Structure based drug design: Development of potent and selective factor IXa (FIXa) inhibitor

Article abstract of DOI:10.1021/jm901476x

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 β and S2-S4 sites. A number of highly selective and potent, factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

Full text of DOI:10.1021/jm901476x

J. Med. Chem. 2010, 53, 1473–1482 1473
DOI: 10.1021/jm901476x
Structure Based Drug Design: Development of Potent and Selective Factor IXa (FIXa) Inhibitors†
Shouming Wang,*,‡ Richard Beck,§ Andrew Burd,‡ Toby Blench,‡ Frederic Marlin,‡ Tenagne Ayele,‡ Stuart Buxton,‡
Claudio Dagostin,‡ Maja Malic,‡ Rina Joshi,‡ John Barry,‡ Mohammed Sajad,‡ Chiming Cheung,‡ Shaheda Shaikh,‡
Suresh Chahwala,‡,§ Chaman Chander,§ Christine Baumgartner, Hans-Peter Holthoff, Elizabeth Murray,‡
Michael Blackney,§ and Amanda Giddings§
‡Department of Medicinal Chemistry, and §Department of Pharmacology, Trigen Ltd., Emmanuel Kaye Building, 1B Manresa Road,
London SW3 6LR, U.K., and Trigen AG, Martinsried, Munich, Germany
Received October 8, 2009
On the basis of our understanding on the binding interactions of the benzothiophene template within the
FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR
strategy by targeting the 4-position of the template to access the S1 β and S2-S4 sites. A number of
highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of
functional groups with conformational changes toward the S2-S4 sites.
FIXaa is a vitamin K-dependent blood coagulation factor
that is essential for the amplification or consolidation phase of
blood coagulation.1,2 Its role in maintaining internal hemos-
tasis in the intrinsic pathway of the clotting cascade sets it
apart from other serine protease enzymes as an alternative
therapeutic target.3 Development of selective inhibitors of
FIXa may provide clinicians safe and effective anticoagulants
in attenuating thrombosis, particularly for myocardial infarc-
tion, ischemic diseases, and common procedures such as
percutaneous coronary intervention, hemodialysis, blood
pheresis, cardiac valve replacement, and extracorporeal cir-
culatory support systems that incite coagulation.4
compound 2, may allow the occupation of the S1 β pocket by
one of the branched groups (R1), while the other (R2) may
point toward the S2-S4 pockets. Notably, simple 4-alkyloxy-
benzothiophene analogues have been demonstrated to show
moderate activity in our early studies. Here, we disclose our
identification of highly potent and selective FIXa inhibitors
through structure based drug design.
Previously, we have reported our early SAR studies of the
benzothiophene template, resulting in a number of highly
potent FIXa inhibitors, though with moderate selectivities
against other serine protease enzymes, such as FXa.5 X-ray
study on one of the analogues, compound 1, revealed dis-
tinctive induced S1 β site opening and a secondary binding of
the molecule in the S3-S4 sites which was believed to be
closed in previous X-ray studies with different ligands.6 Close
examination of the X-ray structure of the 5-aryl substituted
benzothiophene compound 1 revealed the aryl group lies
outside of the S1 β pocket; e.g., the pocket is unoccupied even
though it has been induced to open by the ligand. After a series
of molecular modeling studies on the various substituted
analogues derived from benzothiophene template, in combi-
nation with the knowledge that the S2-S4 sites may be used
for binding interactions, we envisaged that a branched
alkyloxy substitution at 4-position on the template, such as
Chemistry
The general synthesis of 4-substituted benzo[b]thiophene-2-
carboximidine analogues is shown in Scheme 1. Thus, 4-
hydroxy-2-Boc-amidine intermediate 3, prepared according
to the previous report,5 was alkylated with methyl R-bromoa-
cetate and K2CO3 in DMF to give the branched 4-alkoxy
esters 4, which were either reduced to the corresponding
alcohols 5 by LiAlH4 and NaBH4 or converted to the
amides 6 with various amines and HATU/HBTU, after ester
hydrolysis to the acids. The intermediate alcohols 5 were
converted into carbamates 7 via disuccinimidyl carbonate
(DSC) coupling with amines, followed by deprotection of
the Boc amidines by TFA. They could also be converted to the
triflates 8 with trifluoroacetic anhydride and triethylamine,
which was displaced with azide, followed by triphenyl phos-
phine to give the primary amines 9. Similarly, direct amine
displacement of the triflates 8 gave alkylamines 11 after Boc
removal by TFA. The primary amines 9 were treated with
isocyanates, chlorocarboxamides, or chlorocarboxylates to
give ureas or carbamates 10 after deprotection of the t-Boc-
amidine group by TFA. Scheme 2 describes the synthesis of
one example of the amide bioisosteres, oxadiazole 16.
Mandelic acid 12 and hydroxyamidine 13 were heated with
†PDB code for compound 16 cocrystallized with human N-terminally
truncated FIXa is 3LC5.
*To whom correspondence should be addressed. Phone/fax:
þ441628672546. E-mail: shouming_wang@yahoo.co.uk.
a Abbreviations: FIXa, factor IXa, a serine protease enzyme within
the intrinsic pathway of the blood coagulation cascade; uPA, urokinase
plasminogen activator; SAR, structure-activity relationship; FXa,
factor Xa, a serine protease enzyme in the blood coagulation cascade;
aPTT, activated partial thromboplastin time; PT, prothrombin time;
TT, thrombin time.
r
2010 American Chemical Society
Published on Web 02/02/2010
pubs.acs.org/jmc

1474 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
Scheme 1a
a Reagents: (a) (R1)CHBrCO2Me, K2CO3, DMF, room temp, 86%; (b) (i) LiAlH4, THF, -78 ꢀC; (ii) NaBH4, 97%; (c) RNH2, DSC, TEA, DCM,
60 ꢀC; (d) TFA, DCM); (e) LiOH, THF; (f) RNH2, HATU or HBTU, DIPEA, DMF; (g) Tf2O,TEA, DCM, -78 ꢀC; (h) (i) NaN3, DMF; (ii) PPh3, THF,
H2O; (j) RNH2, THF; (k) RNCO or RXCOCl (X = N, O).
Scheme 2a
a Reagents: (a) CDI, diglyme, 90 ꢀC; (b) 3, DEAD, PPh3, THF, 0 ꢀC to room temp, 15%; (c) TFA, DCM, 68%.
CDI in diglyme to give the hydroxyoxazole intermediate 14,
which was coupled with compound 3 under Mitsunobu
condition, yielding compound 15. Compound 16 was ob-
tained after its treatment with TFA. The corresponding chiral
analogues described in Scheme 1 can be accessed from the
chiral alcohol 18, which was prepared according to a chiral
auxiliary approach7 as shown in Scheme 3. Thus, 4-hydroxy-
benzothiophene intermediate 3 was alkylated with (S)-1-oxo-
1-(pyrrolidin-1-yl)propan-2-yl 2-bromo-2-phenylacetate 16
and n-BuLi to give the chiral ester 17, which was reduced to
the chiral alcohol 18 with LiAlH4 at -78 ꢀC first, followed by
NaBH4 in MeOH at 0 ꢀC. Both enantiomers were prepared in
high enantiomeric excess using this method.8 For 4,6-disub-
stituted analogues, Scheme 4 exemplifies the general approach
with the preparation of 4-alkyloxy-6-fluoro analogues via the
key intermediate 25. The formal phenol product 20 from 3,5-
difluorophenol 19 was protected with allyl bromide to give
compound 21. Displacement of 3- fluoro group with thioace-
tonitrile gave the annulated benzothiophene product 22 selec-
tively in situ. Following the established produres5 of
amidination, t-Boc protection, and removal of allyl group
from compound 23, 4-hydroxy-6-fluoro intermediate 24
was obtained and reacted with R-bromophenyl acetate. Sub-
sequent reduction of the ester gave the 4,6-disubstituted

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1475
Scheme 3a
a Reagents: (a) (S)-1-Oxo-1-(pyrrolidin-1-yl)propan-2-yl 2-bromo-2-phenylacetate (16),7 n-BuLi, THF; (b) LiAlH4, -78 ꢀC, THF, NaBH4, 0 ꢀC,
MeOH, 78%.
Scheme 4a
a Reagents: (a) MgCl2, NEt3, (CHO)n, MeCN; (b) allyl bromide, K2CO3, DMF; (c) thioacetonitrile, NaH, THF; (d) (i) LiHMDS, THF, then HCl,
dioxane; (ii) Boc2O, Hunig’s base, THF, H2O; (e) Pd(PPh3)4, PhSiH, CH2Cl2; (f) methyl R-bromophenylacetate, K2CO3, DMF; (g) LiAlH4, THF, then
MeOH, NaBH4.
alcohol 25, which can be further manipulated as compound 5
in Scheme 1.
of compound 30. Our molecular modeling study of these
highly potent and selective FXa inhibitors within the FXa
active site did suggest a very good fit of the 40-substitutents at
the S3-S4 sites. Interestingly, as an amide bioisostere, the
oxadiazole analogue 16 appeared to show better selectivity
(15-fold) against FXa than compound 30. This compound
was then subjected to X-ray crystallography study with
human FIXa protein by the same group,10 which is shown
Results and Discussion
The enzymatic assays for FIXa, FXa, and uPA were carried
out as described earlier5 or by a modified procedure.9 Even
though a large number of amides or carboxylates have been
prepared from compounds 4, Table 1 only shows some of the
typical examples to highlight some significant SAR. To
determine an optimal R1 group for compound 2, we screened
a number of R1 substituted R-halomethyl esters by fixing R2
as the carboxylic ester. Table 1 exemplifies two of the analo-
gues, 26 and 27. Compound 27 with a phenyl R1 group
showed over 10-fold FIXa activity than the methyl analogue
26. Both compounds showed some degree of selectivity
against FXa but not uPA. As the ester group is prone to
hydrolysis, we decided to use phenyl group as R1 and screen a
library of amides easily derived from the ester 27. Simple
alkylamides with either carboxylic group 28 or amine group
29 showed less FIXa activity than 27. The anilide analogue 30
was nearly equipotent against all three enzymes FIXa, FXa,
and uPA. One carbon-extended benzylamide 31 showed less
FIXa activitythan 30. Therefore, we decided to investigate the
SAR of substituted anilides. Those with simple functional
groups show almost flat SAR for FIXa activity, exemplified
by 20-, 30-, or 40-methoxy substituted compounds 32-34, two
of which appeared to be moderately selective FXa inhibitors
32, 34. Indeed, our further exploration of substituents at the
40-position led to highly potent and selective FXa inhibitors
35-37 against both FIXa and uPA. Notably, the FIXa
activity of these compounds remained unimproved over that
˚
inFigure1. Thecocrystallizedstructure with2.62 Aresolution
clearly showed the R-enantiomer of the racemic compound 16
binding at the active site (PDB code 3LC5). As expected,
the amidine group on the benzothiophene template formed
the usualS1 interactionwith Asp189, the S1 β site was induced
open and filled by the phenyl group, and the hydrophobic
S3 site surrounded by Phe174, Trp215, and Tyr99 was
occupied by the phenyl group on the oxadiazole ring.
Moreover, it appeared that one of the oxadiazole N formed
an H bond interaction with NRH of Glu218, which was
significant because it is a glycine residue at the same position
in FXa.
Our molecular modeling study on the corresponding amide
compound 30 suggests no binding interaction exists between
the amide carbonyl and Glu218 residue. Instead the amide
group with a rigid sp2 conformation appeared to have directed
the (substituted) anilide group toward the S3-S4 sites of FXa.
In order to pick up the H bond interaction with Glu218 to
improve the selectivity, we sought to introduce more flexible
sp3 linked groups in place of the amide functional group.
Table 2 shows some of the early examples in this area.
Compound38withaprimarymethylene aminegroupatthe
R-position of the benzyloxy substitutent showed a moderate

1476 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
Table 1. Inhibitory Activitiesa of 4-Alkyloxybenzothiophene Com-
pounds in FIXa, FXa, and uPA Assays
Figure 1. X-ray structure of compound 16 cocrystallized with
human N-terminally truncated FIXa (PDB code 3LC5).
Table 2. Inhibitory Activity of R-Branched 4-Benzyloxy Compounds in
FIXa, FXa, and uPA Assays
Ki (μM),
Ki (μM),
Ki (μM),
compd
R
FIXaa
FXaa
uPAa
38
39
40
41
42
43
44
45
46
47
NH2
NHCOMe
NHBn
0.16
0.34
4.45
1.30
0.25
0.56
0.96
1.74
0.70
0.36
1.81
0.83
0.27
0.22
0.31
0.085
0.25
0.14
0.21
0.15
0.44
0.51
0.10
0.05
NHCO2Ph
NHCONHPh
OH
0.029
0.27
OCONHMe
OCONHEt
OCONHPh
OCONHBn
0.097
0.028
0.010
0.061
a All the IC50 values in this text represent the mean of a minimum of
three experiments. Variations among the results were less than 10%.
any meaningful selectivity against uPA. The hydroxymethylene
analogue 43 also showed a moderate FIXa activity but only
6-fold selectivity against FXa. Its further derivatization to
carbamates gave more potent FIXa inhibitors (44-46), and
compound 46 showed over 180-fold selectivity against FXa and
43-fold against uPA. One carbon-extended benzyl carbamate
47 showed less FIXa acivity and selectivity than compound 46.
Molecular modeling on this compound demonstrated possible
H bond interaction between the carbamate carbonyl and NRH
of Glu218 as shown in Figure 2. Given the superior activity and
selectivity of compound 46, we decided to explore the SAR of
the substituted phenyl carbamates, some of which are shown in
Table 3.
Substitutions at the phenyl ring were generally well tole-
rated, leading to a number of highly potent and selective FIXa
inhibitors, especially those substituted at 20- and 40-positions
exemplified by the fluoro substituted analogues 48-50. In a
majority of cases, the 30-substituted analogue tended to give
slightly poorer FIXa inhibitor with poor selectivity against
a All the IC50 values in this text represent the mean of a minimum of
three experiments. Variations among the results were less than 10%.
b ND: not determined.
FIXa activity and selectivity against FXa (28-fold). Capping
of the primary amine group by acetyl or benzyl group did not
lead to much improved FIXa inhibition or better selectivity
(39, 40). However, the carbamate analogue 41 showed a good
potency against FIXa (Ki = 50 nM) with 11-fold selectivity
against FXa. The corresponding urea analogue 42 showed even
better potency against FIXa (Ki = 29 nM) and selectivity
against FXa (33-fold). None of the above compounds showed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1477
Table 4. Inhibitory Activity for 4-Substituted Branched Benzothio-
phene Heterocarbamate Compounds in FIXa, FXa, and uPA Assays
Figure 2. Molecular docking study of compound 46 in human
truncated FIXa active site.
Table 3. Inhibitory Activity for 4-Alkyloxy(R-phenyl carbamates)-
Substituted Benzothiophene Compounds in FIXa, FXa, and uPA
Assays
a All the IC50 values in this text represent the mean of a minimum of
three experiments. Variations among the results were less than 10%.
selective FIXa inhibitors either substituted at the 40-position
(53, 54) or 20-position (55-59). Compounds 56, 57, and 59
demonstrated high selectivityagainst uPA as well as FXa. Our
attempts to look at the additive effect of the disubstituted
analogues gave more or less the similar potency and selectivity
(60, 61) to those singly substituted (48, 50). However, chiral
resolution of the racemic compound 52 demonstrated that the
(R)-52 enantiomer was 10 times more potent than the (S)-52
enantiomer, which is consistent with the X-ray structure of
compound 16 (Figure 1). As the more potent R form was only
marginally better than the racemic mixture 52, we decided to
continue our ongoing in vitro analogue studies with the
racemic form.
In order to have a wider choice of selection of analogues for
our parallel ADME/PK study, we investigated the bioisostere
replacement of the anilide moiety attached to the carbamate
with large number of heterocyles. Table 4 shows some selected
examples.
The dimethyloxazole analogue 62 showed good FIXa
activity and was over 200-fold selective against FXa, though
the selectivity against uPA was poor. Simple pyrazoles with
carbamate at the 4- or 5-position showed similar high FIXa
activity and uPA selectivity (63, 66). Substitutions at one of
the methyl substitutents of compound 63 with a methoxy or
a All the IC50 values in this text represent the mean of a minimum of
three experiments. Variations among the results were less than 10%.
FXa and uPA 49. With potent and selective FIXa inhibitors
51 and 52 as leads, further analogues with amino basic groups
were explored in an effort to improve solubility and plasma
protein binding properties, which led to more potent and

1478 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
Table 5. Inhibitory Activity for 4,6-Disubstituted Benzothiophene
Carbamate Compounds in FIXa, FXa, and uPA Assays
Table 6. FIXa Compounds in aPTT, PT, and TT Assays
doubling of PD parameter (μM)
compd Ki (nM), FIXa
aPTT
PT
TT
PPB (%)
63
66
68
69
59
10
8
12.8
11.7
7.7
57
63
42
29
17
60
96
95
95
96
95
>80
56
5
3
5.9
5.0
25
20
3
Table 7. Pharmacokinetic Study by iv Dosing (5 mg/kg) in Anesthetized
Rats
Ki (μM), Ki (μM), Ki(FXa)/ Ki (μM), Ki(uPA)/
compd
R
FIXaa FXaa Ki(FIXa) uPAa Ki(FIXa)
Cmax
(μM)
AUC0-¥
T1/2
(h)
CL
(mL/min/kg)
HLM
(% TO)
compd
(μM h)
70
H
0.0155 1.585
102
486
169
2.115
0.828
0.94
137
190
117
3
5.19
71 20-CH2NH2 0.0044 2.115
72 20-CH2NMe2 0.0081 1.363
50
63
10.5
5.19
5.5
2.1
32.4
129.3
11
18
0.951
a All the IC50 values in this text represent the mean of a minimum of
three experiments. Variations among the results were less than 10%.
predominantly involved in the intrinsic pathway. This is re-
flected by nearly 5-fold or more difference of the PD data of
aPTT and those of PT and TT for heterocycle based carba-
mates 63, 66, 68, and 69. The phenyl carbamate 59 showed
more than 3-fold difference in the corresponding measure-
ments. One reason for the significant difference of activities
from FIXa enzymatic assays to the micromolar level of pharmaco-
dynamic measurement could be due to the effect of plasma
protein binding. As shown in Table 6, all of these five com-
pounds are 95-96% plasma protein bound. The activity
data from FIXa assay together with plasma protein binding
data correlate very well with the trend of the pharmacodynamic
data generated, indicating selective FIXa inhibition. Notably,
an early clinical RNA aptamer study by Regado Bioscience
showed that 1.1-fold increase in aPTT in response to FIXa
inhibition is equivalent to 40% loss of FIX activity while a
1.3-fold increase represents a loss of 80% FIX activity.13
As we have obtained potent and selective FIXa inhibitors in
both phenyl and heteroaromatic based carbamates as shown
in Tables 3 and 4, some preliminary pharmacokinetic (PK)
studies were also carried out for both subseries, using anesthe-
tized rats and administering a dose of 5 mg/kg bolus intra-
venously (iv). The data for two early examples are disclosed in
Table 7.
The phenyl carbamate analogue 50 showed a promising
profile with more than 10 μM Cmax and >5 h half-life, while
the pyrazole derived carbamate 63 showed a lower Cmax and
over 2 h half-life. Both compounds showed significant plasma
concentrations after several hours. The clearance (CL) for
compound 50 is significantly lower than that for compound
63. Human liver metabolism (HLM) studies for both com-
pounds demonstrated low turnover (TO), though compound
50 appears to be less metabolized than compound 63, indicat-
ing reasonable initial stability for the carbamate functional
group present in both molecules. This provided us with a
promising platform for our ongoing evaluation and optimiza-
tion in vivo studies, which will be published in due course.
methylamine group led to slightly improved FIXa inhibitors
with selectivity against FXa more or less retained (64, 65).
Alkylations at pyrazole N of compound 66 usually gave slight
reduction of FIXa activity and selectivity exemplified by
compound 67. However, introduction of alkyamine groups
at one of the pyrazole methyl groups gave more potent FIXa
inhibitors with over 470-fold selectivity against FXa (68, 69),
which was consistent with the SAR in the anilide series shown
in Table 3.
As shown by the compounds listed in Tables 2-4, we have
largely overcome the selectivity issue against FXa with a
number of those showing good selectivity(>100-fold) against
uPA as well. However, a significant number of these com-
pounds are potent dual inhibitors of FIXa and uPA, which
could be potentially very useful in treating cardiovascular
patients with restenosis, as uPA has been specifically impli-
cated in stimulating neointima formation and inward arterial
remodeling.11
In our previous communication,5 we reported SAR studies
on the 6-position of the benzothiophene template for S0 site
interactions. Given our understanding of the binding interac-
tions within the S1 β and S2-S4 sites with above 4-substituted
compounds, we also looked at the potential additive effect of
4,6-disubstitutedcompounds for FIXaactivityandselectivity.
Table 5 shows a few preliminary examples with the 6-fluoro
substitutent in this aspect.
As shown by compounds 70-72, introduction of an addi-
tional fluoro group at the 6-position of the active carbamate
compounds led to only marginal reduction of FIXa activity,
but significant improvement of uPA selectivity was observed
with FXa selectivity more or less retained, compared with the
corresponding nonfluorinated analogues 46, 55, and 68. The
reason for the improvement of uPA selectivity was unclear,
though molecular modeling suggests some slight adjustment
of the template vector within the active sites.
In further pharmacodynamic (PD) activity profiling study,
a number of selective FIXa inhibitors were selected for
activated partial thrombin time (aPTT), prothrombin time
(PT), and thrombin time (TT) measurement. It is believed that
better efficacy should be expected in a 2ꢀ aPTT assay for
intrinsicFIXa inhibitorsthan in2ꢀ PTor 2ꢀ TT assays which
measure the effect on the extrinsic pathway.12 Table 6 lists
some selected examples.
Conclusion
Through our understanding of the X-ray binding interac-
tion of the benzothiophene analogue 1 within the FIXa active
site, we developed a structure based drug design chemistry
strategy to explore the binding interactions of S1 β and S2-S4
sites with branched 4-alkyloxy analogues. By modifying the
conformation of the linking side chain, we successfully made
the switch from potent and selective FXa inhibitors to a series
of highly potent and selective FIXa inhibitors, some of which
All compounds shown in Table 6 demonstrated significant
pharmacodynamic activity with inhibiting effects on the factors

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1479
are also potent and selective dual inhibitors for FIXa and
uPA. FurtherX-ray and molecular modelingstudiesindicated
a new potential H-bond binding interaction of Glu218 residue
and carbamate functional group in the highly selective and
potent FIXa inhibitors. Our preliminary in vitro PD and PK
studies demonstrated promising profiles for these benzothio-
phene based FIXa inhibitors. Further in vivo studies with
more optimized analogues in our ongoing development can-
didate selection process will be published elsewhere.
-78 ꢀC for 2 h before the reaction was carefully quenched by
addition of MeOH (100 mL). Sodium borohydride (1.47 g, 38.9
mmol) was then added in one portion, and the resulting solution
was allowed to warm to room temperature and was stirred
at that temperature for 2 h. The reaction mixture was then
quenched by addition of aqueous saturated NH4Cl (50 mL),
and the organic solvents were removed under reduced pressure.
The resulting aqueous mixture was then diluted with H2O
(100 mL). After extraction with EtOAc (3 ꢀ 150 mL), the
combined organic extracts were washed with brine (100 mL),
dried (Na2SO4), filtered, and concentrated under reduced
pressure to give the desired product as a pale-yellow solid,
Experimental Section
1
5a (7.81 g, 97% yield). H NMR (CDCl3, 400 MHz): δ 8.28
Methods and Materials. Reagents, starting materials, and
solvents were purchased from common commercial suppliers
and used as received or distilled from the appropriate drying
agent. Reactions requiring anhydrous conditions were per-
formed under an atmosphere of nitrogen or argon. Precoated
aluminum backed silica gel 60 F254 plates with a layer thickness
of 0.25 mm were used for thin layer chromatography, and the
stationary phase for preparative column chromatography using
medium pressure was silica gel 60, mesh size 40-60 μm from
E. Merck, Darmstadt, Germany.
(s, 1H), 7.37-7.27 (m, 6H), 7.16-7.12 (m, 1H), 6.53 (d, 1H,
J = 7.8 Hz), 5.43 (dd, 1H, J = 8.1, 3.3 Hz), 4.10 (m, 1H), 3.92
(m, 1H), 2.72 (br s, 1H), 1.57 (s, 9H). LCMS (ESþ, M þ H)
m/z 413.
General Procedure B for Amides 6 Preparation. To a solution
of ester 4 (1 equiv) in THF (10 mL/mmol) was added a solution
of aqueous lithium hydroxide (1.0 M, 1 equiv), and the mixture
was stirred at room temperature for 3-5 h. The solvent was
reduced to low volume. The residue was partitioned between
CH2Cl2 and H2O, and the phases were separated. The aqueous
layer was washed with CH2Cl2, acidified to pH 5 with acetic
acid, and then extracted with ethyl acetate (ꢀ3). The ethyl
acetate extracts were combined, dried over MgSO4, and filtered
and the solvent was removed to yield the desired product
acids.
To a solution of the above acids (1 equiv) in DMF (3-5 mL/
mmol) were added HATU or HBTU (1.1 equiv) and diisopropyl-
ethylamine (1.1 equiv). The solution was stirred for 10-30 min.
Then amine (1.1 equiv) was added and the solution stirred at
room temperature for 18 h.
The mixture was partitioned between ethyl acetate and water,
and then the phases were separated. The organics were washed
with brine (ꢀ2) and dried (MgSO4), and the solvent was
removed. The residue was purified via silica chromatography
(EtOAc/hexane or MeOH/CH2Cl2 mix), yielding the t-Boc-
amidine amides, which were deprotected to give the amides 6
by a previously reported procedure.5
General Procedure C for Carbamate 7 Formation. To a
suspension of 5 (1 equiv) in CH2Cl2 (8 mL/mmol) was added
triethylamine (2.4 equiv) and disuccinimidyl carbonate (1.2
equiv). The suspension was stirred for 45 min until a yellow
solution formed, and then the requisite aniline (2 equiv) was
added. The solution was stirred at 60 ꢀC in a sealed tube for 18 h.
The solution was then concentrated and the residue purified via
silica chromatography (ethyl acetate/hexane mix). The residue
was then directly subjected to the general method for deprotec-
tion of BOC amidines with TFA.5
NMR spectra were obtained using a Bruker ACF 400 oper-
ating at 400 MHz, and the H shifts (ppm) were calibrated to
1
residual CHCl3 in CDCl3, at 7.26 ppm. Mass spectra were
obtained in the indicated mode using a Finnigan SSQ 710L
machine. Melting points were determined using an Electrother-
mal 9100 series apparatus.
The purities on all compounds tested in biological systems
were assessed as being >95% using analytical LC-MS, which
was performed using a Waters 600E pump, Waters 2767 auto-
sampler, Waters 2487 IEEE UV detector (set at 254 nm), and
Waters Micromass ZQ detector (ESI mode). Elution was done
with a gradient of 5-95% solvent B in solvent A (solvent A
was 0.1% formic acid in water, and solvent B was 0.1% formic
acid in acetonitrile) over 11 min through a Gemini 5 μm C18
110A (50 mm ꢀ 4.60 mm) column at 1.5 mL min-1. Area %
purity was measured at 254 nm. Preparative LC-MS was
performed using a Waters 600E pump, Waters 515 makeup
pump, Waters 2767 autosampler, Waters 2487 IEEE UV detec-
tor (set at 254 nm), and Waters Micromass ZQ detector (ESI
mode). Elution was done with a gradient of 5-95% solvent B in
solvent A (solvent A was 0.1% formic acid in water, and solvent
B was 0.1% formic acid in acetonitrile) over 8 min through a
X-Terra Prep MS C18 5 μm (19 mm ꢀ 50 mm) column at 20 mL
min-1. The makeup pump was used with methanol. Isolated
fractions yielded pure material as the formate salt after lyophi-
lization.
General Procedure A for Analogues 4 and 5 (Scheme 1). Methyl
2-(2-(N0-(tert-Butoxycarbonyl)carbamimidoyl)benzo[b]thiophen-4-
yloxy)-2-alkylacetates (4) Exemplified by Phenyl Analogue (4a,
When R1 = Ph). To a solution of phenol 3 (1.78 g, 6.10 mmol) in
DMF (60 mL) under argon was added potassium carbonate
(927 mg, 6.71 mmol), followed by methyl R-bromophenylacetate
(1.06 mL, 6.71 mmol). The mixture was stirred at room tempera-
ture for 4 h before it was quenched by addition of H2O (50 mL).
After extraction with Et2O (3 ꢀ 50 mL), the combined organic
extracts were washed with brine (50 mL), dried (Na2SO4), filtered,
and concentrated under reduced pressure. The residue was puri-
fied by flashchromatography using 30% EtOAc in hexanes to give
the desired product as a white solid (2.27 g, 86% yield). 1H NMR
(CDCl3, 400 MHz): δ 8.15 (s, 1H), 7.62 (m, 1H), 7.47-7.43 (m,
4H), 7.29-7.27 (m, 1H), 6.64 (d, 1H, J = 7.8 Hz), 5.80 (s, 1H),
3.76 (s, 3H), 1.57 (s, 9H). LCMS (ESþ, M þ H) m/z 441.
tert-Butyl Amino(4-(2-hydroxy-1-phenylethoxy)benzo[b]thio-
phen-2-yl)methylenecarbamate (5a). A solution of ester 4a
(8.56 g, 19.5 mmol) in THF (100 mL) was cooled to -78 ꢀC
under argon. Lithium aluminum hydride (2 M in THF, 10.2 mL,
20.4 mmol) was added slowly, and the mixture was stirred at
Preparation of Triflates 8 Exemplified by 8a (When R1 = Ph).
To a solution of 5a (1.0 g, 2.4 mmol) in CH2Cl2 (20 mL) at
-78 ꢀC under argon was added triethylamine (406 μL, 2.9
mmol), followed by trifluoroactic acid anhydride (407 μL, 2.4
mmol). The solution was stirred at -78 ꢀC for 1 h, then
quenched with water, diluted with CH2Cl2, and allowed to
warm to room temperature. The phases were separated, the
organics were dried (Na2SO4), and the solvent was removed to
1
yield title compound 8a (1.4 g, 100%). H NMR (400 MHz,
CDCl3): δ 8.17 (s, 1H), 7.44-7.40 (m, 6H), 7.16-7.14 (m, 1H),
6.49 (d, 1H, J = 8 Hz), 5.69-5.67 (m, 1H), 4.93-4.88 (m, 1H),
4.70-4.67 (m, 1H), and 1.56 (s, 9H).
Preparation of Amines 9 Exemplified by 9a (when R1 = Ph).
To a solution of 8a (1.2 g, 2.2 mmol) in DMF (6 mL) was added
sodium azide (184 mg, 2.8 mmol), and the mixture was stirred
at room temperature for 18 h. The mixture was partitioned
between ethyl acetate and water, and the phases were separated.
The organics were washed with water (ꢀ1) and brine (ꢀ1) and
dried (MgSO4), and the solvent was removed. The residue was
purified via silica chromatography (20-30% EtOAc/hexane) to

1480 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
yield the azide intermediate (400 mg, 42%). 1H NMR (400 MHz,
CDCl3): δ 8.18 (s, 1H), 7.41-7.29 (m, 6H), 7.17-7.15 (m, 1H),
6.51 (d, 1H, J = 8 Hz), 6.48-6.46 (m, 1H), 3.84-3.80 (m, 1H),
3.49-3.47 (m, 1H), and 1.56 (s, 9H).
was purified by flash chromatography using 50% EtOAc in
hexanes to give the desired product as a beige solid (389 mg, 47%
yield). 1H NMR (CDCl3, 400 MHz): δ 8.31 (s, 1H), 7.62 (m, 2H),
7.51 (m, 1H), 7.43 (m, 3H), 7.27 (m, 1H), 6.79 (d, 1H, J = 8.0
Hz), 5.73 (s, 1H), 5.21 (q, 1H, J = 7.0 Hz), 3.63-3.30 (m, 4H),
1.99-1.82 (m, 4H), 1.57 (s, 9H), 1.37 (d, 3H, J = 7.0 Hz). LCMS
(ESþ, M þ H) m/z 552.
To a solution of the above azide (400 mg, 0.92 mmol) in THF
(9 mL) and water (4.5 mL) was added triphenylphosphine (265
mg, 1.01 mmol), and the mixture was stirred at room tempera-
ture for 18 h. The solvent was reduced to low volume and the
residue partitioned between ethyl acetate and brine. The phases
were separated, and the aqueous layer was extracted with ethyl
acetate (ꢀ2). The combined organics were dried (Na2SO4), and
the solvent was removed. The residue was triturated with ether
and filtered and the solid collected to yield the title compound 9a
(262 mg, 69%). 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 2H),
8.69 (s, 1H), 7.47-7.44 (m, 3H), 7.39-7.36 (m, 2H), 7.29-7.25
(m, 2H), 6.69 (d, 1H, J = 8 Hz), 6.40 (m, 1H), 3.06-2.94 (m,
2H), and 1.49 (s, 9H).
General Proceduere D for Preparation of 10 by Acylation of
Amines 9. To a solution of amines 9 (1 equiv) in CH2Cl2 (10-20
mL/mmol) was added diisopropylethylamine (1.1-2.2 equiv)
followed by isocyanate or acyl halide (1.1 equiv), and the
solution was stirred for 1-18 h. The solvent was removed
and the residue purified via silica chromatography (ethyl
acetate/hexane mix). Analogues 10 were obtained after TFA
treatment of the above t-Boc amidines using a previously
reported procedure.5
(R)-tert-Butyl
Amino(4-(2-hydroxy-1-phenylethoxy)benzo-
[b]thiophen-2-yl)methylenecarbamate 18. To a solution of 17
(47 mg, 0.09 mmol) in THF (2 mL) at -78 ꢀC was added
dropwise LiAlH4 (2.0 M solution in THF, 45 μL, 0.089 mmol),
and the mixture was stirred at -78 ꢀC for 1 h before quenching
the reaction with MeOH (500 μL). More MeOH (250 mL) was
added, and the reaction mixture was warmed to 0 ꢀC. NaBH4
(6.5 mg, 0.17 mmol) was added and stirred for 3 h at 0 ꢀC. The
mixture was quenched with saturated aqueous NH4Cl and back-
extracted with EtOAc. The combined organic layers were
washed with brine, dried (MgSO4), and concentrated in vacuo.
Purification by silica chromatography, eluting with 20%
1
EtOAc/hexane, gave the chiral alcohol 18 (20 mg, 57%). H
NMR and MS results are as for compound 5a.
2,4-Difluoro-6-hydroxybenzaldehyde 20. To a suspension of
3,5-difluorophenol 19 (3.03 g, 23.3 mmol) in MeCN (100 mL) at
room temperature under argon was added magnesium chloride
(6.66 g, 69.9 mmol) in one portion, followed by triethylamine
(8.12 mL, 58.2 mmol). The mixture was stirred at room tem-
perature for 20 min before paraformaldehyde (3.50 g, 116 mmol)
was added in one portion. The mixture was then heated to 80 ꢀC
and stirred at that temperature for 3 h, then at room temperature
overnight. The mixture was then quenched by pouring onto
aqueous HCl (1 N, 80 mL) and extracted with EtOAc (2 ꢀ
60 mL). The combined organic extracts were washed with brine
(60 mL), dried (Na2SO4), filtered, and then concentrated under
reduced pressure to give the desired product as a pale-orange oil
(3.68 g, 100% yield). 1H NMR (CDCl3, 400 MHz): δ 11.81
(s, 1H), 10.17 (s, 1H), 6.51-6.39 (m, 2H). LCMS (ES-, M - H)
m/z 157.
Preparation of N-Alkylamines 11 Exemplified by 11a (R1 =
Ph, R = Bn). To a solution of 8a (54 mg, 0.1 mmol) in DMF
(1 mL) at room temperature was added K2CO3 under argon,
followed by benzylamine (13 μL, 0.12 mmol) for 18 h . The
solvent was removed and the residue triturated with ether and
filtered. The solid collected was dissolved in DCM, and TFA
was added to deprotect the t-Boc amidine5 to yield the desired
product 11a (22 mg, 49%). 1H NMR (400 MHz, DMSO-d6): δ
9.64 (s, 2H, NH þ TFA), 9.53 (br s, 2H, NH2), 9.40 (s, 2H, NH þ
TFA), 8.72 (s, 1H, ArH), 7.79 (d, 1H, ArH), 7.45-7.62 (m, 11H,
ArH), 6.96 (m, 1H, ArH), 6.07 (m, 1H, CHO), 4.44 (t, 2H,
CH2N), 3.48 (m, 2H, CH2N). MS m/z (ESþ) 402 [M þ H]þ.
Phenyl(3-phenyl[1,2,4]oxadiazol-5-yl)methanol 14. Mandelic
acid 12 (0.848 g, 5.58 mmol) and CDI (0.404 g, 5.58 mmol) in
dyglime (15 mL) were stirred together for 30 min. N-Hydroxy-
benzenecarboxyimidamide 13 (0.76 g, 5.58 mmol) was added,
and the mixture was stirred overnight at room temperature and
then heated at 110 ꢀC under reflux for 2 h. The solution was
concentrated in vacuo. The residue was applied to a silica
chromatography column with EtOAc/hexane (5:1) to give the
title compound, 0.150 g, 11% . 1H NMR (DMSO-d6): δ 8.01 (m,
2H, Ar-H) 7.34 (m, 9H, Ar-H) 6.02 (d, 1H, CH), 3.16 (d, 1H,
OH). MS m/z (ESþ) 253.0. tR = 6.57
2-(Allyloxy)-4,6-difluorobenzaldehyde 21. To a solution of 20
(3.68 g, 23.3 mmol) in DMF (50 mL) at room temperature under
argon was added K2CO3 (3.31 g, 23.9 mmol), followed by allyl
bromide (2.07 mL, 23.9 mmol). The mixture was stirred at room
temperature for 2 h and 30 min. Then it was quenched by
pouring onto aqueous saturated NH4Cl and extracted with
Et2O (3 ꢀ 50 mL). The combined organic extracts were washed
with brine (50 mL), dried (Na2SO4), filtered, and then concen-
trated under reduced pressure. The residue was purified by flash
chromatography using 10% EtOAc in hexanes to give the
1
desired product as a pale-yellow solid (2.12 g, 46% yield). H
NMR (CDCl3, 400 MHz): δ 10.39 (s, 1H), 6.52-6.47 (m, 2H),
6.08-6.01 (m, 1H), 5.52-5.47 (m, 1H), 5.41-5.37 (m, 1H),
4.66-4.64 (m, 1H).
4-[Phenyl(3-phenyl[1,2,4]oxadiazol-5-yl)methoxy]benzo[b]thio-
phene-2-carboxamidine 16. 16 was synthesized from compound
15 by TFA deprotection of the t-Boc group. Compound 15 was
prepared from 14 using Mitsunobu condition reported pre-
viously.5 1H NMR (DMSO-d6): δ 9.20 (bs, 4H, amidine), 8.47
(s, 1H, ArH), 7.85 (d, 2H, ArH), 7.66 (d, 1H, ArH), 7.65 (d, 2H,
ArH), 7.48-7.33 (m, 7H, ArH), 7.32 (s, 1H, CH), 7.06 (d, 1H,
Ar). MS m/z (ESþ) 427.0 [M þ H]þ.
4-(Allyloxy)-6-fluorobenzo[b]thiophene-2-carbonitrile 22. A
solution of thioacetonitrile (778 mg, 10.6 mmol) in THF was
cooled to 0 ꢀC under argon before NaH (60% in mineral oil,
426 mg, 10.6 mmol) was added in one portion. The mixture was
stirred at 0 ꢀC for 10 min, then at room temperature for 15 min
before it was cooled back to 0 ꢀC and 21 was added (2.11 g, 10.6
mmol) in one portion. The mixture was stirred at 0 ꢀC for 30 min,
then at room temperature for 30 min before it was cooled back to
0 ꢀC and NaH (60% in mineral oil, 4 ꢀ 105 mg, 10.6 mmol) was
added portionwise every 10 min. After 1 h at room temperature,
the mixture was quenched by addition of aqueous saturated
NH4Cl, and was extracted with with EtOAc (3 ꢀ 50 mL). The
combined organic extracts were washed with brine (50 mL),
dried (Na2SO4), filtered, and then concentrated under reduced
pressure. The residue was purified by flash chromatography
using 5% EtOAc in hexanes to give the desired product as a pale-
yellow solid (2.08 g, 84% yield). 1H NMR (CDCl3, 400 MHz): δ
8.03 (d, 1H, J = 0.6 Hz), 7.14-7.11 (ddd, 1H, J = 8.2, 2.0, 0.6
Hz), 6.62-6.59 (dd, 1H, J = 10.9 Hz, 2.0 Hz), 6.13-6.06
(R)-(2-t-Boc-carbamimidoylbenzo[b]thiophen-4-yloxy)phenyl-
acetic Acid (S)-1-Methyl-2-oxo-2-pyrrolidin-1-ylethyl Ester 17.
A solution of phenol 3 (511 mg, 1.50 mmol) in THF (5 mL) was
cooled to 0 ꢀC under argon. n-BuLi (1.6 M in heptane, 0.99 mL,
1.58 mmol) was added slowly, and stirring was continued for
20 min at 0 ꢀC. The resulting mixture was added dropwise to a
previously prepared cooled (0 ꢀC) solution of bromide 16
(439 mg, 1.50 mmol) in THF (10 mL) under argon. After 4 h
at 0 ꢀC, the reaction was quenched by careful addition of
aqueous saturated NH4Cl (10 mL), then H2O (10 mL). After
extraction with EtOAc (3 ꢀ 20 mL), the combined organic
extracts were washed with brine (20 mL), dried (Na2SO4),
filtered, and concentrated under reduced pressure. The residue

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1481
(ddt, 1H, J = 18.7, 10.5, 5.3 Hz), 5.51 (ddt, 1H, J = 18.7, 1.3, 1.3
Hz), 5.39 (ddt, 1H, J = 10.5, 1.3, 1.3 Hz), 4.69 (dt, 2H, J = 10.5,
1.3 Hz).
Plasma samples were diluted 1.1 with 100% acetonitrile
(Caution: volatile and precipitate with other substances). After
dilution, the sample was centrifuged for 10 min at 13 200 rpm
(16100g) (centrifuge 5415D, Eppendorf, Germany) at room
temperature. The supernatant was precipitated again with aceto-
nitrile using the same conditions. The second supernatant was
utilized for compound concentration measurements. A concen-
tration versus time curve was plotted, and the pharmacokinetic
parameters, AUC (area under the curve), and apparent
terminal half-life (t1/2) were calculated using a noncompartmen-
tal analysis.
(E)-tert-Butyl (4-(Allyloxy)-6-fluorobenzo[b]thiophen-2-yl)(amino)-
methylenecarbamate 23. 23 was prepared as a beige solid (4.04 g,
100% yield), following the reported procedures.5 1H NMR
(CDCl3, 400 MHz): δ 7.98 (s, 1H), 7.11 (dd, 1H, J = 8.4, 1.8
Hz), 6.54 (dd, 1H, J = 11.0, 2.0 Hz), 6.13-6.06 (m, 1H), 5.48
(dd, 1H, J = 17.2, 1.3 Hz), 5.38 (dd, 1H, J = 10.5, 1.3 Hz), 4.66
(dd, 2H, J = 4.0, 1.3 Hz), 1.56 (s, 9H). LCMS (ESþ, M þ H)
m/z 351.
(E)-tert-Butyl Amino(6-fluoro-4-hydroxybenzo[b]thiophen-2-
yl)methylenecarbamate 24. 24 was prepared as a brown solid
(1.14 g, 61% yield), following the previous procedures.5 1H
NMR (CDCl3, 400 MHz): δ 8.13 (s, 1H), 7.02 (m, 1H), 6.45 (m,
1H), 1.56 (s, 9H).
Acknowledgment. We thank Dr. Roger Dickinson for
providing consultancy in this project.
(E)-tert-Butyl Amino(6-fluoro-4-(2-hydroxy-1-phenylethoxy)-
benzo[b]thiophen-2-yl)methylenecarbamate 25. 25 was prepared
as a white solid (1.1 g, 93% yield), following the same procedure
Supporting Information Available: Experimental and spectro-
scopic information for compounds 28-72. This material is
available free of charge via the Internet at http://pubs.acs.org.
1
as for compound 5a. H NMR (CDCl3, 400 MHz): δ 8.18 (s,
1H), 7.33 (m, 5H), 6.99 (dd, 1H, J = 8.3, 1.8 Hz), 6.30 (dd, 1H,
J = 10.7, 1.8 Hz), 5.37 (dd, 1H, J = 8.1, 3.3 Hz), 4.07 (m, 1H),
3.91 (m,1H), 2.80 (br s, 1H), 1.56 (s, 9H).
References
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(8) The enantiomeric excess was determined by Mosher’s ester forma-
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(9) uPA assay: Urokinase-type plasminogen activator (uPA) was
purchased from Sigma. The amidolytic substrate S-2444 (pyro-
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chemicals were purchased from Sigma and BDH. Amidolytic
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2-(2-Carbamimidoylbenzo[b]thiophen-4-yloxy)propionic Acid
Methyl Ester 26 (as TFA Salt). 26 was prepared as a pale-yellow
solid (74%) from compound 4b (when R1= Me) with TFA,
following the previous procedure.5 1H NMR (DMSO, 400
MHz): δ 9.21 (s, 4H) 8.44 (s, 1H), 7.74 (d, J = 8 Hz, 1H), 7.52
(d, J = 8 Hz, 1H), 6.89 (d, J = 8 Hz, 1H), 5.27 (q, J = 8 Hz, 1H),
3.71 (s, 3H), 1.64 (d, J = 8 Hz, 3H). MS m/z 279 [M þ H]þ.
(2-Carbamimidoylbenzo[b]thiophen-4-yloxy)phenylacetic Acid
Methyl Ester 27 (as TFA Salt). 27 was synthesized as a beige solid
from compound 4a (when R1 = Ph) with TFA, following the
previous procedure.5 1H NMR(CD3OD, 400 MHz): δ 9.52 (s, 2H,
NH2 amidine), 9.08 (s, 2H, NH-TFA amidine), 8.53 (d, 1H, J = 8
Hz, H-Ar), 7.77 (d, 1H, J = 8 Hz, ArH), 7.66-7.56 (m, 2H, ArH),
7.53 (t, 1H, J = 8 Hz, ArH), 7.45-7.41 (m, 3H, ArH), 7.00 (d, 1H,
J = 8 Hz, ArH), 6.34 (s, 1H, CH), 3.68 (s, 3H, CH3). MS m/z
(ESþ) 341.00 [M - H]. tR = 4.93.
PD Measurement. For in vitro coagulation assays, blood was
obtained from healthy volunteers by venipuncture and anti-
coagulated by sodium citrate (9NC Vacutainer, Beckton
Dickinson). Plasma was prepared by centrifugation at 2000g
for 10 min and kept on ice prior to use. Standard clotting assays
were performed in a temperature-controlled automated coagu-
lation analyzer (Sysmex CA-560, Dade Behring, Marburg,
Germany) and individual thrombin time (TT), activated partial
thromboplastin time (aPTT), and prothrombin time (PT) were
determined. For TT, 50 μL of plasma was incubated at 37 ꢀC for
2 min prior to addition of 100 μL of thrombin reagent (Dade
Behring OWHM 13), and duration of clot formation was
monitored at 660 nm. The aPTT assay was performed by
incubating 50 μL of plasma at 37 ꢀC for 1 min before addition
of 50 μL of aPTT reagent (FSL, Dade Behring B4219-2) and
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(Dade Behring ORHO 37) was added and the clot formation
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reagent (Thromborel S, Dade Behring OUHP 29). Clot forma-
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at the following time points: predose, 2, 5, 15, and 30 min and
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by centrifugation (1700g at 20 ꢀC for 5 min) and stored frozen
(-80 ꢀC) until required for analysis of drug concentration.
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