The discovery of glycine and related amino acid-based factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.02.040

Herein, we report on the identification of three potent glycine and related amino acid-based series of FXa inhibitors containing a neutral P1 chlorophenyl pharmacophore. A X-ray crystal structure has shown that constrained glycine derivatives with optimiz

Full text of DOI:10.1016/j.bmc.2006.02.040

Bioorganic & Medicinal Chemistry 14 (2006) 4379–4392
The discovery of glycine and related amino acid-based
factor Xa inhibitors
Jeffrey T. Kohrt,a,* Kevin J. Filipski,a Wayne L. Cody,a Christopher F. Bigge,a
Frances La,a Kathleen Welch,a Tawny Dahring,a John W. Bryant,a Daniele Leonard,a
Gary Bolton,a Lakshmi Narasimhan,a Erli Zhang,a J. Thomas Peterson,a Staci Haarer,a
Vaishali Sahasrabudhe,a Nancy Janiczek,a Shrilakshmi Desiraju,b Mostofa Hena,b
Charles Fiakpui,b Neerja Saraswat,b Raman Sharma,b Shaoyi Sun,b
Samarendra N. Maiti,b Robert Leadleya and Jeremy J. Edmundsa
aPfizer Global Research and Development, Michigan Labs, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
bNaeja Pharmaceutical Inc., #2 Taiho Alberta Center, 4290-91A Street, Edmonton, Alberta, Canada T6E 5V2
Received 21 January 2006; revised 17 February 2006; accepted 21 February 2006
Available online 10 March 2006
Abstract—Herein, we report on the identification of three potent glycine and related amino acid-based series of FXa inhibitors con-
taining a neutral P1 chlorophenyl pharmacophore. A X-ray crystal structure has shown that constrained glycine derivatives with
optimized N-substitution can greatly increase hydrophobic interactions in the FXa active site. Also, the substitution of a pyridone
ring for a phenylsulfone ring in the P4 sidechain resulted in an inhibitor with enhanced oral bioavailability.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
demonstrated to be a therapeutically safe and effective
target for the reduction of the formation of blood
clots.1,2
It has been well established that there is a need for the
replacement of the long-standing antithrombotic warfa-
rin for the treatment and prophylaxis of primary and
secondary venous and arterial thromboembolisms.1
While serving as an effective therapy for the prevention
of blood clot formation, warfarin’s nonspecific activity,
delay in time to reach efficacy, narrow therapeutic win-
dow, and the necessity for constant monitoring leave
considerable room for the discovery of new oral
antithrombotics.1
The inhibition of FXa by small molecules has been
aggressively pursued by the pharmaceutical industry.1a,3
Initial successes have led to the clinical development of
low molecular weight heparins1b and fondaparinux4a,b
both of which are indirect inhibitors of FXa. In addi-
tion, a direct FXa inhibitor, DX-9065a, has also been
advanced to clinical trials.4c While these drugs demon-
strated an improvement in antithrombotic development,
their subcutaneous route of administration may limit
their use in chronic thrombosis disease management.4b
Thus, a continued search for new orally active FXa
inhibitors persists.
Orally bioavailable factor Xa (FXa) inhibitors are cur-
rently being evaluated preclinically and clinically be-
cause they have the potential to address all the
limitations noted above. This key serine protease is at
the central junction of both the intrinsic and extrinsic
coagulation pathways involved in the formation of
blood clots, and the inhibition of FXa has been clinically
Early efforts to develop oral FXa inhibitors were hin-
dered by the need to synthesize molecules that contained
highly charged, strongly basic pharmacophores like phe-
nylguanidine and phenylamidine moieties.5 These basic
pharmacophores anchored the inhibitor in the key S1
subsite of FXa through hydrogen bonding with the car-
boxylic acid of Asp189.1a,6 While these highly basic
compounds were often very potent inhibitors of FXa,
Keywords: Factor Xa inhibitor; Antithrombotics.
*
Corresponding author. Tel.: +1 734 622 1371; fax: +1 734 622
3909; e-mail: jeffrey.kohrt@pfizer.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.040

4380
J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
they usually possessed undesirable pharmacokinetic
properties such as poor oral absorption and short dura-
tion of action.7
In order to improve the pharmacokinetic properties of
these highly basic FXa inhibitors, efforts have focused
on the discovery of compounds with less-basic P1
phenylamidine mimetics such as an aminobenzisoxazole
exemplified in DuPont’s Razaxaban8f or an indole used
in Lilly’s LY-517717.8g The other alternative to improve
pharmacological properties is the use of neutral P1 side-
chains.8–11
Figure 1. Inhibitor rational for the discovery of non-chiral FXa
inhibitors.
A key step in the development of neutral P1 groups was
a patent in 1996 by Zeneca disclosing piperazine naph-
thylsulfonamide compounds containing chloronaphtha-
lene and piperidinylpyridine sidechains.8a While not
specifically stated in the patent, the chloronaphthene
sidechain was assumed in reviews to be binding in the
S4 subsite.6b,8b Subsequently it was reported that molec-
ular modeling studies on this and other related FXa
inhibitors suggested a preferred ‘reversed’ binding mode
in which the chloronaphthylene and other chlorohetero-
cylic sidechains were occupying the S1 pocket with the
basic sidechains binding in the S4 pocket.8c Both Du-
Pont and Lilly added to this body of knowledge with
disclosures of the replacement of a P1 3-amidinophenyl
group with 4-methoxyphenyl and other neutral P1
groups in FXa inhibitors.8d,e Crystal structures of these
inhibitors were not published at this time confirming this
stated binding mode with the enzyme.
2. Chemistry
The synthesis of glycine-based inhibitors related to
N-alkylglycine, cycloalkylglycine, and N-alkylcycloalkyl-
glycine is exemplified by the three examples below
(Schemes 1–3). The synthesis of the cyclopropylglycine
analog 4b was initiated from N-Boc-cyclopropylglycine
carboxylic acid 1, 3-fluoro-20-methylsulfanyl-biphenyl-4-
ylamine 2, and 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihydro-
quinoline (EEDQ)12 to prepare an amide intermediate.
The biphenylmethylsulfide group was then oxidized with
meta-chloroperbenzoic acid (m-CPBA) to provide the
methylsulfone 3. Deprotection of the Boc-group with
trifluoroacetic acid in dichloromethane and then the
addition of 4-chlorophenylisocyanate in the presence
of triethylamine afforded final compound 4b.
Recently, a crystal structure from the Aventis group
showed conclusively that a phenylamidine FXa inhibitor
did not necessarily need this basic group for binding in
the S1 pocket of the enzyme.9 In a series of piperazi-
none-based inhibitors, the phenylamidine portion of
the molecules was shown to bind in the S4 pocket with
a neutral chlorobenzothiophene group occupying the
S1 pocket.9a A subsequent report disclosed an example
of a crystal structure of FXa complexed with a piperaz-
inone inhibitor containing a neutral chlorothiophene
group bound in the S1 pocket and a weakly basic azain-
dole group filling the S4 pocket.9b These results opened
the door to the identification and design of new tem-
plates utilizing neutral P1 sidechains as phenylamidine
replacements in the discovery of FXa inhibitors. The
results of these efforts have been highlighted by recent
reports of the use of the orally active FXa inhibitor
Bay-59-7939 with a chlorothiophene S1 sidechain in
clinical trials.10
Synthesis of the N-(methoxyethyl)glycine analog 8i
proceeded from bromoacetyl bromide that was allowed
to react with aniline 5 to form amide 6. The bromo
group was then displaced by methoxyethylamine to
form 7. The urea derivative was made via the addition
of 4-chlorophenylisocyanate as before to produce the
derivative 8i.
Finally, the synthesis of N-(cyclopropylmethyl)cyclo-
propylglycine analog 11d started from the aminocyclo-
propylglycine amide 9. Reductive amination utilizing
cyclopropylaldehyde, titanium isopropoxide, and sodi-
um cyanoborohydride provided the substituted amine
10.13 Addition of the 4-chlorophenylisocyanate as
before produced the urea derivative 11d.
3. Results and discussion
The design hypothesis for our initial series of FXa inhib-
itors focused on the use of non-chiral, glycine-based
templates such as the a-disubstituted 1-amino-1-cyclo-
propanecarboxylic acid in combination with neutral P1
and P4 pharmacophores. The S4 binding pocket of
FXa contains three aromatic amino acid sidechains
(Phe178, Trp215, and Tyr99) that have been shown to
form favorable interactions with either neutral or posi-
tively charged aromatic groups.6,9 An amino-biphenyl-
sulfonamide was chosen as the initial P4 sidechain
due to reports of its use in other FXa inhibitors.6
As part of a structure-based drug design program to dis-
cover orally active FXa inhibitors, we investigated a
proline-based series of inhibitors that contained multiple
chiral stereocenters.11d Syntheses of these chiral amino
acid templates limited rapid analog development and
resulted in inhibitors of high molecular weight. These is-
sues led us to investigate lower molecular weight non-
chiral amino acids as the core for new inhibitors
(Fig. 1). Herein, we report on the discovery of three ser-
ies of glycine-based FXa inhibitors, in which we have
incorporated a neutral chlorophenyl P1 sidechain.

J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
4381
Scheme 1. Synthesis of cycloalkylglycine analog 4b. Reagents and conditions: (a) 2, EEDQ, triethylamine, CHCl3, reflux, 20 h; (b) m-CPBA, ethyl
acetate, rt, 2.5 h; (c) trifluoroacetic acid, CH2Cl2, rt, 1.5 h; (d) 4-chlorophenylisocyanate, Et3N, CH2Cl2, rt, 2 h.
Scheme 2. Synthesis of N-alkylglycine analog 8i. Reagents and conditions: (a) bromoacetylbromide, CH2Cl2, rt, 15 h; (b) methoxyethylamine,
(i-Pr)2EtNH, CH2Cl2, rt, 2 h; (c) 4-chloro-phenylisocyanate, Et3N, CH2Cl2, rt, 2 h.
Scheme 3. Synthesis of N-alkylcycloalkylglycine analog 11d. Reagents and conditions: (a) cyclopropanecarbaldehyde, Ti(Oi-Pr)4, CH2Cl2,
NaBH3CN, methanol, rt, 20 h; (b) 4-chlorophenylisocyanate, Et3N, CH2Cl2, rt, 2 h.
The 4-chloroaniline group was incorporated as a neutral
P1 sidechain. From molecular modeling studies on a
related series of FXa inhibitors, the 4-chloroaniline sub-
stituent tethered to the amino acid core via a urea link-
age was suggested as an optimal P1 binding element for
the active site of FXa.11d

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J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
Gratifyingly, from the combination of the above phar-
macophores, the first cyclopropylglycine inhibitor 4a
(IC50 = 0.033 lM) provided a good lead to investigate
structure–activity relationship studies for this series of
inhibitors (Table 1). In an examination of the P4 side-
chain, addition of a fluoro group to the aniline ring of
Replacement of the cyclic glycine core with a dialkyl
amino acid such as 2-amino-2-methyl-propionic acid
provided inhibitors 4j and 4k with activities similar to
those of the corresponding cyclic compounds 4b and
4c, respectively (Table 1). In addition, 2-amino-3-
hydroxy-2-hydroxymethyl-propionic acid was used to
synthesize inhibitor 4l that was shown to possess less
than a 2-fold change in potency (IC50 = 0.029 lM) from
compound 4j.
4a provided the more potent inhibitor 4b (IC50
=
0.022 lM). Replacement of the phenylmethylsulfone of
4b with a phenylsulfonamide resulted in the preparation
of 4c (IC50 = 0.036 lM) displaying potency similar to
that of the initial lead 4a. As the chlorophenyl sidechain
was considered an optimal for this template, only its
replacement with 4-chloroaminopyridine14 was exam-
ined. This weakly basic sidechain could potentially alter
the physicochemical and pharmacological properties of
this series. Unfortunately, this pyridine replacement
led to inhibitor 4d (IC50 = 0.255 lM) with greatly dimin-
ished potency from 4c.
Interestingly, incorporating glycine as the core amino
acid resulted in analog 8a (IC50 = 0.053 lM) with only
a 2-fold decrease in potency with respect to the related
cyclopropyl analog 4c (Table 2). This small loss of activ-
ity encouraged us to investigate whether glycine N-sub-
stitution could be used to increase the potency of this
glycine series. Molecular modeling suggested that
N-substitution could provide inhibitors with increased
binding by optimizing interactions in a disulfide contain-
ing, hydrophobic pocket in the FXa active site (Fig. 2).
Previous reports have shown that chiral amino acids
such as phenylglycine can access this binding pocket.15
Following this idea, a more potent analog was realized
with respect to 8a with the incorporation of sarcosine
The replacement of the cyclopropyl ring with larger
rings into the glycine core led to changes in enzyme inhi-
bition (Table 1). For example, the cyclohexyl analog 4e
(IC50 = 0.016 lM) was the most potent inhibitor in this
series. Incorporation of a sulfur or oxygen into the
cyclohexyl ring for analogs 4f and 4g, respectively, led
to 4- and 15-fold decreases in potency with respect to
analog 4e. Replacing the cyclohexyl ring for either the
cyclopentyl or cyclopentenyl rings in derivatives 4h or
4i, respectively, also led to inhibitors with decreased
potency.
Table 2. N-Alkylglycine inhibitor binding data
Cl
O
F
NH
N
N
SO2CH3
R2 R1
O
Table 1. Cycloalkylglycine and dialkylglycine binding data
Compound R1
R2
IC50 (lM)
SEM
Cl
O
A
R1
H
8a
8b
8c
8d
8e
8f
H
H
H
0.053
0.020
0.0030
0.0020
B
N
SO2R2
N
H
N
CH3
H
H
O
CH3 >1.0
Et
Pr
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.021
0.010
0.005
0.110
0.380
0.027
>1.0
0.0020
0.002a
0.0001
0.0050
0.0350
0.0200
Compound R1
R2
A
B
IC50 (lM)
SEM
0.007
Bu
i-Bu
8g
8h
8i
4a
CH3 CH CH 0.033
CH3 CH CF 0.022
NH2 CH CF 0.036
CH2-t-Bu
CH2CH2OCH3
4b
4c
4d
0.001
0.002
0.021
8j
CH2CH2N(CH3)2
CH2–c-Pr
c-Pr
8k
8l
0.004
0.490
0.013
0.017
0.120
0.140
0.003
0.008
0.014
0.027
0.030
0.021
0.023
0.026
0.040
0.064
0.080
0.120
0.00001
0.0370
0.0010
0.0005
0.0050
0.0280
0.0005
0.0004
0.0010
0.0030
0.0010
0.0010
0.0010
0.0030
0.003a
0.0030
0.0070
0.0040
CH3
N
CF 0.26
8m
8n
8o
8p
8q
8r
CH2–c-Bu
CH2–c-Pent
CH2–c-Hx
4e
CH3 CH CH 0.016
NH2 CH CF 0.065
NH2 CH CF 0.25
0.001
0.004
0.022
CH2-3-THP
CH2-2-furyl
CH2-2-thienyl
CH2-2-oxazolyl
CH2-3-thienyl
CH2Ph
S
4f
8s
8t
O
4g
8u
8v
8w
8x
8y
8z
8aa
8bb
3-CH3O–PhCH2
3-F–PhCH2
3-CH3PhCH2
2-CH3O–PhCH2
2-Cl–PhCH2
4-CH3O–PhCH2
4-Cl–PhCH2
4h
4i
CH3 CH CH 0.038
NH2 CH CF 0.066
0.005
0.004
4j
(CH3)2 CH3 CH CF 0.02
(CH3)2 NH2 CH CF 0.044
(CH2OH)2 NH2 CH CH 0.029
0.003
0.001
0.002
c-Pr, cyclopropyl; c-Bu, cyclobutyl; c-Pent, cyclopentyl; c-Hx, cyclo-
hexyl; THP, tetrahydropyranyl.
a Standard deviation for n = 2.
4k
4l

J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
Table 3. N-Alkylglycine binding data
4383
Cl
O
F
H
N
N
N
R1
H
O
R2
FXa IC50 (lM) SEM
Compound R1
R2
O
12
13
CH2CH2OCH3
0.225
0.005
0.01
N
N
O
Figure 2. Proposed N-alkyl interactions in the hydrophobic pocket.
CH2–c-Pr
0.001
in analog 8b (IC50 = 0.020 lM). Not unexpectedly, sub-
stitution of a N-methyl on the aniline portion of the urea
of inhibitor 8c (IC50 > 1 lM) greatly diminished potency
as a result of the loss of a critical hydrogen bond inter-
action with the protein. Based on these results, further
studies were undertaken to increase the potency of the
N-substituted glycine derivatives.
ments (Table 3). The piperidinone analog 12 (IC50
=
0.225 lM) reduced potency from its parent 4i by nearly
10-fold, but interestingly the pyridone analog 13
(IC50 = 0.005 lM) was essentially equipotent to the
P4 phenylsulfone 8k. It is known that sidechains that
are better able to p-p stack with the aromatic amino
acids in the S4 pocket of the enzyme can lead to more
potent inhibitors.6,9 Due to these interactions, the large
increase in potency of the pyridone ring is speculated
to be due to the increased aromatic nature of this side-
chain over that of the non-aromatic piperidone ring.
Thus, the N-methyl was replaced by larger alkyl groups
in the inhibitors 8d–8f. It was shown that the N-butyl
analog 8f (IC50 = 0.005 lM) was the most potent N-al-
kyl inhibitor. Increasing the alkyl branching or incorpo-
ration of a heteroatom into the sidechain resulted in
analogs 8g–8j with significantly reduced potency. Incor-
poration of N-(cycloalkyl)methyl sidechains led to the
preparation of analog 8k (IC50 = 0.004 lM) as the most
potent cycloalkyl derivative. Inhibitors with larger
cycloalkyl rings such as cyclobutyl, cyclopentyl, and
cyclohexyl analogs 8m–8o showed a decrease in potency
from compound 8k.
While modest increases in potency were obtained by
N-substitution of the glycine core, it was realized that
many of the inhibitors while possessing different N-sub-
stitutions had very similar potencies. It was hypothe-
sized that interactions in the disulfide containing
hydrophobic pocket may not be responsible for all of
the observed changes in potency. Modeling suggested
that rotation of the inhibitor’s glycine core could move
the N-substituent out of the hydrophobic pocket and
into the solvent, thus providing for only a small range
of activities for differently N-substituted analogs. This
may help explain why N-benzylic derivatives such as
8v can have the same potency as N-methyl analog 8b.
In order to reduce the potential for rotation and possi-
bly orient the N-substituent into the disulfide pocket, a
new series of inhibitors with a constrained core based
on cyclopropylglycine19 was prepared (Table 4).
A series of N-benzylic and N-heteroaromatic methyl-
glycine inhibitors were then prepared as replacements
for the cyclopropylmethyl group of analog 8k. The most
potent heterocyclic derivative was the 2-furyl analog 8q
(IC50 = 0.003 lM). The 2-thienyl analog 8r and 2-oxaz-
olyl analog 8s were less potent than 8q by approximately
3- and 5-fold, respectively. In addition, the 3-thienyl
analog 8t was the least potent heteroaromatic derivative.
In general, the N-heteroaromatic methyl analogs were
more potent than the N-benzylic analogs. The parent
N-benzyl 8u (IC50 = 0.030 lM) was as potent as the least
potent heteroaromatic derivative 8t. No significant in-
crease in potency was observed with the preparation of
the 3-substituted phenyl analogs 8v–8x. Substitution at
the 2-position of the phenyl ring of analogs 8y and 8z re-
duced potency with respect to 8u, while substitution at
the 4-position of the phenyl ring led to the weakest bind-
ing benzylic analogs 8aa and 8bb.
Table 4. N-Alkylcyclopropylglycine binding data
Cl
O
F
H
N
N
N
R1
SO2CH3
H
O
Compound
R1
Bu
FXa IC50 (lM)
SEM
In order to reduce the overall molecular weight of this
series and potentially increase their absorption and oral
bioavailability,16 various heterocycles and amides17
were used as replacements for the P4 phenylsulfone
sidechain. From molecular modeling studies, it was
hypothesized that N-aryl-substituted piperidones and
pyridones18 would make good phenylsulfone replace-
11a
11b
11c
11d
11e
11f
0.0008
0.017
0.039
0.068
>1
0.0001
0.001
0.005
0.002
CH3
Pr
CH2–c-Pr
CH2–c-Hex
CH2–c-Pent
>1

4384
J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
In most cases, the use of this constrained amino acid
resulted in inhibitors 11b–11f that were equal to or less
potent than the corresponding glycine analogs from Ta-
ble 2. This proposed constraint of rotation19 appeared
to lead to N-substituted inhibitors with reduced or dif-
ferent interactions within the disulfide hydrophobic
pocket and/or increased interactions with solvent. One
exception to this observation was the almost 7-fold in-
crease in potency observed for analog 11a
(IC50 = 0.8 nM) as compared to its glycine homolog 8f.
This was the first glycine-based inhibitor that displayed
subnanomolar potency.
position the N-butyl sidechain in the hydrophobic pock-
et and not allow rotation into solvent. These non-bond-
ing interactions in the hydrophobic disulfide pocket and
a reduction in entropy due to a rigidified template may
account for the increased potency of compound 11a
over the N-butylglycine analog 8f.
3.1. Anticoagulant activity, selectivity, and
pharmacokinetics
Twelve compounds from the three series of inhibitors
were further evaluated for selectivity against serine pro-
teases related to FXa. All compounds tested were highly
selective for FXa. In studies utilizing human trypsin,
none of the compounds had affinity less than 10 lM.
While, in a human thrombin (FIIa) assay, only two
compounds 4l (IC50 = 5.7 lM) and 13 (IC50 = 7.7 lM)
had potency better than 10 lM (Table 5).
Since analog 11a was our most potent inhibitor, an
X-ray crystal structure was obtained of it bound in the
FXa active site (Fig. 3). As expected the 4-chlorophenyl
sidechain was positioned in the S1 pocket of the enzyme,
while the phenylsulfone sidechain was bound into the
aromatic S4 pocket. Strong hydrogen bonds were ob-
served between both the carbonyl of Gly219 and the
Next, the ex vivo anticoagulant activity of key com-
pounds was evaluated via prolongation of fibrin clot for-
mation in human plasma as measured by the doubling
of prothrombin time (2· PT). Evaluation of the inhibi-
tors in the 2· PT assay was disappointing. In most cases,
the concentrations necessary to achieve the doubling
time for fibrin formation (Table 5) were greater than
2 lM, including compound 11a, the most potent in vitro
˚
4-chlorophenylurea NH (3.3 A) and between the car-
bonyl of Gly216 and the cyclopropylglycine amide NH
˚
(3.2 A). Also, the carbonyl of the cyclopropylglycine
formed a hydrogen bond with a water molecule
˚
(2.8 A) that interacts with the sidechains of Ser195
˚
˚
(3.2 A), His57 (3.1 A) and the carbonyl of Ser214
˚
(2.7 A). The constrained amino acid core did help to
Figure 3. X-ray crystal structure of 11a in the active site of FXa.
Table 5. Selectivity, activity, and pharmacokinetic data
FXa % inhibitiona
Aucd
b
Cmax (nM)
c
Tmax (h)
e
App T1/2 (h)
Compound
IC50 (lM)
FIIa IC50 (lM)
2· PT (lM)
Trypsin
4b
4e
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
14.0
ND
13.0
4.5
1.0
0.0
43.0
0.0
1
—
—
—
—
—
—
—
—
—
—
1.5
1.6
—
1.6
—
4
4j
0.0
2.0
130.0
—
—
4l
5.7
—
—
0.5
0.5
4
8f
>10
>10
>10
>10
7.0
7.6
10.0
20.0
30.0
0.0
8k
8q
11a
13
250.0
230.0
13.0
440.0
590
670
9.1
28.0
1.7
—
13,000
7.7
90.0
0.5
a FXa % inhibition, maximum percent inhibition of FXa over 4 or 6 h study.
b Cmax, maximum plasma concentration of inhibitor.
c Tmax, time measured for maximum concentration of inhibitor in plasma.
d Auc, area under the curve—plasma exposure of inhibitor nM/h.
e App T1/2: apparent half-life; ND, not determined.

J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
4385
binding analog. The only compound that demonstrated
a lower 2· PT concentration was the pyridone inhibitor
13.
spectrometry (MS) using atmospheric pressure chemical
ionization (APCI) or electron scatter (ES) ionization
sources. All final compounds were determined to be con-
1
sistent with the proposed structure by H NMR, MS.
Select FXa inhibitors were tested in a conscious rat
pharmacokinetic-pharmacodynamic (PK–PD) model
for an initial evaluation of anti-FXa activity, plasma
exposure, and apparent half-life. Test compounds were
administered intragastrically by oral gavage at a dose
of 5 mg/kg. Plasma samples were taken at set intervals
up to 4 or 6 h post-dose. FXa inhibition and plasma
concentrations were determined by using an ex vivo
FXa chromogenic assay.20 Pharmacokinetic parameters
of plasma exposure and apparent half-life were deter-
mined from a plot of plasma concentrations versus time.
Only one class of compounds, the N-substituted glycine
inhibitors, displayed anti-FXa activity in this assay.
Compounds 8k and 8q were the first compounds to dis-
play modest anti-FXa potency, albeit with low plasma
exposures (Table 5). It was not until the phenylsulfone
group of 8f was replaced with the pyridone group of
13 that an inhibitor displayed good anti-FXa activity
and high plasma exposure indicating increase in absorp-
tion and oral bioavailability. In all cases, the absorbed
analogs displayed a short apparent half-life.
All final compounds also were greater than 95% pure
as determined by analytical RP-HPLC on a Vy-
dac218TP54 C18 column utilizing a gradient of 0.1% tri-
fluoroacetic acid (TFA) in acetonitrile to 0.1% TFA in
water (22–80% over 22 min—method A) or by a Altech
Altima C18 column replacing TFA with 0.1% formic
acid in both water and acetonitrile (22–80% over
10 min—method B) and were within theoretical limits
for elemental analyses (C, H, N) or high resolution mass
spectrometry (HRMS).
5.1.1. 1-[3-(4-Chloro-phenyl)-ureido]-cyclopropanecarb-
oxylic acid (3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-
amide (4b). Step 1. 1-tert-Butoxycarbonylamino-cyclo-
propanecarboxylic acid 1 (1.00 g, 4.97 mmol), 3-fluoro-
20-methylsulfanyl-biphenyl-4-ylamine
2
(1.15 g,
4.97 mmol), and 2-ethoxy-1-(ethoxycarbonyl)-1,2-dihy-
droquinoline (EEDQ) (1.47 g, 5.96 mmol) were dissolved
in dry chloroform (20 mL). Triethylamine (1.04 mL,
7.45 mmol) was added and the solution was heated at re-
flux for 20 h. The reaction was allowed to cool, and an ex-
cess of ethyl acetate was added. The solution was washed
sequentially with 10% aqueous citric acid, 1 N NaOH,
water, and then brine before drying the solution over
MgSO4. Concentration of the solution under reduced
pressure and purification of the crude product by flash
chromatography revealed amide as a white solid (2.10 g,
Differences in physicochemical properties may play a
role in the substantially increased absorption of com-
pound 13 over the related analog 8f. In direct compari-
son to phenylsulfone analog 8f, the pyridone compound
13 showed decreases in logP (3.8 and 2.8, respectively),
polar surface area (91 and 78, respectively), number of
rotatable bonds (11 and 10, respectively), and molecular
weight (530 and 469 g/mol, respectively). Each of these
properties has been shown to be key factors that influ-
ence absorption and oral bioavailability.16,21
1
99%). H NMR (400 MHz, DMSO-d6): d 9.08 (s, 1H),
8.00 (m, 1H), 7.91 (s, 1H), 7.74 (m, 1H), 7.38–7.12 (m,
5H), 2.35 (s, 3H), 1.39 (s, 9H), 1.34 (m, 2H), 1.00 (m,
2H); MS APCI (AP+): 417.1 (M+1)+.
Step 2. To a mixture of the amide (1.80 g, 4.32 mmol) in
ethyl acetate (43 mL) was added m-CPBA (70%, 4.26 g,
17.3 mmol). The solution was stirred for 2.5 h at room
temperature before diluting with excess ethyl acetate.
The solution was washed sequentially with saturated
aqueous Na2S2O3, two portions of saturated aqueous
NaHCO3, water and then brine before drying over
MgSO4. Filtration followed by concentration of the solu-
tion under reduced pressure and purification of the crude
product by flash chromatography revealed 3 as a white
4. Conclusions
In conclusion, we have identified three potent glycine-
based series of FXa inhibitors containing a neutral P1
sidechain. An X-ray crystal structure has shown that rig-
id glycine derivatives with optimized N-substitution can
greatly increase inhibitor potency. Also, the substitution
of a P4 pyridone ring for a phenylsulfone has led to an
inhibitor with enhanced oral bioavailability. Continued
optimization of the physicochemical and pharmacologi-
cal properties of these inhibitors may provide improved
oral FXa anticoagulants.
1
solid (1.72 g, 89%). H NMR (400 MHz, DMSO-d6): d
9.38, 9.10 (2s, 1H), 8.56 (s, 1H), 8.06 (m, 1H), 7.77–7.64
(m, 2H), 7.43 (m, 1), 7.38 (m, 1H), 7.22 (ddd, 1H, J = 2,
9, 9 Hz) 2.89 (s, 3H), 1.67 (m, 2H), 1.50 (s, 9H), 1.39 (m,
2H); MS APCI (AP+): 449.1 (M+1)+.
5. Experimental
5.1. General
Step 3. Into a solution of 3 (0.230 g, 0.515 mmol) in dry
dichloromethane (5 mL) was added trifluoroacetic acid
(TFA) (3 mL), and the solution was stirred at room tem-
perature for 1.5 h. The solution was then concentrated
under reduced pressure and dried under vacuum. The
crude product was dissolved in dry THF (10 mL) and
cooled to 0 ꢁC in an ice bath. Triethylamine
(0.359 mL, 2.58 mmol) was then added followed by 4-
chlorophenyl-isocyanate (0.079 g, 0.515 mmol). The
reaction mixture was stirred at room temperature for
2 h before concentrating under reduced pressure. The
All chemicals, reagents, and solvents were purchased
from commercial sources (e.g., Aldrich Chemical Co.,
Inc., Milwaukee, WI; Mallinckrodt Baker, Inc., Paris,
KY, etc.) where available and used without further puri-
fication. Compound 4l was prepared as outlined in Ref.
22. All intermediates were characterized by proton
nuclear magnetic spectroscopy (1H NMR) and mass

4386
J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
resulting crude product was purified by flash chroma-
tography to reveal 4b as a white solid (0.162 g, 63%).
1H NMR (400 MHz, DMSO-d6): d 9.48 (s, 1H), 8.78
(s, 1H), 8.05 (dd, 1H, J = 1, 8 Hz), 7.83 (t, 1H,
J = 8 Hz), 7.74 (dt, 1H, J = 1, 8 Hz), 7.65 (dt, 1H,
J = 1, 8 Hz), 7.45–7.38 (m, 3H), 7.31–7.24 (m, 3H),
7.17 (dd, 1H, J = 1, 8 Hz), 7.07 (s, 1H), 2.84 (s, 3H),
1.41 (m, 2H), 1.04 (m, 2H); MS APCI (AP+) 502.1
(M+1)+; CHN calcd for C24H21ClFN3O4S: C, 57.36;
H, 4.22; N, 8.36. Found: C, 56.97; H, 4.07; N, 8.05.
ing mixture was stirred for 20 h, quenched with water
(2 mL), and ethyl acetate (100 mL) was added. The pre-
cipitated solid was filtered through Celite, the filtrate
was dried over MgSO4, evaporated, and purified by flash
chromatography (ethyl acetate/hexane, 15:85) to obtain
1
10 as a white solid (0.21 g, 45%). H NMR (400 MHz,
CDCl3): d 10.40 (br s, 1H), 8.55 (t, 1H, J = 8 Hz), 8.20
(t, 1H, J = 8 Hz), 7.70–7.55 (m, 2H), 7.40–7.35 (m, 2H),
7.15 (d, 1H), 2.70 (s, 3H), 2.55–2.45 (m, 2H), 1.50–1.40
(m, 2H), 1.35 (br s, 1H), 1.30–1.20 (m, 1H), 1.00–0.95
(m, 2H), 0.55–0.45 (m, 2H), 0.20–0.10 (m, 2H).
5.1.2.
2-[3-(4-Chloro-phenyl)-1-(2-methoxy-ethyl)-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8i). Step 1. To a solution of 5 (5.0 g, 18.9 mmol)
in dichloromethane (200 mL) at room temperature was
added bromoacetyl bromide (4.20 g, 20.8 mmol) drop-
wise, and the mixture was stirred overnight. It was then
diluted with ethyl acetate (200 mL), washed with 5%
aqueous HCl (30 mL) and then brine. The organics were
concentrated to obtain 6 (7.1 g, 97%). 1H NMR
(400 MHz, CDCl3): d 10.30 (br s, 1H), 8.10 (dd, 1H,
J = 1, 8 Hz), 8.03 (t, 1H, J = 8 Hz), 7.80–7.65 (m, 2H),
7.44 (dd, 1H, J = 1, 8 Hz), 7.37 (dd, 1H, J = 2, 13 Hz),
7.25 (dd, 1H, J = 2, 13 Hz), 4.20 (s, 2H), 2.95 (s, 3H).
Step 2. To a solution of 10 (0.40 g, 1.00 mmol) and tri-
ethylamine (0.33 g, 3.20 mmol) in dichloromethane
(10 mL) was added 4-chlorophenylisocyanate (0.14 g,
0.90 mmol) at room temperature. The mixture was stir-
red for 2 h, quenched with water, and extracted with
ethyl acetate (3· 20 mL). The combined extracts were
washed with 5% aqueous HCl (4 mL), brine, dried over
MgSO4, concentrated under vacuum, and purified by
column chromatography to obtain 11d as a white solid
1
(0.30 g, 54%). H NMR (400 MHz, CDCl3): d 8.95 (br
s, 1H), 8.40 (t, 1H, J = 8 Hz), 8.20 (dd, 1H, J = 1,
8 Hz), 7.70–7.55 (m, 2H), 7.40–7.20 (m, 7H), 7.00 (s,
1H), 3.90 (dd, 1H, J = 5, 8 Hz), 2.90 (dd, 1H, J = 5,
8 Hz), 2.70 (s, 3H), 2.15–2.05 (m, 1H), 1.90–1.80 (m,
1H), 1.55–1.30 (m, 2H), 1.30–1.20 (m, 1H), 0.70–0.55
(m, 2H), 0.45–0.35 (m, 2H); MS (ES) 557.24 (M+1)+;
CHN calcd for C28H27ClFN3O4S: C, 60.48; H, 4.89;
N, 7.56. Found: C, 60.67; H, 5.10; N, 7.38.
Step 2. To a mixture of 6 (1.0 g, 2.6 mmol) and diisopro-
pylethylamine (0.50 g, 3.9 mmol) in dichloromethane
(20 mL) was added 2-methoxy-ethylamine (0.39 g,
5.2 mmol) at room temperature. The mixture was stirred
for 2 h, concentrated under reduced pressure, and puri-
fied by flash chromatography to obtain 7 (0.70 g, 70%).
1H NMR (400 MHz, CDCl3): d 9.85 (br s, 1H), 8.55 (t,
1H, J = 8 Hz), 8.20 (dd, 1H, J = 1, 8 Hz), 7.70–7.50 (m,
2H), 7.40–7.30 (m, 2H), 7.20 (d, 1H, J = 8 Hz), 3.55 (t,
2H, J = 5 Hz), 3.45 (s, 2H), 3.40 (s, 3H), 2.90 (t, 2H,
J = 5 Hz), 2.70 (s, 3H), 1.60 (br s, 1H).
The following compounds were prepared using the gen-
eral procedure for 4b.
5.1.4. 1-[3-(4-Chloro-phenyl)-ureido]-cyclopropanecarb-
oxylic acid (20-methanesulfonyl-biphenyl-4-yl)-amide
(4a). White solid 0.27 g, 80%, 1H NMR (400 MHz,
DMSO-d6): d 9.700 (s, 1H), 8.7 (s, 1H), 8.06 (dd, 1H,
J = 1, 8 Hz), 7.72–7.63 (m, 2H), 7.67 (d, 2H, J = 9 Hz),
7.45 (d, 2H, J = 9 Hz), 7.38 (1H, m), 7.32 (d, 1H,
J = 9 Hz), 7.26 (d, 1H, J = 9 Hz), 6.86 (s, 1H), 2.76 (s,
3H), 1.44–1.43 (m, 1H), 1.02–1.01 (m, 1H); MS APCI
(AP+) 484 (M+1)+; CHN calcd for C24H22ClN3O4S+
0.42H2O: C, 58.64; H, 4.68; N, 8.55. Found: C, 58.25;
H, 4.71; N, 8.34.
Step 3. To a solution of 7 (0.60 g, 1.57 mmol) and trieth-
ylamine (0.31 g, 3.14 mmol) in dichloromethane
(10 mL) was added 4-chlorophenylisocyanate (0.31 g,
1.88 mmol) at room temperature. The mixture was stirred
for 2 h, quenched with an excess of water, and extracted
with ethyl acetate (3· 20 mL). The combined organic ex-
tracts were washed with 5% aqueous HCl (4 mL), brine,
dried over MgSO4, evaporated, and purified by column
chromatography to obtain 8i as a white solid (0.47 g,
57%). 1H NMR (400 MHz, CDCl3): d 9.60 (s, 1H), 8.60
(s, 1H), 8.40 (t, 1H, J = 8 Hz), 8.20 (dd, 1H, J = 1,
8 Hz), 7.70–7.65 (m, 1H), 7.60–7.50 (m, 1H), 7.35–7.28
(m, 6H), 7.20 (d, 1H, J = 8 Hz), 4.15 (s, 2H), 3.75–3.60
(m, 4H), 3.55 (s, 3H), 2.65 (s, 3H); MS (ES): 535.41
(M+1)+; CHN calcd for C25H25ClFN3O5S: C, 56.23; H,
4.72; N, 7.87. Found: C, 56.33; H, 4.75; N, 7.70.
5.1.5. 1-[3-(4-Chloro-phenyl)-ureido]-cyclopropanecarb-
oxylic acid (3-fluoro-20-sulfamoyl-biphenyl-4-yl)-amide
(4c). White solid 0.15 g, 18%, 1H NMR (400 MHz,
DMSO-d6): d 9.41 (s, 1H), 8.78 (s, 1H), 7.99 (dd, 1H,
J = 1, 8 Hz), 7.80 (t, 1H, J = 8 Hz), 7.57 (m, 2H), 7.43
(m, 2H), 7.30 (dd, 1H, J = 2, 8 Hz), 7.24 (m, 5H), 7.13
(dd, 1H, J = 2, 8 Hz), 7.08 (s, 1H), 1.41 (dd, 2H, J = 5,
8), 1.03 (dd, 2H, J = 4, 8); MS APCI (AP+) 503
(M+1)+; CHN calcd for C23H20ClFN4O4S+0.40H2O+
0.45TFA: C, 51.13; H, 3.81; N, 9.98. Found: C, 50.75;
H, 3.58; N, 9.76.
5.1.3.
1-[3-(4-Chloro-phenyl)-1-cyclopropylmethyl-ure-
ido]-cyclopropanecarboxylic acid (3-fluoro-20-metha-
nesulfonyl-biphenyl-4-yl)-amide (11d). Step 1. To a
solution of 9 (0.40 g, 1.15 mmol) in dichloromethane were
added cyclopropanecarbaldehyde (0.08 g, 1.15 mmol)
and titanium isopropoxide (0.41 g, 1.44 mmol) succes-
sively at room temperature. The mixture was stirred for
1 h, diluted with methanol (2 mL), and treated with sodi-
um cyanoborohydride (0.050 g, 0.81 mmol). The result-
5.1.6. 1-[3-(5-Chloro-pyridin-2-yl)-ureido]-cyclopropane-
carboxylic acid (3-fluoro-20-methanesulfonyl-biphenyl-4-
yl)-amide (4d). White solid 0.16 g, 8%, 1H NMR
(400 MHz, DMSO-d6): d 9.57 (s, 1H), 9.32 (s, 1H),
8.23 (dd, 1H, J = 0.5, 3 Hz), 8.13 (s, 1H), 8.05 (dd,

J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
4387
1H, J = 1, 8 Hz), 7.80–7.62 (m, 5H), 7.38 (dd, 1H, J = 1,
8 Hz), 7.28 (dd, 1H, J = 2, 11 Hz), 7.16 (dd, 1H, J = 2,
8 Hz), 2.84 (s, 3H), 1.43 (dd, 2H, J = 5, 8 Hz), 1.06
(dd, 2H, J = 4, 8 Hz); MS APCI (AP+) 503 (M+1)+;
CHN calcd for C23H20ClFN4O4S: C, 54.93; H, 4.01;
N, 11.14. Found: C, 54.68; H, 3.80; N, 10.98.
(app d, 2H), 2.57 (app d, 2H); MS APCI (AP+) 529
(M+1)+; CHN calcd for C25H22ClN4O4S: C, 56.76; H,
4.19; N, 10.59. Found: C, 56.71; H, 4.13; N, 10.21.
5.1.12.
2-[3-(4-Chloro-phenyl)-ureido]-N-(3-fluoro-20-
methanesulfonyl-biphenyl-4-yl)-2-methyl-propionamide
(4j). White solid 0.29 g, 81%, 1H NMR (400 MHz,
DMSO-d6): d 9.39 (s, 1H), 8.77 (s, 1H), 8.03 (dd, 1H,
J = 1, 8 Hz), 7.77 (app t, 1H, J = 8 Hz), 7.71 (ddd 1H,
J = 1, 8, 8 Hz), 7.63 (ddd, 1H, J = 1, 8, 8 Hz), 7.38–
7.34 (m, 3H), 7.28–7.20 (m, 3H), 7.60 (s, 1H), 7.15
(dd, 1H, J = 2, 8 Hz), 2.84 (s, 3H), 1.46 (s, 6H); MS
APCI (AP+) 504.1 (M+1)+; CHN calcd for
C24H23ClFN3O4S: C, 57.20; H, 4.60; N, 8.34. Found:
C, 57.05; H, 4.35; N, 8.17.
5.1.7.
1-[3-(4-Chloro-phenyl)-ureido]-cyclohexanecarb-
oxylic acid (20-methanesulfonyl-biphenyl-4-yl)-amide
(4e). White solid 0.14 g, 56%, 1H NMR (400 MHz,
DMSO-d6): d 9.57 (s, 1H), 8.88 (s, 1H), 8.03 (dd, 1H,
J = 1, 8 Hz), 7.72–7.58 (m, 2H), 7.64 (d, 2H, J = 9 Hz),
7.37 (d, 2H, J = 9 Hz), 7.38 (1H, m), 7.27 (d, 1H,
J = 9 Hz), 7.22 (d, 1H, J = 9 Hz), 6.29 (s, 1H), 2.76 (s,
3H), 2.04–2.01 (m, 2H), 1.75–1.69 (m, 2H), 1.59–1.57
(m, 2H), 1.46–1.43 (m, 2H), 1.23–1.20 (m, 1H); MS
APCI (AP+) 526 (M+1)+; CHN calcd for
C27H28ClN3O4S+0.42H2O: C, 61.65; H, 5.37; N, 7.99.
Found: C, 61.53; H, 5.30; N, 7.90.
5.1.13. 2-[3-(4-Chloro-phenyl)-ureido]-N-(3-fluoro-20-sul-
famoyl-biphenyl-4-yl)-2-methyl-propionamide (4k). White
1
solid 0.17 g, 24%, H NMR (400 MHz, DMSO-d6): d
9.34 (s, 1H), 8.79 (s, 1H), 7.99 (m, 1H), 7.77 (t, 1H,
J = 8 Hz), 7.61–7.53 (m, 2H), 7.38 (m, 2H), 7.31–7.21
(m, 6H), 7.13 (dd, 1H, J = 2, 8 Hz), 6.63 (s, 1H), 1.49
(s, 6H); MS APCI (AP+) 505 (M+1)+; CHN calcd for
C23H22ClFN4O4S+0.15H2O+0.30TFA: C, 52.31; H,
4.20; N, 10.34. Found: C, 52.10; H, 4.09; N, 10.25.
5.1.8. 4-[3-(4-Chloro-phenyl)-ureido]-tetrahydro-thiopy-
ran-4-carboxylic acid (3-fluoro-20-sulfamoyl-biphenyl-4-
yl)-amide (4f). White solid 0.030 g, 61%, 1H NMR
(400 MHz, DMSO-d6): d 9.47 (s, 1H), 8.97 (s, 1H),
8.08 (d, 1H, J = 7 Hz), 7.86 (t, 1H, J = 8 Hz), 7.70–
7.62 (m, 2H), 7.48 (d, 1H, J = 9 Hz), 7.40–7.27 (m,
6H), 7.22 (d, 1H, J = 8 Hz), 6.68 (s, 1H), 2.98–2.84 (m,
2H), 2.65–2.39 (m, 4H), 2.17–2.11 (m, 2H); MS APCI
5.1.14. 2-[3-(4-Chloro-phenyl)-ureido]-3-hydroxy-2-hy-
droxymethyl-N-(20-sulfamoyl-biphenyl-4-yl)-propiona-
mide (4l). See Refs. 11e and 22. White solid 0.14 g, 56%,
1H NMR (400 MHz, DMSO-d6): d 9.60 (s, 1H), 9.17 (s,
1H), 7.98 (dd, 1H, J = 1, 8 Hz), 7.62 (d, 2H, J = 8 Hz),
7.59–7.49 (m, 2H), 7.39 (d, 2H, J = 8 Hz), 7.29–7.20
(m, 3H), 7.16 (s, 2H), 6.40 (s, 1H), 5.13 (t, 2H,
J = 6 Hz) 4.02–3.60 (m, 4H); MS APCI (AP+) 519.5
(M+1)+; HRMS calcd for C23H23ClN4O6SNa adduct:
541.0925. Found: 541.0925. HPLC (method A)
tR = 12.7 min (97.0%). The following compounds were
prepared using the general procedure for 8i.
(AP+)
C25H24ClFN4O4S2: 563.0990. Found: 563.0994; HPLC
563
(M+1)+;
HRMS
calcd
for
(method A) tR = 16.9 min (95.4%).
5.1.9. 4-[3-(4-Chloro-phenyl)-ureido]-tetrahydro-pyran-4-
carboxylic acid (3-fluoro-20-sulfamoyl-biphenyl-4-yl)-am-
ide (4g). White solid 0.20 g, 69%, H NMR (400 MHz,
1
DMSO-d6): d 9.45 (s, 1H), 8.90 (s, 1H), 8.00 (dd, 1H,
J = 1, 8 Hz), 7.81 (t, 1H, J = 8 Hz), 7.62–7.54 (m, 2H),
7.40 (m, 2H), 7.32–7.22 (m, 6H), 7.14 (dd, 1H, J = 2,
8 Hz), 6.75 (s, 1H), 3.76 (m, 2H), 3.58 (t, 2H,
J = 11 Hz), 2.05 (m, 2H), 1.97 (m, 2H); MS APCI
5.1.15.
2-[3-(4-Chloro-phenyl)-ureido]-N-(3-fluoro-20-
methanesulfonyl-biphenyl-4-yl)-acetamide (8a). White
(AP+)
547
(M+1)+;
CHN
calcd
for
1
C25H24ClFN4O5S+0.15H2O+0.10TFA: C, 53.94; H,
4.38; N, 9.99. Found: C, 54.01; H, 4.41; N, 9.78.
solid 0.38 g, 55%; H NMR (400 MHz, DMSO-d6): d
9.95 (s, 1H), 8.98 (s, 1H), 8.05 (dd, 1H, J = 1, 8 Hz),
7.98 (app t, 1H, J = 13 Hz), 7.73 (ddd, 1H, J = 1, 8,
8 Hz), 7.65 (ddd, 1H, J = 1, 8, 8 Hz), 7.42–7.39 (m,
3H), 7.31 (dd, 1H, J = 2, 11 Hz), 7.24 (d, 2H,
J = 13 Hz), 7.17 (dd, 1H, J = 2, 8 Hz), 6.48 (t, 1H,
J = 5 Hz), 4.01 (d, 1H, J = 5 Hz), 2.84 (s, 3H); MS APCI
(AP+) 476.1 (M+1)+; CHN calcd for C22H19ClFN3O4S:
C, 55.52; H, 4.02; N, 8.83. Found: C, 55.44; H, 3.89; N,
8.63.
5.1.10. 1-[3-(4-Chloro-phenyl)-ureido]-cyclopentanecarb-
oxylic acid (20-methanesulfonyl-biphenyl-4-yl)-amide
(4h). White solid 0.12 g, 80%, 1H NMR (400 MHz,
DMSO-d6): d 9.64 (s, 1H), 8.70 (s, 1H), 8.04 (dd, 1H,
J = 1, 8 Hz), 7.72–7.59 (m, 2H), 7.65 (d, 2H, J = 9 Hz),
7.38 (m, 1H), 7.37 (d, 2H, J = 9 Hz), 7.29 (d, 1H,
J = 9 Hz), 7.22 (d, 1H, J = 9 Hz), 6.50 (s, 1H), 2.76 (s,
3H), 2.21–2.14 (m, 2H), 1.87–1.82 (m, 2H), 1.74–1.67
(m, 4H); MS APCI (AP+) 512 (M+1)+; CHN calcd
for C26H26ClN3O4S: C, 60.99; H, 5.12; N, 8.21. Found:
C, 60.94; H, 5.22; N, 7.89.
5.1.16. 2-[3-(4-Chloro-phenyl)-1-methyl-ureido]-N-(3-flu-
oro-20-methanesulfonyl-biphenyl-4-yl)-acetamide
(8b).
1
White solid 0.26 g, 77%; H NMR (400 MHz, CDCl3):
d 8.86 (s, 1H), 8.39 (app t, 1H, J = 8 Hz), 8.21 (dd,
1H, J = 1, 8 Hz), 7.73 (ddd, 1H, J = 1, 8, 8 Hz), 7.56
(ddd, 1H, J = 1, 8, 8 Hz), 7.38–7.25 (m, 6H), 7.18 (br
d, 1H, J = 8 Hz), 6.59 (s, 1H), 4.17 (s, 2H), 3.21 (s,
3H), 2.69 (s, 3H); MS APCI (AP-) 488.1 (Mꢀ1)+;
CHN calcd for C23H21ClFN3O4S+0.03H2O: C, 56.32;
H, 4.33; N, 8.57; H2O, 0.11. Found: C, 56.11; H, 4.27;
N, 8.30; H2O, 0.47.
5.1.11. 1-[3-(4-Chloro-phenyl)-ureido]-cyclopent-3-ene-
carboxylic acid (3-fluoro-20-sulfamoyl-biphenyl-4-yl)-am-
ide (4i). White solid 0.11 g, 43%, H NMR (400 MHz,
1
DMSO-d6): d 9.35 (s, 1H), 8.77 (s, 1H), 7.99 (dd, 1H,
J = 1, 8 Hz), 7.72–7.59 (app t, 1H, J = 8 Hz), 7.61–7.52
(m, 2H), 7.40–7.36 (m, 2H), 7.30–7.21 (m, 4H), 7.14
(dd, 1H, J = 2, 9 Hz), 6.85 (s, 1H), 5.67 (s, 2H), 3.07

4388
J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
5.1.17. 2-[3-(4-Chloro-phenyl)-3-methyl-ureido]-N-(3-flu-
oro-20-methanesulfonyl-biphenyl-4-yl)-acetamide
(8c).
J = 8 Hz), 8.22 (d, 1H, J = 8 Hz), 7.75–7.50 (m, 2H),
7.40–7.24 (m, 6H), 7.19 (d, 1H, J = 8 Hz), 6.91 (s, 1H),
4.20 (s, 2H), 3.35 (s, 2H), 2.73 (s, 3H), 1.10 (s, 9H);
MS (ES) 545.77 (M+H)+; CHN calcd for
C27H29ClFN3O4S: C, 59.40; H, 5.32; N, 7.70. Found:
C, 59.12; H, 5.30; N, 7.61.
The compound 8c was formed via the addition to a
dichloromethane (10 mL) solution of amine (0.20 g,
0.406 mmol), (4-chloro-phenyl)-methyl-carbamoyl chlo-
ride23 (0.083 g, 0.406 mmol), triethylamine (0.226 mL,
3.14 mmol), and DMAP (10 mg). The mixture was stir-
red for 2 h at room temperature, quenched with an ex-
cess of water, and extracted with ethyl acetate. The
organic extract was washed with 5% aqueous HCl
(4 mL), brine, dried over MgSO4, evaporated, and puri-
fied by column chromatography to obtain 8c as a white
solid, which was recrystallized from ethyl acetate/hex-
5.1.22. 2-[3-(4-Chloro-phenyl)-1-cyclopropylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8k). White solid, 0.040 g, 55%; 1H NMR
(400 MHz, CDCl3): d 9.69 (s, 1H), 8.33 (s, 1H), 7.86–
7.81 (m, 2H), 7.54–7.42 (m, 2H), 7.30–7.27 (m, 2H),
7.19–6.94 (m, 6H), 4.11 (s, 2H), 2.64 (s, 3H), 2.26–2.25
(m, 2H), 0.81–0.71 (m, 1H), 0.27–0.22 (m. 2H), 0.04–
0.01 (m, 2H); MS APCI (AP+) 530.0 (M+1)+; HRMS:
calcd for C26H25ClFN3O4S: 530.1272. Found: 530.1310.
1
anes to get white crystals, 0.160 g, 70%; H NMR (400
MHz, CDCl3): d 8.71 (s, 1H), 8.36 (app t, 1H,
J = 8 Hz), 8.21 (dd, 1H, J = 1, 8 Hz), 7.64 (ddd, 1H,
J = 1, 8, 8 Hz), 7.56 (ddd, 1H, J = 1, 8, 8 Hz), 7.43 (m,
2H), 7.35–7.25 (m, 5H), 7.15 (br d, 1H, J = 8 Hz), 4.96
(t, 1H, J = 5 Hz), 4.01 (d, 2H, J = 4H), 3.30 (s, 3H),
2.69 (s, 3H); MS APCI (AP-): 488.1 (Mꢀ1)+; CHN
calcd for C23H21ClFN3O4S: C, 56.28; H, 4.33; N, 8.56.
Found: C, 55.99; H, 4.14; N, 8.31.
5.1.23. 2-[3-(4-Chloro-phenyl)-1-cyclopropyl-ureido]-N-
(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acetamide
(8l). White powder 0.180 g, 75%; H NMR (400 MHz,
1
DMSO-d6): d 9.96 (s, 1H), 8.53 (s, 1H), 8.09 (dd, 1H,
J = 1, 8 Hz), 8.03 (t, 1H, J = 8 Hz), 7.79–7.67 (m, 2H),
7.60 (d, 2H, J = 9 Hz), 7.44–7.31 (m, 2H), 7.30 (d, 2H,
J = 9 Hz), 7.19 (dd, 1H, J = 1, 8 Hz), 4.21 (s, 2H), 2.89
(s, 3H), 2.92–2.84 (m, 1H), 0.97–0.90 (m, 2H), 0.81–
0.74 (m, 2H); MS (ES) 515.78 (M+H)+; CHN calcd
for C25H23ClFN3O4S: C, 58.19; H, 4.49; N, 8.14. Found:
C, 58.07; H, 4.22; N, 7.99.
5.1.18. 2-[3-(4-Chloro-phenyl)-1-ethyl-ureido]-N-(3-fluo-
ro-20-methanesulfonyl-biphenyl-4-yl)-acetamide
(8d).
White solid, 0.210 g, 44%; 1H NMR (400 MHz,
DMSO-d6): d 9.94 (s, 1H), 8.54 (s, 1H), 8.09 (dd, 1H,
J = 1, 8 Hz), 8.06 (t, 1H, J = 8 Hz), 7.79–7.67 (m, 2H),
7.53 (d, 2H, J = 9 Hz), 7.45–7.33 (m, 2H), 7.28 (d, 2H,
J = 9 Hz), 7.20 (dd, 1H, J = 1, 8 Hz), 4.24 (s, 2H), 3.46
(q, 2H, J = 7 Hz), 2.89 (s, 3H), 1.14 (t, 3H, J = 7 Hz);
MS (ES) 501.70 (MꢀH)+; CHN calcd for
C24H23ClFN3O4S: C, 57.20; H, 4.60; N, 8.34. Found:
C, 57.53; H, 4.54; N, 8.14.
5.1.24.
2-[3-(4-Chloro-phenyl)-1-cyclobutylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8m). White solid 0.160 g, 59%; 1H NMR
(400 MHz, CDCl3): d 9.12 (s, 1H), 8.39 (t, 1H,
J = 8 Hz), 8.22 (dd, 1H, J = 1, 8 Hz), 7.68–7.54 (m,
2H), 7.38–7.27 (m, 2H), 7.36 (d, 2H, J = 9 Hz), 7.28
(d, 2H, J = 9 Hz), 7.19 (br d, 1H, J = 8 Hz), 6.65 (s,
1H), 4.15 (s, 2H), 3.47 (d, 2H, J = 7 Hz), 2.78–2.64 (m,
1H), 2.71 (s, 3H), 2.20–2.08 (m, 2H), 2.06–1.76 (m,
4H); MS (ES) 543.8 (M+H)+; CHN calcd for
C27H27ClFN3O4S: C, 59.61; H, 5.00; N, 7.72. Found:
C, 59.60; H 5.03; N, 7.62.
5.1.19. 2-[3-(4-Chloro-phenyl)-1-propyl-ureido]-N-(3-fluo-
ro-20-methanesulfonyl-biphenyl-4-yl)-acetamide
(8e).
1
White solid 0.130 g, 51%; H NMR (400 MHz, CDCl3):
d 9.15 (s, 1H), 8.38 (t, 1H, J = 8 Hz), 8.21 (dd, 1H, J = 1,
8 Hz), 7.67–7.55 (m, 2H), 7.37–7.27 (m, 2H), 7.36 (d,
2H, J = 9 Hz), 7.28 (d, 2H, J = 9 Hz), 7.20 (br d, 1H,
J = 8 Hz), 6.68 (s, 1H), 4.16 (s, 2H), 3.41 (t, 2H,
J = 8 Hz), 2.71 (s, 3H), 1.76 (m, 2H), 1.02 (t, 3H,
J = 7 Hz); MS (ES) 517.75 (M+H)+; CHN calcd for
C25H25ClFN3O4S: C, 56.02; H, 5.08; N, 7.84. Found:
C, 56.57; H, 4.55; N, 7.75.
5.1.25. 2-[3-(4-Chloro-phenyl)-1-cyclopentylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8n). White solid 0.060 g, 44%; 1H NMR
(400 MHz, CDCl3): d 9.20 (br s, 1H), 8.40 (t, 1H,
J = 8 Hz), 8.20 (dd, 1 H, J = 1, 8 Hz), 7.68–7.60 (m,
1H), 7.60–7.52 (m, 1H), 7.38–7.20 (m, 6H), 7.19 (br d,
1H, J = 9 Hz), 6.60 (s, 1H), 4.20 (s, 2H), 3.41 (d, 2H,
J = 5 Hz), 2.75 (s, 3H), 2.32–2.20 (m, 1H), 1.90–1.80
(m, 2H), 1.76–1.52 (m, 4H), 1.35–1.20 (m, 2H); MS
(ES) 557.0 (M)+; CHN calcd for C28H29ClFN3O4S: C,
60.26; H, 5.24; N, 7.53. Found: C, 60.21; H, 5.45; N,
7.08.
5.1.20. 2-[1-Butyl-3-(4-chloro-phenyl)-ureido]-N-(3-fluo-
ro-20-methanesulfonyl-biphenyl-4-yl)-acetamide
(8f).
1
White solid 0.040 g, 55%; H NMR (400 MHz, CDCl3):
d 9.15 (br s, 1H), 8.40 (t, 1H, J = 8 Hz), 8.20 (dd, 2H,
J = 1, 8 Hz), 7.68–7.56 (m, 1H), 7.60 (t, 1H), 7.38–7.24
(m, 4H), 7.20 (d, 1H, J = 8 Hz), 6.60 (s, 3H), 4.75 (s,
2H), 4.10 (s, 2H), 3.40 (t, 2H, J = 5 Hz), 2.62 (s, 3H),
1.75–1.40 (m, 2H), 1.42–1.38 (m, 2H), 1.05–1.00 (m,
3H); MS (ES) m/e 531.0 (M+1)+; CHN calcd for
C29H24ClF2 N3O4S: C, 58.68; H, 5.12; N, 7.90. Found:
C, 58.33; H, 4.87; N, 7.66.
5.1.26. 2-[3-(4-Chloro-phenyl)-1-cyclohexylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8o). Pale yellow solid 0.350 g, 57%; H NMR
1
(400 MHz, DMSO-d6): d 9.94 (s, 1H), 8.50 (s, 1H),
8.09 (dd, 1H, J = 1, 8 Hz), 8.06 (t, 1H, J = 8 Hz),
7.79–7.67 (m, 2H), 7.50 (d, 2H, J = 9 Hz), 7.45–7.32
(m, 2H), 7.28 (d, 2H, J = 9 Hz), 7.20 (dd, 1H, J = 1,
8 Hz), 4.26 (s, 2H), 3.27 (d, 2H, J = 7 Hz), 2.88 (s,
5.1.21. 2-[3-(4-Chloro-phenyl)-1-(2,2-dimethyl-propyl)-
ureido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-
acetamide (8h). White solid 0.074 g; 1H NMR
(400 MHz, CDCl3): d 8.97 (s, 1H), 8.40 (t, 1H,

J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
4389
3H), 1.75–1.55 (m, 6H), 1.25–1.08 (m, 3H), 1.03–0.88
(m, 2H); MS (ES) 571.8 (M+H)+; CHN calcd for
C29H31ClFN3O4S: C, 60.88; H, 5.46; N, 7.34. Found:
C, 60.72; H, 5.44; N, 7.23.
5.1.32. 2-[3-(4-Chloro-phenyl)-1-(3-methoxy-benzyl)-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8v). White solid 0.18 g, 32%; 1H NMR
(400 MHz, CDCl3): d 8.80 (br s, 1H), 8.40 (t, 1H,
J = 8 Hz), 8.20 (dd, 1H, J = 1, 8 Hz), 7.65–7.60 (m,
1H), 7.60–7.52 (m, 1H), 7.40–7.30 (m, 2H), 7.23–7.17
(m, 7H), 6.98–6.80 (m, 3H), 4.62 (s, 2H), 4.20 (s, 2H),
3.80 (s, 3H), 2.75 (s, 3H); MS (ES) 596 (M+1)+; HRMS
calcd for C30H28ClFN3O5S: 596.1422. Found: 596.1435.
HPLC (method B) tR = 6.8 min (97.8%).
5.1.27.
2-[3-(4-Chloro-phenyl)-1-(tetrahydro-furan-3-
ylmethyl)-ureido]-N-(3-fluoro-20-methanesulfonyl-biphe-
nyl-4-yl)-acetamide (8p). White solid 0.110 g, 50%; H
1
NMR (400 MHz, CDCl3): d 9.40 (br s, 1H), 8.38 (t,
1H, J = 8 Hz), 8.22 (d, 1H, J = 8 Hz), 7.80 (s, 1H),
7.70–7.56 (m, 2H), 7.40–7.16 (m, 2H), 7.36 (d, 2H,
J = 9 Hz), 7.24 (d, 2H, J = 9 Hz) 4.40 (d, 1H,
J = 8 Hz), 4.15–4.05 (m, 1H), 3.95–3.85 (m, 2H),
3.64–3.55 (m, 1H), 3.38–3.30 (m, 2H), 2.95 (s, 1H),
2.90 (s, 1H), 2.78 (s, 3H), 2.18–2.08 (m, 1H), 1.70–
1.60 (m, 1H), 1.25 (t, 1H, J = 8 Hz); MS (ES)
559.78 (M+1)+; CHN calcd for C27H27ClFN3O5S:
C, 57.91; H, 4.86; N, 7.50. Found: C, 58.14; H,
4.63; N, 7.78.
5.1.33.
2-[3-(4-Chloro-phenyl)-1-(3-fluoro-benzyl)-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8w). White solid 0.20 g, 33%; 1H NMR
(400 MHz, CDCl3): d 8.68 (br s, 1H), 8.36 (t, 1H,
J = 8 Hz), 8.20 (dd, 2H, J = 1, 8 Hz), 7.68–7.56 (m,
2H), 7.44–7.04 (m, 10H), 6.90 (br s, 1H), 4.75 (s, 2H),
4.24 (s, 2H), 2.70 (s, 3H); MS (ES) 584.12 (M+1)+;
HRMS calcd for C29H25ClF2N3O4S: 584.1222 Found:
584.1248; HPLC (method B) tR = 7.0 min (98.7%).
5.1.28.
2-[3-(4-Chloro-phenyl)-1-furan-2-ylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8q). White solid 0.50 g, 55%; 1H NMR
(400 MHz, CDCl3): d 9.08 (br s, 1H), 8.40 (t, 1H,
J = 8 Hz), 8.20 (dd, 1H, J = 8 Hz), 7.68–7.58 (m, 2H),
7.60–7.52 (m, 1H), 7.46 (d, 1H, J = 1, 8 Hz), 7.40–7.26
(m, 7H), 7.20 (dd, 1H, J = 1, 8 Hz), 6.40 (dd, 1H,
J = 1, 8 Hz), 4.60 (s, 2H), 4.21 (s, 2H), 2.68 (s, 3H);
MS (ES) 555.0 (M)+; CHN calcd for C27H23ClFN3O5S:
C, 58.33; H, 4.17; N, 7.56. Found: C, 58.21; H, 4.30; N,
7.07.
5.1.34. 2-[3-(4-Chloro-phenyl)-1-(3-methyl-benzyl)-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8x). White solid 0.18 g, 41%; 1H NMR (400 MHz,
CDCl3): d 8.84 (br s, 1H), 8.40 (t, 1H, J = 8 Hz), 8.20 (dd,
2H, J = 1, 8 Hz), 7.68–7.54 (m, 2H), 7.38–7.15 (m, 11H),
6.82 (br s, 1H), 4.70 (s, 2H), 4.30 (s, 2H), 2.70 (s, 3H),
2.40 (s, 3H); MS (ES) 579.64 (M+1)+; HRMS calcd for
C30H28ClFN3O4S: 580.1473. Found: 580.1461; HPLC
(method B) tR = 7.5 min (98.3%).
5.1.35. 2-[3-(4-Chloro-phenyl)-1-(2-methoxy-benzyl)-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8y). White solid 0.23 g, 42%; 1H NMR (400 MHz,
CDCl3); d 9.40 (br s, 1H), 8.40 (t, 1H, J = 8 Hz), 8.20 (dd,
1H, J = 1, 8 Hz), 7.94 (br s, 1H), 7.68–7.52 (m, 1H), 7.60–
7.52 (m, 1H), 7.40–7.23 (m, 8H), 7.20 (br d, 1H, J = 8 Hz),
7.10–6.90 (m, 2H), 4.60 (s, 2H), 4.15 (s, 2H), 4.01 (s, 3H),
2.75 (s, 3H); MS (ES) 596 (M+1)+; CHN calcd for
C30H27ClFN3O5S: C, 60.45; H, 4.57; N, 7.05. Found: C,
60.20; H, 4.40; N, 6.94.
5.1.29. 2-[3-(4-Chloro-phenyl)-1-thiophen-2-ylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8r). White solid 0.58 g, 61%; 1H NMR
(400 MHz, CDCl3): d 8.70 (br s, 1H), 8.40 (t, 1H,
J = 9 Hz), 8.21 (dd, 1H, J = 1, 8 Hz), 7.70–7.53 (m,
2H), 7.58 (t, 1H), 7.40–7.00 (m, 10H), 4.82 (s, 2H),
4.23 (s, 2H), 2.71 (s, 3H); MS (ES) 572.27 (M+1)+;
CHN calcd for C27H23ClFN3O4S2: C, 56.69; H, 4.05;
N, 7.35. Found: C, 56.84; H, 3.93; N, 7.35.
5.1.30. 2-[3-(4-Chloro-phenyl)-1-oxazol-2-ylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8s). White solid 0.060 g, 29%; 1H NMR
(400 MHz CDCl3): d 10.60 (br s, 1H), 8.40 (t, 1H,
J = 8 Hz), 8.26 (br s, 1H), 8.22 (d, 1H, J = 8 Hz), 7.75
(s, 1H) 7.68–7.56 (m, 2H), 7.45–7.18 (m, 3H), 7.42 (d,
2H, J = 9 Hz), 7.28 (d, 2H, J = 9 Hz), 4.82 (s, 2H),
4.30 (s, 2H), 2.75 (s, 3H); MS (ES) 556.69 (M+1)+;
CHN calcd for C26H22ClFN4O5S: C, 56.07; H, 3.98;
N, 10.06. Found: C, 56.09; H 4.18; N, 8.83.
5.1.36.
2-[1-(4-Chloro-benzyl)-3-(4-chloro-phenyl)-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8bb). White solid 0.22 g, 24%; 1H NMR
(400 MHz, DMSO-d6): d 9.80 (br s, 1H), 8.70 (s, 1H),
8.12–8.04 (m, 1H), 7.80–7.66 (m, 1H), 7.56–7.28 (m,
4H), 7.54 (d, 2H, J = 8 Hz), 7.42 (d, 2H, J = 8 Hz),
7.36 (d, 2H, J = 8 Hz), 7.30 (d, 2H, J = 8 Hz), 7.20
(dd, 1H, J = 9 Hz), 4.60 (s, 2H), 4.20 (s, 2H), 2.80 (s,
3H); MS (ES) 599.58 (M+1)+; CHN calcd for
C29H24ClFN3O4S+0.5 H2O: C, 57.16; H, 3.97; N, 6.89.
Found: C, 57.12; H, 3.89; % N 6.79.
5.1.31. 2-[3-(4-Chloro-phenyl)-1-thiophen-3-ylmethyl-ure-
ido]-N-(3-fluoro-20-methanesulfonyl-biphenyl-4-yl)-acet-
amide (8t). White solid 0.110 g, 58%; 1H NMR
(400 MHz CDCl3): d 8.78 (br s, 1H), 8.38 (t, 1H,
J = 8 Hz), 8.22 (dd, 1H, J = 1, 8 Hz), 7.70–7.56 (m,
2H) 7.46–7.42 (m, 1H), 7.38–7.20 (m, 8H), 7.10 (dd,
1H, J = 1, 8 Hz), 6.92 (br s, 1H) 4.70 (s, 2H), 4.25 (s,
2H), 2.72 (s, 3H); MS (ES) 571.67 (M+1)+; CHN calcd
for C27H22ClFN3O4S2: C, 56.69; H, 4.05; N, 7.35.
Found: C, 56.35; H, 3.82; N, 6.89.
The following compounds were prepared using the gen-
eral procedure for 11d.
5.1.37. 1-[1-Butyl-3-(4-chloro-phenyl)-ureido]-cyclopro-
panecarboxylic acid (3-fluoro-20-methanesulfonyl-biphe-
nyl-4-yl)-amide (11a). White solid 0.20 g, 60%; 1H NMR
(400 MHz, CDCl3): d 8.55 (br s, 1H), 8.35 (t, 1H,
J = 8 Hz), 8.20 (dd, 1H, J = 1, 8 Hz), 7.70–7.55 (m,
2H), 7.40–7.20 (m, 7H), 6.95 (s, 1H), 3.70–3.55 (m,

4390
J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
1H), 3.40–3.30 (m, 1H), 2.75 (s, 3H), 2.05–1.65 (m, 4H),
1.55–1.20 (m, 4H), 1.00 (t, 3H, J = 8 Hz); MS (ES)
557.24 (M+1)+; CHN calcd for C28H29ClFN3O4S: C,
60.26; H, 5.24; N, 7.53. Found: C, 60.98; H 5.15; N,
7.14.
CHN calcd for C30H31ClFN3O4S: C, 61.69; H, 5.35;
N, 7.19. Found: C, 62.20; H, 5.51; N, 6.97.
5.1.43. 2-[3-(4-Chloro-phenyl)-1-(2-methoxy-ethyl)-ure-
ido]-N-[2-fluoro-4-(2-oxo-piperidin-1-yl)-phenyl]-acet-
amide (12). White solid 0.030 g, 70%; 1H NMR
(400 MHz, CDCl3): d 9.73 (s, 1H), 8.53 (s, 1H), 7.81
(dd, 1H, J = 8, 8 Hz), 7.43–7.30 (m, 2H), 7.25–7.18 (m,
3H), 7.03 (dd, 1H, J = 7, 8 Hz), 4.19 (s, 2H), 3.55–3.44
(m, 6H), 3.20 (s, 3H), 2.32 (t, 2H, J = 6 Hz), 1.82–1.73
(m, 4H); MS (AP+) 477.1 (M+1)+; HRMS calcd for
C23H26ClFN4O4: 477.1660. Found: 477.1709.
5.1.38. 1-[3-(4-Chloro-phenyl)-1-methyl-ureido]-cyclopro-
panecarboxylic acid (3-fluoro-20-methanesulfonyl-biphe-
nyl-4-yl)-amide (11b). White solid 0.060 g, 32%; 1H
NMR (400 MHz, CDCl3): d 8.48 (br s, 1H), 8.30 (t,
1H, J = 8 Hz), 8.20 (dd, 1H, J = 1, 8 Hz), 7.70–7.58
(m, 2H), 7.40–7.18 (m, 6H), 6.90 (br d, 1H, J = 8 Hz),
3.20 (s, 3H), 2.68 (s, 3H), 1.82 (br s, 1H), 1.60–1.54
(m, 2H), 1.24–1.22 (m, 2H); MS (ES) 516 (M+1)+;
CHN calcd for C25H23ClFN3O4S: C, 58.19; H, 4.49;
N, 8.17. Found: C, 57.83; H, 4.59; N, 7.39.
5.1.44. 2-[3-(4-Chloro-phenyl)-1-cyclopropylmethyl-ure-
ido]-N-[2-fluoro-4-(2-oxo-2H-pyridin-1-yl)-phenyl]-acet-
amide (13). White solid 0.47 g, 98%; 1H NMR
(400 MHz, CDCl3): d 9.20 (br s, 1H), 8.40 (t, 1H,
J = 8 Hz), 7.50–7.40 (m, 1H), 7.40–7.15 (m, 7H), 7.15
(d, 1H, J = 8 Hz), 6.65 (d, 1H, J = 8 Hz), 6.25 (t, 1H,
J = 8 Hz), 4.20 (s, 2H), 3.35 (d, 2H, J = 8 Hz), 1.50
(quin, 1H, J = 8 Hz), 0.65 (dt, 2H, J = 6, 8 Hz), 0.35
(dt, 2H, J = 6, 8 Hz). MS (ES) 470.36 (M+1)+; CHN
calcd for C24H22ClFN4O3: C, 61.47; H, 4.73; N, 11.95.
Found: C, 61.30; H, 4.81; N, 11.93.
5.1.39. 1-[3-(4-Chloro-phenyl)-1-propyl-ureido]-cyclopro-
panecarboxylic acid (3-fluoro-20-methanesulfonyl-biphe-
nyl-4-yl)-amide (11c). White solid 0.19 g, 59%; 1H NMR
(400 MHz, CDCl3): d 8.60 (br s, 1H), 8.35 (t, 1H,
J = 8 Hz), 8.20 (dd, 1H, J = 1, 8 Hz), 7.65–7.55 (m,
2H), 7.40–7.20 (m, 7H), 6.95 (s, 1H), 3.65–3.55 (m,
1H), 3.35–3.25 (m, 1H), 2.70 (s, 3H), 2.10–2.00 (m,
1H), 1.85–1.80 (m, 2H), 1.80–1.65 (m, 1H), 1.55–1.35
(m, 2H), 1.00 (t, 3H, J = 8 Hz). MS (ES) 543.33
(M+1)+; CHN calcd for C27H27ClFN3O4S: C, 59.61;
H, 5.00; N, 7.72. Found: C, 59.71; H, 4.92; N, 7.64.
5.2. Crystallization, data, and structural refinement
Binary complex crystals of Human des Gla factor Xab
protein with 11a were produced by hanging drop vapor
diffusion using a reservoir solution composed of 25%
PEG 600 (Fluka), 0.3 M NaCl (Sigma), and 0.1 M
MES (2-morpholinoethanesulfonic acid, Sigma) buffer
(pH 5.9). Des Gla factor Xab protein (8 mg/mL) com-
plexed with 0.1 mM 11a was mixed with an equivalent
volume of reservoir solution and equilibrated over
250 lL of reservoir solution. Rod-shaped, single crystals
(60 · 60 · 240 lm) were obtained in six weeks as a result
of microseeding for two days. Crystals were quickly im-
mersed in oil (70% Paratone-N oil and 30% light, white
mineral oil, Hampton Research) and flash-cooled in
liquid nitrogen before data collection.
5.1.40. 1-[3-(4-Chloro-phenyl)-1-cyclopropylmethyl-ure-
ido]-cyclopropanecarboxylic acid (3-fluoro-20-metha-
nesulfonyl-biphenyl-4-yl)-amide (11d). White solid
0.30 g, 54%; 1H NMR (400 MHz, CDCl3): d 8.95
(br s, 1H), 8.40 (t, 1H, J = 8 Hz), 8.20 (dd, 1H,
J = 1, 8 Hz), 7.70–7.55 (m, 2H), 7.40–7.20 (m, 7H),
7.00 (s, 1H), 3.90 (dd, 1H, J = 5, 8 Hz), 2.90 (dd,
1H, J = 5, 8 Hz), 2.70 (s, 3H), 2.15–2.05 (m, 1H),
1.90–1.80 (m, 1H), 1.55–1.30 (m, 2H), 1.30–1.20 (m,
1H), 0.70–0.55 (m, 2H), 0.45–0.35 (m, 2H). MS (ES)
557.24 (M+1)+; CHN calcd for C28H27ClFN3O4S: C,
60.48; H, 4.89; N, 7.56. Found: C, 60.67; H 5.10;
N, 7.38.
X-ray diffraction intensity data were obtained under
cryogenic conditions at the IMCA-CAT 17-ID beam-
line at the Advanced Photon Source (Argonne Nation-
al Laboratories, Argonne, IL). The crystals diffracted
5.1.41. 1-[3-(4-Chloro-phenyl)-1-cyclohexylmethyl-ure-
ido]-cyclopropanecarboxylic acid (3-fluoro-20-metha-
nesulfonyl-biphenyl-4-yl)-amide (11e). White solid
1
˚
0.21 g, 40%; H NMR (400 MHz, CDCl3): d 8.60 (br
to a resolution of 1.9 A and are a member of the space
s, 1H), 8.30 (t, 1H, J = 8 Hz), 8.20 (dd, 1H, J = 1,
8 Hz), 7.65–7.55 (m, 2H), 7.45–7.15 (m, 7H), 7.05 (s,
1H), 3.45–3.15 (m, 2H), 2.70 (s, 3H), 2.05–1.35 (m,
11H), 1.35–0.85 (m, 4H); MS (ES) 598.29 (M+1)+;
HRMS calcd for C31H33ClFN3O4S: 598.1942; Found:
598.1954. HPLC (method B) tR = 6.2 min (99.2%).
group P212121 with one molecule per asymmetric unit.
The structure of the FXa–11a binary complex was re-
fined starting with previously refined FXa model struc-
tures, without their inhibitor ligand, with program
package CNX.24 A rigid-body rotation–translation
refinement was initially performed to align the modeled
structure accurately in the FXa–11a unit cell. Further
crystallographic refinement was performed by conju-
gated-gradient minimization and individual B factor
refinement with CNX, and model/ligand building was
performed with the program ‘QUANTA.’ There is no
electron density observed for the first EGF domain,
thus models for the EGF1 domain were not included
in subsequent refinement steps. The final model includ-
ed 279 residues, 99 water molecules, 2 Ca2+ ions, and
11a.
5.1.42. 1-[3-(4-Chloro-phenyl)-1-cyclopentylmethyl-ure-
ido]-cyclopropanecarboxylic acid (3-fluoro-20-metha-
nesulfonyl-biphenyl-4-yl)-amide (11f). White solid
1
0.27 g, 45%; H NMR (400 MHz, CDCl3): d 8.75 (br
s, 1H), 8.35 (t, 1H, J = 8 Hz), 8.20 (dd, 1H, J = 1,
8 Hz), 7.70–7.50 (m, 2H), 7.40–7.20 (m, 7H), 7.00 (s,
1H), 3.75–3.65 (m, 1H), 3.20–3.15 (m, 1H), 2.70 (s,
3H), 2.35–2.25 (m, 1H), 2.00–1.40 (m, 8H), 1.35–1.20
(m, 2H), 0.90–0.80 (m, 2H); MS (ES) 584.31 (M+1)+;

J. T. Kohrt et al. / Bioorg. Med. Chem. 14 (2006) 4379–4392
4391
5.3. Enzyme inhibition assays
of plasma concentration at 0, 0.5, 1, 2, and 4 or 0, 0.5,
1, 4, and 6 h post-dose. 0.9 mL of blood was drawn into
a syringe containing 0.1 mL of 3.8% sodium citrate.
Samples were centrifuged twice and the final plasma
was stored at ꢀ80 ꢁC until analyzed. At the conclusion
of the study rats were euthanized with an overdose of
Beuthanasia D (Schering Plough, Kennilworth, NJ).
Compounds were tested for the inhibition of thrombin
and trypsin according to a modified protocol of Chu
et al.27a All enzymes were purchased from Enzyme Re-
search Laboratories (South Bend, Indiana) including
purified human FXa, thrombin, and trypsin. Com-
pounds were tested for the inhibition of FXa via a fluor-
ogenic assay for which the fluorogenic substrate for
FXa, FS-2765, was purchased from California Peptide
(Napa, California).27b The chromogenic substrates for
thrombin and trypsin were purchased from Roche
Molecular Biochemicals (Mannheim, Germany) and
Chromogenix (Molndal, Sweden), respectively. Buffer
A containing 10.0 mM HEPES, 150 mM sodium chlo-
ride, and 0.1% (bovine serum albumin) BSA at pH 7.4
was prepared for use in the FXa and trypsin assays.
Ex vivo anti-FXa activity of test compounds was deter-
mined by a chromogenic assay previously described.20
In brief, antithrombin III buffer (0.05 M Tris–HCl,
0.175 M sodium chloride, and 7.5 mM EDTA, pH
8.4), bovine FXa (20 nKat),28 and SPECTROZYMEꢂ
FXa (methoxycarbonyl-D-cyclohexyglycyl-L-glycyl-D-
arginine p-nitroanilide acetate, 4 lM) were prepared
according to the manufacturer’s instructions (American
Diagnostica Inc.). A standard curve was prepared with
fixed concentrations of test compounds in rat plasma
with sodium citrate. Plasma samples were thawed and
kept at 4 ꢁC. Plasma samples and standards (20 lL of
each) were added to each well of a 96-well microplate.
Antithrombin III buffer (60 lL), bovine FXa (60 lL),
and SPECTROZYMEꢂ FXa (60 lL) were added via
pipette into the wells of the plate with 60 s intervals of
mixing at room temperature between additions of each
reagent. Immediately following the addition of the sub-
strate, the plate was read kinetically for 5 min at 405 nm
on a SPECTRAmax Plus384 spectrophotometer (Molec-
ular Devices Corporation) set at 37 ꢁC. SOFTmaxꢂ Pro-
software (Molecular Devices Corporation) was used to
record and analyze the maximal velocity of the reaction
for each well. Factor Xa inhibition was calculated for
Thrombin was assayed in Buffer
B
containing
10.0 mM HEPES, 100 mM NaCl, 0.1% PEG 8000, and
0.05% BSA at pH 7.4. Factor Xa was tested at a final
concentration of 30 pM. Thrombin and trypsin were
both tested at 500 pM. Compounds were serially diluted
to final concentrations of 1.0 lM–900 fM for the FXa
inhibition assay and 200 lM–3.4 nM in the selectivity
assays. The reactions were initiated by the addition of
prewarmed substrate and immediately placed in the
microplate reader (SpectraMAX Gemini XS fluoromet-
ric plate reader and SpectraMAX 190 chromogenic plate
reader) prewarmed to 37 ꢁC. Concentration curves were
run in triplicate and IC50s were calculated by following
the 4-parameter logistic curve fitting equation. IC50s
are reported as means SEM (standard error of the
mean).
each plasma sample or standard using the following
equation: ð1 ꢀ V smaamxple=V
control
max
Þ ꢁ 100. The control value
control
max
5.3.1. Prothrombin time (PT) assay. Citrated, pooled
normal human plasma was purchased from George
King Bio-Medical Inc. Inhibitors were serially diluted
in DMSO and spiked into the pooled human plasma
at final assay concentrations of 13.2–0.26 lM. Samples
were run in duplicate using the recombinant thrombo-
plastin reagent Innovin (Dade Behring Inc, Deerfield,
Illinois), and PT was measured on the ACLTM 9000 Sys-
tem (Beckman Coulter Inc, Fullerton, California)
according to the manufacturers’ protocol.25 The results
were reported as the concentration of inhibitor that dou-
bled the time (2· PT) to initial fibrin strand formation.
ðV
Þ was the predose sample for an individual ani-
mal or the no drug standard for the standard curve.
SOFTmaxꢂ Pro interpolated the final assay concentra-
tion for each sample from the standard curve. Values
were reported as percent inhibition and plasma concen-
tration (nM). All plasma samples and standards were
run in duplicate.
Acknowledgments
The authors thank Mehran Jalaie for help in preparing
the X-ray crystallographic binding diagrams used in
the manuscript, Michael Stier for his help in compiling
experimental data and procedures, Wayne Cody for
his help in the editing of the manuscript, and Dana De-
John for the HRMS determinations.
5.3.2. In vivo assay—rat PK–PD. Rats were housed in an
AALAC accredited facility in climate-controlled rooms
on a fixed light cycle and had access to normal rat chow
and water for one to seven days before the study. This
study protocol was approved by the Pfizer Ann Arbor
Laboratories Animal Care and Use Committee, and
conforms to the guide for the Care and Use of Labora-
tory Animals26 published by the US National Institute
of Health (NIH Publication No. 85-23, revised 1996).
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