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

Article abstract of DOI:10.1021/jm060870c

Factor Xa inhibitory activities for a series of N-{(3S)-1-[(1S)-1-methyl-2- morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}sulfonamides with different P1 groups are described. These data provide insight into binding interactions within the S1 primary spe

Full text of DOI:10.1021/jm060870c

1546
J. Med. Chem. 2007, 50, 1546-1557
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†
Chuen Chan,*,‡ Alan D. Borthwick,‡ David Brown,§ Cynthia L. Burns-Kurtis,| Matthew Campbell,‡ Laiq Chaudry,
Chun-wa Chung,# Ma´ire A. Convery,# J. Nicole Hamblin,‡ Lisa Johnstone,‡ Henry A. Kelly,‡ Savvas Kleanthous,‡
Angela Patikis,X Champa Patel, Anthony J. Pateman,X Stefan Senger,# Gita P. Shah,‡ John R. Toomey,| Nigel S. Watson,‡
Helen E. Weston,‡ Caroline Whitworth,‡ Robert J. Young,‡ and Ping ZhouX
CVU UK Medicinal Chemistry Department, Screening and Compound Profiling UK, Assay DeVelopment UK, Computational and Structural
Chemistry, CVU UK DMPK Department, GlaxoSmithKline Research and DeVelopment, Medicines Research Centre, Gunnels Wood Road,
SteVenage, Hertfordshire SG1 2NY, United Kingdom, and Vascular Inflammatory Diseases, GlaxoSmithKline, 709 Swedeland Road,
King of Prussia, PennsylVania 19406
ReceiVed July 24, 2006
Factor Xa inhibitory activities for a series of N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-
oxopyrrolidin-3-yl}sulfonamides with different P1 groups are described. These data provide insight into
binding interactions within the S1 primary specificity pocket; rationales are presented for the derived SAR
on the basis of electronic interactions through crystal structures of fXa-ligand complexes and molecular
modeling studies. A good correlation between in vitro anticoagulant activities with lipophilicity and the
extent of human serum albumin binding is observed within this series of potent fXa inhibitors. Pharmacokinetic
profiles in rat and dog, together with selectivity over other trypsin-like serine proteases, identified 1f as a
candidate for further evaluation.
Introduction
intervention. Indeed, fondaparinux,6 a selective indirect fXa
inhibitor suitable for subcutaneous administration, has been
approved for the prevention of venous thromboembolism in
patients undergoing major orthopaedic (hip or knee replacement)
surgery. Furthermore, direct thrombin and fXa inhibitors have
also shown encouraging results for the prevention of throm-
boembolic events in clinical studies.7a-d Ximelagatran, the first
registered, orally active thrombin inhibitor, was approved in
Europe for the prevention of thrombosis in patients undergoing
hip or knee replacement, but was withdrawn from the market
due to concerns over toxicity in the liver.7e Currently a number
of orally active thrombin [e.g., MCC-977, dabigatran etexilate]8
and fXa [e.g., rivaroxaban, LY-517717, apixaban, and DU-
176b]9a-g inhibitors are being investigated in clinical trials for
similar conditions. In addition, DX-9065a is currently in clinical
trials for parenteral indications.9h-i As part of our program to
develop efficacious and orally bioavailable anticoagulants, we
have identified highly selective and potent thrombin10 and fXa
inhibitors with good anticoagulant activities and promising
profiles for chronic oral administration. Factor Xa inhibitors
containing the 3-aminopyrrolidin-2-one scaffold and highly basic
benzamidine P1 substituents have been disclosed previously.11a
Our own studies11b with this scaffold have led to a nonbasic
series of novel and orally active fXa inhibitors incorporating
6-chloro-N-[(3S)-1-substituted-2-oxo-3-pyrrolidinyl]-2-naphtha-
lenesulfonamides and identified 1a as our lead compound. The
Thrombosis is a multifactorial disorder initiated by intravas-
cular blood clotting leading to thrombus formation in an artery
or vein. The resultant thrombi can be obstructive or dislodged
into smaller blood vessels, depriving vital organs of oxygen
(ischemia), which can be catastrophic. Manifestation of these
life threatening conditions includes myocardial infarction,
pulmonary embolism, and stroke. Drugs that inhibit the blood
clotting process can prevent these events, and the development
of antithrombotics is a major focus of pharmaceutical research.1
A number of antithrombotics such as aspirin, heparin, low
molecular weight heparins,2 and warfarin3 are used clinically,
but these drugs suffer from limited efficacy, low oral bioavail-
ability, or a narrow therapeutic window. Indeed, administration
of warfarin, a vitamin K epoxide reductase inhibitor, requires
individual treatment with continuous dose adjustment and
laboratory monitoring because of its interactions with other drugs
and dietary vitamin K. Furthermore, warfarin has a slow onset
of action.
A key driver of thrombus formation is the dysregulation of
blood coagulation, which consists of the interconnected intrinsic
and extrinsic pathways4 that converge onto the formation of
activated factor X (fXa). When bound to factor Va, calcium,
phospholipid, and prothrombin (prothrombinase complex), fXa
converts prothrombin into thrombin which in turn proteolytically
converts fibrinogen into fibrin. Ultimately, fibrin chains are
cross-linked into an insoluble matrix (the blood clot). Thus,
thrombin and fXa are attractive targets5 for pharmacological
† PDB ID codes for 1a, 1b, 1d, 1e, and 1x complexed with factor Xa
are 2cji, 2j95, 2j38, 2j34, and 2j94, respectively.
* To whom correspondence should be addressed. Tel.: (+44) 1438
768445. Fax: (+44) 1438 764869. E-mail: chuen.8.chan@gsk.com.
‡ CVU UK Medicinal Chemistry Department.
§ Screening and Compound Profiling UK.
| Vascular Inflammatory Diseases.
Assay Development UK.
# Computational and Structural Chemistry.
X CVU UK DMPK Department.
10.1021/jm060870c CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/06/2007

Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1547
by base-induced cyclization12 with the hydroxide form of Dowex
ion-exchange resin, established the pyrrolidin-2-one system,
which was deprotected with TFA in DCM to provide the
carboxylic acid 4. Amide coupling with morpholine using
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluo-
roborate (TBTU) and i-Pr2NEt, followed by deprotection under
palladium-catalyzed hydrogenolysis, thereby yielded the key
amine intermediate 2.
The secondary sulfonamides 1a-1h, 1j-1p, and 1s-1y were
prepared by sulfonylation of the (3S)-aminopyrrolidin-2-one
alanylamide 2 with appropriate sulfonyl chlorides, R1SO2Cl, 6,
or suitably protected derivatives thereof under standard condi-
tions (see Method A in General Procedures). R1s are defined in
Table 1. When Boc was used as a protecting group, its removal
was effected with TFA in DCM (for 1s and 1t). With the
exception of 6h, 6n, and 6u-6y, all other sulfonyl chlorides
were either available commercially (6m) or prepared according
to literature procedures (6a-6g,13,14 6j-6l,14,15 6o,16 6p,16 N-Boc
of 6s,17 and N-Boc of 6t17). Sulfonyl chlorides 6h, 6n, and 6u-
6w were synthesized from the corresponding chloroheteroaro-
matics by direct lithiation with n-BuLi at -78 °C, followed by
sulfinylation with SO2 and chlorinative oxidation with NCS
(Scheme 2). In the case of 6y, lithiation was carried out by
metal-halogen exchange of 5-bromo-2-(5-chloro-2-thienyl)-
pyridine18 with t-BuLi, followed by sulfinylation and chlorina-
tive oxidation as above to provide the requisite sulfonyl chloride.
The chloro-bis(heteroaryls) were prepared by palladium-
catalyzed coupling reactions. Thus, 5-(5-chloro-2-thienyl)-1,3-
thiazole, 2-(5-chloro-2-thienyl)-1,3-thiazole, and 5-chloro-2-(2-
thienyl)-1,3-thiazole were prepared by Suzuki coupling of either
2- or 5-bromothiazole19a with 5-chlorothiophene-2-boronic acid
or 2-bromo-5-chlorothiazole19b with thiophene-2-boronic acid.
2-(5-Chloro-2-thienyl)furan was synthesized via Stille coupling
of the tri-n-butyl-2-(furanyl)stannane with 2-bromo-5-chlo-
rothiophene. 3-(5-Chloro-2-thienyl)-1H-1,2,4-triazole-5-sulfonyl
chloride 6x was prepared by the reaction of thiourea with
5-chloro-2-thiophenecarbohydrazide20 at 160 °C in the absence
of solvent, followed by careful oxidation of the resultant
mercaptotriazolyl derivative with chlorine in AcOH (Scheme
3). The sulfonyl chlorides described above were generally used
for sulfonylation with 2 without purification.
Due to complications in the synthesis of 6q,21 an alternative
and indirect synthesis for 1q and 1r was employed. Thus,
S-chlorination of the appropriate chloro-1,3-benzothiazole-
2(3H)-thione22 using NCS, followed by sulfenylation with 2 and
oxidation with KMnO4, provided the 5- and 6-chloro-1,3-
benzothiazole-2-sulfonamido derivatives 1q and 1r (Scheme 4).
Compound 1i was synthesized via the sulfonylation of 2 with
4-iodophenylsulfonyl chloride to give 7. Sonagashira coupling
with trimethylsilylacetylene in the presence of Et3N and catalytic
amounts of CuI and PdCl2(PPh3)2 in DMF at room temperature,
followed by the removal of the trimethylsilyl group with TBAF
yielded the acetylenic compound 8. Hydroborative chlorination23
with t-hexylborane and cupric chloride afforded the required
1i (Scheme 5).
Figure 1. Crystal structure of 1a bound to fXa showing a water-
mediated hydrogen bonding interaction of pyrrolidin-2-one carbonyl
to Ser214 carbonyl group. (A) Solvent accessible surface of fXa binding
site depicting the 6-chloronaphthyl group binding into the S1 pocket
and the alanyl morpholinamide into the S4 pocket while the methyl
group binds into a subpocket within S4. (B) Alternative view without
the solvent accessible surface showing the bound conformation of
6-chloronaphth-2-ylsulfonamido group in the S1 pocket and the key
amino acid residues.
crystal structure of 1a bound to fXa (Figure 1) clearly showed
that the 6-chloronaphth-2-yl group bound in the S1 primary
specificity pocket and the morpholino amide occupies the
aromatic box (Tyr99, Try215, and Phe174) of the S4 pocket.
The pendent methyl group of the alanyl motif binds to a small
subpocket formed by Phe174 and the carbon atoms of the side-
chain of Glu217. Herein, we extend our study and describe a
systematic investigation into the effects of sulfonamido R1
substituents on fXa inhibitory activities and anticoagulant
activities within the N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-
yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}sulfonamide series. Our
understanding of the tolerance of sulfonamido groups within
the S1 pocket through X-ray crystallography and molecular
modeling studies is compared and contrasted with those reported
in the literature for alternative series of fXa inhibitors. Cor-
relation of anticoagulant activities with lipophilicity and the
extent of human serum albumin (HSA) binding are highlighted
together with pharmacokinetic data that led to the identification
of 1f as a candidate for further evaluation.
Results and Discussion
Chemistry
The key intermediate amine 2 was readily prepared from
N-CBZ-L-methionine (Scheme 1) by coupling with L-alanine
t-butyl ester under standard conditions using 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-
hydroxybenzotriazole (HOBt) in the presence of triethylamine
to give as an intermediate the protected dipeptide 3. Activation
of the methylthioether moiety of 3 with iodomethane, followed
The influence of sulfonamido R1 substituents on fXa inhibi-
tory activities has been investigated within a series of N-{(3S)-
1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-
3-yl}sulfonamides (Table 1). 6-Chloronaphthyl, 5′-chloro-2,2′-
bithienyl, 4-chlorostyryl, 5- and 6-chlorobenzothienyl, and
5-chlorothienylethenyl analogues 1a-1f show good to excellent
fXa inhibitory activities (Ki ) 4-47 nM). Related findings for

1548 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Chan et al.
Scheme 1a
a Reagents and conditions: (a) L-alanine t-butyl ester, HOBt, Et3N, EDC; (b) MeI, acetone, then Dowex (HO- form) ion-exchange resin; (c) TFA, DCM;
(d) morpholine, i-Pr2NEt, TBTU; (e) 10% Pd-C, H2, EtOH.
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) for the precursor to 6v, ArX )
2-bromothiazole, Y ) B(OH)2, Z ) CH: Pd2dba3‚CHCl3, PPh3, Na2CO3,
H2O, DME; for the precursor to 6w, ArX ) 5-bromothiazole,19a Y )
B(OH)2, Z ) CH: PdCl2dppf‚CH2Cl2, K2CO3, DME; for the precursor to
6n, ArX ) thiophene-2-boronic acid, Y ) Br, Z ) N:19b Pd(PPh3)4, Cs2CO3,
H2O, DME; for the precursor to 6u, ArX ) tri-n-butyl(2-furanyl)stannane,
Y ) Br, Z ) CH: Pd2dba3‚CHCl3, PPh3, THF; (b) (i) n-BuLi, THF, -78
°C, SO2; (ii) NCS, DCM; *For 6y, 5-bromo-2-(5-chloro-2-thienyl)pyridine18
was used: (i) t-BuLi, THF, -78 °C, SO2; (ii) NCS, DCM.
a Reagents and conditions: (a) NCS, DCM; (b) pyridine, 2, MeCN; (c)
KMnO4.
Scheme 5a
Scheme 3a
a Reagents and conditions: (a) 4-iodophenylsulfonyl chloride, pyridine,
MeCN; (b) (i) trimethylsilylacetylene, CuI, PdCl2(PPh3)2, Et3N, DMF; (ii)
n-Bu4NF, THF; (c) t-hexylborane, CuCl2.23
a Reagents and conditions: (a) thiourea, 160 °C; (b) Cl2, AcOH, 10 °C.
these and other chloroheteroaryl P1s have been reported13c,24,25
with other structural classes of fXa inhibitors. Basic P1 groups
such as 3- and 4-substituted benzamidines in first generation
fXa inhibitors26 have been reported to interact with Asp189 in
the S1 pocket. In contrast, 1a-1f are nonbasic and, at the outset
of our program, before X-ray crystal structures of our com-
pounds bound to fXa were available, their binding orientation
was uncertain. Evidence that chloroaromatic groups could bind
in the S1 pocket was obtained by elegant 19F NMR studies of
a 6-fluoronaphthyl containing inhibitor bound to fXa with and
without Gd-EDTAa complex as well as competitive displace-
ment studies with a 6-chloronaphthyl containing fXa inhibitor.27
Confirmation of a similar binding mode in the present series
was established by X-ray crystal structures of these compounds
complexed with fXa (Figures 1-3), which showed clearly that
the chloroaromatics in 1a, 1b, 1d, and 1e bound within the S1
pocket. The morpholino group occupied the S4 pocket with the
alanyl methyl group bound to a small hydrophobic pocket near
to S4. In contrast to a related series28 in which 3R stereochem-
istry is preferred and the pyrrolidin-2-one CdO is hydrogen-
bonded to Gly219,28 the corresponding carbonyl in our series
is indirectly hydrogen-bonded to the carbonyl of Ser214 via a
water-mediated interaction. These interactions are similar to that
reported for 1a (Figure 1) in our previous communication.11b
In contrast to the flat and rigid chlorobenzothienyl and chlo-
ronaphthyl P1s present in 1d, 1e, and 1a, respectively, the two
thienyl rings in 1b are out of coplanarity (S-C-C-S torsion
ca. 159°), which is in good agreement with that reported for
the lowest energy conformer of bithiophene.29 Our crystal
structures show the chlorine atom is 3.7-4.2 Å from the centroid
a Abbreviations: Gd-EDTA, gadoliniumsethylenediaminetetraacetic acid
complexes; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic
acid; Mes, 2-morpholinoethanesulfonic acid; PEG, polyethylene glycol; SD,
standard deviation.

Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1549
Table 1. Affinities of 1a-1y in the Fluorogenic Factor Xa Assay
Figure 2. Crystal structure of 1b bound to fXa showing the
conformation of bithienyl P1 (thick gray stick) with the 6-chloronaphth-
2-yl P1 of 1a (thin gray stick) as comparison.
Figure 3. Crystal structures of 1d (thick wheat stick) and 1e (thick
gray stick) bound to fXa showing the bound conformations of
chlorobenzothienyl P1s. 6-Chloronaphth-2-yl P1 of 1a is shown (thin
gray stick) as reference.
hypothesis arose by replacing the Cl atom with the isosteric
methyl group, which resulted in over 10-fold loss in activity
[1g vs 1a]. Furthermore, polarization of the aromatic proton
facing Asp189 by the adjacent chlorine atom could provide an
additional interaction (nonclassical hydrogen bonding),31 which
could augment the binding affinity of chloroaromatics. However,
this latter interaction is unlikely to be present in 1h and 1i, the
thieno[3,2-b]thiophene analogue and the reversed analogue of
1c, respectively. In the bioactive conformation of 1h, the sulfur
atom in the chlorothieno moiety is expected to occupy the
equivalent volume of space that faces Asp189 and, therefore,
cannot attain the nonclassical hydrogen bonding interaction
mentioned above. When bound to fXa, the proton attached to
the same carbon as the Cl atom in the chlorovinyl moiety of 1i
is unlikely to align directly with the carboxylate of Asp189 and
is also over 4 Å away to contribute any significant binding
affinity. These considerations could provide an explanation for
the moderate potency of 1h and 1i.
Replacement of the aromatic carbon atom by nitrogen in the
chloroaryl ring is generally detrimental to their fXa inhibitory
activities (1j-1n), which may be explained following close
analysis of X-ray crystal structures of 1b, 1d, and 1e (Figures
2 and 3) bound to fXa. Analogous to 1a, the chloroaromatics
in 1b, 1d, and 1e are bound to the S1 pocket and display a
similar vector for the Cl-Tyr228 interaction. Assuming all
chloroheteroaryl analogues maintain this highly favorable Cl-
Tyr228 interaction, it is possible to predict the position of the
a SD values are derived from at least n g 2.
of the phenolic ring of Tyr228 in the S1 pocket. This interaction
has been reported in the literature as hydrophobic for other series
of fXa inhibitors.24 However, we believe there is an electrostatic
component to this interaction based on electrostatic potential
maps for these chloroaromatics, which showed a slight positive
charge at the center of the Cl atom.30 Indeed, support for this

1550 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Chan et al.
Table 2. Anticoagulant Activities and Human Serum Albumin Binding
of Potent Factor Xa Inhibitors
Ki
(nM)
1.5XPT
(µM)
logD @
pH 7.4
HSA
binding %
cmpd
SD
n
1a
1b
1c
1d
1e
1f
1v
1w
1y
6
4
11
47
15
4
5
2
24
5.4
10.4
2.7
13.1
7.5
0.46
0.08
0.5
0.34
0.9
0.04
1.4
4
3
2
2
2
5
3
2
2
2
1.79
2.03
1.49
2.07
1.85
1.47
2.18
2.1
79
95
63
85
88
59
93
95
89
1.2
24.7
49.2
27.2
1.3
2.24
Table 3. Selectivity Profiles of 1a-1f against Other Trypsin-Like
Serine Proteasesa
TF/
cmpd Thrb fVIIab
fXIIab
aPCb
Plasb
Trypb
Kallb
1a
1b
1c
1d
1e
1f
37 >4500
>4500
>1000
>1000
>1000
>2000
>700
>300
>1500
>1300
nde
298 >12 000 >12 000 >25 000 >7000
19 >3500
10c >350c,d nde
22 nde nde
90 >30 000 >20 000 >4000
>3500
>1000
>700
>350c,d >750c
>500c
>3000c >1500c >1000c nde
>20 000 >20 000 >1500
a Unless otherwise stated, fluorogenic assays were performed on these
compounds. b Ratio of Ki values (nM) to fXa. TF/fVIIa ) tissue factor -
factor VIIa; aPC ) activated protein C; Plas ) plasmin; Tryp ) trypsin;
and Kall ) kallikrein. c These ratios were derived from chromogenic assays.
d These ratios are based on IC50 values. e nd ) not determined.
nitrogen in the chloroheteroaromatic rings of compounds 1j-
1n. The lone pair of electrons on the nitrogen atom in the ring
of 1k-1n is placed in a highly electron-rich environment formed
by the backbone carbonyl of Gly219 and the carboxylate of
Asp189. Such a highly unfavorable interaction is reflected in
their poor intrinsic activities. In contrast, 1j is moderately potent
(Ki ) 163 nM) and is predicted to bind with the nitrogen facing
the other side of the S1 pocket, which is likely less electron
rich and, therefore, a more favorable environment.
Modifications to the aromatic ring distal to the chlorine atom
(i.e., directly attached to SO2 of the sulfonamido group) are
shown in 1o-1y. In the 5,6-fused aromatic systems, 1o-1t are
moderate to weakly active. Interestingly, within each isomeric
pair (5- and 6-chloro isomers) of benzofurans and benzothia-
zoles, one member (1p, 1q) is significantly less active than the
other (1o, 1r). However, both chloroindolyl analogues (1s and
1t) have similarly moderate activities. Ab initio calculations for
the conformational preference of the 5-membered ring het-
eroaryl-2-sulfonamides, as model systems for 1o-1t, showed32
that one of the SdO bonds prefers to be coplanar with the Cs
H, planar NsH, or S in these aromatic systems. When this
conformational preference is translated into the 5,6-fused
chlorobenzoheteroaryl systems and the important Cl-Tyr228
interaction is maintained, there are significant energy penalties
for the bioactive conformations of 1p and 1q. Hence, 5-ben-
zofuryl (1p) and 6-chlorobenzothiazolyl (1q) analogues are less
potent than their corresponding isomers 1o and 1r. The similar
activities of 5- and 6-chloroindolyl isomers (1s and 1t) could
also be explained by the little difference in energy between the
conformers of the sulfonamido SdO that eclipse the CsH or
NsH of the pyrrole ring in 1H-pyrrole-2-sulfonamide.
Figure 4. Crystal structure of 1x (thick stick) in fXa binding site.
Compound 1b (thin stick) is shown for comparison. (A) P1 orientation
of 1x showing the perturbed conformation of the triazolyl moiety
relative to the bithienyl P1 in 1b. (B) S4 orientation in solvent accessible
surface showing major changes in the bound conformation of the
3-sulfonamidopyrrolidin-2-one moiety in 1x relative to that in 1b.
Table 4. Pharmacokinetic Parameters of 1f in Male Rats and Dogs
Following Intravenous and Oral Administration at Nominal Doses of 1
mg/kg i.v. and 2.5 mg/kg p.o.
a
c
t1/2
(h)
Clpb
(mL/min/kg)
Vss
(L/kg)
Fd
(%)
species
Spague Dawley 0.7 ( 0.1 8.0 ( 1.7
rats
0.29 ( 0.02 75 ( 23
Beagle dogs
1.2 ( 0.3 4.6 ( 1.7
0.42 ( 0.03 53 ( 9
a t1/2 ) half-life of the test compound expressed in hours. b Clp ) plasma
c
clearance of the test compound expressed mL/min/kg. Vss ) steady-state
volume of distribution of test compound expressed as L/kg. d F ) oral
bioavailability of test compound expressed as percentage.
above), and replacement of aromatic carbons by nitrogen are
expected to be better tolerated. Furthermore, the rotatable bond
between the two thienyl rings in 1b can be twisted to alleviate
any possible unfavorable interactions. Indeed both the thiazolyl
analogues (1v and 1w) retain potent fXa inhibitory activities
(Ki ) 5 and 2 nM, respectively) in contrast to the benzothiazolyl
analogues (1q and 1r, Ki ) 4160 and 112 nM, respectively).
Even replacing the thiazolyl ring by a 6-membered pyridyl ring
(1y) retains good potency (Ki ) 24 nM). However, both the
furyl (1u) and triazolyl (1x) analogues are significantly less
potent. The weak fXa inhibitory activity of 1u may be
rationalized by the preferred conformation of the P1 sulfonamido
group in which one of the SdO bonds eclipses the CH of the
furan ring (vide supra) while maintaining the highly favorable
Cl-Tyr228 interaction.32 This would position the furyl oxygen
atom on the same side as the sulfur atom of the chlorothienyl
unit and could change to a less favorable bioactive conformation
of 1u relative to 1b. A comparison of the crystal structures of
The thienylsulfonamido heteroaromatic ring in the chloro-
bithienyl P1 analogue 1b is positioned further away from the
bottom of the S1 pocket than the ring distal to the chlorine atom
in the benzothienyl analogues (vide supra: 1b in Figure 2 vs
1d and 1e in Figure 3). Therefore, this thienyl ring is likely to
be further away from potential unfavorable interactions in the
lower half of the S1 binding pocket (cf. rationales for 1k-1n

Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1551
Table 5. Crystallographic and Refinement Data
compound
RCSB code
resolution
cell dims
1aa
1ba
1da
1ea
1xb
2cji
2.1
56.968
72.633
79.784
2.1
2j95
2
56.914
72.731
79.901
2
2j38
2.1
56.794
72.731
79.895
2.1
2j34
2
56.867
73.147
80.125
2
2j94
2.1
56.739
72.563
78.924
2.1
resolution
(high shell)
Rmerge
(2.1-2.18)
0.077
(0.531)
19 989
99
(2.0-2.07)
0.050
(0.381)
22 752
99.5
(2.1-2.18)
0.068
(0.288)
18 209
94.1
(2.0-2.07)
0.070
(0.486)
21 497
94.2
(2.1-2.21)
0.109
(0.431)
18 402
94
(high shell)
unique reflection
completeness
multiplicity
Rfactor
Rfree
rms bond
rms angle
total no. atoms
avg B
4.0
4.0
3.8
3.8
3.8
0.192
0.242
0.016
1.52
0.193
0.242
0.017
1.571
2443
36.95
32.338
40.723
36.117
0.211
0.302
0.016
1.697
2485
24.28
24.018
31.682
26.585
0.178
0.220
0.017
1.513
2462
24.837
24.57
31.588
28.143
0.181
0.234
0.015
1.588
2441
34.27
33.778
44.206
39.735
2397
31.259
31.128
37.337
32.524
avg B for catalytic domain
avg B for inhibitor
avg B for solvent
a The X-ray intensity data was collected at the Synchrotron Radiation Source (Daresbury, UK) on station 9.6. b The X-ray intensity data was collected at
the European Synchrtoron Radiation Facility (Grenoble, France) on beamline ID29.
the triazolyl analogue 1x with 1b (Figure 4) in fXa revealed
such a distortion, which could account for the weaker intrinsic
activity of 1x. While the S atom of the thiophene ring proximal
to the sulfonamido group in 1b is orientated toward Gly219,
the N atom at the 1 position of the 1,3,4-triazolyl group in 1x
appears to be indirectly hydrogen-bonded to Ser214 via a water
molecule. This distortion is amplified in the binding of the
pyrrolidin-2-one and the morpholinoalanylamide in which the
previously observed water-mediated interaction of the pyrrolidin-
2-one CdO with Ser214 is now absent in 1x.
The pharmacokinetic profiles of compounds with good
anticoagulant activities were initially ranked using either one
to two animals or a cassette dosing regiment with four animals.
Compounds 1a-1f showed promising pharmacokinetic proper-
ties (plasma clearance 3-18 mL/min/kg, volumes of distribution
0.17-0.6 L/kg, half-lives 0.57-1.5 h) and high oral bioavail-
abilities (69-100%) in rat. Definitive studies (n ) 3) in both
rat and dog were then carried out with 1f, which showed
encouraging pharmacokinetic profiles with high oral bioavail-
abilities (Table 4). Based on a combination of attractive in vitro
and pharmacokinetic profiles, 1f was selected as a candidate
for further evaluation. Its antithrombotic profile and further
pharmacological evaluation will be reported separately.34
The anticoagulant activities of compounds with fXa Ki values
e47 nM (1a-1f, 1v, 1w, and 1y) together with their logD
values, determined at pH 7.4, and binding to immobilized human
serum albumin (HSA) in a high throughput HPLC assay33 are
shown in Table 2. These compounds showed a broad range of
anticoagulant activities (1.5xPT 1.2-49.2 µM), which did not
correlate with their intrinsic fXa affinities. Indeed the fall off
in plasma-based activities relative to their fXa inhibitory
activities ranged from 2 to 4 orders of magnitude. The biggest
fall off in 1.5xPT values was observed with the bithienyl and
related bi(heteroaromatic) P1s (1b, 1v and 1w). In particular,
there is over 20 000-fold difference between fXa affinity and
anticoagulant activity for 1w. These more lipophilic analogues
(logD 2.03-2.18 at pH 7.4) showed high protein binding (>90%
bound to HSA), which could account for their weaker than
expected anticoagulant activities (1.5xPT 10.4-49.2 uM).
Indeed there is a good correlation of the degree of HSA binding
with lipophilicity (logD at pH 7.4) for this series of compounds.
Thus, good anticoagulant activities (1.5xPT 1.2-7.5 uM) were
observed for compounds (1a, 1c, 1e, and 1f) that are more
hydrophilic (logD 1.47-1.85 at pH 7.4) and showed <90%
HSA binding, while having similar potent fXa inhibitory
activities (Ki ) 5-16 nM).
Conclusions
Systematic modifications to a series of N-{(3S)-1-[(1S)-1-
methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}-
sulfonamides have resulted in a number of highly potent fXa
inhibitors (1a-1f, 1s, 1v, 1w, and 1y). X-ray crystallography
of selected ligands in fXa and molecular modeling studies has
provided rationales for the derived SAR. The importance of both
electrostatic and steric interactions of the chlorine atom to the
phenolic centroid of Tyr228 has been highlighted. Modifications
to the chloroaromatic ring through the replacement of C-H by
nitrogen atom are generally detrimental due to the presence of
polar groups (e.g., carboxylate of Asp189 and carbonyl back-
bone of Gly219) which form one side of the S1 pocket.
However, there is a greater scope to modify the aromatic ring
attached to the sulfonamido group, which is also influenced by
the torsional preference of the NSO2-heteroaryl bond. Relative
to their fXa affinities, good anticoagulant activities have been
identified for the more hydrophilic analogues that show <90%
binding to HSA. Against a panel of other trypsin-like serine
proteases, these compounds showed excellent selectivities
against trypsin, kallikrein, and the fibrinolytic enzyme, plasmin.
With respect to other proteases in the coagulation process,
analogues with good selectivities against thrombin and no
significant activities against activated protein C, TF/factor VIIa,
and factor XIIa have been identified. Together with the good
pharmacokinetic profile in rats and dogs, 1f was selected as a
candidate for further evaluation.
Selected compounds (1a-1f) with good anticoagulant activi-
ties were evaluated against a panel of other trypsin-like serine
proteases (Table 3). These compounds have excellent selectivity
over trypsin, kallikrein, and the fibrinolytic enzyme plasmin.
Against other proteases in the coagulation process, thrombin
activities were weaker with good selectivities seen for 1a, 1b,
and 1f, while no significant activities against activated protein
C, TF/factor VIIa, and factor XIIa were detected.

1552 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Chan et al.
was added followed by HOBt (1.2 equiv, 5.72 g, 42.33 mmol) and
triethylamine (4 equiv, 19.7 mL, 141.34 mmol). The mixture was
stirred for 1 h, L-alanine t-butyl ester (7.7 g, 53 mmol) was then
added, and stirring was continued for 18 h. The mixture was
concentrated under reduced pressure and partitioned between diethyl
ether and water. The separated organic phase was washed with 1
M hydrochloric acid, saturated aqueous sodium bicarbonate solution,
and brine, dried (MgSO4), and concentrated under reduced pressure
to give 3 (11.9 g, 82%) as an orange oil, which crystallized on
Experimental Section
General Procedures. All general purpose solvents used were
Fisons analytical reagents. Anhydrous solvents (Sureseal) were
purchased from Aldrich and Fluka. All other reagents were usually
obtained from Aldrich, Fluka, or Lancaster. All reactions involving
organometallic reagents were carried out under nitrogen atmosphere.
Reactions were monitored either by LC-MS or TLC using Merck
60 F254 silica gel glass backed plates (5 × 10 cm), eluted with
suitable solvent mixtures and visualized by UV light, followed by
heating with aqueous phosphomolybdic acid. LC-MS were run on
a Hewlett-Packard 1050 coupled with a Micromass Platform Series
II or Waters ZQ [using electrospray (ES) positive or negative
ionization modes] equipped with a Supelcosil ABZ+PLUS column
(3 µm, 3.3 cm × 4.6 mm ID). Standard conditions were eluent
systems A (H2O, 0.1% formic acid, 10 mmol ammonium acetate)
and B (95% MeCN and 0.05% formic acid in H2O): gradient 100%
A 0.7 min, 100% A-100% B 4.2 min, 100% B 1.1 min, 100-0%
B 0.2 min; flow rate ) 3 mL/min). Purifications were performed
on silica SPE (solid-phase extraction) eluted under suction or using
an OPTIX-10 system, Biotage chromatography using prepacked
Kieselgel columns, preparative hplc or mass-directed autoprep. SPE
refers to the use of cartridges sold by International Sorbent
Technology, Ltd. OPTIX-10 refers to the ISCO OPTIX 10
automated chromatography system. This uses pumps to give
gradient control of solvents and variable wavelength UV detection
and trigger for fraction collection (λ ) 254 nm, unless specified).
Biotage chromatography refers to purification carried out using
equipment sold by Dyax Corporation (either the Flash 40i or Flash
150i) and cartridges prepacked with KPSil. Preparative HPLC refers
to methods where the material was purified by high performance
liquid chromatography on a Supelcosil ABZ+ PLUS column (5
µm, 10 cm × 21.2 mm i.d.) with 0.1% HCO2H in water (A) and
MeCN (0.5% HCO2H; B) utilizing the generic gradient elution
conditions expressed as “x to y” gradient with a gradient system
as follows: 0-1.45 min x %B, 1.45-20 min x to y %B, 20-24
min y to 95% B, 24-30 min 95% B, 32-34 min 95 to x %B at a
flow rate of 8 mL/min. The Gilson 233 fraction collector was
triggered by UV (254 nm unless specified). Mass directed autoprep
refers to methods where the material was purified by high
performance liquid chromatography on a HPLCABZ+ 5 µm
column (5 cm × 10 mm i.d.) with 0.1% HCO2H in water and 95%
MeCN, 5% water (0.5% HCO2H) utilizing the following gradient
elution conditions: 0-1.0 min 5% B, 1.0-8.0 min 5 to 30% B,
8.0-8.9 min 30% B, 8.9-9.0 min 30-95% B, 9.0-9.9 min 95%
B, 9.9-10 min 95-0% B at a flow rate of 8 mL/min (System 2).
The Gilson 202-fraction collector was triggered by a Micromass
ZMD Mass Spectrometer on detecting the mass of interest. Where
appropriate, test compounds were isolated from their aqueous
solutions, if necessary, in the presence of 1,4-dioxan, by freeze-
drying using Edwards Freeze Dryer Modulyo. GC-MS was
performed on a HP5973MSD with CTC Combipal injector, using
1/100 split injection and chemical ionization with ammonia or
methane as collision gas, equipped with HP5 5% phenyl methyl
siloxane column (30 m × 0.25 mm × 0.25 mm). Standard GC
condition was 80-320 °C at 50 °C/min over 10 min using helium
as carrier gas at 1.5 mL/min. Mass spectra were run by an
electrospray Hewlett-Packard 5989B instrument. All NMR spectra
were run on a Bruker DPX 400 MHz instrument in an appropriate
deuterated solvent using tetramethylsilane or residual hydrogenated
solvent as standard. Elemental microanalyses were determined by
Butterworth Laboratories Limited, Teddington, Middlesex, U.K.
High-resolution mass spectra were obtained as positive ion accurate
mass centroided data using a Micromass Q-Tof 2 hybrid quadrupole
time-of-flight mass spectrometer, equipped with a Z-spray interface,
over a mass range of 80-1200 Da, with a scan time of 0.95 s, and
an interscan delay of 0.07 s, using reserpine as the external mass
calibrant ([M + H]+ ) 609.2812 Da).
1
standing. H NMR (CDCl3): δ 1.37 (d, 3H, J ) 7 Hz), 1.46 (s,
9H), 1.92-2.16 (m, 2H), 2.11 (s, 3H), 2.59 (br t, 2H, J ) 7 Hz),
4.33-4.48 (m, 2H), 5.11 (s, 2H), 5.55 (br d, 1H, J ) 7.5 Hz), 6.62
(br d, 1H, J ) 6.5 Hz), 7.28-7.40 (m, 5H).
(2S)-2-((3S)-3-{[(Benzyloxy)carbonyl]amino}-2-oxopyrrolidin-
1-yl) Propanoic Acid 4. A solution of 3 (11.9 g, 29 mmol) in
acetone (75 mL) was treated with methyl iodide (10 equiv, 18 mL,
0.29 mol) and stirred at room temperature for 72 h. The reaction
mixture was then concentrated under reduced pressure to give an
orange solid that was dissolved in MeCN (200 mL). Dowex resin
(hydroxide form, 19.42 g) was added, and the mixture stirred for
18 h at room temperature. The mixture was filtered and the resin
was washed with EtOAc. The filtrate was concentrated under
reduced pressure to afford a yellow oil that was purified by Biotage
chromatography (eluting with cyclohexane/EtOAc 3:2) to give the
t-butyl (2S)-2-((3S)-3-{[(benzyloxy)carbonyl]amino}-2-oxopyrro-
1
lidin-1-yl)propanoate (2.92 g, 28%) as a colorless oil. H NMR
(CDCl3): δ 1.39 (d, 3H, J ) 7.5 Hz), 1.45 (s, 9H), 1.86-1.97 (m,
1H), 2.64-2.76 (m, 1H), 3.27-3.36 (m, 1H), 3.48 (t, 1H, J ) 9
Hz), 4.25-4.36 (m, 1H), 4.72 (q, H, J ) 7 Hz), 5.13 (s, 2H), 5.36
(br s, 1H), 7.28-7.38 (m, 5H).
t-Butyl (2S)-2-((3S)-3-{[(benzyloxy)carbonyl]amino}-2-oxopy-
rrolidin-1-yl)propanoate (0.5 g, 1.38 mmol) was dissolved in DCM
(7 mL), and TFA (4.7 mL) was added. The mixture was stirred at
room temperature for 4 h and then concentrated under reduced
pressure to give 4 (0.423 g, 99.9%) as a colorless oil, which after
1
azeotroping with toluene, crystallized to a white solid. H NMR
(MeOH-d4): δ 1.43 (d, 3H, J ) 7.3 Hz), 1.88-2.02 (m, 1H), 2.37-
2.50 (m, 1H), 3.34-3.52 (m, 1H), 4.43 (dd, 1H, J ) 10.5 and 9
Hz), 4.67 (q, 1H, J ) 7.3 Hz), 5.09 (s, 2H), 7.25-7.50 (m, 5H).
LC-MS: tR 2.43 min; m/z (ES) 307 (MH+), 305 (M - H)-.
Benzyl (3S)-1-[(1S)-1-Methyl-2-morpholin-4-yl-2-oxoethyl]-
2-oxopyrrolidin-3-ylcarbamate 5. Compound 4 (84.5 g, 0.276mol)
was dissolved in DMF (2 L) and TBTU (1.8 equiv, 161 g, 0.5 mol)
was added, followed by N,N-diisopropylethylamine (2.4 equiv, 92
mL, 0.66 mol) and morpholine (1.9 equiv, 46 mL, 0.527 mol). The
mixture was stirred under nitrogen for 2.5 h, and saturated aqueous
ammonium chloride was added. The mixture was stirred for 15
min then partitioned between water and EtOAc. The separated
organic phase was washed with aqueous lithium chloride (10% w/v),
followed by saturated sodium bicarbonate and brine. The organic
layer was dried (over Na2SO4) and concentrated under reduced
pressure to give 5 (65 g, 63%) as a yellow solid. IH NMR (MeOH-
d4): δ 1.28 (d, 3H, J ) 7.1 Hz), 1.95-2.08 (m, 1H), 2.34-2.44
(m, 1H), 3.31-3.55 (m, 4H), 3.56-3.72 (m, 6H), 4.23 (t, 1H, J )
9.5 Hz), 5.03-5.12 (m, 3H), 5.09 (s, 2H), 7.25-7.39 (m, 5H). LC-
MS: tR 2.39 min; m/z (ES) 376 (MH+), 374 (M - H)-.
(3S)-3-Amino-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-
pyrrolidin-2-one 2. A mixture of 5 (20 g, 53.3 mmol), 10%
palladium on carbon (10% w/w, 2 g), and ethanol (1.3 L) was
stirred under an atmospheric pressure of hydrogen for 16 h. The
reaction mixture was filtered through Celite, and the filtrate was
concentrated under reduced pressure to give 2 (12.3 g, 96%) as a
pale white oil. 1H NMR (CDCl3): δ 1.31 (d, 3H, J ) 7 Hz), 1.64-
1.76 (m, 3H), 2.38-2.49 (m, 1H), 3.30 (td, 1H, J ) 9.5 and 6.5
Hz), 3.39 (td, 1H, J ) 9.5 and 1.7 Hz), 3.47-3.73 (m, 9H), 5.07
(q, 2H, J ) 7 Hz).
General Procedures. Method A: Sulfonylation of 2 with
Sulfonyl Chlorides 6. To a solution of 2 (typically 50-100 mg)
in anhydrous MeCN were added the appropriate sulfonyl chloride
6 (1.05-1.2 equiv) in MeCN or DCM and a mild base (2.2 equiv)
such as pyridine or N,N-di-isopropylethylamine, and the mixture
t-Butyl N-[(Benzyloxy)carbonyl]-L-methionyl-L-alaninate 3.
N-(benzyloxycarbonyl)-L-methionine (10 g, 35.3mmol) was dis-
solved in DMF (200 mL) and EDC (1.2 equiv, 8.13 g, 42.4 mmol)

Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1553
was stirred at room temperature overnight (typically 18 h). Saturated
ammonium chloride solution was added, and the resultant mixture
was stirred at room temperature for 30 min. The mixture was
concentrated under reduced pressure, and the residue was partitioned
between chloroform and a 1:1 mixture of hydrochloric acid (2 M)
and water. The organic layer was washed with a 1:1 mixture of
saturated aqueous sodium bicarbonate and water and brine. The
organic layer was separated, dried (MgSO4), and concentrated under
reduced pressure to give the corresponding sulfonamide, which may
have required purification by SPE and/or (mass-directed) autoprep
before being freeze-dried in an aqueous dioxan solution.
Method B: Preparation of Sulfonyl Chlorides 6 from Parent
or Bromo-chloro(hetero)aromatics. n-Butyl lithium (1.6 M in
hexanes, 1.1 equiv) was added to a cooled (-78 °C) solution of
the appropriate chloro(hetero)aromatics (typically 100 mg) in
anhydrous THF (typically 5-10 mL) over 15 min. The reaction
was stirred for 20 min. Sulfur dioxide gas was then condensed into
the reaction for 10 min. The reaction was allowed to warm to room
temperature, stirred for 1 h, and then evaporated in vacuo. The
resultant residue was stirred at room temperature (typically for 2-18
h) with N-chlorosuccinimide (1.23 equiv) in anhydrous DCM
(typically 6-10 mL). The solution was filtered, and the filtrate was
concentrated under reduced pressure to give the corresponding
sulfonyl chloride as a crude product, which was used in Method A
without further purification.
washed with water (3 × 30 mL), dried (MgSO4), and filtered.
Removal of solvent gave a light brown residue that was purified
using SPE (10 g SiO2, EtOAc) to give 8 (276.5 mg, 83%) as a
1
white solid. H NMR (CDCl3): δ 1.30 (d, 3H, J ) 7 Hz), 1.95-
2.08 (m, 1H), 2.54-2.63 (m, 1H), 3.27 (s, 1H), 3.31 (td, 1H, J )
10 and 6 Hz), 3.43-3.77 (m, 10H), 5.01 (q, 1H, J ) 7 Hz), 5.27
(br s, 1H), 7.63 (d, 2H, J ) 8.6 Hz), 7.87 (d, 2H, J ) 8.6 Hz).
LC-MS: tR 2.46 min; m/z (ES) 406 MH+, 404 (M - H)-.
4-[(E)-2-Chloroethenyl]-N-{(3S)-1-[(1S)-1-methyl-2-(4-mor-
pholinyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}benzenesulfona-
mide 1i. To a slurry of 8 (132.7 mg, 0.327 mmol) in anhydrous
THF (4 mL) at 0 °C under nitrogen, t-hexylborane (prepared from
0.69 mL of each of THF solution of 1 M borane and 2 M 2-methyl-
2-butene according to standard procedure) was added and stirred
for 4 h. Copper(II) chloride (2.4 equiv, 105.6 mg, 0.785 mmol)
followed by 1,3-dimethyl-2-imidazolidinone (0.5 mL) and water
(0.014 mL, 0.785mmol) were added.23 The mixture was stirred at
0 °C to room temperature for 18 h and quenched with water (5
mL). Extraction with EtOAc (3×) gave a yellow oil that was
purified using autopreparative reverse phase HPLC to give 1i (49.6
1
mg, 34%) as a creamy white solid. H NMR (CDCl3): δ 1.30 (d,
3H, J ) 7 Hz), 1.96-2.08 (m, 1H), 2.53-2.64 (m, 1H), 3.26-
3.36 (m, 1H), 3.43-3.75 (m, 9H), 3.74 (dd, 1H, J ) 10.5 and 8
Hz), 5.01 (q, 1H, J ) 7 Hz), 5.15-5.60 (br s, 1H), 6.84 (ABq, 2H,
J ) 13.6 Hz), 7.44 (d, 1H, J ) 8 Hz), 7.86 (d, 1H, J ) 8 Hz).
LC-MS: tR 2.70 min, 98% purity; m/z (ES) 442/444 MH+, 440/
442 (M - H)-. HRMS calcd for C19H23N3O5ClS m/z (MH+),
442.1203; found, 442.1213.
(E)-2-(5-Chlorothien-2-yl)-N-{(3S)-1-[(1S)-1-methyl-2-mor-
pholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}ethenesulfona-
mide 1f. Compound 1f (19.3 g, 70% yield) as a white solid was
synthesized from 2 (14.9 g, 61.8mmol) and (E)-2-(5-chloro-2-
thienyl)ethenesulfonyl chloride13c (16.5 g, 67.9 mmol) using Method
5-(5-Chloro-1,3-thiazol-2-yl)-N-{(3S)-1-[(1S)-1-methyl-2-(4-
morpholinyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}-2-thiophene-
sulfonamide 1n. To a mixture of thiophene-2-boronic acid (1.1
equiv, 0.71 g, 5.54 mmol), 2-bromo-5-chlorothiazole19b (1 g, 5.04
mmol), and Cs2CO3 (2.6 equiv, 4.27 g, 13.1 mmol) under nitrogen,
degassed water (12 mL) was added and stirred for 10 min. DME
(12 mL) followed by Pd(PPh3)4 (5 mol%, 291 mg, 0.25 mmol)
were then added. The mixture was heated at 70 °C overnight. The
crude reaction mixture was extracted with Et2O (3 × 90 mL), dried,
and evaporated to give a dark brown oil (1.152 g). SPE (20 g SiO2,
cyclohexane, 3 to 10% EtOAc-cyclohexane) followed by further
purification on OPTIX 10 (2 × 10 g SiO2, 5% EtOAc-cyclohex-
ane) gave the 5-chloro-2-(2-thienyl)-1,3-thiazole as a yellow solid
(527 mg, 51.9%). 1H NMR (CDCl3): δ 7.07 (dd, 1H, J ) 5 and 4
Hz), 7.40-7.44 (m, 2H), 7.55 (s, 1H). LC-MS: tR 3.37 min; m/z
(ES) 202/204 MH+.
Using Method A under General Procedures, 1n (3.3 mg) was
synthesized from 2 (101.6 mg, 0.42 mmol) and 5-(5-chloro-1,3-
thiazol-2-yl)-2-thiophenesulfonyl chloride 6n [139.5 mg; prepared
from 5-chloro-2-(2-thienyl)-1,3-thiazole (104 mg, 0.52 mmol) using
Method B under General Procedures and used without further
purification]. 1H NMR (CDCl3): δ 1.33 (d, 3H, J ) 7 Hz), 2.04-
2.16 (m, 1H), 2.62-2.73 (m, 1H), 3.32-3.40 (m, 1H), 3.45-3.73
(m, 9H), 3.92 (br t, 1H, J ) 9 Hz), 5.03 (q, 1H, J ) 7 Hz), 5.48
(br s, 1H), 7.37 (d, 1H, J ) 4 Hz), 7.61 (d, 1H, J ) 4 Hz), 7.63 (s,
1H). LC-MS: tR 2.83 min, 95% purity; m/z (ES) 505/507 MH+,
503/505 (M - H)-. HRMS calcd for C18H21ClN4O5S3 m/z (MH+),
505.0441; found, 505.0431.
6-Chloro-N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoet-
hyl]-2-oxopyrrolidin-3-yl}-1,3-benzothiazole-2-sulfonamide 1q.
N-Chlorosuccinimide (0.37 g, 2.77 mmol) was added to 6-chloro-
1,3-benzothiazole-2(3H)-thione22 (0.5 g, 2.48 mmol) in DCM (15
mL) under nitrogen, and stirred at room temperature for 3 h. A
solution of 2 (0.569 g, 2.36 mmol) and triethylamine (1.04 mL,
7.49 mmol) in anhydrous DCM (5 mL) were added, and the
resulting mixture was stirred at room temperature under nitrogen
for 2 h. The solution was filtered, and the filtrate was diluted with
DCM. The organic solution was washed with water and brine, dried
(MgSO4), and concentrated under reduced pressure. The residue
was purified by SPE (silica, eluting with cyclohexane-EtOAc 1:1,
increasing polarity to EtOAc-MeOH 19:1) to give (3S)-3-{[(6-
chloro-1,3-benzothiazol-2-yl)thio]amino}-1-[(1S)-1-methyl-2-mor-
pholin-4-yl-2-oxoethyl]pyrrolidin-2-one (300 mg, 29%) as a white
1
A under General Procedures. H NMR (CDCl3): δ 1.35 (d, 3H, J
) 7 Hz), 1.99-2.12 (m, 1H), 2.59-2.68 (m, 1H), 3.31-3.40 (m,
1H), 3.47-3.74 (m, 9H), 3.96 (ddd, 1H, J ) 11, 8 and 4 Hz), 5.02
(br d, 1H, J ) 3 Hz), 5.07 (q, 1H, J ) 7 Hz), 6.55 (d, 1H, J ) 15
Hz), 6.91 (d, 1H, J ) 4 Hz), 7.09 (d, 1H, J ) 4 Hz), 7.49 (d, 1H,
J ) 15 Hz). LC-MS: tR 2.70 min; m/z (ES) 448/450 MH+, 446/
448 (M - H)-. Anal. (C17H22ClN3O5S2): C, H, N.
4-Iodo-N-{(3S)-1-[(1S)-1-methyl-2-(4-morpholinyl)-2-oxoethyl]-
2-oxo-3-pyrrolidinyl}benzenesulfonamide 7. Compound 7 (3.217
g, 71%) was synthesized from 2 (2.163 g, 8.96 mmol) and
4-iodophenylsulfonyl chloride (1.1 equiv, 2.983 g, 9.86 mmol) using
1
Method A under General Procedures. H NMR (CDCl3): δ 1.29
(d, 3H, J ) 7 Hz), 1.91-2.05 (m, 1H), 2.45-2.65 (m, 1H), 3.29
(td, 1H, J ) 10 and 6 Hz), 3.42-3.74 (m, 9H), 3.82 (dd, 1H, J )
10, and 8 Hz), 5.03 (q, 1H, J ) 7 Hz), 6.27 (br s, 1H), 7.64 (d, 2H,
J ) 8.6 Hz), 7.87 (d, 2H, J ) 8.6 Hz). LC-MS: tR 2.60 min; m/z
(ES) 508 MH+, 506 (M - H)-.
4-Ethynyl-N-{(3S)-1-[(1S)-1-methyl-2-(4-morpholinyl)-2-oxo-
ethyl]-2-oxo-3-pyrrolidinyl}benzenesulfonamide 8. A mixture of
7 (547 mg, 1.08 mmol), CuI (3 mol%, 6.2 mg, 0.032 mmol),
dichlorobis(triphenylphosphine)palladium(II) (3 mol%, 22.7 mg,
0.032 mmol), trimethylsilylacetylene (3 equiv, 0.458 mL, 3.24
mmol), and triethylamine (2 mL) in DMF (2 mL) was stirred at
room temperature for 22 h. The reaction mixture was evaporated
in vacuo to give a dark brown residue that was purified using SPE
(20 g SiO2, 50 to 70% EtOAc-cyclohexane, EtOAc then 5%
MeOH-EtOAc) to give N-{(3S)-1-[(1S)-1-methyl-2-(4-morpholi-
nyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}-4-[(trimethylsilyl)ethynyl]-
benzenesulfonamide as a brown foam (514 mg, 99.9%). 1H NMR
(CDCl3): δ 0.26 (s, 9H), 1.30 (d, 3H, J ) 7 Hz), 1.92-2.05 (m,
1H), 2.49-2.59 (m, 1H), 3.29 (td, 1H, J ) 10 and 6 Hz), 3.43-
3.77 (m, 10H), 5.01 (q, 1H, J ) 7 Hz), 5.56 (bd, 1H, J ) 3 Hz),
7.59 (d, 2H, J ) 8.6 Hz), 7.84 (d, 2H, J ) 8.6 Hz). LC-MS: tR
3.20 min; m/z (ES) 478 MH+, 476 (M - H)-.
To a solution of N-{(3S)-1-[(1S)-1-methyl-2-(4-morpholinyl)-2-
oxoethyl]-2-oxo-3-pyrrolidinyl}-4-[(trimethylsilyl)ethynyl]benze-
nesulfonamide (391 mg, 0.82 mmol) in dry THF (4 mL) at
-78 °C, tetra n-butylammonium fluoride (1 M in THF, 0.819 mL,
0.82 mmol) was added. The mixture was stirred at -78 °C for 1 h
and then at room temperature for another hour and evaporated in
vacuo. The resultant residue was dissolved in EtOAc (80 mL) and

1554 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Chan et al.
1
solid. H NMR (CDCl3): δ 1.34 (d, 3H, J ) 6.6 Hz), 2.06-2.20
Using Method A under General Procedures, 1u (37 mg, 19%
based on 2) as a light brown foam was synthesized from 2 (97.4
mg, 0.4mmol) and 5-(5-chloro-2-thienyl)-2-furansulfonyl chloride
6u [226 mg; prepared from 2-(5-chloro-2-thienyl)furan (167.5 mg,
0.91mmol) using Method B under General Procedures and used
(m, 1H), 2.40-2.51 (m, 1H), 3.30-3.44 (m, 2H), 3.47-3.78 (m,
8H), 4.03 (ddd, 1H, J ) 12, 8 and 3.5 Hz), 4.35 (d, 1H, J ) 3.5
Hz), 5.10 (q, 1H, J ) 6.6 Hz), 7.37 (dd, 1H, J ) 9 and 2 Hz), 7.72
(d, 1H, J ) 9 Hz), 7.77 (d, 1H, J ) 2 Hz). LC-MS: tR 2.82 min;
m/z (ES) 441/443 MH+, 439/441 (M - H)-.
1
without further purification]. H NMR (CDCl3): δ 1.33 (d, 3H, J
) 7 Hz), 1.99-2.13 (m, 1H), 2.59-2.68 (m, 1H), 3.35 (td, 1H, J
) 10 and 6 Hz), 3.42-3.74 (m, 9H), 3.96 (dd, 1H, J ) 10.6 and
8.1 Hz), 5.04 (q, 1H, J ) 7 Hz), 5.63 (br s, 1H), 6.50 (d, 1H, J )
3.5 Hz), 6.91 (d, 1H, J ) 4 Hz), 7.12 (d, 1H, J ) 3.5 Hz), 7.18 (d,
1H, J ) 4). LC-MS: tR 2.93 min; m/z (ES) 488/490 MH+, 486/
488 (M - H)-. Anal. (C19H22ClN3O6S2‚0.55CH2O2): C, H, N.
2-(5-Chloro-2-thienyl)-N-{(3S)-1-[(1S)-1-methyl-2-(4-mor-
pholinyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}-1,3-thiazole-5-sul-
fonamide 1v. To a mixture of 2-bromothiazole (0.55 mL, 6.1 mmol)
and 5-chlorothiophene-2-boronic acid (991 mg, 6.1 mmol) in DME
(10 mL) under nitrogen, a solution of sodium carbonate (1.68 g,
15.86 mmol) in water (10 mL), bubbled with nitrogen for 10 min,
followed by Pd2dba3‚CHCl3 (2.5 mol%, 109 mg, 0.105 mmol) and
a solution of PPh3 (10 mol%, 110.5 mg, 0.421 mmol) in DME (10
mL) were added. The mixture was heated at 80 °C under nitrogen
for 16 h and evaporated in vacuo. The resultant aqueous mixture
was extracted with DCM (3 × 20 mL), dried (MgSO4), and filtered.
Removal of solvent gave a brown oil that was purified by SPE (20
g SiO2, 3-5% EtOAc-cyclohexane) to give the 2-(5-chloro-2-
thienyl)-1,3-thiazole (374 mg, 30%) as a light brown oil. 1H NMR
(CDCl3): δ 6.91 (d, 1H, J ) 4 Hz), 7.26 (d, 1H, J ) 3 Hz), 7.28
(d, 1H, J ) 4 Hz), 7.76 (d, 1H, J ) 3 Hz). LC-MS: tR 3.26 min;
m/z (ES) 202/204 MH+.
(3S)-3-{[(6-Chloro-1,3-benzothiazol-2-yl)thio]amino}-1-[(1S)-1-
methyl-2-morpholin-4-yl-2-oxoethyl]pyrrolidin-2-one (0.1 g, 0.227
mmol) was stirred at room temperature in anhydrous acetone (3
mL) and 5% aqueous potassium permanganate (1.35 mL) for 3 h,
after which additional acetone (3 mL) and 5% aqueous potassium
permanganate (1.35 mL) were added. The reaction mixture was
stirred for a further 18 h and filtered through Celite. The filtrate
was concentrated under reduced pressure, and the residue was
purified by mass directed preparative HPLC to give 1q (6.2 mg,
6%) as a white solid. 1H NMR (CDCl3): δ 1.32 (d, 3H, J ) 7 Hz),
2.08-2.21 (m, 1H), 2.70-2.81 (m, 1H), 3.37 (td, 1H, J ) 10.5
and 6 Hz), 3.43-3.74 (m, 9H), 4.31 (dd, 1H, J ) 10.8 and 8 Hz),
5.01 (q, 1H, J ) 7 Hz), 5.86-6.03 (br s, 1H), 7.68 (dd, 1H, J )
8.8 and 2 Hz), 7.97 (d, 1H, J ) 2 Hz), 8.10 (d, 1H, J ) 8.8 Hz).
LC-MS: tR 2.98 min, 98% purity; m/z (ES) 473 MH+. HRMS calcd
for C18H21ClN4O5S2 m/z (MH+), 473.0720; found, 473.0719.
5-Chloro-N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoet-
hyl]-2-oxopyrrolidin-3-yl}-1H-indole-2-sulfonamide 1s. t-Butyl
5-chloro-2-[({(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-
2-oxopyrrolidin-3-yl}amino)sulfonyl]-1H-indole-1-carboxylate (13
mg, 25%) as a colorless glass was synthesized from 2 (44 mg,
0.18mmol) and 1,1-dimethylethyl 5-chloro-2-(chlorosulfonyl)-1H-
indole-1-carboxylate17 (33 mg, 0.094mmol) using Method A under
General Procedures. 1H NMR (CDCl3): δ 1.27 (d, 3H, J ) 7 Hz),
1.75 (s, 9H), 2.06-2.18 (m, 1H), 2.64-2.73 (m, 1H), 3.32 (td,
1H, J ) 10 and 6.5 Hz), 3.41-3.73 (m, 9H), 3.90 (ddd, 1H, J )
11, 8, and 3 Hz), 5.00 (q, 1H, J ) 7 Hz), 6.75 (d, 1H, J ) 3 Hz),
7.42 (dd, 1H, J ) 8 and 2 Hz), 7.42 (s, 1H), 7.62 (d, 1H, J ) 2
Hz), 7.95 (d, 1H, J ) 8 Hz). LC-MS: tR 3.41 min; m/z (ES) 555/
557 MH+, 553/555 (M - H)-.
Using Method A under General Procedures, 1v (66 mg, 32%)
was synthesized from 2 (98 mg, 0.41 mmol) and 2-(5-chloro-2-
thienyl)-1,3-thiazole-5-sulfonyl chloride 6v [165 mg, prepared from
2-(5-chloro-2-thienyl)-1,3-thiazole (133.2 mg, 0.66 mmol) using
Method B under General Procedures and used without further
1
purification]. H NMR (MeOH-d4): δ 1.27 (d, 3H, J ) 7 Hz),
1.80-1.93 (m, 1H), 2.39-2.49 (m, 1H), 3.33-3.41 (m, 2H), 3.43-
3.70 (m, 8H), 4.26 (dd, 1H, J ) 10.4 and 8.6 Hz), 4.99 (q, 1H, J
) 7 Hz), 7.08 (d, 1H, J ) 4 Hz), 7.66 (d, 1H, J ) 4 Hz), 8.16 (s,
1H). LC-MS: tR 2.96 min; m/z (ES) 505/507 MH+, 503/505 (M -
H)-. Anal. (C18H21ClN4O5S3‚0.6H2O): C, H, N.
t-Butyl 5-chloro-2-[({(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-
oxoethyl]-2-oxopyrrolidin-3-yl}amino)sulfonyl]-1H-indole-1-car-
boxylate (11 mg, 0.02 mmol) was dissolved in 1:1 TFA/DCM (0.5
mL) and allowed to stand at room temperature for 1 h. The mixture
was concentrated under reduced pressure, and the residue was
partitioned between saturated aqueous sodium bicarbonate and
DCM. The separated organic phase was dried (MgSO4) and
concentrated under a stream of nitrogen to give 1s (8.2 mg, 91%)
5-(5-Chloro-2-thienyl)-N-{(3S)-1-[(1S)-1-methyl-2-(4-mor-
pholinyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}-1,3-thiazole-2-sul-
fonamide 1w. To a mixture of 5-chlorothiophene-2-boronic acid
(1.041 g, 6.41mmol) and 5-bromothiazole19a (92% in ether, 1.143
g, 6.41mmol) in DME (30 mL, bubbled with N2 for 10 min before
use), a solution of sodium carbonate (2.6 eq., 1.77 g, 16.67mmol)
in water (15 mL, bubbled with N2 for 20 min before use) followed
by tetrakis(triphenylphosphine)palladium(0) (10 mol%, 741 mg,
0.64 mmol) were added. The mixture was heated at 70-80 °C under
nitrogen for 5.5 h. The cooled reaction mixture was evaporated in
vacuo, extracted with ether (4 × 30 mL), dried (MgSO4), and
filtered. Removal of solvent gave a brown oil (1.7 g). Purification
on a SPE (20 g SiO2) and eluted with cyclohexane followed by
5-10% EtOAc-cyclohexane gave the 5-(5-chloro-2-thienyl)-1,3-
1
as a white solid. H NMR (CDCl3): δ 1.36 (d, 3H, J ) 7 Hz),
2.05-2.17 (m, 1H), 2.58-2.68 (m, 1H), 3.37 (td, 1H, J ) 10 and
6.5 Hz), 3.39-3.70 (m, 9H), 4.17-4.28 (m, 1H), 5.03 (q, 1H, J )
7 Hz), 5.69 (d, 1H, J ) 6.5 Hz), 7.06 (d, 1H, J ) 2 Hz), 7.21-
7.30 (m, 2H), 7.63 (d, 1H, J ) 1 Hz), 10.9 (s, 1H). LC-MS: tR
2.95 min, 96% purity; m/z (ES) 455/457 MH+, 453/455 (M - H)-.
HRMS calcd for C19H23ClN4O5S m/z (MH+), 455.1156; found,
455.1158.
5-(5-Chloro-2-thienyl)-N-{(3S)-1-[(1S)-1-methyl-2-(4-mor-
pholinyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}-2-furansulfona-
mide 1u. A mixture of 2-bromo-5-chlorothiophene (0.307 mL,
2.8 mmol), tri-n-butyl(2-furanyl)stannane (0.882 mL, 2.8 mmol),
tris(dibenzylideneacetone)dipalladium(0)-chloroform adduct (Pd2-
dba3‚CHCl3; 2.5 mol%, 72.5 mg, 0.07 mmol), and PPh3 (10 mol%,
73.4 mg, 0.28 mmol) in THF (15 mL) was heated under nitrogen
at 75 °C for 21.5 h. KF (10 g) and water (5 mL) were added to the
cooled reaction mixture and stirred at room temperature for 7 h.
Aqueous NaOH (2 M, 5 mL) was then added. The resultant mixture
was evaporated in vacuo to remove most THF, diluted with water
(20 mL), extracted with ether (3 × 30 mL), and dried (MgSO4).
Removal of solvent gave an oily brown solid (1.31 g) that was
purified by SPE (20 g SiO2, cyclohexane) to give 2-(5-chloro-2-
1
thiazole as a yellow solid (708 mg, 51%). H NMR (CDCl3): δ
6.89 (d, 1H, J ) 3.8 Hz), 6.99 (d, 1H, J ) 3.8 Hz), 7.90 (s,1H),
8.71 (s, 1H). LC-MS: tR 3.24 min; m/z (ES) 202/204 MH+.
Using Method A under General Procedures, 1w (140 mg, 66.5%)
was synthesized from 2 (100.6 mg, 0.42 mmol) and 5-(5-chloro-
2-thienyl)-1,3-thiazole-2-sulfonyl chloride 6w [175 mg; prepared
from 5-(5-chloro-2-thienyl)-1,3-thiazole (103 mg, 0.51 mmol) using
Method B under General Procedures and used without further
purification]. 1H NMR (CDCl3): δ 1.33 (d, 3H, J ) 7 Hz), 2.06-
2.19 (m, 1H), 2.65-2.74 (m, 1H), 3.37 (td, 1H, J ) 10 and 6 Hz),
3.45-3.74 (m, 9H), 4.27 (dd, 1H, J ) 10.8 and 8 Hz), 5.04 (q,
1H, J ) 7 Hz), 6.20 (br s, 1H), 6.93 (d, 1H, J ) 4 Hz), 7.07 (d,
1H, J ) 4 Hz), 7.89 (s, 1H). LC-MS: tR 2.97 min; m/z (ES) 505/
507 MH+, 503/505 (M - H)-. Anal. (C18H21ClN4O5S3‚0.5H2O):
C, H. For N: calcd, 10.90%; found, 10.34%.
1
thienyl)furan (299 mg, 58%) yellow liquid. H NMR (CDCl3): δ
6.43-6.48 (m, 2H), 6.86 (t, 1H, J ) 3.5 Hz), 7.01 (t, 1H, J ) 3.5
Hz), 7.40 (s, 1H). GC-MS: tR 7.07 min; m/z (APCI for C8H5ClOS)
184/186 M+.
3-(5-Chloro-2-thienyl)-1H-1,2,4-triazole-5-sulfonyl Chloride
6x. A mixture of 5-chloro-2-thiophenecarboxylic acid hydrazide

Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1555
(197 mg, 1.12 mmol) and thiourea (90.3 mg, 1.12 mmol) was heated
at 160 °C20 under nitrogen without solvent for 6.5 h. The cooled
mixture was stirred with aqueous NaOH (5%, 20 mL) for 4 h and
filtered to remove any insolubles. The basic solution was acidified
with acetic acid (1.65 mL) to pH 5. The resultant precipitate was
collected and dried in vacuo at 50 °C overnight to give the 5-(5-
chloro-2-thienyl)-1,2-dihydro-3H-1,2,4-triazole-3-thione as a creamy
white solid (198 mg, 82%). 1H NMR (DMSO-d6): δ 7.69 (d, 1H,
J ) 3.8 Hz), 7.85 (d, 1H, J ) 3.8 Hz). LC-MS: tR 3.04 min; m/z
(ES) 218/220 MH+, 216/218 (M - H)-.
(Accelrys). All other data manipulation was carried out using
programs from the CCP4 suite,38 including crystallographic refine-
ment using REFMAC.39 All nonglycine residues fall within the
energetically favorable regions of the Ramachandran plot. All of
the atoms of the inhibitors and residues in the vicinity of the active
site lie in well-defined electron density. The majority of the rest of
the protein is in well-determined electron density, except for the
EGF1 domain, which was not visible. The statistics of the
crystallographic refinement are shown in Table 5. Atomic coordi-
nates have been deposited with the RCSB Protein Data Bank (2cji
for 1a, 2j95 for 1b, 2j38 for 1d, 2j34 for 1e, and 2j94 for 1x).
In Vitro Assay for Inhibition of Factor Xa. The ability of test
compounds to inhibit human fXa in vitro was determined in a
fluorescence assay, using rhodamime 110, bis-(Boc-L-glycylglycyl-
L-arginine amide) as the fluorogenic substrate. Test compounds were
diluted from a 10 mM DMSO solution to the final assay concentra-
tion range of 166 µM to 0.09 nM. Assay was performed at room-
temperature using buffer consisting of 50 mM HEPESa, 150 mM
NaCl, and 5 mM CaCl2 at pH 7.4 containing human factor Xa (0.2
nM final concentration). Test compound and enzyme were prein-
cubated for 15 min prior to addition of the substrate (0.01 mM
final concentration). Incubation was for 3 h, after which a LJL
Analyst (now marketed by Molecular Devices Corporation) was
used to monitor the fluorescence at 485/535 nm wavelength.
Analysis of the derived data using Activity Base, a curve fitting
software from IDBS, gave the pIC50 and pKi values from which Ki
values were derived.
Prothrombin Time (PT) Assay.40 Blood from two female and
two male donors, supplied by Biological Specialty Corporation
(Colmar, PA), was collected into 3.8% sodium citrate vacutainers
and mixed gently. Plasma was generated by centrifugation of the
citrated blood samples at 1200 g for 20 min at room temperature
and stored at -20 °C until use. Prior to assay, plasma from four
different human subjects (two male and two female) was thawed
and pooled.
PT assays were performed at 37 °C using the Dade Behring BCS
Coagulation System (n ) 2 trials). An amount equal to 50 µL of
plasma containing test compound at concentrations ranging from
0.03 to 100 µM (made from a 100 µM plasma stock containing
1% DMSO and prepared by the coagulation analyzer) was combined
with 100 µL of thromboplastin C Plus (Dade Behring, Newark,
DE). Absorbance at 405 nm was then monitored and time (sec) to
clot formation at each concentration of test compound was
determined and compared to time to clot formation of control
(human plasma in the absence of test compound) and reported as
the fold-increase in PT.
To a solution of 5-(5-chloro-2-thienyl)-1,2-dihydro-3H-1,2,4-
triazole-3-thione (45.5 mg, 0.21 mmol) in acetic acid (2 mL) at
about 10 °C (bath temperature), a solution of chlorine in acetic
acid (0.11 g/mL, prepared by bubbling chlorine gas into 10 mL of
acetic acid in ice-water bath for 15 min, 3.3 equiv, 0.444 mL,
0.63 mmol) was added. The mixture was allowed to warm to room
temperature slowly, stirred for 3 h, and evaporated to dryness to
afford 3-(5-chloro-2-thienyl)-1H-1,2,4-triazole-5-sulfonyl chloride
6x, which was used without purification. An amount of this crude
product was treated with excess methylamine in THF. LC-MS
confirmed the formation of the corresponding N-methyl sulfonamide
[tR 2.68 min; m/z (ES) 279/281 MH+, 277/279 (M - H)-].
3-(5-Chloro-2-thienyl)-N-{(3S)-1-[(1S)-1-methyl-2-(4-mor-
pholinyl)-2-oxoethyl]-2-oxo-3-pyrrolidinyl}-1H-1,2,4-triazole-5-
sulfonamide 1x. Compound 1x (23 mg, 23% based on 2), as a
white solid, was synthesized from 2 (50 mg, 0.21 mmol) and 3-(5-
chloro-2-thienyl)-1H-1,2,4-triazole-5-sulfonyl chloride 6x (crude,
obtained as above) using Method A under General Procedures. 1H
NMR (CDCl3): δ 1.39 (d, 3H, J ) 7 Hz), 2.13-2.26 (m, 1H),
2.67-2.76 (m, 1H), 3.39-3.74 (m, 11H), 4.58 (dd, 1H, J ) 10.3
and 8.6 Hz), 5.03 (q, 1H, J ) 7 Hz), 6.87 (d, 1H, J ) 4 Hz), 7.42
(d, 1H, J ) 4 Hz). LC-MS: tR 2.55 min; m/z (ES) 489/491 MH+,
487/489 (M - H)-. Anal. (C17H21ClN6O5S2‚1.2H2O): C, H, N.
FXa Crystal Structures with Bound Ligands. Preparation of
the Crystals. Purified des-Gla fXaâ was purchased from Enzyme
Research Laboratories (South Bend, IN). The protein was further
purified on a weak cation-exchange resin (carboxymethyl (CM)
sepharose, Amersham Biosciences, Uppsala, Sweden) equilibrated
in a solution containing 25 mM sodium acetate (pH 5.0) from which
protein was eluted with a NaCl gradient. Protein was then buffer
exchanged into a solution containing 5 mM Mesa-NaOH (pH 6.0),
5 mM CaCl2, and up to 1 µM crystallization tool compound
RPR12851535 prior to concentration to approximately 5 mg/mL for
crystallization experiments in a Vivaspin ultracentrifugation unit
(Vivascience, Hanover, Germany). Crystallizaton was carried out
using the hanging drop vapor diffusion method in 2 µl drops
containing a 1:1 mixture of protein and well solution. Well solution
contained 16-20% PEGa 6 K, 50 mM Mesa-NaOH (pH 5.7-6.0),
5 mM CaCl2, and 50 mM NaCl. Streak seeding followed by
macroseeding, using the derived small crystals, routinely gave
needle-shaped crystals (size 200 × 50 × 50 µm) suitable for
synchrotron X-ray data collection.
The concentration of test compound, which yield 1.5-fold
extension in PT in the human plasma, was estimated using a
mathematical model.41 The extrapolated 1.5× concentration was
validated by spiking the plasma with this exact concentration of
test compound, and the resulting PT was compared to that of control
plasma. The 1.5× concentration was considered accurate if they
caused the plasma to clot 1.5-fold slower than control plasma.
Pharmacokinetic Studies. The formulation used for both i.v.
and p.o. dosing to rat and dog was a 5:95% (v/v) mixture of DMSO
and 50:50 PEGa-200/sterile water. For all animal studies, serial
blood samples were collected into heparinised containers at various
time-points and blood centrifuged to yield plasma.
X-ray Data Collection and Processing. Compound was pre-
pared as a 100 mM stock in DMSO for soaking experiments.
Crystals were soaked in 20 µl well solution containing 1-5 mM
compound and 5-10% DMSO for up to 1 week. Prior to freezing
at 100 K, the crystals were briefly transferred to well solution with
20% glycerol included. The X-ray intensity data was collected at
either the Synchrotron Radiation Source (Daresbury, UK) on station
9.6 or the European Synchrotron Radiation Facility (Grenoble,
France) on beamlines ID29 or ID14 eh4. Data processing and
scaling was carried out using DENZO and SCALEPACK,36 except
for 1x, which was processed using MOSFLM.37 All crystals have
P212121 symmetry and similar cell dimension, a ≈ 56.8 Å, b ≈
72.3 Å, and c ≈ 79.5 Å.
Acknowledgment. The authors would like to thank the
Physicochemical Characterisation group of Analytical Chem-
istry, GSK Stevenage, for carrying out human serum albumin
binding and log D measurements for compounds highlighted
in Table 2 and Mark E. Burgert (Principal Statistician at CVU
CEDD, Upper Merion) for the design of a mathematical model
for the analysis of the effect of concentrations of test compounds
on the coagulation parameters.
Structure Solution and Crystallographic Refinement. The
initial structure was solved by molecular replacement using the
coordinates of 1ezq,35 a fXa complex structure from the RCSB
Protein Data Bank with ligand removed as the search model.
Subsequent structures were solved using difference Fouriers. Model
building, including ligand fitting, was carried out in QUANTA2000
Supporting Information Available: Electrostatic potential
maps of selected N-methyl P1 sulfonamide and additional experi-

1556 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Chan et al.
[(1R)-2-[4-(1-methyl-4-piperidinyl)-1-piperazinyl]-2-oxo-1-phenyl-
ethyl]-1H-Indole-6-carboxamide, CAS number 313489-71-3, cur-
rently in Phase II): (c) LY-517717: A Novel Prophylactic for VTE
following Major Orthopedic Surgery. Prous; 02 Jan 2006. (d) Saiah,
E.; Soares, C. Small Molecule Coagulation Cascade Inhibitors in the
Clinic. Curr. Top. Med. Chem. 2005, 5 (16), 1677-1695. DU-176b
(CAS number 912273-65-5, no structure available): (e) Daiichi
Sankyo (JP): Phase II Clinical Trial. Pharmaprojects; 03 Apr 2006.
(f) Cardiovasculars Take Centre Stage in Daiichi-Sankyo’s R&D Plan.
Scrip; 30 Mar 2006. Apixaban: (g) Apixaban Enters Phase II/III
Trials as a Preventive Therapy for Venous and Pulmonary Throm-
bosis. Prous; 16 Sep 2005. DX-9065a ([S-(R*,R*)]-7-(Aminoimi-
nomethyl)-R-[4-[[1-(1-iminoethyl)-3-pyrrolidinyl]oxy]phenyl]-2-naph-
thalenepropanoic Acid, Monohydrochloride, Pentahydrate, CAS
number 155204-81-2, currently in Phase II): (h) Kaiser, B. DX-
9065a Daiichi. Curr. Opin. InVest. Drugs 2003, 4 (9), 1105-1112.
(i) Daiichi Sankyo (JP): Phase II Clinical Trial. Pharmaprojects;
Monday, 24 Jul 2006.
mental data are available. This material is available free of charge
via the Internet at http://pubs.acs.org.
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(42) These molecular graphics were generated using DeLano’s PyMOL
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JM060870C