Exploration of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides as potent, orally active Factor Xa inhibitors with extended duration of action

Article abstract of DOI:10.1016/j.bmc.2009.01.063

Aiming to improve upon previously disclosed Factor Xa inhibitors, a series of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides were explored with the intent of increasing the projected human half-life versus 5 (projected human t_1/2 = 6 h). A stereospecific route to compounds containing a 4-aryl-4-hydroxypyrrolidine scaffold was developed, resulting in several compounds that demonstrated an increase in the half-life as well as an increase in the in vitro potency compared to 5. Reported herein is the discovery of 26, containing a (2R,4S)-4-hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, which is a selective, orally bioavailable, efficacious Factor Xa inhibitor that appears suitable for a once-daily dosing (projected human t_1/2 = 23 h).

Full text of DOI:10.1016/j.bmc.2009.01.063

Bioorganic & Medicinal Chemistry 17 (2009) 2501–2511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides as potent,
orally active Factor Xa inhibitors with extended duration of action
*
Chad A. Van Huis, Agustin Casimiro-Garcia , Christopher F. Bigge, Wayne L. Cody, Danette A. Dudley,
Kevin J. Filipski, Ronald J. Heemstra, Jeffrey T. Kohrt, Robert J. Leadley Jr., Lakshmi S. Narasimhan,
Thomas McClanahan, Igor Mochalkin, Michael Pamment, J. Thomas Peterson, Vaishali Sahasrabudhe,
Robert P. Schaum, Jeremy J. Edmunds
Michigan Laboratories, Ann Arbor Campus, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Aiming to improve upon previously disclosed Factor Xa inhibitors, a series of 4,4-disubstituted pyrroli-
dine-1,2-dicarboxamides were explored with the intent of increasing the projected human half-life ver-
Received 13 November 2008
Revised 22 January 2009
Accepted 26 January 2009
Available online 3 February 2009
sus
5 (projected human t1/2 = 6 h). A stereospecific route to compounds containing a 4-aryl-4-
hydroxypyrrolidine scaffold was developed, resulting in several compounds that demonstrated an
increase in the half-life as well as an increase in the in vitro potency compared to 5. Reported herein
is the discovery of 26, containing a (2R,4S)-4-hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, which
is a selective, orally bioavailable, efficacious Factor Xa inhibitor that appears suitable for a once-daily dos-
ing (projected human t1/2 = 23 h).
Keywords:
Factor Xa
Factor Xa inhibitor
Antithrombotic agents
Pyrrolidine-1,2-dicarboxamides
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
concentrations of warfarin, which has an inherent risk due to
metabolism, drug–drug interactions and food effects. Thus safety
In recent years there has been an intensive research effort to
develop orally bioavailable antithrombotic agents. Such agents
are expected to reduce cardiovascular complications and mortal-
ity associated with several life threatening conditions. Among
these are disease states precipitated by abnormal coagulation
and inappropriate thrombus formation within blood vessels such
as myocardial infarction, unstable angina, stroke, pulmonary
embolism, deep vein thrombosis and venous thromboembolism
associated with surgery and cancers. This research has led to
several compounds that have reached late stage clinical develop-
ment.1,2 These new agents must address the limitations associated
with warfarin (CoumadinÒ) therapy. Regular coagulation
monitoring is necessary to maintain safe and therapeutic plasma
and ease of use are of primary importance for a new antithrom-
botic agent.
Recently, direct thrombin inhibitors (DTI) in the form of pro-
drugs have advanced to the threshold of market launch, or have
actually reached the market. Unfortunately, the promising DTI
ximelagatran was withdrawn in 2005.1,3 The decision was due to
a significant risk of liver damage upon chronic use of the drug
and an increased risk of heart attack.4 Ximelagatran also showed
some rebound thrombosis which has also been observed with
other DTIs.5 Another orally-active prodrug DTI, dabigatran etexi-
late (1, Fig. 1) is now in the market.1,6
Factor Xa (FXa) is a serine protease that acts at the interface of
the intrinsic and extrinsic coagulation cascade and converts pro-
thrombin to thrombin.2 As further elaborated below, evidence
indicates that FXa inhibition will have an enhanced risk-benefit
margin compared to warfarin therapy as FXa is primarily responsi-
ble for clot-associated pro-coagulant activity.
Abbreviations: FXa, Factor Xa; DTI, direct thrombin inhibitor; Cmax, maximum
plasma concentration; Cmin, minimum plasma concentration; Cmax/Cmin, peak-to-
trough plasma concentration ratio; PT, prothrombin time; 2 Â PT, concentration
Direct FXa inhibition has been demonstrated to disaggregate
preformed platelet thrombi in vivo and produce long-term reduc-
tion of thrombus procoagulant activity (passivation) in animal
models of thrombosis even with short-term FXa inhibition.7 In
contrast to the ‘rebound thrombosis’ observed shortly after termi-
nation of current antithrombin and antiplatelet therapies, direct
required to
give a doubling of the prothrombin time; PK/PD, pharmacokinetic/
pharmacodynamic; AV, arteriovenous; IV, intravenous administration; PO, oral
administration; QD, once daily dosing; ND, not determined; Vdss volume of
,
distribution at steady state; Ka, absorption rate constant; F, oral bioavailability; t1/2
,
elimination half-life; CYP, cytochrome P450.
* Corresponding author. Tel.: +1 860 686 9155; fax: +1 860 715 4483.
E-mail address: agustin.casimiro-garcia@pfizer.com (A. Casimiro-Garcia).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.063

2502
C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
CH3
N
O
O
NH2
O
N
N
O
HN
CH3
O
HN
N
O
H3C
O
N
O
S
N
O
N
O
Cl
1
2
H3CO
HN
F
O
O
H
N
H2N
O
H
N
O
N
N
N
O
N
H
N
N
O
N
N
O
N
O
N
CH3
Cl
OCH3
3
4
5
Figure 1. Structures of new oral anticoagulants in development: 1 = dabigatran etexilate, 2 = rivaroxaban, 3 = apixaban, 4 = LY-517717, 5 = eribaxaban.
FXa inhibition provides the potential for sustained antithrombotic
prevention after therapy is terminated and may provide a unique
advantage over currently available anticoagulants. Indirect inhib-
itors of FXa such as fondaparinux (ArixtraÒ) have demonstrated
efficacy and safety which provides additional evidence that
inhibitors of FXa may overcome current limitations of oral anti-
thrombotic therapy.1 The first generation of small molecule FXa
inhibitors relied upon a benzamidine moiety for potency and
consequently exhibited high clearance and lacked acceptable oral
bioavailability.2 Subsequently, poor pharmacokinetic and phar-
macodynamic properties have been successfully addressed
through the replacement of the benzamidine moiety with a neu-
tral P1 element, as several FXa inhibitors are in late stage clinical
trials. Prominent among these FXa inhibitors are rivaroxaban 2,8
apixaban 3,9 LY-517717 41 and eribaxaban 510,11 as shown in
Figure 1.
For an ideal anticoagulant therapy, the plasma concentration
must remain within the therapeutic window to afford maximal
protection without increasing the risk of bleeding. As the dose
of an anticoagulant is increased, the incidence of clotting events
decreases. However, a threshold of exposure exists where the
risk of serious bleeding exceeds its benefit. Maintaining a plasma
concentration within the therapeutic window may be affected by
the peak-to-trough ratio (Cmax/Cmin) that is a function of the
pharmacokinetic profile of the specific agent and its dosing reg-
imen. Thus, the Cmax of an anticoagulant could be higher and/or
the Cmin lower than ideal for the therapeutic window. Using
some baseline assumptions (e.g., similar absorption rates), com-
pounds with half-lives of 6 h, 12 h and 24 h have Cmax/Cmin ra-
tios of 9.4, 3.0 and 1.8, respectively, when dosed every 24 h.12
This effect of elimination half-life on Cmax/Cmin ratio for a once
daily dosing regimen is presented graphically in Figure 2. This
example demonstrates how a compound with extended half-life
can minimize large fluctuations in drug plasma concentrations.
An anticoagulant with a half-life appropriate for once daily dos-
ing would lead to improved clinician confidence in dosing as
well as enhanced convenience and patient compliance. Herein
2. Molecular modeling and design
A co-crystal X-ray structure of Factor Xa protein and 5 was ob-
tained in our laboratories (Fig. 3a).10 It was observed that the C-4
hydrogen of the pyrrolidine ring was directed towards the unoccu-
pied FXa active-site subpocket formed by the disulfide region
(Cys191–Cys220) and the fully-extended side chains of residues
Arg143, Glu147 and Gln192 of the enzyme. To probe this hydro-
phobic subpocket, possible replacements for the C-4 hydrogen,
such as small alkyl groups and aryl rings, were modeled in the
FXa active site and a select number of compounds were synthe-
sized. It was hypothesized that further substitution at the C-4 site
was not likely to adversely impact potency as the strong H-bond
between Gly219 of FXa and the lone pairs of the 4-methoxy group
or 4-hydroxy group would be preserved. Further details about the
orientation of one of these compounds, the 4-hydroxy-4-phenyl
analog 17 were revealed in a X-ray co-crystal structure (Fig. 3b).
The crystal structure showed that the disulfide bridge region was
still not fully occupied which suggested that space may still exist
for additional substitution at the ortho position of the phenyl ring.
A number of these analogs were subsequently synthesized. One of
these analogs, the 2,4-difluorophenyl derivative 26, was shown via
X-ray crystallography, to bind to the enzyme with its ortho substi-
tuent occupying the disulfide region (Fig. 3c). Further discussion
about the X-ray crystallography results are given in Section 4.
3. Chemistry
The general synthetic route for the above 4,4-disubstituted pyr-
rolidine compounds is outlined in Scheme 1. Ketone 7 was ob-
tained from commercially available cis-hydroxy-D-proline via
protection as the t-butylcarbamate and oxidation of the alcohol
with trichloroisocyanuric acid and catalytic TEMPO in dichloro-
methane.13 The ketoacid 7 was treated with an excess of the appro-
priate Grignard reagent to produce the tertiary alcohol 8. Use of the
carboxylic acid intermediate was preferred over an ester as the
resulting carboxylate anion prevented racemization at the proline
is described
a number of 4,4-disubstituted pyrrolidine-1,2-
a-carbon. Interestingly, the methyl and ethyl Grignard reagents re-
dicarboxamide derivatives that were explored in an effort to de-
velop a compound with a projected human half-life suitable for
once daily dosing.
sulted in an approximate 50:50 diastereomeric mixture of inter-
mediate 8, whereas aryl Grignard reagents proceeded
stereoselectively to give the desired cis-hydroxyl product. When

C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
2503
Figure 2. Simulated drug concentration versus time profiles illustrating the effect of elimination half-life on Cmax/Cmin ratio for a once daily dosing regimen (assuming
Vdss = 1 L/kg, Ka = 0.7 h–1, F = 100%). Blue line: t1/2 = 6 h, Cmax/Cmin = 9.4. Magenta line: t1/2 = 12 h, Cmax/Cmin = 3.0. Green line: t1/2 = 24 h, Cmax/Cmin = 1.8.
R1 was a small alkyl moiety, the mixture of diastereomers was cou-
pled directly with the aniline or 2-aminopyridine derivative used
as the moiety to bind in the S4 pocket (P4 binding moiety) using
EEDQ.14 The diastereomers were later separated by column chro-
matography after the final step. In the case of the ether analogs
12, 15, and 16, intermediate 8 was alkylated with methyliodide
or ethylbromide to give the corresponding methyl or ethyl ether.
Selective alkylation was accomplished by forming the di-anion of
8 with excess sodium hydride in THF, followed by the addition of
the alkylating agent. Only the alcohol reacts under these condi-
tions, even with a large excess of alkylating agent. In the case of
4-aryl products, activation of the carboxylic acid with various cou-
pling reagents resulted exclusively in intramolecular cyclization
with the cis-4-hydroxy group to form lactone 9.
Treatment of the aniline or 2-aminopyridine derivative used as
the P4 binding moieties with trimethylaluminum in toluene fol-
lowed by addition of lactone 9 with heating effected the ring open-
ing to the desired product 10.15 While the use of these forcing
conditions led to moderate yields of the needed intermediate, only
the desired diastereomer was identified from the reaction mixture.
Most of the by-products encountered were either decomposition
by-products or unreacted lactone starting material. The final com-
pounds were obtained by first removing the t-butylcarbamate with
TFA in methylene chloride or by addition of 10 to freshly prepared
HCl in methanol. Secondly, the deprotected amine was reacted
with 4-chlorophenylisocyanate in methylene chloride with trieth-
ylamine, or in a biphasic mixture of sodium bicarbonate in water
and dichloromethane to give the desired products 11–26.
This initial route was suitable for small scale preparation of 26.
However, Grignard addition to the ketone and the opening of the
lactone using trimethylaluminum posed potential safety issues
on large scale. First, it is known that an ortho-halogen substituted
aryl Grignard reagent can form unstable benzynes with unknown
energetics.16 Spectroscopic analysis and thermal stability studies
were performed during the preparation of 2,4-difluorophenylmag-
nesium bromide. Although the benzyne related byproducts were
observed at temperatures above room temperature (>23 °C), no
such evidence of benzyne formation was detected when the Grig-
nard reagent was formed below 0 °C. For this reason, large scale
preparation of 2,4-difluorophenylmagnesium bromide was accom-
plished using transfer methodology utilizing isopropylmagnesium
bromide with 2,4-difluorobromobenzene while keeping reaction
temperatures below 0 °C.17
A second potential hazard of the large scale synthesis of 26 was
the highly pyrophoric properties of trimethylaluminum. Direct
opening of the lactone 9 (R1 = 2,4-difluorophenyl) by heating with
1-(6-aminopyridin-3-yl)pyridin-2(1H)-one and without further
activation was not accomplished. To increase the reactivity, poten-
tial bases to generate the corresponding anion of the aminopyri-
dine P4 binding moiety were screened which revealed that anion
formation with lithium hexamethyldisilazide in THF followed by
the addition of lactone 9 gave the desired amide 10. The compound
obtained using these conditions was found to be identical by 1H
NMR and HPLC to 10 prepared using trimethylaluminum
conditions.
4. Results and discussion
A table of 4,4-disubstituted pyrrolidines is shown with the
in vitro FXa binding affinity and half-life from rat IV cassette dosing
studies (Table 1). In all cases the cis-hydroxy or cis-alkoxy deriva-
tives are shown. As suggested by our molecular modeling results,
replacement of the C-4 hydrogen with a methyl group, while main-
taining a cis-4-hydroxy or cis-4-methoxy substituent did not ad-
versely impact potency, as seen in 11 and 12. These results also
suggested that even with the addition of the methyl group at C-
4, the lone pair of the oxygen atom was likely forming an H-bond
with Gly219 similar to that observed with 5. As would be expected
based on lipophilicity, increasing the size of the R1 group was ob-
served to have an effect in the potency. However, it was noted that
small changes in the size of the R1 group (like methyl to ethyl as in
11 to 13 or 12 to 15) did not yield noticeable changes in potency.
Larger alterations in the size of R1 (like ethyl to phenyl as in 14
to 17) led to a modest (ꢀ2-fold) improvement of potency.
The anticoagulant activity of several compounds was evaluated
via prolongation of fibrin clot formation in human plasma as mea-
sured by the doubling of prothrombin time (2 Â PT) and the results
are shown in Table 1. Several compounds achieved doubling of PT
at concentrations of <1
lM. As previously observed by other
groups,18 the anticoagulant activity seemed to be correlated with

2504
C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
Figure 3. Co-crystal X-ray structures of FXa protein and 5 (white), 17 (magenta), and 26 (cyano) in the active site with a resolution of 2.3, 1.9, and 2.05 Å, respectively.
Enzyme shown with Connolly surface (probe radius 1.4 Å). Surface color represents hydrophobicity, ranging from orange (lipophilic) to blue (hydrophilic).
the lipophilicity (as measured by clogD at pH 7.4) of the new com-
pounds. It was observed that more lipophilic compounds like 17
(clogD = 2.34) required FXa inhibitory potency <0.1 nM to achieve
As seen from 6 (Table 1), replacing the 4-fluorophenyl moiety
with 2-aminopyridine while maintaining the same C-4 substitu-
tion as in 5 caused a 10-fold reduction in binding potency
(IC50 = 4.60 nM versus 0.57 nM, respectively). However, the discov-
ery of 17, with a 7-fold increase in potency and increase in half-life
compared to 5, enabled the combination of the 4-hydroxy-4-phe-
nylpyrrolidine core with the 2-aminopyridine P4 binding moiety.
This combination surprisingly resulted in an inhibitor with subn-
anomolar potency (18, Table 2). While 18 had a shorter rat half life
than 17, the 2-aminopyridine replacement in 18 resulted in a de-
crease in clearance and an increase in the volume of distribution
in rat as compared to 5 (6.73 mL/min/kg and 1490 mL/kg vs 12
mL/min/kg and 1100 mL/kg, respectively). In addition, 18 was
measured to have a rat oral bioavailability of 47%. As a result, the
2-aminopyridine P4 binding moiety of 18 became a focus of the
discovery effort due to the opportunity to achieve a reduction in
molecular weight and lipophilicity.
anticoagulant activity of <1 lM.
At first glance, replacing the C-4 hydrogen with other more lipo-
philic groups appeared to positively impact the PK profile. For
example, comparing 5 with 13, an encouraging increase in half-life
was achieved by replacing the C-4 hydrogen with an ethyl group.
However the effect of C-4 substitution on half-life is not straight-
forward since the rat IV cassette half-life of 13 was surprisingly
long when compared to 14 and 17. Unfortunately, this apparently
long half-life for 13 was not validated in a definitive rat single dose
IV PK study (t1/2 = 2 h). While 17 had a shorter half life than 13
(t1/2 = 4 h versus 5.2 h, respectively) the half-life of 17 (t1/2 = 4 h) in
single dosed rat was approximately twice that of 5 (t1/2 = 2 h).10
Due to the increase in both half-life and potency, the 4-hydroxy-4-
aryl substitution pattern became the focus of further research.

C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
2505
(FXa IC50 = 0.10 nM) versus the unsubstituted phenyl 18. The rat
and dog oral bioavailabilities for 26 were 19% and 24%, respectively
(Table 3). Clearance was low in both rat and dog with volumes of
distribution slightly greater than the volume of total body water
suggesting moderate tissue distribution. Utilizing allometric scal-
ing calculations,19 26 was estimated to have a human half-life of
23 h, and human oral bioavailability was predicted to be 23%. In
addition, 26 was measured to have >10,000-fold selectivity based
upon Ki determinations against a number of related serine prote-
ases (activated protein C, plasmin, thrombin, tissue plasminogen
activator, and trypsin). Furthermore, 26 showed considerably weak
R1
O
HO
HO
a, b
c
OH
OH
OH
N
Boc
N
H
N
Boc
O
O
O
7
8
d, e
f
R1
O
R1
O
O
N
O
H
N
Y
R3
g
R2
inhibition of four of the major drug-metabolizing CYPs at 3
lM
N
Boc
N
(CYP1A2: 0%; CYP2C9: 9.53%; CYP2D6: 0.5%; CYP3A4: 3.5%).20
O
10
h, i
Boc
The increased FXa inhibitory potency obtained with the mono-
fluorinated 24 and di-fluorinated 25 and 26 led us to review the
potential formation of polar interactions between these com-
pounds and the protein (fluorine–protein interactions).21 The
X-ray crystal structure of 26 was used to look for this type of inter-
actions. Although, it has been recognized that distinct fluorophilic
environments in proteins include the multipolar C–FÁÁÁC@O, C–
9
R1
O
N
O
H
N
Y
R3
R2
FÁÁÁH–N and C–FÁÁÁH–Ca interactions with peptide bonds, particu-
N
larly those in hydrophobic environments, the C-2 fluorine atom
of 26 is located away from the backbone atoms of the enzyme
(>3.9 Å distance), pointing into the side chain amide residue of
Gln192 (FÁÁÁN distance: 3.6 Å). Interaction between C–F and side-
chain amide residues of Gln and Asn have been observed.22 The
guanidinium group of Arg have also been reported to interact with
C–F.22 Interestingly, the C-2 fluorine atom of 26 is also located in
the vicinity of the guanidinium group of Arg143 (distance between
F and the central C of the guanidinium group: 4.1 Å). On the basis
of this analysis, it appears that introduction of the 2,4-difluoro-
phenyl group into the lipophilic subpocket may lead to the forma-
tion of a number of fluorine–protein interactions with the side
chains of residues Arg143 and Gln192 that ultimately had a favor-
able effect on the binding affinity.
O
HN
O
Cl
11-26
Scheme 1. General route to 4,4-disubstituted pyrrolidine FXa inhibitors. Reagents
and conditions: (a) cis-hydroxy- -proline, Boc2O, 1 M NaOH, THF, 0 °C to room
temperature, 81%; (b) trichloroisocyanuric acid, TEMPO, CH2Cl2, 0 °C to room
D
temperature, 97%; (c) R1MgBr, THF, for R1 = Ph, 53%; (d) 1-(4-amino-3-fluoro-
phenyl)pyridin-2(1H)-one,
1-(4-amino-3-fluorophenyl)-3-methylpyridin-2(1H)-
one, or 1-(6-aminopyridin-3-yl)pyridin-2(1H)-one,10 EEDQ, Et3N, CHCl3, reflux; (e)
R2X, NaH, THF; (f) BOP, Et3N, DMF, for R1 = Ph, quantitative yield; (g) P4 amine (see
step d), trimethylaluminum, toluene, 0 °C to 100 °C, for R1 = Ph, 49%; (h) TFA, CH2Cl2
or HCl, MeOH; (i) 4-chlorophenylisocyanate, saturated sodium bicarbonate, CH2Cl2
or Et3N, CH2Cl2.
The in vivo antithrombotic effect of 26 was evaluated in a rabbit
arteriovenous (AV) shunt model of thrombosis. The model em-
ployed is a modification of the method of Wong et al.23 with
thrombus weight used as the primary endpoint of this efficacy
study. The antithrombotic effect is expressed as the percent reduc-
tion of thrombus weight. The calculation is simply the difference in
thrombus weight between the baseline thrombus weight and trea-
ted subject (i.e., the thrombus weight from the first shunt) divided
by the baseline thrombus weight. The percent reductions in throm-
bus weight were 38 12%, 74 8%, and 93 1% at the doses of 1, 3,
Surprisingly, it was found that while 18 had an increase in rat
half-life compared to 5, the half-life of 18 in dog was actually lower
(2.9 h for 18 vs 4.9 h for 5). The decrease in dog half-life was
hypothesized to be a result of the added phenyl ring providing a
site of metabolism. Small ring substituents, such as methyl or hal-
ogens, were targeted in an attempt to hinder possible metabolism
(Table 2). First, methyl-substituted phenyl rings were investigated.
Accordingly, the 3-methyl 20 showed a decrease in both potency
and half-life. Both the 4-methyl 19 and 2-methyl 21 had increased
in vitro potency with decreased half-life. The methyl group itself
could be a site of metabolism, therefore the 2-trifluoromethyl ana-
log 22 was examined. This change did not show a significant in-
crease in half-life. A further gain in potency was achieved with
the 2-chloro substituent incorporated in 23 (IC50 = 0.02 nM). An
X-ray co-crystal structure later confirmed this chloro was binding
in the disulfide bridge region as suggested by the original modeling
studies. Compound 23 had a rat half-life similar to that of 5, how-
ever the half-life in the dog was less than 1 h. Several fluoro-substi-
tuted phenyl analogs were examined. Similar to 19, the 4-fluoro
group of 24 increased potency but not half-life. Retaining the 4-flu-
oro group and incorporating a 3-fluoro group in 25 showed in-
creased rat half-life relative to 24, but not to the extent needed
for predicted QD dosing. Finally, the 2,4-difluoro analog 26 demon-
strated an increase in rat half-life (3.3 h) and a substantial increase
in the dog half-life (7.3 h) as determined by IV cassette studies
(12.6 h in single dose dog experiments, Table 3). In addition, the
2,4-difluorophenyl ring was found to increase the potency 5-fold
and 10 lg/kg/min via intravenous administration, respectively.
The relationship between plasma concentrations and thrombus
mass was modeled and 26 was determined to have an
EC50 = 118 nM. Subsequent studies following oral administration
at doses of 10 and 30 mg/kg produced a dose-dependent reduction
in thrombus weight of 56 6% and 84 6% respectively. These re-
sults indicated that 26 produced a reliable dose-dependent anti-
thrombotic effect in the rabbit A–V shunt model of thrombosis
by both intravenous and oral administration.
The species-dependent inhibition of 26 towards purified FXa
was in agreement with the results previously reported for 5.10 Inhi-
bition of human FXa (IC50 = 0.211 nM) by 26 was similar to rabbit
(IC50 = 0.108 nM) but greater than dog (IC50 = 0.887 nM) and rat
(IC50 = 1.32 nM). These results provided further support for the
use of rabbit as an appropriate animal model for the pharmacolog-
ical characterization of this inhibitor.
5. Conclusions
To improve upon previously discovered orally-active,
direct FXa inhibitors, we designed 26, containing a (2R,4S)-4-

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C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
Table 1
In vitro FXa binding, half-life, and 2 Â PT data
R1
O
O
H
N
Y
R3
R2
N
N
O
HN
O
Cl
Compound
R1
R2
R3
Y
FXa IC50 (nM)
Rat t1/2 (h)
2 Â PT (
lM)
clogDd
a
b
5
6
H
H
Me
Me
Et
Et
Et
Et
Ph
Me
Me
H
Me
H
H
Me
Et
H
H
Me
H
Me
H
H
H
H
C–F
N
0.57
4.60
0.54
0.31
0.50
0.18
0.22
0.05
0.08
2.6 (2.0)c
2.0
0.58
0.55
1.08
N.D.
1.39
0.50
N.D.
N.D.
0.73
1.04
0.89
1.53
1.58
2.06
1.47
2.11
2.64
2.34
11
12
13
14
15
16
17
C–F
C–F
C–F
C–F
C–F
C–F
C–F
1.6
1.7
5.2
2.4
1.7
1.4
H
4.0c
a
IC50 values are means of multiple measurements (n P 2) according to Ref. 10.
Determined by rat IV cassette studies unless noted otherwise.
Determined by rat IV singleton studies. Values for 5 were taken from Ref. 10.
b
c
d
clogD values were calculated using ACD/LogD Suite, v. 9.03, Advanced Chemistry Development, Inc: Toronto, Ontario, Canada.
Table 2
In vitro FXa binding, and PK parameters
R
O
HO
H
N
N
N
N
O
HN
O
Cl
Compound
R
FXa IC50 (nM)
t1/2 (h)
Clb (mL/min/kg)
Vdss (mL/kg)
clogDe
a
b
b
5
18
19
20
21
22
23
24
25
26
0.57
0.50
0.18
1.20
0.11
0.34
0.02
0.16
0.08
0.10
2.0c (4.9)d
2.9c (2.9)
1.8
1.4
1.3
1.9 (1.0)
2.9 (0.8)
1.8
2.1 (2.1)
3.3 (7.3)
12c
1100c
1490c
1580
438
468
393
1.04
2.19
2.65
2.65
2.65
2.76
2.79
2.24
2.21
2.33
H
6.73c
10.3
6.78.
5.12
2.89
4.16
9.44
13.1
7.64
4-Me
3-Me
2-Me
2-CF3
2-Cl
4-F
3,4-Di-F
2,4-Di-F
923
1970
1600
1810
a
IC50 values are means of multiple measurements (n P 2) according to Ref. 10.
Determined by rat IV cassette unless otherwise noted, values in parentheses under t1/2 are results of dog IV cassette studies.
Determined by rat IV singleton studies.
Determined by dog IV singleton studies. Values for 5 were taken from Ref. 10.
clog D values were calculated using ACD/Log D Suite, v. 9.03, Advanced Chemistry Development, Inc: Toronto, Ontario, Canada.
b
c
d
e
hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, that showed
a marked increase in half-life in both rats and dogs when com-
pared to our previously described orally efficacious 5.10 The pre-
dicted human half-life of 26 (23 h) modeled for once-daily
dosing with an estimated peak-to-trough ratio of two. In the
rabbit AV shunt model of thrombosis, both IV and oral adminis-
tration of 26 produced a dose-dependent reduction in thrombus
weight.
6. Experimental
6.1. General chemistry
All chemicals, reagents and solvents were purchased from com-
mercialsources(e.g., AldrichChemicalCo., Inc., Milwaukee, WI;Mal-
linckrodt Baker, Inc., Paris, KY, etc.) where available and
used without further purification. All final compounds were

C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
2507
Table 3
Pharmacokinetic parameters of 26 following single-dose administration in male animals
*
Species
Dose (mg/kg)
Route
Cmax (ng/mL)
Tmax (h)
AUCinf (ng h/mL)
Cl (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
Fd (%)
Rata
Rat
0.5 (N = 2)
3c (N = 3)
0.5 (N = 3)
3c (N = 2)
IV
PO
IV
—
236
—
—
1.7
—
978
9.5
—
2.1
—
2.4
—
2.0
—
3.3
—
12.6
—
—
19
—
1010
4870
6450
Dogb
Dog
PO
397
2.0
24
a
Rat species = Sprague Dawley.
Dog species = Beagle.
Dosed as a suspension of the final crystalline form.
F = Absolute oral (PO) bioavailability.
b
c
d
characterized by proton nuclear magnetic spectroscopy (1H NMR)
with a 400 MHz Varian spectrometer and mass spectrometry (MS)
using atmospheric pressure chemical ionization (APCI) or electron
scatter (ES) ionization sources. All final compounds were deter-
mined to be consistent with the proposed structure by 1H NMR
and MS. Microanalyses were performed by Quantitative Technolo-
gies Inc. and were within 0.4% of the calculated values. All final com-
pounds were purified by silica gel flash chromatography. A typical
eluting system consisted of an ethyl acetate (100%)–ethyl acetate/
methanol(90%:10%)gradient. Allfinalcompoundsweregreaterthan
95% pure as determined by analytical reverse-phase HPLC (Vydac
218TP54 C18 column; Mobile phase A: 0.1% TFA in water, B: 0.1%
TFA in acetonitrile). The synthesis for compounds 5 and 6 has been
previously published.10 The synthesis of 14, 15, and 18 exemplifies
the typical conditions utilized for the preparation of 11–26 and is de-
tailed below. For intermediates 8–10, a single compound number
represents several distinct intermediates containing different R1
and R2 groups. Where this occurs, the R groups are defined (e.g., 8,
R1 = ethyl). Alternative conditions for the large scale preparation of
intermediates 8 (R1 = 2,4-difluorophenyl) and 10 (R1 = 2,4-difluoro-
phenyl, R2 = H) are described following the general conditions.
(DMSO-d6, d) 11.10 (s, 1H), 4.48 (t, J = 8.42 Hz, 1H), 3.68–3.85 (m,
1H), 3.56–3.67 (m, 1H), 2.96–3.15 (m, 1H), 2.46–2.51 (m, 1H),
1.35 (m, 9H); MS: APCI (APÀ): 228.1 (MÀH)+.
6.1.2. 1-(tert-Butoxycarbonyl)-4-ethyl-4-hydroxypyrrolidine-2-
carboxylic acid (8, R1 = ethyl)
A solution of 7 (6.3 g, 28 mmol) in 100 mL anhydrous THF under
an argon atmosphere was first cooled to À78 °C, then added a 1 M
solution of ethylmagnesium bromide (60 mL, 60 mmol) in THF
dropwise over 30 min. Stirred the solution at À78 °C for 1 h, then
removed cooling bath and stirred at ambient temperature over-
night. The solution was quenched with 30 mL of satd ammonium
chloride. Acidified the solution with 1.2 M HCl, extracted with
ethyl acetate, dried combined organics with anhydrous magne-
sium sulfate, filtered and concentrated in vacuo. The resulting dark
oil was purified by column chromatography eluting with a gradient
of CHCl3–MeOH–AcOH (98:2:2 to 92:6:2). Combined and concen-
trated desired fractions to yield 8 (R1 = ethyl) as an approximate
50:50 mixture of diastereomers. MS: APCI (APÀ): 258.2 (MÀH)+.
6.1.3. Large scale preparation of (2R,4S)-1-(tert-butoxy
carbonyl)-4-(2,4-difluorophenyl)-4-hydroxypyrrolidine-2-
carboxylic acid 8 (R1 = 2,4-difluorophenyl)
6.1.1. (R)-1-(tert-Butoxycarbonyl)-4-oxopyrrolidine-2-
carboxylic acid (7)
6.1.1.1. Step 1: (2R,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyr-
To a flask equipped with a mechanical stirrer, nitrogen inlet,
and thermocouple was added 2 M isopropyl magnesium bromide
in THF (497 mL, 996 mmol) followed by THF (100 mL). The solution
was cooled to À26 °C and 2,4-difluorobromobenzene in THF (180
mL) was added dropwise maintaining an internal temperature be-
low À14 °C over approximately 30 min. The solution was stirred
for 1 h warming to À7 °C at which point 7 (95 g, 414 mmol) was
charged as a solid in portions while maintaining an internal tem-
perature below 0 °C. After complete addition the reaction was stir-
red at 0 °C for an additional hour then cooled to À30 °C. To the
reaction was added 3 N HCl (240 mL). The aqueous layer was ex-
tracted with THF (250 mL), the organic layers combined and con-
densed to an off white solid which was carried on without
purification.
rolidine-2-carboxylic acid.
A suspension of cis-hydroxy-D-
proline (30.0 g, 229 mmol) in 300 mL THF was cooled to 0 °C, added
1 M NaOH (275 mL, 275 mmol) followed by the portionwise addi-
tion of di-tert-butyl-dicarbonate (49.9 g, 229 mmol). The cooling
bath was removed and the resulting solution was stirred at ambi-
ent temperature overnight. The volume was reduced in vacuo,
acidified with 1 M HCl, then extracted twice with ethyl acetate.
Washed combined organics with brine, dried with anhydrous mag-
nesium sulfate, filtered and concentrated to yield (2R,4R)-1-(tert-
butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid as an oil
that solidifies over time. (42.6 g, 81%) 1H NMR (DMSO-d6, d)
4.11–4.24 (m, 1H), 4.02–4.11 (m, 1H), 3.39–3.54 (m, 1H), 3.02–
3.14 (m, 1H), 2.19–2.37 (m, 1H), 1.72–1.84 (m, 1H), 1.26–1.41
(m, 9H); MS: APCI (APÀ): 230.1 (MÀH)+.
6.1.4. tert-Butyl 4-ethyl-2-((2-fluoro-4-(2-oxopyridin-1(2H)-
yl)phenyl)carbamoyl)-4-hydroxypyrrolidine-1-carboxylate (10,
R1 = ethyl, R2 = H, R3 = H, Y = C–F)
6.1.1.2. Step 2: (R)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-
carboxylic acid (7).
(2R,4R)-1-(tert-Butoxycarbonyl)-4-hydro
The diastereomeric mixture 8 (R1 = ethyl, 0.52 g, 1.9 mmol)
was dissolved in 10 mL chloroform, followed by the addition of
1-(4-amino-3-fluorophenyl)pyridin-2(1H)-one10 (0.39 g, 1.9
mmol), triethylamine (0.8 mL, 5.7 mmol), and EEDQ (0.71 g, 2.9
mmol). The solution was refluxed overnight. The solution was
cooled to ambient temperature, diluted with ethyl acetate,
washed with 1.2 M HCl, satd sodium bicarbonate, brine, dried
with anhydrous magnesium sulfate, filtered and concentrated in
vacuo. Purified resulting material by column chromatography
(0–10% MeOH in ethyl acetate). Combined and concentrated de-
sired fractions to yield 10 (R1 = ethyl, R2 = H, R3 = H, Y = C–F) as
an approximate 50:50 mixture of diastereomers. (0.41 g, 47%)
MS: APCI (APÀ): 458.1 (MÀH)+.
xypyrrolidine-2-carboxylic acid (19.1 g, 82.6 mmol) was then dis-
solved in 400 mL dichloromethane, added trichloroisocyanuric acid
(19.2 g, 82.6 mmol), then cooled the suspension to 0 °C. Added
TEMPO (0.65 g, 4.1 mmol) and continued stirring at 0 °C for
30 min. The cooling bath was removed and the solution was stirred
at ambient temperature for 2.5 h. Added 200 mL water and stirred
for 15 min. The organics were removed in vacuo, diluted with 300
mL ethyl acetate, then filtered through a plug of Celite, washing
with ethyl acetate. The filtrate was acidified with 40 mL of 1M
HCl, separated layers, washed organics with water (4 Â 200 mL),
brine (100 mL), dried with magnesium sulfate, filtered, and con-
centrated to yield 7 as a white solid (18.4 g, 97%): 1H NMR

2508
C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
6.1.5. (2R,4R)-N1-(4-Chlorophenyl)-4-ethyl-N2-(2-fluoro-4-(2-
oxopyridin-1(2H)-yl)phenyl)-4-hydroxypyrrolidine-1,2-
dicarboxamide (14)
utes. Added 9 (R1 = phenyl, R2 = H, 2.47 g, 8.54 mmol) and heated
solution to 100 °C for 5 h. The solution was cooled to ambient tem-
perature then carefully poured into 200 mL ice water. Partitioned
between 200 mL ethyl acetate and 100 mL 1 M HCl, separated lay-
ers, washed organics with 1 M HCl (2 Â 200 mL), re-extracted
aqueous layers with 150 mL ethyl acetate. Combined organic ex-
tracts, washed with brine, dried with anhydrous magnesium sul-
fate, filtered and concentrated in vacuo. Purified crude material
by column chromatography (gradient = 0–10% MeOH in ethyl ace-
tate) Combined and concentrated pure fractions to yield 10
(R1 = phenyl, R2 = H, R3 = H, Y = N) as an off-white solid (2.0 g,
49%): 1H NMR (DMSO-d6, d) 10.24–10.55 (m, 1H), 8.33 (s, 1H),
8.17 (d, J = 8.79 Hz, 1H), 7.80–7.95 (m, 1H), 7.61–7.71 (m, J = 5.62
Hz, 1H), 7.41–7.54 (m, 3H), 7.32 (t, J = 7.45 Hz, 2H), 7.23 (t,
J = 7.32 Hz, 1H), 6.45 (d, J = 8.79 Hz, 1H), 6.29 (t, J = 7.32 Hz, 1H),
5.83–6.00 (m, 1H), 4.39–4.55 (m, 1H), 3.58–3.70 (m, 2H), 2.69
(dd, J = 13.43, 9.77 Hz, 1H), 2.21 (dd, J = 13.43, 3.42 Hz, 1H),
1.23–1.39 (m, 9H); MS: APCI (AP+): 477.2 (M+H)+, (APÀ): 475.2
(MÀH)+.
To 10 (R1 = ethyl, R2 = H,, R3 = H, Y = C–F, 0.45 g, 1.0 mmol) in 20
mL DCM was added 10 mL TFA and stirred at ambient temperature
for 1.5 h. The solution was concentrated in vacuo. Redissolved in 30
mL DCM, cooled to 0 °C, added triethylamine (0.70 mL, 5.1 mmol)
slowly, followed by 4-chlorophenylisocyanate (0.16 g, 1.0 mmol)
which was predissolved in 3 mL DCM. The cooling bath was re-
moved and the solution was stirred at ambient temperature for 1
h. The solution was concentrated in vacuo, redissolved in 150 mL
ethyl acetate, washed with water, 10% citric acid, satd sodium
bicarbonate, brine, dried with anhydrous magnesium sulfate, fil-
tered and concentrated. Purified on a silica gel column eluting with
gradient of 0–10% MeOH in ethyl acetate. Combined and concen-
trated pure fractions to yield 14 (0.21 g, 43%): 1H NMR (DMSO-
d6, d) 9.84 (s, 1H), 8.47 (s, 1H), 8.08 (t, J = 8.66 Hz, 1H), 7.59–7.64
(m, 1H), 7.52–7.57 (m, 2H), 7.45–7.51 (m, 1H), 7.42 (dd, J = 11.59,
2.32 Hz, 1H), 7.23–7.29 (m, 2H), 7.20 (dd, J = 7.81, 2.20 Hz, 1H),
6.41–6.48 (m, 1H), 6.25–6.33 (m, 1H), 5.09 (s, 1H), 4.56 (dd, J =
9.39, 3.29 Hz, 1H), 3.62 (d, J = 9.76 Hz, 1H), 3.43 (d, J = 10.00 Hz,
1H), 2.17–2.27 (m, 1H), 1.98 (dd, J = 13.18, 2.93 Hz, 1H), 1.59 (q,
J = 7.32 Hz, 2H), 0.93 (t, 3H); MS: APCI (AP+): 499.1, 501.1
(M+H)+, (APÀ): 497.1, 499.1 (MÀH)+. Anal. Calcd for
C25H24ClFN4O4Á0.20H2O: C, 60.18; H, 4.85; N, 11.23. Found: C,
59.83; H, 4.70; H, 11.08.
6.1.9. Large scale preparation of (2R,4S)-tert-butyl 4-(2,4-
difluorophenyl)-4-hydroxy-2-((5-(2-oxopyridin-1(2H)-
yl)pyridin2-yl)carbamoyl)pyrrolidine-1-carboxylate 10
(R1 = 2,4-difluorophenyl, R2 = H, R3 = H, Y = N)
1-(6-Aminopyridin-3-yl)pyridin-2(1H)-one (23 g, 120 mmol)
was charged to a 250 mL three-necked, round bottom flask
equipped with a mechanical stirrer, thermocouple, and nitrogen
inlet. To the flask was added anhydrous THF (140 mL) and stirring
begun. The resulting grayish slurry was cooled to an internal tem-
perature of 5 °C and a solution of 1 M lithium hexamethyldisilazide
in THF (120 mL, 120 mmol) was added at a rate to keep the internal
temperature below 10 °C. The resulting slurry was stirred for
25 min warming to 15 °C. The reaction was cooled to À2 °C and 9
(R1 = 2,4-difluorophenyl, 20 g, 61 mmol) was added as a solid in
3 equiv portions keeping the internal temperature below 3 °C.
The mixture was stirred for 1 h at which point the reaction was
quenched with 100 mL of 3 N HCl keeping below 18 °C during
the addition. The reaction was diluted with 185 mL of methyl t-bu-
tyl ether and the organic layer washed successively with 2 Â 60 mL
1N HCl, 60 mL saturated aqueous sodium bicarbonate, and 60 mL
saturated aqueous sodium chloride. The aqueous layers were ex-
tracted with a mixture of 110 mL methyl t-butyl ether/35 mL
THF. The organic layers were combined and dried over anhydrous
magnesium sulfate and condensed in vacuo to yield 10 (R1 = 2,4-
difluorophenyl, R2 = H, R3 = H, Y = N) as a tan solid (32 g, 102%).
This product was used without further purification.
6.1.6. (2R,4S)-1-(tert-Butoxycarbonyl)-4-hydroxy-4-
phenylpyrrolidine-2-carboxylic acid (8, R1 = phenyl, R2 = H)
A solution of 7 (2.90 g, 12.7 mmol) in 30 mL anhydrous THF was
added dropwise to a 1.0 M solution of phenylmagnesium bromide
in tetrahydrofuran (35.0 mL, 35.0 mmol) at 0 °C. After addition was
complete the solution was stirred at 0 °C for another 5 min, then
quenched with 30 mL of satd ammonium chloride. The organics
were removed in vacuo. Partitioned between ethyl acetate and
1.2 M HCl, washed organics with brine, dried with anhydrous mag-
nesium sulfate, then filtered. The organics were concentrated to a
volume of 50 mL, then slowly added hexanes to the stirring solu-
tion. A total of 150 mL hexanes were added. The resulting solid
was filtered off, washing with hexanes to yield 8 (R1 = phenyl) as
a single diastereomer (2.07 g, 53%): 1H NMR (DMSO-d6, d) 7.44
(d, J = 7.56 Hz, 2H), 7.27–7.36 (m, 2H), 7.23 (t, J = 6.83 Hz, 1H),
5.48 (s, 1H), 4.20–4.35 (m, 1H), 3.47–3.65 (m, 2H), 2.54–2.63 (m,
1H), 2.16–2.28 (m, 1H), 1.33–1.41 (m, 9H); MS: APCI (APÀ):
306.2 (MÀH)+.
6.1.7. (1R,4S)-tert-Butyl 6-oxo-4-phenyl-5-oxa-2-aza-
bicyclo[2.2.1]heptane-2-carboxylate (9, R1 = phenyl)
6.1.10. (2R,4S)-N1-(4-Chlorophenyl)-4-hydroxy-N2-(5-(2-
oxopyridin-1(2H)-yl)pyridin-2-yl)- 4-phenylpyrrolidine-1,2-
dicarboxamide (18)
To a solution of 8 (R1 = phenyl, 0.50 g, 1.6 mmol) in 10 mL DMF
was added triethylamine (0.34 mL, 2.4 mmol) and BOP (0.79 g, 1.8
mmol). Stirred at ambient temperature overnight. The solution was
diluted with ethyl acetate, washed with 1 M HCl, sat. sodium bicar-
bonate, brine, dried with anhydrous magnesium sulfate, filtered
and concentrated in vacuo to yield 9 (R1 = phenyl) as a sticky oil
(0.47 g, quant. yield): 1H NMR (DMSO-d6, d) 7.49–7.60 (m, 2H),
7.39–7.49 (m, 3H), 4.63 (s, 1H), 3.76 (d, J = 10.49 Hz, 1H), 3.59
(dd, J = 10.49, 1.71 Hz, 1H), 2.41–2.62 (m, 2H), 1.41 (s, 9H).
A fresh solution of HCl in methanol was prepared by the addi-
tion of acetyl chloride (1.0 mL, 14.5 mmol) to 10 mL anhydrous
methanol at 0 °C under an argon atmosphere. Removed cooling
bath and stirred at ambient temperature for 5 min. This solution
was then transferred via cannula to a flask containing 10 (R1 = phe-
nyl, R2 = H, R3 = H, Y = N, 0.46 g, 0.97 mmol). Stirred reaction at
ambient temperature for 3.5 h. The solution was concentrated in
vacuo. The resulting solid was used directly in the next step. MS:
APCI (AP+): 377.2 (M+H)+, (APÀ): 375.2 (MÀH)+. The solid (0.97
mmol) was dissolved in 25 mL DCM and 10 mL satd sodium bicar-
bonate followed by the addition of 4-chlorophenylisocyanate (0.15
g, 0.97 mmol). The biphasic mixture was stirred at ambient tem-
perature for 2 h. Diluted the mixture with 100 mL DCM, separated
layers, then dried organics with anhydrous magnesium sulfate, fil-
tered and concentrated in vacuo. Purified material by column chro-
matography (gradient = 0–10% MeOH in ethyl acetate). The pure
6.1.8. (2R,4S)-tert-Butyl 4-hydroxy-2-((5-(2-oxopyridin-1(2H)-
yl)pyridin-2-yl)carbamoyl)-4-phenylpyrrolidine-1-carboxylate
(10, R1 = phenyl, R2 = H, R3 = H, Y = N)
1-(6-Aminopyridin-3-yl)pyridin-2(1H)-one10 (4.00 g, 21.3
mmol) was suspended in 75 mL anhydrous toluene under an argon
atmosphere, cooled to 0 °C, then added 2.0 M trimethylaluminum
in toluene (10.7 mL, 21.3 mmol) slowly. After addition the cooling
bath was removed and stirred at ambient temperature for 10 min-

C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
2509
fraction were combined and concentrated in vacuo to yield 18 as a
6.1.14. (2R,4R)-N1-(4-Chlorophenyl)-4-ethyl-N2-(2-fluoro-4-(3-
white solid (0.20 g, 39%): 1H NMR (DMSO-d6, d) 10.33 (s, 1H), 8.55
(s, 1H), 8.37 (d, J = 3.17 Hz, 1H), 8.23 (d, J = 8.78 Hz, 1H), 7.91 (dd,
J = 8.91, 2.81 Hz, 1H), 7.67–7.73 (m, 1H), 7.55–7.62 (m, 4H), 7.48–
7.56 (m, 1H), 7.35–7.43 (m, 2H), 7.24–7.34 (m, 3H), 6.50 (dd,
J = 9.39, 2.07 Hz, 1H), 6.28–6.40 (m, 1H), 5.95 (s, 1H), 4.73 (dd,
J = 9.64, 2.56 Hz, 1H), 3.98 (d, J = 10.00 Hz, 1H), 3.86 (d, J = 10.00
Hz, 1H), 2.77 (dd, J = 13.05, 9.88 Hz, 1H), 2.27–2.38 (m, 1H). Anal.
calcd for C28H24ClN5O4Á0.65H2O: C, 62.08; H, 4.71; N, 12.93. Found:
C, 62.00; H, 4.59; N, 12.63.
methyl-2-oxopyridin-1(2H)-yl)phenyl)-4-hydroxypyrrolidine-
1,2-dicarboxamide (13)
This compound was synthesized in a similar manner to 14
(0.090 g, 22%): 1H NMR (DMSO-d6, d) 9.85 (s, 1H), 8.49 (s, 1H),
8.10 (t, J = 8.66 Hz, 1H), 7.57 (d, 2H), 7.35–7.52 (m, 3H), 7.28 (d,
J = 8.78 Hz, 2H), 7.21 (d, J = 9.03 Hz, 1H), 6.23 (t, J = 6.83 Hz, 1H),
5.12 (s, 1H), 4.58 (dd, J = 9.64, 3.05 Hz, 1H), 3.64 (d, J = 9.27 Hz,
1H), 3.45 (d, J = 10.00 Hz, 1H), 2.18–2.29 (m, 1H), 2.03 (s, 3H),
1.95–2.01 (m, 1H), 1.61 (q, J = 7.40 Hz, 2H), 0.95 (t, 3H); MS: APCI
(AP+): 513.1,515.1 (M+H)+, (AP-): 511.1,513.1 (M-H)+. Anal. Calcd
for C26H26ClFN4O4Á1.85H2O: C, 57.16; H, 5.48; N, 10.26. Found: C,
56.77; H, 5.43; N, 10.19.
6.1.11. (2R,4R)-N1-(4-Chlorophenyl)-4-ethyl-N2-(2-fluoro-4-(2-
oxopyridin-1(2H)-yl)phenyl)-4-methoxypyrrolidine-1,2-
dicarboxamide (15)
The 50:50 diastereomeric mixture 8 (R1 = ethyl, 0.50 g, 1.9
mmol) was dissolved in 10 mL anhydrous THF under an argon
atmosphere, cooled to 0 °C, then added sodium hydride (60% in
mineral oil, 0.19 g, 4.8 mmol) in portions. Removed cooling bath
and stirred at ambient temperature for 15 min. Added methylio-
dide (0.24 mL, 3.9 mmol) in one portion. Stirred the reaction at
ambient temperature overnight. Some starting material was ob-
served by MS. Recooled solution to 0 °C, added methyliodide
(0.25 mL, 4.0 mmol) followed by sodium hydride (0.1 g, 2.5 mmol).
Stirred at ambient temperature overnight. The solution was care-
fully quenched with 1.2 M HCl, extracted with ethyl acetate, dried
with anhydrous magnesium sulfate, filtered and concentrated in
vacuo to yield a dark oil that was carried on without purification.
MS: APCI (APÀ): 272.1 (MÀH)+. This material was further elabo-
rated to 15 in a similar manner as 14 (0.11 g, 24%): 1H NMR
(DMSO-d6, d) 9.54 (s, 1H), 8.51 (s, 1H), 8.00 (t, J = 8.66 Hz, 1H),
7.64 (dd, J = 7.08, 1.71 Hz, 1H), 7.55–7.60 (m, 2H), 7.47–7.54 (m,
1H), 7.44 (dd, J = 11.59, 2.32 Hz, 1H), 7.26–7.33 (m, 2H), 7.17–
7.25 (m, 1H), 6.47 (d, J = 9.27 Hz, 1H), 6.27–6.35 (m, 1H), 4.58
(dd, J = 9.39, 3.05 Hz, 1H), 3.77 (d, J = 10.25 Hz, 1H), 3.44 (d,
J = 10.49 Hz, 1H), 3.10 (s, 3H), 2.30 (dd, J = 13.66, 2.93 Hz, 1H),
2.09–2.19 (m, 1H), 1.57–1.79 (m, 2H), 0.88 (t, J = 7.44 Hz, 3H);
MS: APCI (AP+): 513.1, 515.1 (M+H)+, (APÀ): 511.1,513.1 (MÀH)+.
Anal. Calcd for C26H26ClFN4O4Á0.17H2O: C, 60.52; H, 5.14; N,
10.86. Found: C, 60.17; H, 5.06; N, 10.60.
6.1.15. (2R,4R)-N1-(4-Chlorophenyl)-4-ethoxy-4-ethyl-N2-(2-
fluoro-4-(2-oxopyridin-1(2H)-yl)phenyl)pyrrolidine-1,2-
dicarboxamide (16)
This compound was synthesized in a similar manner to 14. 1H
NMR (DMSO-d6, d) 9.50 (s, 1H), 8.51 (s, 1H), 8.03 (t, J = 8.66 Hz,
1H), 7.59–7.64 (m, 1H), 7.52–7.58 (m, 2H), 7.45–7.51 (m, 1H),
7.42 (dd, J = 11.59, 2.32 Hz, 1H), 7.25–7.31 (m, 2H), 7.17–7.23 (m,
1H), 6.45 (dd, J = 9.27, 2.20 Hz, 1H), 6.24–6.31 (m, 1H), 4.57 (dd,
J = 9.27, 2.93 Hz, 1H), 3.73 (d, J = 10.49 Hz, 1H), 3.42 (d, J = 10.49
Hz, 1H), 2.31 (dd, J = 13.05, 2.56 Hz, 1H), 2.11 (dd, J = 13.30, 9.39
Hz, 1H), 1.67–1.75 (m, 2H), 1.56–1.65 (m, 2H), 0.97 (t, J = 6.83
Hz, 3H), 0.87 (t, J = 7.44 Hz, 3H); MS: APCI (AP+): 527.2,529.2
(M+H)+, (APÀ): 525.2,527.2 (MÀH)+; HRMS ([M+H]+) for
C27H28ClFN4O4: calcd 527.1856; found 527.1852.
6.1.16. (2R,4S)-N1-(4-Chlorophenyl)-N2-(2-fluoro-4-(2-
oxopyridin-1(2H)-yl)phenyl)-4-hydroxy-4-phenylpyrrolidine-
1,2-dicarboxamide (17)
This compound was synthesized in a similar manner to 18. 1H
NMR (DMSO-d6, d) 9.88 (d, J = 1.22 Hz, 1H), 8.54 (s, 1H), 8.16 (t,
J = 8.66 Hz, 1H), 7.65 (dd, J = 7.20, 1.83 Hz, 1H), 7.54–7.61 (m,
4H), 7.43–7.55 (m, 2H), 7.40 (t, J = 7.56 Hz, 2H), 7.26–7.34 (m,
3H), 7.21–7.26 (m, 1H), 6.48 (d, J = 8.78 Hz, 1H), 6.27–6.35 (m,
1H), 4.73 (dd, J = 9.64, 2.81 Hz, 1H), 3.92–4.00 (m, 1H), 3.85 (d,
J = 10.00 Hz, 1H), 2.76 (dd, J = 12.93, 9.76 Hz, 1H), 2.35 (dd,
J = 12.93, 2.20 Hz, 1H); MS: APCI (AP+): 547.1,549.0 (M+H)+,
(AP-): 545.0,547.0 (M-H)+. Anal. Calcd C29H24ClFN4O4Á0.39 ethyl
acetate: C, 63.14, H, 4.70; N, 9.64. Found: C, 62.74, H, 4.45; N,
9.62.
6.1.12. (2R,4R)-N1-(4-Chlorophenyl)-N2-(2-fluoro-4-(3-methyl-
2-oxopyridin-1(2H)-yl)phenyl)-4-hydroxy-4-
methylpyrrolidine-1,2-dicarboxamide (11)
This compound was synthesized in a similar manner to 14. 1H
NMR (DMSO-d6, d) 9.85 (s, 1H), 8.46 (s, 1H), 8.06 (t, J = 8.78 Hz,
1H), 7.54 (d, 2H), 7.35–7.47 (m, 3H), 7.25 (d, J = 9.0 Hz, 2H), 7.18
(m, 1H), 6.21 (t, J = 6.83 Hz, 1H), 5.28 (s, 1H), 4.55 (dd, J = 9.03,
4.15 Hz, 1H), 3.61 (d, J = 9.52 Hz, 1H), 3.43 (d, J = 9.52 Hz, 1H),
2.20–2.25 (m, 1H), 2.03 (m, 4H), 1.31 (s, 3H); MS: APCI (AP+):
499.1,501.1 (M+H)+, (APÀ): 497.1,499.1 (MÀH)+. Anal. Calcd for
C25H24ClFN4O4Á0.14H2O: C, 59.88; H, 4.88; N, 11.17. Found: C,
59.85; H, 4.84; N, 11.09.
6.1.17. (2R,4S)-N1-(4-Chlorophenyl)-4-hydroxy-N2-(5-(2-
oxopyridin-1(2H)-yl)pyridin-2-yl)-4-p-tolylpyrrolidine-1,2-
dicarboxamide (19)
This compound was synthesized in a similar manner to 18 (0.13
g, 37%): 1H NMR (CDCl3, d) 10.58 (br s, 1H), 8.40 (d, 1H), 8.20 (d,
1H), 7.70 (d, 1H), 7.40–7.12 (m, 9H), 7.00 (br s, 1H), 6.62 (d, 1H),
6.30 (t, 1H), 5.28 (d, 1H), 4.84 (d, 1H), 3.92 (d, 1H), 3.78 (d, 1H),
2.79–2.60 (m, 2H), 2.34 (s, 3H); MS (ES+): 544.04 (M+H)+. Anal.
calcd for C29H26ClFN5O4Á0.5H2O: C, 62.99, H, 4.92, N, 12.66. Found:
C, 63.04, H, 4.67; N, 12.28.
6.1.13. (2R,4R)-N1-(4-Chlorophenyl)-N2-(2-fluoro-4-(2-
oxopyridin-1(2H)-yl)phenyl)-4-methoxy-4-methylpyrrolidine-
1,2-dicarboxamide (12)
6.1.18. (2R,4S)-N1-(4-Chlorophenyl)-4-hydroxy-N2-(5-(2-
oxopyridin-1(2H)-yl)pyridin-2-yl)-4-m-tolylpyrrolidine-1,2-
dicarboxamide (20)
This compound was synthesized in a similar manner to 18 (0.21
g, 35%): 1H NMR (CDCl3, d) 10.50 (br s, 1H), 8.35 (d, 1H), 8.26 (s,
1H), 7.73 (m, 1H), 7.44 (m, 1H), 7.35–7.11 (m, 9H), 6.90 (br s,
1H), 6.63 (d, 1H), 6.28 (m, 1H), 5.24 (s, 1H, OH), 4.91 (d, 1H),
3.90 (d, 1H), 3.79 (d, 1H), 2.70 (m, 2H), 2.35 (s, 3H); MS (ES+):
544.01 (M+H)+. Anal. Calcd for C29H26ClN5O4: C, 64.03, H, 4.82, N,
12.87. Found: C, 63.58, H, 4.89, N, 12.60.
This compound was synthesized in a similar manner to 14. 1H
NMR (DMSO-d6, d) 9.57 (s, 1H), 8.49 (s, 1H), 7.98 (t, J = 8.54 Hz,
1H), 7.61 (m, 1H), 7.55 (d, J = 9.02 Hz, 2H), 7.40–7.50 (m, 2H),
7.27 (d, J = 9.03 Hz, 2H), 7.18 (m, 1H), 6.45 (m, 1H), 6.28 (t,
J = 5.37 Hz, 1H), 4.56 (m, 1H), 3.61 (d, J = 10.25 Hz, 1H), 3.45 (d,
J = 10.00 Hz, 1H), 3.29 (s, 3H), 2.50 (m, 1H), 2.25 (m, 1H), 1.31 (s,
3H); MS: APCI (AP+): 499.1, 501.1 (M+H)+, (APÀ): 497.1,499.1
(MÀH)+. Anal. Calcd for C25H24ClFN4O4Á0.32H2O: C, 59.49; H,
4.92; N, 11.10. Found: C, 59.10; H, 4.85; N, 10.96.

2510
C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
6.1.19. (2R,4S)-N1-(4-Chlorophenyl)-4-hydroxy-N2-(5-(2-
oxopyridin-1(2H)-yl)pyridin-2-yl)-4-o-tolylpyrrolidine-1,2-
dicarboxamide (21)
This compound was synthesized in a similar manner to 18
(0.148 g, 49%): 1H NMR (CDCl3, d) 8.35 (m, 2H), 7.84 (dd, 1H),
7.62 (m, 2H), 7.45 (m, 3H), 7.20 (m, 4H), 6.63 (dd, 1H), 6.48 (ddd,
1H), 4.76 (dd, 1H), 4.32 (dd, 1H), 3.91 (d, 1H), 2.97 (dd, 1H), 2.74
(d, 1H); MS (ES+): 544 (M+H)+. Anal. Calcd for
C29H26N5O4ClÁ0.6CHCl3: C, 57.74, H, 4.36, N, 11.37. Found: C,
57.88, H, 4.13, N, 11.34.
8.2 (d, J = 9.0 Hz, 1H), 7.9 (dd, J = 8.9, 2.6 Hz, 1H), 7.6 (dd, J = 6.9,
1.5 Hz, 1H), 7.5 (d, J = 9.0 Hz, 2H), 7.5 (dd, 1H), 7.2 (d, J = 9.0 Hz,
2H), 7.2 (d, J = 9.0 Hz, 1H), 7.1 (m, 1H), 6.4 (d, J = 9.2 Hz, 1H), 6.0
(s, 1H), 6.3 (m, 1H), 4.7 (dd, J = 9.0, 1.4 Hz, 1H), 3.9 (m, 2H), 2.8
(m, 1H), 2.4 (d, J = 14.0 Hz, 1H); MS: APCI (AP+): 566.0, 568.0
(M+H)+. Anal. Calcd for C28H22ClF2N5O4Á0.40H2O: C, 58.67, H,
4.01, N, 12.22. Found: C, 58.29, H, 3.89, N, 12.12.
6.2. Enzyme inhibition assays
All enzyme inhibition assays (FXa, thrombin, trypsin, activated
protein C, plasmin, and tissue plasminogen activator) were per-
formed as previously described.10
6.1.20. (2R,4S)-N1-(4-Chlorophenyl)-4-hydroxy-N2-(5-(2-
oxopyridin-1(2H)-yl)pyridin-2-yl)-4-(2-(trifluoromethyl)
phenyl)pyrrolidine-1,2-dicarboxamide (22)
This compound was synthesized in a similar manner to 18
(0.215 g, 39%): 1H NMR (CDCl3, d) 10.40 (br s, 1H), 8.35 (d, 1H),
8.31 (d, 1H), 7.82 (d, 1H), 7.75 (dd, 1H), 7.71 (d, 1H), 7.57 (t, 1H),
7.47 (t, 1H), 7.45–7.18 (m, 6H), 6.74 (br s, 1H), 6.64 (d, 1H), 6.27
(t, 1H), 4.97 (d, 1H), 4.91 (s, 1H, OH), 4.12 (d, 1H), 3.89 (d, 1H),
2.99 (d, 1H), 2.85 (dd, 1H); MS (ES+): 562.12 (M+H)+. Anal. Calcd
for C29H23ClF3N5O4: C, 58.25, H, 3.88, N, 11.71. Found: C, 57.90,
H, 3.69, N, 11.50.
6.3. X-ray crystallography
X-ray diffraction data from the crystals were collected at the
Advanced Photon Source facility on beamline 17ID. Intensity data
were measured at about À180 °C. One X-ray data set was collected
for each crystal. Auto-indexing and processing of the measured
intensity data were carried out with the HKL2000 software pack-
age.24 The intensity data-collection statistics are summarized in
Supplementary data Table 1 (see Supplementary data). The crystal
structures of FXa complexes were solved by molecular replace-
6.1.21. (2R,4S)-4-(2-Chlorophenyl)-N1-(4-chlorophenyl)-4-
hydroxy-N2-(5-(2-oxopyridin-1(2H)-yl)pyridin-2-yl)pyrrolidine-
1,2-dicarboxamide (23)
25
ment using MOLREP with the FXa protein coordinates (PDB code
2PHB) as the search model. The molecular replacement solution
This compound was synthesized in a similar manner to 18
(0.348 g, 40%): 1H NMR (CD3OD, d 8.35 (m, 2H), 7.85 (dd, 1H),
7.75 (dd, 1H), 7.60 (m, 2H), 7.45 (m, 4H), 7.34 (m, 2H), 7.22 (m,
2H), 6.62 (dd, 1H), 6.47 (ddd, 1H), 4.90 (m, 1H), 4.28 (d, 1H), 4.15
(d, 1H), 3.28 (dd, 1H), 2.60 (d, 1H); MS (ES+): 564 (M+H)+. Anal.
Calcd for C28H23N5O4Cl2Á0.7CHCl3: C, 53.19, H, 3.69, N, 10.80.
Found: C, 52.92, H, 3.43, N, 10.61.
was further optimized by rigid-body, coordinates and B-value min-
26
imization using REFMAC
.
Calculated (2Fo–Fc) and (Fo–Fc) electron
density maps were utilized for interactive fitting of protein struc-
tures into electron density using the COOT software program.27
Placement of the ligands into electron density maps was carried
out with X-LIGAND28 implemented in QUANTA (Accelrys Inc.).
The coordinates and structure factors may be found in the Protein
Data Bank under ID code 2W3K for 17 and 2W3I for 26.
6.1.22. (2R,4S)-N1-(4-Chlorophenyl)-4-(4-fluorophenyl)-4-
hydroxy-N2-(5-(2-oxopyridin-1(2H)-yl)pyridin-2-yl)pyrrolidine-
1,2-dicarboxamide (24)
Acknowledgements
This compound was synthesized in a similar manner to 18. 1H
NMR (DMSO-d6, d) 10.35 (s, 1H), 8.56 (s, 1H), 8.37 (d, J = 2.69 Hz,
1H), 8.22 (d, J = 8.79 Hz, 1H), 7.87–7.94 (m, J = 8.91, 2.56 Hz, 1H),
7.66–7.73 (m, J = 6.72, 2.08 Hz, 1H), 7.48–7.64 (m, 6H), 7.17–7.33
(m, 5H), 6.50 (d, J = 8.79 Hz, 1H), 6.35 (t, J = 7.45 Hz, 1H), 6.02 (s,
1H), 4.72 (d, J = 9.52 Hz, 1H), 3.96 (d, J = 10.01 Hz, 1H), 3.84 (d,
J = 9.77 Hz, 1H), 2.70–2.81 (m, 1H), 2.25–2.36 (m, J = 13.43, 1.71
Hz, 1H); MS: APCI (AP+): 548.0, 551.0 (M+H)+, (APÀ): 546.0,
548.0 (MÀH)+. Anal. Calcd for C28H23ClFN5O4Á0.23H2O: C, 60.91,
H, 4.28, N, 12.68. Found: C, 60.52, H, 4.13, N, 12.46.
The authors would like to thank John Bryant and Kathy Welch
for their in vitro biology support, Erli Zhang for collecting the X-
ray crystallography data, Mark Hadd for the large scale synthesis
of 26.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.01.063.
References and notes
6.1.23. (2R,4S)-N1-(4-Chlorophenyl)-4-(3,4-difluorophenyl)-4-
hydroxy-N2-(5-(2-oxopyridin-1(2H)-yl)pyridin-2-yl)pyrrolidine-
1,2-dicarboxamide (25)
1. Eriksson, B. I.; Quinlan, D. J. Drugs 2006, 66, 1411.
2. Casimiro-Garcia, A.; Dudley, D. A.; Heemstra, R. J.; Filipski, K. J.; Bigge, C. F.;
Edmunds, J. J. Exp. Opin. Ther. Pat. 2006, 16, 119.
This compound was synthesized in a similar manner to 18 (0.35 g,
26%): 1H NMR (DMSO-d6, d) 10.35 (s, 1H), 8.55 (s, 1H), 8.37 (d, 1H),
8.22 (d, J = 9.28 Hz, 1H), 7.91 (dd, J = 8.79, 2.69 Hz, 1H), 7.70 (dd,
J = 6.84, 1.95 Hz, 1H), 7.39–7.66 (m, 6H), 7.26–7.32 (m, 2H), 6.46–
6.54 (m, J = 9.28 Hz, 1H), 6.32–6.37 (m, 1H), 6.12 (s, 1H), 4.74 (dd,
J = 9.65, 2.56 Hz, 1H), 3.81–3.98 (m, 2H), 2.76 (dd, J = 12.70, 9.77 Hz,
1H), 2.28–2.36 (m, 1H); MS: APCI (AP+): 566.0, 568.0 (M+H)+, (AP-):
564.0, 565.0 (MÀH)+. Anal. Calcd for C28H22ClF2N5O4Á0.38H2O: C,
58.71, H, 4.01, N, 12.23. Found: C, 58.67, H, 3.66, N, 12.01.
3. Gustafsson, D. J. Intern. Med. 2003, 254, 322.
4. (a) ExantaÒ (ximelagatran) Tablets NDA 21-686, FDA Advisory Committee
Briefing Document, 10 September 2004, Cardiovascular and Renal Drugs
Advisory Committee, Center for Drug Evaluation & Research. Washington, D.C.
Available from URL: http://www.fda.gov/ohrms/dockets/ac/04/briefing/2004-
4069B1_01_AstraZeneca-Backgrounder.pdf.; (b) Press Release, February 14th,
2006. Available from URL: http://astrazeneca.com/pressrelease/5217.aspx.
5. Spyropoulos, A. C. Exp. Opin. Invest. Drugs 2007, 16, 431.
6. (a) RE-NOVATE: a randomized, double-blind study to investigate the efficacy
and safety of two different dose regimens of dabigatran etexilate compared to
subcutaneous enoxaparin 40 mg once daily for 28–35 days, in prevention of
venous thromboembolism after primary elective total hip replacement surgery.
Available from URL: http://www.clinicaltrials.gov/ct/gui/show/NCT00168818.;
(b) RE-MODEL (thromboembolism prevention after knee surgery): two
different dose regimens of orally administered dabigatran etexilate capsules
[150 or 220 mg once daily starting with a half dose (i.e., 75 or 110 mg) on the
day of surgery] compared to subcutaneous enoxaparin 40 mg once daily for 6–
10 days. Available from URL: http://www.clinicaltrials.gov/ct/gui/show/
NCT00168805.; (c) RE-MOBILIZE: a phase III study investigating the efficacy
6.1.24. (2R,4S)-N1-(4-Chlorophenyl)-4-(2,4-difluorophenyl)-4-
hydroxy-N2-(5-(2-oxopyridin-1(2H)-yl)pyridin-2-yl)pyrrolidine-
1,2-dicarboxamide (26)
This compound was synthesized in a similar manner to 18. 1H
NMR (DMSO-d6, d) 10.3 (s, 1H), 8.5 (s, 1H), 8.3 (d, J = 2.3 Hz, 1H),

C. A. Van Huis et al. / Bioorg. Med. Chem. 17 (2009) 2501–2511
2511
and safety of two different dose regimens of dabigatran etexilate compared to
enoxaparin in prevention of venous thromboembolism in patients with
primary elective total knee replacement. Available from URL: http://
www.clinicaltrials.gov/ct/gui/show/NCT00152971.
14. Franchini, C.; Corbo, F.; Lentini, G.; Bruno, G.; Scilimati, A.; Tortorella, V.;
Camerino, D. C.; De Luca, A. J. Med. Chem. 2000, 43, 3792.
15. Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 18, 4171.
16. (a) Furukawa, N.; Shibutani, T.; Fujihara, H. Tetrahedron Lett. 1987, 28, 2727; (b)
Coe, P. L.; Waring, A. J.; Yarwood, T. D. J. Chem. Soc., Perkin Trans. 1 1995, 2729.
17. Abarbi, M.; Dehmel, F.; Knochel, P. Tetrahedron Lett. 1999, 40, 7449.
18. (a) Chan, C.; Borthwick, A. D.; Brown, D.; Burns-Kurtis, C. L.; Campbell, M.;
Chaudry, L.; Chung, C.; Convery, M. A.; Hamblin, J. N.; Johnstone, L.; Kelly, H. A.;
Kleanthous, S.; Patikis, A.; Patel, C.; Pateman, A. J.; Senger, S.; Shah, G. P.;
Toomey, J. R.; Watson, N. S.; Weston, H. E.; Whitworth, C.; Young, R. J.; Zhou, P.
J. Med. Chem. 2007, 50, 1546; (b) Imaeda, Y.; Kuroita, T.; Sakamoto, H.;
Kawamoto, T.; Tobisu, M.; Konishi, N.; Hiroe, K.; Kawamura, M.; Tanaka, T.;
Kubo, K. J. Med. Chem. 2008, 51, 3422.
19. (a) Ward, K. W.; Smith, B. R. Drug Metab. Dispos 2004, 32, 603; (b) Ward, K. W.;
Smith, B. R. Drug Metab. Dispos. 2004, 32, 612.
20. CYP inhibitory activities were determined by a reported method: Zientek, M.;
Miller, H.; Smith, D.; Dunklee, M. B.; Heinle, L.; Thurston, A.; Lee, C.; Hyland, R.;
Fahmi, O.; Burdette, D. J. Pharm. Tox. Methods 2008, 58, 206.
7. Karnicki, K.; McBane, R.; Miller, R. S.; Leadley, R. J., Jr.; Morser, J.; Owen, W. G.;
Chesebro, J. H. J. Thrombosis Haemostasis 2004, 2, 2162.
8. (a) Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, J.; Schlemmer,
K-H.; Reinemer, P.; Perzborn, E. J. Med. Chem. 2005, 48, 5900; (b) Eriksson, B. I.;
Borris, L. C.; Dahl, O. E.; Haas, S.; Huisman, M. V.; Kakkar, A. K.; Muehlhofer, E.;
Dierig, C.; Misselwitz, F.; Kälebo, P. Circulation 2006, 114, 2374.
9. (a) Lassen, M. R.; Davidson, B. L.; Gallus, A.; Pineo, G.; Ansell, J.; Deitchman, D.
Blood 2006, 108, 574; (b) Luettgen, J. M.; Bozarth, T. A.; Bozarth, J. M.; Barbera,
F. A.; Lam, P. Y.; Quan, M. L.; Pinto, D. J.; Wexler, R. R.; Rendina, A. R.; Knabb, R.
M. Blood 2006, 108, 4130.
10. (a) Kohrt, J. T.; Bigge, C. F.; Bryant, J. W.; Casimiro-Garcia, A.; Chi, L.; Cody, W.
L.; Dahring, T.; Dudley, D. A.; Filipski, K. J.; Haarer, S.; Heemstra, R.; Janiczek, N.;
Narasimhan, L.; McClanahan, T.; Peterson, J. T.; Sahasrabudhe, V.; Schaum, R.;
Van Huis, C. A.; Welch, K. M.; Zhang, E.; Leadley, R. J.; Edmunds, J. J. Chem. Biol.
Drug Des. 2007, 69, 100; (b) Bigge, C. F.; Dudley, D. A.; Edmunds, J. J.; Van Huis,
C. A.; Casimiro-Garcia, A.; Filipski, K. J.; Kohrt, J. T. U.S. Patent 7,030,141, 2006.
11. For our first report in this series, see the following: Van Huis, C. A.; Bigge, C. F.;
Casimiro-Garcia, A.; Cody, W. L.; Dudley, D. A.; Filipski, K. J.; Heemstra, R. J.;
Kohrt, J. T.; Narasimhan, L. S.; Schaum, R. P.; Zhang, E.; Bryant, J. W.; Haarer, S.;
Janiczek, N.; Leadley, R. J., Jr.; McClanahan, T.; Peterson, J. T.; Welch, K. M.;
Edmunds, J. J. Chem. Biol. Drug Des. 2007, 69, 444.
21. Hagmann, W. K. J. Med. Chem. 2008, 51, 4359.
22. Müller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
23. Wong, P. C.; Quan, M. L.; Crain, E. J.; Watson, C. A.; Wexler, R. R.; Knabb, R. M. J.
Pharmacol. Exp. Ther. 2000, 292, 351.
24. Otwinoski, Z.; Minor, W.. In Methods in Enzymology; Carter, C. W., Jr., Sweet, R.
M., Eds.; Academic Press: New York, 1997; Vol. 276A, pp 307–326.
25. Vagin, A.; Teplyakov, A. J. Appl. Crystallogr. 1997, 30, 1022.
12. Basic assumptions and calculation for Cmax/Cmin: (Vdss = 1 L/kg, Ka = 0.7 hÀ1
F = 100%).
,
26. Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr. 1997, D53, 240.
27. Emsley, P.; Cowtan, K. Acta Crystallogr. 2004, D60, 2126.
13. De Luca, L.; Giacomelli, G.; Porcheddu, A. Org. Lett. 2001, 3, 3041.
28. Oldfield, T. J. Acta Crystallogr. 2001, D57, 696.