Design of small-molecule thrombin inhibitors based on the cis-5-phenylproline scaffold

Article abstract of DOI:10.1007/s11172-011-0107-x

The design of novel organic compounds containing no strongly basic amidine or guanidine functional groups typical of serine protease inhibitors was performed to develop an oral anticoagulant drug. A three-dimensional computational model for thrombin active site was constructed and optimized for docking of small-molecule organic compounds and calculating the energies of inhibitor-enzyme interactions. Novel racemic derivatives of 1-[2-(4- chlorophenylthio)acetyl]-5-phenylpyrrolidine-2,4-dicarboxylic acids were synthesized for which Cl-π interactions between the inhibitors and the S1 pocket of thrombin active site are predicted by modeling. The compounds synthesized deactivate thrombin in vitro and the inhibition properties show good correlations with the results of calculations.

Full text of DOI:10.1007/s11172-011-0107-x

Russian Chemical Bulletin, International Edition, Vol. 60, No. 4, pp. 685—693, April, 2011
685
Design of smallꢀmolecule thrombin inhibitors
based on the cisꢀ5ꢀphenylproline scaffold
K. V. Kudryavtsev,a D. A. Shulga,a V. I. Chupakhin,b A. V. Churakov,c
N. G. Datsuk,a D. V. Zabolotnev,a and N. S. Zefirova,b
aDepartment of Chemistry, M. V. Lomonosov Moscow State University,
Bldg. 3, 1 Leninskie Gory, 119991 Moscow, Russian Federation
Fax: +7 (495) 932 8846. Eꢀmail: kudr@org.chem.msu.ru
bInsitute of Physiologically Active Compounds, Russian Academy of Sciences,
1 Severnyi proezd, 142432 Chernogolovka, Moscow Region, Russian Federation
cN. S. Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences,
31 Leninskiy prosp., 119991 Moscow, Russian Federation
The design of novel organic compounds containing no strongly basic amidine or guanidine
functional groups typical of serine protease inhibitors was performed to develop an oral anticoꢀ
agulant drug. A threeꢀdimensional computational model for thrombin active site was constructꢀ
ed and optimized for docking of smallꢀmolecule organic compounds and calculating the enerꢀ
gies of inhibitor—enzyme interactions. Novel racemic derivatives of 1ꢀ[2ꢀ(4ꢀchlorophenylꢀ
thio)acetyl]ꢀ5ꢀphenylpyrrolidineꢀ2,4ꢀdicarboxylic acids were synthesized for which Cl—π interꢀ
actions between the inhibitors and the S1 pocket of thrombin active site are predicted by
modeling. The compounds synthesized deactivate thrombin in vitro and the inhibition properꢀ
ties show good correlations with the results of calculations.
Key words: anticoagulant therapy, thrombin, smallꢀmolecule inhibitors, molecular docking,
1,3ꢀdipolar cycloaddition, azomethine ylides.
Cardiovascular diseases are the major cause of mortalꢀ
ity in modern world. Usually, these pathologies lead to
thrombosis which limits the delivery of nutrients and oxyꢀ
gen to tissues and eventually causes cell loss. The final
clinical outcomes of blood vessel occlusion include cardiꢀ
ac arrest, acute coronary syndrome, stroke, and peripheral
embolism accompanying arterial thrombosis, acute deep
vein thrombosis, pulmonary embolism, and paradoxical
arterial embolism in the case of vein thrombosis. The reaꢀ
sons for these clinical outcomes include rupture of atheroꢀ
sclerotic plaques, cardiac embolism as a result of fibrillaꢀ
tion or left ventricular aneurysm, stasis and immobility in
the postoperative period, hypercoagulation states (e.g.,
protein C deficiency, malignant tumors), and a variety of
more rare diseases. In addition, thrombosis is a possible
cardiovascular surgery complication and may lead to unꢀ
stable functioning of implants including artificial heart
valves, arterial shunts, and vein filters. Thus, thromboꢀ
prophylaxis and treatment of thrombosis are key therapeuꢀ
tic goals in the treatment of cardiovascular diseases. Antiꢀ
thrombotic therapy involves the use of drugs that can be
divided into three groups with respect to their therapeutic
action, viz., anticoagulants inhibiting blood coagulation
by influencing the concentration of endogenous thrombin
or the enzymatic activity of thrombin; inhibitors of thromꢀ
bocyte aggregation in hemostasis and thrombosis; and fiꢀ
brinolytic agents that dissolve the existing thrombi.
A thrombus is formed from a fibrin clot produced as a
result of fibrin polymerization under the action of factor
XIIIa. In turn, fibrin is formed from fibrinogen under the
action of thrombin. Thrombin (factor IIa) is the lastꢀstage
enzyme in the complex cascade of biochemical reactions
in the blood coagulation system and executes important
procoagulant and anticoagulant functions.1 The use of
smallꢀmolecule direct thrombin inhibitors is considered
as a basic strategy of treatment of various types of thromꢀ
bosis.2 At present, anticoagulant therapy in clinical pracꢀ
tice involves the use of two smallꢀmolecule thrombin inꢀ
hibitors, viz., parenteral anticoagulant argatroban (1) and
dabigatran etexilate (2) for peroral administration. The
latter, also known as Pradaxa™, was approved by the Euꢀ
ropean Commission for prevention of thromboembolic
phenomena in elder patients after orthopedic surgery
(Spring 2008) and by the U.S. Food and Drug Adminisꢀ
tration for prevention of strokes in patients with atrial
fibrillation (October 2010).3
In organism, transformations of dabigatran etexilate
(2) under the action of enzymes lead to hydrolysis of two
functional groups (arrowed) and to the formation of dabigꢀ
atran, an actual thrombin inhibitor.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 671—679, April, 2011.
1066ꢀ5285/11/6004ꢀ685 © 2011 Springer Science+Business Media, Inc.

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Kudryavtsev et al.
Asp102, His57, and Ser195. A distinctive feature of thrombin
is an insertion loop (Tyr60A, Pro60B, Pro60C, Trp60D),
which is absent in other serine proteases and restricts acꢀ
cess of peptide substrates and inhibitors to the active site.
The thrombin active site comprises the catalytic site with
nucleophilic Ser195 residue, selective (S1) pocket, large
hydrophobic distal (D or S3) pocket, and a small proximal
(P or S2) pocket (Fig. 1). The substrateꢀbinding pockets
S1, S2, and S3 of the active site interact with the corresꢀ
ponding fragments P1, P2, and P3 of the ligand. The enꢀ
zyme is highly specific toward peptide hydrolysis after the
basic P1 residue (mainly arginine) in which the positively
charged guanidine group interacts with the Asp189 resiꢀ
due at the bottom of the S1 pocket. The S2 pocket is
formed by the side chains of the Tyr60A and Trp60D resiꢀ
dues localized within the insertion loop characteristic of
thrombin and mainly binds to hydrophobic amino acids in
the P2 position of the substrate. The S3 pocket containing
lipophilic and aromatic fragments interacts with the P3
aromatic groups of the ligand. Figure 1 schematically
shows the binding of a synthetic tricyclic inhibitor to the
thrombin active site4 according to Xꢀray data.
Usually, thrombin ligands contain guanidine (argaꢀ
troban) or amidine (dabigatran) fragment which forms
a rather strong bond with the Asp189 residue located in
the specific S1 pocket of thrombin active site and deterꢀ
mines the strength of the inhibitor—thrombin binding.
The molecule of human thrombin is comprised of the
Aꢀchain (36 amino acid residues) and Bꢀchain (259 amino
acid residues) linked by disulfide bridges. Similarly to other
serine proteases, thrombin includes a catalytic triad
Fig. 1. Binding of tricyclic inhibitor to thrombin active site according to Xꢀray data4: a schematic representation.

5ꢀPhenylproline thrombin inhibitors
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687
However, high basicity of guanidine and amidine groups
limit the bioavailability of such drugs. In the case of dabigꢀ
atran, the bioavailability is enhanced by conversion of the
inhibitor to a double prodrug, dabigatran etexilate. Recent
studies have demonstrated that the P1 fragment of the
inhibitor should not necessarily contain a functional group
of high basicity (e.g., guanidine or amidine) for efficient
inhibition of thrombin. Xꢀray studies of some inhibitors
revealed a deep insertion of the chlorophenyl fragment of
the inhibitor molecule into the S1 pocket of thrombin
active site. In addition to complementary hydrophobic
interactions, studies of tripeptidomimetic 3 (it inhibits
thrombin through unusual electrostatic interactions and
entropy factors) and some other compounds revealed locaꢀ
tion of the chlorine atom near the aromatic ring of the side
chain of the Tyr228 residue of thrombin.5
The contribution of such a Cl—π interaction to the
total energy of the inhibitor—enzyme interaction can be
as large as 8.4 kJ mol–1 6
, and may be considered as
a promising feature for an increase in the thrombin affinity
of the compounds being designed. The aim of the present
work was to design smallꢀmolecule thrombin inhibitors
with low basicity using the conformationally rigid cisꢀ5ꢀ
phenylproline scaffold and methods of computer simulaꢀ
tion of ligand—thrombin interactions.
The new molecular scaffold for the design of thrombin
inhibitors includes (Scheme 1) derivatives of 5ꢀarylpyrrolꢀ
idineꢀ2,4ꢀdicarboxylic acids 5 which can be obtained with
ease by 1,3ꢀdipolar cycloaddition of tertꢀbutyl acrylate to
azomethine ylides generated from imino esters 4.7
Conformational rigidity of the central pyrrolidine ring
and the exactly determined spatial arrangement of substitꢀ
uents7 in compounds 5 facilitates the simulation and reꢀ
duces the entropy factors of the interaction with biological
targets. Also, this molecular scaffold was chosen based on
the available data on the thrombinꢀinhibiting properties of
smallꢀmolecule compounds containing the structural fragꢀ
ment 5.8 To check the possibility of design of thrombin
inhibitors with low basicity, we planned to convert pyrrolꢀ
idineꢀ2,4ꢀdicarboxylic acid derivatives 5 to polysubstitutꢀ
ed pyrrolidines 6 and 7 (see Scheme 1) in which the
4ꢀchlorophenylsulfanyl fragment should be the P1 fragꢀ
Scheme 1
R1 = R2 = H (a); R1 = 4ꢀBr, R2 = H (b); R1 = H, R2 = Me (c); R1 = 2ꢀMeO, R2 = Me (d)
i. Et3N, MgSO4, CH2Cl2; ii. tertꢀbutyl acrylate, LiBr, Et3N, THF; iii. 1) ClCH2COCl, Et3N, THF; 2) 4ꢀClPhSH, DMF, K2CO3;
iv. HCl, ether.

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Kudryavtsev et al.
ment of the ligand and interact with the S1 pocket of
thrombin active site. The substituents R1 and R2 were
chosen taking into account the availability of the starting
reactants and the need to assess how structural factors
influence the biological activity.
AutoDock software does not include the Cl—π interacꢀ
tions6 in explicit form, and in our model the key role is
seemingly played by the hydrophobic interactions and the
shape match of the chlorophenyl fragment of the inhibitor
and the S1 pocket of thrombin active site.
A computer model for thrombin active site was conꢀ
structed using the Xꢀray data5 for a complex of thrombin
with tripeptidomimetic 3. To carry out molecular dockꢀ
ing, water molecules were removed from the protein strucꢀ
ture and hydrogen atoms were added to all amino acid
residues. The molecular structures of compounds 6 and 7
were optimized using the MMFF94 force field9 (within
the framework of the OpenBabel program10) and then preꢀ
processed by the AutoDockTools software.11 Since we
planned to synthesize some racemic derivatives (see
Scheme 1), calculations of the ligand—thrombin interacꢀ
tion were performed for both enantiomeric series. The
most energetically favorable conformations of the pyrroꢀ
lidines 6 and 7 are the enantiomeric structures labeled "I"
and "IV". Calculations were carried out taking into acꢀ
count not only the differences in the interaction of the
enantiomers with the chiral environment of the thrombin
active site, but also possible conformers labeled "II" and
"III", which differ from the structures I and IV in position
of the amide bond.
Correctness of the thrombin active site model thus conꢀ
structed was confirmed by the results of docking of the
structure of tripeptidomimetic 3. The calculated conforꢀ
mation of compound 3 and its arrangement in the thromꢀ
bin active site were in excellent agreement with the crysꢀ
tallographic data.5 According to our calculations, the enꢀ
ergy of the 3—thrombin interaction (AutoDock 4.2) is
–29.7 kJ mol–1 (Ki = 6250 nmol L–1), being 8.8 kJ mol–1
higher than the experimental value –38.5 kJ mol–1
(Ki = 180 140 nmol L–1).5 The parameterization of the
Ligandꢀprotein Cl—π interactions are typical of the
crystal structures;6 however, the known molecular modelꢀ
ing software does not include them in explicit form. In
this connection, the development of methods for taking
into account these interactions in molecular docking
and virtual screening software seems to be promising.
There are grounds to believe that if the structure of the
ligand—thrombin complex obtained from docking matches
the available crystal structure (PDB : 2ZC9), one can exꢀ
pect an additional stabilization energy of these binding
modes through the Cl—π interactions ignored by the
AutoDock4.2 software. For most conformers of the polyꢀ
substituted pyrrolidines 6 and 7 presented here, calculaꢀ
tions predict the highest stability for such modes in which
the 4ꢀchlorophenyl fragment of the smallꢀmolecule comꢀ
pound is within the S1 pocket of thrombin active site.
Also, often the coordinates of the 4ꢀchlorophenyl fragꢀ
ment of compounds 6 and 7 in the thrombin active site are
close to the crystallographic coordinates of the 3ꢀchloroꢀ
phenyl fragment of ligand 3 in the complex PDB : 2ZC9.
This can be explained by good shape match of the chloroꢀ
phenyl fragment and the rather narrow S1 pocket. Acꢀ
cording to docking results, the highest thrombin affinity
has structure 6c(III) (Fig. 2, a; Table 1) in which the
ligand shape almost perfectly matches that of the S1 pocket
and the 4ꢀchlorophenyl fragment is in the S1 pocket and
directed at the hydroxybenzyl side chain of the Tyr228
residue at the bottom of the pocket. The 5ꢀphenyl substitꢀ
uent in the central pyrrolidine fragment of 6c(III) is diꢀ
rected at the S2 pocket between the aromatic side chains
a
b
Fig. 2. The most energetically favorable interactions of enantiomers (2R,4R,5S)ꢀ6c (a) and (2S,4S,5R)ꢀ6c (b) with thrombin active site
predicted by molecular docking. Inhibitor 3 is shown in ballꢀandꢀstick view and the enantiomers of the Nꢀ(2ꢀ(4ꢀchloroꢀ
phenylthio)acetyl)ꢀ5ꢀphenylpyrrolidineꢀ2,4ꢀdicarboxylic acid derivative 6c are shown in thick stick view.

5ꢀPhenylproline thrombin inhibitors
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 4, April, 2011
689
of the Tyr60A and Trp60D residues (see Fig. 2, a). The
S atom of the 4ꢀchlorophenylsulfanyl fragment in conꢀ
former 6c(III) is near the Cys191—Cys220 disulfide bond
(5.6 and 4.5 Å, respectively), which can lead to energetiꢀ
cally favorable van der Waals interactions. According to
calculations, in most conformers mentioned above the
S3 pocket of the thrombin active site is filled with the
tertꢀbutoxycarbonyl group (for enantiomers (2R,4R,5S)ꢀ6)
or 5ꢀphenyl substituent (for (2S,4S,5R)ꢀ6 and (2S,4S,5R)ꢀ7).
Further optimization of the inhibitors may lead to addiꢀ

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Kudryavtsev et al.
tional strengthening of interactions in this region owing to
the larger contact area and to more specific interactions
with the Asn98 residue. According to calculations, some
interactions of thrombin with polysubstituted pyrrolidines
6 and 7 which are characterized by higher energies correꢀ
spond to localization of the 4ꢀchlorophenylsulfanyl fragꢀ
ment of the inhibitor in the distal S3 pocket of thrombin
active site. This interaction may be the starting point of
a molecular recognition process resulting in a more stable
complex with the filled S1 pocket. A postꢀdocking analyꢀ
sis of the geometries of the complexes of polysubstituted
pyrrolidines 6 and 7 with thrombin suggests that the cisꢀ5ꢀ
proline molecular scaffold allows permits efficient filling
of the S1 and S3 pockets of thrombin active site. The
interaction of inhibitors with the S2 pocket of thrombin
active site is an important factor for the selectivity of the
compounds being designed toward related trypsin proteasꢀ
es. Docking of structure 6b(III) involves a complementary
filling of the S2 pocket of the 5ꢀ(4ꢀbromophenyl) substituꢀ
ent, and a halogen bonding12 with polar heteroatoms of
the His57 and Lys60F residues becomes possible. Accordꢀ
ing to docking results for enantiomeric structure 6b(I), the
interaction involves the S1 and S3 pockets of thrombin
active site, whereas the S2 pocket remains unfilled. One
can assume that the design of thrombin inhibitors based
on the (2S,4S,5R)ꢀ5 molecular scaffold will result in comꢀ
pounds with low selectivity toward trypsin. Docking
showed that orthoꢀsubstitution of the 5ꢀphenyl fragment
Table 1. Inhibition of thrombin by Nꢀ[2ꢀ(4ꢀchlorophenylthio)acetyl]ꢀ5ꢀphenylpyrrolidineꢀ2,4ꢀdicarboxylic acid derivatives
in buffer solution and calculated thrombinꢀinhibiting abilitya
Comꢀ
pound
M
E/kJ mol–1
Ki/μmol L–1
Imax (%)
(c/μmol L–1
)
I
II
III
IV
I
II
III
IV
b
6a
6b
6c
6d
7a
7c
490.02
568.92
504.05
534.08
433.91
447.94
–23.7
–26.5
–24.3
–23.6
–26.7
–25.8
–25.2
–25.5
–25.7
–21.9
–24.3
–23.9
–25.0
–26.7
–27.2
–21.2
–24.0
–24.5
–27.2
–25.6
–21.4
–20.6
–26.0
–25.0
71
23
56
74
21
31
37
35
31
145
56
66
41
22
17
193
63
53
18
33
182
245
28
—
b
—
53 (180)
b
—
51 (4300)
27 (400)
41
a M is the molecular weight of inhibitor. E is the calculated energy of the interaction with thrombin conformers (I—IV). Ki is
the calculated constant of thrombin inhibition by the conformers I—IV; Imax is the maximum inhibition of thrombin in the
buffer solution.
b Not measured.

5ꢀPhenylproline thrombin inhibitors
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 4, April, 2011
691
Table 2. Selected bond lengths (d/Å) and bond angles (ω/deg) in
molecule of (2R,4R,5S)ꢀ6d
in (2R,4R,5S)ꢀ6d leads to violation of the shape correꢀ
spondence between the molecular fragments of the inꢀ
hibitor and the specific protein pockets. The stronger
binding was predicted for the enantiomeric structure 6d(I)
(74 μmol L–1) in which the 5ꢀ(2ꢀmethoxyphenyl) fragꢀ
ment molecule interacts with the S3 pocket of thrombin
active site. Thus, an important feature of our model for
thrombin active site and the docking protocol is differenꢀ
tiation of chiral smallꢀmolecule organic compounds.
To experimentally check the predicted interactions,
we synthesized novel 5ꢀphenylpyrrolidineꢀ2,4ꢀdicarboxyꢀ
lic acid derivatives 6 and 7 (see Scheme 1). The cycloaddiꢀ
tion of tertꢀbutyl acrylate to azomethine ylides generated
from imino esters 4 under the action of Lewis acids proꢀ
ceeds stereospecifically to give the racemic pyrrolidine 5
in which both carboxyl groups and the 5ꢀphenyl substituent
are on the one side of the pyrrolidine ring (see Scheme 1).7
Further synthetic modifications of the molecular scaffold
5 involving the introduction of the 2ꢀ(4ꢀchlorophenylꢀ
thio)acetyl substituent at the endocyclic atom of the pyrꢀ
rolidine ring and resulting in compounds 6 do not involve
stereogenic centers. Nevertheless, to exactly determine the
molecular structure of this class of compounds, we carried
out an Xꢀray study of crystals of the polysubstituted pyrꢀ
rolidine 6d (Fig. 3, Table 2). The crystal studied belonged
to the space group P212121 which includes only three symꢀ
metry elements, viz., the translational axes 21 passing along
three mutually perpendicular directions. Therefore, the
crystal structure of pyrrolidine 6d has no symmetry eleꢀ
ments that transform one molecular configuration to
a mirror molecular configuration. According to Xꢀray data,
the unit cell of compound 6d in the crystal contains only
one independent molecule, i.e., crystallization led to
Bond
d/Å
Angle
ω/deg
S(1)—C(21) 1.7574(16)
S(1)—C(10) 1.7968(14)
Cl(1)—C(24) 1.7431(16)
C(21)—S(1)—C(10) 103.01(7)
C(9)—N(1)—C(1)
C(9)—N(1)—C(4)
C(1)—N(1)—C(4)
C(6)—O(3)—C(7)
N(1)—C(1)—C(5)
N(1)—C(1)—C(6)
C(5)—C(1)—C(6)
N(1)—C(1)—C(2)
C(5)—C(1)—C(2)
C(6)—C(1)—C(2)
C(3)—C(2)—C(1)
C(8)—C(3)—C(2)
C(8)—C(3)—C(4)
C(2)—C(3)—C(4)
119.02(10)
126.68(11)
113.66(10)
114.95(11)
111.29(11)
111.03(10)
109.53(11)
102.04(10)
111.83(11)
110.96(10)
102.85(10)
116.88(11)
111.69(10)
104.42(10)
N(1)—C(9)
N(1)—C(1)
N(1)—C(4)
O(1)—C(9)
1.3495(17)
1.4751(16)
1.4814(15)
1.2266(16)
O(2)—C(12) 1.3615(17)
O(2)—C(27) 1.4204(17)
O(3)—C(6)
O(3)—C(7)
O(4)—C(6)
O(5)—C(8)
1.3340(17)
1.4511(16)
1.1984(16)
1.3317(16)
O(5)—C(17) 1.4885(16)
O(6)—C(8)
C(1)—C(5)
C(1)—C(6)
C(1)—C(2)
C(2)—C(3)
C(3)—C(8)
C(3)—C(4)
1.2062(16)
1.5303(18)
1.5314(17)
1.5400(18)
1.5305(17)
1.5168(18)
1.5593(18)
N(1)—C(4)—C(11) 113.24(10)
N(1)—C(4)—C(3) 101.76(10)
C(11)—C(4)—C(3) 112.61(10)
O(3)—C(6)—C(1)
O(5)—C(8)—C(3)
112.53(11)
111.63(11)
O(1)—C(9)—N(1) 121.10(12)
O(1)—C(9)—C(10) 122.24(12)
N(1)—C(9)—C(10) 116.64(11)
C(4)—C(11) 1.5220(17)
C(9)—C(10) 1.5257(19)
separation of enantiomers. The absolute configuration of
molecules 6d constituting the crystal under study was exꢀ
actly determined owing to the presence of heavy atoms
(sulfur and chlorine, see Fig. 3). Spontaneous separation
of enantiomers of the derivatives of Nꢀ(4ꢀchlorophenylꢀ
thio)acetylꢀ5ꢀphenylpyrrolidineꢀ2,4ꢀdicarboxylic acids 6
under crystallization will be used in further studies to obꢀ
tain enantiomerically pure thrombin inhibitors based on
the molecular scaffold 5. The inhibiting properties of the
compounds synthesized were tested taking hydrolysis of
a thrombin substrate in a buffer solution in the presence of
thrombin (see the rightmost column in Table 1)*. All the
three compounds studied (6c, 7a, 7c) appeared to be
thrombin inhibitors. The enzymatic activity of thrombin
is more than halved in the presence of polysubstituted
pyrrolidine 6c at a concentration of 180 μmol L–1
(see Table 1). This experimentally determined value agrees
well with the calculated values of the constant of thrombin
inhibition by compound 6c, which lies in the range
17—182 μmol L–1 (see Table 1). This suggests a high preꢀ
dictive power of the constructed model for thrombin acꢀ
tive site and the docking protocol.
C(19)
C(17)
C(20)
C(27)
C(13)
C(18)
O(5)
O(6)
C(8)
C(3)
O(2)
C(12)
C(11)
C(4)
C(14)
C(15)
C(2)
C(16)
C(1)
O(3)
C(5)
N(1)
C(10)
C(22)
C(9)
O(1)
C(23)
C(24)
C(6)
Cl(1)
O(4)
S(1)
C(21)
C(26)
* Tested at the Physical Biochemistry Laboratory (Hematology
Research Center, Ministry of Health and Social Development
of the Russian Federation, Moscow, Russia) headed by Prof.
F. I. Ataullakhanov.
C(7)
C(25)
Fig. 3. Molecular structure of compound 6d.

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Kudryavtsev et al.
128.19, 157.04, 172.59, 176.48. Found (%): C, 65.43; Н, 7.71;
N, 3.89. C19H27NO5. Calculated (%): C, 65.31; Н, 7.79; N, 4.01.
Synthesis of 5ꢀarylꢀ1ꢀ[2ꢀ(4ꢀchlorophenylthio)acetyl]pyrrolꢀ
idineꢀ2,4ꢀdicarboxylic acid esters 6aꢀd (general procedure). To
a solution of pyrrolidineꢀ2,4ꢀdicarboxylic acid diester 5 (20 mmol)
in THF (50 mL), Et3N (2.63 g, 26 mmol, 3.63 mL) was added in
argon atmosphere. The reaction mixture was cooled and a soluꢀ
tion of chloroacetyl chloride (2.71 g, 24 mmol, 1.91 mL) in THF
(20 mL) was added with stirring at such a rate that the temperaꢀ
ture was at most +5 °C. The mixture was stirred for 5 h at room
temperature and the Et3N·HCl residue was filtered off. The filꢀ
trate was concentrated in vacuo with a rotary evaporator. The
residue (Nꢀchloroacetyl derivative of compound 5) was dissolved
in DMF (120 mL) in argon atmosphere. To this solution, K2CO3
(2.76 g, 20 mmol) was added and a solution of 4ꢀchlorothiopheꢀ
nol (2.90 g, 20 mmol) in DMF (40 mL) was added on stirring.
The combined solution was stirred for 24 h at room temperature;
then, DMF was evaporated in vacuo. Water (60 mL) and 1 M
NaOH (60 mL) was added and extracted with EtOAc (3×60 mL).
The combined organic extracts were washed with saturated NaCl
solution (20 mL) and dried with Na2SO4. The drying agent was
filtered off and the filtrate was concentrated in vacuo with
a rotary evaporator. The residue was chromatographed on
60 silica gel (Lancaster, particle size 0.040—0.063 mm) using
CHCl3—MeOH (20 : 1) as eluent.
(2S*,4S*,5R*)ꢀ4ꢀtertꢀButoxycarbonylꢀ1ꢀ[2ꢀ(4ꢀchlorophenylꢀ
thio)acetyl]ꢀ2ꢀmethoxycarbonylꢀ5ꢀphenylpyrrolidine (6a). The
yield was 65%, colorless crystals, m.p. 135 °C. 1H NMR (CDCl3,
δ, J/Hz): 1.14 (s, 9 H, (CH3)3); 2.35—2.42 (m, 1 H, H(3));
2.48—2.58 (m, 1 H, H(3)); 3.37 (s, 2 H, CH2S); 3.40—3.48 (m, 1 H,
H(4)); 3.84 (s, 3 H, COOCH3); 4.41 (dd, 1 H, H(2), J = 11.4,
J = 6.8); 5.30 (d, 1 H, H(5), J = 9.1); 7.20—7.26 (m, 4 Нarom);
7.32—7.38 (m, 3 Нarom); 7.61 (d, 2 Нarom, J = 6.8). 13C NMR
(CDCl3, δ): 27.62 (3 C), 28.91, 36.98, 50.53, 52.46, 59.69, 63.29,
81.83, 128.34 (2 C), 128.64, 128.72 (2 C), 129.10 (2 C), 131.20
(2 C), 132.90, 133.39, 137.61, 167.53, 167.63, 171.87. Found (%):
C, 61.25; Н, 5.92; N, 2.99. C25H28ClNO5S. Calculated (%):
C, 61.28; Н, 5.76; N, 2.86.
(2S*,4S*,5R*)ꢀ5ꢀ(4ꢀBromophenyl)ꢀ4ꢀtertꢀbutoxycarbonylꢀ
1ꢀ[2ꢀ(4ꢀchlorophenylthio)acetyl]ꢀ2ꢀmethoxycarbonylpyrrolidine
(6b). The yield was 71%, colorless crystals, m.p. 124—125 °C.
1H MR (CDCl3, δ, J/Hz): 1.18 (s, 9 H, (CH3)3); 2.34—2.49
(m, 2 H, H(3)); 3.29—3.37 (m, 2 H, CH2S); 3.42 (ddd, 1 H,
H(4), J = 12.7, J = 8.6, J = 6.8); 3.83 (s, 3 H, COOCH3); 4.41
(dd, 1 H, H(2), J = 11.1, J = 7.1); 5.26 (d, 1 H, H(5), J = 8.6);
7.22—7.27 (m, 4 Нarom); 7.47—7.52 (m, 4 Нarom). 13C NMR
(CDCl3, δ): 27.70 (3 C), 28.81, 37.12, 50.46, 52.54, 59.64, 62.63,
82.13, 122.82, 129.17 (2 C), 130.00 (2 C), 131.61 (2 C), 131.82
(2 C), 133.04, 133.26, 136.69, 167.33, 167.56, 171.90. Found (%):
C, 52.89; Н, 4.82; N, 2.39. C25H27BrClNO5S. Calculated (%):
C, 52.78; Н, 4.78; N, 2.46.
(2S*,4S*,5R*)ꢀ4ꢀtertꢀButoxycarbonylꢀ1ꢀ[2ꢀ(4ꢀchlorophenylꢀ
thio)acetyl]ꢀ2ꢀmethoxycarbonylꢀ2ꢀmethylꢀ5ꢀphenylpyrrolidine
(6c). The yield was 72%, colorless crystals, m.p. 73—74 °C.
1H NMR (CDCl3, δ, J/Hz): 1.11 (s, 9 H, (CH3)3); 1.55 (s, 3 H,
2ꢀCH3); 1.99 (dd, 1 H, H(3), J = 12.8, J = 6.4); 2.77 (t, 1 H,
H(3), J = 13.3); 3.23 (s, 2 H, CH2S); 3.66 (ddd, 1 H, H(4),
J = 13.3, J = 9.3, J = 6.4); 3.82 (s, 3 H, COOCH3); 5.38 (d, 1 H,
H(5), J = 9.3); 7.22 (s, 4 Нarom); 7.30—7.39 (m, 3 Нarom); 7.64
(d, 2 Нarom, J = 6.8). 13C NMR (CDCl3, δ): 20.49, 27.56 (3 C),
36.98, 37.60, 49.45, 52.66, 63.22, 66.41, 81.75, 128.45 (2 C),
Summing up, a combination of a molecular docking
study and analysis of ligandꢀprotein interactions followed
by multistage targeted syntheses and biochemical studies
gave a series of novel nonbasic smallꢀmolecule organic
compounds based on the cisꢀ5ꢀphenylproline molecular
scaffold. These compounds exhibit inhibiting properties
toward thrombin, the key serine protease for hemostasis.
Experimental
Molecular docking was carried out individually for each
structure on the SKIF—MSU "Chebyshev" supercomputer clusꢀ
ter facilities using the AutoDock4.2 software.11
Compounds used in the experiments include tertꢀbutyl acrylꢀ
ate, aromatic aldehydes, chloroacetyl chloride, 4ꢀchlorothioꢀ
phenol, and silica gel (Lancaster). Prior to use, benzaldehyde
was purified by distillation in vacuo. The course of reactions and
the purity of the compounds was monitored using Sorbfil PTSKhꢀ
AFꢀAꢀUF TLC plates with CHCl3—MeOH (10 : 1) as eluent.
1Н and 13C NMR spectra were recorded on a Bruker Avance
400 instrument (operating frequency is 400 MHz for 1H and
100 MHz for 13C) at T = 303 K in solutions in DMSOꢀd6 or
CDCl3 using the residual solvent signals as internal standards.
An Xꢀray study of compound 6d was carried out on a Bruker
SMART APEX II automated diffractometer at 150.0(2) K
(MoꢀKα radiation, λ = 0.71073 Å, graphite monochromator).
The structure was solved by the direct methods and refined using
the fullꢀmatrix leastꢀsquares method with respect to F 2 in
the anisotropic approximations for all nonꢀhydrogen atoms.13
All hydrogen atoms were located objectively and refined isoꢀ
tropically.
The effect of compounds 6c, 7a, and 7c on the enzymatic
activity of thrombin was studied photometrically.14
Diesters of pyrrolidineꢀ2,4ꢀdicarboxylic acids 5a,15 5b,8 and
5c (see Ref. 7) were synthesized from imino esters 4a—c followꢀ
ing the known procedures and identified spectroscopically. Imino
ester 4d was synthesized by a known procedure.16 Compound 5d
was obtained from imino ester 4d and tertꢀbutyl acrylate, as deꢀ
scribed for structural analogs.7
Methyl 2ꢀ(2ꢀmethoxybenzilidenamino)propanoate (4d) was
synthesized from DLꢀalanine methyl ester hydrochloride and
2ꢀmethoxybenzaldehyde and used in the subsequent stage withꢀ
out additional purification. The yield was 89%. An oily subꢀ
stance. 1H NMR (DMSOꢀd6, δ, J/Hz): 1.37 (d, 3 Н, CН3,
J = 6.8); 3.64 (s, 3 Н, OCН3); 3.86 (s, 3 Н, COOCН3); 4.24 (q, 1 Н,
H(2), J = 6.8); 6.99 (t, 1 Нarom, J = 7.5); 7.11 (d, 1 Нarom
,
J = 8.0); 7.44—7.49 (m, 1 Нarom); 7.82 (d, 1 Нarom, J = 7.5); 8.70
(s, 1 Н, CH=).
(2S*,4S*,5R*)ꢀ4ꢀtertꢀButoxycarbonylꢀ2ꢀmethoxycarbonylꢀ
5ꢀ(2ꢀmethoxyphenyl)ꢀ2ꢀmethylpyrrolidine (5d). Obtained from
compound 4d and tertꢀbutyl acrylate. The yield was 81%. Colorꢀ
1
less crystals, m.p. 73—74 °C. H NMR (CDCl3, δ, J/Hz): 0.98
(s, 9 H, (CH3)3); 1.50 (s, 3 H, 2ꢀCH3); 2.06 (dd, 1 H, H(3),
J = 13.6, J = 8.1); 2.60 (dd, 1 H, H(3), J = 13.6, J = 3.5); 3.43—3.48
(m, 1 H, H(4)); 3.81 (s, 3 H, OCH3); 3.85 (s, 3 H, COOCH3);
4.71 (d, 1 H, H(5), J = 7.1); 6.85 (d, 1 Нarom, J = 8.1); 6.92
(d, 1 Нarom, J = 7.5); 7.20—7.25 (m, 1 Нarom); 7.32 (d, 1 Нarom
,
J = 7.5). 13C NMR (CDCl3, δ): 27.03, 27.46 (3 C), 41.65, 49.07,
52.44, 55.18, 59.72, 65.10, 79.90, 109.80, 120.29, 126.43, 126.83,

5ꢀPhenylproline thrombin inhibitors
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 4, April, 2011
693
128.61 (2 C), 128.76 (2 C), 129.06 (2 C), 131.44 (2 C), 133.10,
138.16, 166.61, 167.88, 173.47. Found (%): C, 62.25; Н, 5.93;
N, 2.98. C26H30ClNO5S. Calculated (%): C, 61.96; Н, 6.00;
N, 2.78.
(2S*,4S*,5R*)ꢀ4ꢀtertꢀButoxycarbonylꢀ1ꢀ[2ꢀ(4ꢀchlorophenylꢀ
thio)acetyl]ꢀ2ꢀmethoxycarbonylꢀ5ꢀ(2ꢀmethoxyphenyl)ꢀ2ꢀmethꢀ
ylpyrrolidine (6d). The yield was 68%, colorless crystals, m.p.
153—154 °C. 1H NMR (CDCl3, δ, J/Hz): 1.08 (s, 9 H, (CH3)3);
1.56 (s, 3 H, 2ꢀCH3); 2.03 (dd, 1 H, H(3), J = 12.8, J = 6.7); 2.82
(t, 1 H, H(3), J = 13.1); 3.20 (d, 1 H, CH2S, J = 14.6); 3.31
(d, 1 H, CH2S, J = 14.6); 3.66 (ddd, 1 H, H(4), J = 13.1, J = 9.5,
J = 6.7); 3.80 (s, 3 H, OCH3); 3.84 (s, 3 H, COOCH3); 6.00
(2R*,3S*,5S*)ꢀ1ꢀ[2ꢀ(4ꢀChlorophenylthio)acetyl]ꢀ5ꢀ(methꢀ
oxycarbonyl)ꢀ5ꢀmethylꢀ2ꢀphenylpyrrolidineꢀ3ꢀcarboxylic acid
(7c). The yield was 94%, colorless crystals, m.p. 108—109 °C.
1H MR (DMSOꢀd6, δ, J/Hz): 1.48 (s, 3 H, 2ꢀCH3); 1.95 (dd, 1 H,
H(4), J = 12.4, J = 6.2); 2.46—2.53 (m, 1 H, H(4)); 3.09 (d, 1 H,
CH2S, J = 15.7); 3.69 (s, 3 H, COOCH3); 3.74 (d, 1 H, CH2S,
J = 15.7); 3.81 (ddd, 1 H, H(3), J = 13.6, J = 9.0, J = 6.2); 5.50
(d, 1 H, H(2), J = 9.0); 7.02—7.06 (m, 2 Нarom); 7.25—7.29
(m, 3 Нarom); 7.33—7.37 (m, 2 Нarom); 7.64—7.66 (m, 2 Нarom).
13C NMR (DMSOꢀd6, δ): 20.62, 36.85, 37.19, 48.88, 52.70,
62.99, 66.28, 128.31, 128.45 (2 C), 128.69 (2 C), 129.15 (2 C),
129.92 (2 C), 130.91, 135.11, 139.53, 165.90, 171.04, 173.71.
Found (%): C, 59.17; Н, 4.92; N, 3.30. C22H22ClNO5S. Calcuꢀ
lated (%): C, 58.99; Н, 4.95; N, 3.13.
(d, 1 H, H(5), J = 9.5); 6.84 (d, 1 Нarom, J = 8.3); 7.02 (d, 1 Нarom
,
J = 7.5); 7.17—7.21 (m, 4 Нarom); 7.26—7.30 (m, 1 Нarom); 7.96
(dd, 1 Нarom, J = 7.5, J = 1.6). 13C NMR (CDCl3, δ): 20.50,
27.38 (3 C), 37.17, 38.03, 48.80, 52.60, 54.82, 55.33, 66.16, 81.12,
109.92, 121.41, 126.99, 128.91 (2 C), 129.52 (2 C), 130.90 (2 C),
132.50, 133.90, 156.37, 166.59, 168.22, 173.50. Found (%):
C, 61.01; Н, 5.91; N, 2.89. C27H32ClNO6S. Calculated (%):
C, 60.72; Н, 6.04; N, 2.62.
This work was financially supported by the Russian
Foundation for Basic Research (Project Nos 11ꢀ03ꢀ00630ꢀa,
11ꢀ03ꢀ91375ꢀST_a and 08ꢀ04ꢀ01800ꢀa).
References
Xꢀray study of compound 6d.
A crystal (dimensions
0.25×0.20×0.15 mm) was grown by slow evaporation of solvents
from a dilute solution of 6d in a hexane—AcOEt (1 : 1) mixture.
Crystals of (2R,4R,5S)ꢀ6d (C27H32Cl1N1O6S1, M = 534.05)
are orthorhombic, space group P212121, a = 10.1984(5), b =
= 13.1163(6), c = 19.9614(9) Å, V = 2670.1(2) Å3, Z = 4,
dcalc = 1.328 g cm–3, μ(MoꢀKα) = 0.263 mm–1, and F(000) =
= 1128. The intensities of 27808 reflections (6549 independent
reflections, Rint = 0.0310) were measured using ωꢀscan with
a 0.5° increment in the interval 2.24 < θ < 28.00° (–13 ≤ h ≤ 13,
–17 ≤ k ≤ 17, –26 ≤ l ≤ 26). The final reliability factor was
R1 = 0.0289, Rw = 0.0732 for 6094 reflections with I > 2σ(I) and
453 refinement parameters. The absolute structure parameter
is –0.01(4).
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Synthesis of 1ꢀ[2ꢀ(4ꢀchlorophenylthio)acetyl]ꢀ5ꢀ(methoxyꢀ
carbonyl)ꢀ2ꢀphenylpyrrolidineꢀ3ꢀcarboxylic acids 7a,c (general
procedure). Through a solution of 4ꢀtertꢀbutyl ester of substitutꢀ
ed pyrrolidineꢀ2,4ꢀdicarboxylic acid 6 (2.0 mmol) in anhydrous
CHCl3 (30 mL), gaseous HCl was passed for 2 h and the solution
was stirred at room temperature for 24 h. Then, 5 mL of saturatꢀ
ed NaHCO3 solution was added to pН 5, the organic phase of
the washed with saturated NaCl solution, dried with Na2SO4,
and the solvents were evaporated in vacuo. The product was
purified by recrystallization from Et2O—MeOH, 20 : 1.
(2R*,3S*,5S*)ꢀ1ꢀ[2ꢀ(4ꢀChlorophenylthio)acetyl]ꢀ5ꢀ(methꢀ
oxycarbonyl)ꢀ2ꢀphenylpyrrolidineꢀ3ꢀcarboxylic acid (7a). The
yield was 87%, colorless crystals, m.p. 189—190 °C. 1H NMR
(DMSOꢀd6, δ, J/Hz): 2.20 (q, 1 H, H(4), J = 12.5); 2.34 (dt, 1 H,
H(4), J = 12.5, J = 6.5); 3.23 (d, 1 H, CH2S, J = 15.9); 3.62
(ddd, 1 H, H(3), J = 12.5, J = 8.9, J = 6.5); 3.71 (s, 3 H,
COOCH3); 3.96 (d, 1 H, CH2S, J = 15.9); 4.36 (dd, 1 H, H(5),
J = 11.3, J = 6.8); 5.34 (d, 1 H, H(2), J = 8.9); 7.08 (d, 1 H,
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44, 384 [Russ. J. Org. Chem. (Engl. Transl.), 2008, 44, 378].
2 Нarom, J = 8.6); 7.26—7.32 (m, 3 Нarom); 7.37 (t, 2 Нarom
,
J = 7.5); 7.64 (d, 2 Нarom, J = 7.5); 12.48 (br.s, COOH).
13C NMR (DMSOꢀd6, δ): 29.00, 36.41, 49.40, 52.49, 59.64,
62.68, 128.28 (2 C), 128.43, 128.70 (2 C), 129.17 (2 C), 129.79
(2 C), 130.86, 135.29, 138.99, 167.11, 170.63, 172.29. Found (%):
C, 58.05; Н, 4.71; N, 3.02. C21H20ClNO5S. Calculated (%):
C, 58.13; Н, 4.65; N, 3.23.
Received March 15, 2011;
in revised form April 11, 2011