Rational design of potent, small, synthetic allosteric inhibitors of thrombin

Article abstract of DOI:10.1021/jm2005767

Thrombin is a key enzyme targeted by the majority of current anticoagulants that are direct inhibitors. Allosteric inhibition of thrombin may offer a major advantage of finely tuned regulation. We present here sulfated benzofurans as the first examples of

Full text of DOI:10.1021/jm2005767

ARTICLE
pubs.acs.org/jmc
Rational Design of Potent, Small, Synthetic Allosteric Inhibitors of
Thrombin
Preetpal Singh Sidhu,†,‡ Aiye Liang,†,‡ Akul Y. Mehta,†,‡ May H. Abdel Aziz,†,‡ Qibing Zhou,†,§ and
Umesh R. Desai*,†,‡
†Department of Medicinal Chemistry and ‡Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University,
Richmond, Virginia 23219, United States
§Institute for Materia Medica, College of Life Science and Technology, Huazhong University of Science and Technology,
Wuhan, Hubei 430074, P. R. China
S Supporting Information
b
ABSTRACT: Thrombin is a key enzyme targeted by the majority of
current anticoagulants that are direct inhibitors. Allosteric inhibition
of thrombin may offer a major advantage of finely tuned regulation.
We present here sulfated benzofurans as the first examples of potent,
small allosteric inhibitors of thrombin. A sulfated benzofuran library of
15 sulfated monomers and 13 sulfated dimers with different charged,
polar, and hydrophobic substituents was studied in this work. Synth-
esis of the sulfated benzofurans was achieved through a multiple step,
highly branched strategy, which culminated with microwave-assisted
chemical sulfation. Of the 28 potential inhibitors, 11 exhibited reason-
able inhibition of human R-thrombin at pH 7.4. Structureꢀactivity
relationship analysis indicated that sulfation at the 5-position of the
benzofuran scaffold was essential for targeting thrombin. A tert-butyl
5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC50 of 7.3 μM under physiologically
relevant conditions. MichaelisꢀMenten studies showed an allosteric inhibition phenomenon. Plasma clotting assays indicate that
the sulfated benzofurans prolong both the activated partial thromboplastin time and prothrombin time. Overall, this work puts
forward sulfated benzofurans as the first small, synthetic molecules as powerful lead compounds for the design of a new class of
allosteric inhibitors of thrombin.
’ INTRODUCTION
biphenyls, and benzimidazoles, are preferred because of their
high thrombin selectivity. Fundamentally, these designed inhi-
bitors attempt to mimic the D-Phe-Pro-Arg recognition sequence
through the incorporation of a guanidine or amidine moiety.
Recently, however, newer molecules have been designed with a
P-1 halophenyl substituent to interact with Tyr228 in the S1
pocket to improve the poor oral bioavailability of most P-1
guanidine-based inhibitors.3,7ꢀ9
An exciting alternative to competitive inhibition of thrombin is
allosteric inhibition through either exosite I or exosite II. It is well
established that binding of ligands in these exosites can induce
conformational changes in the active site of thrombin.10ꢀ13 An
example of this is thrombomodulin, which interacts with exosite I
to change the substrate specificity of thrombin from fibrinogen to
protein C.12 Likewise, hirugen binding in exosite I increases or
decreases the catalytic efficiency of thrombin, depending on the
nature of the chromogenic substrate.10,11 Similarly, exosite Iꢀactive
site allosteric coupling is also the reason why peptides such as
Thrombin is a key enzyme of the coagulation cascade exhibit-
ing important roles in both procoagulation and anticoagulation
processes. Most clinically used anticoagulant drugs, including
polymeric heparin, warfarin, hirudin, argatroban, and the recently
approved dabigatran aim to reduce thrombin activity.1ꢀ3 Although
these drugs have been successful in treating many thrombotic
disorders, the anticoagulation therapy continues to suffer from
bleeding risk, narrow therapeutic index, and other drug-specific
adverse effects, which highlight the critical need to develop agents
with better therapeutic profile.
Structurally, thrombin is a complex serine protease of the
trypsin family that displays multiple ligand-binding domains, which
modulate its activity and interactions with other molecules. These
domains include the active site, anion-binding exosites I and II,
and the sodium binding site. The design of thrombin inhibitors
has focused almost exclusively on blocking the substrate’s access
to the active site through a competitive process.3 This paradigm
has resulted in the design of thousands of molecules distributed
across a large number of scaffolds.3ꢀ6 Of these, a select group
of scaffolds, including the pyrazoles, napthylamidines, indoles,
Received: May 6, 2011
Published: June 29, 2011
r
2011 American Chemical Society
5522
dx.doi.org/10.1021/jm2005767 J. Med. Chem. 2011, 54, 5522–5531
|

Journal of Medicinal Chemistry
ARTICLE
diversity to these molecules, which nevertheless bind to exosite II
of thrombin and allosterically induce thrombin inhibition.18
Sulfated LMWLs represent the first examples of an exclusive
exosite II-mediated inhibition of the key coagulation protease.
In this work, we present a study of 28 monomeric and dimeric
sulfated benzofurans, molecules derived from the sulfated
LMWL structure, as inhibitors of thrombin. Enzyme inhibition
studies show that the molecules display a range of inhibition
potential, while MichaelisꢀMenten kinetic studies show the
inhibition to arise from an allosteric process. Collectively, this
work establishes the potential of allosteric inhibition of thrombin
through the interesting and unusual features of sulfated benzo-
furans that direct the interaction with thrombin.
Figure 1. Design of sulfated benzofurans on the basis of the structure of
sulfated lignins, which were found to be potent inhibitors of thrombin.
Sulfated lignins are a three-dimensional network of chemoenzymatically
coupled 4-hydroxycinnamic acid monomers. Four different intermono-
meric linkages are possible in these oligomers including βꢀ5, βꢀO4,
βꢀβ, and 5ꢀ5 linkages. Chiral centers are identified using curvy bonds.
The chain length of sulfated lignins is typically 5ꢀ15 monomer units.
Sulfated benzofurans studied in this work were designed from one
specific structure generated by βꢀ5 linked monomers, as shown by the
structure in box. Positions 2, 3, 5, and 6 are numbered. See text for
details.
’ EXPERIMENTAL PROCEDURES
Chemicals, Reagents, and Analytical Chemistry. Microwave-
based sulfation reactions were performed using a CEM-Discover
synthesizer (Matthews, NC) in sealed reaction vessels (7 mL) at 50
W. The temperature of the reaction was maintained at the desired setting
using cooled nitrogen (45 psi) flow. Reagents and chemicals used in
reactions were purchased from either Sigma-Aldrich (Milwaukee, WI)
or Fisher (Pittsburgh, PA) and used as supplied. Analytical thin layer
chromatography (TLC) was performed using UNIPLATE silica gel
GHLF 250 μm precoated plates (ANALTECH, Newark, DE) that were
analyzed by fluorescence (254 nm). Compound purity was evaluated
independently through gradient reverse-phase HPLC. The purity of
each compound synthesized in this work was greater than 95% and is
listed in the Supporting Information.
bothrojaracin inhibit thrombin.14,15 Nevertheless, no small or-
ganic molecule has been designed that utilizes exosite I to effect
physiological thrombin inhibition.
Exosite II ligands include heparin, chondroitin sulfate, hae-
madin, and fibrinogen γ0. These can be broadly classified as either
highly anionic polysaccharides or traditional peptides. Interaction of
heparin with exosite II is known to induce greater reactivity with
antithrombin, and recent work shows that formation of antith-
rombinꢀthrombin complex disrupts exosite II.16 Also, exosite
IIꢀactive site coupling is suggested by the change in fluorescence
properties of para-aminobenzamidine, a noncovalent active
site probe, upon heparin binding to exosite II.17 However, no
exclusive exosite II ligand has been found to date that reduces the
catalytic efficiency of thrombin.
Allosteric regulation is a promising strategy for thrombin inhibi-
tion. Nature tends to utilize allosterism to confer specificity of
recognition and also to effect regulation. For thrombin, the possi-
bility of regulation, or controlled inhibition, is of considerable
importance because nearly all current anticoagulants are asso-
ciated with risk of bleeding. An appropriately designed allosteric
regulator that does not fully nullify the procoagulant signal may
maintain a finely tuned balance between procoagulant and
anticoagulant signal, resulting in reduced bleeding complications.
In pursuit of such allosteric regulators, we had earlier designed
sulfated low molecular weight lignins (LMWLs) as functional
mimetics of heparin targeted to exosite II-like regions of coagula-
tion enzymes, including thrombin.18,19 Sulfated LMWLs, che-
moenzymatic variants of the naturally available lignin (Figure 1),
were found to potently inhibit thrombin with an IC50 of 18ꢀ34
nM under physiological conditions.18 More importantly, sulfated
LMWLs also inhibited blood clotting under ex vivo conditions
with potency comparable to that of the LMW heparins.20
Despite their excellent in vitro and ex vivo anticoagulant proper-
ties, sulfated LMWLs are challenging because of their polydispersity
and microheterogeneity, which parallel that observed with the
heparins. Sulfated LMWLs represent a large library of structures
arising from multiple intermonomeric linkages, variable sulfation
and chain length, and the presence of many chiral centers
(Figure 1).21 These variables introduce considerable structural
Column chromatography was performed using Teledyne ISCO
(Lincoln, NE) Combiflash RF system and disposable normal silica
cartridges of 30ꢀ50 μm particle size, 230ꢀ400 mesh size, and 60 Å pore
size. Sodium exchange chromatography was performed using SP Se-
phadex C-25 sodium cation exchange columns from GE Healthcare Life
Sciences (Piscataway, NJ). Approximately 100 mg of sulfated sample
was loaded onto 10 g of the cation exchanger in a 10 mm ꢁ 460 mm
column and eluted with either water or 20% EtOH/H2O at 0.5 mL/min.
Anhydrous reactions were carried out under nitrogen atmosphere in
glassware dried at high temperature. Reagent solutions and solvents
were handled under nitrogen atmosphere using syringe techniques.
HPLC purification of sulfated benzofurans was carried out on a Jasco
chromatography system (Easton, MD) using a 250 mm ꢁ 10 mm Varian’s
Dynamax Microsorb C4 column. An acetonitrileꢀwater gradient
(5ꢀ100% acetonitrile in 20 min) at 3 mL/min was used to purify the
compounds on milligram scale. The eluents were detected at 300 nm.
1H and 13C NMR spectra were recorded at either 300 or 400 MHz
(Varian Mercury or Bruker Ultrashield Plus) in appropriate deuterated
solvents including CDCl3, DMSO-d6, CD3OD, acetone-d6, or D2O.
All signals are reported in ppm with the internal chloroform, DMSO,
acetone, and D2O signals at 7.26, 2.50, 3.31, 2.05, and 4.79, respectively,
as standards. The data are reported as chemical shifts (ppm), multiplicity
(s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant(s)
(Hz), and integration. Mass spectrometry was performed on all synthe-
sized molecules using a Micromass ZMD4000 single quadrupole mass
spectrometer with ESI ionization probe operating in negative ion mode
(Waters Corp., Milford, MA). The samples, dissolved in acetonitrile
containing formic acid (5% v/v), were infused at 10 μL/min. The source
block temperature and the probe temperature were typically held at 100 and
120 °C, respectively, while corona and cone voltages were selected through
manual optimization. The desolvation nitrogen flow was 500 L/h. Mass
spectra were acquired in the mass range from 110 to 1000 Da at 400 amu/s.
Synthesis of Benzofuran Monomer and Dimer Precursors
for Subsequent Sulfation at 5-, 50-, and Other Positions. The
5523
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
detailed protocol for the synthesis of each benzofuran precursor is
provided in Supporting Information. The reactions used in the synthesis
are fairly common and hence not described here.
6: 1H NMR (CD3OD, 300 MHz) δ 2.61 (s, 3H), 3.86 (s, 3H), 6.88
(s, 2H), 7.15 (s, 1H), 7.86 (s, 1H). 13C NMR (CD3OD) δ 10.85, 55.78,
59.93, 95.76, 110.69, 112.67, 120.71, 138.55, 150.48, 151.65, 154.22.
7: 1H NMR (CD3OD, 300 MHz) δ 0.44 (s, 6H), 1.02 (s, 9H), 2.75
(s, 3H), 3.96 (s, 3H), 6.68 (s, 2H), 6.98 (s, 1H), 7.36 (s, 1H). 13C NMR
(CD3OD) δ 4.48, 12.29, 18.70, 26.02, 55.88, 56.04, 95.47, 109.94,
114.31, 120.84, 142.03, 149.32, 149.40, 151.81.
Sulfation of Appropriate Precursors To Yield Sulfated
Benzofuran Monomers and Dimers. The synthesis of sulfated
benzofurans was developed following our earlier report on the synthesis
of sulfated flavonoids.22 Briefly, to a solution of unsulfated benzofuran
monomer or dimer (typically 50 mg) in acetonitrile/DMF (4:1, 0.45 mL)
in a microwave tube were added triethylamine (10 equiv) and trimethy-
lamineꢀsulfur trioxide complex (15 equiv). The reaction mixture was
exposed to microwaves (50 W) for 40 min at 100 °C followed by vacuum
concentration to remove all solvent. The solid so obtained was directly
loaded on a Sephadex C-25 cation exchange resin and eluted with water.
Fractions containing the sulfated product were lyophilized to obtain a
solid residue, which was further purified on reverse-phase gradient HPLC to
remove residual unsulfated contaminants.
9a: 1H NMR (D2O, 400 MHz) δ 2.47 (S, 3H), 3.55ꢀ3.90 (m, 8H),
3.91 (s, 3H), 7.25 (s, 1H), 7.41 (s, 1H). 13C NMR (D2O, 400 MHz)
δ 12.48, 42.76, 47.82, 56.43, 66.41, 66.79, 96.68, 110.52, 112.70,
118.31, 137.35, 149.74, 151.36, 156.79, 166.23. ESI HRMS (ꢀve)
m/z calcd for C15H16NO8SNa [(M ꢀ Na)ꢀ)] 370.0544, found
370.0652 (M ꢀ Na)ꢀ.
9b: 1H NMR (D2O, 400 MHz) δ 0.89 (d, J = 17 Hz, 3H), 1.01 (m,
1H), 1.61ꢀ1.81 (m, 4H), 2.39 (s, 3H), 2.92 (m, 1H), 3.08 (m, 1H), 3.56
(m, 1H), 3.92 (s, 3H), 4.45 (m, 1H), 7.27 (s, 1H), 7.64 (s, 1H). 13C
NMR (D2O) δ 12.47, 12.55, 14.88, 20.89, 30.17, 55.67, 56.59, 96.67,
111.46, 112.63, 118.67, 137.67, 149.82, 151.26, 155.45, 165.32. ESI
HRMS (ꢀve) m/z calcd for C17H20NO7SNa [(M ꢀ Na)ꢀ)] 482.1022,
found 382.0847 (M ꢀ Na)ꢀ.
5a: 1H NMR (D2O, 400 MHz) δ 1.45 (t, J = 6 Hz, 3H), 2.51 (s, 3H),
3.76 (s, 3H), 4.36 (q, J = 6 Hz, 2H), 5.38 (s, 1H), 7.15 (s, 1H), 7.57 (s, 1H).
5b: 1H NMR (D2O, 400 MHz) δ 2.48 (s, 3H), 3.89 (s, 3H), 4.21 (s,
3H), 6.98 (s, 1H), 6.61 (s, 1H). ESI MS (ꢀve) m/z calcd for
C12H11O8SNa [(M ꢀ Na)ꢀ)] 315.02, found 315.01(M ꢀ Na)ꢀ.
5c: 1H NMR (D2O, 400 MHz) δ 1.62 (s, 9H), 2.49 (s, 3H), 3.89 (s,
3H), 6.98 (s, 1H), 6.61 (s, 1H). 13C NMR (D2O) δ 13.40, 21.22, 61.33,
73.87, 99.93, 107.79, 114.00, 118.54, 139.23, 147.28, 150.45, 164.37,
165.49. ESI MS (ꢀve) m/z calcd for C13H17O8SNa [(M ꢀ Na)ꢀ)]
357.05, found 357.08 (M ꢀ Na)ꢀ.
10: 1H NMR (D2O, 400 MHz) δ 1.47 (t, J =18 Hz, 3H), 2.71 (m,
4H), 3.81 (m, 4H), 3.95 (s, 3H), 4.02 (s, 2H), 4.39 (q, J = 18 Hz, 2H),
7.25 (s, 1H), 7.74 (s, 1H). 13C NMR (D2O) δ 13.45, 52.47, 52.64, 56.34,
61.82, 66.07, 96.24, 111.83, 114.82, 117.14, 137.95, 150.45, 151.64,
159.30, 164.77. ESI HRMS (ꢀve) m/z calcd for C17H20NO9SNa [(M ꢀ
Na)ꢀ)] 414.0933, found 414.0847 (M ꢀ Na)ꢀ.
12a: 1H NMR (CD3OD, 300 MHz) δ 1.32 (J = 6 Hz, t, 3H), 2.65
(s, 3H), 3.82 (s, 3H), 3.86 (s, 3H), 4.29 (J = 6 Hz, q, 2H), 5.52 (s, 2H),
7.12 (s, 1H, ArꢀH), 7.22 (s, 1H, ArꢀH), 7.43 (s, 1H, ArꢀH), and 8.04
(s, 1H, ArꢀH). 13C NMR (CD3OD) δ 13.12, 13.53, 13.54, 55.65, 60.20,
60.67, 63.93, 95.54, 95.63, 109.19, 112.32, 115.57, 117.40, 118.31,
139.89, 145.50, 149.74, 152.08, 152.20, 158.70, 162.67, 163.51,
164.48. ESI HRMS (ꢀve) m/z calcd for C26H25O13SNa [(M ꢀ Na)ꢀ)]
577.1011, found 577.1146 (M ꢀ Na)ꢀ.
1
5d: H NMR (D2O, 400 MHz) δ 2.48 (s, 3H), 3.21 (s, 3H), 3.62
(t, J = 12 Hz, 2H), 3.72 (s, 3H), 4.26 (t, J = 12 Hz, 2H), 6.91 (s, 1H), 7.59
(s, 1H). ESI MS (ꢀve) m/z calcd for C13H13O9SNa [(M ꢀ Na)ꢀ)]
345.02, found 345.01 (M ꢀ Na)ꢀ.
5e: 1H NMR (D2O, 400 MHz) δ 1.70 (m, 6H), 2.99 (s, 3H), 4.41
(q, J = 17 Hz, 2H), 4.66 (q, J = 17 Hz, 2H), 7.55 (s, 1H), 8.21 (s, 1H).
13C NMR (D2O) δ 16.36, 16.39, 16.76, 63.15, 67.53, 99.69, 110.66,
116.09, 119.96, 141.73, 146.05, 151.33, 152.58, 165.90, 167.00. ESI
HRMS (ꢀve) m/z calcd for C14H16O8SNa [(M ꢀ Na)ꢀ)] 343.0512,
found 343.0357 (M ꢀ Na)ꢀ.
12b: 1H NMR (CD3OD, 300 MHz) δ 2.67 (s, 3H), 3.83 (s, 3H), 3.84
(s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 5.55 (s, 2H), 7.13 (s, 1H), 7.24 (s,
1H), 7.47 9s, 1H), 8.03 (s, 1H). ESI HRMS (ꢀve) m/z calcd for
C24H21O13SNa [(M ꢀ Na)ꢀ)] 549.0723, found 549.0795 (M ꢀ Na)ꢀ.
12c: 1H NMR (CD3OD, 300 MHz) δ 1.49 (s, 9H), 1.51 (s, 9H), 2.65
(s, 3H), 3.83 (s, 3H), 3.88 (s, 3H), 5.47 (s, 2H), 7.12 (s, 1H), 7.22 (s,
1H), 7.37 (s, 1H), 8.09 (s, 1H). 13C NMR (CD3OD) δ 13.09, 27.30,
27.45, 55.59, 55.66, 64.26, 81.00, 81.62, 95.44, 95.56, 109.89, 110.21,
113.55, 115.59, 117.70, 118.42, 139.79, 145.31, 149.83, 151.87, 152.05,
158.16, 162.26, 162.63, 163.80. ESI MS (ꢀve) m/z calcd for
C30H33O13SNa [(M ꢀ Na)ꢀ)] 633.16, found 633.19 (M ꢀ Na)ꢀ.
12d: 1H NMR (D2O, 400 MHz) δ 2.30 (s, 3H), 3.26 (s, 3H), 3.32 (s,
3H), 3.57 (m, 2H), 3.63 (m, 5H), 3.77 (s, 3H), 4.18 (m, 2H), 4.27 (m,
2H), 5.17 (s, 2H), 6.49 (s, 1H), 6.89 (s, 1H), 6.93 (s, 1H), 7.67 (s, 1H).
13C NMR (D2O) δ 13.49, 55.40, 56.07, 58.13, 58.24, 62.74, 62.96, 63.79,
69.76, 69.89, 93.22, 95.76, 105.85, 107.40, 111.06, 114.98, 116.73,
117.17, 138.28, 144.55, 147.64, 148.10, 150.78, 151.62, 158.55,
162.96, 163.49, 164.40. ESI HRMS (ꢀve) m/z calcd for C28H29O115S-
Na [(M ꢀ Na)ꢀ)] 637.1223, found 637.1161 (M ꢀ Na)ꢀ.
5f: 1H NMR (D2O, 400 MHz) δ 1.37 (m, 9H), 2.36 (s, 3H), 4.23
(q, J =18 Hz, 2H), 4.54 (m, 1H), 6.96 (s, 1H), 7.60 (s, 1H). 13C NMR
(D2O) δ 13.40, 13.48, 21.22, 61.33, 73.87, 99.93, 107.79, 114.00, 118.54,
139.23, 147.28, 150.45, 164.37, 165.49. ESI HRMS (ꢀve) m/z calcd for
C14H15O8SNa [(M ꢀ Na)ꢀ)] 357.0532, found 357.0600 (M ꢀ Na)ꢀ.
5g: 1H NMR (D2O, 400 MHz) δ 1.45 (t, J =18 Hz, 3H), 2.50 (s, 3H),
3.64 (s, 3H), 4.31 (q, J = 18 Hz, 2H), 5.33 (s, 2H), 7.15 (s, 1H), 7.64 (s,
1H). ESI HRMS (ꢀve) m/z calcd for C14H15O9SNa [(M ꢀ Na)ꢀ)]
359.0420, found 359.0395 (M ꢀ Na)ꢀ.
5h: 1H NMR (D2O, 400 MHz) δ 1.39 (t, J =18 Hz, 3H), 2.57 (s, 3H),
3.87 (m, 8H), 4.29 (q, J =18 Hz, 2H), 7.45 (s, 1H), 7.57 (s, 1H). 13C
NMR (D2O) δ 13.48, 13.72, 44.11, 44.94, 61.53, 61.58, 66.25, 105.77,
106.60, 108.11, 114.53, 123.18, 139.99, 140.26, 149.66, 154.36, 165.33,
166.19. ESI HRMS (ꢀve) m/z calcd for C17H18O10SNa [(M ꢀ Na)ꢀ)]
428.0332, found 428.0889 (M ꢀ Na)ꢀ.
5i: 1H NMR (D2O, 400 MHz) δ 2.51 (s, 3H), 3.63 (s, 3H), 4.65 (d,
2H), 5.02 (d, 1H), 5.16 (d, 2H), 5.91 (m, 1H), 6.81 (s. 1H), 7.56 (s, 1H).
13C NMR (D2O) δ 13.40, 55.05, 61.19, 70.68, 97.79, 107.91, 113.75,
114.30, 118.36, 128.61, 128.99, 138.55, 148.09, 150.16, 158.52, 163.59,
165.06. ESI HRMS (+ve) m/z calcd for C20H19O9SNa [(M + Na)+)]
481.0632, found 481.1400 (M + Na)+.
12e: 1H NMR (D2O, 400 MHz) δ 1.14 (m, 12H), 2.32 (s, 3H), 3.72
(m, 4H), 4.06 (m, 4H), 5.15 (s, 2H), 6.51 9s, 1H), 6.71 9s, 1H), 6.98
(s, 1H), 7.56 (s, 1H). ESI HRMS (ꢀve) m/z calcd for C28H29O13SNa
[(M ꢀ Na)ꢀ)] 605.1312, found 605.0708 (M ꢀ Na)ꢀ.
12f: 1H NMR (D2O, 400 MHz) δ 1.07 (m, 18H), 2.27 (s, 3H), 3.99
(m, 4H), 4.20 (m, 2H), 5.05 (s, 2H), 6.64 (s, 1H), 6.67 (s, 1H), 6.99 (s,
1H), 7.81 (s, 1H). 13C NMR (D2O) δ 13.29, 13.50, 13.69, 13.78, 20.94,
21.19, 21.40, 60.34, 61.09, 63.81, 71.86, 72.97, 98.90, 108.13, 111.04,
114.71, 117.74, 118.33, 139.92, 146.62, 146.72, 148.33, 148.60, 151.24,
158.69, 162.57, 163.34, 164.08. ESI HRMS (ꢀve) m/z calcd for
C30H33O13SNa [(M ꢀ Na)ꢀ)] 633.1236, found 633.1494 (M ꢀ Na)ꢀ.
5j: 1H NMR (D2O, 400 MHz) δ 1.28 (t, J =18 Hz, 3H), 2.09 (s, 3H),
3.54 (s, 3H), 4.12 (q, J = 18 Hz, 2H), 4.78 (s, 2H), 6.57 (s, 1H), 6.68
(d, J = 21 Hz, 2H), 7.17 (d, J = 21 Hz, 2H), 7.67 (s,1H). 13C NMR
(D2O) δ 13.40, 55.05, 61.19, 70.68, 97.79, 107.91, 113.75, 114.30,
118.36, 128.61, 128.99, 138.55, 148.09, 150.16, 158.23, 163.59, 165.06.
ESI MS (ꢀve) m/z calcd for C14H13O8SNa [(M ꢀ Na)ꢀ)] 325.03,
found 325.04 (M ꢀ Na)ꢀ.
5524
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
13: 1H NMR (CD3OD, 400 MHz) δ 1.38 (m, 6H,), 2.60 (m, 3H,),
3.71 (m, 4H,), 3.91 (s, 3H,), 3.93 (s, 3H), 4.37 (m, 6H,), 5.62 (s, 2H),
7.02 (s, 1H), 7.05 (s, 1H), 7.49 (s, 1H), 8.01 (s, 1H). ESI MS (ꢀve) m/z
calcd for C30H32NO14SNa[(Mꢀ Na)ꢀ)] 659.14, found 659.15 (M ꢀ Na)ꢀ.
14: 1H NMR (D2O, 400 MHz) δ 2.40 (s, 3H,), 3.24 (m, 8H), 3.67
(m, 8H), 3.81 (s, 3H,), 3.92 (s, 3H), 5.30 (s, 2H,), 6.85 (s, 1H,), 7.14 (s,
1H), 7.33 (s.1H,), 7.43 (s, 1H). 13C NMR (D2O) δ 12.36, 42.55, 56.00,
56.40, 63.52, 66.17, 66.36, 96.23, 96.73, 107.16, 110.44, 113.33, 115.29,
116.94, 117.92, 138.10, 143.04, 148.81, 149.45, 151.21, 151.81, 152.46,
156.38, 164.20, 165.93. ESI HRMS (ꢀve) m/z calcd for C30H31N2O13S-
Na [(M ꢀ Na)ꢀ)] 659.1422, found 659.1531 (M ꢀ Na)ꢀ.
from Valley Biomedical (Winchester, VA). Thromboplastin and ellagic
acid were obtained from Fisher Diagnostics (Middletown, VA).
Inhibition of Thrombin. Direct inhibition of thrombin by sulfated
benzofuran derivatives was measured through a chromogenic substrate
hydrolysis assay.18,19 The buffer used in these experiments was 20 mM
Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 2.5 mM CaCl2, and
0.1% polyethylene glycol (PEG) 8000. Benzofuran derivatives (2ꢀ30 μL)
at concentrations ranging from 4 μg/mL to 5 mg/mL were diluted with
appropriate volume of assay buffer in PEG 20000-coated acrylic cuvettes
at 25 °C. To this solution was added 5 μL of thrombin solution to give
approximately 5 nM initial thrombin concentration. After 10 min of
incubation, 20 μL of 1 mM Spectrozyme TH was rapidly added and
the residual thrombin activity was measured from the initial rate of
increase in absorbance at 405 nm. Relative residual thrombin activity
at each concentration of the inhibitor was calculated from the ratio
of thrombin activity in the presence and absence of inhibitor. Log-
istic eq 1 was used to fit the dose-dependence of residual proteinase
activity to obtain the IC50 and the efficacy ΔY (=YM ꢀ Y0) of
inhibition.
17: 1H NMR (D2O, 400 MHz) δ 1.23 (m, 6H), 2.36 (s, 3H), 3.70 (s,
3H), 4.18 (m, 4H), 5.23 (s, 2H), 6.96 (s, 1H), 7.03 (s, 1H), 7.53 (s, 1H),
7.62 (s, 1H). 13C NMR (D2O) δ 13.4, 13.51, 13.59, 56.30, 61.53, 62.08,
63.51, 96.28, 99.06, 108.05, 111.73, 114.48, 115.06, 117.13, 119.54,
138.31, 138.82, 147.96, 150.23, 150.94, 152.02, 158.07, 164.50, 164.87,
165.60. ESI HRMS (ꢀve) m/z calcd for C25H22O14S2Na [(M ꢀ Na)ꢀ)]
665.0121, found 665.0366 (M ꢀ Na)ꢀ.
18: 1H NMR (D2O, 400 MHz) δ 1.55 (t, J =18 Hz, 3H),1.59 (t, J =18
Hz, 3H), 2.41 (s, 3H), 3.81 (s, 3H), 4.18 (q, J =18 Hz, 2H), 4.23 (q, J =18
Hz, 2H), 5.18 (s, 2H), 6.71 (s, 1H), 6.89 (s, 1H), 7.65 (s, 1H), 7.91 (s,
1H). 13C NMR (D2O) δ 13.47, 13.61, 13.63, 56.32, 61.53, 62.18, 63.61,
96.28, 99.16, 108.01, 111.53, 114.48, 115.06, 117.13, 119.54, 138.31,
138.82, 147.96, 150.23, 150.84, 152.12, 158.17, 164.53, 164.77, 165.60.
ESI HRMS (ꢀve) m/z calcd for C25H22O14S2Na [(M ꢀ Na)ꢀ)]
665.0121, found 665.0277 (M ꢀ Na)ꢀ.
YM ꢀ Y0
Y ¼ Y0
þ
ð1Þ
ꢀ logIC50ÞðHSÞ
1 þ 10ðlog½Iꢂ
0
In this equation, Y is the ratio of residual thrombin activity in the
presence of inhibitor to that in its absence (fractional residual
activity), YM and Y0 are the maximum and minimum possible values
of the fractional residual proteinase activity, IC50 is the concentration
of the inhibitor that results in 50% inhibition of enzyme activity, and
HS is the Hill slope, which was set constant at 1. A current version of
SigmaPlot (SPSS, Inc., Chicago, IL) was used to perform nonlinear
curve fitting in which YM, Y0, and IC50 were allowed to float.
19: 1H NMR (CDCl3, 300 MHz) δ 1.39 (m, 6H), 2.67 (s, 3H), 3.85
(s, 3H), 3.88 (s, 3H), 4.36 (m, 4H), 5.55 (s, 2H), 7.22 (s, 1H), 7.23 (s,
1H), 7.43 (s, 1H), 8.07 (s, 1H). 13C NMR (CD3OD) δ 13.25, 13.38,
13.52, 55.66, 55.77, 60.25, 60.79, 63.16, 95.63, 99.17, 103.95, 108.76,
112.48, 115.58, 117.27, 120.11, 139.90, 146.40, 148.00, 148.45, 152.15,
152.23, 158.27, 163.04, 163.59, 164.61. ESI HRMS (ꢀve) m/z calcd for
C26H25O13SNa [(M ꢀ Na)ꢀ)] 577.1011, found 577.1100 (M ꢀ Na)ꢀ.
Synthesis of Benzofuran Dimer 15 from 12a. To a solution of
12a (100 mg, 0.16 mmol) in 2 mL of methanol was added LiOH (20 mg,
0.8 mmol), and the mixture was stirred for 5 h at room temperature.
After completion, the reaction mixture was concentrated and purified
using HPLC. δ 1H NMR (CD3OD, 300 MHz) δ 2.66 (s, 3H), 3.84 (s,
3H), 3.89 (s, 3H), 5.52 (s, 2H), 7.11 (s, 1H, ArꢀH), 7.27 (s, 1H,
ArꢀH), 7.41 (s, 1H, ArꢀH), and 8.01 (s, 1H, ArꢀH). 13C NMR
(CD3OD) δ 13.43, 61.67, 63.73, 95.54, 95.63, 109.19, 112.32, 115.57,
117.40, 118.31, 139.89, 145.50, 149.74, 152.08, 152.20, 158.54, 162.57,
164.51, 165.48. ESI HRMS (ꢀve) m/z calcd for C17H20NO9SNa
[(M ꢀ Na)ꢀ)] 521.0322, found 521.1496 (M ꢀ Na)ꢀ.
MichaelisꢀMenten Kinetics of Spectrozyme TH Hydroly-
sis. The initial rate of Spectrozyme TH hydrolysis by 5ꢀ10 nM
thrombin was monitored from the linear increase in absorbance at
405 nm corresponding to less than 10% consumption of the substrate, as
described earlier.18,19 The initial rate was measured as a function of
various concentrations of the substrate (10ꢀ600 μM) in the presence of
fixed concentration of dimer 12a (0ꢀ30 μM) in 20 mM Tris-HCl buffer,
pH 7.4, at 37 °C. The data were fitted by MichaelisꢀMenten eq 2 to
determine KM and VMAX
.
VMAX½Sꢂ
Vi ¼
ð2Þ
KM þ ½Sꢂ
Prothrombin Time and Activated Partial Thromboplastin
Time. Clotting time was measured in a standard one-stage recalcifica-
tion assay with a BBL Fibrosystem fibrometer (Becton-Dickinson,
Sparles, MD), as described previously.20,21 For PT assays, thromboplas-
tin was reconstituted according to manufacturer’s directions and
warmed to 37 °C. A 10 μL sample of the sulfated benzofuran, to give
the desired concentration, was brought up to 100 μL with citrated
human plasma, incubated for 30 s at 37 °C followed by addition of
200 μL prewarmed thromboplastin. Clotting time in the absence of an
anticoagulant was determined using 10 μL of deionized water. For the
APTT assay, 10 μL of inhibitor was mixed with 90 μL of citrated human
plasma and 100 μL of prewarmed APTT reagent (0.2% ellagic acid).
After incubation for 4 min, clotting was initiated by adding 100 μL of
25 mM CaCl2 (37 °C) and time to clot noted. Each clotting assay was
performed in duplicate. The data were fit to a quadratic trendline, which
was used to determine the concentration of the inhibitor necessary to
double the clotting time, 2 ꢁ APTT or 2 ꢁ PT.
Synthesis of Benzofuran Dimer 16 from 11a. To a solution of
11a (200 mg, 0.32 mmol) in 10 mL of anhydrous THF was added
LiAlH4 (65 mg, 1.6 mmol), and the mixture was stirred for 6 h under
nitrogen. After completion, the reaction was quenched by adding 30 mL
of water and extracted with 100 mL of diethyl ether twice. The organic
layer was concentrated and purified using gradient flash chromatography
(0ꢀ50% ethyl acetate in hexane). The reduced form of 11a was sulfated
using the general sulfation procedure described above. 1H NMR
(CD3OD, 300 MHz) δ 0.17 (s, 6H), 1.01 (s, 9H), 2.71 (s, 3H), 3.87
(s, 3H), 3.91 (s, 3H)), 5.51 (s, 2H), 6.65 (s, 2H), 6.69 (s, 2H), 7.01 (s,
1H, ArꢀH), 7.04 (s, 1H,), 7.37 (s, 1H), 7.45 (s, 1H). 13C NMR
(CDCl3) δ ꢀ4.47, 12.31, 18.70, 26.00, 55.45, 55.81, 55.94, 56.58, 94.60,
95.48, 103.35, 114.35, 118.34, 120.39, 121.24, 142.32, 142.65, 144.82,
148.21, 149.73, 152.05.
Thrombin and Chemicals for Biological Experiments. Hu-
man R-thrombin was from Haematologic Technologies (Essex Junction,
VT). Stock solutions of thrombin were prepared in 20 mM sodium
phosphate buffer, pH 7.4, containing 100 mM NaCl. Chromogenic
substrate Spectrozyme TH was from American Diagnostica (Greenwich,
CT). Citrated human plasma for coagulation time assays was purchased
’ RESULTS AND DISCUSSION
Design of the Sulfated Benzofuran Scaffold. Sulfated LMWLs,
which recognize exosite II of thrombin, are based on an aromatic
5525
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
scaffold, a scaffold dramatically different from the highly anionic
polysaccharide scaffold of heparin.21 Nevertheless, sulfated LMWLs
retain one characteristic of heparin: structural complexity arising
from the presence of multiple intermonomeric linkages, such as
βꢀ5, βꢀβ, βꢀO4, and 5ꢀ5 (Figure 1). A simple calculation
suggests that LMWL molecules as small as a tetramer can exhibit
nearly 1400 distinct structures even if sulfation and inter-residue
linkage are the only variables considered. To uncover distinct
structures that may be the seat of anticoagulant activity, we visualized
the lignin structure as concatenated, aromatic units instead of
repetitions of 4-hydroxycinnamic acid monomers joined together
through different inter-residue linkages. In this alternative analysis,
oligomeric lignin can be considered as a combinatorial library of
dihydrobenzofuran, phenoxypropanoic acid, biphenyl, and fused
furan monomers (Figure 1). We reasoned that one or more of these
aromatic units with appropriate sulfate groups could be the seat of
thrombin recognition and inhibition.
To begin assessing the contribution of these individual aro-
matic units, we focused on the benzofuran unit, which is a
common scaffold used in many synthetic studies. In a preliminary
investigation, we had prepared a library of sulfated and carboxy-
lated benzofuran monomers, of which a small group of molecules
were found to inhibit thrombin, albeit with IC50 in the millimolar
range. Interestingly, mechanistic studies had suggested allosteric
inhibition by these molecules.23 This promise led us to explore a
more diverse set of sulfated benzofuran monomers and dimers as
potential inhibitors of thrombin (Figure 2).
Overall Description of the Sulfated Benzofuran Library.
The benzofuran unit of sulfated LMWLs contains substitutions
at the 2, 3, and 5 positions (Figure 1). Of these, the 2 and 5
positions are utilized for intermonomeric linkage and the 3 position
may bear a carboxylate or an alkylsulfate group. Considering
these variations, charged, polar, and hydrophobic substitutions
were engineered on this scaffold to obtain a structurally diverse
library. Figure 2 describes the multimodal approach developed to
synthesize 15 sulfated monomers and 13 sulfated dimers. This
library reflects significant variations in (1) the number of negatively
charged groups (one to three anions), (2) the positions of anionic
and polar substitutions from the terminal to the center of the
scaffold, (3) the size and nature of hydrophobic groups, and (4)
inter-residue linkage geometry. A characteristic feature is that
each member of the library is nearly fully water-soluble because
of the presence of one or more sulfate groups, despite the
primarily hydrophobic benzofuran backbone. This combination
of water solubility and hydrophobicity has been known to introduce
novel physicochemical characteristics in the sulfated lignin-based
scaffold, e.g., higher than expected partition coefficient24 and
greater proportion of nonionic forces in binding to proteins.25
Synthesis of Sulfated Benzofuran Monomers. The first step
in the synthesis of the library was the generation of base structures,
5,6-dihydroxybenzofuran-3-carboxylic acid esters (1aꢀe, Figure 2),
from catechol in one step using oxidative Michael addition
conditions following the report of Pei et al.26 The reaction was
strongly susceptible to substituent effects, which required ex-
tensive optimization of reaction conditions, especially the or-
ganic base, reaction time, and solvent. Eventually, products 1aꢀe
were obtained in reasonably good yields (50ꢀ65%). Monoalk-
ylation or acylation of dihydroxybenzofurans 1 proceeded
smoothly to selective 6-substituted derivatives 2aꢀj. That the
substitution occurred at the 6-position of the benzofuran ring was
confirmed through two-dimensional nuclear Overhauser spectros-
copy experiment (ROESY), which showed a correlation between
the protons of the 3-alkyl ester and 6-alkoxy groups (not shown).
The monomeric benzofurans 2aꢀj so synthesized in two steps
formed one major group of precursors for library construction.
Another set of monomer precursors 3aꢀg and 4aꢀg were
synthesized from 2aꢀj through silyl protection and bromination,
respectively (Figure 2).
Monosulfated benzofurans 5aꢀj were prepared by direct
sulfation of precursors 2aꢀj. Phenolic molecules are typically
more challenging to sulfate than alcoholic molecules.27 We had
previously developed a microwave-based sulfation protocol for
introducing multiple sulfate groups in a crowded environment
using triethylamineꢀsulfur trioxide complex at 100 °C.22,23 This
method gave good yields of monosulfated benzofurans 5aꢀj in
their ammonium salt forms, which were exchanged for sodium
using a Sephadex C-25 exchange column. To test whether the
number and location of sulfate groups on the benzofuran scaffold
play a significant role in recognizing thrombin, we introduced a
sulfate group at the 3-position also. Thus, benzofurans 2a and 3a
were reduced with LAH and sulfated to yield 6 and 7, respec-
tively. However, both 6 and 7 were found to degrade within a
couple of hours after dissolution in water at room temperature.
The reason for the reduced aqueous stability of these 3-methy-
lene sulfated benzofurans is not clear, although the dual combi-
nation of electron pull by sulfate groups and electron push by ring
oxygen may be the cause of more rapid desulfation.
To assess the importance of the nature of the carbonyl group at
the 3-position, silyl-protected benzofuran ester 3 was directly
converted to morpholinyl and 4-methylpiperidinyl amides 8a
and 8b, respectively, using a procedure reported by Weinreb and
co-workers in the late 1970s.28 Desilylation and sulfation gave
monosulfated benzofuran amides 9a and 9b in excellent yields.
Finally, 2-morpholino monomer 10 was prepared from 4a to
study the effect of varying the 2-substituent.
Synthesis of Sulfated Benzofuran Dimers. The construc-
tion of the library of dimers relied on the nucleophilic displace-
ment of the allylic bromide at the 2 position by free phenolic
group at the 5-position. The apparent simplicity of this reaction29
and the availability of 10 nucleophilic monomers 2aꢀj and 7
allylic bromides 4aꢀg enticed us to construct a combinatorial
library (Figure 2). However, the coupling was challenging on
both synthesis and purification fronts. Steric crowding around
both the nucleophile and the electrophile resulted in less than
optimal yields (<50%) of 11aꢀf. In addition, the synthesized
dimers typically coeluted with monomers in a wide range of
multisolvent systems, creating significant purification issues. We
suspected that coelution may be occurring because of π-stacking
at high concentrations. Thus, a higher yielding dimerization reaction
was desirable, which was made possible through implementation
of microwave coupling. By use of this strategy, silylated dimers
11aꢀf were synthesized in excellent yields (>90%). These were
then deprotected and sulfated to yield monosulfated benzofuran
dimers 12aꢀf. Likewise, monosulfated benzofuran dimers 13 and
14, which contain a morpholine ring at the 2 and 3 positions,
respectively, were synthesized starting from monomers 4a and 8a,
respectively, through a series of silyl protection, allylic bromination,
deprotection, nucleophilic coupling, and sulfation steps (Figure 2).
To evaluate the role of multiple anionic groups on the small
hydrophobic scaffold, 12a was hydrolyzed in alcoholic LiOH30 to
yield 15, a monosulfated, dicarboxylated dimer. Likewise, 11a was
reduced with LAH and sulfated to yield 16, which is a disulfated
dimer, but as with monomers 6 and 7, 16 was found to degrade
rapidly in water. Finally, coupling of 4a with 1a followed by
5526
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
Figure 2. Synthesis of a library of sulfated benzofurans as potential allosteric regulators of thrombin. The library consists of 15 monomers (5aꢀj, 6, 7,
9a, 9b, and 10) and 13 dimers (12aꢀf, 13ꢀ19). R and R0 groups are defined in Table 1. Synthesis of 19 is described in the text and Supporting
Information. Reagents and conditions for the reactions are as follows: (a) Cs2CO3, alkyl or acyl halide, anh DMF; (b) TBDMSCl, imidazole, DMF; (c)
NBS, CCl4; (d) Cs2CO3, 2, DMF; (e) KF, CH3COOH, DMF; (f) Et3N/SO3, CH3CN/DMF (4:1), microwave; (g) Me3Al, R-H, toluene; (h) Cs2CO3,
1a, DMF; (i) LAH, anh THF; (j) Cs2CO3, morpholine, DMF; (k) Cs2CO3, 4a, DMF; (l) Cs2CO3, DMF, desilylated 8a; (m) alc LiOH. See text for
details.
desilylation and sulfation gave 17, which is also a disulfated dimer
but with a structure completely different from 16. Likewise,
another variant of disulfated dimer, i.e., 18, was synthesized by
coupling 1a and 2a in five steps (Figure 2). In combination,
dimers 15ꢀ18 display anionic groups around three key positions
(3, 5, and 6) of the benzofuran scaffold. These contain either two
or three negative charges, which approximate the number of
charges typically found on a heparin disaccharide.
Thrombin Inhibition Potential of Designed Sulfated Ben-
zofuran Monomers and Dimers. Inhibition of thrombin by the
designed benzofurans was measured through Spectrozyme TH
hydrolysis assay, as described earlier.18,19 This assay relies on the
decrease in the initial rate of substrate hydrolysis in the presence
of the sulfated benzofuran derivative as a quantitative measure of
the inhibition. The fractional decrease in initial rate of hydrolysis
typically shows a sigmoidal profile on a semilog plot, which is
5527
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
fitted by doseꢀresponse eq 1 to derive IC50, HS, Y0, and YM
parameters for each potential inhibitor.
monomers. Of the 15 sulfated monomers studied in this work
and 17 sulfated and unsulfated monomers studied earlier,23 only
5i inhibited thrombin with measurable potency (∼500 μM,
Table 1). By contrast, most sulfated dimers displayed thrombin
inhibition (Table 2). This suggested that an oligomeric scaffold
may exhibit higher potency and rationalizes the nanomolar
potency observed for sulfated LMWL inhibition of thrombin.18
The distinct groups at the 2, 3, 5, and 6 positions on the
benzofuran ring result in interesting structureꢀactivity relation-
ships. The sulfate group at the 5 position was found to be the key
basis for thrombin inhibition, since all unsulfated precursors
studied in this work, monomers as well as dimers, did not inhibit
thrombin. Such high level of dependence on one sulfate group is
striking. It also appears to be unusual because thrombin is known
to be considerably forgiving in recognizing ligands. For example,
ligands as different as heparin and RNA are known to bind in
Figure 3 shows the semilog inhibition curves observed for
selected benzofuran monomers and dimers. Of the 28 potential
inhibitors studied in this work, only 11 molecules exhibited
reasonable inhibition of human R-thrombin at pH 7.4 and 25 °C.
These included sulfated monomer 5i and dimers 12aꢀf, 13, and
17ꢀ19 (Tables 1 and 2). Within this group, inhibitors 12b, 12c,
and 12f were the only molecules exhibiting IC50 less than 50 μM.
The tert-butyl derivative 12c was found to be the most potent
thrombin inhibitor with an IC50 of 7.3 μM under physiologically
relevant conditions.
The range of inhibition efficacy was found to vary considerably
with the structure of the inhibitor. For example, sulfated dimer
12e displayed essentially complete inhibition of thrombin (95%,
Table 2) while dimer 15 with two carboxylic acid groups inhibited
to the extent of only 14% (Table 2). Nearly 85% of the inhibitors
studied inhibited thrombin to more than 60%, suggesting good
overall efficacy in this series of sulfated benzofurans.
Table 2. Inhibition of Human r-Thrombin by Sulfated
Benzofuran Dimersa
Comparison of the two series of benzofuran molecules shows
that dimers tend to inhibit thrombin several-fold better than
R
R0
log IC50 (M) IC50 (μM) ΔY
12a ꢀOCH2CH3
12b ꢀOCH3
ꢀCH3
ꢀCH3
ꢀCH3
ꢀ4.22 ( 0.16b 60 ( 22
ꢀ4.43 ( 0.12 37 ( 11
0.75
0.68
12c ꢀOC(CH3)3
ꢀ5.14 ( 0.18 7.3 ( 3.1 0.84
ꢀ3.42 ( 0.13 375 ( 109 0.80
12d ꢀOCH2CH2OCH3 ꢀCH3
12e ꢀOCH2CH3
12f ꢀOCH2CH3
13 nac
ꢀCH2CH3 ꢀ3.92 ( 0.05 121 ( 14 0.95
ꢀCH(CH3)2 ꢀ4.52 ( 0.19 30 ( 13
0.72
nac
ꢀ3.89 ( 0.20 129 ( 60 0.62
14 N-morpholino
15 ꢀOH
ꢀCH3
ꢀCH3
nac
incompleted
>7100
nac
ꢀ3.43 ( 0.37 ∼374e
0.14
0.95
17 nac
18 nac
19 nac
ꢀ3.12 ( 0.35 ∼764e
nac
nac
ꢀ3.44 ( 0.06 365 ( 57 0.79
ꢀ4.05 ( 0.26 89 ( 54
0.70
a Thrombin inhibition was measured in spectrophotometric assay
through initial rate of Spectrozyme TH hydrolysis as described in
Experimental Procedures. b Standard error shown is (1 SE. c Not
applicable. See complete structures of 13, 14, 15, 17, 18, and 19 in
Figure 2. d Full inhibition profile was not measured. e The error in IC50
could not be accurately defined because of insufficient number of data
points.
Figure 3. Direct inhibition of thrombin by designed sulfated benzofur-
an monomers and dimers. The inhibition of human thrombin was
determined spectrophotometrically through a Spectrozyme TH hydro-
lysis assay at pH 7.4 and 25 °C. Solid lines represent sigmoidal fits to the
data to obtain IC50, YM, and Y0 (eq 1), as described in the Experimental
Procedures. See text for details.
Table 1. Inhibition of Human r-Thrombin by Sulfated Benzofuran Monomersa
compd
R
R0
log IC50 (M)
IC50 (μM)
>2500c
>3000c
>3000c
>2000c
ΔY
5a
5b
5c
5d
5e
5f
ꢀOCH2CH3
ꢀOCH3
ꢀCH3
ꢀCH3
ꢀCH3
ꢀCH3
ndb
ndb
ndb
ndb
ꢀ1.7 ( 0.2d
ꢀ1.9 ( 0.3
ndb
ꢀOC(CH3)3
ꢀOCH2CH2OCH3
ꢀOCH2CH3
ꢀOCH2CH3
ꢀOCH2CH3
ꢀOCH2CH3
ꢀOCH2CH3
ꢀOCH2CHdCH2
N-morpholino
N-piperidino
ꢀCH2CH3
ꢀCH2CH3
ꢀCH(CH3)2
ꢀCH2OCH3
morpholine-4-carbonyl
benzoyl-p-methoxy
ꢀCH3
20560 ( 8500
13650 ( 10400
>16000c
>25000c
520 ( 280
>3000
>2000c
>2000c
>7000c
0.60
0.75
5g
5h
5i
ndb
ꢀ3.3 ( 0.2
0.54
5j
ndb
9a
9b
10e
ꢀCH3
ndb
ndb
ndb
ꢀCH3
ꢀCH3
a Thrombin inhibition was measured in spectrophotometric assay through initial rate of Spectrozyme TH hydrolysis as described in Experimental
Procedures. b Not determined because of incomplete and/or poor inhibition. c Estimates on the basis of the highest concentration used in experiment.
d Standard error shown is (1 SE. e 10 contains a morpholine group at the allylic position (instead of a ꢀCH3 group). See Figure 2 for the full structure.
5528
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
exosite II.31,32 Similarly, hirudin, which contains a sulfated tyrosine,
and desulfated hirudin, i.e., desirudin, bind in exosite I with
excellent affinities.2,10,13,33
The stringent requirement for 5-sulfate led to the expectation
that multiple sulfate groups on the benzofuran scaffold may
induce higher potency. Yet dimers 17 and 18, which bear two
sulfate groups (5 and 50, and 5 and 6 positions, respectively)
inhibited thrombin approximately 6- to 17-fold weaker (Table 2).
Furthermore, the IC50 of 15, which contains the 5-sulfate as well
as a carboxylate at each of the 3-positions of the benzofuran rings,
also decreases 6.2-fold from its parent 12a. This implies that any
additional anionic group on the benzofuran scaffold is counter-
productive, which is contrary to what one would predict on the
basis of the thrombinꢀheparin system.17,31,32
Figure 4. MichaelisꢀMenten kinetics of Spectrozyme TH hydrolysis
by human thrombin in the presence of dimer 12a. The initial rate of
hydrolysis at various substrate concentrations was measured spectro-
photometrically in pH 7.4 buffer as described in Experimental Proce-
dures. The concentrations of 12a chosen for study include 0 (O),
3.0 ([), 10.5 (2), and 30 μM (]). Solid lines represent nonlinear
regressional fits to the data by the standard MichaelisꢀMenten equation
Comparison of IC50 of 12a,12b,and12c shows that as lipophilicity
of the 3-ester increases, the inhibition potency improves 8.2-fold
(Table 2). Likewise, change in substitution from ethyl to isopropyl
at the 6-position, as in 12e and 12f, also increases the potency nearly
4-fold. This implies that hydrophobicity of the scaffold plays an
important role in thrombin recognition. In support of this,
increase in hydrophilicity of groups markedly decreases potency.
For example, the 3-methoxyethyl ester containing 12d is a 6.2-
fold weaker inhibitor of thrombin than 3-ethyl containing 12a
(Table 2). More importantly, introduction of an N-morpholinyl
ring (dimer 14) increased IC50 to >2000 μM, which represents a
more than 270-fold loss of affinity from a comparable tert-butyl
ester containing dimer 2c.
to yield KM and VMAX
.
and, by analogy, other sulfated benzofuran molecules. The reduction
in catalytic efficiency by these novel inhibitors is primarily brought
about by the slow conversion of the thrombinꢀSpectrozyme TH
Michaelis complex, which may arise from the intrinsic change in
the rate of chemical reaction or by a change in the structure of the
catalytic triad induced by the allosteric inhibitors.
While the above discussion suggests fairly stringent structural
requirements on the benzofuran scaffold, two interesting results
highlight the possible variances. Dimer 13 containing a morpho-
line ring at the 2-allylic position displays IC50 of 129 μM, which is
only 2.2-fold higher than that of 12a. This suggests that introduction
of a hydrophilic and sterically bulkier group at the terminus opposite
the 5-sulfate is not detrimental. Likewise, dimer 19 displays no
change in IC50 from its comparable parent 12a (Table 2). Dimer
19 contains a 2-CH2O-6 intermonomeric linkage compared to a
2-CH2O-5 linkage present in dimer 12a. An essentially identical
inhibition potency of 19 compared to 12a indicates that a change
as drastic as a ring flip, or regioisomerism, is well tolerated.
Mechanism of Thrombin Inhibition by Sulfated Benzofur-
an Dimer 12a. To elucidate the nature of thrombin inhibition by
the designed sulfated benzofurans, the kinetics of Spectrozyme
TH hydrolysis by thrombin at pH 7.4 in the presence and
absence of 12a was studied. Spectrozyme TH has been routinely
used as a chromogenic substrate of thrombin.18 The initial rate of
amidolysis as a function of the substrate concentration displayed
a hyperbolic profile, as expected (Figure 4), from which the
Michaelis constant (KM) and maximal velocity of the reaction
(VMAX) were derived. The KM and VMAX for Spectrozyme TH in
the absence of 12a was found to be 18.9 μM and 100 mAU minꢀ1
μMꢀ1, respectively. In the presence of 1.5, 3.0, 10.5, and 30 μM
12a, the KM of substrate was found to be 12.6 ( 4.7, 15.5 ( 5.5,
15.9 ( 7.4, and 15.5 ( 3.7 μM. In contrast, the VMAX was
measured to be 97 ( 13, 107 ( 14, 73 ( 13, and 66 ( 6 mAU
minꢀ1 μMꢀ1 in these experiments. This corresponds to ∼34%
decrease in maximal velocity of reaction upon approximately
50% saturation of the enzyme. This suggests that the presence of
12a does not significantly affect the binding of the chromogenic
substrate to the active site of the enzyme while significantly
reducing its catalytic efficiency.
Prolongation of Plasma Clotting Time by Sulfated Benzo-
furans. Two plasma clotting assays are typically used to assess
the anticoagulant potential of new coagulation inhibitors. These
are the prothrombin and activated partial thromboplastin time
assays (PT and APTT, respectively), which attempt to measure
the effect of an inhibitor on the extrinsic and intrinsic flux of
coagulation. The concentrations of the inhibitors required to
double the PT and APTT were measured, as described earlier for
sulfated LMWLs.20,21 The prolongation of plasma clotting time
as a function of the concentration of the sulfated benzofurans
followed a pattern typical of other well-studied anticoagulants
(Figure 5) except for the range of active concentrations. A 2-fold
increase in PT required 850ꢀ2100 μM concentration of 5i, 12a,
or 12b (Table 3). In a similar manner, the doubling of APTT
required 355ꢀ1250 μM 5i, 12a, 12c, or 12e (Table 3). Most
other monomers or dimers were found to be not effective. These
results suggest that the designed small, sulfated benzofurans are
about 100- to 250-fold less potent in human plasma than the
oligomeric sulfated LMWLs from which they were designed.21
This is not too unexpected considering that thrombin inhibition
by the smaller synthetic molecules is nearly 400-fold weaker than
that of the parent oligomers. Yet interesting similarities and
differences are discernible. For example, dimers are better antic-
oagulants in plasma than monomers. Within the group of dimers,
12a, 12c, and 12e, which inhibit thrombin well, are also the best
in terms of prolonging APTT. Interestingly, 12b, which is
comparable to 12a in thrombin inhibition, does not affect APTT
at all (Table 3), and the reason for this behavior is not clear.
Overall, the plasma studies suggest the feasibility of designing
sulfated benzofurans that will function as anticoagulants in vivo.
’ SIGNIFICANCE
The above MichaelisꢀMenten kinetics result indicates a non-
The range of thrombin inhibition potency and efficacy discov-
ered for sulfated benzofurans suggest that the designed sulfated
competitive, allosteric mechanism of thrombin inhibition by 12a
5529
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
This work presents small, synthetic, sulfated aromatic structures
as novel allosteric modulators of thrombin function. Although it is
too early to speculate on the site of binding of sulfated benzofur-
ans, the parent oligomeric sulfated LMWLs are known to engage
exosite 2 of thrombin.18 Allosteric, exosite 2-based modulators of
thrombin are of special interest because they afford the possibility
of controlled inhibition of this key protease that takes part in a
number of physiological processes.13 Excess inhibition could, in
principle, be reversed by highly sulfated small molecules, e.g.,
sucrose octasulfate, that are also expected to bind in this region
without inducing significant reduction in thrombin’s catalytic
activity. Allosteric, exosite 2-mediated inhibitors with nanomolar
potency, other than sulfated LMWLs,18 have not been designed
as yet, and thus, sulfated benzofurans represent first molecules in
a new class of thrombin modulators.
Although the potency of our first allosteric inhibitors is not
high, it is not a major concern because rational design of these
molecules from the parent polymers invoked a rather drastic
reduction in molecular weight. Whereas sulfated LMWLs are
oligomeric chains with an average molecular weight of 5000,
sulfated benzofurans range from 500 to 1000.21 Thus, a relatively
easy approach to higher potency would be to prepare appropriate
trimers and tetramers of sulfated benzofurans.
Finally, this work also highlights the opportunity of exploring
allosteric modulators of other coagulation enzymes, e.g., factors
Xa, IXa, and XIa, through sulfated lignin scaffolds. Considering
the wealth of structural diversity accessible with the lignin scaffold,
it may be possible to design allosteric inhibitors that target these
enzymes in a highly specific manner.
Figure 5. Prolongation of clotting time as a function of concentration of
sulfated benzofuran monomers and dimers in either activated partial
thromboplastin time assay (A) or prothrombin time assay (B). Solid
lines are trend lines and not nonlinear regressional fits. Error bars in the
range of symbol size have been omitted. APTT and PT assays were
performed as described in the Experimental Procedures.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis and spectral proper-
b
Table 3. Effect of Designed Sulfated Benzofurans on Human
Plasma Clotting Timea
ties of molecules depicted in Figure 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
compd
APPT (μM)
PT (μM)
’ AUTHOR INFORMATION
5i
1250
>1200
>2000
740
1200
neb
neb
5g
Corresponding Author
5c
*Address: Department of Medicinal Chemistry, Virginia Common-
wealth University, 800 East Leigh Street, Suite 212, P.O. Box
980133, Richmond, VA 23219. Phone: 804-828-7328. Fax: 804-
827-3664. E-mail: urdesai@vcu.edu.
12a
12b
12c
12d
12e
14
2100
850
neb
neb
neb
ndc
neb
>1000
355
>1000
530
’ ACKNOWLEDGMENT
>1000
>1000
This work was supported by Grants HL099420 and HL090586
from the National Institutes of Health. We thank Dr. H. Tonie
Wright for helpful discussions and reading of the manuscript.
15
a PT and APTT values were deduced in in vitro human plasma experiments
where the clot initiator is either thromboplastin or ellagic acid, respectively.
See Methods section for details. b No effect. c Not determined.
’ ABBREVIATIONS USED
benzofuran scaffold may be an excellent lead in developing
anticoagulants radically different from those studied to date.
The βꢀ5 intermonomer linkage is only one of the many units
present in sulfated LMWLs,18,21 which implies that computa-
tional structure-based design may be necessary to identify the
most appropriate “small” structure from the large number of
potential molecules. One such strategy, e.g., combinatorial
virtual library screening, has been put forward for highly
sulfated molecules34 and also been found useful for sulfated
lignins binding to antithrombin.25
APTT, activated partial thromboplastin time; LMWL, low
molecular weight lignin; PT, prothrombin time
’ REFERENCES
(1) Bauer, K. A. New anticoagulants. Hematology (Am. Soc. Hematol.,
Educ. Program Book) 2006, 450–456.
(2) Henry, B. L.; Desai, U. R. Anticoagulants. In Burger’s Medicinal
Chemistry, Drug Discovery and Development, 7th ed.; Abraham, D. J.,
Rotella, D. P., Eds.; John Wiley: Hoboken, NJ, 2010; pp 365ꢀ408.
5530
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531

Journal of Medicinal Chemistry
ARTICLE
(3) Straub, A.; Roehrig, S.; Hillisch, A. Oral, direct thrombin and
factor Xa inhibitors: the replacement of warfarin, leeches and pig
intestines? Angew. Chem., Int. Ed. 2011, 50, 4574–4590.
(4) Steinmetzer, T; Sturzebecher, J. Progress in the development of
synthetic thrombin inhibitors as new orally active anticoagulants. Curr.
Med. Chem. 2004, 11, 2297–2321.
direct and indirect inhibitors of coagulation proteinases. Bioorg. Med.
Chem. 2006, 14, 7988–7998.
(22) Raghuraman, A.; Riaz, M.; Hindle, M.; Desai, U. R. Rapid, high-
yielding microwave-assisted per-sulfation of organic scaffolds. Tetrahe-
dron Lett. 2007, 48, 6754–6758.
(23) Verghese, J.; Liang, A.; Sidhu, P. S.; Hindle, M.; Zhou, Q.;
Desai, U. R. First steps in the direction of synthetic, allosteric, direct
inhibitors of thrombin and factor Xa. Bioorg. Med. Chem. Lett. 2009,
19, 4126–4129.
(24) Liang, A.; Thakkar, J. N.; Desai, U. R. Study of physico-chemical
properties of novel highly sulfated, aromatic, mimetics of heparin and
heparan sulfate. J. Pharm. Sci. 2010, 99, 1207–1216.
(25) Henry, B. L.; Connell, J.; Liang, A.; Krishnasamy, C.; Desai,
U. R. Interaction of antithrombin with sulfated, low molecular weight
lignins. Opportunities for potent, selective modulation of antithrombin
function. J. Biol. Chem. 2009, 284, 20897–20908.
(26) Pei, L.; Li, Y.; Bu, X.; Gu, L.; Chan, A. S. C. One-pot synthesis of
5,6-dihydroxylated benzo[b]furan derivatives. Tetrahedron Lett. 2006,
47, 2615–2618.
(27) Al-Horani, R. A.; Desai, U. R. Chemical sulfation of small
molecules—advances and challenges. Tetrahedron 2010, 66, 2907–2918.
(28) Basha, A.; Lipton, M.; Weinreb, S. A mild, general method for
conversion of esters to amides. Tetrahedron Lett. 1977, 48, 4171–4174.
(29) Flessner, T.; Doye, S. Cesium carbonates: a powerful inorganic
base in organic synthesis. J. Prakt. Chem. 1999, 341, 186–190.
(30) Dayal, B.; Salen, G.; Toome, B.; Tint, G. S.; Shefer, S.; Padia, J.
Lithium hydroxide/aq. methanol: a mild reagent for hydrolysis of bile
acids methyl esters. Steriods 1990, 55, 233–237.
(31) Carter, W. J.; Cama, E.; Huntington, J. A. Crystal structure of
thrombin bound to heparin. J. Biol. Chem. 2005, 280, 2745–2749.
(32) Jeter, M. L.; Ly, L. V.; Fortenberry, Y. M.; Whinna, H. C.;
White, R. R.; Rusconi, C. P.; Sullenger, B. A.; Church, F. C. RNA
aptamer to thrombin binds anion-binding exosite-2 and alters protease
inhibition by heparin-binding serpins. FEBS Lett. 2004, 568, 10–14.
(33) Liu, C. C.; Brustad, E.; Liu, W.; Schultz, P. G. Crystal structure
of a biosynthetic sulfo-hirudin complexed to thrombin. J. Am. Chem. Soc.
2007, 129, 10648–10649.
(5) Saiah, E.; Soares, C. Small molecule coagulation cascade inhibi-
tors in the clinic. Curr. Top. Med. Chem. 2005, 5, 1677–1695.
(6) Smallheer, J. M.; Quan, M. L. Recent advances in coagulation
serine protease inhibitors. Annu. Rep. Med. Chem. 2009, 44, 189–208.
(7) Tucker, T. J.; Brady, S. F.; Lumma, W. C.; Lewis, S. D.; Gardell,
S. J.; Naylor-Olsen, A. M.; Yan, Y. W.; Sisko, J. T.; Stauffer, K. J.; Lucas,
B. J.; Lynch, J. J.; Cook, J. J.; Stranieri, M. T.; Holahan, M. A.; Lyle, E. A.;
Baskin, E. P.; Chen, I. W.; Dancheck, K. B.; Krueger, J. A.; Cooper, C. M.;
Vacca, J. P. Design and synthesis of a series of potent and orally
bioavailable noncovalent thrombin inhibitors that utilize nonbasic
groups in the P1 position. J. Med. Chem. 1998, 41, 3210–3219.
(8) Burgey, C. S.; Robinson, K. A.; Lyle, T. A.; Nantermet, P. G.;
Selnick, H. G.; Isaacs, R. C. A.; Lewis, S. D.; Lucas, B. J.; Krueger, J. A.;
Singh, R.; Miller-Stein, C.; White, R. B.; Wong, B.; Lyle, E. A.; Stranieri,
M. T.; Cook, J. J.; McMasters, D. R.; Pellicore, J. M.; Pal, S.; Wallace,
A. A.; Clayton, F. C.; Bohn, D.; Welsh, D. C.; Lynch, J. J.; Yan, Y. W.;
Chen, Z. G.; Kuo, L.; Gardell, S. J.; Shafer, J. A.; Vacca, J. P. Pharma-
cokinetic optimization of 3-amino-6-chloropyrazinone acetamide
thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver
an orally bioavailable series containing P1N-benzylamides. Bioorg. Med.
Chem. Lett. 2003, 13, 1353–1357.
(9) Brånalt, J.; Gustafsson, D.; Nilsson, I.; Polla, M. New Hetero-
cyclic Carboxamides for Use as Thrombin Inhibitors. Int. Pat. Appl. WO
2009/157860 A1, 2009; AstraZeneca AB.
(10) Liu, L. W.; Vu, T. K.; Esmon, C. T.; Coughlin, S. R. The region
of the thrombin receptor resembling hirudin binds to thrombin and
alters enzyme specificity. J. Biol. Chem. 1991, 266, 16977–16980.
(11) Hortin, G. L.; Trimpe, B. L. Allosteric changes in thrombin’s
activity produced by peptides corresponding to segments of natural
inhibitors and substrates. J. Biol. Chem. 1991, 266, 6866–6871.
(12) Ye, J.; Liu, L. W.; Esmon, C. T.; Johnson, A. E. The fifth and
sixth growth factor-like domains of thrombomodulin bind to the anion-
binding exosite of thrombin and alter its specificity. J. Biol. Chem. 1992,
267, 11023–11028.
(34) Raghuraman, A.; Mosier, P. D.; Desai, U. R. Finding needle in a
haystack. Development of a combinatorial virtual screening approach for
identifying high specificity heparin/heparan sulfate sequence(s). J. Med.
Chem. 2006, 49, 3553–3562.
(13) Huntington, J. A. Molecular recognition mechanisms of throm-
bin. J. Thromb. Haemostasis 2005, 3, 1861–1872.
(14) Monteiro, R. Q.; Rap^oso, J. G.; Wisner, A.; Guimar~aes, J. A.;
Bon, C.; Zingali, R. B. Allosteric changes of thrombin catalytic site
induced by interaction of bothrojaracin with anion-binding exosites I
and II. Biochem. Biophys. Res. Commun. 1999, 262, 819–822.
(15) Monteiro, R. Q. Targeting exosites on blood coagulation
proteases. An. Acad. Bras. Cienc. 2005, 77, 275–280.
(16) Li, W.; Johnson, D. J.; Adams, T. E.; Pozzi, N.; De Filippis, V.;
Huntington, J. A. Thrombin inhibition by serpins disrupts exosite II.
J. Biol. Chem. 2010, 285, 38621–38629.
(17) Olson, S. T.; Halvorson, H. R.; Bjork, I. Quantitative character-
ization of the thrombinꢀheparin interaction. Discrimination between
specific and nonspecific binding models. J. Biol. Chem. 1991, 266, 6342–6352.
(18) Henry, B. L.; Monien, B. H.; Bock, P. E.; Desai, U. R. A novel
allosteric pathway of thrombin inhibition. Exosite II mediated potent
inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers
of 4-hydroxycinnamic acids. J. Biol. Chem. 2007, 282, 31891–31899.
(19) Henry, B. L.; Abdel Aziz, M.; Zhou, Q.; Desai, U. R. Sulfated,
low molecular weight lignins are potent inhibitors of plasmin, in addition
to thrombin and factor Xa: novel opportunity for controlling complex
pathologies. Thromb. Haemostasis 2010, 103, 507–515.
(20) Henry, B. L.; Thakkar, J. N.; Martin, E. J.; Brophy, D. F.; Desai,
U. R. Characterization of the plasma and blood anticoagulant potential
of structurally and mechanistically novel oligomers of 4-hydroxycin-
namic acids. Blood Coagulation Fibrinolysis 2009, 20, 27–34.
(21) Monien, B. H.; Henry, B. L.; Raghuraman, A.; Hindle, M.;
Desai, U. R. Novel chemo-enzymatic oligomers of cinnamic acids as
5531
dx.doi.org/10.1021/jm2005767 |J. Med. Chem. 2011, 54, 5522–5531