The discovery of new human coagulation factor XIa (FXIa) inhibitors by synthesis, biological evaluation, and structure-based modeling

Article abstract of DOI:10.1002/bkcs.10831

Factor XIa (FXIa) is an enzyme that is activated during the earliest stage of initiation of the intrinsic pathway of the blood coagulation mechanism. In this study, we attempted to discover a new FXIa inhibitor based on structure-based molecular modeling. We found that compound 16 exhibits satisfactory predicted properties while maintaining important binding interactions with FX1a.

Full text of DOI:10.1002/bkcs.10831

Article
DOI: 10.1002/bkcs.10831
BULLETIN OF THE
M. H. Lee et al.
KOREAN CHEMICAL SOCIETY
The Discovery of New Human Coagulation Factor XIa (FXIa)
Inhibitors by Synthesis, Biological Evaluation, and Structure-based
Modeling
Myeong Hwi Lee,†,§ Ho Young Song,‡,§ Hyoungrae Kim,‡ Kyung Eun Park,‡ Jinyeong Kim,‡ Tae
Kyo Park,‡ Yong Ju Kim,‡ and Nam Sook Kang†,
*
†Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon
305-764, Korea. *E-mail: nskang@cnu.ac.kr
‡LegoChem Biosciences Inc., Daejeon, Korea
Received February 25, 2016, Accepted May 12, 2016
Factor XIa (FXIa) is an enzyme that is activated during the earliest stage of initiation of the intrinsic path-
way of the blood coagulation mechanism. In this study, we attempted to discover a new FXIa inhibitor
based on structure-based molecular modeling. We found that compound 16 exhibits satisfactory predicted
properties while maintaining important binding interactions with FX1a.
Keywords: Factor X1a, SBDD, Blood coagulation, Thromboembolic diseases, Inhibitor
Thromboembolic diseases are the major cause of morbidity
and mortality in developed countries.1,2 Deep vein throm-
bosis, acute myocardial infarction, heart attack, pulmonary
embolism, and ischemic stroke are all classified as throm-
boembolic diseases. Several studies have reported that cur-
rent medications against thromboembolic diseases use
nonspecific inhibitors that cause a high risk of bleeding side
effects by interfering with the activities of other blood clot-
ting factors.3
The blood coagulation process involves numerous serine
proteases and cofactors. Factor XIa (FXIa) is an enzyme
that is activated during the earliest stage of the initiation of
the intrinsic pathway of the blood coagulation mechanism.
The intrinsic pathway of the blood coagulation mechanism
is initiated by a process known as contact activation. Con-
tact activation leads to Factor XIIa (FXIIa) converting Fac-
tor XI (FXI) into active Factor Xia (FXIa), which serves as
the starting point for the activation of FX1a and Factor Xa
(FXa) through a chain reaction. At the intersection of the
extrinsic pathway of the blood coagulation mechanism,
FXa subsequently generates thrombin (FIIa) and thus gen-
erates the final hemostatic clots.4 The intrinsic pathway, in
which FXIa intervenes, is responsible for the amplification
phase of the blood coagulation process and serves to further
strengthen the hemostatic clots once they are formed in the
injured area through an extrinsic pathway.5
antibodies against FXI, the number of blood clots decreased
considerably, whereas the bleeding time was unaffected.7
In addition, in a carotid artery occlusion experiment using
FeCl3 conducted on mice without FXI enzymes, it was
reported that there were no changes in the bleeding times.8
Hence, the need for FXIa inhibitors has risen, and many
research groups are carrying out research to develop FXIa
inhibitors.9–13 Nonetheless, FXIa inhibitors of small-
molecule chemicals that are undergoing clinical tests have
yet to be reported. In this context, BMS262084 (Figure 1
(b)) under development by Bristol-Myers Squibb (BMS)
exhibits FXIa inhibition activation with an IC50 value of
2.8 nM.14 However, BMS262084 is still under study for
anti-allergic effects, and it is an irreversible inhibitor
because the β-lactam rings of molecules and FXIa form
covalent bonds.15
The FXIa peptide inhibitor (Figure 1(a)) reported by
Daiichi Asubio Medical Research Laboratories, LLC
(DAIAMED)16 has strong in vitro efficacy (IC50 = 6 nM)
and outstanding medicinal effects in rats in an animal
thrombus model. However, as with BMS262084 (Figure 1
(b)), this peptide is an irreversible inhibitor that forms cova-
lent bonds with FXIa. Such irreversible inhibitors are likely
to give unpredictable side effects, because there are many
nucleophiles in the body that can react with them.
Consequently, new compounds with excellent inhibition
activation against FXIa and possessing reversible properties
are urgently needed. In this study, we have discovered new
FXIa inhibitors based on structure-based molecular modeling.
The inhibition of FXIa has been reported to prevent the
generation of blood clots and has been found to be very
safe with respect to hemorrhagic side effects, the most sig-
nificant side effect of antithrombotics, when compared with
that of thrombin and FXa inhibitors.6 Results of an experi-
ment involving rabbits reported that after processing
Experimental
Docking Study. To find a new FX1a inhibitor, we carried
§ Contributed equally to this work.
out a docking study of selected molecules using the
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H2N
NH
(Macherey-Nagel 0.25 mm silica gel 60 with fluorescent
indicator UV254, MACHEREY-NAGEL GmbH & Co.,
Bethlehem, PA, USA), and the spots were visualized under
UV light (254 nm) and/or charring after dipping the TLC plate
into ninhydrin or Ce–Mo staining solution. Column chroma-
tography was performed on silica gel (Merck 9385 silica gel
60; Merck, Darmstadt, Germany). The designed compounds
were synthesized according to the following Schemes (1–7).
OH
NH
N
O
O
H
N
S
N
N
N
H
H
H
O
O
Br
Procedure for the Synthesis of 1-(4-Chloro-2-ethynyl-
phenyl)-1H-tetrazole (Scheme 1). To 4-chloro-2-iodo ani-
line, 1 (30 g, 118.4 mmol) in acetic acid (300 mL), sodium
azide (23 g, 355.1 mmol) and trimethyl orthoformate
(38.85 mL, 355.1 mmol) were added, and the solution was
stirred at room temperature (rt) for 4 h, and then extracted
with ethyl acetate (50 mL × 2). The organic layer was
washed with brine, dried (MgSO4), and filtered. The sol-
vent was evaporated under reduced pressure, and the resid-
ual mixture was poured into diethyl ether (30 mL) to afford
the white solid 1-(4-chloro-2-iodophenyl)-1H-tetrazole,
2 (1 g, 85%). To 2 (10 g, 32.6 mmol) in acetonitrile (ACN;
180 mL), ethynyltrimethylsilane (4.5 mL, 32.6 mmol),
(a) DAIAMED (IC50=6 nM)
NH
H2
N
CO2
H
O
N
H
N
N
N
H
N
O
O
(b) BMS262084 (IC50=2.8 nM)
Figure 1. Structures of known FXIa inhibitors.
released crystal structure complex (PDB ID:3SOS)17,18 of
FXIa from the RCSB protein databank (www.rcsb.org).
The protein was subjected to the “Prepare protein”19 proc-
ess using the CHARMm force field and default conditions.
The prepared protein was then defined as a receptor for the
docking calculation. The binding site of FXIa was defined
as a 13.2 Å sphere around the original ligand of the 3SOS
structure. In order to prepare the input molecules, each con-
formation of the designed molecules was obtained through
energy minimization calculations under the CHARMm
force field.19 The designed molecules were docked into the
binding site of the FXIa by the LigandFit20 protocol of Dis-
covery Studio 4.5. Each docked conformation was evalu-
ated and ranked using the scoring functions of LigScore1,
LigScore2,21 piecewise linear potential (PLP), PLP2,22
Jain,23 and potential of mean force.24
triethylamine
(TEA;
13.6 mL,
97.9 mmol)
bis(triphenylphosphine)palladium(II) dichloride (PdCl2
(PPh3)2, 230 mg, 0.326 mmol), and copper iodide (62 mg,
0.326 mmol) were added, and the solution was stirred for
3 h under a nitrogen atmosphere. The reaction mixture was
evaporated under reduced pressure, and column chromatog-
raphy was used to obtain 1-(4-chloro-2-((trimethylsilyl)
ethynyl)phenyl)-1H-tetrazole, 3 (3.8 g, 42%). Compound
3 (3.8 g, 13.7 mmol) in tetrahydrofuran (THF; 50 mL) was
mixed with tetra-n-butylammonium fluoride (TBAF;
16.5 mL, 16.5 mmol) and stirred at −78ꢀC for 1 h under a
nitrogen atmosphere. Ammonium chloride (20 mL) was
added, and the sample was extracted with ethyl acetate
(20 mL × 2). To obtain 1-(4-chloro-2-ethynyl-phenyl)-1H-
tetrazole, 4 (2.1 g, 75%), the residual mixture was poured
into diethyl ether (5 mL) and recrystallized. Compound 2:
1H-NMR (600 MHz, CDCl3): δ 8.92 (s, 1 H), 8.05 (d,
J = 2 Hz, 1 H), 7.56 (dd, J = 2.4, 8.4 Hz, 1 H), 7.38 (d,
J = 8.4 Hz, 1 H). Compound 3: 1H-NMR (600 MHz,
CDCl3): δ 9.33 (s, 1 H), 7.67–7.69 (m, 2 H), 7.51–7.53 (m,
1 H), 0.22 (s, 9 H). Compound 4: 1H-NMR (600 MHz,
CDCl3): δ 9.29 (s, 1 H), 7.71 (d, J = 2.4 Hz, 1H), 7.68 (d,
J = 9.0 Hz, 1 H), 7.57 (dd, J = 2.4, 9.0 Hz, 1 H), 3.41
(s, 1H).
Chemical Synthesis. All chemicals used were purchased
from Sigma-Aldrich (Munich, Germany, Waters, MA, USA).
1H NMR spectra were recorded using a Varian Inova
(600 MHz; Santa Clara, CA, USA) spectrometer. Mass spec-
tra were recorded on a Waters 2695 Separation Module
(Waters, MA, USA) and micromass ZQ spectrometer
(Waters, MA, USA) using the electron impact method. The
progress of the reaction was checked on TLC plates
c
b
a
1
3
4
2
Scheme 1. Synthesis of 1-(4-chloro-2-ethynyl-phenyl)-1H-tetrazole. (a) NaN3, TMOF, AcOH, rt; (b) Ethynyl-trimethyl-silane, CuI, PdCl2
(PPh3)2, TEA, ACN, rt; (c) TBAF, THF, −78ꢀC.
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Procedure for Synthesis of tert-Butyl (1-Azido-3-phenyl-
propan-2-yl)carbamate (Scheme 2). tert-Butyl (1-
281 mg, 0.54 mmol) and N,N-diisopropylethylamine
(DIPEA; 188 μL, 1.08 mmol) were added to 8 in DMF
(3 mL) and stirred for 6 h. The reaction mixture was added
to an ammonium solution (20 mL) and distilled water
(20 mL), and extracted with ethyl acetate (20 mL × 2).
hydroxy-3-phenylpropan-2-yl)
carbamate,
5
(3 g,
11.9 mmol) in THF (30 mL) was mixed with TEA
(4.98 mL, 3.57 mmol) and Ms2O (2.5 g, 14.28 mmol), and
stirred at rt for 1 h. Distilled water (20 mL) was added to
the reaction mixture and then extracted with ethyl acetate
(20 mL × 2). The extracted product was dissolved in
dimethylformamide (DMF; 30 mL), and sodium azide
(3.9 mL, 59.5 mmol) was added. This solution was stirred
at 50ꢀC, extracted with ethyl acetate (50 mL × 2), poured
into diethyl ether (5 mL), and recrystallized to afford the
1
Compound 7 H-NMR (600 MHz, DMSO-d6) δ 8.77 (s,
1 H), 8.07 (s, 1 H), 7.55 (s, 1 H), 7.42–7.17 (m, 7 H), 6.85
(s, 1 H), 4.66 (s, 1 H), 4.43–4.15 (m, 2 H), 2.76 (d,
J = 7.8 Hz, 2 H), 1.40 (s, 9 H).; LCMS (m/z): 481.2 (M
+H+).; compound 8 1H-NMR (600 MHz, DMSO-d6) δ 9.73
(s, 1 H), 8.38 (br s, 2 H), 8.11 (s, 1 H), 8.03 (s, 1 H), 7.73
(s, 2 H), 7.37–7.26 (m, 5 H), 4.58 (s, 2 H), 3.91 (br s, 1 H),
1
tert-butyl
(1-azido-3-phenylpropan-2-yl)carbamate,
2.99–2.94 (m, 1 H), 2.83–2.78 (m, 1 H). Compound 9: H-
6 (21 mg, 95%). Compound 6: 1H-NMR (600 MHz,
DMSO-d6): δ 7.72 (d, J = 44.4 Hz, 5 H), 6.99 (s, 1H),
3.76 (s, 1 H), 3.28 (s, 2 H), 2.66–2.72 (m, 2 H), 1.27
(s, 9 H).
NMR (600 MHz, DMSO-d6) δ 9.64 (s, 1 H), 8.56 (d,
J = 8.4 Hz, 1 H), 8.01–7.97 (m, 3 H), 7.74–7.71 (m, 3 H),
7.28–7.17 (m, 5 H), 4.62–4.46 (m, 3 H), 3.87 (s, 3 H), 3.01
(m, 1 H), 2.94–2.84 (m, 2 H); LCMS (m/z): 542.93 (M
+H+). Compound 10: 1H-NMR (600 MHz, DMSO-d6) δ
9.64 (s, 1 H), 8.21 (d, J = 8.4 Hz, 1 H), 7.98 (s, 1 H), 7.94
(s, 1 H), 7.73–7.69 (m, 2 H), 7.63 (d, J = 7.2 Hz, 2 H),
7.27–7.14 (m, 5 H), 6.95 (d, J = 9.0 Hz, 2 H), 4.59–4.46
(m, 3 H), 3.79 (s, 3 H), 2.86 (d, J = 7.2 Hz, 2 H); LCMS
(m/z): 514.94 (M+H+). Compound 11: 1H-NMR (600 MHz,
DMSO-d6) δ 9.63 (s, 1 H), 8.61 (d, J = 9.0 Hz, 1 H),
7.98–7.92 (m, 4 H), 7.76–7.71 (m, 4 H), 7.29–7.18 (m,
5 H), 4.64–4.46 (m, 3 H), 2.92–2.73 (m, 2 H); LCMS (m/
z): 509.95 (M+H+).
Procedure for the Synthesis of Compounds 9–11
(Scheme 3). The products, compounds 4 and 6 obtained
from Scheme 1 (500 mg, 2.44 mmol) were dissolved in
dimethyl sulfoxide (DMSO) and distilled water (1:1), and
sodium ascorbate (48 mg, 0.24 mmol) and copper(II) sulfate
(30 mg, 0.12 mmol) were added. This solution was stirred
for 2 h under a nitrogen atmosphere. The reaction was
extracted with ethyl acetate (10 mL × 2), and the extracted
product was poured into diethyl ether (10 mL) and recrystal-
lized to obtain 7 (1.1 g, 90%). Compound 7 in dichloro-
methane (DCM) was mixed with HCl (4 M, 5 mL), stirred
for 1 h, and evaporated under reduced pressure. 1-(4-(5-
Chloro-2-(1H-tetrazol-1-yl)ph enyl)-1H-1,2,3-triazol-1-yl)-
3-phenylpropan-2-amine hydrochloride, 8 (433 mg, 96%)
was obtained. R1-benzoic acid, benzotriazol-1-yl-oxytripyr-
Procedure for the Synthesis of 4-(3-Azido-2-benzylpro-
panamido)-Benzoic Acid Methyl Ester (Scheme 4).
2-Benzyl-3-hydroxypropanoic acid, 12 (5 g, 27.70 mmol)
in DCM (200 mL) was added to t-butyldimethylsilyl chlo-
ride (9.2 g, 61.0 mmol) and imidazole (8.5 g,
124.70 mmol), stirred for 5 h, and extracted, and then
ammonium chloride was added (20 mL) and again
extracted with DCM (20 mL × 2). After LiOH (554 mg,
13.2 mmol) was added, the reaction mixture was stirred for
2 h, extracted with ethyl acetate (20 mL × 2), recrystallized
with diethyl ether (5 mL), and 13 (8.7 mg, 99%) was
obtained. And then, DIPEA (188 μL, 1.08 mmol) and
PyBOP (2.3 g, 4.40 mmol) were added to 13 (1 g,
3.39 mmol) in DCM (20 mL). This solution was stirred for
6 h, and the reaction mixture was extracted with ethyl ace-
tate (20 mL × 2). TBAF (0.5 mL, 0.48 mmol) was added
rolidinophos-phonium
hexafluorophosphate
(PyBOP;
Scheme 2. Synthesis of tert-butyl (1-azido-3-phenylpropan-2-yl)
carbamate. (a) Ms2O, TEA, NaN3, rt.
Scheme 3. Synthesis of compounds 9–11. (a) (1-Azidomethyl-3-methyl-pent-3-enyl)-carbamic acid tert-butyl ester, CuSO4, sodium ascor-
bate, DMSO:H2O, rt; (b) 4 M HCl, DCM, rt; (d) p-R-benzoic acid, PyBOP, DIPEA, DMF, rt.
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Scheme 4. Synthesis of 4-(3-azido-2-benzylpropanamido)-benzoic acid methyl ester. (a) TBSCl, Imidazole; LiOH; (b) methyl 4-amino-
benzoate, PyBOP, DIPEA, DCM; TBAF,THF; (c) Ms2O,TEA,NaN3, rt.
to the isolated compound in THF, stirred at 0ꢀC for 1 h,
and then extracted with ethyl acetate (20 mL × 2). Com-
pound 14 (94 mg, 0.30 mmol) in THF (5 mL) was mixed
with TEA (0.13 mL, 0.89 mol) and Ms2O (63 mg,
0.36 mmol), and stirred for 1 h and extracted with ethyl
acetate (20 mL × 2). The reaction mixture was dissolved in
DMF (5 mL), sodium azide added, and the mixture was
stirred for 3 h and extracted with ethyl acetate (20 mL × 2)
to give methyl 4-(3-azido-2-benzylpropanamido)benzoate,
15 (82 mg, 81%). Compound 13: 1H-NMR (600 MHz,
DMSO-d6) δ 7.32–7.29 (m, 2 H), 7.23–7.21 (m, 3 H),
3.74–3.69 (m, 2 H), 2.85–2.78 (m, 1 H), 2.77–2.72 (m,
2.91–2.88 (m, 1 H), 2.75 (s, 1 H), 2.47 (s, 1 H). Com-
pound 15: 1H NMR (600 MHz, CDCl3) δ 7.96 (d,
J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.30–7.14 (m,
5H), 3.89 (s, 3H), 3.76–3.73 (m, 1H), 2.91–2.88 (dd,
J = 12 Hz, J = 4.2 Hz 1H), 3.02–2.97 (dd, J = 13.2 Hz,
J = 6.0 Hz, 1H), 2.71 (m, 1H).
Procedure for Synthesis of Compound 16 (Scheme 5)-
Sodium ascorbate (12 mg, 0.06 mmol) and copper(II) sul-
fate (8 mg, 0.03 mml) were added to 1-(4-chloro-2-ethynyl-
phenyl)-1H-tetrazole, 4 (128 mg, 0.63 mmol) and 15
(279 mg, 0.63 mmol) in a DMSO/H2O (1:1) solution. This
mixture was stirred for 2 h, extracted with ethyl acetate
(20 mL × 2), and recrystallized with diethyl ether to yield
16 (267 mg, 66%). Compound 16: 1H-NMR (600 MHz,
CD3OD): δ 9.27 (s, 1 H), 8.26 (s, 1 H), 7.88 (d,
J = 7.8 Hz, 1 H), 7.82–7.86 (m, 4 H), 7.61 (s, 1 H), 7.39
(d, J = 7.2 Hz, 2 H), 7.17–7.31 (m, 5 H), 5.46 (m, 1 H),
4.67 (dd, J = 9.6, 13.8 Hz, 1 H), 4.54 (dd, J = 4.8,
13.8 Hz, 1 H), 3.02 (dd, J = 9.0, 13.8 Hz, 1 H), 2.86 (dd,
J = 6.0, 13.8 Hz, 1 H).
1
2 H), 0.88 (s, 15 H). Compound 14: H-NMR (600 MHz,
DMSO-d6) δ 7.96 (s, 2 H), 7.69 (s, 1 H), 7.44 (s, 2 H),
7.30–7.14 (m, 5 H), 3.89 (s, 3 H), 3.10–3.07 (m, 1 H),
a
Procedure for Synthesis of Benzyl-(2-hydroxyimino-
ethyl)-Carbamic Acid tert-Butyl Ester/(2-hydroxyimino-
ethyl)-phenethyl-Carbamic Acid tert-Butyl Ester
(Scheme 6). (2-Bromoethyl)benzene, 17 (1 g, 5.40 mmol),
ethyl glycinate (130 g, 8.11 mmol), and DIPEA (2.83 mL,
16.2 mmol) were dissolved in ACN, stirred for 48 h, and
extracted with ethyl acetate (30 mL × 2) to yield com-
pounds 18 (1.41 g, 37%). Compounds 18 in 1,4-dioxane
Scheme 5. Synthesis of compound 16. (a) 4-(3-Azido-2-benzyl-
propionylamino)-benzoic acid, CuSO4, sodium ascorbate, DMSO:
H2O, rt.
n
n
n
a
c
b
n
d
e
n
n
Scheme 6. Synthesis of benzyl-(2-hydroxyimino-ethyl)-carbamic acid tert-butyl ester/(2-hydroxyimino-ethyl)-phenethyl-carbamic acid
tert-butyl ester. (a) Ethyl glycinate, DIPEA, ACN, rt; (b) Boc2O, Na2CO3; (c) LAH, THF, −20ꢀC; (d) SO3, Pyr, TEA, DMSO:DCM, 0ꢀC;
(e) NH2OH, HCl, Na3C, EtOH, rt. n = 2.
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(20 mL) were mixed with Na2CO3 (1.08 g, 10.2 mmol)
and Boc2O (1.78 g, 8.16 mmol), stirred for 5 h, and
extracted with ammonium chloride (100 mL) and ethyl ace-
tate (50 mL) to give 19. The products were dissolved in
THF (30 mL), cooled to −20ꢀC, lithium aluminum hydride
(8.84 mL, 8.84 mmol) was added slowly, and this mixture
was stirred for 1 h to give 20 (2.43 g, 99%) through col-
umn chromatography. The products were dried and then
dissolved in DCM/DMSO (20 mL). The reaction mixture
was cooled to 0ꢀC and stirred for 3 h after adding the
SO3Ápyridine complex (2.94 g, 18.3 mmol) and TEA
(5.15 mL, 36.6 mmol). The mixture was extracted with
1 N HCl solution (30 mL) and ethyl acetate (50 mL × 2)
to yield 21. These products were dissolved in ethanol
(30 mL), NH2OHÁHCl salt and NaCO3 were added, and the
mixture was stirred for 8 h. The reaction mixture was
extracted with ethyl acetate (50 mL) and distilled water
(50 mL) to yield 22 (2.17 g, 76%). Compound 18: 1H-
NMR (600 MHz, CDCl3) δ 7.30–7.19 (m, 5 H), 4.16 (q,
J = 7.2 Hz, 2 H), 3.39 (s, 2 H ), 2.89 (t, J = 2.7 Hz, 2 H),
2.81 (t, J = 12.4 Hz, 2 H), 1.25 (t, J = 7.2 Hz, 3 H). Com-
4-methoxybenzyl, 123 mg, 0.479 mmol) and carbonyldii-
midazole (81.6 mg, 0.503 mmol) in THF (5 mL). The
resulting solution was stirred at 0ꢀC for 16 h and concen-
trated. After column chromatography, the resulting com-
pound was diluted with DCM (3 mL), and then phenol
(366 mg, 3.89 mmol) and trifluoroacetic acid (0.144 mL,
1.95 mmol) were added sequentially. After 24 h at rt, the
reaction was concentrated and triturated with diethyl ether
(10 mL) to afford 27 (36 mg) as a white solid. Compounds
24–26 and 28 were obtained using the same procedure as
1
above. Compound 24: H-NMR (600 MHz, DMSO-d6) δ
9.81 (s, 1 H), 9.00 (s, 1 H), 8.20 (s, 1 H), 8.10(m, 1H),
7.88 (dd, J = 2.4, 8.4 Hz, 2 H), 7.84–7.80 (m, 2 H), , 7.56
(d, J = 7.8 Hz, 1 H), 7.39–7.24 (m, 6 H), 6.52 (s, 1 H),
4.61 (s, 2 H), 4.54 (s, 2 H); LCMS (m/z): 529.92 (M+H+).
1
Compound 25: H-NMR (600 MHz, DMSO-d6) δ 9.81 (s,
1 H), 9.00 (s, 1 H), 8.20 (s, 1 H), 7.88 (dd, J = 2.4, 8.4 Hz,
2 H), 7.84 (d, J = 9.0 Hz, 2 H) 7.63 (d, J = 9.0 Hz, 2 H),
7.36–7.33 (m, 5 H), 6.55 (s, 1 H), 4.60 (s, 2 H), 4.54 (s,
2 H); LCMS (m/z): 529.92 (M+H+). Compound 26: 1H-
NMR (600 MHz, DMSO-d6) δ 9.81 (s, 1 H), 9.31 (s, 1 H),
8.23 (s, 1 H), 7.88 (dd, J = 2.4, 8.4 Hz, 2 H), 7.84 (d,
J = 9.0 Hz, 2 H), 7.58 (d, J = 9.0 Hz, 2 H), 7.30–7.15 (m,
5 H), 6.49 (s, 1 H), 4.56 (s, 2 H), 3.59 (t, J = 7.8 Hz, 2 H),
2.80 (t, J = 7.8 Hz, 2 H); LCMS (m/z): 543.97 (M+H+).
1
pound 19: H-NMR (600 MHz, CDCl3) δ 7.29–7.15 (m,
5 H), 4.16 (q, J = 14.4 Hz, 2 H), 3.70 (s, 2 H ), 2.82 (m,
2 H), 1.47 (s, 9 H), 1.25 (t, J = 18 Hz, 3 H). Compound
20: 1H-NMR (600 MHz, CDCl3) δ 7.30–7.17 (m, 5 H),
3.70 (s, 2 H), 3.49 (s, 2 H ), 3.44 (s, 2 H), 2.87 (s, 2 H),
1
Compound 27: H-NMR (600 MHz, DMSO-d6) δ 9.82 (s,
1
1.29 (s, 9 H). Compound 21: H-NMR (600 MHz, CDCl3)
1 H), 8.65 (s, 1 H), 8.20 (d, J = 2.4 Hz, 1 H), 7.90 (dd,
J = 2.4 , 8.4 Hz, 1 H), 7.85 (d, J = 8.4 Hz, 1 H), 7.41–7.34
(m, 5 H), 7.27–7.24 (m, 3 H), 7.15 (d, J = 9.0 Hz, 2 H),
6.50 (s, 1 H), 4.58 (s, 2 H), 4.51 (s, 2 H), 3.48(s, 2 H);
LCMS (m/z): 543.85 (M+H+). Compound 28: 1H-NMR
(600 MHz, DMSO-d6) δ 9.82 (s, 1 H), 8.89 (s, 1 H), 8.20
(d, J = 2.4 Hz, 1 H), 7.89 (dd, J = 2.4, 9.0 Hz, 1 H), 7.84
(d, J = 8.4, Hz, 1 H), 7.79 (d, J = 9.0 Hz, 2 H), 7.57 (d,
J = 9.0 Hz, 2 H ), 7.38–7.20 (m, 5 H), 6.51 (s, 1 H), 4.60
(s, 2 H), 4.54 (s, 2 H); LCMS (m/z): 528.85 (M+H+).
δ 9.47 (S, 1 H), 7.30–7.14 (m, 5 H), 4.12 (s, 1 H), 3.82 (s,
2 H ), 3.54–3.47 (m, 2 H), 2.87–2.80 (m, 2 H), 1.26 (s,
1
9 H). Compound 22: H-NMR (600 MHz, CDCl3) δ 8.47
(s, 1 H), 7.43–7.15 (m, 5 H), 3.91 (m, 2 H), 3.44 (m, 2 H
), 2.82 (m, 2 H), 1.26 (s, 9 H).
Procedure for the Synthesis of Compounds 24–28
(Scheme 7). t-Butyl 2-(hydroxyimino-ethyl)phenethyl-
carbamate and 22 (2.17 g, 6.93 mmol) were dissolved in
DMF (10 mL). N-Chlorosuccinimide (1 g, 7.70 mmol) was
added to this mixture, and then 4 dissolved in DMF was
added. This solution was stirred for 2 h at 0ꢀC and then
extracted with ethyl acetate (60 mL × 2). The organic layer
was washed with distilled water, dried with MgSO4, and fil-
tered. HCl (4 M in 1,4-dioxane, 10 mL) was added slowly
into the filtered compound in DMF (10 mL) and then stir-
red for 4 h at rt. The solvent was concentrated under
reduced pressure. The prepared compounds and DIPEA
(0.086 mL, 0.479 mmol) were added to the pre-reacted
solution of R-phenylamine (R = p-CH2CO2PMB, PMB =
Assay for Biological Activity Against FXIa. We used the
chromogenic substrate S-2366 (PyroGlu-Pro-Arg-pNA.HCl,
from Chromagenix (Instrumentation Laboratory, MA,
USA)) and FXIa, which was purchased from Enzyme
Research Laboratories (South Bend, IN, USA). The assay
was carried out in a buffer containing 100 mM Tris–HCl
(pH 7.8), 150 mM NaCl, and 0.1% PEG8000 (poly(ethyl-
ene glycol)). The enzyme and substrate concentrations were
3.75 and 0.4 mM, respectively. The assay was incubated at
37ꢀC for 10 min and the released p-nitroaniline was
Scheme 7. Synthesis of compounds 24–28. (a) NCS, TEA, DMF; HCl; p-R-Tolyamine, CDI, DIPEA, THF, 0ꢀC to rt.
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Table 1. Biological activities and predicted docking scores of the synthesized compounds.
Compound
Structure
Ki (FXIa) (μM)a
1.0
DOCK scoreb
81.61
9
N
N
N
O
N
N
N
N
N
H
CO2Me
Cl
10
5.7
76.20
N
N
N
O
N
N
N
N
N
H
OMe
Cl
N
11
6.0
84.35
N
N
O
N
N
N
N
N
H
N
Cl
16
0.9
80.19
N
N
N
N
N
N
H
N
N
O
CO2
H
Cl
24
6.7
77.68
N
N
N
N
N
O
H
N
N
CO2H
O
Cl
25
2.2
75.16
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Table 1 (continued)
Compound
Structure
Ki (FXIa) (μM)a
DOCK scoreb
N
N
N
N
N
O
H
N
N
O
CO2H
Cl
26
10.0
70.971
N
N
N
N
N
O
H
N
N
O
CO2H
Cl
27
7.9
75.483
N
N
N
N
N
O
H
N
N
O
CO2H
Cl
N
28
2.3
72.472
N
N
N
N
O
H
N
N
O
NH2
O
Cl
O61c (3SOS)
0.02
76.744
N
N
N
O
N
H
N
N
H
O
HN
S
Cl
O
a
Calculated using ACD/percepta25 Consensus Log P module.
Docked using Discovery Studio 4.5 LigandFit protocol.
b
c
The compound in the reference complex structure (PDB ID:3SOS).
measured at 405 nm by a microplate reader (Spectra
Max190; Molecular Devices, Sunnyvale, CA, USA). The
data were fitted to a one-site dose–response curve by non-
linear least-squares regression to determine the IC50 values.
Results and Discussion
The following steps were performed to identify a new
structure capable of acting as an inhibitor of FXIa. The
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Table 2. Predicted physicochemical propertiesa of synthesized 16
and the reference compound O61.
Compound
Log P
Log S
16
O61
4.32
3.14
−5.21 mol/L
−5.95 mol/L
a
ACD/Percepta 14.0.0.25
In particular, we largely changed the ethylamide linker,
showing the plane-like conformation, into a ring structure.
Various compounds were designed and synthesized, as
described in Schemes 1–7. The biological activities of these
compounds were examined, and they are reported in
Table 1, along with docking scores.
The expected binding mode of compound 16 with the
best activity compared with the known O61 compound is
displayed in Figure 2. For compound 16, the triazole ring
replacing the ethylamide linker forms a hydrogen bond
with Gly573 and undergoes cation–π interactions with
His431. The benzyl group of the S1 site forms stacking
interactions with His431, while the carboxylic benzyl group
of the S2 site forms a hydrogen bond with Arg413. On the
other hand, the isoxazole that replaced the ethylamide
linker does not form many hydrogen bond interactions
compared with that of the triazole. For compound 26 (not
shown here), the ethylphenyl group in S1 seems to be too
long to form a cation–π interaction with His431.
Figure 2. Binding mode of compound (a) the inhibitor O61 in
3SOS and (b) 16. The green and violet lines represent hydrogen
bonds and stacking interactions, respectively. S1 and S2 denote
the enzyme substrate binding sites and the residues involved in
this binding are shown.13
As described in Table 2, the identified compound 16
exhibits properties similar to the O61 compound, maintain-
ing the main interactions in the S1 site.
complex structure of human FXIa and an inhibitor (O61)
were obtained initially from the Protein Data Bank (PDB
ID:3SOS). Using this structure, the main interactions of
the existing compound were identified.17 The inhibitor
O61 of this complex contains a chlorophenyl-tetrazole
and ethylamide group, a benzyl group, and a benzothiazi-
none ring. The chlorophenyl-tetrazole scaffold is posi-
tioned deep within the S1 binding pocket, and a C–Cl
bond is perpendicular to the phenol ring of Tyr608. The
tetrazole ring of the ligand is in close contact with the
disulfide bond formed between Cys571 and Cys599, pos-
sibly forming an interaction between the π system of the
tetrazole ring and the lone pairs of the S atoms. The
amide of the ethylamide group forms hydrogen bonds
with the backbone of Ser575 and Gly573. The benzyl
group binds to S1 and comes into contact with His431,
which is part of the catalytic triad, which also includes
Tyr434 and Leu415. The S2 site was filled by the ben-
zothiazinone ring, which forms a hydrogen bond with the
backbone of His414. Arg413 is located approximately
3.2 Å above the junction between the phenyl and thiazi-
none rings, further stabilizing the ligand through a
cation–π interaction.
Conclusion
In this study, we have discovered potential new FXIa inhi-
bitors based on structure-based molecular modeling. The
identified compound 16 as having the highest potential with
regard to both activity and properties as an early new hit
compound.
Acknowledgment. This study was financially supported
by a research fund from the Chungnam National University
in 2014.
References
1. K. G. Mann, S. Butenas, K. Brummel, Arterioscler. Thromb.
Vasc. Biol. 2003, 23, 17.
2. N. S. Jenny, K. G. Mann, In Thrombosis and Hemorrhage,
J. Loscalzo, A. I. Schafer Eds., Williams & Wilkins, Balti-
more, USA, 1998, p. 3.
3. J. Hirsh, J. E. Dalen, D. R. Anderson, L. Poller, H. Bussey,
J. Ansell, D. Deykin, Chest 2001, 119, 8S.
4. J. P. Riddel, B. E. Aouizerat, C. Miaskowski, D. P. Lillicrap,
J. Pediatr. Oncol. Nurs. 2007, 24, 123.
Based on these analyses, we attempted to design a new
compound with pharmacophore features to support the
main interactions while also maintaining the chlorophenyl-
tetrazole scaffold and the overall conformation from the
binding information.
5. D. Gailani, G. J. Broze, Science 1991, 253, 909.
6. C. M. Kessler, Seminars in Hematology, Vol. 41, WB Saun-
ders, Philadelphia, USA, 2004, p. 44.
Bull. Korean Chem. Soc. 2016
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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BULLETIN OF THE
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7. A. Yamashita, K. Nishihira, T. Kitazawa, K. Yoshihashi,
T. Soeda, K. Esaki, T. Imamura, K. Hattori, Y. Asada,
J. Thromb. Haemost. 2006, 4, 1496.
8. X. Wang, Q. Cheng, L. Xu, G. Z. Feuerstein, M. Y. HSU,
P. L. Smith, D. A. Seiffert, W. A. Schumacher,
M. L. Ogletree, D. Gailani, J. Thromb. Haemost. 2005, 3, 695.
9. J. Sanchez, G. Elgue, J. Riesenfeld, P. Olsson, Thromb. Res.
1998, 89, 41.
15. P. C. Wong, E. J. Crain, C. A. Watson, W. A. Schumacher,
J. Thromb. Thrombolysis 2011, 32, 129.
16. J. Lin, H. Deng, L. Jin, P. Pandey, J. Quinn, S. Cantin,
M. J. Rynkiewicz, J. C. Gorga, F. Bibbins, C. A. Celatka,
P. Nagafuji, T. D. Bannister, H. V. Meyers, R. E. Babine,
N. J. Hayward, D. Weaver, H. Benjamin, F. Stassen,
S. S. Abdel-Meguid, J. E. Strickler, J. Med. Chem. 2006,
49, 7781.
10. W. Gan, L. Deng, C. Yang, Q. He, J. Hu, H. Yin, L. Peng,
FEBS Lett. 1976, 2009, 583.
11. W. A. Schumacher, J. M. Luettgen, M. L. Quan,
D. A. Seiffert, Arterioscler. Thromb. Vasc. Biol. 2010,
30, 388.
17. X. Fradera, B. Kazemier, E. Carswell, A. Cooke, A. Oubrie,
W. Hamilton, M. Dempster, S. Krapp, S. Nagel, A. Jestel,
Acta Cryst. 2012, F68, 404.
18. Pinto, D. J. P.; Quan, M. L.; Smith, I. L. M.; Orwat, M. J.;
Gilligan, P. J. Patent WO2008157162 A1; 2008.
12. Z. Hu, P. C. Wong, P. J. Gilligan, W. Han, K. B. Pabbisetty,
J. M. Bozarth, E. J. Crain, T. Harper, J. M. Luettgen,
J. E. Myers, V. Jr. Ramamurthy, K. A. Rossi, S. Sheriff,
C. A. Watson, A. Wei, J. J. Zheng, D. A. Seiffert,
R. R. Wexler, M. L. Quan, ACS Med. Chem. Lett. 2015,
6, 590.
13. O. Fjellström, S. Akkaya, H. G. Beisel, P. O. Eriksson,
K. Erixon, D. Gustafsson, U. Jurva, D. Kang, D. Karis,
W. Knecht, V. Nerme, I. Nilsson, T. Olsson, A. Redzic,
R. Roth, J. Sandmark, A. Tigerström, L. Öster, PLoS One
2015, 10, e0113705.
19. B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States,
S. Swaminathan, M. Karplus, J. Comput. Chem. 1983, 4, 187.
20. C. M. Venkatachalam, X. Jiang, T. Oldfield, M. Waldman,
J. Mol. Graph. Model. 2003, 21, 289.
21. A.
Krammer,
P.
D.
Kirchhoff,
X.
Jiang,
C. M. Venkatachalam, M. Waldman, J. Mol. Graph. Model.
2005, 23, 395.
22. D. K. Gehlhaar, G. M. Verkhivker, P. A. Rejto,
C. J. Sherman, D. B. Fogel, L. J. Fogel, S. T. Freer, Chem.
Biol. 1995, 2, 317.
23. A. N. Jain, J. Comput. Aided Mol. Des. 1996, 10, 427.
24. I. Muegge, J. Med. Chem. 2006, 49, 5895.
25. www.acdlabs.com/percepta: 2015.
14. W. A. Schumacher, S. E. Seiler, T. E. Steinbacher,
A. B. Stewart, J. S. Bostwick, K. S. Hartl, E. C. Liu,
M. L. Ogletree, Eur. J. Pharmacol. 2007, 570, 167.
Bull. Korean Chem. Soc. 2016
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