVII deficient pro-
thrombin time increase due to reduced protein binding.
Figure 4. (a) Crystal structure of 54 bound to FVIIa (PDB ID: 5L2Y), solved at 1.82 Å.
The omit Fo–Fc electron density is displayed at 5 rmsd. (b) Crystal structure of 56
bound to FVIIa (PDB ID: 5L2Z), solved at 1.79 Å. The omit Fo–Fc electron density is
displayed at 3.5 rmsd. (c) Overlay of 54 with 56 at the FVIIa binding site with
surface. Graphics were generated with the program PyMOL.27
To further understand the effect of the novel P10 groups on com-
pounds’ overall properties, two acid-containing macrocycles 54
and 55 were advanced into i.v. PK studies (Table 1). Disappoint-
ingly, both compounds showed high clearance in dog, likely due
to glucuronidation of the carboxylic acid moiety. We then decided
to dose tetrazolyl analog 59 to investigate if an acid surrogate can
exert a positive effect on the overall pharmacokinetic profile. We
were pleased to discover that tetrazole 59 displayed a much lower
clearance of 7 mL/min/kg after i.v. administration in dogs, however
low oral bioavailability (F% = 2) was observed. The aforementioned
data suggests that tetrazole derivative 59 may have less propensity
for glucuronidation, and could be considered an excellent starting
(54) and cyclohexyl (56) groups are oriented nicely into the S10
Cys42-Cys58 pocket (3.8–5.1 Å), forming a hydrophobic edge to
edge interaction with the disulfide bridge. Overlay of cyclobutyl
and cyclohexyl P10 groups is depicted in Figure 4c. As can be
seen from the superimposition, the six-membered ring occupies
the binding pocket more fully, which may explain the
exceptional FVIIa binding affinity observed for 56. The

5056
V. Ladziata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5051–5057
5. Bauer, K. A. Hematol. Am. Soc. Hematol. Educ. Program 2013, 2013, 464.
6. Priestley, E. S. Drug Discovery Today 2014, 19, 1440.
7. (a) Steffel, J.; Lüscher, T. F.; Tanner, F. C. Circulation 2006, 113, 722; (b) Petrillo,
G.; Cirillo, P.; D’Ascoli, G. L.; Maresca, F.; Ziviello, F.; Chiariello, M. Curr. Cardiol.
Rev. 2010, 6, 325.
point for further potency and ADME optimization to reach oral
bioavailability.
In conclusion, we have successfully designed and synthesized
macrocyclic TF-FVIIa inhibitors bearing novel 1-(1H-tetrazol-5-yl)-
cyclopropyl and 1-carboxycycloalkyl P10 moieties as cyclopropyl
sulfone replacements. The discovered scaffolds allowed for
remarkable improvements both in FVIIa inhibitory potency
(25–300 fold increase) as well as in overall protease selectivity
(>1000 fold enhancement). Important highlights from the X-ray
crystallography and SAR studies include: (1) carboxylic acid sub-
stituent interacts with both Lys60A and Lys192, (2) lipophilic
cycloalkyl moieties are preferred, and bulkier alkyls interface more
efficiently with Cys42-Cys58 motif resulting in potency boost, (3)
tetrazolyl moiety performed as an acid bioisotere, allowing for
similar interactions with FVIIa enzyme and yielding outstanding
inhibitory potency. Ultimately, compound 59 had the optimal bal-
ance of TF-FVIIa potency, selectivity, physicochemical properties
and clearance, representing an optimized and structurally diverse
alternative to cyclopropyl sulfone P10.
8. (a) Himber, J.; Kirchhofer, D.; Riederer, M.; Tschopp, T.; Steiner, B.; Roux, S.
Thromb. Haemost. 1997, 78, 1142; (b) Himber, J.; Refino, C. J.; Burcklen, L.; Roux,
S.; Kirchhofer, D. Thromb. Haemost. 2001, 85, 475; (c) Golino, P.; Ragni, M.;
Cirillo, P.; D’Andrea, D.; Scognamiglio, A.; Ravera, A.; Buono, C.; Ezban, M.;
Corcione, N.; Vigorito, F. Circ. Res. 1998, 82, 39; (d) Szalony, J. A.; Suleymanov,
O. D.; Salyers, A. K.; Panzer-Knodle, S. G.; Blom, J. D.; LaChance, R. M.; Case, B.
L.; Parlow, J. J.; South, M. S.; Wood, R. S.; Nicholson, N. S. Thromb. Res. 2003, 112,
167; (e) Suleymanov, O. D.; Szalony, J. A.; Salyers, A. K.; LaChance, R. M.;
Parlow, J. J.; South, M. S.; Wood, R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther.
2003, 306, 1115; (f) Salyers, A. K.; Szalony, J. A.; Suleymanov, O. D.; Parlow, J. J.;
Wood, R. S.; South, M. S.; Nicholson, N. S. Pharmacology 2004, 70, 100; (g)
Olivero, A. G.; Eigenbrot, C.; Goldsmith, R.; Robarge, K.; Artis, D. R.; Flygare, J.;
Rawson, T.; Sutherlin, D. P.; Kadkhodayan, S.; Beresini, M.; Elliott, L. O.;
DeGuzman, G. G.; Banner, D. W.; Ultsch, M.; Marzec, U.; Hanson, S. R.; Refino,
C.; Bunting, S.; Kirchhofer, D. J. Biol. Chem. 2005, 280, 9160; (h) Zbinden, K. G.;
Banner, D. W.; Hilpert, K.; Himber, J.; Lavé, T.; Riederer, M. A.; Stahl, M.;
Tschopp, T. B.; Obst-Sander, U. Biorg. Med. Chem. 2006, 14, 5357; (i) 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, 2037;
(j) Arnold, C. S.; Parker, C.; Upshaw, R.; Prydz, H.; Chand, P.; Kotian, P.; Bantia,
S.; Babu, Y. S. Thromb. Res. 2006, 117, 343; (k) Wong, P. C.; Luettgen, J. M.;
Rendina, A. R.; Kettner, C. A.; Xin, B.; Knabb, R. M.; Wexler, R.; Priestley, E. S.
Thromb. Haemost. 2010, 104, 261; (l) Nagakura, T.; Tabata, K.; Kira, K.; Hirota, S.;
Clark, R.; Matsuura, F.; Hiyoshi, H. Thromb. Res. 2013, 132, 271.
Acknowledgements
The authors would like to thank Dr. Rick Ewing and Dr. Donald
Pinto for their valuable advice during preparation of this manu-
script. Jeffrey Bozarth, Randi Brown, Tara Peterson, Mojgan Abous-
leiman, Frank Barbera and Yiming Wu are acknowledged for
obtaining enzyme inhibition and clotting assay data. We also
would like to thank Dr. Steven Sheriff for the help with preparation
and PDB deposition of coordinates and data for FVIIa complexes
with compounds 54, 56 and the des-F aminoisoquinoline analog
of 59. X-ray data were collected by Rick Walter and Gina Ranieri
of Shamrock Structures LLC using the IMCA beamlines, 17-BM
and 17-ID, at the Advanced Photon Source. 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.
9. (a) Liu, Y.; Jiang, P.; Capkova, K.; Xue, D.; Ye, L.; Sinha, S. C.; Mackman, N.; Janda,
K. D.; Liu, C. Cancer Res. 2011, 71, 6492; (b) Sampson, M. T.; Kakkar, A. K.
Biochem. Soc. Trans. 2002, 30, 201; (c) Meuller, B. M.; Ruf, W. J. Clin. Invest. 1998,
101, 1372.
10. (a) Taylor, F. B.; Chang, A. C.; Peer, G.; Li, A.; Ezban, M.; Hedner, U. Blood 1998,
91, 1609; (b) Camerer, E.; Huang, W.; Coughlin, S. R. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 5255.
11. Quan, M. L.; Glunz, P. W.; Smallheer, J. M. Advances in Anticoagulants. In
Comprehensive Medicinal Chemistry III, Rotella, D., Chackalamannil, S., Ward, S.,
Eds.; Elsevier Science, 2016.
12. (a) Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157; (b)
Mahesh, S. A.; Gabrail, N. Y.; Gandhi, J. G.; Khorana, A. A.; Manges, R.; Shah, S.;
Thomas, G. W.; Hamdy, A. M.; Stevens-Brogan, M.; Zhou, C. J. Clin. Oncol. 2013,
31, e15014; (c) Arnold, C. S.; Parker, C.; Upshaw, R.; Prydz, H.; Chand, P.; Kotian,
P.; Bantia, S.; Babu, Y. S. Thromb. Res. 2006, 117, 343; (d) Kotian, P. L.; Krishnan,
R.; Rowland, S.; El-Kattan, Y.; Saini, S. K.; Upshaw, R.; Bantia, S.; Arnold, S.; Babu,
Y. S.; Chand, P. Bioorg. Med. Chem. 2009, 17, 3934; (e) Glunz, P. W.; Cheng, X.;
Cheney, D. L.; Weigelt, C. A.; Wei, A.; Luettgen, J. M.; Wong, P. C.; Wexler, R. R.;
Priestley, E. S. Bioorg. Med. Chem. Lett. 2015, 25, 2169; (f) Hu, H.; Kolesnikov, A.;
Riggs, J. R.; Wesson, K. E.; Stephens, R.; Leahy, E. M.; Shrader, W. D.; Sprengeler,
P. A.; Green, M. J.; Sanford, E.; Nguyen, M.; Gjerstad, E.; Cabuslay, R.; Young, W.
B. Bioorg. Med. Chem. Lett. 2006, 16, 4567; (g) 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, 160; (h) Cheney, D. L.; Bozarth, J. M.; Metzler, W. J.; Morin, P. E.;
Mueller, L.; Newitt, J. A.; Nirschl, A. H.; Rendina, A. R.; Tamura, J. K.; Wei, A.;
Wen, X.; Wurtz, N. R.; Seiffert, D. A.; Wexler, R. R.; Priestley, E. S. J. Med. Chem.
2015, 58, 2799; (i) 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.
13. Wong, P. C.; Luettgen, J. M.; Rendina, A. R.; Kettner, C. A.; Xian, B.; Knabb, R. M.;
Wexler, R. R.; Priestley, E. S. Thromb. Haemostasis 2010, 104, 261.
References and notes
1. (a) Braunwald, E.; Califf, R. M.; Cannon, C. P.; Fox, K. A.; Fuster, V.; Gibler, W. B.;
Harrington, R. A.; King, S. B., 3rd.; Kleiman, N. S.; Theroux, P.; Topol, E. J.; Van de
Werf, F.; White, H. D.; Willerson, J. T. Am. J. Med. 2000, 108, 41; (b) Cushman,
M.; Tsai, A. W.; White, R. H.; Heckbert, S. R.; Rosamond, W. D.; Enright, P.;
Folsom, A. R. Am. J. Med. 2004, 117, 19.
14. 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.
15. Zhang, X.; Glunz, P. W.; Johnson, J. A.; Jiang, W.; Jacutin-Porte, S.; Ladziata, V.;
Zou, Y.; 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. E.; Priestley,
E. S. J. Med. Chem. 2016, 59, 7125.
2. Ansell, J.; Hirsh, J.; Poller, L.; Bussey, H.; Jacobson, A.; Hylek, E. Chest 2004, 126,
204S.
3. De Caterina, R.; Husted, S.; Wallentin, L.; Andreotti, F.; Arnesen, H.; Bachmann,
F.; Baigent, C.; Huber, K.; Jespersen, J.; Kristensen, S. D.; Lip, G. Y.; Morais, J.;
Rasmussen, L. H.; Siegbahn, A.; Verheugt, F. W.; Weitz, J. I. J. Am. Coll. Cardiol.
2012, 59, 1413.
16. McNaney, C. A.; Dexler, D. M.; Hnatyshyn, S. Y.; Zvyaga, T. A.; Knipe, J. O.;
Belcastro, J. V.; Sanders, M. ASSAY Drug Dev. Technol. 2008, 6, 121.
17. van Breemen, R. B.; Li, Y. Expert Opin. Drug Metab. Toxicol. 2005, 1, 175.
18. (a) Zhang, X.; Jiang, W.; Jacutin-Porte, S.; Glunz, W. P.; Zou, Y.; Cheng, X.; Nirschl,
H. A.; Wurtz, R. N.; Luettgen, M. J.; Rendina, R. A.; Luo, G.; Harper, M. T.; Wei, A.;
Anumula, R.; Cheney, L. D.; Knabb, M. R.; Wong, C. P.; Wexler, R. R.; Priestley, E. S.
ACS Med. Chem. Lett. 2014, 5, 188; (b) Glunz, P. W.; Zhang, X.; Zou, Y.; Nirschl, A.
H.; Cheng, X.; Weigelt, C. A.; Cheney, D. L.; Wei, A.; Wen, X.; Bozarth, J. M.;
Normandin, D. E.; Hartl, K. S.; Rendina, A. R.; Barbera, F. A.; Luettgen, J. M.; Brown,
R. L.; Peterson, T.; Zhang, G.; Wong, P. C.; Harpel, M.; Shen, L.; M., K. R.; Wexler, R.
R.; Priestley, E. S. Bioorg. Med. Chem. Lett. 2013, 23, 5244.
4. (a) Hauel, N. H.; Nar, H.; Priepke, H.; Ries, U.; Stassen, J. M.; Wienen J. Med.
Chem. 2002, 45, 1757; (b) Weitz, J. I.; Linkins, L. A. Expert Opin. Invest. Drugs
2007, 16, 271; (c) Haas, S.; Schellong, S. Hamostaseologie 2007, 27, 41; (d)
Eriksson, B. I.; Smith, H.; Yasothan, U.; Kirkpatrick, P. Nat. Rev. Drug Disc. 2008,
7, 557; (e) Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, J. J.
Med. Chem. 2005, 48, 5900; (f) Lassen, M. R.; Ageno, W.; Borris, L. C.; Lieberman,
J. R.; Rosencher, N.; Bandel, T. J.; Misselwitz, F.; Turpie, A. G. G. N. Engl. J. Med.
2008, 358, 2776; (g) Straub, A.; Roehrig, S.; Hillisch, A. Curr. Top. Med. Chem.
2010, 10, 257; (h) Perzborn, E.; Roehring, S.; Straub, A.; Kubitza, D.; Misselwitz,
F. Nat. Rev. Drug Disc. 2011, 10, 61; (i) Pinto, D.; Orwat, M.; Koch, S.; Rossi, K.;
Alexander, R.; Smallwood, A.; Wong, P.; Rendina, A.; Luettgen, J. M.; Knabb, R.
M.; Xin, B.; Wexler, R. R.; Lam, P. J. Med. Chem. 2007, 50, 5339; (j) He, K.;
Luettgen, J. M.; Zhang, D.; He, B.; Grace, J. E., Jr.; Xin, B.; Pinto, D. J. P.; Wong, P.
C.; Knabb, R. M.; Lam, P. Y. S.; Wexler, R. R.; Grossman, S. J. Eur. J. Drug Metab.
Pharm. 2011, 36, 129; (k) Pinto, D. J. P.; Smallheer, J. M.; Cheney, D. L.; Knabb, R.
M.; Wexler, R. R. J. Med. Chem. 2010, 53, 6243; (l) Furugohri, T.; Isobe, K.;
Honda, Y.; Kamisato-Matsumoto, C.; Sugiyama, N.; Nagahara, T.; Morishima, Y.;
Shibano, T. J. Thromb. Haemost. 2008, 6, 1542; (m) Bounameaux, H.; Camm, A. J.
Drugs 2014, 74, 1209.
19. Wallace, D. J.; Chen, C.-Y. Tetrahedron Lett. 2002, 43, 6987.
20. Ma, D.; Cai, Q. Acc. Chem. Res. 2008, 41, 1450.
21. (a) Krapcho, A. P.; Weimaster, J. F.; Eldridge, J. M.; Jahngen, E. G. E., Jr.; Lovey, A.
J.; Stephens, W. P. J. Org. Chem. 1978, 43, 138; (b) Krapcho, A. P.; Ciganek, E. Org.
React. 2013, 81, 1.
22. (a) Corey, E. J.; Chaykovsky, M. J. Am. Chem. Soc. 1962, 84, 867; (b) Corey, E. J.;
Chaykovsky, M. J. Am. Chem. Soc. 1962, 84, 3782; (c) Corey, E. J.; Chaykovsky, M.
J. Am. Chem. Soc. 1965, 87, 1353.

V. Ladziata et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5051–5057
5057
23. Gao, X.; Fu, H.; Qiao, R.; Jiang, Y.; Zhao, Y. J. Org. Chem. 2008, 73, 6864.
24. 53: MS (ESI) m/z: 612.2 [M+1]+; 1H NMR (400 MHz, CD3OD) d ppm 8.09 (dd,
J = 9.3, 2.0 Hz, 1H), 7.65 (dd, J = 8.0, 1.8 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.42 (d,
J = 5.0 Hz, 1H), 7.28 (dd, J = 9.3, 2.3 Hz, 1H), 7.21 (s, 1H), 7.16 (d, J = 8.3 Hz, 1H),
6.97 (s, 1H), 6.67 (dd, J = 8.0, 2.3 Hz, 1H), 5.97 (d, J = 2.0 Hz, 1H), 5.77 (s, 1H),
5.54 (d, J = 16.8 Hz, 1H), 4.64 (t, J = 10.9 Hz, 1H), 3.89-4.00 (m, 2H), 3.43-3.53
(m, 1H), 3.28 (s, 3H), 2.32 (s, 3H), 1.64 (br. s., 2H), 1.31 (d, J = 7.0 Hz, 3H,), 1.20
(br. s., 2H). 54: MS (ESI) m/z: 626.3 [M+1]+; 1H NMR (400 MHz, CD3OD) d ppm
8.09 (dd, J = 9.2, 1.9 Hz, 1H), 7.62 (dd, J = 7.9, 1.63 Hz, 1H), 7.40–7.47 (m, 2H),
7.28 (dd, J = 9.3, 2.5 Hz, 1H), 7.22 (d, J = 1.5 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.97
(s, 1H), 6.73 (dd, J = 8.2, 2.1 Hz, 1H), 5.93 (d, J = 2.01 Hz, 1H), 5.70–5.79 (m, 1H),
5.36 (d, J = 16.6 Hz, 1H), 4.61 (t, J = 10.9 Hz, 1H), 3.97 (dd, J = 10.8, 4.3 Hz, 1H),
3.81 (d, J = 16.6 Hz, 1H), 3.42–3.55 (m, 1H), 3.20 (s, 2H), 2.72–2.86 (m, 2H),
2.46–2.61 (m, 2H), 2.10–2.24 (m, 1H), 2.03 (s, 3H), 1.78–1.91 (m, 1H), 1.31 (d,
J = 7.0 Hz, 3H). 55: MS (ESI) m/z: 640.3 [M+1]+; 1H NMR (400 MHz, CD3OD) d
ppm 8.86 (s, 1H), 8.08 (dd, J = 9.3, 1.8 Hz, 1H), 7.62 (dd, J = 8.0, 1.5 Hz, 1H),
7.38–7.47 (m, 2H), 7.25–7.34 (m, 2H), 7.22 (s, 1H), 6.96 (br. s., 1H), 6.72 (dd,
J = 8.3, 2.3 Hz, 1H), 6.01 (d, J = 2.0 Hz, 1H), 5.75 (s, 1H), 5.46 (d, J = 16.3 Hz, 1H),
4.59 (t, J = 10.9 Hz, 1H), 3.97 (dd, J = 10.8, 4.3 Hz, 1H), 3.88 (d, J = 16.3 Hz, 1H),
3.42–3.55 (m, 1H), 3.20 (s, 3H), 2.59 (dt, J = 12.7, 6.3 Hz, 1H), 2.43–2.53 (m, 1H),
2.33 (s, 3H), 1.90–2.09 (m, 2H), 1.65–1.84 (m, 4H), 1.31 (d, J = 7.0 Hz, 3H). 56:
MS (ESI) m/z: 654.3 [M+1]+; 1H NMR (400 MHz, CD3OD) d ppm 8.10 (dd, J = 9.2,
1.9 Hz, 1H), 7.60 (dd, J = 7.8, 1.8 Hz, 1H), 7.36–7.47 (m, 3H), 7.26–7.32 (m, 1H),
7.21 (s, 1H), 6.97 (1 H, s), 6.75 (dd, J = 8.5, 2.3 Hz, 1H), 6.04 (1 H, s), 5.74 (s, 1H),
5.51 (d, J = 16.3 Hz, 1H), 4.57 (t, J = 10.9 Hz, 1H), 3.93–4.03 (m, 2H), 3.44–3.54
(m, 1H), 3.18 (s, 3H), 2.41 (d, J = 15.3 Hz, 1H), 2.35 (s, 3H), 2.30 (d, J = 13.8 Hz,
1H), 1.75–1.88 (m, 2H), 1.55–1.74 (m, 5H), 1.34–1.42 (m, 1H), 1.32 (d,
J = 7.0 Hz, 3H) 57: MS (ESI) m/z: 656.2 [M+1]+; 1H NMR (400 MHz, CD3OD) d
ppm 8.92 (s, 1H), 8.10 (dd, J = 9.3, 1.8 Hz, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.40–7.48
(m, 2H), 7.37 (d, J = 8.5 Hz, 1H), 7.29 (dd, J = 9.3, 2.3 Hz, 1H), 7.19 (1 H, s), 6.96
(1 H, br. s.), 6.77 (dd, J = 8.4, 2.1 Hz, 1H), 6.09 (s, 1H), 5.75 (s, 1H), 5.50 (d,
J = 16.3 Hz, 1H), 4.58 (t, J = 10.9 Hz, 1H), 3.93–4.03 (m, 2H), 3.71–3.91 (m, 3H),
3.42–3.54 (m, 1H), 3.20 (s, 3H), 2.46 (d, J = 15.1 Hz, 1H), 2.34 (s, 3H), 2.03–2.15
(m, 1H), 1.89–2.01 (m, 1H), 1.32 (d, J = 7.0 Hz, 3H). 59: MS (ESI) m/z: 636.2 [M
+1]+; 1H NMR (400 MHz, CD3OD) d ppm 9.03 (s, 1H), 8.09 (dd, J = 9.4, 1.77 Hz,
1H), 7.65 (dd, J = 8.1, 1.8 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 4.8 Hz, 1H),
7.36 (d, J = 8.1 Hz, 1H), 7.29 (dd, J = 9.4, 2.3 Hz, 1H), 7.14 (s, 1H), 6.96 (s, 1H),
6.77 (dd, J = 8.1, 2.0 Hz, 1H), 6.09–6.15 (m, 1H), 5.76 (s, 1H), 5.27 (d, J = 16.9 Hz,
1H), 4.67 (t, J = 11.1 Hz, 1H), 3.97 (dd, J = 10.7, 4.4 Hz, 1H), 3.81 (d, J = 16.7 Hz,
1H), 3.44–3.56 (m, 1H), 2.31 (s, 3H), 1.65–1.84 (m, 2H), 1.48–1.61 (m, 2H), 1.32
(d, J = 7.1 Hz, 3H).
25. Relative atomic coordinates for the crystallographic structures of 54, 56, and
the des-F aminoisoquinoline analog of 59 complexed with factor VIIa have been
deposited in the PDB; their deposition numbers are 5L2Y, 5L2Z, 5L30,
respectively.
26. For procedures for crystallization, data collection, and structure refinement,
please see Ref. 12j.
27. The PyMOL Molecular Graphics System, Version 1.7 Schrödinger, LLC.
28. (a) Carini, D. J.; Christ, D. D.; Duncia, J. V.; Pierce, M. E. Pharm. Biotechnol. 1998,
11, 29; (b) Naylor, E. M.; Chakravarty, P. K.; Costello, C. A.; Chang, R. S.; Chen, T.-
B.; Faust, K. A.; Lotti, V. J.; Kivlighn, S. D.; Zingaro, G. J.; Siegl, P. K. S.; Wong, P.
C.; Carini, D. J.; Wexler, R. R.; Patchett, A. A.; Greenlee, W. J. Bioorg. Med. Chem.
Lett. 1993, 3, 69.