Studies of benzothiophene template as potent factor IXa (FIXa) inhibitors in thrombosis

Article abstract of DOI:10.1021/jm901475e

FIXa is a serine protease enzyme involved in the intrinsic pathway of the coagulation cascade. The upstream intervention of the coagulation cascade in selectively inhibiting FIXa would leave hemostasia intact via the extrinsic pathway, leading to an optim

Full text of DOI:10.1021/jm901475e

J. Med. Chem. 2010, 53, 1465–1472 1465
DOI: 10.1021/jm901475e
Studies of Benzothiophene Template as Potent Factor IXa (FIXa) Inhibitors in Thrombosis†
Shouming Wang,*,‡ Richard Beck,§ Toby Blench,‡ Andrew Burd,‡ Stuart Buxton,‡ Maja Malic,‡ Tenagne Ayele,‡
Shaheda Shaikh,‡ Suresh Chahwala,§ Chaman Chander,§ Richard Holland,‡ Sandrine Merette,‡ Lihua Zhao,‡
Michael Blackney,§ and Alexandra Watts‡
‡Department of Medicinal Chemistry and §Department of Pharmacology, Trigen Ltd., Emmanuel Kaye Building,
1B Manresa Road, London SW3 6LR, U.K.
Received October 8, 2009
FIXa is a serine protease enzyme involved in the intrinsic pathway of the coagulation cascade. The upstream
intervention of the coagulation cascade in selectively inhibiting FIXa would leave hemostasis intact via the
extrinsic pathway, leading to an optimum combination of efficacy and safety with low incidence of bleeding.
We have identified 2-amindinobenzothiophene template as a lead scaffold for FIXa inhibiton based on its
homology with urokinase plasminogen activator (uPA). Subsequent SAR work on the template revealed a
number of highly potent FIXa inhibitors, though with moderate selectivity against FXa. X-ray study with one
of the analogues demonstrated active site binding interaction with the induced opening of the S1 β pocket and
a secondary binding at the S2-S4 sites, which is in direct contrast with the previous finding.
Despite tremendous efforts to prevent and treat thrombotic
events, arterial and venous thromboses continue to be the major
cause of mortality. Current medical strategy for combating
thrombosis involves aspirin as the primary antiplatelet agent
for chronic, secondary prevention of arterial thrombosis,1,2
whereas ticlopidine and clopidgrel add marginal benefits over
aspirin.3,4 Heparin and warfarin are proven anticoagulants for
acute and chronic thrombotic disorders, respectively.5-7 How-
ever, these anticoagulants have restricted therapeutic utility due
to significant adverse effect, such as bleeding, thrombocytope-
nia, and need for careful patient monitoring.7 Therefore, there is
a significant medical need for superior antithrombotic agents
with a wider therapeutic index. In the past number of years,
research efforts have been focused on targeting the specific
enzymes of the extrinsic/common pathway in the coagulation
cascade, such as thrombin, FXa,a and TF/FVIIa.8 Although
many selective and potent thrombin and FXa inhibitors have
been advanced to the clinical development, the potential risk of
bleeding and a narrow therapeutic window remain for these
newer anticoagulants and their safety profiles are yet to be
established in clinical trials.9
A recent study shows that patients who are heterozygous gene
carriers of FIXa have decreased coagulability and are protected
from ischemic heart diseases.13 As FIX is activated both by the
stimulation of the intrinsic system and by low level of TF, it is
believed that selective inhibition of FIXa would be effective in
antithrombosis in low tissue factor sites but not in high tissue
factor environments,14 which will allow for intravascular anti-
coagulation with maintenance of extravascular hemostasis.15
Therefore, the upstream inhibition of FIXa at the initiation
stage of the cascade would represent an excellent approach for
developing selective and safe anticoagulants.
Early approaches to FIXa inhibition have been attempted with
the use of antibodies (SB24914),16 synthetic active site blocked
competitive inhibitor,15 and RNA aptamers.17 Recently, several
groups have reported their small molecule approaches but with
limited success, as most of the leads were derived from FXa
inhibitor scaffolds.18 Notably, Transtech has selected a partial
FIXa inhibitor, TTP889, for clinical development, although the
mechanism of action of this compound has not been published.19
We herby report our work in the small molecule approach
to the inhibition of FIXa.
FIXa is a key coagulation factor in the intrinsic pathway of
the cascade, essential for amplification of coagulation that
results in thrombus formation via FX activation to FXa.10
Enzymology studies have shown that activation of factor X by
IXa/VIIIa is nearly 50 times more efficient than activation by
factor VIIa/TF.11 Furthermore, deficiency in FIXa leads to
mild to moderate bleeding tendency in hemophilia B patients.12
As the active sites of FIXa, FXa, and uPA are very similar,18,20
we evaluated a number of templates reported in literature to
seek an initial lead for FIXa inhibitors. After a series of synthetic
efforts and molecular modeling studies, we decided to focus
on the 2-amidinobenzothiophene template, exemplified by
2-amidino-4-iodobenzothiophene (1) as the lead.
†PDB code of compound 16 in the FIXa active site is 3LC3.
*To whom correspondence should be addressed. Phone/fax:
þ441628672546. E-mail: shouming_wang@yahoo.co.uk.
aAbbreviations: 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;
TF/FVIIa, tissue factor and factor VIIa complex.
Notably, benzothiophenetemplate derivedcarboxylic acids
or their amides have been reported as bronchiodialators.21
r
2010 American Chemical Society
Published on Web 02/02/2010
pubs.acs.org/jmc

1466 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
Scheme 1a
Scheme 2a
a Reagents: (a) HSCH2CN, DBU, DMF, 60 °C, 71-85%; (b)
LiHMDS, THF, 0 °C, then HCl, 99%.
Compound 1 has been reported as a uPA inhibitor with an
IC50 of 210 nM;20 it shows a Ki of 0.95 μM against FIXa in our
FIXa assay. We envisaged that the FIXa activity and selec-
tivity may be improved through a systematic SAR investiga-
tion, as the lead molecule 1 is relatively simple and has a low
molecular weight.
The lead optimization strategy for this series was initially
aimed at substitutions at the benzo[b] ring to establish key
functional groups at optimal positions for potency. We
decided to leave the selectivity issue to a later stage after
gaining further understanding on how the template binds at
the FIXa active site. It is known that the S1 sites of uPA and
FIXa are structurally very similar to but different from that of
FXa, with a serine residue and an alanine residue at position
190, respectively. Our modeling study suggests that substitu-
tion at the thiophene ring of the molecule 1 would impede the
binding interaction of the amidine group with Asp189 in the
smaller S1 pocket.
a Reagents: (a) BBr3, DCM, -78 °C to room temp, 92%; (b) allyl
bromide, NaH, DMSO, 0 °C to room temp, 98%; (c) LiHMDS (2 equiv),
THF, 0 °C to room temp, then HCl, 100%; (d) t-Boc2O, DIPEA,
dioxane-H2O, 100%; (e) PhSiH3, Pd(Ph3P)4, DCM, 0 °C, 68%; (f) 8
(when X = Cl, Br), K2CO3, DMF, or MeCN; (g) DEAD, Ph3P, 8 (when
X = OH), THF, room temp; (h) TFA, DCM, room temp; (j) Mg(ClO4)2,
MeCN, 80 °C.
and palladium catalyst gave the 6-hydroxy-t-Boc amidine
intermediate 7. This key intermediate could be prepared in
multigram quantity and coupled with a varietyof alkyl halides
oralkylalcoholsunder Mitsunobu conditionsto give the ether
linked products 9. The final removal of the t-Boc group was
achieved by either trifluoroacetic acid (TFA) or magnesium
perchlorate Mg(ClO4)2.22
Results and Discussion
Chemistry
The FIXa inhibitory activities of the analogues prepared
according to Schemes 1 and 2 were evaluated in an amidolytic
assay using chromogenic substrate S-2366 and human recom-
binant FIXa (see Experimental Section). FXa assay was
carried out as described in the literature.24 Initial round
screening on the analogues substituted with a variety of simple
functional groups around the b-ring of the benzothiophene
template uncovered a number of active compounds that
showed moderate inhibitory activity against FIXa and a
limited degree of selectivity against FXa. Some representative
examples are illustrated in Table 1.
The general synthesis of the substituted benzo[b]thiophene-
2-carboximidine analogues is shown in Scheme 1. Thus, sub-
stituted 2-fluoro-/nitrobenzaldehydes (2) were treated with
thioacetonitrile (stabilized with amberlyst) and 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) in dimethylformamide (DMF)
at 60 °C to give the annulated product 2-cyanobenzothiophenes
(3) in over 70% yield after purification. The conversion of the
2-cyano group to the amidine was readily achieved in high
yield by treating compounds 3 with over 1 equiv of lithium
hexamethyldisilazide (LiHMDS), followed by the addition of
hydrochloric acid (HCl).
Although many functional groups were examined, it appeared
that only analogues with O- or aryl-linked substitutents showed
any degree of activity against FIXa, particularly those substi-
tuted at the 4-, 5-, or 6-position. No significant activity was
observed for any compounds substituted at the 7-position. This
was substantiated by modeling studies that suggest 7-substituted
compounds are not well tolerated in the FIXa active site.
At 4-position, the initial SAR indicated a preference for
lipophilic groups (compounds 11-13) with gradual enhance-
ment of FIXa activity, though none showed any significant
improvement over the lead compound 1. The phenyl substi-
tuted analogue 14 showed the similar potency to compound
12. The selectivities against FXa were generally poor. At the
5-position, the phenyl substituted analogue 15 was 5-fold less
To generate a library of O-alkylated analogues efficiently, a
more convergent synthetic route with an allyloxy for hydroxy
and t-BOC for amidine protection strategy was developed.
Scheme 2 exemplifies the preparation of 6-alkyoxybenzothio-
phene analogues from the corresponding 6-methoxyben-
zothiophene-2-nitrile (3a).
Thus, 6-methoxy analogue 3a was demethylated by boron
tribromide (BBr3) at -78 °C in dichloromethane (DCM),
followed by its reprotection by ally bromide in the presence of
sodium hydride (NaH) and dimethyl sulfoxide (DMSO) to
give the allyloxy intermediate 5. The 2-amidine compound 6
was furnished with the treatment of 5 by LiHMDS in quanti-
tative yield. The amidine protection with t-Boc anhydride and
subsequent deprotection of the ally group with phenylsilane

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1467
Table 1. Inhibitory Activitiesa of Substituted Benzothiophene Com-
pounds in FIXa and FXa Assays
Figure 1. Molecular docking of compound 18 in FIXa active site
(1RFN).
Table 2. Inhibitory Activity of Substituted 6-Benzyloxy Compounds in
FIXa and FXa Assays
compd
X
Ki (μM), FIXaa
Ki (μM), FXaa
18
21
22
23
24
25
26
27
28
29
30
31
32
33
34
H
3.8
14.8
NDb
6.2
20-F
30-F
40-F
23.0
0.68
0.7
7.3
20-Cl
7.2
0.9
NDb
4.3
30-Cl
40-Cl
0.45
26.9
0.45
0.25
0.9
2.9
20-MeO
30-MeO
40-MeO
40-Me
NDb
4.3
13.1
7.9
NDb
5.7
40-CO2Me
30,40-F,F
30,40-F,Cl
30,40-Cl,F
11.3
0.45
0.075
0.031
0.105
0.055
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.
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.
potent than the 4-phenyl analogue 14. Further exploration on
the substitutions of the phenyl ring showed only marginal
improvement in FIXa activity, exemplified by 30,40-dimethoxy-
phenyl compound 16. The 5-benzyloxy analogue 17 showed
similar magnitude of activities to the corresponding 4-benzy-
loxy analogue 12 against both FIXa and FXa. At the 6-posi-
tion, both O-linked analogues 18 and 19 demonstrated
moderate FIXa activities, though with different selectivities
against FXa, whereas the 6-phenyl substituted analogue 20
was less potent inhibitor than the 5-phenyl analogue 16.
A number of 4-, 5-, and 6-substituted analogues were
subjected to a series of modeling and docking studies within
the FIXa active site, using the published crystal structure of
human FIXa.25 It became apparent that 4- and 5-substituted
analogues offered little noticeable interactions except that of
the amidine and Asp189. Most of the 4- and 5-substitutents
appeared to point toward the solvent front, while the
6-substitutents appeared to point toward the S0 pocket. This
is exemplified by our modeling of compound 18 within the
FIXa active site, as shown in Figure 1.
Given the potential functional residues, such as Lys98,
His57, and Tyr99 within the vicinity of the S0 site available
for potential binding interactions, we decided to focus on the
SAR work at the 6-position, using analogue 18 as a new lead.
Table 2 exemplifies some of the active analogues from our
effort in substituting the phenyl ring of compound 18.
Substitutions at the 2-position of the phenyl ring with
halogens (F, Cl) and methoxy groups led to a significant
reduction of the FIXa activity (compounds 21, 24, 27), while
the same substitutions at either the 3- or 4-position gave more
potent FIXa inhibitors (22, 23, 25, 26, 28, 29) than compound
18. All these compounds showed a moderate selectivity
against FXa, with that of compound 29 reaching greater than
50-fold. Further screening of basic functional groups at the
4-position did not yield many significantly more potent FIXa
inhibitors; for example, 4-methyl analogue 32 showed similar

1468 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
Table 3. Inhibitory Activity for 6-Arylalkyloxy-Substituted Benzothio-
phene Compounds in FIXa and FXa Assays
Table 4. Inhibitory Activity for 6-Substituted Branched Benzothio-
phene Compounds in FIXa and FXa Assays
compd
R
Ki (μM), FIXaa
Ki (μM), FXaa
35
36
37
PhCH2
3-Py
2-thiophen
21.6
1.05
0.35
NDb
3.35
1.15
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.
activity to compound 23, while 4-carboxylate methyl ester 32
showed less activity than the lead molecule 18. However,
analogues with 3,4-disubstitutions appeared to be more
potent than the corresponding singly substituted analogues,
ranging from moderately improved (compound 32) to the
more dramatically improved (compound 33, Ki = 75 nM;
compound 34, Ki = 31 nM), though no selectivity improve-
ment was observed against FXa.
Next we also examined the extension of the benzyloxy side
chain of compound 18 and the bioisostere replacement of its
phenyl ring with variousheterocycles. As the majority of those
screened did not lead to significant improvement over the lead
molecule 18, Table 3 only shows a few typical examples.
One carbon extension of the benzyloxy side chain on the
lead 18 resulted in a compound (35) with 6-fold less activity,
whereas the 3-pyridyl replacement of the phenyl group gave
some improvement of activity (36). The more dramatic
improvement of activity was observed with the 2-thiophene
analogue (37), showing over 10-fold greater FIXa activity
than the lead molecule (18).
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.
As shown in Table 1, compound 19 with an O-linked
acetate group showed equipotent FIXa activity to the benzy-
loxy analogue 18, and we envisaged that a combination of
both groups, preferably with those more optimal aryl groups
asshowninTables 2 and 3, would enhancethe activityfurther.
Therefore, weinvestigated a number of branched analogues at
the benzylic position of compound 18, including those with
carboxylate groups. Table 4 shows some selected examples.
Introduction of a methyl or amide group at the benzylic
position of compound 18 gave compounds 38 and 39 with
much reduced FIXa activity when compared with analogue 23.
However, introduction of methyl carboxylate gave an over
17-fold more potent FIXa inhibitor (40) than compound 23,
though it was equipotent against FXa. Its hydrolyzed product
acid 41 showed a further 2-fold improvement of FIXa activity
over the ester analogue 40 but with 44-fold better selectivity
against FXa. Similarly, the corresponding 4-chlorophenyl
analogue ester 42 was also 2-fold more potent than its 4-fluoro
analogue 40, but our attempts to assay the corresponding
carboxylic acid of this compound failed because of its insolu-
bility. Further SAR studies with the extended esters 43 and 44
based on ester 42 gave a similar magnitude of activities against
both FIXa and FXa. Interestingly, introduction of an ethyl
carboxylate ester group to the potent 2-thiophene analogue
(37) gave a very potent FIXa inhibitor (45, Ki = 3 nM). Again,
no selectivity was observed against FXa. Our attempts to
hydrolyze this ester also met with difficulty in solubility, which
was difficult to characterize and assay.
Figure 2. X-ray structure of compound 16 in the FIXa active site
(PDB code 3LC3).
analogues substituted at the 4-, 5-, or 6-position for X-ray
crystallography studies, using a truncated form of human
factor IXa,26 Compound 16 was successfully cocrystallized
˚
with FIXa, yielding a high resolution (1.9 A) crystal structure
(3LC3) as shown in Figure 2.
It appeared that two molecules of compound 16 were
present in the crystal structure; one in the primary binding
site (S1, S10), the other at the S3-S4 site. At the S1 site,
hydrogen bondswereformedasexpectedbetween the amidine
moiety and the carboxylate of Asp189 (distance between
˚
nitrogen and oxygen, 2.9 and 3.0 A). The benzothiophene
template clearly laid in the hydrophobic S1 pocket. Com-
In order to understand how this benzothiophene template
binds at the FIXa active site, we selected a number of
pound 16 with 5-(30,40-dimethoxyphenyl) substitution at the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1469
stationary phase for preparative column chromatography using
medium pressure was silica gel 60, mesh size 40-60 μm, from
E. Merck, Darmstadt, Germany.
NMR spectra were obtained using a Bruker ACF 400 oper-
ating at 400 MHz, and the H shifts (ppm) were calibrated to
1
that of 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 Electro-
thermal 9100 series apparatus.
The purities of 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 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 8 min through a X-Terra Prep MS C18
5 μm (19mm ꢀ 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 lyophilization.
Figure 3. Selected view of compound 16 at primary binding S1 site
with an induced opening of S1 β site.
benzothiophene template appeared to have induced the open-
ing of the S1 β pocket as shown more clearly in Figure 3. This
is in direct contrast with the previous X-ray crystal structure
(1RFN),25 using p-aminobenzamidine as theligand, where the
S1 β site was seen closed.
The secondary binding of compound 16 at S3/S4 site was
unexpected, as it was observed previously that S2 or S3/S4
sites are blocked by Tyr99.25 As shown in Figure 2, the Tyr99
residue appeared to have moved away from its previous
reported position because of a dramatic rearrangement of
the 99-loop, leaving open the S2-S4 sites for the secondary
molecule binding. The amidine group of the second molecule
(16) formed hydrogen bonding interaction with the carbonyl
group of Lys98. The benzothiophene template lay directly
within the lipophilic pocket surrounded by Trp215, Phe174,
and Tyr99. The shift of Tyr99 residue and secondary binding of
molecule 16 at the S3/S4 site is significant, as it has been suggested
that blockage of either the S2 or S3/S4 site by Tyr99 may be one of
the reasons that no potent low molecular weight active site FIXa
inhibitors have been reported so far.25 Our findings above suggest
it is possible to develop potent FIXa inhibitors.
Microanalyses of representive compounds were performed by
MEDAC Ltd. UK. Where analyses are indicated only by the
symbols of the elements, results obtained were within 0.4% of
the theoretical values. All compounds (3-45) quoted in this
paper have been patented.23
General Procedure A for Substituted Benzothiophene-2-
carboximindine Analogues (Scheme1). 2-Cyanobenzothiophene
(3) Synthesis. To a solution of arylaldehydes (1 equiv) in DMF
(3 mL/mmol) and DBU (2 equiv) was added thioacetonitrile
(1.2 equiv) dropwise at 0 °C. The solution was stirred at 0 °C for
20 min, then heated to 40 °C for 16-20 h. The reaction mixture
was allowed cool to room temperature, the solvent was reduced
in vacuo, and the product was purified via silica chromato-
graphy (EtOAc/hexane mix), yielding the intermediates 3.
Amidines (4) Formation. To a solution of nitriles 3 (1 equiv) in
THF (3 mL/mmol) at 0 °C under argon was added a solution of
lithium hexamethyldisilazide (1 M in THF, 1.2 equiv) dropwise.
The solution was stirred at 0 °C for 1.5 h, then allowed to warm
room temperature for 45 min.
Conclusion
In the present report we have described some synthesis and
initial rounds of SAR studies on the benzothiophene template
as FIXa inhibitors. Our initial focus on its 6-position led to a
number of highly potent FIXa inhibitors, albeit the selectivity
against FXa was moderate (up to 50-fold). We have demon-
strated that a moderately active compound (16) binds at the
FIXa active site through X-ray study with an induced S1 β site
opening and a secondary binding interaction at the S3/S4 site,
due to Tyr99 residue shift and a dramatic 99-loop rearrange-
ment. These findings are in direct contrast with those reported
previously in literature,25 paving the way for our modified
optimization strategy in the development of potent and
selective FIXa inhibitors, which will be the subject of our next
publications.
The reaction was quenched with methanol and the solvent
removed. The residue was redissolved in THF (3 mL/mmol).
Then HCl (4 M in dioxane, 2-3 equiv) was added slowly. The
mixture was stirred at room temperature for 30 min and then
filtered and the solid collected to yield desired amidines 4.
6-Allyloxy-2-cyanobenzothiophene (5). To a solution of
6-methoxy-2-cyano-benzothiophene (3a) (17.1 g, 90.5 mmol)
in DCM (200 mL) at -78 °C under argon was added BBr3
(0.1 M, 180.9 mL, 180.9 mmol) dropwise over 1 h. After
addition was complete the mixture was stirred for a further
2 h, then allowed to warm to room temperature and stirred
overnight. The reaction was quenched with saturated aqu-
eous NaHCO3 (500 mL) and back-extracted with EtOAc. The
organic layers were washed with H2O (2 ꢀ 200 mL) and brine
(1 ꢀ 200 mL), dried (MgSO4), and concentrated in vacuo to
afford the intermediate 6-hydroxy-2-cyanobenzothiophene
compound as a dark-orange/brown solid, 14.6 g, 92%, which
was taken directly to the next step. 1H NMR (MeOH-d4,
400 MHz): δ 7.93 (s, 1H), 7.74-7.76 (m, 1H), 7.26 (s, 1H),
6.96-7.01 (m, 1H). MS m/z (ESþ) 175.9 [M þ H]þ.
Experimental Section
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
The above intermediate (14.5 g, 82.8 mmol) was stirred in
DMSO (130 mL) at 0 °C under argon, and NaH (60% disper-
sion, 7.89 mL, 91.1 mmol) was added portionwise. The reaction

1470 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Wang et al.
mixture was stirred for 15 min before addition of allyl bromide
(7.89 mL, 91.1 mmol). The reaction mixture was warmed to room
temperature and stirred for 48 h before pouring onto H2O/ice
(600 mL). This was extracted with EtOAc (400 mL), and the
organic layer was washed with 1 M aqueous NaOH, H2O, and
brine, dried (MgSO4), and concentrated to give the title compound
General Procedure D for the Deprotection of BOC Amidines
(9) with TFA Preparation of 6-Alkoxybenzothiophene-2-carbox-
imidines (10). The BOC protected amidines 9 were dissolved in
10-20% TFA/CH2Cl2 (10-15 mL/mmol) and stirred at room
temperature for 2-6 h. The solvent was removed, the residue
triturated with excess diethyl ether and filtered, and the solid
collected to give the desired product 10 as the TFA salt. If
required, further purification was performed via LC-MS.
General Procedure E for the Deprotection of BOC Amidines
(9) with Mg(ClO4) Preparation of 6-Alkoxybenzothiophene-2-
carboximidines (10). To a solution of BOC protected amidine
9 (1 equiv) in acetonitrile (10 mL/mmol) was added MgClO4
(1.5 equiv) under an argon atmosphere, and the mixture was
heated to 70 °C for 16-20 h. The mixture was allowed to cool to
room temperature and the solvent concentrated in vacuo. The
crude residue was purified by silica chromatography (methanol/
CH2Cl2 mix) to yield the desired products 10.
General Procedure F for the Hydrolysis of Esters. To a solution
of ester 10 (when R1 = CO2Me) (1 equiv) in THF (10 mL/mmo)
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 carboxylic
acid products (10, when R1 = CO2H).
1
5 as a beige solid, 17.6 g, 98%. H NMR (CDCl3, 400 MHz):
δ 7.67-7.72 (m, 2H, ArH), 7.20 (m, 1H, ArH), 7.35 (m, 1H, ArH),
6.01 (m, 1H, CHd), 5.38 (dd, 1H, CHd), 5.27 (dd, 1H, CHd), 4.55
(d, 2H, CH2O). MS m/z (ESþ) 215.95 [M þ H]þ.
6-Allyloxybenzothiophene-2-carboximidine (6). To a solution
of compound 5 (5.35 g, 25.0 mmol) in THF (70 mL) at -10 °C
under argon was added LiHMDS (1.0 M, 27.4 mL, 27.3 mmol)
dropwise over 60 min. After the mixture was warmed to room
temperature and stirred for 5 h, an additional 1 equiv of LiHMDS
was added. HCl in dioxane (4.0 M, 6.84 mL, 27.3 mmol) was added
dropwise and the reaction mixture stirred at room temperature
overnight. The reaction mixture was concentrated, and addition of
ether precipitated the title compound 6 as the HCl salt, 6.68 g,
100%. 1H NMR (DMSO-d6, 400 MHz): δ 7.77 (s, 1H, ΑrH), 7.71
(m, 1H, ArH), 7.15 (s, 1H, ArH), 7.01 (m, 1H, ArH), 6.72 (br, 4H,
amidine þ HCl), 6.08 (m, 1H, CHd), 5.43 (m, 1H, CHd), 5.28 (m,
1H, CHd), 4.64 (s, 2H, CH2O). MS m/z (ESþ), 233.0 [M þ H]þ.
6-Hydroxybenzothiophene-2-t-Boc-carboximidine (7). To a
solution of 6 (7.00 g, 30.1 mmol) in dioxane/H2O (1:1, 300 mL)
at room temperature under argon was added DIPEA (8.50 mL,
60.3 mmol), and the reaction mixture was stirred for 30 min.
(BOC)2O (7.27 g, 33.2 mmol) was added and the mixture stirred
for a further 1 h. The reaction mixture was extracted with EtOAc
(300 mL), and the organics were washed with H2O (2 ꢀ 200 mL)
and saturated aqueous NaCl (2 ꢀ 200 mL), dried (MgSO4), and
concentrated in vacuo. Trituration with diethyl ether yielded the
6-allyloxybenzothiophene-2-t-Boc-carboximidine intermediate
4-Benzyloxybenzo[b]thiophene-2-carboxamidine (12). 12 was
synthesized in an analogous fashion to general procedure A to give
the title compound. 1H NMR (DMSO-d6): δ 8.14 (s, 1H, H3) 7.55
(m, 3H, 3ArH), 7.42 (m, 4H, 4ArH), 7.04 (d, 1H, J = 7.70 Hz, Ar-
H7), 5.30 (s, 2H, CH2). MS m/z (ESþ) 283 [M þ H]þ.
1
(9.77 g, 100%), which was taken directly to the next step. H
5-(3,4-Dimethoxyphenyl)benzo[b]thiophene-2-carboxamidine
Hydrochloride (16). 16 was synthesized according to the general
procedure A (23% over two steps). 1H NMR (400 MHz,
DMSO-d6): δ 9.59 (s, 2H), 9.22 (s, 2H), 8.41 (s, 1H), 8.33 (s,
1H), 8.25 (d, 1H, J = 8 Hz), 7.92 (dd, 1H, J = 1 Hz and J =
8 Hz), 7.32 (d, 2H, J = 8 Hz), 7.19-7.03 (m, 2H), 3.88 (s, 3H),
and 3.82 (s, 3H). MS m/z: 349.85 (M þ H)þ.
NMR (DMSO-d6, 400 MHz): δ 9.08 (s, 2H, NH2), 8.23 (s, 1H,
ArH), 7.79 (m, 1H, ArH), 7.56 (d, 1H, ArH), 7.06 (m, 1H, ArH),
5.45 (m, 1H, CHd), 5.30 (m, 1H, CHd), 5.27 (m, 1H, CHd), 4.67
(s, 2H, CH2O), 1.46 (s, 9H, t-Bu). MS m/z (ESþ) 333 [M þ H]þ.
To a solution of the above product (9.77 g, 29.4 mmol) in
DCM (110 mL) at room temperature under argon was slowly
added Pd(PPh3)4 (847 mg, 0.70 mmol). After being stirred for
10 min, the reaction mixture was cooled to 0 °C and phenylsilane
(11.0 mL, 58.9 mmol) was added dropwise. The resulting
solution was stirred at room temperature for 30 min. Then the
reaction mixture was quenched with H2O (300 mL) and
the aqueous layers were back-extracted with DCM. The com-
bined organic layers were dried (MgSO4) and concentrated in
vacuo. Purification by silica chromatography, eluting with a
solvent gradient of 15-50% ethyl acetate in hexane, yielded the
title compound 7 as a yellow semisolid (6.0 g, 68%). 1H NMR
(DMSO-d6, 400 MHz): δ 10.0 (br, 1H, ΟH), 9.04 (br, 2H, NH2),
8.18 (s, 1H, ArH), 7.71 (d, 1H, ArH), 7.25 (d, 1H, ArH), 6.91 (m,
1H, ArH), 1.46 (s, 9H, t-Bu). MS m/z (ESþ) 293 [M þ H]þ.
General Procedure B for the Preparation of 6-Alkoxyben-
zothiophene-2-t-Boc-carboximidines (9). To a solution of com-
pound 7 (1.0 equiv, 150 mg, 0.51 mmol) in DMF (5.0 mL per
0.5 mmol of 7) was added the alkyl bromides 8 (1.1 equiv),
followed by K2CO3 (1.1 equiv). The reaction mixture was heated
under reflux until completion was evident by TLC analysis. The
residue was purified by silica chromatography, yielding the title
compounds 9.
6-Benzyloxybenzo[b]thiophene-2-carboxamidine (18). 18 was
synthesized in an analogous fashion to general procedure A to
1
give the title compound. H NMR (DMSO-d6): δ 8.01 (s, 1H,
Ar-H3) 7.85 (d, 1H, J = 8.8 Hz, Ar-H5), 7.74 (s, 1H, Ar-H7),
7.49 (d, 1H, J = 7.6 Hz, Ar-H-20), 7.40 (t, 2H, J = 7.0 Hz,
Ar-H30), 7.34 (d, 1H, J = 6.6 Hz, Ar-H40), 7.14 (d, 1H, J = 8.8
Hz Ar-H4), 5.20 (s, 2H, CH2). MS m/z (ESþ) 283 [M þ H]þ.
6-(3-Chloro-4-fluorobenzyloxy)benzo[b]thiophene-2-carbox-
amidine. Compound with Trifluoroacetic Acid (34). The syn-
thesis was done according to general procedure C followed by
general procedure D to yield the title compound as a beige
solid, 24% over two steps. 1H NMR (DMSO, 400 MHz): δ 9.5
(bs, 1H, NH), 8.3 (s, 1H, ArH), 8.01 (dd, 1H, ArH), 7.9 (s, 1H,
ArH), 7.73 (dd, 1H, ArH), 7.64 (m, 2H, ArH), 7.31 (d, 1H,
ArH), and 5.2 (s, 2H, methylene H). LC-MS m/z (ESþ) 335
[M þ H]þ.
6-(Thiophen-2-ylmethoxy)benzo[b]thiophene-2-carboxamidine
(37). 37 was synthesized in an analogous fashion to general
procedure A to give the title compound. 1H NMR (DMSO-d6):
δ 8.54 (s, 2H,NH2), 8.15 (s, 1H, ArH), 7.89 (d, 1H, J = 8.88 Hz,
ArH), 7.65 (d, 1H, J = 2.02 Hz, ArH), 7.42 (dd, 1H, J = 1.02
and 5.05 Hz, ArH), 7.22 (m, 2H, 2 ArH), 7.01 (m, 1H, ArH), 5.39
(s, 2H, CH2). MS m/z (ESþ) 288.9 [M þ H]þ.
General Procedure C for the Preparation of 6-Alkoxyben-
zothiophene-2-t-Boc-carboximidines (9). To a solution of com-
pound 7 (1.0 equiv, 150 mg, 0.51 mmol), the alkyl alcohols (8,
X = OH) (1.5 equiv), and PPh3 (1.5 equiv) in THF (2 mL per
0.5 mmol of 7) at room temperature was added a solution of
DEAD (1.5 equiv) in THF dropwise. After being stirred at room
temperature until completion, the reaction mixture was concen-
trated in vacuo and purified by silica chromatography, yielding
the title compounds 9.
3-(2-Carbamimidoylbenzo[b]thiophen-6-yloxy)-3-(4-chloro-
phenyl)propionic Acid Methyl Ester Trifluoroacetic Acid Salt
(43). 43 was synthesized according to general procedures C
and D with the preparation of the alcohol as follows. Sodium
borohydride was added to a solution of methyl (4-chlorophenyl)-
acetate in methanol at room temperature and stirred for 30 min.
The mixture was partitioned between ethyl acetate and water,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1471
extracted with ethyl acetate (30 mL ꢀ 3), dried over magnesium
sulfate, filtered, and solvent was removed. MPLC (4:1 hexane/
ethyl acetate) was conducted to yield a colorless oil (489 mg, 2.3
mmol, 48%). 1H NMR (DMSO-d6, 400 MHz): δ 7.38 (s, 4H), 5.61
(d, 1H, J = 4.9 Hz), 4.97 (m, 1H), 3.58 (s, 3H), 2.64 (m, 2H).
This compound was then used in the general Mitsunobu
procedure C to yield a colorless gum (30 mg, 0.062 mmol,
112%). MS m/z: 489.03 (M þ H)þ. This residue was then BOC
deprotected according to the generalTFA/DCM procedure D to
yield the desiredproduct, a white crystalline solid (1.0 mg, 0.0019
mmol, 3%). 1H NMR (DMSO-d6 400 MHz): δ 9.14 (br s, 4H),
8.32 (s, 1H), 8.03 (d, 1H, J = 8.8 Hz), 7.84 (s, 1H), 7.68 (d, 2H,
J = 8.4 Hz), 7.55 (d, 2H, J = 8.4 Hz), 7.28 (m, 1H), 6.07 (m, 1H),
3.74 (s, 3H), 3.19 (m, 2H). MS m/z: 389.03 (M þ H)þ.
was refolded over 4 days at 4 °C in a solution containing 500 mM
L-arginine and 0.5 mM L-cysteine, then dialyzed into a Tris/
NaCl buffer in two overnight steps.
Activation of Purified Factor IX. Recombinant factor IX was
activated by overnight incubation at 11 °C with factor XIa
(HTI, 0.5% w/v) and then further purified by Ni-NTA affinity
chromatography before final dialysis into 20 mM Tris, 50 mM
NaCl (pH 8.0).
Assay. The factor IXa amidolytic assay was performed in
50 mM Tris, 100 mM NaCl, 5 mM CaCl2, 0.5% PEG 6000 (pH 7.4)
in a VersaMax plate reader. The S-2366 chromagenic substrate
(Quadratech) was used at a final concentration of 500 μM, and the
change in absorption was recorded at 405 nm using SoftMax
software. Test compounds were assayed at least three times at a
range of concentrations, and an IC50 value was established using
Prism software (GraphPad).
4-(2-Carbamimidoylbenzo[b]thiophen-6-yloxy)-4-(4-chloro-
phenyl)butyric Acid Methyl Ester Trifluoroacetic Acid Salt
(44). Methyl iodide (0.32 mL, 5.2 mmol) was added to a solu-
tion of 3-(4-chlorobenzoyl)propionic acid (1.00 g, 4.7 mmol)
and potassium carbonate (716 mg, 5.2 mmol) in dry acetoni-
trile (30 mL) under inert atmosphere and stirred at 85 °C for
16 h. The mixture was partitioned between DCM (10 mL) and
NaOH (1.0 mol solution, 10 mL) and extracted with DCM
(10 mL ꢀ 3). The organics were dried and filtered and solvent
was removed to yield a white crystalline solid (429 mg,
1.9 mmol, 40%). This was then dissolved in MeOH (20 mL).
NaBH4 (77 mg, 2.1 mmol) was added portionwise, and the
mixture was stirred at room temperature for 1.5 h, water
(15 mL). DCM (15 mL) was added and the mixture stirred for
30 min. The sample layer was extracted with DCM (10 mL ꢀ
3). The organics were dried and filtered, and solvent was
removed. MPLC (4:1 hexane/ethyl acetate) was conducted
to yield the hydroxyl compound as a colorless oil (259 mg,
1.1 mmol, 60%). MS m/z: 228.92 (M þ H)þ. This compound
was then coupled with 7 via the general Mitsunobu procedure
Molecular Modeling. Molecular modeling of compounds was
performed with Chemical Computing Group’s MOE software
(Montreal, Canada).
Acknowledgment. We thank Dr. Roger Dickinson for
providing consultancy on the project.
Supporting Information Available: Experimental and spectro-
scopic information for additional compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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