Design, synthesis, and anti-thrombotic evaluation of some novel fluorinated thrombin inhibitor derivatives

Article abstract of DOI:10.1002/ardp.201400460

Computer-aided simulation was used to design and synthesize nine novel fluorinated thrombin inhibitor derivatives. These compounds were confirmed by spectral analyses (¹H NMR, ¹³C NMR, and FT-ICR-MS). Their inhibitory activities agai

Full text of DOI:10.1002/ardp.201400460

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Archiv der Pharmazie
Full Paper
Design, Synthesis, and Anti-Thrombotic Evaluation of Some Novel
Fluorinated Thrombin Inhibitor Derivatives
Haifeng Chen and Yujie Ren
College of Chemistry and Environmental Engineering, Shanghai Institute of Technology, Shanghai, China
Computer-aided simulation was used to design and synthesize nine novel fluorinated thrombin inhibitor
derivatives. These compounds were confirmed by spectral analyses (1H NMR, 13C NMR, and FT-ICR-MS).
Their inhibitory activities against thrombin enzyme were evaluated by chromogenic assay. All the
derivatives demonstrated thrombin inhibitory activity in vitro. Five of these compounds exerted more
potent effects against thrombin enzyme compared with the reference drug argatroban. Compound 3-(2-
(((4-carbamimidoylphenyl)amino)methyl)-1-ethyl-N-(2-fluoro-phenyl)-1H-benzo[d]imidazole-5-carbox-
amido)propanoic acid (IC50 ¼ 3.52 ꢀ 0.32 nmol/L) was a more potent inhibitor of thrombosis than
argatroban (IC50 ¼ 9.46 ꢀ 0.92 nmol/L).
Keywords: Argatroban / Chromogenic assay / Docking / Fluorine / Thrombin inhibitor
Received: December 16, 2014; Revised: February 21, 2015; Accepted: February 27, 2015
DOI 10.1002/ardp.201400460
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
oral bioavailability and the probability of hemorrhage when
used in large doses.
Introduction
Thrombosis occurs in the arterial or venous circulation; this
condition has become one of the medical highlights. The
pathophysiology of arterial thrombosis differs from that of
venous thrombosis; this difference is reflected by the different
ways by which these conditions are treated. Arterial
thrombosis is generally treated with drugs that target
platelets, whereas venous thrombosis is treated with drugs
that target protein of the coagulation cascade [1].
A variety of thrombin inhibitory drugs have been listed on
the stock market recently, including rivaroxaban (Bayer) [2–4],
argatroban (Mitsubishi) [5], and dabigatran etexilate (Boeh-
ringer Ingelheim) [6–9] (Fig. 1). In particular, dabigatran
etexilate was the first oral direct thrombin inhibitor, which
broke the earlier 50-year monopoly of the anti-
coagulant drug market. However, the new series of anti-
coagulants have several disadvantages, including their low
To date, fluorinated drugs account for a considerable
proportion of the drugs used in clinical treatment [10–15].
With the introduction of fluorine atoms or fluoride groups,
the physical and chemical properties of drug molecules could
be changed, which, in turn, alters the pharmacokinetic
properties of drug molecules. For instance, the distribution
of a drug in the target tissue could be improved, which
consequently increases its bioavailability. The changes in the
structural conformation of compounds may enhance their
capacity to bind with ligands and target proteins as well as
their selectivity for target proteins. Fluorination could block
the metabolic pathways of drugs in a particular site, change
the rate of their metabolism, prolong their effects in the body,
and improve their metabolic stability, among others. Further-
more, the introduction of fluorine in drug molecules may
enhance the membrane permeability [16–19] and the specific
loci formed with target proteins during hydrophobic interac-
tion [20]. Since the introduction of 5-fluorouracil, the addition
of fluorine atoms or fluoride groups into drug molecules has
gradually become an important research strategy in drug
design and structure transformation.
Correspondence: Prof. Yujie Ren, College of Chemistry and
Environmental Engineering, Shanghai Institute of Technology,
No. 100 Haiquan Road, Fengxian District, 201400 Shanghai, China.
E-mail: clab@sit.edu.cn
Therefore, to combine the benefits of anti-thrombin drugs
and fluorine atoms during treatment, fluoride groups were
introduced into thrombin inhibitory drugs to modify their
Fax: þ021-6087-7210
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
408

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Fluorinated Thrombin Inhibitors
Archiv der Pharmazie
thereby providing more efficient and safer novel anti-
clotting drug candidate compounds.
The research and development of novel compounds has
become more efficient with the development of computer-
aided simulation and design. In the present study, we
designed and synthesized a series novel of fluoride anti-
thrombin derivatives using dabigatran etexilate as the
reference molecule, studied their anti-thrombin activities,
and validated their activity using molecular modeling
approaches (Fig. 2). Initially, docking experiments with
designed structures at the active site (PDB entry code:
4JN2) [21] were performed in silico to assess their binding
(docking scores) with respect to that of dabigatran. Dabiga-
tran etexilate has
a basic benzimidazole ring and a
benzamidine group that is interlinked using amino linker.
However, a 3-(pyridyl-2-amino)propionic acid ethyl ester is
connected to the benzimidazole ring through carboxide.
According to the literature [22], 3-(pyridyl-2-amino)propionic
acid ethyl ester and its analogs account for its bioactivity.
Therefore, we replaced the pyridine ring with a benzene ring
and introduced various fluoride groups at different sites
(Fig. 3). The effects of the introduction of fluorine atoms on
the anti-coagulant activity were determined.
Figure 1. Structures of commercial thrombin inhibitory drugs.
structures. This modification was expected to generate a
series of fluoride-modified anti-thrombin derivatives, which
could maximize the selected characteristics of fluorine atoms
and improve the shortcomings of thrombin inhibitory drugs,
The designed compounds were synthesized and evaluated
for their capacity to inhibit thrombin activity. Furthermore,
Figure 2. The outline of the various experimental and modeling techniques used in the study.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
409

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
H. Chen and Y. Ren
Archiv der Pharmazie
molecular modeling studies were conducted by molecular
docking approaches to understand the inhibitory mechanism.
Results and discussion
Chemistry
Given the high pharmacological activity profile of thrombin
inhibitory drugs and fluorinated compounds, we designed
and synthesized fluorinated thrombin inhibitor derivatives
through the synthetic process depicted in Scheme 1. The main
intermediate ethyl 3-((fluorinated-phenyl)amino)propanoate
(3) was synthesized from fluorinated anilines (1) in excess
ethyl acrylate with the catalyst trifluoromethanesulfonic acid
at 100°C. The synthesis of 4-(ethylamino)-3-nitrobenzoic acid
(6) was achieved in good yield by the reaction between 4-
chloro-3-nitrobenzoic acid and ethylamine in water (60%)
without
a solvent. The intermediate 4-(ethylamino)-3-
nitrobenzoyl chloride (7) was synthesized from compound 6
and thionyl chloride in dichloromethane using N,N-
dimethylformamide as a catalyst. The important intermediate
ethyl 3-(N-(fluorinated-phenyl)-4-(ethylamino)-3-nitrobenza-
mido)propanoate (8) was synthesized from the intermediate
Figure 3. Design strategy of fluorinated thrombin inhibitor
derivaties.
Scheme 1. Reagents, conditions, and range of yields: a) trifluoromethanesulfonic acid, yields: 38–98%; b) thionyl chloride, N,N-
dimethylformamide, dichloromethane, yields: 100%; c) triethylamine, dichloromethane, yields: 24–79%; d) Zn powder, ammonium
chloride, tetrahydrofuran/water, yields: 59–98%; e) (i) 2-((4-cyanophenyl)amino)acetic acid, EDCI, HoBt, tetrahydrofuran/N,N-
dimethylformamide, (ii) acetic acid, ammonium hydroxide, yields: 46–89%; f) (i) hydroxylamine hydrochloride, triethylamine,
ethanol, (ii) Pd/C, ammonium formate, acetic acid, nitrogen, yields: 54–88%; g) sodium hydroxide, ethanol/water, yields: 42–99%.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
410

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Fluorinated Thrombin Inhibitors
Archiv der Pharmazie
Table 1. Thrombin inhibitor activities of the synthesized compounds.
No.
Compound
Rf
IC50 (Mean ꢀ SD)a) (nmol/L)
1
2
3
4
5
6
7
8
9
10
12a
12b
12c
12d
12e
12f
12g
12h
2-F
3-F
4-F
3,4-F2
3,5-F2
2-CF3
3-CF3
4-CF3
4-OCF3
3.52 ꢀ 0.32
7.49 ꢀ 0.7
4.26c ꢀ 0.3
9.55 ꢀ 0.81
7.9 ꢀ 0.56
6.21 ꢀ 0.62
26.3 ꢀ 0.33
199 ꢀ 16.48
117.7 ꢀ 10.77
9.46 ꢀ 0.92
12i
Argatrobanb)
Bold face indicates significant anti-thrombin inhibitory activity.
a)Values are expressed as the mean ꢀ standard error of the mean of six experiments.
b)Reference drug.
compounds 3 and 7 in dichloromethane using triethylamine
as the base. The main intermediate ethyl 3-(3-amino-N-
(fluorinated-phenyl)-4-(ethylamino)benzamido)propanoate
(9) was obtained by the reductant of zinc powder from
compound 8 in tetrahydrofuran with water as the solvent. The
main intermediate compound 10 was obtained from com-
pounds 9a–i by condensation reactions. However, the most
important intermediate compound 11 was achieved from
intermediate 10 through the Pinner reaction. The target
compound 12 was obtained from compounds 11a–i by
hydrolysis.
the case of 12f–h. Compound 12d showed moderate thrombin
inhibitory potency when (–F2) was attached at the 3,4-position;
the inhibitory activity increased when this group was found in
the 3,5-position (12e; 7.9 nmol/L). Compound 12i has a strong
electron donating (–OCF3) group at the para position and a
remarkably decreased IC50 value of 117.7 nmol/L.
Molecular modeling
The molecular modeling studies were performed using the
molecular modeling package SYBYL-X2.0 (Tripos, Inc., USA).
The 3D structures of all compounds were constructed with the
Sketch Molecule module. Energy minimization was per-
formed by the Powell gradient algorithm with the Tripos
The structures of products 12a–i were determined from
their 1H NMR and 13C NMR spectra as well as by FT-ICR-MS.
€
force field [23] and GasteigerꢁHuckel charge [24]. The
Anti-thrombin activity in vitro
maximum number of iterations for the minimization was
set to 10000. The minimization was terminated when the
The potency of the anti-thrombin activity of the synthesized
fluorinated thrombin inhibitor analogs was measured by
chromogenic assay [6] to determine potential thrombin
inhibitors of interest. Argatroban was used as the reference
compound for the comparison of the results. The thrombin
inhibitory activities of thesynthesized compounds areshown in
Table 1. Most of the designed compounds clearly exhibited
moderate inhibitory activity toward thrombin. Compounds
12a–f were found to be more potent than argatroban for
thrombin inhibition. Their IC50 values differed because of the
different attached substituents and different positions of the
phenyl ring in 12a–i. The substituent fluorinated thrombin
inhibitor derivatives, including 12a–f, showed significant
activity with IC50 values of 3.52, 7.49, 4.26, 7.9, and
6.21 nmol/L, respectively. Among the newly synthesized deriv-
atives, compound 12a (3.52 nmol/L) showed the most potent
thrombin inhibition; this compound had a (–F) group at the
ortho position. When this (–F) group was attached at the meta
and para positions, the thrombin inhibition activity was
decreased, as observed in 12b and 12c with IC50 values of
7.49 and 4.26 nmol/L, respectively. However, when (–CF3) was
placed in the ortho, meta, and para positions, the thrombin
inhibitory activity decreased to 6.21, 26.3, and 199nmol/L, as in
̊
energy gradient convergence criterion of 0.005 kcal/mol ꢂ A
was reached [25].
Molecular docking
To study the binding mode of the inhibitors in the active site
of thrombin protein, molecular docking was performed with
the Surflex-Dock module of SYBYL-X 2.0. The crystal structure
of the thrombin protein receptor complex was retrieved from
the RCSB Protein Data Bank (PDB entry code: 4JN2). The
ligands were docked to the binding site of the corresponding
proteins by an empirical scoring function and a patented
search engine in Surflex-Dock. Before the docking process,
one natural ligand was extracted while the other natural
ligands and water molecules were removed from the crystal
structure. Subsequently, the protein was prepared with the
Biopolymer module implemented in Sybyl. The polar hydro-
€
gen atoms were added and the GasteigerꢁHuckel charges
were assigned to the protein atoms. Automated docking was
applied in the present work. Other parameters were
established by default in the software. The Surflex-
Dock total scores, which were expressed in –log10(Kd) units
to represent binding affinities, were applied to estimate the
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
411

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
H. Chen and Y. Ren
Archiv der Pharmazie
Figure 4. a) The comparison of dabigatran (red) in the crystal structure (PDB code 4JN2) and compound 12a (cyan) in the docked
complex by superimposing the coordinates of protein together. The ribbon is color-coded by lipophilic potential. b) A molecular
surface of the active site of inhibitor–thrombin complexes depicted by transparent.
ligandꢁreceptor interactions of the newly designed
successfully docked in the binding cavity (Fig. 5a). This
compound shares similar binding mode with other
molecules.
a
compounds, as reported in the literature. The benzimidazole
ring forms p–p stacking interactions with the conserved
Tyr32, which is crucial for the binding of dabigatran
derivatives with the thrombin protein. The central template
is bound to thrombin by a hydrophobic interaction with the
key residue Tyr40. The substituted benzene ring is positioned
between Tyr104, Tyr107, and Asp108 in the same pocket. The
carboxyl group and benzimidazole ring of compound 12a
exhibit van der Waals interaction with the key residues:
Tyr40, His99, Thr98, and Phe100. Electrostatic interactions
were observed between the amidino and carboxyl groups
and the following important amino acids: Trp48, Phe110,
Tyr107, and His102. Two hydrogen bonds were formed
between the amidino group and the Asp36 and His102
residues, while two more hydrogen bonds were formed
between the O and H atoms of the hydroxyl group and the
Trp53 and Tyr101 residues, as shown in Fig. 5b. The observed
Docking analysis
Dabigatran was re-docked to validate the docking reliability,
as shown in Fig. 4. The binding mode of the re-docked and co-
crystallized compound 12a is almost similar in the active site of
the thrombin receptor (PDB entry code: 4JN2) (Table 2). For
example, the H atom of acetamidine interacts with the
carbonyl group of His102 and the O atom of Trp53 by water-
mediated hydrogen bonds. The O atom of the carbonyl group
interacts with the hydroxyl group of Ser57. The benzimid-
azole rings of both compounds are bound to thrombin by a
hydrophobic interaction in the same pocket. The pyridine ring
is also positioned in the same pocket, except for the slight
rotation of bonds. After validating the docking reliability, the
compound with the highest activity was selected for exploring
the probable binding conformation.
The interaction of compound 12a with the binding site of
the thrombin receptor is shown in Fig. 5. Compound 12a was
̊
hydrogen bond distances were 1.94 A (Asp36–O…H–N–),
Table 2. Binding scores, amino acid interactions, and hydrogen bond length of the docked compounds on the active site
of 4JN2.
Compound no.
Total score
Amino acid residue
H bond length A
12a
12b
12c
12d
12e
12f
12g
10.0853
11.2988
10.2847
10.4239
10.0810
11.0723
9.0756
Asp36, His102, Trp53, Tyr101
Asp36, His102, Trp53, Tyr101
Asp36, His102, Trp53, Tyr104
Asp36, His102, Ser57
1.94, 2.18, 2.06, 2.02
1.92, 2.17, 2.06, 2.06
2.02, 2.09, 2.16, 1.91
1.87, 1.97, 2.05
2.09, 2.22, 1.92, 2.60
2.04, 2.11, 2.82, 2.04, 2.04
2.04, 2.26
Asp36, His102, Trp53, Ser57
Asp36, His102, Tyr32, Trp53,Tyr101
Asp36, His102
12h
12i
Dabigatran
11.7752
10.1096
11.0146
Asp36, His102, Trp53
Asp36, His102, Phe100
1.97, 2.29, 1.90
2.01, 2.13, 2.16
2.01, 1.77
Asp36, Tyr104
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
412

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Fluorinated Thrombin Inhibitors
Archiv der Pharmazie
Figure 5. a) A surface of compound 12a in complex with thrombin receptor. The lipophilic potential is mapped on the protein
surface. b) Docking result of the compound 12a into the binding site of thrombin protease. Hydrogen bonds are shown as pink
̊
dashed lines, with distance unit of A. The inhibitor (cyan) and the important residues (atom type) are shown as stick model.
̊
̊
2.18 A (His102–C–O…H–N–), 2.02 A (Tyr101–O–H…H–N),
reference. Chemical shifts are reported in d scale (ppm). Mass
spectra of the synthesized compounds were recorded by a
Bruker FT-ICR-MS.
̊
and 2.06 A (Trp53–N–H…O–H). These hydrogen bond inter-
actions may have important roles in the interaction of
dabigatran derivatives with the residues in the active site of
thrombin.
General procedure for the synthesis of ethyl 3-
((fluorinated-phenyl)amino)propanoates 3a–i
To a solution of commercial fluorinated benzenamine (1a–i)
(0.05 mol) and ethyl acrylate (0.09 mol) without solvent,
trifluoromethanesulfonic acid (0.005 mol) was added. The
resulting mixture was heated to 100°C for 24 h. The progress
of the reaction was monitored by thin layer chromatography
using petroleum ether/dichloromethane (2:1) as solvent
system. The crude mixture was purified by silica gel column
chromatography eluting 5:1 petroleum ether/dichlorome-
thane to give the immediate compounds 3a–g.
Conclusion
A novel series of fluorinated thrombin inhibitor derivatives
were designed and synthesized on the basis of a docking study
at the 4JN2 site. The synthesized compounds were character-
ized and evaluated in vitro for thrombin inhibitory activity.
The present investigation demonstrated the significant anti-
thrombin activity in some of these novel compounds, which
was comparable to argatroban. The results suggested that
these novel compounds could represent alternative lead
compounds for the synthesis of alternative thrombin
inhibitors.
Ethyl 3-((2-fluorophenyl)amino)propanoate (3a)
Red liquid; yield: 86%; 1H NMR (500 MHz, chloroform-d) d 6.91
(t, J ¼ 8.7 Hz, 2H), 6.61–6.55 (m, 2H), 4.18 (q, J ¼ 7.2 Hz, 2H),
3.95 (s, 1H), 3.42 (t, J ¼ 6.3 Hz, 2H), 2.62 (t, J ¼ 6.3 Hz, 2H), 1.29
(t, J ¼ 7.2 Hz, 3H). FT-MS m/z Calcd. for C11H14FNO2 ([MþH]þ):
212.10813. Found: 212.10697.
Experimental
General
Ethyl 3-((3-fluorophenyl)amino)propanoate (3b)
All the chemicals and solvents were purchased from Darui and
Titan Corporation and used without further purification.
Melting points were measured on WRS-1B and were
uncorrected. NMR spectra were carried out using a Bruker
Avance 500 MHz NMR spectrometer, using TMS as internal
Brown-yellow liquid; yield: 92%; 1H NMR (500 MHz, chloro-
form-d) d 6.91 (t, J ¼ 8.7 Hz, 2H), 6.61–6.55 (m, 2H), 4.18 (q,
J ¼ 7.2 Hz, 2H), 3.95 (s, 1H), 3.42 (t, J ¼ 6.3 Hz, 2H), 2.62 (t,
J ¼ 6.3 Hz, 2H), 1.29 (t, J ¼ 7.2 Hz, 3H). FT-MS m/z Calcd. for
C
11H14FNO2 ([MþH]þ): 212.10813. Found: 212.10819.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
413

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
H. Chen and Y. Ren
Archiv der Pharmazie
Ethyl 3-((4-fluorophenyl)amino)propanoate (3c)
Red liquid; yield: 98%; 1H NMR (500 MHz, chloroform-d) d 6.91
(t, J ¼ 8.7 Hz, 2H), 6.61–6.55 (m, 2H), 4.18 (q, J ¼ 7.2 Hz, 2H),
3.95 (s, 1H), 3.42 (t, J ¼ 6.3 Hz, 2H), 2.62 (t, J ¼ 6.3 Hz, 2H), 1.29
(t, J ¼ 7.2 Hz, 3H). FT-MS m/z Calcd. for C11H14FNO2 ([MþH]þ):
212.10813. Found: 212.10811.
80°C for 5 h. The solution was adjusted to pH 4–5 with acetic
acid. The precipitations of yellow solids were filtered to get
products 5 (19.6 g, 100%).
Synthetic procedure for 4-(ethylamino)-3-
nitrobenzoyl chloride (7)
A 250 mL round-bottom flask was charged with 4-(ethyl-
amino)-3-nitrobenzoic acid (9.0 g, 0.046 mol). Thirty milliliters
of dichloromethane and five drops of N,N-dimethyl-
formamide were added in one portion. The mixture was
stirred at room temperature (30°C). Thionyl chloride (20 mL,
0.23 mol) was added dropwise via addition funnel. The
reaction solution was stirred at room temperature for 5–
6 h. The progress of the reaction was monitored by thin layer
chromatography using ethyl acetate/petroleum ether (1:1) as
solvent system. The reaction solvent was evaporated in vacuo
to dryness to give the product 6 without further operation.
Ethyl 3-((3,4-difluorophenyl)amino)propanoate (3d)
Orange-yellow liquid; yield: 96%; 1H NMR (500 MHz, chloro-
form-d) d 6.95 (q, J ¼ 9.1 Hz, 1H), 6.40 (ddd, J ¼ 12.7, 6.6, 2.8 Hz,
1H), 6.28 (d, J ¼ 8.9 Hz, 1H), 4.19–4.13 (m, 2H), 3.37 (t,
J ¼ 6.3 Hz, 2H), 2.59 (t, J ¼ 6.3 Hz, 2H), 1.27 (t, J ¼ 7.1 Hz, 3H).
FT-MS m/z Calcd. for C11H13F2NO2 ([MþH]þ): 230.09871.
Found: 230.09822.
Ethyl 3-((3,5-difluorophenyl)amino)propanoate (3e)
Yellow-green solid; yield: 93%; m.p.: 42.1–43.2°C; 1H NMR
(500 MHz, chloroform-d) d 6.15 (tt, J ¼ 9.2, 2.2 Hz, 1H), 6.11
(dd, J ¼ 10.0, 2.1 Hz, 2H), 4.37 (s, 1H), 4.18 (q, J ¼ 7.1 Hz, 2H),
3.47–3.37 (m, 2H), 2.62 (t, J ¼ 6.2 Hz, 2H), 1.29 (t, J ¼ 7.2 Hz,
3H). FT-MS m/z Calcd. for C11H13F2NO2 ([MþH]þ): 230.09871.
Found: 230.09858.
General procedure for the synthesis of ethyl 3-(N-
(fluorinated-phenyl)-4-(ethylamino)-3-nitrobenzamido)-
propanoates 8a–i
To a vigorously stirred mixture of compound 3 (0.03 mol) and
triethylamine (0.03 mol) in anhydrous dichloromethane
(50 mL) at room temperature 4-(ethylamine)-3-nitrobenzoyl-
chloride (0.03 mol) in anhydrous dichloromethane was added
dropwise over 30 min. The reaction mixture was further
stirred for 5 h at room temperature. The progress of the
reaction was monitored by thin layer chromatography using
ethyl acetate/petroleum ether (1:1) as solvent system. The
reaction mixture was washed with aqueous sodium chloride
solution three times and dried over anhydrous sodium sulfate.
The crude product was purified by silica gel column
chromatography eluting with 2:1 petroleum ether/ethyl
acetate to give the product 8a–i.
Ethyl 3-((2-(trifluoromethyl)phenyl)amino)propanoate (3f)
1
Brown-yellow oil; yield: 62%; H NMR (500 MHz, DMSO-d6) d
7.42 (t, J ¼ 7.5 Hz, 2H), 6.86 (d, J ¼ 8.3 Hz, 1H), 6.75–6.68 (m,
1H), 5.38 (s, 1H), 4.07 (q, J ¼ 6.8 Hz, 2H), 3.45 (q, J ¼ 6.1 Hz, 2H),
2.60 (t, J ¼ 6.4 Hz, 2H), 1.17 (t, J ¼ 7.0 Hz, 3H). FT-MS m/z Calcd.
for C12H14F3NO2 ([MþH]þ): 262.10494. Found: 262.10449.
Ethyl 3-((3-(trifluoromethyl)phenyl)amino)propanoate (3g)
Red liquid; yield: 85%; 1H NMR (500 MHz, chloroform-d) d 7.30–
7.27 (m, 1H), 6.96 (d, J¼ 7.6 Hz, 1H), 6.83 (s, 1H), 6.77 (dd, J¼ 8.2,
2.0 Hz, 1H), 4.31 (s, 1H), 4.19 (q, J¼ 7.1 Hz, 2H), 3.49 (t, J¼ 6.2 Hz,
2H), 2.64(t, J¼ 6.3 Hz, 2H), 1.29 (t, J¼ 7.1 Hz, 3H). FT-MSm/z Calcd.
for C12H14F3NO2 ([MþH]þ): 262.10494. Found: 262.10401.
Ethyl 3-(4-(ethylamino)-N-(2-fluorophenyl)-3-
nitrobenzamido)propanoate (8a)
Ethyl 3-((4-(trifluoromethyl)phenyl)amino)propanoate (3h)
Gray-green solid; yield: 38%; m.p.: 60.5–61.2°C; 1H NMR
(500 MHz, chloroform-d) d 6.91 (t, J ¼ 7.0 Hz, 2H), 6.62–6.54 (m,
2H), 4.18 (q, J ¼ 5.7 Hz, 2H), 3.95 (s, 1H), 3.42 (t, J ¼ 5.0 Hz, 2H),
2.62 (t, J ¼ 5.0 Hz, 2H), 1.29 (t, J ¼ 5.7 Hz, 3H). FT-MS m/z Calcd.
for C12H14F3NO2 ([MþH]þ): 262.10494. Found: 262.10416.
Yellow solid; yield: 65%; m.p.: 91.2–92.3°C; 1H NMR (500 MHz,
chloroform-d) d 8.12 (d, J ¼ 1.9 Hz, 1H), 8.03 (s, 1H), 7.48 (dd,
J ¼ 9.0, 1.7 Hz, 1H), 7.25–7.21 (m, 1H), 7.11 (t, J ¼ 7.6 Hz, 1H),
7.02 (t, J ¼ 9.1 Hz, 1H), 6.64 (d, J ¼ 9.0 Hz, 1H), 4.16–4.09 (m,
2H), 4.05 (q, J ¼ 7.1 Hz, 2H), 3.34–3.24 (m, 2H), 2.71 (d,
J ¼ 35.8 Hz, 2H), 1.31 (t, J ¼ 7.2 Hz, 3H), 1.20 (t, J ¼ 7.1 Hz, 3H).
FT-MS m/z Calcd. for C20H22FN3O5 ([MþH]þ): 404.16163.
Found: 404.16413.
Ethyl 3-((4-(trifluoromethoxy)phenyl)amino)propanoate (3i)
Red liquid; yield: 85%; 1H NMR (500 MHz, chloroform-d) d 7.11
(q, J ¼ 8.0 Hz, 1H), 7.06 (d, J ¼ 8.7 Hz, 1H), 6.59 (d, J ¼ 8.9 Hz,
1H), 6.41 (ddd, J ¼ 13.8, 8.2, 2.0 Hz, 1H), 4.18 (q, J ¼ 7.1 Hz, 2H),
3.45 (t, J ¼ 6.2 Hz, 2H), 2.63 (t, J ¼ 6.3 Hz, 2H), 1.29 (t, J ¼ 7.1 Hz,
3H). FT-MS m/z Calcd. for C12H14F3NO3 ([MþH]þ): 278.09985,
Found: 278.10087.
Ethyl 3-(4-(ethylamino)-N-(3-fluorophenyl)-3-
nitrobenzamido)propanoate (8b)
Red-orange solid; yield: 79%; m.p.: 95.8–96.3°C; 1H NMR
(500 MHz, chloroform-d) d 8.16 (d, J ¼ 2.0 Hz, 1H), 7.42 (dd,
J ¼ 9.0, 1.9 Hz, 1H), 7.30–7.24 (m, 1H), 6.93 (td, J ¼ 8.2, 2.0 Hz,
2H), 6.86 (dt, J ¼ 9.5, 2.1 Hz, 1H), 6.63 (d, J ¼ 9.1 Hz, 1H), 4.19 (t,
J ¼ 7.2 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 3.34–3.27 (m, 2H), 2.70
(t, J ¼ 7.2 Hz, 2H), 1.33 (t, J ¼ 7.2 Hz, 3H), 1.22 (t, J ¼ 7.1 Hz, 3H).
FT-MS m/z Calcd. for C20H22FN3O5 ([MþH]þ): 404.16163.
Found: 404.16131.
Synthetic procedure for 4-(ethylamino)-3-nitrobenzoic acid (6)
A 250 mL round-bottom flask was charged with 4-chloro-3-
nitrobenzoic acid (20 g, 0.1 mol) and a solution of ethylamine
in water (60%, 86 mL, 0.8 mol). The mixture was refluxed at
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
414

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Fluorinated Thrombin Inhibitors
Archiv der Pharmazie
Ethyl 3-(4-(ethylamino)-N-(4-fluorophenyl)-3-
nitrobenzamido)propanoate (8c)
1H), 6.34 (dd, J¼ 6.5, 1.4 Hz, 1H), 6.12 (d, J ¼ 6.6 Hz, 1H), 4.06 (t,
J¼ 5.6 Hz, 2H), 3.95 (q, J ¼ 5.7 Hz, 2H), 2.98 (dt, J ¼ 9.8, 4.9 Hz, 2H),
2.56 (t, J¼ 5.6 Hz, 2H), 1.13 (dt, J¼ 13.0, 5.7 Hz, 6H). FT-MS m/z
Calcd. for C21H22F3N3O5 ([MþH]þ): 454.15843. Found: 454.16151.
Yellow solid; yield: 65%; m.p.: 85.3–86.9°C; 1H NMR (500 MHz,
chloroform-d) d 8.18–7.99 (m, 2H), 7.33 (d, J ¼ 8.8 Hz, 1H),
7.18–7.04 (m, 2H), 7.00–6.91 (m, 1H), 6.58 (d, J ¼ 9.0 Hz, 1H),
4.21–4.10 (m, 2H), 4.02 (q, J ¼ 6.9 Hz, 2H), 3.24 (dd, J ¼ 13.1,
6.1 Hz, 2H), 2.64 (t, J ¼ 7.0 Hz, 2H), 1.33–1.24 (m, 3H), 1.17 (q,
Ethyl 3-(4-(ethylamino)-3-nitro-N-(4-(trifluoromethoxy)-
phenyl)benzamido)propanoate (8i)
J ¼ 9.9, 7.1 Hz, 3H). FT-MS m/z Calcd. for
([MþH]þ): 404.16163. Found: 404.16423.
C
20H22FN3O5
Yellow oil; yield: 48%; 1H NMR (500 MHz, chloroform-d) d 8.14
(d, J ¼ 2.1 Hz, 1H), 7.38 (dd, J ¼ 9.0, 1.9 Hz, 1H), 7.16 (s, 4H), 6.63
(d, J ¼ 9.1 Hz, 1H), 4.18 (t, J ¼ 7.1 Hz, 2H), 4.06 (q, J ¼ 7.1 Hz,
2H), 3.34–3.27 (m, 2H), 2.70 (t, J ¼ 7.1 Hz, 2H), 1.32 (t,
J ¼ 7.2 Hz, 3H), 1.20 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for
Ethyl 3-(N-(3,4-difluorophenyl)-4-(ethylamino)-3-
nitrobenzamido)propanoate (8d)
Yellow solid; yield: 75%; m.p.: 110.3–110.7°C; 1H NMR
(500 MHz, chloroform-d) d 8.15 (d, J ¼ 1.7 Hz, 1H), 7.41–7.36
(m, 1H), 7.09 (q, J ¼ 8.9 Hz, 1H), 7.02 (td, J ¼ 7.4, 3.5 Hz, 1H),
6.89 (d, J ¼ 8.4 Hz, 1H), 6.65 (d, J ¼ 9.0 Hz, 1H), 4.14 (t,
J ¼ 7.0 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 3.36–3.25 (m, 2H),
2.69 (t, J ¼ 7.0 Hz, 2H), 1.33 (t, J ¼ 7.2 Hz, 3H), 1.22 (t, J ¼ 7.1 Hz,
3H). FT-MS m/z Calcd. for C20H21F2N3O5 ([MþH]þ): 422.15220.
Found: 422.15308.
C
21H22F3N3O5 ([MþH]þ): 470.15335. Found: 470.15766.
General procedure for the synthesis of ethyl 3-(3-amino-N-
(fluorinated-phenyl)-4-(methylamino)benzamido)-
propanoates 9a–i
A round bottom flask was charged with a solution of
compound 8 (20 mmol) in tetrahydrofuran/water (40/80 mL).
Zn powder (0.1 mol) and NH4Cl (10 mmol) were added in turn.
The reaction mixture was refluxed at 80°C for overnight under
the atmosphere of nitrogen. The reaction process was
monitored by thin layer chromatography using ethyl ace-
tate/petroleum ether (1:1) as solvent system. The mixture was
filtered through a pad to remove the Zn powder. The residue
solution was extracted with dichloromethane three times. The
combined organic layer was washed with an aqueous solution
of sodium chloride solution three times and dried over
anhydrous sodium sulfate. The crude product was purified by
silica gel chromatography eluting with 1:1 ethyl acetate/
petroleum ether to give product 9.
Ethyl 3-(N-(3,5-difluorophenyl)-4-(ethylamino)-3-
nitrobenzamido)propanoate (8e)
Yellow solid; yield: 59%; m.p.: 99.0–99.1°C; 1H NMR (500 MHz,
chloroform-d) d 8.19 (d, J ¼ 1.6 Hz, 1H), 8.10 (s, 1H), 7.42 (d,
J ¼ 9.0 Hz, 1H), 6.71–6.65 (m, 3H), 4.15 (t, J ¼ 7.1 Hz, 2H), 4.09
(q, J ¼ 7.1 Hz, 2H), 3.32 (p, J ¼ 7.1 Hz, 2H), 2.69 (t, J ¼ 7.1 Hz,
2H), 1.33 (t, J ¼ 7.2 Hz, 3H), 1.22 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z
Calcd. for
422.15140.
C
20H21F2N3O5 ([MþH]þ): 422.15220. Found:
Ethyl 3-(4-(ethylamino)-3-nitro-N-(2-(trifluoromethyl)-
phenyl)benzamido)propanoate (8f)
Ethyl 3-(3-amino-4-(ethylamino)-N-(2-fluorophenyl)-
benzamido)propanoate (9a)
Orange-yellow solid; yield: 49%; m.p.: 78.4–78.8°C; 1H NMR
(500 MHz, DMSO-d6) d 7.50 (d, J ¼ 3.8 Hz, 2H), 7.47 (d, J ¼ 6.5 Hz,
1H), 7.39–7.34 (m, 1H), 6.70 (d, J ¼ 1.4 Hz, 1H), 6.28 (dd, J ¼ 6.6,
1.4 Hz, 1H),6.10(d,J ¼ 6.6 Hz, 1H),4.07(t, J ¼ 5.5 Hz, 2H),3.94(q,
J ¼ 5.7 Hz,2H),2.98(q,J ¼ 5.4 Hz,2H),2.56(t,J ¼ 5.5 Hz,2H),1.12
(dt, J ¼ 11.3, 5.7 Hz, 6H). FT-MS m/z Calcd. for C21H22F3N3O5
([MþH]þ): 454.15843. Found: 454.15842.
Black solid; yield: 98%; m.p.: 118.8–120.1°C; 1H NMR (500 MHz,
DMSO-d6) d 7.32 (t, J ¼ 7.9 Hz, 1H), 7.25 (q, J ¼ 6.8, 6.1 Hz, 1H),
7.14 (t, J ¼ 8.4 Hz, 2H), 6.71 (d, J ¼ 1.7 Hz, 1H), 6.36 (d, J ¼ 8.0 Hz,
1H), 6.08 (d, J ¼ 8.3 Hz, 1H), 4.75 (s, 1H), 4.58 (s, 2H), 3.95 (q,
J ¼ 7.1 Hz,4H),2.97(q,J ¼ 7.0 Hz,2H),2.57(t,J ¼ 6.6 Hz,2H),1.13
(dt, J ¼ 13.9, 7.1 Hz, 6H). FT-MS m/z Calcd. for C20H24FN3O3
([MþH]þ): 374.18745. Found: 374.18959.
Ethyl 3-(4-(ethylamino)-3-nitro-N-(3-(trifluoromethyl)-
phenyl)benzamido)propanoate (8g)
Ethyl 3-(3-amino-4-(ethylamino)-N-(3-fluorophenyl)-
benzamido)propanoate (9b)
Orange-yellow solid; yield: 63%; m.p.: 81.2–82.1°C; 1H NMR
(500 MHz, chloroform-d) d 8.12 (d, J ¼ 2.0 Hz, 1H), 8.07 (s, 1H),
7.48 (d, J ¼ 7.8 Hz, 1H), 7.44 (t, J ¼ 7.9 Hz, 1H), 7.40 (d,
J ¼ 8.6 Hz, 1H), 7.33–7.28 (m, 1H), 6.65 (d, J ¼ 9.1 Hz, 1H),
4.23 (t, J ¼ 7.1 Hz, 2H), 4.07 (q, J ¼ 7.1 Hz, 2H), 3.36–3.25 (m,
2H), 2.73 (t, J ¼ 7.1 Hz, 2H), 1.33 (t, J ¼ 7.2 Hz, 3H), 1.21 (t,
J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C21H22F3N3O5 ([MþH]þ):
454.15843. Found: 454.15764.
Brown solid; yield: 82%; m.p.: 114.6–115.4°C; 1H NMR
(500 MHz, chloroform-d) d 7.19 (q, J ¼ 7.4 Hz, 1H), 6.85 (d,
J ¼ 10.1 Hz, 4H), 6.73 (d, J ¼ 8.1 Hz, 1H), 6.34 (d, J ¼ 8.3 Hz, 1H),
4.17 (t, J ¼ 7.2 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 3.10 (q,
J ¼ 7.1 Hz, 2H), 2.71 (t, J ¼ 7.2 Hz, 2H), 1.24 (dt, J ¼ 17.8,
7.1 Hz, 6H). FT-MS m/z Calcd. for C20H24FN3O3 ([MþH]þ):
374.18745. Found: 374.18941.
Ethyl 3-(4-(ethylamino)-3-nitro-N-(4-(trifluoromethyl)-
phenyl)benzamido)propanoate (8h)
Ethyl 3-(3-amino-4-(ethylamino)-N-(4-fluorophenyl)-
benzamido)propanoate (9c)
Orange-yellow oil; yield: 74%; 1H NMR (500MHz, DMSO-d6) d
7.63 (d, J ¼ 6.7 Hz, 2H), 7.30 (d, J ¼ 6.6 Hz, 2H), 6.68 (d, J¼ 1.5 Hz,
Gray solid; yield: 84%; m.p.: 112.7–113.8°C; 1H NMR
(500 MHz, DMSO-d6) d 7.17–7.07 (m, 4H), 6.68 (d, J ¼ 1.8 Hz,
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
415

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
H. Chen and Y. Ren
Archiv der Pharmazie
1H), 6.33 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.11 (d, J ¼ 8.3 Hz, 1H), 4.73
(s, 1H), 4.55 (s, 2H), 3.98 (p, J ¼ 7.0 Hz, 4H), 2.99 (q, J ¼ 6.9 Hz,
2H), 2.54 (t, J ¼ 7.1 Hz, 2H), 1.14 (q, J ¼ 7.2 Hz, 6H). FT-MS m/z
Ethyl 3-(3-amino-4-(ethylamino)-N-(4-(trifluoromethoxy)-
phenyl)benzamido)propanoate (9i)
Dark green oil; yield: 79%; 1H NMR (500 MHz, DMSO-d6) d 7.26
(d, J ¼ 8.7 Hz, 2H), 7.22 (d, J ¼ 8.9 Hz, 2H), 6.69 (s, 1H), 6.32 (d,
J ¼ 8.2 Hz, 1H), 6.11 (d, J ¼ 8.3 Hz, 1H), 4.76 (s, 1H), 4.57 (s, 2H),
4.02 (t, J ¼ 6.9 Hz, 2H), 3.95 (q, J ¼ 7.1 Hz, 2H), 2.99 (q,
J ¼ 7.1 Hz, 2H), 2.56 (t, J ¼ 6.9 Hz, 2H), 1.13 (dt, J ¼ 14.4,
7.1 Hz, 6H). FT-MS m/z Calcd. for C21H24F3N3O4 ([MþH]þ):
440.17917. Found: 440.14178.
Calcd. for
374.18883.
C
20H24FN3O3 ([MþH]þ): 374.18745. Found:
Ethyl 3-(3-amino-N-(3,4-difluorophenyl)-4-(ethylamino)-
benzamido)propanoate (9d)
Red-brown solid; yield: 66%; m.p.: 112.7–113.8°C; 1H NMR
(500 MHz,DMSO-d6) d 7.39–7.28 (m, 2H), 6.89 (d, J ¼ 8.8 Hz,
1H), 6.64 (d, J ¼ 1.9 Hz, 1H), 6.38 (dd, J ¼ 8.2, 1.9 Hz, 1H), 6.15
(d, J ¼ 8.3 Hz, 1H), 4.77 (s, 1H), 4.61 (s, 2H), 4.00 (d, J ¼ 7.1 Hz,
2H), 3.99–3.96 (m, 2H), 3.00 (q, J ¼ 7.0 Hz, 2H), 2.55 (t,
J ¼ 7.1 Hz, 2H), 1.17–1.11 (m, 6H). FT-MS m/z Calcd. for
General procedure for the synthesis of compounds 10a–i
A round bottom flask was charged with a solution of 2-((4-
cyanophenyl)amino)acetic acid (12mmol), EDCI (12 mmol),
and HoBt (12 mmol) in tetrahydrofuran/N,N-dimethylforma-
mide (70/10mL). Thereactionmixture was stirred atice bath for
35 min. Then a solution of compound 9 (10 mmol) in tetrahy-
drofuran (50 mL) was added dropwise with stirring at room
temperature. The reaction solution was stirred for overnight.
The reaction solution was evaporated in vacuo to dryness. The
residual was dissolved in dichloromethane (100 mL), washed
with water three times, and dried over anhydrous sodium
sulfate.The organic layer was evaported in vacuo to dryness.
Then the residual was dissolved in acetic acid (70 mL) and
refluxed at 120°C for 2–3 h. The reaction solution was adjusted
to pH 9–10 with ammonium hydroxide and stirred for 30 min.
The reaction solution was extracted with dichloromethane
three times, washed with water three times, and dried over
anhydrous sodium sulfate. The reaction process was monitored
by thin layer chromatography using dichloromethane/methyl
alcohol (20:1) as solvent system. The crude product was
purified by silica gel chromatography eluting with 2:1 ethyl
acetate/petroleum ether to give product 10.
C
20H23F2N3O3 ([MþH]þ): 392.17802. Found: 392.17966.
Ethyl 3-(3-amino-N-(3,5-difluorophenyl)-4-(ethylamino)-
benzamido)propanoate (9e)
Gray solid; yield: 95%; m.p.: 122.5–123.5°C; 1H NMR (500 MHz,
DMSO-d6) d 7.04 (d, J ¼ 9.2 Hz, 1H), 6.92–6.86 (m, 2H), 6.68 (d,
J ¼ 1.8 Hz, 1H), 6.43 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.19 (d, J ¼ 8.3 Hz,
1H), 4.82 (s, 1H), 4.61 (s, 2H), 4.06–4.01 (m, 2H), 4.01–3.96 (m,
2H), 3.02 (q, J ¼ 6.5 Hz, 2H), 2.57 (t, J ¼ 7.0 Hz, 2H), 1.15 (dt,
J ¼ 11.2, 7.1 Hz, 6H). FT-MS m/z Calcd. for C20H23F2N3O3
([MþH]þ): 392.17802. Found: 392.17923.
Ethyl 3-(3-amino-4-(ethylamino)-N-(2-(trifluoromethyl)-
phenyl)benzamido)propanoate (9f)
Dark green oil; yield: 59%; 1H NMR (500 MHz, DMSO-d6) d
7.52–7.46 (m, 3H), 7.36 (d, J ¼ 5.8 Hz, 1H), 6.72–6.67 (m, 1H),
6.28 (dd, J ¼ 6.6, 1.4 Hz, 1H), 6.10 (d, J ¼ 6.6 Hz, 1H), 4.78 (s, 1H),
4.60 (s, 2H), 4.07 (t, J ¼ 5.5 Hz, 2H), 3.94 (q, J ¼ 5.7 Hz, 2H), 2.98
(q, J ¼ 5.4 Hz, 2H), 2.56 (t, J ¼ 5.5 Hz, 2H), 1.12 (dt, J ¼ 11.3,
5.7 Hz, 6H). FT-MS m/z Calcd. for C21H24F3N3O3 ([MþH]þ):
424.18425. Found: 424, 18649.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(2-
fluorophenyl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoate (10a)
Ethyl 3-(3-amino-4-(ethylamino)-N-(3-(trifluoromethyl)-
phenyl)benzamido)propanoate (9g)
Red-brown solid; yield: 89%; m.p.: 176.8–177.6°C; 1H NMR
(500 MHz, DMSO-d6)
d
7.48 (t, J ¼ 7.6 Hz, 3H), 7.43 (d,
Dark red oil; yield: 84%; 1H NMR (500 MHz, DMSO-d6) d 7.51–
7.45 (m, 3H), 7.36 (d, J ¼ 7.3 Hz, 1H), 6.70 (d, J ¼ 1.8 Hz, 1H),
6.28 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.10 (d, J ¼ 8.3 Hz, 1H), 4.78 (s, 1H),
4.60 (s, 2H), 4.07 (t, J ¼ 6.9 Hz, 2H), 3.94 (q, J ¼ 7.1 Hz, 2H), 2.98
(q, J ¼ 6.8 Hz, 2H), 2.56 (t, J ¼ 6.9 Hz, 2H), 1.12 (dt, J ¼ 14.1,
7.1 Hz, 6H). FT-MS m/z Calcd. for C21H24F3N3O3 ([MþH]þ):
424.18425. Found: 424.18649.
J ¼ 8.4 Hz, 1H), 7.30 (t, J ¼ 5.2 Hz, 1H), 7.24 (d, J ¼ 8.1 Hz,
2H), 7.13 (dt, J ¼ 23.1, 8.2 Hz, 2H), 6.82 (d, J ¼ 8.7Hz, 2H), 4.59
(d, J ¼ 5.3 Hz, 2H), 4.24 (q, J ¼ 6.8 Hz, 2H), 4.04 (s, 2H), 3.96 (q,
J ¼ 7.1 Hz, 2H), 2.71–2.54 (m, 2H), 1.24 (t, J ¼ 7.0 Hz, 3H), 1.12
(t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H28FN5O3 ([MþH]þ):
514.22489. Found: 514.22582.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(3-
fluorophenyl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoate (10b)
Ethyl 3-(3-amino-4-(ethylamino)-N-(4-(trifluoromethyl)-
phenyl)benzamido)propanoate (9h)
Dark gray oil; yield: 92%; 1H NMR (500 MHz,DMSO-d6) d 7.63
(d, J ¼ 8.4 Hz, 2H), 7.30 (d, J ¼ 8.3 Hz, 2H), 6.68 (d, J ¼ 1.9 Hz,
1H), 6.34 (dd, J ¼ 8.2, 1.7 Hz, 1H), 6.12 (d, J ¼ 8.3 Hz, 1H), 4.80 (t,
J ¼ 4.9 Hz, 1H), 4.60 (s, 2H), 4.06 (t, J ¼ 7.0 Hz, 2H), 3.95 (q,
J ¼ 7.1 Hz, 2H), 2.98 (p, J ¼ 7.0 Hz, 2H), 2.56 (t, J ¼ 7.0 Hz, 2H),
1.13 (dt, J ¼ 16.3, 7.1 Hz, 6H). FT-MS m/z Calcd. for
Red-brown oil; yield: 73%; 1H NMR (500 MHz, DMSO-d6) d 7.54
(s, 1H), 7.48 (d, J ¼ 8.7 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.29–7.24
(m, 2H), 7.20 (d, J ¼ 10.2 Hz, 1H), 6.99 (d, J ¼7.5 Hz, 2H), 6.84 (d,
J ¼ 8.7 Hz, 2H), 4.61 (d, J ¼ 5.4 Hz, 2H), 4.25 (q, J ¼ 7.0 Hz, 2H),
4.13 (t, J ¼ 7.1 Hz, 2H), 4.00 (q, J ¼ 7.1 Hz, 2H), 2.64 (t, J ¼ 7.1 Hz,
2H), 1.25 (t, J ¼ 7.2 Hz, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z
Calcd.forC29H28FN5O3 ([MþH]þ):514.22489.Found:514.22602.
C
21H24F3N3O3 ([MþH]þ): 424.18425. Found: 424.18480.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
416

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Fluorinated Thrombin Inhibitors
Archiv der Pharmazie
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(4-
fluorophenyl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoate (10c)
3H). FT-MS m/z Calcd. for C30H28F3N5O3 ([MþH]þ): 564.22170.
Found: 564.21980.
White solid; yield: 74%; m.p.: 161.7–162.4°C; 1H NMR (500 MHz,
chloroform-d)d7.62(s, 1H),7.40(d,J ¼ 8.6 Hz,2H),7.29–7.24(m,
1H), 7.11 (d, J ¼ 8.4 Hz, 1H), 7.04 (dd, J ¼ 8.6, 4.7 Hz, 2H), 6.88 (t,
J ¼ 8.4 Hz,2H),6.70(d,J ¼ 8.6 Hz,2H),5.70(t,J ¼ 4.7 Hz,1H),4.47
(d, J ¼ 4.6 Hz, 2H), 4.20 (t, J ¼ 7.2 Hz, 2H), 4.12 (q, J ¼ 7.2 Hz, 2H),
4.07 (q, J ¼ 7.1 Hz, 2H), 2.70 (t, J ¼ 7.2 Hz, 2H), 1.36 (t, J ¼ 7.2 Hz,
3H), 1.21 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H28FN5O3
([MþH]þ): 514.22489. Found: 514.22627.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(4-
(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (10h)
Yellow solid; yield: 68%; m.p.: 148.2–149.1°C; 1H NMR
(500 MHz, chloroform-d) d 7.69 (s, 1H), 7.46 (t, J ¼ 8.2 Hz,
4H), 7.33–7.27 (m, 1H), 7.19 (dd, J ¼ 15.9, 8.4 Hz, 3H), 6.72 (d,
J ¼ 8.6 Hz, 2H), 4.51 (d, J ¼ 4.4 Hz, 2H), 4.28 (t, J ¼ 7.1 Hz, 2H),
4.17 (q, J ¼ 7.2 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H), 2.74 (t,
J ¼ 7.1 Hz, 2H), 1.41 (t, J ¼ 7.3 Hz, 3H), 1.22 (t, J ¼ 7.1 Hz, 3H).
FT-MS m/z Calcd. for C30H28F3N5O3 ([MþH]þ): 564.22170.
Found: 564.22271.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-N-(3,4-
difluorophenyl)-1-ethyl-1H-benzo[d]imidazole-5-
carboxamido)propanoate (10d)
Red-brown oil; yield: 69%; 1H NMR (500 MHz, DMSO-d6) d 7.53
(s, 1H), 7.49–7.44 (m, 3H), 7.30 (dt, J ¼ 15.2, 9.2 Hz, 2H), 7.21 (d,
J ¼ 8.5 Hz, 1H), 7.01 (d, J ¼ 8.5 Hz, 1H), 6.83 (d, J ¼ 8.7 Hz, 2H),
4.61 (d, J ¼ 5.4 Hz, 2H), 4.25 (q, J ¼ 6.7 Hz, 2H), 4.08 (t,
J ¼ 7.0 Hz, 2H), 4.00 (q, J ¼ 7.1 Hz, 2H), 2.65–2.59 (m, 2H),
1.25 (t, J ¼ 7.2 Hz, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd.
for C29H27F2N5O3 ([MþH]þ): 532.21547. Found: 532.21860.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(4-
(trifluoromethoxy)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (10i)
Pink solid; yield: 46%; m.p.: 162.3–163.1°C; 1H NMR (500 MHz,
DMSO-d6) d 7.51 (s, 1H), 7.44 (dd, J ¼ 16.8, 8.6 Hz, 3H), 7.32 (t,
J ¼ 8.0 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz, 2H), 7.21 (d, J ¼ 8.5 Hz, 1H),
6.82 (d, J ¼ 8.7 Hz, 2H), 4.61 (d, J ¼ 5.3 Hz, 2H), 4.25 (q,
J ¼ 7.0 Hz, 2H), 4.12 (t, J ¼ 6.9 Hz, 2H), 3.96 (q, J ¼ 7.1 Hz, 2H),
2.62 (t, J ¼ 7.0 Hz, 2H), 1.24 (t, J ¼ 7.2 Hz, 3H), 1.11 (t, J ¼ 7.1 Hz,
3H). FT-MS m/z Calcd. for C30H28F3N5O4 ([MþH]þ): 580.21662.
Found: 580.22290.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-N-(3,5-
difluorophenyl)-1-ethyl-1H-benzo[d]imidazole-5-
carboxamido)propanoate (10e)
Pink solid; yield: 93%; m.p.: 128.4–128.9°C; 1H NMR (500 MHz,
chloroform-d) d 7.67 (s, 1H), 7.43 (d, J ¼ 8.7 Hz, 2H), 7.37–7.32
(m, 1H), 7.20 (d, J ¼ 8.4 Hz, 1H), 6.72 (d, J ¼ 8.7 Hz, 2H), 6.67–
6.63 (m, 2H), 6.61–6.56 (m, 1H), 5.61 (s, 1H), 4.51 (d, J ¼ 4.5 Hz,
2H), 4.21 (t, J ¼ 7.1 Hz, 2H), 4.18–4.13 (m, 2H), 4.13–4.07 (m,
2H), 2.73 (t, J ¼ 7.1 Hz, 2H), 1.40 (t, J ¼ 7.2 Hz, 3H), 1.23 (t,
J ¼ 7.2 Hz, 3H). FT-MS m/z Calcd. for C29H27F2N5O3 ([MþH]þ):
532.21547. Found: 532.21610.
General procedure for the synthesis of compounds 11a–i
A round bottom flask was charged with a solution of
compound 10 (5 mmol), hydroxylamine hydrochloride
(10 mmol), and triethylamine (10 mmol) in ethanol (50 mL).
The reaction mixture was refluxed at 80°C for 4–5 h. Then the
reaction mixture was evaporated in vacuo to dryness to obtain
the crude intermediate which was used without further
purification in the next step. The crude intermediate was
dissolved in acetic acid (50 mL). Then Pd/C (5%, 1.0 g) and
ammonium formate (15 mmol) were added in turn. The
reaction mixture was refluxed at 120°C for 5–6 h under the
protection of nitrogen. The reaction process was monitored
by thin layer chromatography using dichloromethane/methyl
alcohol (10:1) as solvent system. The crude product was
purified by silica gel chromatography eluting with 7:1
dichloromethane/methyl alcohol to give product 11.
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(2-
(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (10f)
Red solid; yield: 80%; m.p.: 152.3–152.9°C; 1H NMR (500 MHz,
DMSO-d6) d 7.72–7.67 (m, 1H), 7.61 (s, 2H), 7.47 (d, J ¼ 8.1 Hz,
3H), 7.41 (s, 1H), 7.28 (s, 1H), 7.22 (d, J ¼ 7.3 Hz, 1H), 6.82 (d,
J ¼ 7.5 Hz, 2H), 4.58 (s, 2H), 4.48 (s, 1H), 4.27–4.17 (m, 2H),
4.07–3.95 (m, 2H), 3.77–3.42 (m, 2H), 2.84–2.69 (m, 2H), 1.23 (s,
3H), 1.14 (s, 3H). FT-MS m/z Calcd. for C30H28F3N5O3 ([MþH]þ):
564.22170. Found: 564.22165.
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(2-fluorophenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (11a)
Ethyl 3-(2-(((4-cyanophenyl)amino)methyl)-1-ethyl-N-(3-
(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (10g)
White solid; yield: 51%; m.p.: 99.9–101.5°C; 1H NMR (500 MHz,
DMSO-d6) d 8.83 (s, 3H), 7.67 (d, J ¼ 8.9 Hz, 2H), 7.50–7.46 (m,
2H), 7.43 (d, J ¼ 8.3 Hz, 1H), 7.22 (d, J ¼ 8.1 Hz, 2H), 7.13 (dt,
J ¼ 24.1, 8.1 Hz, 2H), 6.86 (d, J ¼ 8.9 Hz, 2H), 4.64 (d, J ¼ 5.3 Hz,
2H), 4.26 (q, J ¼ 6.8 Hz, 2H), 4.07–3.99 (m, 2H), 3.96 (q,
J ¼ 7.1 Hz, 2H), 2.71–2.56 (m, 2H), 1.23 (t, J ¼ 7.1 Hz, 3H), 1.12
(t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H31FN6O3 ([MþH]þ):
531.25144. Found: 531.24776.
Brown-yellow oil; yield: 77%; 1H NMR (500 MHz, chloroform-
d) d 7.67 (s, 1H), 7.46 (d, J ¼ 8.6 Hz, 2H), 7.38 (d, J ¼ 11.2 Hz, 2H),
7.33 (t, J ¼ 7.8 Hz, 1H), 7.31–7.25 (m, 2H), 7.17 (d, J ¼ 8.4 Hz,
1H), 6.73(d, J ¼ 8.6 Hz, 2H), 4.51 (d, J ¼ 4.3 Hz, 2H), 4.28 (t,
J ¼ 7.0 Hz, 2H), 4.16 (q, J ¼ 7.2 Hz, 2H), 4.08 (q, J ¼ 7.1 Hz, 2H),
2.76 (t, J ¼ 7.1 Hz, 2H), 1.39 (t, J ¼ 7.2 Hz, 3H), 1.22 (t, J ¼ 7.1 Hz,
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
417

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
H. Chen and Y. Ren
Archiv der Pharmazie
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(3-fluorophenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (11b)
(m, 2H), 1.23 (t, J¼ 5.7 Hz, 3H), 1.12 (t, J¼ 5.7 Hz, 3H). FT-MS m/z
Calcd. for C30H31F3N6O3 ([MþH]þ): 581.24825. Found: 581.25478.
White solid; yield: 42%; m.p.: 194.7–196.1°C; 1H NMR
(500 MHz, DMSO-d6) d 8.80 (s, 3H), 7.64 (d, J ¼ 8.8 Hz, 2H),
7.51 (s, 1H), 7.44 (d, J ¼ 8.1 Hz, 1H), 7.28–7.17 (m, 3H), 7.03–
6.96 (m, 2H), 6.86 (d, J ¼ 8.8 Hz, 2H), 4.64 (d, J ¼ 5.3 Hz, 2H),
4.26 (q, J ¼ 6.7 Hz, 2H), 4.10 (t, J ¼ 7.0 Hz, 2H), 3.99 (q,
J ¼ 7.1 Hz, 2H), 2.62 (t, J ¼ 7.0 Hz, 2H), 1.24 (t, J ¼ 7.0 Hz, 3H),
1.14 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H31FN6O3
([MþH]þ): 531.25144. Found: 531.25389.
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(3-(trifluoromethyl)phenyl)-1H-benzo[d]-
imidazole-5-carboxamido)propanoate (11g)
White solid; yield: 44%; m.p.: 126.1–126.9°C; 1H NMR (500 MHz,
DMSO-d6) d 8.77 (s, 4H), 7.76–7.71 (m, 1H), 7.70–7.66 (m, 1H),
7.64 (d, J ¼ 8.6 Hz, 2H), 7.51 (s, 1H), 7.45 (d, J ¼ 5.1 Hz, 2H), 7.44–
7.40 (m, 1H), 7.19 (d, J ¼ 8.5 Hz, 1H), 6.86 (d, J ¼ 8.7 Hz, 2H), 4.65
(d, J ¼ 5.4 Hz, 2H), 4.26 (q, J ¼ 8.6, 7.7 Hz, 2H), 4.15 (t, J ¼ 6.8 Hz,
2H), 3.96 (q, J ¼ 7.1 Hz, 2H), 2.63 (t, J ¼ 6.9 Hz, 2H), 1.24–1.19 (m,
3H), 1.12 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C30H31F3N6O3
([MþH]þ): 581.24825. Found: 581.25271.
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(4-fluorophenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoate (11c)
White solid; yield: 51%; m.p.: 122.4–123.3°C; 1H NMR
(500 MHz, DMSO-d6) d 8.93 (s, 2H), 8.76 (s, 2H), 7.68 (d,
J ¼ 8.8 Hz, 2H), 7.51–7.48 (m, 1H), 7.41 (d, J ¼ 8.4 Hz, 1H), 7.24
(dd, J ¼ 8.7, 5.0 Hz, 2H), 7.18 (d, J ¼ 8.3 Hz, 1H), 7.10 (t,
J ¼ 8.7 Hz, 2H), 6.87 (d, J ¼ 8.8 Hz, 2H), 4.64 (d, J ¼ 5.5 Hz, 2H),
4.27 (q, J ¼ 6.9 Hz, 2H), 4.08 (t, J ¼ 7.0 Hz, 2H), 3.99 (q,
J ¼ 7.1 Hz, 2H), 2.60 (t, J ¼ 7.1 Hz, 2H), 1.24 (t, J ¼ 7.1 Hz, 3H),
1.14 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H31FN6O3
([MþH]þ): 531.25144. Found: 531.25429.
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(4-(trifluoromethyl)phenyl)-1H-benzo[d]-
imidazole-5-carboxamido)propanoate (11h)
White solid; yield: 48%; m.p.: 109.4–111.1°C; 1H NMR (500 MHz,
DMSO-d6)d8.91(s, 3H), 7.64(t, J¼ 8.0 Hz, 4H), 7.53 (s, 1H), 7.45 (d,
J¼ 8.4 Hz, 1H), 7.41 (d, J¼ 8.2 Hz, 2H), 7.22 (d, J¼ 8.5 Hz, 1H), 6.86
(d, J¼ 8.6 Hz, 2H), 4.65 (d, J¼ 5.2 Hz, 2H), 4.27 (q, J¼ 6.6 Hz, 2H),
4.15 (t, J ¼ 6.9 Hz, 2H), 3.97 (q, J¼ 7.1 Hz, 2H), 2.63 (t, J¼ 6.9 Hz,
2H), 1.24 (t, J ¼ 7.0 Hz, 3H), 1.12 (t, J¼ 7.1 Hz, 3H). FT-MS m/z
Calcd. for C30H31F3N6O3 ([MþH]þ): 581.24825. Found: 581.24708.
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-N-
(3,4-difluorophenyl)-1-ethyl-1H-benzo[d]imidazole-5-
carboxamido)propanoate (11d)
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]-
imidazole-5-carboxamido)propanoate (11i)
White solid; yield: 57%; m.p.: 105.6–106.3°C; 1H NMR (500 MHz,
DMSO-d6)d7.61(d,J ¼ 8.8 Hz,2H),7.54(s,1H),7.45(d,J ¼ 8.4 Hz,
1H), 7.31 (q, J ¼ 9.2 Hz, 2H), 7.21 (d, J ¼ 9.5 Hz, 1H), 7.01 (d,
J ¼ 8.8 Hz, 1H), 6.85 (d, J ¼ 8.8 Hz, 2H), 4.64 (d, J ¼ 5.4 Hz, 2H),
4.27 (q, J ¼ 7.0 Hz, 2H), 4.08 (t, J ¼ 7.1 Hz, 2H), 4.00 (q, J ¼ 7.1 Hz,
2H), 2.62 (t, J ¼ 7.1 Hz, 2H), 1.25 (t, J ¼ 7.2 Hz, 3H), 1.14 (t,
J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for C29H30F2N6O3 ([MþH]þ):
549.24202. Found: 549.24301.
White solid; yield: 55%; m.p.: 142.6–145.3°C; 1H NMR (500 MHz,
DMSO-d6)d10.91(s,1H),8.47(s,2H),7.60(s,2H),7.52(s,1H),7.42
(d, J ¼ 8.4 Hz, 1H), 7.33 (d, J ¼ 8.8 Hz, 2H), 7.26 (d, J ¼ 8.5 Hz, 2H),
7.18 (d, J ¼ 8.4 Hz, 1H), 6.85 (d, J ¼ 8.8 Hz, 2H), 4.63 (d, J ¼ 5.3 Hz,
2H), 4.26 (q, J ¼ 6.8 Hz, 2H), 4.11 (t, J ¼ 6.9 Hz, 2H), 3.96 (q,
J ¼ 7.1 Hz, 2H), 2.62 (t, J ¼ 6.9 Hz, 2H), 1.71 (s, 2H), 1.24 (t,
J ¼ 7.1 Hz, 3H), 1.12 (t, J ¼ 7.1 Hz, 3H). FT-MS m/z Calcd. for
C
30H31F3N6O4 ([MþH]þ): 597.24317. Found: 597.24712.
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-N-
(3,5-difluorophenyl)-1-ethyl-1H-benzo[d]imidazole-5-
carboxamido)propanoate (11e)
General procedure for the synthesis of compounds 12a–i
Aroundbottomflaskwaschargedwithasolutionofcompound
11 (0.2 mmol), sodium hydroxide (0.6 mmol) in 6 mL of water
and 3 mL of ethanol. The reaction mixture was stirred at
ambient temperature for 2 h. The mixture was neutralized
with acetic acid. The precipitate was isolated and washed with
water and ether to afford the zwitterionic title compound 12.
1
White solid; yield: 55%; m.p.: 101.2–103.0°C; H NMR (500 MHz,
DMSO-d6) d 8.87 (s, 1H), 8.63 (s, 2H), 7.65 (d, J¼ 8.8 Hz, 2H), 7.56 (s,
1H), 7.46 (dd, J¼ 12.7, 7.0 Hz, 2H), 7.26–7.22 (m, 1H), 7.09–7.02 (m,
2H), 6.87 (d, J¼ 8.9 Hz, 2H), 4.66 (d, J¼ 5.5 Hz, 2H), 4.28 (q,
J¼ 6.9 Hz, 2H),4.10(t, J¼ 7.0 Hz, 2H), 4.00 (q, J¼ 7.1 Hz, 2H), 2.64(t,
J¼ 7.0 Hz, 2H), 1.26 (t, J¼ 7.2 Hz, 3H), 1.17–1.14 (m, 3H). FT-MS m/z
Calcd. for C29H30F2N6O3 ([MþH]þ): 549.24202. Found: 549.24629.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(2-fluorophenyl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoic acid (12a)
Ethyl 3-(2-(((4-carbamimidoylphenyl)amino)methyl)-1-
ethyl-N-(2-(trifluoromethyl)phenyl)-1H-benzo[d]-
imidazole-5-carboxamido)propanoate (11f)
White solid; yield: 51%; m.p.: 238–238.9°C; 1H NMR (500 MHz,
DMSO-d6) d 9.10 (s, 1H), 8.88 (s, 1H), 7.88 (d, J ¼ 7.8 Hz, 1H),
7.74 (d, J ¼ 8.7 Hz, 2H), 7.68 (s, 1H), 7.55 (t, J ¼ 8.5 Hz, 1H), 7.48
(d, J ¼ 7.1 Hz, 1H), 7.30–7.21 (m, 1H), 7.15 (dt, J ¼ 24.6, 7.5 Hz,
2H), 6.92 (d, J ¼ 8.8 Hz, 2H), 5.03 (s, 2H), 4.51–4.46 (m, 2H), 4.01
(s, 2H), 2.58 (d, J ¼ 31.2 Hz, 2H), 1.36 (t, J ¼ 7.1 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6) d 172.35, 170.17, 164.25, 164.17, 164.10,
1
White solid; yield: 48%; m.p.: 105.3–106.4°C; H NMR (500 MHz,
DMSO-d6) d 8.82 (s, 4H), 7.67 (d, J¼ 7.1 Hz, 2H), 7.49 (q, J¼ 5.3,
4.7 Hz, 2H), 7.43 (d, J¼ 6.7 Hz, 1H), 7.22 (d, J¼ 6.5 Hz, 2H), 7.13 (dt,
J¼ 19.2, 6.5 Hz, 2H), 6.86 (d, J¼ 7.1 Hz, 2H), 4.64 (d, J¼ 4.3 Hz, 2H),
4.26(q, J¼ 5.4 Hz, 2H), 4.03(s, 2H), 3.96(q, J¼ 5.7 Hz, 2H), 2.70–2.55
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
418

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
Fluorinated Thrombin Inhibitors
Archiv der Pharmazie
155.71, 152.76, 152.65, 135.25, 130.25, 129.81, 129.67, 129.54,
129.46, 125.26, 122.95, 118.00, 116.37, 116.17, 113.44, 11277,
111.71, 110.02, 45.79, 32.08, 14.64. FT-MS m/z Calcd. for
J ¼ 6.8 Hz, 2H), 4.06 (t, J ¼ 7.2 Hz, 2H), 2.60 (t, J ¼ 7.3 Hz, 2H),
1.37 (t, J ¼ 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) d 172.43,
168.18, 164.13, 163.31, 163.16, 160.86, 160.71, 152.86, 152.05,
133.64, 132.44, 129.78, 129.26, 125.77, 114.54, 112.52, 112.17,
111.91, 46.07, 40.47, 31.99, 13.95. FT-MS m/z Calcd. for
C
27H27FN6O3 ([MþH]þ): 503.22014. Found: 503.22069.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(3-fluorophenyl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoic acid (12b)
C
27H26F2N6O3 ([MþH]þ): 521.21072. Found: 521.20974.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(2-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoic acid (12f)
White solid; yield: 42%; m.p.: 226.9–229.4°C; 1H NMR (500 MHz,
DMSO-d6) d 9.11 (s, 1H), 8.84 (s, 1H), 7.89 (d, J ¼ 8.6 Hz, 1H), 7.73
(d, J ¼ 9.4 Hz, 3H), 7.48 (d, J ¼ 8.4 Hz, 1H), 7.26 (d, J ¼ 8.1 Hz, 2H),
7.06–6.99 (m, 2H), 6.91 (d, J ¼ 8.6 Hz, 2H), 5.03 (s, 2H), 4.49 (d,
J ¼ 7.0 Hz, 2H), 4.04(t, J ¼ 7.0 Hz, 2H), 2.55(t,J ¼ 7.1 Hz, 2H), 1.35
(t,J ¼ 7.1 Hz,3H).13CNMR(125 MHz,DMSO-d6)d172.46,168.28,
163.58, 163.06, 160.62, 152.70, 151.96, 133.96, 132.23, 130.87,
130.77, 129.78, 128.99, 125.89, 124.50, 115.27, 115.04, 114.35,
114.09, 112.47, 112.17, 46.12, 40.55, 31.96, 13.92. FT-MS m/z
Calcd.forC27H27FN6O3 ([MþH]þ):503.22014.Found:503.21804.
White solid; yield: 80%; m.p.: 258.3–259.0°C; 1H NMR
(500 MHz, DMSO-d6) d 9.10 (s, 1H), 8.86 (s, 1H), 7.89 (d,
J ¼ 8.6 Hz, 1H), 7.74 (d, J ¼ 9.0 Hz, 2H), 7.70 (d, J ¼ 7.9 Hz, 1H),
7.68–7.63 (m, 2H), 7.61 (s, 1H), 7.53–7.44 (m, 2H), 6.91 (d,
J ¼ 8.4 Hz, 2H), 5.01 (s, 2H), 4.63 (s, 2H), 3.49 (dt, J ¼ 14.1,
7.4 Hz, 2H), 2.72 (qt, J ¼ 15.8, 6.6 Hz, 2H), 1.34 (t, J ¼ 6.9 Hz,
3H). 13C NMR (125 MHz, DMSO-d6) d 172.51, 168.65, 164.19,
152.85, 152.32, 139.81, 133.77, 133.28, 132.90, 132.04, 129.74,
129.16, 127.97, 124.84, 115.63, 114.26, 112.95, 112.02, 111.52,
46.66, 31.82, 14.16. FT-MS m/z Calcd. for C28H27F3N6O3
([MþH]þ): 553.21695. Found: 553.21460.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(4-fluorophenyl)-1H-benzo[d]imidazole-5-carboxamido)-
propanoic acid (12c)
White solid; yield: 51%; m.p.: 249.8–251.9°C; 1H NMR
(500 MHz, DMSO-d6) d 9.02 (s, 1H), 8.79 (s, 1H), 7.69 (dd,
J ¼ 17.4, 8.4 Hz, 3H), 7.62 (s, 1H), 7.34 (d, J ¼ 8.3 Hz, 1H), 7.30
(dd, J ¼ 7.9, 5.2 Hz, 2H), 7.10 (t, J ¼ 8.5 Hz, 2H), 6.90 (d,
J ¼ 8.6 Hz, 2H), 4.87 (s, 2H), 4.40 (q, J ¼ 6.4 Hz, 2H), 4.04 (t,
J ¼ 7.2 Hz, 2H), 2.56 (t, J ¼ 7.2 Hz, 2H), 1.31 (t, J ¼ 7.1 Hz, 3H).
13C NMR (125 MHz, DMSO-d6) d 172.44, 168.28, 164.12, 161.62,
159.19, 152.63, 152.00, 134.13, 132.11, 130.34, 130.25, 129.78,
129.13, 125.83, 116.20, 115.98, 114.64, 114.40, 112.36, 112.16,
46.10, 40.47, 31.93, 13.95. FT-MS m/z Calcd. for C27H27FN6O3
([MþH]þ): 503.22014. Found: 503.21761.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(3-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoic acid (12g)
White solid; yield: 44%; m.p.: 225.1–226.0°C; 1H NMR
(500 MHz, DMSO-d6) d 9.11 (s, 1H), 8.85 (s, 1H), 7.89 (d,
J ¼ 7.5 Hz, 1H), 7.73 (s, 4H), 7.48 (d, J ¼ 26.9 Hz, 4H), 6.90 (d,
J ¼ 8.5 Hz, 2H), 5.04 (s, 2H), 4.53–4.47 (m, 2H), 4.07 (s, 2H), 2.56
(s, 2H), 1.33 (d, J ¼ 13.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) d
172.41, 168.47, 164.07 (t, J ¼ 7.7 Hz), 152.63, 151.92, 133.94,
132.29, 132.13, 130.40, 129.98, 129.77, 129.66, 129.01, 125.75,
124.70, 123.76, 122.09, 114.65–114.47 (m), 114.29, 112.52,
112.15, 46.15, 40.55, 31.98, 13.91. FT-MS m/z Calcd. for
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-N-(3,4-
difluorophenyl)-1-ethyl-1H-benzo[d]imidazole-5-
carboxamido)propanoic acid (12d)
C
28H27F3N6O3 ([MþH]þ): 553.21695. Found: 553.21798.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoic acid (12h)
White solid; yield: 57%; m.p.: 228.5–229.0°C; 1H NMR (500 MHz,
DMSO-d6) d 9.12 (s, 1H), 8.89 (s, 1H), 7.91 (d, J ¼ 8.5 Hz, 1H), 7.74
(d, J ¼ 8.6 Hz, 3H), 7.59 (ddd, J ¼ 10.0, 7.3, 2.2 Hz, 1H), 7.49 (d,
J ¼ 7.9 Hz, 1H), 7.33 (q, J ¼ 9.3 Hz, 1H), 7.11 (d, J ¼ 7.3 Hz, 1H),
6.93(d,J ¼ 8.8 Hz,2H), 5.05(s,2H), 4.51(d,J ¼ 7.1 Hz, 2H),4.04(t,
White solid; yield: 99%; m.p.: 213.8–215.1°C; 1H NMR (500 MHz,
DMSO-d6) d 9.09(s, 1H), 8.86(s, 1H), 7.88(d, J ¼ 8.7 Hz, 1H), 7.78–
7.71 (m, 3H), 7.65 (d, J ¼ 8.5 Hz, 2H), 7.48 (dd, J ¼ 15.8, 8.5 Hz,
3H), 6.92 (d, J ¼ 8.8 Hz, 2H), 5.03 (s, 2H), 4.50 (q, J ¼ 7.0 Hz, 2H),
3.43 (q, J ¼ 7.0 Hz, 2H), 2.58 (t, J ¼ 7.3 Hz, 2H), 1.36 (t, J ¼ 7.2 Hz,
3H). 13C NMR (125 MHz, DMSO-d6) d 172.37, 168.50, 164.16 (t,
J ¼ 7.6 Hz), 152.84, 152.14, 146.33, 133.10, 132.91, 130.83,
129.74, 128.59, 127.05, 126.74, 126.46–126.21 (m), 125.54,
125.16, 115.42,114.44(d,J ¼ 6.7 Hz), 112.09, 55.91, 46.28,32.06,
18.40, 14.06. FT-MS m/z Calcd. for C28H27F3N6O3 ([MþH]þ):
553.21695. Found: 553.21682.
J ¼ 6.4 Hz, 2H), 2.57 (t, J ¼ 7.0 Hz, 2H), 1.36 (t, J ¼ 7.2 Hz, 3H). 13
C
NMR (125MHz, DMSO-d6) d 172.44, 168.23, 164.14, 152.72,
152.03, 149.41, 149.28, 147.78, 147.64, 146.95, 146.83, 133.76,
132.31, 129.78, 129.34, 125.73, 117.77, 117.60, 114.59, 112.44,
112.16, 46.09, 40.45, 31.94, 13.97. FT-MS m/z Calcd. for
C
27H26F2N6O3 ([MþH]þ): 521.21072. Found: 521.21102.
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-N-(3,5-
difluorophenyl)-1-ethyl-1H-benzo[d]imidazole-5-
carboxamido)propanoic acid (12e)
3-(2-(((4-Carbamimidoylphenyl)amino)methyl)-1-ethyl-N-
(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazole-5-
carboxamido)propanoic acid (12i)
White solid; yield: 99%; m.p.: 254.5–257.0°C; 1H NMR
(500 MHz, DMSO-d6) d 9.09 (s, 1H), 8.86 (s, 1H), 7.91 (d,
J ¼ 8.6 Hz, 1H), 7.78–7.71 (m, 3H), 7.52 (d, J ¼ 8.7 Hz, 1H), 7.19–
7.08 (m, 3H), 6.93 (d, J ¼ 8.7 Hz, 2H), 5.03 (s, 2H), 4.51 (q,
White solid; yield: 55%; m.p.: 219.6–226.2°C; 1H NMR
(500 MHz, DMSO-d6) d 9.02 (s, 1H), 8.80 (s, 1H), 7.71 (d,
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
419

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2015, 348, 408–420
H. Chen and Y. Ren
Archiv der Pharmazie
J ¼ 8.5 Hz, 3H), 7.67 (s, 1H), 7.37 (dd, J ¼ 16.8, 8.6 Hz, 3H), 7.27
(d, J ¼ 8.3 Hz, 2H), 6.91 (d, J ¼ 8.6 Hz, 2H), 4.90 (s, 2H), 4.42 (q,
J ¼ 6.1 Hz, 2H), 4.06 (t, J ¼ 7.2 Hz, 2H), 2.57 (t, J ¼ 7.2 Hz, 2H),
1.32 (t, J ¼ 7.1 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) d 172.38,
168.26, 164.11 (q, J ¼ 7.6 Hz), 152.72, 152.02, 146.54, 141.47,
133.80, 132.33, 130.00, 129.76, 129.54, 125.76, 121.75, 121.09,
118.54, 114.70, 112.27, 112.13, 55.91, 46.24, 31.98, 18.39,
[3] A. Straub, S. Roehrig, A. Hillisch, Curr. Top. Med. Chem.
2010, 10, 257–269.
[4] F. Misselwitz, S. D. Berkowitz, E. Perzborn, Ann. N. Y.
Acad. Sci. 2011, 1222, 64–75.
[5] M. W. Jeanine, Pathophysiol. Haemost. Thromb. 2002,
32, 9–14.
[6] N. H. Hauel, H. Nar, H. Priepke, U. Ries, J. M. Stassen,
W. Wienen, J. Med. Chem. 2002, 45, 1757–1766.
[7] M. Ieko, Curr. Opin. Invest. Drugs 2007, 8, 758–768.
[8] J. V. Ryn, J. Stangier, S. Haertter, K. H. Liesenfeld,
W. Wienen, M. Feuring, Thromb. Haemost. 2010, 103,
1116–1127.
[9] G. J. Hankey, J. W. Eikelboom, Circulation 2011, 123,
1436–1450.
[10] W. K. Hagmam, J. Med. Chem. 2008, 51, 4359–4369.
[11] S. Purser, P. R. Moore, S. Swallow, V. Gouverner, Chem.
Soc. Rev. 2008, 37, 320–330.
13.95. FT-MS m/z Calcd. for
C
28H27F3N6O4 ([MþH]þ):
569.21186. Found: 569.20958.
Thrombin assay
Different dilutions of the test compounds dissolved in DMSO
were preincubated for 10 min at 37°C. The national standard,
lyophilized human thrombin (5.4 mg/mL), was purified from
human blood. After that, Ac-FVR-AMC (5 mM), a specific
fluorogenic thrombin substrate, was added to above system.
We detected the dynamic changes in relative fluorescence
intensity using an Envision microplate reader (PerkinElmer) at
room temperature within 10 min. The slope of the linear
enzyme dynamics curve during the initial stage of the reaction
was referred to as the initial velocity of enzyme reaction. The
known thrombin inhibitor argatroban was used as a positive
control. Instrument settings included the following: excita-
tion wavelength, 355 nm and emission wavelength, 460 nm.
Each well was measured 20 times every 20 s for about 10 min.
The change in fluorescence within a predetermined time was
measured under these conditions. The reaction kinetic curve
slope (vmax) was used as an activity indicator.
[12] K. L. Kirk, J. Fluorine. Chem. 2006, 127, 1013–1029.
[13] C. Isanbor, D. O’Hagan, J. Fluorine. Chem. 2006, 127,
303–319.
€
[14] K. Muller, C. Faeh, F. Dicderich, Science 2007, 317, 1881–
1886.
[15] K. L. Kirk, Org. Process. Res. Dev. 2008, 12, 305–321.
[16] H. Choo, Y. Chong, Y. Choi, J. Mathew, R. F. Schinazi,
C. K. Chu, J. Med. Chem. 2003, 46, 389–398.
[17] M. D. Johnson, J. Chen, B. D. Anderson, Drug Metabol.
Dispos. 2002, 30, 191–198.
[18] F. Tanaka, T. Fukuse, H. Wada, M. Fukushima, Curr.
Pharm. Biotechnol. 2000, 1, 137–164.
[19] J. D. McCarter, M. J. Adam, N. G. Hartman, S. G. Withers,
Biochem. J. 1994, 301(2), 343–348.
The concentration that induced an IC50 was calculated. All
measurements were performed in duplicate; the mean values
of both determinations are presented.
[20] J. Wang, H. Liu, Chin. J. Org. Chem. 2011, 31, 1785–1798.
[21] F. Schiele, J. V. Ryn., K. Canada, C. Newsome, E. Sepulveda,
J. Park, H. Nar, T. Litzenburger, Blood. 2013, 121, 3554–
3562.
This work is supported by the Science and Technology
Commission of Shanghai Municipality (No. 13142201001,
13DZ1930402, 13DZ 1930403).
[22] J. K. Moon, Y. S. Keun, E. C. Hwang, B. S. Park,
H. R. Chang, Q. X. Li, J. K. Kim, J. Agric. Food Chem.
2007, 55, 5416–5422.
The authors have declared no conflict of interest.
[23] M. Clark, R. D. Cramer III, N. V. Opdenbosch, J. Comput.
Chem. 1989, 10, 982–1012.
[24] J. Gasteiger, M. Marsili, Tetrahedron. 1980, 36, 3219–
3222.
References
[1] N. Mackman, Nature 2008, 451, 914–918.
[2] E. Perzborn, S. Roehring, A. Straub, D. Kubitza,
F. Misselwitz, Nat. Rev. Drug Discov. 2011, 10, 61–75.
[25] J. Sun, S. Cai, N. Yan, H. Mei, Eur. J. Med. Chem. 2010, 45,
1008–1014.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
420