Design, structure-activity relationships, X-ray crystal structure, and energetic contributions of a critical P1 pharmacophore: 3-Chloroindole-7-yl- based factor Xa inhibitors

Article abstract of DOI:10.1021/jm800855x

An indole-based P1 moiety was incorporated into a previously established factor Xa inhibitor series. The indole group was designed to hydrogen-bond with the carbonyl of Gly218, while its 3-methyl or 3-chloro substituent was intended to interact with Tyr228. These interactions were subsequently observed in the X-ray crystal structure of compound 18. SAR studies led to the identification of compound 20 as the most potent FXa inhibitor in this series (IC₅₀ = 2.4 nM, EC_2xPT = 1.2 μM). An in-depth energetic analysis suggests that the increased binding energy of 3-chloroindole-versus 3-methylindole- containing compounds in this series is due primarily to (a) the more hydrophobic nature of chloro- versus methyl-containing compounds and (b) an increased interaction of 3-chloroindole versus 3-methylindole with Gly218 backbone. The stronger hydrophobicity of chloro- versus methyl-substituted aromatics may partly explain the general preference for chloro- versus methyl-substituted P1 groups in FXa, which extends beyond the current series.

Full text of DOI:10.1021/jm800855x

J. Med. Chem. 2008, 51, 7541–7551
7541
Design, Structure-Activity Relationships, X-ray Crystal Structure, and Energetic Contributions
,‡
of a Critical P1 Pharmacophore: 3-Chloroindole-7-yl-Based Factor Xa Inhibitors
Yan Shi,* Doree Sitkoff,* Jing Zhang, Herbert E. Klei, Kevin Kish, Eddie C.-K. Liu, Karen S. Hartl, Steve M. Seiler,§
Ming Chang, Christine Huang, Sonia Youssef, Thomas E. Steinbacher, William A. Schumacher, Nyeemah Grazier,
Andrew Pudzianowski, Atsu Apedo, Lorell Discenza, Joseph Yanchunas, Jr., Philip D. Stein, and Karnail S. Atwal†
Bristol-Myers Squibb Research & DeVelopment, P.O. Box 5400, Princeton, New Jersey 08543-5400
ReceiVed July 11, 2008
An indole-based P1 moiety was incorporated into a previously established factor Xa inhibitor series. The
indole group was designed to hydrogen-bond with the carbonyl of Gly218, while its 3-methyl or 3-chloro
substituent was intended to interact with Tyr228. These interactions were subsequently observed in the
X-ray crystal structure of compound 18. SAR studies led to the identification of compound 20 as the most
potent FXa inhibitor in this series (IC50 ) 2.4 nM, EC2xPT ) 1.2 µM). An in-depth energetic analysis suggests
that the increased binding energy of 3-chloroindole-versus 3-methylindole-containing compounds in this
series is due primarily to (a) the more hydrophobic nature of chloro- versus methyl-containing compounds
and (b) an increased interaction of 3-chloroindole versus 3-methylindole with Gly218 backbone. The stronger
hydrophobicity of chloro- versus methyl-substituted aromatics may partly explain the general preference
for chloro- versus methyl-substituted P1 groups in FXa, which extends beyond the current series.
Introduction
We have reported some of our efforts in developing orally
active and selective FXa inhibitors.11 The ketene aminal
chemotype 2 (Scheme 1), which was derived from a high
throughput screening (HTS) hit (compound 1) by SAR develop-
ment through parallel syntheses and optimization via medicinal
chemistry, was attractive because of its nonpeptidic nature and
charge neutrality. Many early FXa inhibitors containing charged
benzamidine or guanidine moieties had poor oral bioavailability,
and we hoped to avoid this problem with a neutral FXa
inhibitor.12 Compound 2 is a selective FXa inhibitor relative to
related trypsin-like serine proteases and is active in rats after
intravenous administration by measurement of ex vivo clotting
time.13
While 2 and related FXa inhibitors containing a 2-methyl-
benzofuran group have many desirable properties, their potential
for mutagenicity and/or CYP inhibition due to the benzofuran
moiety limited our interest in these compounds.14 In addition,
the concentrations to double the prothrombin time (EC2xPT) are
on the order of 103 times higher than the corresponding IC50
values for this series.11a Several recent literature reports suggest
that highly nonpolar coagulation factor inhibitors can have a
tendency toward high protein binding.15 This tendency could
be associated with the relatively high concentrations required
to achieve efficacy in plasma-based assays and presumably in
vivo.7a,12,16 The 2-methylbenzofuran moiety may contribute to
the hydrophobic nature of compound 2 and result in its observed
high EC2xPT (19 µM). We thus sought to replace the 2-meth-
ylbenzofuran with a more hydrophilic pharmacophore while
retaining the FXa inhibition activity, oral bioavailability, and
other favorable properties of chemotype 2.11a
Thromboembolic events are a leading cause of mortality
worldwide.1 However, only warfarin2 is approved as an oral
antithrombotic treatment in the U.S. Warfarin administration
requires careful monitoring, as its plasma concentration levels
can vary with patients’ CYP2C9 genotype, diet, and coadmin-
istration of other drugs, leading to an increased risk of bleeding
or thrombosis.3 There is clearly an unmet need for oral
anticoagulants with a better safety profile.
Factor Xa (FXaa), one of the serine proteases in the
coagulation pathway, has been a target for antithrombotic
development for some time.4,5 FXa is involved in the initiation
of coagulation when it is activated by the tissue factor/factor
VIIa complex upon vascular injury. It also participates in the
propagation stage of coagulation, catalyzing the cleavage of
prothrombin to form the large burst of thrombin that is necessary
for the final coagulation stage, clot formation.6 Direct FXa
inhibitors have been shown to be effective in animal models
without causing a significant increase in bleeding,7e,8b and
several compounds have progressed into late stage clinical
trials.7-10 Recent clinical trial disclosures on oral, direct FXa
inhibitors such as razaxaban,7 apixaban,8 rivaroxaban,9 and
N-[(1R)-2-[4-(1-methyl-4-piperidinyl)-1-piperazinyl]-2-oxo-1-
phenylethyl]-1H-indole-6-carboxamide (LY517717)10 have pro-
vided clinical support for preclinical findings.
Dedicated to Professor William D. Wulff on the occasion of his 60th
birthday.
‡
The X-ray crystal structure coordinates of 18 in human factor Xa have
been deposited in the Protein Data Bank (PDB code 3ens).
* To whom correspondence should be addressed. For Y.S.: phone, 609-
818-4124; fax, 609-818-3456; e-mail, yan.shi@bms.com. For D.S.: phone,
609-818-5046; fax, 609-818-3545; e-mail, doree.sitkoff@bms.com.
Rational Design
§
On the basis of the X-ray crystal structure of a compound
closely related to 2, we used computer modeling to assist in
identifying a replacement moiety. The starting point of design
was the model of 2 in FXa (see Experimental Section).11a In
this model (Figure 1), compound 2 adopts the commonly seen
L-shaped conformation in the FXa active site. The acylpyrro-
Deceased March 31, 2003.
Deceased December 29, 2006.
†
a Abbreviations: FXa, factor Xa; CYP, cytochrome P450; PDB, Protein
Data Bank; EC2xPT, the concentration of inhibitor required to double the
prothrombin based clotting time in human plasma; EDCI, 1-[3-(dimethy-
lamino)propyl]-3-ethylcarbodiimide hydrochloride; tPA, tissue plasminogen
activator.
10.1021/jm800855x CCC: $40.75
2008 American Chemical Society
Published on Web 11/11/2008

7542 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Shi et al.
Scheme 1
lidine fills the hydrophobic S4 pocket, and the caprolactam
carbonyl oxygen forms a weak hydrogen bond with the
backbone NH of Gly216 (N-O distance of 3.3 Å). One of the
ligand’s nitrile groups packs against the disulfide bond between
Cys220 and Cys191 side chains. The 2-methylbenzofuran group
fits in the S1 pocket of the active site, with the methyl group
just above Tyr228. This interaction appears similar to that seen
for chloroaryl P1 groups in several recently published crystal
structures of serine proteases17 and will be discussed later in
further detail. The oxygen of the 2-methylbenzofuran does not
have an explicit hydrogen bond partner, but its electron-
withdrawing nature could enable the 3-CH to interact favorably
with Asp189.18
We focused on satisfying two major interaction sites in the
S1 pocket in our design. The first is the by now well-known
hydrophobic interaction site above Tyr228, which is fully filled
by the methyl group of the 2-methylbenzofuran in 2 and is often
occupied by a nonpolar moiety in potent inhibitors of trypsin-
like serine proteases that have a neutral P1 group.5 The second
interaction site, not satisfied in 2, involves the carbonyl backbone
of Gly218,19 which could potentially accept a hydrogen bond
from an appropriately placed donor in the ligand. Inhibitors
containing basic P1 groups such as benzamidines commonly
form a hydrogen bond to this carbonyl. At the time of our
design, there were no known P1 pharmacophores that simul-
taneously achieved both the hydrophobic interaction with
Tyr228 and a direct (non-water-mediated) hydrogen bonding
interaction with Gly218 carbonyl.20 We envisioned that a P1
pharmacophore with these properties could impart potency
through the above interactions, while also improving pharma-
cological properties of the ligand relative to 2, by increasing
polarity (due to the hydrogen bond donor) and therefore
potentially reducing protein binding.
As a result of these considerations, we designed compound
3 (Scheme 1), which has a 3-methylindole in place of the
2-methylbenzofuran. The proposed binding model of 3 in FXa
is shown in Figure 2. As can be seen in Figure 2a, the methyl
group of the 3-methylindole overlays with the methyl group of
2-methylbenzofuran in 2 and should thus make similar interac-
tions with Tyr228 in the S1 pocket of FXa, although it does
not extend into the S1 pocket quite as far. Also, as intended by
the design, the NH of the indole forms a slightly elongated
hydrogen bond with the carbonyl group of Gly218 (3.3 Å, Figure
2b). In addition, compound 3 has a calculated log P of 2.42.21
This value is 0.57 units lower than that of compound 2, and
therefore, 3 is predicted to be more hydrophilic than 2 (Table
1).
Chemistry
All the compounds reported in this work were prepared from
their corresponding amines 6a-g or their isothiocyanates 7a-g
and the caprolactam amine 8.11b The amines 6a and 6c-g were
obtained from their corresponding nitro compounds, which are
either commercially available or readily prepared via known
procedures.22 The 3-methyl-7-aminoindole 6b was synthesized
by a two-step sequence from 7-nitro-1H-indole 4 as shown in
Scheme 2. Formylation of indole 4 and one-step reduction of
both the formyl and nitro groups with NaBH4 afforded 6b in
80% yield. The dicyano ketene aminals (3, 14, and 15) were
prepared by sequential displacement of the two methylthio
groups of 2-(bis(methylthio)methylene)malononitrile with 7-ami-
noindoles 6a-c and the caprolactam amine 8 (Scheme 3). The
ketene aminals 16-23 were prepared according to a one-pot
procedure we developed for N,N′-disubstituted ketene aminals
(Scheme 3).23 For example, treatment of methyl 2-cyanoacetate
(10) with sodium hydride and reaction of the resulting anion
with 3-chloroindole-7-isothiocyanate 7c afforded an intermediate
thioamide anion (12c). Subsequent reaction of 12c with capro-
lactam amine 8 and EDCI provided 18 (X ) CO2Me, Y ) CH,
Z ) Cl) in 85% yield. The cyanoguanidine analogues 25 and
26 were prepared from a similar sequence according to a
literature procedure (Scheme 4).24 Treatment of the indole-7-
isothiocyanates (7b and 7c) with sodium cyanoamide gave the
intermediates 24b and 24c, which were reacted with amine 8
Figure 1. Binding model of 2 in FXa. Key residues are shown in
stick representation, and the hydrogen bond between the ligand and
Gly216 amide NH is highlighted. This and the following figures were
created using PyMOL (http://www.pymol.org).

3-Chloroindole-7-yl-Based Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7543
Figure 2. Model of the proposed compound 3 in FXa: (a) bound models of 2 (magenta carbons) and 3 (cyan carbons) superimposed in the FXa
protein (not shown); (b) model of 3 in FXa. Key residues are shown in stick representation. The hydrogen bond between the ligand and Gly216
amide NH is highlighted, as is the designed hydrogen bond between the indole NH and Gly218 carbonyl.
and EDCI to afford 25 and 26, respectively. The anti-FXa
activity for compounds 2, 3, 14-23, 25, and 26 are shown in
Table 1.
and the size and polarity of the 3-substituent of the indole have
a strong impact on anti-FXa activity. (4) The linker between
the indole and caprolactam amine also has an important impact
on anti-FXa activity. The ketene aminal compounds are more
potent than their cyanoguanidine analogues. For example, all
the 3-chloroindole ketene aminal compounds 15 (X ) CN, IC50
) 9.3 nM), 18 (X ) CO2Me, IC50 ) 8.9 nM), and 20 (X )
CONMe2, IC50 ) 2.4 nM) are more potent than the 3-chlor-
oindole cynanoguanidine 26 (IC50 ) 46 nM). In addition, the
activities of the chloroindole compounds are more sensitive to
changes in the linker; replacing the ketene linker with a
cyanoguanidine in the chloroindole case decreases potency by
about 20-fold (compounds 20 and 26), whereas in the meth-
ylindole case, potency is decreased by only about 3-fold
(compounds 19 and 25). Compound 20 is the most potent FXa
inhibitor in this series, having IC50 ) 2.4 nM and EC2xPT ) 1.2
µM.
X-ray Crystal Structure of 18 Bound to FXa. Figure 3
depicts the crystal structure of 18 in FXa, measured at 2.3 Å
resolution. The ketene aminal adopts a typical syn-anti con-
formation23 with the methyl ester anti to the 3-chloroindole
group. The ketene and the aminal planes are significantly twisted
(40°) along the elongated CdC bond (1.42 Å). As was intended
by the design, the NH of the indole forms a hydrogen bond to
the backbone carbonyl of Gly218 (2.8 Å). Also as expected
from the modeling, the chlorine is slightly less deeply placed
than the methyl group of 2-methylbenzofuran compound 2 and
in other FXa compounds that contain a chlorine at the base of
the S1 pocket (4.2 Å distance between chlorine and the Tyr228
C-OH atom for the crystal of 18 compared with a typical
distance of 3.6 Å). This is consistent with the SAR observation
that a hydrophobic and much larger 3-bromine atom (compound
21, IC50 ) 9 nM) can also be accommodated at this position,
but the larger hydrophilic 3-cyano (compound 22) and 3-car-
boxamide groups (compound 23) are disfavored. The ester lies
along the disulfide bond, with the carbonyl forming a hydrogen
bond with the backbone NH of Gln192 (3.0 Å), in addition to
its intramolecular interaction with the indole aminal NH (2.6
Å). The lactam carbonyl forms a hydrogen bond with Gly216
NH (3.0 Å) and with a crystallographic water molecule (3.1
Å); this water also participates in a hydrogen bond with the
Results and Discussion
Although the proposed 3-methylindole compound 3 (IC50
)
114 nM) is about 4-fold less potent in FXa IC50 than 2, it shows
slight improvement in anticoagulation activity (EC2xPT ) 13
µM) compared to 2 (EC2xPT ) 19 µM). The des-methyl analogue
14 is about 4-fold less potent than 3, which is similar to what
was observed in the 2-methylbenzofuran series.11a The 3-chloro
substituted indole compound 15 (IC50 ) 9.3 nM) shows over
10-fold improvement in IC50 compared to 3 and is about 5-fold
more potent in EC2xPT (2.4 µM). The indole compound 3 (clogP
) 2.42) was designed to be more polar than 2 (clogP ) 2.99);
this probably leads to a lower protein binding value for 3 (77%)
in human plasma compared to 2 (92%). While the clogP of the
3-chloroindole compound 15 (clogP ) 2.93) is very close to
that of compound 2, it has a slightly lower protein binding value
(88%). The EC2xPT/IC50 ratios for both compounds 3 (114) and
15 (258) are substantially lower than for 2 (633).
The effect of the different substituents at the 3 position of
indole on anti-FXa activity was further examined with other
ketene aminal linker series (compounds 16-23) and in cyano-
guanidine compounds 25 and 26. The SAR are summarized as
follows: (1) The 3-chloroindole compounds are always more
potent than their 3-methylindole analogues (compare 15 to 3,
20 to 19, and 26 to 25), with the 3-methylindole compounds
having lower plasma protein binding values and EC2xPT/IC50
ratios in each pair. (2) The indazole compound 17 (IC50 ) 13700
nM) is about 30-fold less potent than the corresponding indole
analogue 16 (IC50 ) 650 nM), indicating the 2-nitrogen atom
of indazole has an unfavorable interaction with FXa protein.
(3) While the change of the 3-methylindole in compound 19
(IC50 ) 118 nM) to 3-chloro- (20, IC50 ) 2.4 nM) and
3-bromoindole (21, IC50 ) 9 nM) improves FXa inhibition
activity, the larger and more polar 3-cyano- (22, IC50 ) 290
nM) and 3-carboxamide indole (23, IC50 ) 17000 nM)
analogues are far less potent. This is consistent with our
modeling (vide supra) and X-ray crystal structure analysis of
18 (vide infra) in which the 3-chlorine atom is near Tyr228

7544 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Shi et al.
Table 1. Human FXa Inhibitory Activity and Plasma Protein Bindings of 2, 3, 14-23, 25, and 26
compd
X
Ar
IC50 (nM)a
EC2xPT (µM)b
EC2xPT/IC50
clogPc
protein binding (%)d
2
3
CN
CN
CN
CN
COOMe
COOMe
COOMe
CONMe2
CONMe2
CONMe2
CONMe2
CONMe2
2-methylbenzofuran-5-yl
3-methyl-1H-indol-7-yl
1H-indol-7-yl
3-Cl-1H-indol-7-yl
1H-indol-7-yl
30
114
430
9.3
650
13700
8.9
118
2.4
9
19
13
>50
2.4
50
>50
4.9
17
1.2
6
42
>50
45
633
114
2.99
2.42
92
77
14
15
16
17
18
19
20
21
22
23
25
26
258
2.93
88
1H-indazol-7-yl
3-Cl-1H-indol-7-yl
3-Me-1H-indol-7-yl
3-Cl-1H-indol-7-yl
3-Br-1H-indol-7-yl
3-cyano-1H-indol-7-yl
3-carboxamide-1H-indol-7-yl
3-Me-1H-indol-7-yl
3-Cl-1H-indol-7-yl
550
144
500
667
143
2.40
2.91
80
84
290
17000
336
46
133
326
2.65
3.09
80
84
15
a IC50 values are measured against human FXa utilizing the cleavage of a synthetic substrate S-2222. b Concentration of inhibitor required to double the
prothrombin based clotting time in human plasma. c Biobyte clogP, version 4.2. d Protein binding in human serum by equilibrium dialysis (n ) 3).
Scheme 2. Synthesis of Amine 6b and Isothiocyanates 7a-ga
Scheme 3. Synthesis of 3 and 14-23a
a Reagents and conditions: (a) 2-(bis(methylthio)methylene)malononitrile,
EtOH, 70 °C; (b) 8, EtOH, 70 °C (40-85% for two steps); (c) NaH, DMF;
(d) 7a-g, 60 °C; (e) 8, EDCI (35-91%).
a Reagents and conditions: (a) DMF, POCl3; (b) NaBH4 (80% for two
steps); (c) 1,1′-thiocarbonyldi-2(1H)-pyridone, CH2Cl2 (50-95%).
Scheme 4. Synthesis of 25 and 26a
side chain of Ser214. The pyrrolidine lies squarely in the S4
pocket. A surprising observation is that the Ser195 side chain
oxygen is rotated toward His57 side chain rather than toward
the ligand binding site, in a rare position/torsion angle.25,26 This
is presumably to accommodate the six-membered ring portion
of the chloroindole, which is in proximity because of the linkage
of the indole at its 7-position.
Chloro- versus Methyl-Substituted Indoles. In general,
there is a dramatic increase in the anti-FXa activity of 3-chloro
substituted indole compounds versus 3-methyl substituted indole
compounds (compare 15 with 3, 20 with 19, 26 with 25). This
increased potency due to substitution of a methyl with a chlorine
varies from 7- to 50-fold, corresponding to a difference in
binding energy of 1-2.3 kcal/mol at room temperature. A
preference for chlorine over methyl at the position above Tyr228
has been noted in other chemotypes as well.16c,18b,27 Signifi-
cantly, there are vastly more examples of FXa inhibitors in the
literature that contain chlorine atoms at this position in the P1
a Reagents and conditions: (a) NaNHCN, DMF; (b) 8, EDCI (83-87%
for two steps).
pocket versus a methyl group, although the two groups are
commonly considered to be of fairly similar size and hydro-
phobicity. The source of the binding energy associated with the
chlorine atom has been ascribed to (a) lipophilic interactions,17b,28
(b) a positive electrostatic potential at the tip of the chlorine
atom which interacts favorably with the π-electrons of Tyr
228,18b,20 (c) polarization of the C-H bond ortho to the chloro

3-Chloroindole-7-yl-Based Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7545
Figure 3. (a) Crystal structure of 18 bound in FXa. Hydrogen bonds between the ligand and protein partners Gly218 backbone carbonyl, Gln192
backbone amide NH, and Gly216 backbone amide NH are highlighted, as is a hydrogen bond between the ligand’s lactam carbonyl and a
crystallographic water molecule (red sphere). (b) Alternative view of bound X-ray crystal structure of 18. The 2Fo - Fc electron density contoured
at 1σ is shown for Ser195 and surrounding residues, supporting the unusual torsion of Ser195 side chain oxygen toward His57 rather than toward
the ligand. The hydrogen bond interactions between the crystallographic water and the ligand/protein are also shown. (c) X-ray crystal structure of
18 (orange carbons) overlaid with the bound models of 2 (magenta carbons) and 3 (cyan carbons) (protein not shown).
Table 2. MP2 Quantum Chemical Interaction Energies (kcal/mol)
substituent, leading to a favorable interaction with the nearby
between Isolated FXa Residue Surrogates and the 3-Chloroindole or
3-Methylindole Fragment of 18 in Its Bound Conformation
Asp189,18 and (d) favorable dispersion interactions with the
π-electrons of Tyr228.29a As yet, however, few studies have
a
b
FXa residue surrogate
∆Hchloroindole
∆Hmethylindole
∆∆Hc
critically and quantitatively examined or rank-ordered the
reasons for the apparent preference for chlorine over methyl in
the FXa S1 pocket. In the following sections, we present
quantitative calculations and examine relevant experimental data
with the goal of rationalizing the difference between the
activities of the methyl- and chloro-substituted indole com-
pounds in the current series.
Quantum Chemical Calculations. To probe which residues
in FXa are responsible for the difference in binding energy of
the chloro versus methyl compounds, quantum chemical interac-
tion energies were calculated between an isolated chloroindole
or methylindole ligand fragment and various nearby residue
fragments from FXa. The residue fragments selected for analysis
were the Tyr228 side chain (modeled by p-cresol), the Asp189
side chain (modeled by acetic acid), and the backbone carbonyl
of Gly218 (modeled by N-methylacetamide). The calculations
involving the Asp189 side chain were performed for both the
ionized and neutral forms of the side chain. According to the
crystal structure of 18, the carboxylate is in very close contact
with the ligand but does not have a salt bridge or ion partner,
so the neutral form could be energetically favored when the
ligand is bound due to desolvation penalties for burying charged
species. The results of these calculations, shown in Table 2,
Tyr228 side chain
-0.67
-11.62
-1.03
-5.43
-0.63
-12.11
-0.31
-4.39
-0.04
0.49
-0.72
-1.04
Asp189 side chain (charged)
Asp189 side chain (neutral)
Gly218 carbonyl
a Calculated heat of formation of the 3-chloroindole-residue complex
minus heats of formation of the 3-chloroindole and FXa residue alone.
b Calculated heat of formation of the 3-methylindole-residue complex minus
heats of formation of the 3-methylindole and FXa residue alone. c ∆∆H )
∆Hchloroindole - ∆Hmethylindole
.
demonstrate that all of the FXa residue fragments interact
favorably with both the 3-chloro- and 3-methylindole (see
∆Hchloroindole and ∆Hmethylindole interaction energy columns). The
residue fragment with the largest interaction energies is the
charged Asp189 side chain, followed by the Gly218 carbonyl;
the Tyr228 and Asp189 neutral side chains have the smallest
interaction energies. As noted in the relative interaction energies
column (∆∆H) of Table 2, the Tyr228 side chain shows virtually
no preference for 3-chloroindole versus 3-methylindole. Instead,
the FXa residue fragment most responsible for the preferred
binding of 3-chloroindole over 3-methylindole is the Gly218
backbone carbonyl; presumably the hydrogen bond between the
Gly218 backbone carbonyl and the indole NH, or the electro-
static interaction in general is strengthened when the electron-

7546 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Shi et al.
Table 3. The log P and clogP Values for Compounds Containing an
Arylmethyl versus an Arylchlorine Group
withdrawing chlorine is substituted for the electron-donating
methyl group on the indole ring. This is consistent with the
observation that changing the ketene linker to a cyanoguanidine
linker has a more dramatic effect on FXa potency in chloroin-
dole (20 and 26) versus methylindole (19 and 25) compounds
(vide supra); differences in linker geometry could influence the
indole NH-Gly218CdO interaction. The Asp189 charged side
chain actually shows a preference for 3-methylindole over
3-chloroindole, in contrast to the expectation based on Cl- versus
Me-induced polarization of the indole 2C-H bond. However,
the neutral version of the Asp189 side chain favors 3-chloroin-
dole over 3-methylindole, in agreement with the experimental
findings. In summary, the quantum calculations suggest that the
preference for 3-chloroindole over 3-methylindole is due to
enhanced interactions between the ligand and the backbone
carbonyl of Gly218 but not due to changes in interactions with
Tyr228. In addition, calculations indicate the chloroindole to
interact more favorably with the neutral form of Asp189 side
chain than does the methylindole, raising the possibility of a
significant pKa shift of Asp189 when these ligands are bound.
a
compd exptl log Poct
toluene
chlorobenzene
3-methylindole
3-chloroindole
clogPb
2.69
2.84
2.60
2.64
2.86
2.63
3.07
a Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in
Chemistry and Biology; Wiley: New York, 1979. b Biobyte clogP, version
4.2.
Table 4. Selectivity Profiles of 15 and 20
IC50 (nM) or % inhibition at 33 µM
human enzymes
15
20
factor Xa
trypsin
thrombin
tryptase
9.3
10813
7360
2.4
1043
1100
27%
13%
activated protein C
factor IXa
factor VIIa
urokinase
plasmin
>33000
>33000
32%
17%
>33000
>33000
11%
>33000
>33000
32%
It should be noted that the chlorine or methyl group on the
7-linked 3-substituted indole ligand studied here is considerably
farther from Tyr228 than are the chlorines in most chloro-P1
groups (4.2 Å versus 3.6 Å), which could explain the insensitiv-
ity of the calculated Tyr228-ligand interaction energy to the
chloro versus methyl substitution. Interestingly, in a recent study
of Cl-π interactions in simplified systems,29a dispersion was
highlighted as the major contributor for the Cl-ethyne/benzene
interaction at ∼3.5 Å, i.e., near the minimum of the interaction
energy curve. The calculated interactions decrease with distance
for both the Cl-ethyne/benzene and the methane/benzene
pairs;29a-c however, more detailed comparisons to that work
are difficult, as the model systems differ from those studied
here. Notably, a previous, similar assessment of a more typically
positioned chlorine-containing P1 ligand in FXa found that
interactions with Tyr228 actually slightly disfavored binding
of the chlorine- versus methyl-containing ligands; in that case,
interactions with the charged Asp189 residue favored the chloro-
versus methyl-P1 group.18a
tPA
3990
45%
20, 25, 26, and several relevant small molecules in Tables 1
and 3. The log P and clogP differences between each matched
pair of chlorine versus methyl-containing compounds in this
group range from 0.15 to 0.51, corresponding to a 0.2-0.7 kcal/
mol desolvation contribution that consistently favors binding
of the chlorine-containing compounds. This amount is less than
the observed 1-2.3 kcal/mol preference based on measured FXa
IC50 values in the current series; thus, desolvation by itself only
partially explains why chloro compounds are favored in FXa
over their methyl-containing counterparts for this series. How-
ever, we note that a stronger hydrophobicity, and thus a more
favorable desolvation contribution, is likely at least part of the
reason that chloro-, rather than methyl-, substituted aromatics
are commonly preferred in the S1 pocket in general.
In Vivo Activity. The ketene aminals described above are
selective FXa inhibitors relative to related trypsin-like serine
proteases. Table 4 shows the activity profiles of compounds 15
and 20. Furthermore, compound 15 partially protected against
lethality in mice induced by intravenous injection of Russel’s
viper venom (which causes FXa activation and lethality due to
pulmonary thrombosis). Vehicle-treated mice died within 2.4
( 0.3 min (n ) 22) after the Russel’s viper venom injection,
while survival times were increased to 18.0 ( 5.2 min (n ) 4)
after oral administration of 5 mg/kg 15.30
As mentioned above, the preference for chlorine over methyl
substituents in the S1 pocket has previously been attributed to
a positive electrostatic potential at the tip of the chlorine in
chlorobenzene which might interact favorably with the π-elec-
trons of Tyr228. Rather than estimating interactions based on
ligand surface potentials, the quantum method used here
computes total interaction energies between the two molecular
fragments. Additionally, the quantum energies are calculated
at a higher level of theory, permitting a more reliable quantitative
estimate of the interaction between Tyr228 and the chloro versus
the methyl substituent.
Conclusion
We have reported here the design and incorporation of an
indole-based P1 moiety onto a previously established FXa
inhibitor series. The indole group was designed and subsequently
observed in an X-ray crystal structure to achieve a hydrogen
bond with the backbone carbonyl of Gly218. While there have
been previous reports of indoles as P1 groups,20,31 this is the
first report of a series of FXa inhibitors linked at the 7-position
of indole, which causes the primary hydrogen bond partner to
be Gly218 in place of Asp189, and the Ser195 side chain to
move into a rarely seen torsional position. The new torsion angle
creates a slightly larger and more hydrophobic pocket which
could be capitalized upon in future ligand designs. An in-depth
energetic analysis suggests that the increased binding energy
of 3-chloroindole versus 3-methylindole-containing compounds
in this series is due primarily to (a) the more hydrophobic nature
Desolvation Contributions. The experimental measurements
of protein binding (Table 1) are higher for the 3-chloroindole
compounds versus their 3-methylindole counterparts, indicating
that the chloroindole-containing compounds are likely more
hydrophobic (compare 15, 20, and 26 with 3, 19, and 25). Higher
hydrophobicity indicates that desolvation contributions favor
removal from water and therefore binding of the chlorine-
containing ligands, in agreement with the observed increased
binding of chlorine- versus methyl-containing compounds to
FXa. Additionally, the ratios of EC2xPT to IC50 increase upon
replacement of a methyl with a chloro group (Table 1), again
suggesting that the chlorine-containing ligands are more hy-
drophobic and are likely easier to desolvate. Experimental and
calculated log P values are shown for compounds 3, 15, 19,

3-Chloroindole-7-yl-Based Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7547
on an absolute scale with TRUNCATE37 from the CCP438 suite.
An earlier in-house structure, with its inhibitor and waters removed,
was used as the initial model. The structure was first refined to 2.3
Å resolution with CNX39 (Accelrys, Inc., San Diego, CA) and
modeled with QUANTA (Accelrys, Inc., San Diego, CA). The
refinement was completed with PHENIX,40 modeled with COOT,41
and checked with MolProbity.42 Ligand parametrization was done
with eLBOW;40 however, the target length of the vinyl bond was
set to 1.41 Å based on the structures of similar compounds23 and
QChem 3.043 RI-MP2/6-31+G(d,p) ab initio calculations on the
truncated structure (Z)-methyl 2-cyano-3-(methylamino)-3-(pheny-
lamino)acrylate. The measured structure factors and refined coor-
dinates were deposited in the Protein Data Bank44 as PDB entry
3ens.
of chloro- versus methyl-containing compounds, which makes
them easier to desolvate, and (b) increased interactions of
3-chloro- versus 3-methyl-containing indoles with Gly218
backbone. Interactions with Asp189 may play a secondary role.
The Tyr228 interacts well with both 3-chloroindoles and
3-methylindoles without special preference. The stronger hy-
drophobic nature of chloro- versus methyl-substituted aromatic
rings may explain in part the general preference for chloro-
versus methyl-substituted P1 groups in FXa, which extends
beyond the current series. These calculations have led to a deeper
understanding of the role played by chlorine in indole- as well
as nonindole-containing ligand-FXa interactions.
Chemistry. All reagents and solvents were of commercial quality
and used without further purification. Column chromatography was
performed using Merck silica gel 60 (230-400 mesh). Proton NMR
spectra (500 MHz) and carbon NMR (126 MHz) were obtained on
a Joel 500 spectrometer. Chemical shifts are reported in parts per
million relative to CDCl3 (7.26 ppm for proton and 77.00 ppm for
carbon NMR). All new final SAR compounds were determined to
Experimental Section
Generation of Binding Models. Models were obtained by
minimizing ligands 2 and 3 in the published FXa crystal structure
1fjs32 using a distance-dependent dielectric in Discover, version
98.0, in the InsightII package (Accelrys, Inc., San Diego, CA) with
the CFF force field.33 The ligand was initially placed in the FXa
active site based on an in-house crystal structure of a compound
identical to 2 but with an acylguanidine linker in place of the ketene
aminal.11a The ligand and all residues in the protein were held fixed
with the exception of Tyr99, Phe174, Cys191, Cys220, Trp215,
Ser195, Gln192, Arg143, Glu146, and Gly218. Following conver-
gence, the minimization was repeated with the ligand free to move.
Calculation of Quantum Mechanical Interaction Energies.
All molecules were minimized separately in the gas phase in the
Jaguar program34 (Schro¨dinger, LLC) using ab initio density
functional theory B3LYP/6-31G*. Subsequently, either chloroindole
or methylindole was placed near the molecule representing the side
chain or backbone moiety of interest in a geometry taken from the
crystal structure of 18 (for 3-methylindole, the indole was fixed to
its position in the crystal structure of 18). The interaction energies
were calculated with fixed geometries at the MP2 level with basis
set 6-311+G** using Gaussian35 with counterpoise (CP) corrections.
Crystallization of FXa with Compound 18. When complexed
with the inhibitor, the protein stock solution consisted of FXa at
9.8 mg/mL (0.27 mM based on the calculated molecular weight of
36.1 kDa) in 20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5% v/v
DMSO, and 100 µM compound 18. All co-crystallization trials of
FXa were prepared by the hanging-drop, vapor-diffusion method.
The initial crystallization screens, which were prepared on a
Cyberlab/Gilson C-240 (Middleton, WI) in NeuroProbe (Gaithers-
burg, MD) CT300L2/5 trays, consisted of several commercial kits
from Hampton Research (Aliso Viejo, CA).
The initial crystals were observed from solution 26 of Crystal
Screen 2 and were further optimized on siliconized cover slips in
24-well Linbro plates. The 1 mL reservoir solution consisted of
15-22% w/v PEG MME 5000, 10 mM calcium acetate, 350 mM
sodium acetate, 100 mM lithium sulfate, and 100 mM MES pH
6.0. Drops were formed from 1 µL of the protein solution and 1
µL of the reservoir solution (total initial drop volume of 2 µL),
mixed, and equilibrated at room temperature. Crystals appeared
within 1 week. Single crystals were removed and prepared for data
collection with 20% v/v glycerol added to the reservoir solution as
the cryoprotectant. The diffraction pattern from crystals gave unit
cell parameters of a ) 62.3 Å, b ) 78.9 Å, c ) 73.9 Å, and ꢀ )
102.8°. The symmetry was consistent with space group P21. On
the basis of the unit cell and space group, the asymmetric unit was
estimated to contain two FXa molecules (50% solvent fraction).
Structure Refinement. Diffraction images were recorded on a
R-axis IV++ image plate detector mounted on a Rigaku RU-200
X-ray generator run at 50 kV and 100 mA (5 kW) with Osmic
“blue” confocal optics. A collimator with 0.3 mm × 0.6 mm
pinholes was used to collimate the Cu KR beam. An Oxford
Cryosystems model 600 liquid-nitrogen cooler maintained the
mounted crystal at 100 K. The diffraction images were processed
with DENZO and SCALEPACK from the HKL36 suite. The
intensities were converted to structure-factor amplitudes and placed
1
be consistent with the proposed structures by H and 13C NMR,
MS, HRMS, and combustion elemental analysis.
General Procedure for the Synthesis of Compounds 2, 3,
14, and 15. A solution of 2-(bis(methylthio)methylene)malononitrile
(42.5 mg, 0.25 mmol) and 1H-indol-7-amine 6a (36.3 mg, 0.28
mmol) in absolute ethanol (1.5 mL) was stirred at 70 °C for 6 h.
The caprolactam amine 8 (60 mg, 0.25 mmol) was added, and the
reaction mixture was stirred at 70 °C overnight. The mixture was
then concentrated to dryness and the residue was purified by flash
chromatography on silica gel (0-5% methanol in ethyl acetate) to
afford compound 14 as a yellow solid (44.5 mg, 40% yield).
General Procedure for the Synthesis of Compounds
16-23, 25, and 26. To a solution of methyl 2-cyanoacetate (24.8
mg, 0.25 mmol) in dry DMF (1.5 mL) was added NaH (95%, 8.2
mg, 0.33 mmol). The mixture was stirred for 10 min at room
temperature, upon which time 3-chloro-7-isothiocyanato-1H-indole
7c (47 mg, 0.23 mmol) was added in one portion. The mixture
was stirred for 30 min at 60 °C, and the HPLC analysis showed
the disappearance of 3-chloro-7-isothiocyanato-1H-indole. The
caprolactam amine 8 (60 mg, 0.25 mmol) and 1-(3-dimethylami-
nopropyl)-2-ethylcarbodiimide hydrochloride (58 mg, 0.30 mmol)
were added in that order. The reaction mixture was stirred at room
temperature overnight. The reaction was then quenched by addition
of 10% of lithium chloride solution (2 mL) and extracted with ethyl
acetate three times (3 × 10 mL). The combined organic fractions
were washed once with brine, dried over MgSO4, and evaporated.
The residue was purified by flash chromatography on silica gel
(0-5% methanol in ethyl acetate) to afford compound 18 as a
yellow solid (116 mg, 91% yield). The products were analyzed
with HPLC with following method: column-YMC S-5 ODS-A, 4.6
mm × 50 mm; detection at 220 nm; flow rate 4 mL/min; 4 min
linear gradient from 10:90 methanol/water (containing 0.2%
phosphoric acid) to 90:10 methanol/water (containing 0.2% phos-
phoric acid).
(S)-2-((2-Methylbenzofuran-5-ylamino)(2-oxo-1-(2-oxo-2-
(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)methylene)malononi-
trile (2). This compound was prepared in 95% yield from
2-methylbenzofuran-5-amine, 2-(bis(methylthio)methylene)ma-
lononitrile, and 8. 1H NMR (CDCl3) δ 7.40 (d, 1H, J ) 8.2
Hz), 7.25 (s, 1H), 7.09 (s, 1H), 6.96 (d, 2H, J ) 6.6 Hz), 6.36
(s, 1H), 4.90 (br s, 1H), 4.06 (s, 2H), 3.60 (t, 1H, J ) 11.8 Hz),
3.40 (t, 2H, J ) 6.9 Hz), 3.30 (t, 2H, J ) 6.9 Hz), 3.16 (dd, 1H,
J ) 13.2, 3.3 Hz), 2.43 (s, 3 H), 2.15 (d, 1H, J ) 13.2 Hz),
2.06 (br s, 1H), 1.55-1.95 (m, 8H) ppm; 13C NMR (126 MHz,
CDCl3) δ 171.86, 165.47, 160.01, 157.66, 153.78, 130.73 (2C),
128.62, 121.37, 118.21, 117.93, 112.03, 102.88 (2C), 53.99,
51.64, 50.57, 46.04, 45.60, 31.02, 26.33, 26.10, 25.62, 23.98,
14.11 ppm. HRMS for C25H28N6O3 calcd for (M + H)+,
461.2301; measured, 461.2303. Anal. (C25H28N6O3) C, H, N.

7548 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Shi et al.
(S)-2-((3-Methyl-1H-indol-7-ylamino)(2-oxo-1-(2-oxo-2-(pyr-
rolidin-1-yl)ethyl)azepan-3-ylamino)methylene)malononitrile
(3). This compound was prepared in 85% yield from 6b, 8, and
2-(bis(methylthio)methylene)malononitrile. 1H NMR (CDCl3) δ
9.38 (s, 1H), 7.47 (d, 1H, J ) 8.2 Hz), 7.30 (s, 1H), 7.01 (t, 1H, J
) 7.7 Hz), 6.92 (s, 1H), 6.81 (d, 1H, J ) 7.7 Hz), 6.32 (d, 1H, J
) 5.0 Hz), 4.72 (br s, 1H), 4.0-4.12 (m, 2H), 3.69 (s, 3H), 3.47
(br s, 1H), 3.33-3.40 (m, 2H), 3.30 (t, 2H, J ) 6.6 Hz), 3.12 (d,
1H, J ) 6.0 Hz), 2.26 (s, 3 H), 1.40 -1.92 (m, 10H) ppm; 13C
NMR (126 MHz, CDCl3) δ 172.12, 165.81, 164.92, 131.25, 130.91,
123.23, 119.36, 119.26, 119.06, 116.86, 116.65, 116.03, 112.35,
54.74, 52.01, 50.37, 46.12, 45.72, 30.49, 26.22, 26.10, 26.00, 24.00,
9.66 ppm. HRMS for C25H29N7O2 calcd for (M + H)+, 460.2461;
measured, 460.2463. Anal. (C25H29N7O2) C, H, N.
(S)-2-((1H-Indol-7-ylamino)(2-oxo-1-(2-oxo-2-(pyrrolidin-1-
yl)ethyl)azepan-3-ylamino)methylene)malononitrile (14). This
compound was prepared in 40% yield from 6a, 8, and malononitrile.
1H NMR (500 MHz, CDCl3) δ 9.93 (s, 1 H), 7.59 (d, J ) 7.9 Hz,
1 H), 7.48 (s, 1 H), 7.21 (s, 1 H), 7.07 (t, J ) 7.5 Hz, 1H), 6.92 (d,
J ) 7.5 Hz, 1 H), 6.54 (s, 1 H), 6.35 (s, 1H), 5.25 (s, 1H), 4.75 (s,
1H), 4.12 (s, 1 H), 3.19-3.60 (m, 6 H),1.39-1.90 (m, 10 H) ppm.
HRMS for C24H27N7O2 calcd for (M + H)+, 446.2305; measured,
446.2304. Anal. (C24H27N7O2) C, H, N.
(S)-2-((3-Chloro-1H-indol-7-ylamino)(2-oxo-1-(2-oxo-2-(pyr-
rolidin-1-yl)ethyl)azepan-3-ylamino)methylene)malononitrile
(15). This compound was prepared in 79% yield from 6c, 8, and
2-(bis(methylthio)methylene)malononitrile. 1H NMR (500 MHz,
CDCl3) δ 10.10 (s, 1H), 7.61 (s, 1H), 7.52 (d, 1H, J ) 8.3 Hz),
7.04-7.12 (m, 2H), 6.92 (d, 1H, J ) 7.2 Hz), 6.34 (s, 1H), 4.73
(s, 1H), 4.15 (d, 1H, J ) 16.5 Hz), 3.96 (d, 1H, J ) 14.9 Hz), 3.49
(m, 1H), 3.26-3.34 (m, 1H), 3.19-3.27 (m, 2H), 3.05-3.19 (m,
2H, J ) 16.8 Hz), 1.39-1.90 (m, 10H) ppm; 13C NMR (126 MHz,
CDCl3) δ 172.34, 165.81, 165.59, 129.94, 129.58, 127.69, 122.66,
120.90, 120.56, 119.95, 118.37, 117.87, 117.64, 106.60, 55.02,
52.15, 50.49, 46.12, 45.74, 30.53, 26.26, 26.16, 26.02, 23.94 ppm.
HRMS for C24H26ClN7O2 calcd for (M + H)+, 480.1915; measured,
480.1910. Anal. (C24H26ClN7O2) C, H, Cl, N.
(CDCl3) δ 172.91, 170.93, 165.71, 162.54, 130.32, 129.90, 127.53,
122.54, 120.78, 120.44, 119.01, 118.21, 106.68, 99.89, 55.12, 52.21,
51.54, 50.29, 46.06, 45.70, 36.48, 30.63, 26.29, 26.10, 24.00 ppm.
HRMS for C25H29ClN6O4 calcd for (M + H)+, 513.2017; measured,
513.2010. Anal. (C25H29ClN6O4) C, H, Cl, N.
(S)-2-Cyano-N,N′-dimethyl-3-(3-methyl-1H-indol-7-ylamino)-3-
(2-oxo-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)acryl-
amide (19). This compound was prepared in 81% yield from 7b,
8, and 2-cyano-N,N′-dimethylacetamide. 1H NMR (CDCl3) δ
11.58 (s, 0.5H), 9.41 (s, 0.5H), 7.99 (s, 1H), 7.46 (d, 1H, J )
8.2 Hz), 7.08 (t, 1H, J ) 7.4 Hz), 6.99 (d, 1H, J ) 7.7 Hz),
6.96 (s, 1H), 6.07 (br s, 0.5H), 4.94 (br s, 0.5H), 4.18 (br s,
1H), 4.02 (d, 1H, J ) 15.3 Hz), 3.32-3.45 (m, 6H), 3.15 (br s,
1H), 3.06 (s, 6H), 2.29 (s, 3H), 1.39-1.98 (m, 10H) ppm; 13C
NMR (CDCl3) δ 172.87, 165.81, 160.74, 152.16, 146.64, 139.99,
130.97, 130.38, 122.80, 119.32, 118.33, 117.83, 111.98, 99.41,
54.76, 51.72, 50.25, 46.04, 45.72, 38.34, 38.22, 30.29, 26.53,
26.26, 24.02, 9.72 ppm. HRMS for C27H35N7O3 calcd for (M +
H)+, 506.2880; measured, 506.2883. Anal. (C27H35N7O3) C, H,
N.
(S)-3-(3-Chloro-1H-indol-7-ylamino)-2-cyano-N,N′-dimethyl-3-
(2-oxo-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)acry-
lamide (20). This compound was prepared in 90% yield from
1
7c, 8, and 2-cyano-N,N′-dimethylacetamide. H NMR (CDCl3)
δ 11.55 (br s, 1H), 10.30 (br s, 0.6H), 7.52 (d, 1H, J ) 7.7 Hz),
7.13-7.17 (m, 2H), 7.05 (d, 1H, J ) 7.7 Hz), 5.68 (br s, 0.4H),
4.95 (br s, 1H), 4.20 (br s, 1H), 4.04 (d, 1H, J ) 16.5 Hz), 3.71
(s, 1H), 3.34-3.45 (m, 4H), 3.13-3.20 (m, 1H), 3.05 (s, 7H),
1.97 (quintet, 2H, J ) 6.6 Hz), 1.85 (quintet, 2H, J ) 6.6 Hz),
1.45-1.80 (m, 6H) ppm; 13C NMR (CDCl3) δ 173.11, 167.13,
160.59, 152.51, 148.79, 137.18, 131.19, 127.35, 122.23, 120.66,
119.51, 117.16, 111.27, 106.42, 56.88, 52.01, 51.93, 50.39,
46.10, 45.72, 38.30, 31.77, 30.29, 26.67, 26.16, 24.04 ppm.
HRMS for C26H32ClN7O3 calcd for (M + H)+, 526.2333;
measured, 526.2328. Anal. (C26H32ClN7O3) C, H, Cl, N.
(S)-3-(3-bromo-1H-indol-7-ylamino)-2-cyano-N,N′-dimethyl-3-
(2-oxo-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)acryl-
amide (21). This compound was prepared in 93% yield from 7e,
8, and 2-cyano-N,N′-dimethylacetamide. 1H NMR (CDCl3) δ
11.49 (br s, 0.5H), 10.40 (br s, 0.5H), 7.42 (d, 1H, J ) 7.7 Hz),
7.16 (s, 1H), 7.11 (t, 1H, J ) 7.4 Hz), 7.00 (d, 1H, J ) 7.1 Hz),
5.70 (br s, 0.5H), 4.90 (br s, 0.5H), 4.14 (br s, 1H), 3.98 (d, 1H,
J ) 14.9 Hz), 3.52 (br s, 1H), 3.28-3.43 (m, 5H), 3.12 (d, 1H,
J ) 12.1 Hz), 3.00 (s, 6H), 1.37-1.95 (m, 10H) ppm; 13C NMR
(CDCl3) δ 173.07, 171.23, 165.63, 160.60, 131.31, 129.94,
128.81, 125.33, 124.74, 121.61, 120.76, 119.46, 118.09, 91.04,
55.06, 51.97, 50.39, 50.25, 46.08, 45.70, 43.41, 38.32, 30.27,
26.67, 26.14, 24.02 ppm. HRMS for C26H32BrN7O3 calcd for
(M + H)+, 570.1828; measured, 570.1823. Anal. (C26H32BrN7O3)
C, H, N.
(S)-Methyl-3-(1H-indol-7-ylamino)-2-cyano-3-(2-oxo-1-(2-oxo-
2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)acrylate (16). This
compound was prepared in 67% yield from 7a, 8, and methyl
1
2-cyanoacetate. H NMR (CDCl3) δ 11.03 (s, 1H), 9.64 (s, 1H),
7.60 (d, 1H, J ) 8.3 Hz), 7.13 (t, 1H, J ) 7.4 Hz), 7.02 (d, 1H, J
) 7.7 Hz), 6.56 (s, 1H), 6.17 (br s, 1H), 5.21 (br s, 1H), 4.14-4.34
(m, 1H), 3.96 (d, 1H, J ) 15 Hz), 3.77 (s, 3H), 3.30-3.47 (m,
6H), 3.12-3.18 (m, 1H), 1.21-1.96 (m, 10H) ppm; 13C NMR
(CDCl3) δ 173.82, 170.22, 165.85, 164.17, 131.07, 130.20, 125.59,
120.66, 120.25, 120.08, 119.42, 117.20, 116.90, 103.23, 54.90,
52.01, 51.52, 50.19, 46.06, 45.74, 42.78, 40.99, 30.71, 26.16, 24.04
ppm. HRMS for C25H30N6O4 calcd for (M + H)+, 479.2407;
measured, 479.2401. Anal. (C25H30N6O4) C, H, N.
(S)-Methyl-3-(1H-indazol-7-ylamino)-2-cyano-3-(2-oxo-1-(2-
oxo-2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)acrylate (17). This
compound was prepared in 50% yield from 7d, 8, and methyl
2-cyanoacetate. 1H NMR (CDCl3) δ 11.07 (s, 1H), 9.76 (s, 0.5H),
8.06 (s, 1H), 7.47-7.68 (m, 1H), 7.00-7.16 (m, 2H), 6.03 (br
s, 0.5H), 5.05 (s 1H), 4.02-4.05 (m, 1H), 3.78 (s, 3H),
6.60-3.68 (m, 1H), 3.22-3.45 (m, 6H), 3.03-3.17 (m, 1H),
1.21-1.95 (m, 10H) ppm; 13C NMR (CDCl3) δ 172.73, 170.95,
159.26, 155.60, 137.76, 133.36, 123.95, 121.61, 120.72, 119.61,
116.37, 110.43, 104.60, 54.96, 51.64, 50.63, 50.05, 46.16, 45.80,
30.63, 30.35, 29.70, 26.12, 24.02 ppm. HRMS for C24H29N7O4
calcd for (M + H)+, 480.2359; measured, 480.2359. Anal.
(C24H29N7O4) C, H, N.
(S)-Methyl-3-(3-chloro-1H-indol-7-ylamino)-2-cyano-3-(2-
oxo-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)acrylate
(18). This compound was prepared in 91% yield from 7c, 8, and
methyl 2-cyanoacetate. 1H NMR (CDCl3) δ 10.99 (s, 0.5H), 10.06
(s, 1H), 7.98 (s, 0.5H), 7.58 (d, 1H, J ) 7.7 Hz), 7.13-7.24 (m,
2H), 6.95-7.05 (m, 1H), 5.95 (br s, 0.5H), 5.14 (br s, 0.5H), 4.25
(br s, 1H), 3.96 (d, 1H, J ) 16 Hz), 3.75 (s, 3H), 3.23-3.46 (m,
6H), 3.05-3.17 (m, 1H), 1.25-1.95 (m, 10H) ppm; 13C NMR
(S)-3-(3-Cyano-1H-indol-7-ylamino)-2-cyano-N,N′-dimethyl-3-
(2-oxo-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)-
acrylamide (22). This compound was prepared in 85% yield
from 7f, 8, and 2-cyano-N,N′-dimethylacetamide. 1H NMR
(CDCl3) δ 11.68 (br s, 0.6H), 11.51 (br s, 0.6H), 7.70 (s, 1H),
7.65 (d, 1H, J ) 7.7 Hz), 7.14-7.24 (m, 2H), 7.11 (d, 1H, J )
7.7 Hz), 5.41 (br s, 0.4H), 5.01 (br s, 0.4H), 4.03-4.20 (m, 1H),
3.07-3.70 (m, 7H), 3.04 (s, 6H), 2.90-2.99 (m, 1H), 1.45-1.97
(m, 10H) ppm; 13C NMR (CDCl3) δ 173.37, 165.53, 157.74,
133.26, 129.19, 129.01, 128.20, 125.27, 124.22, 122.64, 121.32,
120.52, 118.41, 115.70, 87.39, 55.36, 52.23, 50.55, 46.20, 45.74,
38.24, 30.24, 26.79, 26.12, 24.02 ppm. HRMS for C27H32N8O3
calcd for (M + H)+, 517.2676; measured, 517.2665. Anal.
(C27H32N8O3) C, H, N.
(S)-7-(2-Cyano-3-(dimethylamino)-3-oxo-1-(2-oxo-1-(2-oxo-
2-(pyrrolidin-1-yl)ethyl)azepan-3-ylamino)prop-1-enylamino)-
1H-indole-3-carboxamide (23). This compound was prepared in
35% yield from 7g, 8, and 2-cyano-N,N′-dimethylacetamide. 1H
NMR (CDCl3) δ 11.55 (br s, 1H), 10.30 (br s, 1H), 7.70 (s,
1H), 7.65 (d, 1H, J ) 7.7 Hz), 7.14-7.24 (m, 2H), 7.11 (d, 1H,
J ) 7.7 Hz), 6.56 (br s, 1H), 5.41 (br s, 1H), 4.03-4.20 (m,

3-Chloroindole-7-yl-Based Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7549
Hoffman, M.; Monroe, D. M., III. A cell-based model of hemostasis.
Thromb. Haemostasis 2001, 85, 958–965.
1H), 3.07-3.70 (m, 8H), 3.04 (s, 6H), 1.45-1.97 (m, 10H) ppm.
HRMS for C27H34N8O4 calcd for (M + H)+, 535.2781; measured,
535.2777. Anal. (C27H34N8O4) C, H, N.
(7) (a) Quan, M. L.; Lam, P. Y. S.; Han, Q.; Pinto, D. J. P.; He, M. Y.;
Li, R.; Ellis, C. D.; Clark, C. G.; Teleha, C. A.; Sun, J.-H.; Alexander,
R. S.; Bai, S.; Luettgen, J. M.; Knabb, R. M.; Wong, P. C.; Wexler,
R. R. Discovery of 1-(3′-aminobenzisoxazol-5′-yl)-3-trifluoromethyl-
N-[2-fluoro-4-[(2′-dimethylaminomethyl)imidazol-1-yl]phenyl]-1H-
pyrazole-5-carboxyamide hydrochloride (razaxaban), a highly potent,
selective, and orally bioavailable factor Xa inhibitor. J. Med. Chem.
2005, 48, 1729–1744. (b) Pinto, D. J. P.; Orwat, M. J.; Wang, S.;
Fevig, J. M.; Quan, M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.;
Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.; Liang, L.;
Aungst, B. J.; Wright, M. R.; Knabb, R. M.; Wong, P. C.; Wexler,
R. R.; Lam, P. Y. S. Discovery of 1-[3-(aminomethyl)phenyl]-N-[3-
fluoro-2′-(methylsulfonyl)-[1,1′-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-
pyrazole-5-carboxamide (DPC423), a highly potent, selective, and
orally bioavailable inhibitor of blood coagulation factor Xa. J. Med.
Chem. 2001, 44, 566–578. (c) Lam, P. Y. S.; Clark, C. G.; Li, R.;
Pinto, D. J. P.; Orwat, M. J.; Galemmo, R. A.; Fevig, J. M.; Teleha,
C. A.; Alexander, R. S.; Smallwood, A. M.; Rossi, K. A.; Wright,
M. R.; Bai, S. A.; He, K.; Luettgen, J. M.; Wong, P. C.; Knabb, R. M.;
Wexler, R. R. Structure-based design of novel guanidine/benzamidine
mimics: potent and orally bioavailable factor Xa inhibitors as novel
anticoagulants. J. Med. Chem. 2003, 46, 4405–4418. (d) Lassen, M. R.;
Davidson, B. L.; Gallus, A.; Pineo, G.; Ansell, J.; Deitchman, D. A
phase II randomized, double-blind, five-arm, parallel-group, dose-
response study of a new oral directly-acting factor Xa inhibitor,
razaxaban, for the prevention of deep vein thrombosis in knee
replacement surgeryson behalf of the razaxaban investigators. Blood
2003, 102, Abs 41. (e) Wong, P. C.; Crain, E. J.; Watson, C. A.;
Wexler, R. R.; Lam, P. Y. S.; Quan, M. L.; Knabb, R. M. Razaxaban,
a direct factor Xa inhibitor, in combination with aspirin and/or
clopidogrel improves low-dose antithrombotic activity without enhanc-
ing bleeding liability in rabbits. J. Thromb. Thrombolysis 2007, 24,
43–51.
(8) (a) Pinto, D. J. P.; Orwat, M. J.; Koch, S.; Rossi, K. A.; Alexander,
R. S.; Smallwood, A.; Wong, P. C.; Rendina, A. R.; Luettgen, J. M.;
Knabb, R. M.; He, K.; Xin, B.; Wexler, R. R.; Lam, P. Y. S. Discovery
of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-
4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (Apixa-
ban, BMS-562247), a highly potent, selective, efficacious, and orally
bioavailable inhibitor of blood coagulation factor Xa. J. Med. Chem.
2007, 50, 5339–5356. (b) Wong, P. C.; Crain, E. J.; Xin, B.; Wexler,
R. R.; Lam, P. Y.; Pinto, D. J.; Luettgen, J. M.; Knabb, R. M.
Apixaban, an oral, direct and highly selective factor Xa inhibitor in
vitro, antithrombotic and antihemostatic studies. J. Thromb. Haemo-
stasis 2008, 6, 820–829.
(9) (a) Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer,
J.; Schlemmer, K.-H.; Reinemer, P.; Perzborn, E. Discovery of the
novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomor-
pholin-4-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)thiophene-2-carboxa-
mide (BAY 59-7939): an oral, direct factor Xa inhibitor. J. Med. Chem.
2005, 48, 5900–5908. (b) Eriksson, B. I.; Borris, L.; Dahl, O. E.; Haas,
S.; Huisman, M. V.; Kakkar, A. K.; Misselwitz, F.; Kaelebo, P. Oral,
direct factor Xa inhibition with BAY 59-7939 for the prevention of
venous thromboembolism after total hip replacement. J. Thromb.
Haemostasis 2006, 4, 121–128. (c) Kubitza, D.; Becka, M.; Wensing,
G.; Voith, B.; Zuehlsdorf, M. Safety, pharmacodynamics, and phar-
macokinetics of BAY 59-7939san oral, direct factor Xa inhibitorsafter
multiple dosing in healthy male subjects. Eur. J. Clin. Pharmacol.
2005, 61, 873–880. (d) Turpie, A. G. G.; Fisher, W. D.; Bauer, K. A.;
Kwong, L. M.; Irwin, M. W.; Kalebo, P.; Misselwitz, F.; Gent, M.
BAY 59-7939: an oral, direct factor Xa inhibitor for the prevention
of venous thromboembolism in patients after total knee replacement.
A phase II dose-ranging study. J. Thromb. Haemostasis 2005, 3, 2479–
2486.
(S)-1-(3-Methyl-1H-indol-7-yl)-2-cyano-3-(2-oxo-1-(2-oxo-2-
(pyrrolidin-1-yl)ethyl)azepan-3-yl)guanidine (25). This compound
was prepared in 83% yield from 7b, 8, and sodium cyanoamide.
1H NMR (CDCl3) δ 9.87 (s, 1H), 8.03 (s, 1H), 7.47 (d, 1H, J )
7.7 Hz), 7.06 (t, 1H, J ) 7.7 Hz), 6.99 (d, 1H, J ) 7.7 Hz), 6.98
(s, 1H), 6.36 (d, 1 H, J ) 6.0 Hz), 4.58-4.62 (m, 1H), 4.11 (d,
1H, J ) 16.5 Hz), 4.06 (d,1H, J ) 16.5 Hz), 3.65 (dd, 1H, J )
14.5, 10.4 Hz), 3.24-3.38 (m, 5H), 3.22 (br d, 1H, J ) 12.6 Hz),
2.30 (s, 3H), 1.99 (d, 1H, J ) 12.6 Hz), 1.91 (quintet, 2H, J ) 6.6
Hz), 1.55-1.81 (m, 6H), 1.41-1.48 (m, 1H) ppm; 13C NMR
(CDCl3) δ 173.01, 166.03, 157.24, 131.33, 130.83, 123.14, 119.24,
118.96, 118.43, 118.21 (2C), 111.82, 54.49, 51.54, 50.45, 46.02,
45.70, 31.20, 27.54, 26.57, 26.10, 23.98, 9.66 ppm. HRMS for
C23H29N7O2 calcd for (M + H)+, 436.2461; measured, 436.2455.
Anal. (C23H29N7O2) C, H, N.
(S)-1-(3-Chloro-1H-indol-7-yl)-2-cyano-3-(2-oxo-1-(2-oxo-2-
(pyrrolidin-1-yl)ethyl)azepan-3-yl)guanidine (26). This compound
was prepared in 87% yield from 7c, 8, and sodium cyanoamide.
1H NMR (CDCl3) δ 10.54 (s, 1H), 8.24 (s, 1H), 7.52 (d, 1H, J )
8.3 Hz), 7.17 (d, 1H, J ) 2.2 Hz), 7.11 (t, 1H, J ) 7.7 Hz), 7.05
(d, 1H, J ) 7.7 Hz), 6.44 (d, 1 H, J ) 6.1 Hz), 4.58-4.66 (m,
1H), 4.11 (d, 1H, J ) 16.0 Hz), 4.03 (d,1H, J ) 16.0 Hz), 3.66
(dd, 1H, J ) 15.1, 11.3 Hz), 3.27-3.41 (m, 5H), 3.19 (dd, 1H, J
) 15.1, 2.8 Hz), 2.04 (d, 1H, J ) 11.6 Hz), 1.91 (quintet, 2H, J )
6.6 Hz), 1.40-1.81 (m, 7H) ppm; 13C NMR (CDCl3) δ 173.03,
165.89, 156.98, 130.00, 127.67, 122.44, 120.46, 119.83, 119.02,
118.09, 117.62, 106.62, 54.57, 51.62, 50.61, 46.02, 45.68, 31.26,
27.54, 26.57, 26.08, 23.96 ppm. HRMS for C22H26ClN7O2 calcd
for (M
(C22H26ClN7O2) C, H, Cl., N.
+
H)+, 456.1915; measured, 456.1910. Anal.
Acknowledgment. We thank Drs. P. T. Cheng, P. Y. S. Lam,
D. L. Cheney, and R. R. Wexler for insightful review of this
work.
Supporting Information Available: Details of the enzymatic
assays, EC2xPT measurements, and plasma protein binding measure-
ments, combustion analysis data for compounds 2, 3, 14-23, 25,
and 26, and crystallization details of FXa with 18. This material is
available free of charge via the Internet at http://pubs.acs.org.
References
(1) (a) Murray, C. J. L.; Lopez, A. D. Mortality by cause for eight regions
of the world: Global Burden of Disease Study. Lancet 1997, 349,
1269–1276. (b) White, R. H. The epidemiology of venous throm-
boembolism. Circulation 2003, 107, I4–I8.
(2) Warfarin: (RS)-4-hydroxy-3-(3-oxo-1-phenylbutyl)coumarin.
(3) Herman, D.; Locatelli, I.; Grabnar, I.; Peternel, P.; Stegnar, M.; Mrhar,
A.; Breskvar, K.; Dolzan, V. Influence of CYP2C9 polymorphisms,
demographic factors and concomitant drug therapy on warfarin
metabolism and maintenance dose. Pharmacogenomics J. 2005, 5,
193–202.
(4) (a) Lormeau, J. C.; Goulay, J.; Choay, J. Pharmaceutical Mucopolysac-
charide Fraction. DE 2944792, 1980. (b) Glover, G. I.; Schasteen,
C. S. Serine Protease Inhibitors. EP 238473, 1987.
(10) (a) Agnelli, G.; Haas, S. K.; Krueger, K. A.; Bedding, A. W.; Brandt,
J. T. A phase II study of the safety and efficacy of a novel oral FXa
inhibitor (LY517717) for the prevention of venous thromboembolism
following TKR or THR. ASH Annu. Meet. Abstr. 2005, 106, 278. (b)
Hampton, T. New oral anticoagulants show promise. JAMA, J. Am.
Med. Assoc. 2006, 295, 743–744. (c) Liebeschuetz, J. W.; Jones, S. D.;
Wiley, M. E.; Young, S. C. Iterative structure-based screen of virtual
chemical libraries and factor Xa: finding the oral available antithrom-
botic candidate LY517717. Struct.-Based Drug DiscoVery 2006, 173–
192.
(11) (a) Shi, Y.; Zhang, J.; Stein, P. D.; Shi, M.; O’Connor, S. P.; Bisaha,
S. N.; Li, C.; Atwal, K. S.; Bisacchi, G. S.; Sitkoff, D.; Pudzianowski,
A. T.; Liu, E. C.; Hartl, K. S.; Seiler, S. M.; Youssef, S.; Steinbacher,
T. E.; Schumacher, W. A.; Rendina, A. R.; Bozarth, J. M.; Peterson,
T. L.; Zhang, G.; Zahler, R. Ketene aminal-based lactam derivatives
as a novel class of orally active FXa inhibitors. Bioorg. Med. Chem.
Lett. 2005, 15, 5453–5458. (b) Bisacchi, G. S.; Stein, P. D.; Gougoutas,
(5) For reviews, see the following: (a) Zhu, B.-Y.; Scarborough, R. M.
Recent advances in inhibitors of factor Xa in the prothrombinase
complex. Curr. Opin. CardioVasc., Pulm., Renal InVest. Drugs 1999,
1, 63–87. (b) Vacca, J. P. Thrombosis and coagulation. Annu. Rep.
Med. Chem. 1998, 33, 81–90. (c) Kunitada, S.; Nagahara, T.; Hara,
T. Inhibitors of factor Xa. Handb. Exp. Pharmacol. 1999, 132, 397–
420. (d) Ewing, W. R.; Pauls, H. W.; Spada, A. P. Progress in the
design of inhibitors of coagulation factor Xa. Drugs Future 1999, 24,
771–787. (e) Fevig, J. M.; Wexler, R. R. Anticoagulants: Thrombin
and factor Xa inhibitors. Annu. Rep. Med. Chem. 1999, 34, 81–100.
(f) Betz, A. Recent advances in factor Xa inhibitors. Expert Opin.
Ther. Pat. 2001, 11, 1007–1017. (g) Casimiro-Garcia, A.; Dudley,
D. A.; Heemstra, R. J.; Filipski, K. J.; Bigge, C. F.; Edmunds, J. J.
Progress in the discovery of factor Xa inhibitors. Expert Opin. Ther.
Pat. 2006, 16, 119–145.
(6) (a) Walsh, P. N. Roles of factor XI, platelets and tissue factor-initiated
blood coagulation. J. Thromb. Haemostasis 2003, 1, 2081–2086. (b)

7550 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Shi et al.
J. Z.; Hartl, K. S.; Lawrence, R. M.; Liu, E. C.; Pudzianowski, A. T.;
Schumacher, W. A.; Sitkoff, D.; Steinbacher, T. E.; Sutton, J.; Zhang,
Z.; Seiler, S. M. Initial structure-activity relationships for a capro-
lactam-based series of neutral factor Xa inhibitors: lead identification.
Lett. Drug Des. DiscoVery 2005, 2, 625–630. (c) Stein, P. D.; Shi,
Y.; O’Connor, S. P.; Li, C. Preparation of Lactam Inhibitors of Factor
Xa. WO 2001096331, 2001.
P. Factor Xa inhibitors: S1 binding interactions of a series of N-{(3S)-
1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-
yl}sulfonamides. J. Med. Chem. 2007, 50, 1546–1557.
(19) Gly218 is sometimes referred to as Gly219 in the literature depending
on how the FXa sequence was aligned with that of chymotripsinogen,
on which the numbering system is based.
(20) Subsequent to our discovery of the 7-linked 3-chloroindole P1
pharmacophore,11c several reports and crystal structures of a 2-linked-
5-chloroindole P1 group that fills the base of the S1 pocket and
hydrogen bonds to Gly218 carbonyl have appeared: Komoriya, S.;
Haginoya, N.; Kobayashi, S.; Nagata, T.; Mochizuki, A.; Suzuki, M.;
Yoshino, T.; Horino, H.; Nagahara, T.; Suzuki, M.; Isobe, Y.;
Furugoori, T. Design, synthesis, and biological activity of non-basic
compounds as factor Xa inhibitors: SAR study of S1 and aryl binding
sites. Bioorg. Med 2005, 13, 3927–3954. Nagata, T.; Yoshino, T.;
Haginoya, N.; Yoshikawa, K.; Isobe, Y.; Furugohri, T.; Kanno, H.
Cycloalkanediamine derivatives as novel blood coagulation factor Xa
inhibitors. Bioorg. Med. Chem. Lett. 2007, 17, 4683–4688. Addition-
ally, there are several examples of amide-linked para-chlorophenyl
P1 groups in which the NH of the amide linker forms a hydrogen
bond to Gly218 carbonyl (earliest examples are pdb entries 1mk5 and
1mk6 from 2003). Recently, Qiao et al. reported a ligand with a
6-linked 3-chloroindole P1 group crystallized in FXa. As expected,
the primary hydrogen bond is to Asp189; there is, however, a
secondary, weak (3.5 Å) interaction to Gly218 carbonyl. See the
following: Qiao, J. X.; Chang, C.-H.; Cheney, D. L.; Morin, P. E.;
Wang, G. Z.; King, S. R.; Wang, T. C.; Rendina, A. R.; Luettgen,
J. M.; Knabb, R. M.; Wexler, R. R.; Lam, P. Y. S. SAR and X-ray
structures of enantiopure 1,2-cis-(1R,2S)-cyclopentyldiamine and cy-
clohexyldiamine derivatives as inhibitors of coagulation factor Xa.
Bioorg. Med. Chem. Lett. 2007, 17, 4419–4427.
(21) The clogP values are calculated with Biobyte clogP, version 4.2, using
version 22 of the associated fragment database. For both compounds
2 and 3, the software used an approximated fragment value (error
code 30). For comparison, the clogP values for 3-methylindole and
2-methylbenzofuran are 2.6 and 3.2 with no errors codes reported,
following the same trend as the values for the complete compounds 3
and 2. This suggests that the approximated fragment is likely in the
non-S1, or constant portion of the compounds, and that difference in
clogP for the complete compounds is measuring the 3-methylindole
versus 2-methylbenzofuran contribution.
(22) Owa, T.; Yoshino, H.; Okauchi, T.; Yoshimatsu, K.; Ozawa, Y.; Sugi,
N. H.; Nagasu, T.; Koyanagi, N.; Kitoh, K. Discovery of novel
antitumor sulfonamides targeting G1 phase of the cell cycle. J. Med.
Chem. 1999, 42, 3789–3799.
(23) Shi, Y.; Zhang, J.; Grazier, N.; Stein, P. D.; Atwal, K. S.; Traeger,
S. C.; Callahan, S. P.; Malley, M. F.; Galella, M. A.; Gougoutas, J. Z.
One-pot synthesis and conformational features of N,N′-disubstituted
ketene aminals. J. Org. Chem. 2004, 69, 188–191.
(24) Atwal, K. S.; Ahmed, S. Z.; O’Reilly, B. C. A facile synthesis of
cyanoguanidines from thioureas. Tetrahedron Lett. 1989, 30, 7313–
7316.
(25) Among all currently available pdb structures of FXa, only structure
1iqe has its Ser195 side chain modeled in a position similar to that
observed in the crystal structure of 18. 1iqe is a 2.9 Å resolution
structure; unfortunately, there is no associated publication to document
and discuss the finding, and the structure factors were not submitted
to the pdb.
(12) Masters, J. J.; Franciskovich, J. B.; Tinsley, J. M.; Campbell, C.;
Campbell, J. B.; Craft, T. J.; Froelich, L. L.; Gifford-Moore, D. S.;
Hay, L. A.; Herron, D. K.; Klimkowski, V. J.; Kurz, K. D.; Metz,
J. T.; Ratz, A. M.; Shuman, R. T.; Smith, G. F.; Smith, T.; Towner,
R. D.; Wiley, M. R.; Wilson, A.; Yee, Y. K. Non-amidine-containing
1,2-dibenzamidobenzene inhibitors of human factor Xa with potent
anticoagulant and antithrombotic activity. J. Med. Chem. 2000, 43,
2087–2092.
(13) Compound 2 is inactive (IC50 > 33 µM) when tested against thrombin,
trypsin, factor IXa, factor VIIa, plasmin, and urokinase; intravenous
bolus injection of 10 mg/kg of 2 led to a 1.8-, 1.4-, and 1.1-fold
increase in prothrombin time (PT) after 5, 20 and 60 min, respectively.
(14) (a) IARC. Monographs on the EValuation of the Carcinogenic Risk
of Chemicals to Man; IARC Press: Lyon, France, 1995; p 431. (b)
Burka, L. T.; Boyd, M. R. Furans. Bioact. Foreign Compd. 1985, 243–
257.
(15) Kratochwil, N. A.; Huber, W.; Mueller, F.; Kansy, M.; Gerber, P. R.
Predicting plasma protein binding of drugssrevisited. Curr. Opin.
Drug DiscoVery DeV. 2004, 7, 507–512.
(16) (a) Tucker, T. J.; Lumma, W. C.; Lewis, S. D.; Gardell, S. J.; Lucas,
B. J.; Baskin, E. P.; Woltmann, R.; Lynch, J. J.; Lyle, E. A.; Appleby,
S. D.; Chen, I. W.; Dancheck, K. B.; Vacca, J. P. Potent noncovalent
thrombin inhibitors that utilize the unique amino acid D-dicyclohexy-
lalanine in the P3 position. Implications on oral bioavailability and
antithrombotic efficacy. J. Med. Chem. 1997, 40, 1565–1569. (b)
Zhang, P.; Bao, L.; Zuckett, J. F.; Goldman, E. A.; Jia, Z. J.; Arfsten,
A.; Edwards, S.; Sinha, U.; Hutchaleelaha, A.; Park, G.; Lambing,
J. L.; Hollenbach, S. J.; Scarborough, R. M.; Zhu, B.-Y. Design,
synthesis, and SAR of anthranilamide-based factor Xa inhibitors
incorporating substituted biphenyl P4 motifs. Bioorg. Med. Chem. Lett.
2004, 14, 983–987. (c) Chou, Y.-L.; Davey, D. D.; Eagen, K. A.;
Griedel, B. D.; Karanjawala, R.; Phillips, G. B.; Sacchi, K. L.; Shaw,
K. J.; Wu, S. C.; Lentz, D.; Liang, A. M.; Trinh, L.; Morrissey, M. M.;
Kochanny, M. J. Structure-activity relationships of substituted
benzothiophene-anthranilamide factor Xa inhibitors. Bioorg. Med.
Chem. Lett. 2003, 13, 507–511. (d) Hirayama, F.; Koshio, H.;
Katayama, N.; Kurihara, H.; Taniuchi, Y.; Sato, K.; Hisamichi, N.;
Sakai-Moritani, Y.; Kawasaki, T.; Matsumoto, Y.; Yanagisawa, I. The
discovery of YM-60828: a potent, selective and orally-bioavailable
factor Xa inhibitor. Bioorg. Med. Chem. 2002, 10, 1509–1523. (e)
Sinha, U.; Lin, P. H.; Edwards, S. T.; Wong, P. W.; Zhu, B.;
Scarborough, R. M.; Su, T.; Jia, Z. J.; Song, Y.; Zhang, P.; Clizbe,
L.; Park, G.; Reed, A.; Hollenbach, S. J.; Malinowski, J.; Arfsten,
A. E. Inhibition of purified factor Xa amidolytic activity may not be
predictive of inhibition of in vivo thrombosis. Arterioscler., Thromb.,
Vasc. Biol. 2003, 23, 1098–1104. (f) Shi, Y.; Sitkoff, D.; Zhang, J.;
Han, W.; Hu, Z.; Stein, P. D.; Wang, Y.; Kennedy, L. J.; O’Connor,
S. P.; Ahmad, S.; Liu, E. C.-K.; Seiler, S. M.; Lam, P. Y.S.; Robl,
J. A.; Macor, J. E.; Atwal, K. S.; Zahler, R. Amino(methyl)
pyrrolidines as novel scaffolds for factor Xa inhibitors. Bioorg. Med.
Chem. Lett. 2007, 17, 5952–5958.
(26) An additional notable finding is that in one of the two structures in
the asymmetric unit of the crystal structure of 18, the conserved water
that is usually found in S4 is missing; there is no obvious reason for
its absence.
(27) Herron, D. K.; Goodson, T. J.; Wiley, M. R.; Weir, L. C.; Kyle, J. A.;
Yee, Y. K.; Tebbe, A. L.; Tinsley, J. M.; Mendel, D.; Masters, J. J.;
Franciskovich, J. B.; Sawyer, J. S.; Beight, D. W.; Ratz, A. M.; Milot,
G.; Hall, S. E.; Klimkowski, V. J.; Wikel, J. H.; Eastwood, B. J.;
Towner, R. D.; Gifford-Moore, D. S.; Craft, T. J.; Smith, G. F. 1,2-
Dibenzamidobenzene inhibitors of human factor Xa. J. Med. Chem.
2000, 43, 859–872.
(28) (a) Matter, H.; Will, D. W.; Nazare, M.; Schreuder, H.; Laux, V.;
Wehner, V. Structural requirements for factor Xa inhibition by
3-oxybenzamides with neutral P1 substituents: combining X-ray
crystallography, 3D-QSAR, and tailored scoring functions. J. Med.
Chem. 2005, 48, 3290–3312. (b) Fre´de´rick, R.; Robert, S; Charlier,
C.; Wouters, J.; Masereel, B.; Pochet, L. Mechanism-based thrombin
inhibitors: design, synthesis, and molecular docking of a new selective
2-oxo-2H-1-benzopyran derivative. J. Med. Chem. 2007, 50, 3645–
3650.
(29) (a) Imai, Y. N.; Inoue, Y.; Nakanishi, I.; Kitaura, K. Cl-π interactions
in protein-ligand complexes. Protein Sci. 2008, 17, 1129–1137. (b)
Tsuzuki, S.; Honda, K.; Uchimaru, T.; Mikami, M.; Tanabe, K. The
magnitude of the CH/π interaction between benzene and some model
hydrocarbons. J. Am. Chem. Soc. 2000, 122, 3746–3753. (c) Ringer,
(17) For early examples, see the following: (a) Tucker, T. J.; Brady, S. F.;
Lumma, W. C.; Lewis, S. D.; Gardell, S. J.; Naylor-Olsen, A. M.;
Yan, Y.; Sisko, J. T.; Stauffer, K. J.; Lucas, B. J.; Lynch, J. J.; Cook,
J. J.; Stranieri, M. T.; Holahan, M. A.; Lyle, E. A.; Baskin, E. P.;
Chen, I.-W.; Dancheck, K. B.; Krueger, J. A.; Cooper, C. M.; Vacca,
J. P. Design and synthesis of a series of potent and orally bioavailable
noncovalent thrombin inhibitors that utilize nonbasic groups in the
P1 position. J. Med. Chem. 1998, 41, 3210–3219. (b) Maignan, S.;
Guilloteau, J.-P.; Choi-Sledeski, Y. M.; Becker, M. R.; Ewing, W. R.;
Pauls, H. W.; Spada, A. P.; Mikol, V. Molecular structures of human
factor Xa complexed with ketopiperazine inhibitors: preference for a
neutral group in the S1 pocket. J. Med. Chem. 2003, 46, 685–690. (c)
Adler, M.; Kochanny, M. J.; Ye, B.; Rumennik, G.; Light, D. R.;
Biancalana, S.; Whitlow, M. Crystal structures of two potent nona-
midine inhibitors bound to factor Xa. Biochemistry 2002, 41, 15514–
15523.
(18) (a) Sitkoff, D. Probing the source of the exceptional role played by
chlorine in the FXa S1 pocket. Abstr. Pap.sAm. Chem. Soc. 2006,
232, COMP-075. (232nd National Meeting of the American Chemical
Society, Division of Computational Chemistry, San Fancisco, CA,
September, 2006). (b) Chan, C.; Borthwick, A. D.; Brown, D.; Burns-
Kurtis, C. L.; Campbell, M.; Chaudry, L.; Chung, C.-w.; Convery,
M. A.; Hamblin, N.; Johnstone, L.; Kelly, H. A.; Kleanthous, S.;
Patikis, A.; Patel, C.; Pateman, A. J.; Senger, S.; Shah, G. P.; Toomey,
J. R.; Watson, N. S.; Weston, H. E.; Whitworth, C.; Youg, R. J.; Zhou,

3-Chloroindole-7-yl-Based Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7551
A. L.; Figgs, M. S.; Sinnokrot, M. O.; Sherrill, C. D. Aliphatic C-H/π
interactions: methane-benzene, methane-phenol, and methane-
indole complexes. J. Phys. Chem. A 2006, 110, 10822–10828.
W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision
D.01; Gaussian, Inc.: Wallingford, CT, 2004.
(36) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data
Collected in Oscillation Mode. In Methods in Enzymology, Macro-
molecular Crystallography, Part A; Carter, C. W., Sweet, R. M., Eds.;
Academic Press, Inc.: New York, 1997; Vol. 276, pp 307-326.
(37) French, S.; Wilson, K. On the treatment of negative intensity
observations. Acta Crystallogr. 1978, A34, 517–525.
(30) Swiss Webster mice (24-38 g) were anesthetized with sodium
pentobarbital (100 mg/kg, ip). Russell’s viper venom (Sigma Chemical
Co., St. Louis, MO) was prepared in 0.9% saline and injected into
the tail vein at a dose of 7 µg/mouse in a volume of 0.1 mL given 15
min after induction of anesthesia. This dose of venom was found to
be uniformly fatal in 105 mice with death occurring in an average of
1.5 ( 1.9 min ((SD, range was 1-12 min with 88% of mice dying
within 2 min). Histological evaluation of several venom-treated mice
revealed that early death was due to occlusion of pulmonary capillaries
with fibrin aggregates, probably caused by initiation of blood coagula-
tion by venom activation of factor X to factor Xa. A 5 mg/kg dose of
test compounds or saline vehicle was administered by oral gavage 45
min before the venom injection. Survival was monitored out to 30
min after venom injection.
(31) Quan, M. L.; Smallheer, J. M. The race to an orally active factor Xa
inhibitor: recent advances. Curr. Opin. Drug DiscoVery DeV. 2004,
7, 460–469.
(32) Adler, M.; Davey, D. D.; Phillips, G. B.; Kim, S.-H.; Jancarik, J.;
Rumennik, G.; Light, D. R.; Whitlow, M. Preparation, characterization,
and the crystal structure of the inhibitor ZK-807834 (CI-1031)
complexed with factor Xa. Biochemistry 2000, 39, 12534–12542.
(38) Bailey, S. The CCP4 suite: programs for protein crystallography. Acta
Crystallogr. 1994, D50, 760–763.
(39) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J.-S.; Kuszewski, J; Nilges, M;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: a new software suite for macromo-
lecular structure determination. Acta Crystallogr. 1998, D54, 905–
921.
(40) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L.-W.; Ioerger, T. R.;
McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter,
N. K.; Terwilliger, T. C. PHENIX: building new software for
automated crystallographic structure determination. Acta Crystallogr.
2002, D58, 1948–1954.
(41) Emsley, P.; Cowtan, K. COOT: model-building tools for molecular
graphics. Acta Crystallogr. 2004, D60, 2126–2132.
(42) Lovell, S. C.; Davis, I. W.; Arendall, W. B., III; de Bakker, P. I. W.;
Word, J. M.; Prisant, M. G.; Richardson, J. S.; Richardson, D. C.
Structure validation by CR geometry: ꢁ, ψ, and Cꢀ deviation. Proteins:
Struct., Funct., Genet. 2003, 50, 437–450.
(43) Shao, Y.; Fusti-Molnar, L.; Jung, Y.; Kussmann, J.; Ochsenfeld, C.;
Brown, S. T.; Gilbert, A. T. B.; Slipchenko, L. V.; Levchenko, S. V.;
O’Neill, D. P.; Distasio, R. A., Jr.; Lochan, R. C.; Wang, T.; Beran,
G. J. O.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.; Van Voorhis, T.;
Chien, S. H.; Sodt, A; Steele, R. P.; Rassolov, V. A.; Maslen, P. E.;
Korambath, P. P.; Adamson, R. D.; Austin, B.; Baker, J.; Byrd,
E. F. C.; Dachsel, H.; Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.;
Dutoi, A. D.; Furlani, T. R.; Gwaltney, S. R.; Heyden, A.; Hirata, S.;
Hsu, C.-P.; Kedziora, G.; Khalliulin, R. Z.; Klunzinger, P.; Lee, A. M.;
Lee, M. S.; Liang, W.; Lotan, I.; Nair, N.; Peters, B.; Proynov, E. I.;
Pieniazek, P. A.; Rhee, Y. M.; Ritchie, J.; Rosta, E.; Sherrill, C. D.;
Simmonett, A. C.; Subotnik, J. E.; Woodcock, H. L., III; Zhang, W.;
Bell, A. T.; Chakraborty, A. K.; Chipman, D. M.; Keil, F. J.; Warshel,
A.; Hehre, W. J.; Schaefer, H. F., III; Kong, J.; Krylov, A. I.; Gill,
P. M. W.; Head-Gordon, M. Advances in methods and algorithms in
a modern quantum chemistry program package. Phys. Chem. Chem.
Phys. 2006, 8, 3172–3191.
(33) (a) Dinur, U.; Hagler, A. T. New Approaches to Empirical Forcefields.
In ReViews in Computational Chemistry; Lipkowitz, K. B., Boyd,
D. B., Eds; VCH Publishers: New York, 1991; Vol. 2, pp 99-164.
(b) Maple, J. R.; Hwang, M. J.; Stockfisch, T. P.; Dinur, U.; Waldman,
M.; Ewig, C. S.; Hagler, A. T. Derivation of class II forcefields. I.
Methodology and quantum forcefield for the alkyl function group and
alkane molecules. J. Comput. Chem. 1994, 15, 162–182.
(34) Jaguar, version 6.5; Schrodinger, LLC: New York, 2005.
(35) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin,
K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone,
V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa,
J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene,
M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev,
O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala,
P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas,
O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.;
Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.;
Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen,
(44) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. Protein data bank. Nucleic
Acids Res. 2000, 28, 235–242.
JM800855X