Design, synthesis and biological activity evaluation of novel methyl substituted benzimidazole derivatives

Article abstract of DOI:10.1016/j.tet.2020.131027

Ten new dabigatran derivatives (7a–j) with high docking scoring were designed, synthesised and biologically evaluated. The inhibitory in vitro activity of these compounds on thrombin was evaluated on the basis of preliminary activity screening results. The IC₅₀ values of compounds 7a, 7d and 7j were 1.92, 2.17 and 1.54 nM, respectively, and are equivalent to the dabigatran (IC₅₀ = 1.20 nM). Therefore, the most active compound, 7j, was selected to further investigate the anticoagulant activity in rats. Compound 7j presented excellent in vivo inhibitory effects on arteriovenous thrombosis, and the inhibition rate was (84.19 ± 1.14) %. The anticoagulant activity of compound 7k synthesised in the previous work was evaluated in vivo, and its inhibition rate was (85.58 ± 2.89) %. This rate was nearly equivalent to that of dabigatran (85.07 ± 0.61) %. Results indicated that compounds 7a, 7d, 7j and 7k can be further studied as novel antithrombin drug candidates.

Full text of DOI:10.1016/j.tet.2020.131027

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Design, synthesis and biological activity evaluation of novel methyl substituted
benzimidazole derivatives
Tianrong Zhang, Qianqian Liu, Yujie Ren
PII:
S0040-4020(20)30134-4
https://doi.org/10.1016/j.tet.2020.131027
TET 131027
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 20 November 2019
Revised Date: 5 February 2020
Accepted Date: 6 February 2020
Please cite this article as: Zhang T, Liu Q, Ren Y, Design, synthesis and biological activity evaluation
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Design, Synthesis and Biological Activity Evaluation of Novel Methyl Substituted
Benzimidazole Derivatives
Tianrong Zhang, Qianqian Liu, Yujie Ren*,
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China
* Corresponding author. College of Chemical and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan
Road, Shanghai 201418, China.
E-mail address: clab@sit.edu.cn (Y.-J. Ren).
Abstract
Ten new dabigatran derivatives (7a–j) with high docking scoring were designed,
synthesised and biologically evaluated. The inhibitory in vitro activity of these
compounds on thrombin was evaluated on the basis of preliminary activity screening
results. The IC50 values of compounds 7a, 7d and 7j were 1.92, 2.17 and 1.54 nM,
respectively, and are equivalent to the dabigatran (IC50 = 1.20 nM). Therefore, the
most active compound, 7j, was selected to further investigate the anticoagulant
activity in rats. Compound 7j presented excellent in vivo inhibitory effects on
arteriovenous thrombosis, and the inhibition rate was (84.19±1.14) %. The
anticoagulant activity of compound 7k synthesised in the previous work was
evaluated in vivo, and its inhibition rate was (85.58±2.89) %. This rate was nearly
equivalent to that of dabigatran (85.07 ± 0.61) %. Results indicated that compounds
7a, 7d, 7j and 7k can be further studied as novel antithrombin drug candidates.
Keywords: Design ꢀSynthesis ꢀMethyl Substituted Benzimidazole ꢀbiological
eva-luationꢁMolecular docking
1. Introduction
Cardiovascular diseases (CVDs) are the leading cause of death worldwide. The
World Health Organisation reported that 17.5 million people die of CVDs annually,
accounting for approximately 31% of the world’s death population [1]. Therefore,
scientists have exerted considerable effort into researching different kinds of drugs to
treat CVDs. Arterial and venous thrombotic diseases represent a major pathogenic
factor of CVDs and can be fatal if improperly managed. Excessive stimulation of the
coagulation cascade is closely linked to thrombotic diseases [2]. Thrombin is a
multifunctional specific trypsin-like serine proteinase that is crucial in the coagulation
cascade. Thrombin also plays an important role in anticoagulation processes by
converting soluble fibrinogen to insoluble fibrin. Therefore, thrombin is an attractive
target for CVD therapy. Antithrombotic drugs have been recognised as the best for the
treatment of CVDs and their complications [3-5]. Antithrombotic drugs are mainly
[6]
classified into indirect thrombin inhibitors and direct thrombin inhibitors (DTIs)
.
Thrombin is crucial in the blood coagulation process in these inhibitors and facilitates
coagulation by specifically hydrolysing fibrinogen to fibrin [7]. At present, indirect
thrombin inhibitors mainly involve unfractionated heparin. However, unfractionated
heparin would interfere with platelet function and raise the risk of increased bleeding
due to its large molecular weight [8], These conditions would result in the gradual
withdrawal of unfractionated heparin from the market. Direct thrombin inhibitors
1

(DTIs) (Fig. 1), which can directly bind to thrombin, are the most promising new
anticoagulants [9], mainly include argatroban [10, 11], ximelagatran [12] and dabigatran
etexilate [13]. These DTIs have been extensively studied due to their unique
anticoagulant properties [14]. DTIs are a group of small molecules that can directly
inhibit thrombin activity without cofactors, which can effectively inhibit free and
bound thrombins to blood clots. Moreover, DTIs do not bind to plasma proteins,
thereby resulting in predictable pharmacodynamic and pharmacokinetic
characteristics [15]. However, the therapeutic effect of argatroban is limited because of
its low oral bioavailability. Meanwhile, ximelagatran has been withdrawn from
clinical development due to drug-induced severe liver damage and low oral
bioavailability [16, 17]. Therefore, dabigatran etexilate has become the only oral, safe
and effective DTI available on the market [18-20]. Dabigatran was initially approved in
Europe and later in 2010 by the United States Food and Drug Administration (FDA)
[21-24]
for reducing the risk of stroke in non-valvular atrial fibrillation. (AF)
.
Dabigatran is orally administered as dabigatran etexilate and converted into its active
form by dabigatran etexilate [25]. Monoclonal anti-idarucizumab has been recently
found to reverse the anticoagulant activity induced by dabigatran by rapidly binding
to the latter. This reversal can effectively resolve dyspepsia, liver damage and
bleeding events [26-28]. Therefore, dabigatran etexilate currently remains a safe and
effective drug for the treatment of CVDs. Over 75% of CVD deaths occurred in low
and middle-income countries [1], but the high price of dabigatran etexilate imposes a
heavy financial burden on patients and families. However, dabigatran etexilate
efficacy is limited by poor bioavailability and the bleeding risk at high does [29]. Its
bioavailability is only 6.5% [30,31]. Therefore, the structure of dabigatran could be
modified to identify active candidate compounds with comparable or superior
antithrombin activity and bioavailability. Finding additional dabigatran analogues
may be vital to alleviate the economic burden of patients, improve bioavailability,
reduce the rise of bleeding.
Hauel [32] reported that the benzamidine ring of dabigatran interacted with residues,
such as Leu99, Ile174 and Trp215, in the thrombin active site cleft and formed a
bidentate salt bridge with the carboxylate of residue Asp189; the pyridine ring of
dabigatran is not essential for bioactivity. N-methyl is highly suitable for the P-pocket
located in the active site of the enzyme, and benzamidine is docked to the D-pocket
[34]
via hydrophobic interactions [33]. Guo-qiang Lin
obtained a novel non-peptide
thrombin inhibitor by replacing the pyridine ring in dabigatran etexilate with a
[35]
benzene ring. Johnson et al.
established that the introduction of a single methyl
group with a lipophilic efficiency can effectively increase the efficacy of a compound
commonly known as ‘magic methyl’. The introduced methyl group optimally
occupies the lipophilic pocket on the target protein, and the van der Waals force
between the ligand and the protein increases, thereby effectively increasing the
binding efficiency [36-39]. Therefore, given the above research conclusions, the
pyridine ring of dabigatran was replaced with a benzene ring and a methyl group was
simultaneously introduced at different positions to explore a remarkably effective
thrombin inhibitor as a candidate compound (Fig. 2).
2

This study employed a molecular model and a scoring technology to avoid
blindness and save resources during the synthesis and predict the bioactivity of the
designed compounds. Targeted compounds with a total score higher than the positive
control dabigatran were selected for synthesis (Table 1). The synthesised compounds
were screened by their thrombin inhibitory activities in vitro. Most of the compounds
exhibit promising inhibitory activities within the range of the reference drug
dabigatran. On this basis, the most active compound in vitro was further selected to
complete the antithrombotic activity test in vivo. The results of the present study will
lay the foundation for the design, synthesis and biological evaluation of novel
dabigatran derivatives and further provide candidate compounds for new
anticoagulant drugs.
2. Results and discussions
HO
O
O
NH
NH2
O
O
HN
HN
H
N
N
H
N
O
HN
O
HO NH
S
H
O
N
ximelagatran
argatran
O
O
O
O
N
N
N
O
N
NH2
N
N
HO
in vivo converted to
HN
O
O
NH2
NH
N
N
HN
N
dabigatran
dabigatran etexilate
Fig.1. Chemical structures of direct thrombin inhibitors.
Fig. 2. Design strategy of dabigatran derivatives.
3

Table 1 The results obtained from docking studies of compounds 7a-j.
Compound
Total-score
12.3874
11.2158
11.7354
12.8474
12.8667
11.8716
10.8182
10.9033
12.4723
12.5338
10.3345
Key Residues
Asp189; Gly219; Gly216; Thr172
Asp189(2); Gly219
7a
7b
Asp189(2); Gly216; Thr172(2)
Asp189; Gly219; Gly216
Asp189; Gly219
7c
7d
7e
Asp189; Gly219; Glu97A
Asp189(2); Gly216; Thr172
Asp189(2); Gly219; Gly216; Asn98
Asp189;Gly219; Glu217; Thr172
Asp189(2); Gly219; Gly216; Thr172
Asp189(2); Gly219; Gly216
7f
7g
7h
7i
7j
Dabigatran
2.1 Chemistry
The synthesis of compounds 7a–j using a convenient synthetic route is shown in
Scheme 1. Ten target compounds with different substituents 2a–j were synthesised
using ethyl acrylate and corresponding amines 1a–j with trifluoromethanesulfonic
acid as a catalyst at 100 °C by refluxing for 16 h. Amongst these compounds, 2i–j
were white solid with low yield (44%–50%), whilst 2g–h were brown-red oil with
high yield (77%–89%). Electron-donating methoxy-substituted aniline may be
beneficial for the Michael addition reaction. Compounds 2a–j reacted with
4-(methylamino)-3-nitrobenzoyl chloride to generate important intermediates 3a–j.
[32]
Hauel
used 10% Pd/C and H2 in methanol/dichloromethane at room temperature.
Meanwhile, the reductant of zinc and ammonium chloride in tetrahydrofuran with
water at 80 °C was previously explored for 5 h under N2 to reach the reduction
reactions of compounds 3a–j to 4a–j. Zinc in acetic acid was used with water as a
solvent at room temperature only for 1 h to prepare compounds 4a–j. This method
was simple and did not require any gas piping. Interestingly, the reductant can be
easily recycled, and the yields were all more than 90%. This conventional reduction
method of the nitro could be applied for the technical study of dabigatran. Compounds
5a–j were prepared by treating 4a–j with 2-[(4-cyanophenyl)-amino]-acetic acid in
the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and
1-hydroxybenzotriazole. Cyano compounds 5a–j with hydroxylamine hydrochloride
and triethylamine under ethanol provided the corresponding hydroxamic acids by
4

refluxing at 80 °C for 3 h, followed by 10% Pd/C reduction under an N2 atmosphere
to obtain the amidino compounds 6a–j. These amidino compounds were hydrolysed
in the presence of sodium hydroxide in ethanol with water at room temperature for 1 h
to obtain the target compounds 7a–j. These target compounds were then characterised
by 1H-NMR, 13C-NMR and HR-MS.
Scheme 1 Synthesis of dabigatran derivatives. Reagents and conditions: (a) TfOH, N2, 100 °C, reflux, 12h; (b) CH3NH2, 75 °C,
reflux, 5h; (c) DCM, SOCl2, DMF, 45 °C, reflux,1h; (d) DCM, Et3N, rt, 4h; (e) Zn, AcOH/H2O, rt, 1h; (f) ꢂ EDCI, HOBt,
4-cyano-2-substituted-phenylamino)-acetic acid, THF/DMF, rt, 5h; ꢃAcOH, 120 °C, reflux, 2.5h; (g) ꢂ NH2OH·HCl, Et3N,
EtOH, 80 °C, reflux, 3h; ꢃ HCOONH4, Pd/C, AcOH, 120 °C, reflux, 5h; (h) NaOH, EtOH/H2O, rt, 1h.
2.2 Antithrombin activity in vitro
The thrombin inhibitory activities of the synthesised dabigatran analogues 7a–j
were measured with the chromogenic assay in vitro, with dabigatran as the positive
control. All these compounds exhibited more than 50% inhibition effects after
preliminarily screening for inhibitory activity against thrombin at 1 ꢀg/mL
concentration (Fig. 3). The IC50 values of these compounds for thrombin inhibition
are shown in Table 2. Most of the compounds exhibit excellent activity against
thrombin in vitro, except for compounds 7g–h. The activity data were consistent with
the results predicted by molecular docking simulation.
Amongst the synthesised compounds, 7a, 7d, 7i and 7j showed the most potent
thrombin inhibition with respective IC50 values of 1.92, 2.17, 2.58 and 1.54 nM.
5

These values are comparable to those of dabigatran (1.20 nM). Table 2 indicates that
the positions of the electron-donating methyl group on the ring may affect the
antithrombin activity. Compound 7a with the methyl group at the para position
exhibited lower activities relative to 7b. Compounds 7i–7j with a methyl group on the
pyridine ring exhibited inhibitory activities in the following order: 7i (5-CH3) < 7j
(6-CH3). Therefore, the methyl group at a position farther from the nitrogen atom in
the pyridine ring may weaken antithrombin activities. When two methyl groups were
placed on the ring and one methyl group was at the para position, the thrombin
inhibitory activity of compounds 7c and 7e decreased to 4.62 and 4.37 nM,
respectively, regardless of another methyl position. Overall, the modification of the
para position may be detrimental to thrombin inhibitory activities. The introduction of
a methoxy group to the rings of 7g and 7h was more detrimental than that observed in
the ring of 7a. Therefore, a strong electron-donating effect on the ring could
negatively influence the antithrombin activity.
Fig. 3 Thrombin inhibitory activity of synthesized compounds at 1 ꢀg/mL concentration
Table 2 Thrombin inhibitory activities of twelve compounds.
Compound
R
X
IC50(nM)a,b
1.92±0.25
4.44±0.60
4.62±0.24
2.17±0.24
4.37±0.48
5.90±1.28
-
3-CH3
4-CH3
2,4-CH3
2,5-CH3
3,4-CH3
3,5-CH3
3-OCH3
4-OCH3
5-CH3
6-CH3
2-CH3
CH
CH
CH
CH
CH
CH
CH
CH
N
7a
7b
7c
7d
7e
7f
7g
-
7h
2.58±0.41
1.54±0.12
2.74±0.20
1.20±0.09
7i
N
7j
CH
7k
Dabigatran
6

a) The concentrations that causes a 50% inhibition of thrombin;
b) Results were expressed as means±SD of six independent experiments.
2.3 Inhibitory effects on rat arteriovenous thrombosis in vivo
An arteriovenous thrombosis model was used to evaluate the antithrombotic
activities of intravenous administration of compounds 7j and known compound 7k.
Dabigatran served as a reference drug. Compounds 7j and 7k were given as a
pre-treatment and exerted significant effects on rat arteriovenous thrombosis with
inhibition rates of (84.19 ± 1.14) % and (85.58 ± 2.89) %, respectively. Such
outcomes are comparable to those of dabigatran (85.07 ± 0.61) %. The results are
shown in Table 3.
Table 3 Antithrombotic activities of intravenous administration of synthesized compounds
Compounds
Concentrations
Thromboembolic
weight(mg)
The inhibition rate (%)a
CMC-Na
Dabigatran
7j
-
42.04±5.09
0.00±12.11
0.5 mg/mL
0.5 mg/mL
0.5 mg/mL
6.28±0.26**
6.65±0.48**
6.06±1.22**
85.07±0.61**
84.19±1.14**
85.58±2.89**
7k
**ꢄp<0.01 versus the CMC-Na group;
a) Results were expressed as means±SD of eight independent experiments.
2.4 Computer-aided simulation
2.4.1 Activity predicted by Computer simulation
By introduction of giving-electronics substituent into the benzene ring to replace
the pyridine ring of dabigatran, ten new dabigatran derivatives were designed.
Molecular docking and scoring technologies were applied to discover drugs and
predict the activities of these new derivatives. Targeted compounds with a total score
higher than dabigatran (10.3345) were selected for synthesis. Except for compounds
7g and 7h, other compounds showed considerable antithrombin activities. It is
noteworthy that the R group of compounds 7g and 7h linked to a methoxyl, which
may be one of the main reasons for no activities.
2.4.2 Molecular docking analysis
To further validate the rationality of our designed compounds, the molecular
docking study was performed using the Surflex-docking module in SYBYL-X 2.0.
The cognate ligand (Dabigatran) and compound 7j with good antithrombotic activity
were docked into the active site of the thrombin receptor (PDB code: 1KTS), as
shown in Fig. 4. It was found that the compound 7j we synthesized formed two
significant hydrogen bond interactions with the active pocket from Fig. 4D. The
distance of hydrogen bonds observed were 2.99Å (Glu97=O···H-N-) and 2.91 Å
(Trp215-OH···O=). Compound 7j was tightly fixed around the receptor through these
two hydrogen bonds. Moreover, residue His57 and Ile174 formed T-shaped π-π
stacking and π- Alkyl interactions, respectively, which illustrated that the
multi-interactions enhanced the degree of binding pattern. Docking results and
7

interactions map of Dabigatran were shown in Fig. 4A and 4C. Four important
hydrogen bond interactions between Dabigatran and pocket were formed. The
distances were 2.91 Å (Phe227=O···H-N-), 3.10Å (Phe227=O•••H-N-), 2.96Å
(Cys220-S•••H-N-) and 2.82 Å (Gly219=O···H-N-), respective. Furthermore, Pi-Pi
stacked, carbon hydrogen bond and Pi-Alkyl interactions also enhanced the pesticide
effect of Dabigatran. Different weak interactions make the binding conformations of
7j-1KTS and Dabigatran-1KTS different. Meanwhile, compounds 7g and 7k were
docked into 1KTS pocket and the results were illustrated in Fig. S1.
Fig. 4 Docking results and 2D interaction maps of Dabigatran (A and C) and selected compound 7j (B and D) in the binding site
of thrombin receptor (PDB code: 1KTS).
Conclusion
Ten dabigatran derivatives (7a–7j) were synthesised in this paper by introducing
methyl and methoxy groups at different positions in dabigatran. The thrombin
inhibitory activities in vitro were then evaluated. In addition to compounds 7g and 7h,
other compounds showed effective inhibitory activity against thrombin during the
initial screening in vitro. The preliminary relationship between the structure and
activity of these compounds revealed that the introduction of methyl groups on the
benzene ring can enhance the anticoagulant activity. Moreover, the methyl group can
effectively activate the docking pocket and increase the lipophilic efficiency.
Comparative studies indicated that the introduction of methoxy at these positions
8

reduces inhibitory potency. The order of thrombin inhibition activities in vitro is as
follows: compounds 7j > 7a. In particular, the IC50 values of compounds 7a, 7d and 7j
were 1.92, 2.17 and 1.54 nM, respectively. These outcomes are comparable to the
anticoagulant activity of the reference drug dabigatran. The anticoagulant activities in
rats of compounds 7j and 7k were further studied. Both activities demonstrated
effective in vivo activities in inhibiting arteriovenous thrombosis. The inhibition rates
of compounds 7j and 7k were (84.19 ± 1.14) % and (85.58 ± 2.89) %, respectively,
and these values are nearly equivalent to those of dabigatran (85.07 ± 0.61) %.
Molecular docking studies were conducted to generate valuable insights into the
binding of active compounds to receptors and help clarify their inhibitory mechanisms.
The in vitro results were consistent with those from molecular docking. Hence,
compounds 7a, 7d, 7j and 7k can be further studied as antithrombin drug candidates.
4 Experimental
4.1. Materials and methods
Unless otherwise noted, all the chemicals and solvents were purchased from
Darui and Titan Corporation, and used without further purification. Reaction
course was routinely monitored by thin-layer chromatography on silica gel
under UV light (254 nm). The products were purified by column
chromatography (200–300 mesh) with designated solvents. The melting points
were measured using a WPS-2A digital melting point apparatus and were
uncorrected. High-resolution mass spectra (HRMS) were obtained using a
SolariX-70FT-MS Bruker spectrometer equipped with electrospray ionisation
(ESI) equipment. 1H and 13C NMR spectra were obtained in CDCl3 or
DMSO-d6 solution using a Bruker Avance 400 magnetic resonance
spectrometer at 400 MHz, respectively, with tetramethylsilane (TMS) as the
internal reference.
4.2. General procedure for preparation of compounds 2a-2j
Compounds 1a-1j(50mmol) and ethyl acrylate
2 (6.0g, 60mmol) were mixed
in a 100 mL dry round bottom flask. TfOH (5mmol) as catalyst was added. The
mixture was stirred for 16h at reflux temperature in a nitrogen atmosphere. The
reaction was detected by TLC. The unreacted ethyl acrylate was concentrated
under reduced pressure. The residue was purified by silica gel chromatography
(PE/ EtOAc = 6:1-1:1) to afford pure compounds 2a-2j as brown red oil or
white solid in 44-89% yield.
4.3. General procedure for preparation of compounds 3a-3j
4-Chloro-3-nitrobenzoic acid (9.0 g, 45 mmol) and aqueous methylamine
(865 mmol) were mixed in 250 mL dry round bottom flask. The mixture was
heated to reflux for 6h at 75 °C. Upon completion, PH value of the mixture was
adjusted with acetic acid to 4~5. The solids were filtrated and washed carefully
with
water
and
methanol,
and
dried
to
afford
yellow
4-(methylamino)-3-nitrobenzoic acid. To a DCM (50 mL) solution of
4-(methylamino)-3-nitrobenzoic acid (6.8 g, 35 mmol), five drops of DMF were
added. SOCl2 (20 mL, 0.23 mol) was added slowly via addition funnel. The
mixture was stirred at room temperature (30°C) for 5h. The reaction was
9

detected by TLC. After completion of the reaction, the mixture was
concentrated under reduced pressure to obtain bright yellow solid
4-methylamino-3-nitrobenzoyl chloride. To a DCM (50 mL) solution of
compound 2a-2j (35 mmol) containing triethylamine (TEA) (35 mmol), a DCM
(40 mL) solution of the yellow solid chloride was slowly added at room
temperature and then the mixture was stirred for 5h until completion of the
reaction. Upon completion, the reaction mixture was washed with water and
extracted with DCM in the separatory funnel, dried by anhydrous sodium
sulfate (Na2SO4) and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (PE/ EtOAc =3:1-1:1) to afford pure
yellow solid compounds 3a-3j in 69-82% yield.
4.4. General procedure for preparation of compounds 4a-4j
To a solution of compounds 3a-3j (21mmol) in 100 mL acetic acid and 50
mL water was added Zn (6.6g, 105mmol). This mixture was stirred for 1h at
room temperature. After completion of the reaction, the Zn was filtered, and
the mixture was extracted with DCM (3×40 mL). The DCM layer was dried by
anhydrous Na2SO4, and concentrated under reduced pressure. The residue was
purified by silica gel chromatography (PE/ EtOAc =1:1) to obtain white solid
compounds 4a-4j
.
4.4.1.
1
acid ethyl ester (4a). Yield: 94.0%; mp: 138.9-139.6 °C. H NMR (400
MHz, CDCl3) δ 7.06 (t, = 7.7 Hz, 1H, Ar-H), 6.92 (d, = 7.6 Hz, 1H, Ar-H),
6.89 (s, 1H, Ar-H), 6.84 (s, 1H, Ar-H), 6.79 (d, = 7.9 Hz, 1H, Ar-H), 6.70 (d,
= 8.3 Hz, 1H, Ar-H), 6.25 (d, = 8.3 Hz, 1H, Ar-H), 4.13 (t, = 7.4 Hz, 2H,
-CH2-), 4.03 (q, = 7.2 Hz, 2H, -CH2-), 2.74 (s, 3H, -NCH3), 2.66 (t, = 7.4
J
J
J
J
J
J
J
J
Hz, 2H, -CH2-), 2.24 (s, 3H, Ar-CH3), 1.17 (t, J = 7.1 Hz, 3H, -CH3).
4.4.2.
3-[(3-Amino-4-methylamino-benzoyl)-(4-methyl-phenyl)-amino]-propionic
1
acid ethyl ester (4b). Yield: 92.8%; mp: 134.4-135.4 °C. H NMR (400 MHz,
CDCl3) δ 7.00 (d,
(s, 1H, Ar-H), 6.71 (d,
4.12 (t, = 7.4 Hz, 2H, -CH2-), 4.03 (q,
J
= 8.1 Hz, 2H, Ar-H), 6.91 (d,
= 8.2 Hz, 1H, Ar-H), 6.26 (d,
= 7.1 Hz, 2H, -CH2-), 2.75 (s, 3H,
= 7.4 Hz, 2H, -CH2-), 2.25 (s, 3H, Ar-CH3), 1.17 (t, = 7.1
J
= 8.2 Hz, 2H, Ar-H), 6.82
J
J
= 8.3 Hz, 1H, Ar-H),
J
J
-NCH3), 2.65 (t,
Hz, 3H, -CH3).
4.4.3.
J
J
3-[(3-Amino-4-methylamino-benzoyl)-(2,4-dimethyl-phenyl)-amino]-propio
nic acid ethyl ester (4c). Yield: 94.9%; mp: 121.7-122.8 °C. 1H NMR (400
MHz, CDCl3) δ 6.92 (s, 1H, Ar-H), 6.89 (m, 2H, Ar-H), 6.83 (s, 1H, Ar-H),
6.66 (d, J = 7.7 Hz, 1H, Ar-H), 6.24 (d, J = 8.1 Hz, 1H, Ar-H), 4.28 (m, 1H,
-CH2-), 4.03 (q, J = 7.2 Hz, 2H, -CH2-), 3.75 (m, 1H, -CH2-), 2.75 (s, 3H,
-NCH3), 2.68 (d, J = 15.0, 6.9 Hz, 2H, -CH2-), 2.24 (s, 3H, Ar-CH3), 2.11 (s,
3H, Ar-CH3), 1.18 (t, J = 7.1 Hz, 3H, -CH3).
4.4.4.
10

3-[(3-Amino-4-methylamino-benzoyl)-(2,5-dimethyl-phenyl)-amino]-propio
1
nic acid ethyl ester (4d). Yield: 97.0%; mp: 125.6-126.3 °C. H NMR (400
MHz, CDCl3) δ 6.98 (d,
6.86 (d, = 7.0 Hz, 2H, Ar-H), 6.65 (d,
Hz, 1H, Ar-H), 4.23 (d, = 13.7, 7.0 Hz, 1H, -CH2-), 4.03 (q,
J
= 7.6 Hz, 1H, Ar-H), 6.91 (d,
= 7.7 Hz, 1H, Ar-H), 6.23 (d,
= 7.1 Hz, 2H,
J
= 7.9 Hz, 1H, Ar-H),
J
J
J
= 8.0
J
J
-CH2-), 3.82(m, 1H, -CH2-), 2.75 (s, 3H, -NCH3), 2.69 (t,
J
= 7.3 Hz, 2H,
-CH2-), 2.23 (s, 3H, Ar-CH3), 2.07 (s, 3H, Ar-CH3), 1.18 (t,
J = 7.1 Hz, 3H,
-CH3).
4.4.5.
3-[(3-Amino-4-methylamino-benzoyl)-(3,4-dimethyl-phenyl)-amino]-propio
nic acid ethyl ester (4e). Yield: 96.3%; mp: 126.9-128.2 °C. 1H NMR (400
MHz, CDCl3) δ 6.93 (d,
(m, 1H, Ar-H), 6.71 (d,
4.10 (t,
J
J
= 8.0 Hz, 1H, Ar-H), 6.85 (s, 2H, Ar-H), 6.76 – 6.72
= 2.1 Hz, 1H, Ar-H), 6.27 (d, = 8.3 Hz, 1H, Ar-H),
J = 7.5 Hz, 2H, -CH2-), 4.03 (q, J = 7.1 Hz, 2H, -CH2-), 2.75 (s, 3H,
J
-NCH3), 2.65 (t,
Ar-CH3), 1.18 (t,
4.4.6.
J
J
= 7.5 Hz, 2H, -CH2-), 2.16 (s, 3H, Ar-CH3), 2.15 (s, 3H,
= 7.1 Hz, 3H, -CH3).
3-[(3-Amino-4-methylamino-benzoyl)-(3,5-dimethyl-phenyl)-amino]-propio
nic acid ethyl ester (4f). Yield: 91.7%; mp: 134.6-135.7 °C. 1H NMR (400
MHz, CDCl3) δ 6.87 (s, 1H, Ar-H), 6.75 (s, 1H, Ar-H), 6.72 (d,
Ar-H), 6.65 (s, 2H, Ar-H), 6.27 (d, = 8.3 Hz, 1H, Ar-H), 4.10 (t,
2H, -CH2-), 4.04 (q, = 7.1 Hz, 2H, -CH2-), 2.76 (s, 3H, -NCH3), 2.65 (t,
J
= 8.2 Hz, 1H,
J
J
= 7.5 Hz,
J
J =
7.5 Hz, 2H, -CH2-), 2.19 (s, 6H, Ar-CH3), 1.18 (t, J = 7.1 Hz, 3H, -CH3).
4.4.7.
3-[(3-Amino-4-methylamino-benzoyl)-(3-methoxy-phenyl)-amino]-propioni
c acid ethyl ester (4g). Yield: 92.8%; mp: 144.8-145.9 °C. 1H NMR (400 MHz,
CDCl3) δ 7.10 (t, J = 8.3 Hz, 1H, Ar-H), 6.83 (s, 1H, Ar-H), 6.75 (d, J = 6.4 Hz,
1H, Ar-H), 6.66 (d, J = 8.7 Hz, 1H, Ar-H), 6.61 (d, J = 7.0 Hz, 2H, Ar-H), 6.28
(d, J = 8.3 Hz, 1H, Ar-H), 4.14 (t, J = 7.4 Hz, 2H, -CH2-), 4.04 (q, J = 7.1 Hz,
2H, -CH2-), 3.68 (s, 3H, -OCH3), 2.76 (s, 3H, -NCH3), 2.67 (t, J = 7.4 Hz, 2H,
-CH2-), 1.18 (t, J = 7.1 Hz, 3H, -CH3).
4.4.8.
3-[(3-Amino-4-methylamino-benzoyl)-(4-methoxy-phenyl)-amino]-propioni
1
c acid ethyl ester (4h). Yield: 97.1%; mp: 112.9-114.0 °C. H NMR (400
MHz, CDCl3) δ 6.93 (d,
8.6 Hz, 2H, Ar-H), 6.65 (d,
Ar-H), 4.07 (t, = 7.3 Hz, 2H, -CH2-), 4.01 (d,
3.70 (s, 3H, -OCH3), 2.70 (s, 3H, -NCH3), 2.61 (t,
(t, = 7.0 Hz, 3H, -CH3).
4.4.9.
J
= 8.7 Hz, 2H, Ar-H), 6.79 (s, 1H, Ar-H), 6.71 (d,
= 6.8 Hz, 1H, Ar-H), 6.22 (d, = 8.3 Hz, 1H,
= 14.8, 7.5 Hz, 2H, -CH2-),
= 7.4 Hz, 2H, -CH2-), 1.15
J =
J
J
J
J
J
J
3-[(3-Amino-4-methylamino-benzoyl)-(5-methyl-pyridin-2-yl)-amino]-prop
1
ionic acid ethyl ester (4i). Yield: 96.0%; mp: 114.5-115.8 °C. H NMR (400
MHz, CDCl3) δ 8.22 (s, 1H, Py-H), 7.17 (d,
J
= 8.2 Hz, 1H, Py-H), 6.80 (s, 1H,
= 8.2 Hz, 1H, Py-H), 6.28 (d, J
Ar-H), 6.70 (d, = 9.4 Hz, 1H, Ar-H), 6.59 (d,
J
J
11

= 8.2 Hz, 1H, Ar-H), 4.30 (t,
J
= 7.4 Hz, 2H, -CH2-), 4.02 (q,
J = 7.1 Hz, 2H,
-CH2-), 2.77 (s, 3H, -NCH3), 2.70 (t,
J
= 7.3 Hz, 2H, -CH2-), 2.23 (s, 3H,
Py-CH3), 1.17 (t, J = 7.1 Hz, 3H, -CH3).
4.4.10.
3-[(3-Amino-4-methylamino-benzoyl)-(6-methyl-pyridin-2-yl)-amino]-prop
1
ionic acid ethyl ester (4j). Yield: 97.4%; mp: 119.6-120.0 °C. H NMR (400
MHz, CDCl3) δ 7.18 (t,
6.75 (s, 1H, , Ar-H), 6.62 (d,
Py-H), 6.20 (d, = 8.3 Hz, 1H, Ar-H), 4.28 (t,
J
= 7.7 Hz, 1H, Py-H), 6.78 (d,
J
= 7.5 Hz, 1H, Py-H),
= 7.9 Hz, 1H,
= 7.3 Hz, 2H, -CH2-), 3.97 (q,
= 7.4 Hz, 2H, -CH2-),
J
= 8.2 Hz, 1H, Ar-H), 6.39 (d,
J
J
J
J
= 7.1 Hz, 2H, -CH2-), 2.68 (s, 3H, -NCH3), 2.65 (d,
J
2.44 (s, 3H, Py-CH3), 1.12 (t,
J
= 7.1 Hz, 3H, -CH3).
4.4.11.
3-[(3-Amino-4-methylamino-benzoyl)-(2-methyl-phenyl)-amino]-propionic
1
acid ethyl ester (4k). Yield: 92.2%; mp: 138.3-139.5 °C. H NMR (400 MHz,
CDCl3) δ 7.05 (d, J = 8.2 Hz, 3H, Ar-H), 7.00 (s, 1H, Ar-H), 6.75 (s, 1H,
Ar-H), 6.58 (d, J = 7.5 Hz, 1H, Ar-H), 6.14 (d, J = 7.7 Hz, 1H, Ar-H), 3.98 (q, J
= 7.1 Hz, 2H, -CH2-), 2.64 (s, 3H, -NCH3), 2.11 (s, 3H, Ar-CH3), 1.13 (t, J =
7.1 Hz, 3H, -CH3).
4.5. General procedure for preparation of compounds 5a-5j
To a solution of (4-cyano-2-substitutedphenylamino)-acetic acid (3.3g, 19
mmol), HOBt (2.5g, 19 mmol) and EDCI (3.6g, 19 mmol) in 70 mL
tetrahydrofuran (THF) and 10 mL DMF was stirred under the ice baths. After
30 min, the compounds 4a-4o (16mmol) were dissolved in 40 mL
tetrahydrofuran (THF), and slowly added into this system. The mixture was
stirred at room temperature for 5h. The solvent was concentrated and extracted
with DCM (3×40 mL). The combined organic layers were washed with brine,
dried over anhydrous Na2SO4 and concentrated under reduced pressure. To
solution of the residue, 40 mL acetic acid was added. The mixture was heated
to reflux for 2.5h at 120 °C. After cooled to room temperature, the acetic acid
was removed under reduced pressure, and the residue was neutralized to
PH=7-8 with concentrated aqueous ammonia (NH3·H2O). Then the mixture was
extracted with DCM (3×40 mL). The DCM layer was dried by anhydrous
Na2SO4, and concentrated under reduced pressure. The residue was purified by
silica gel chromatography (DCM/ MeOH=100:1) to obtain white solid
compounds 5a-5j.
4.5.1.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(3-methyl-phenyl)-amino)-propionic acid ethyl ester (5a). Yield:
61.8%; mp: 206.7-207.8 °C; 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H, Ar-H),
7.42 (d,
Ar-H), 7.00 (m, 1H, Ar-H), 6.88 (d,
Ar-H), 6.77 (d, = 7.8 Hz, 1H, Ar-H), 6.68 (d,
= 4.5 Hz, 2H, -CH2-), 4.19 (t,
J
= 8.6 Hz, 2H, Ar-H), 7.26 (d,
= 6.9 Hz, 1H, Ar-H, s,
= 8.6 Hz, 2H, Ar-H), 4.43 (d, J
J
= 8.4 Hz, 1H, Ar-H), 7.04 (s, 1H,
J
J
= 6.9 Hz, 1H,
J
J
J
= 7.4 Hz, 2H, -CH2-), 4.05 (q, J = 7.1 Hz, 2H,
12

-CH2-), 3.64 (s, 3H, -NCH3), 2.69 (t,
J = 7.4 Hz, 2H, -CH2-), 2.20 (s, 3H,
Ar-CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
4.5.2.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(4-methyl-phenyl)-amino)-propionic acid ethyl ester (5b). Yield:
61.2%; mp: 199.8-200.4 °C; 1H NMR (400 MHz, CDCl3) δ 7.65 (s, 1H, Ar-H),
7.42 (d,
Hz, 1H, Ar-H), 6.95 (d,
6.69 (d, = 8.5 Hz, 2H, Ar-H), 4.44 (d,
Hz, 2H, -CH2-), 4.05 (q, = 7.1 Hz, 2H, -CH2-), 3.64 (s, 3H, -NCH3), 2.68 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.25 (d,
= 8.2 Hz, 2H, Ar-H), 6.90 (d,
= 4.5 Hz, 2H, -CH2-), 4.18 (t,
J
= 10.0 Hz, 2H, Ar-H), 7.04 (d,
= 8.2 Hz, 2H, Ar-H),
= 7.3
J = 8.5
J
J
J
J
J
J
J
= 7.3 Hz, 2H, -CH2-), 2.19 (s, 3H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
4.5.3.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(2, 4-dimethyl-phenyl)-amino)-propionic acid ethyl ester (5c). Yield:
79.3%; mp: 199.8-201.2 °C; 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 1H, Ar-H),
7.42 (d, J = 8.5 Hz, 2H, Ar-H), 7.26 (d, J = 8.4 Hz, 1H, Ar-H), 7.03 (d, J = 8.4
Hz, 1H, Ar-H), 6.89 (d, J = 7.8 Hz, 1H, Ar-H), 6.86 (s, 1H, Ar-H), 6.82 (d, J =
7.8 Hz, 1H), 6.68 (d, J = 8.5 Hz, 2H, Ar-H), 4.42 (d, J = 3.9 Hz, 2H, -CH2-),
4.05 (q, J = 7.0 Hz, 2H, -CH2-), 3.76 (d, J = 15.8, 8.5 Hz, 2H, -CH2-), 3.63 (s,
3H, -NCH3), 2.71 (dd, J = 15.9, 9.0 Hz, 2H, -CH2-), 2.17 (s, 3H, Ar-CH3),
2.14 (s, 3H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
4.5.4.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(2, 5-dimethyl-phenyl)-amino)-propionic acid ethyl ester (5d). Yield:
51.7%; mp: 201.7-202.5 °C; 1H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H, Ar-H),
7.42 (d,
Hz, 1H, Ar-H), 6.93 (d,
(d, = 8.5 Hz, 1H, Ar-H), 4.43 (d,
4.05 (q, = 7.1 Hz, 2H, -CH2-), 3.85 (m, 1H, -CH2-), 3.63 (s, 3H, -NCH3), 2.73
(t, = 7.0 Hz, 2H, -CH2-), 2.17 (s, 3H, Ar-CH3), 2.10 (s, 3H, Ar-CH3), 1.20 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.27 (d,
= 7.9 Hz, 1H, Ar-H), 6.86 (d,
= 4.3 Hz, 2H, -CH2-), 4.34 (m, 1H, -CH2-),
J
= 8.4 Hz, 1H, Ar-H), 7.02 (d,
J = 8.5
J
J
= 6.9 Hz, 2H), 6.68
J
J
J
J
J
= 7.1 Hz, 3H, -CH3).
4.5.5.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(3, 4-dimethyl-phenyl)-amino)-propionic acid ethyl ester (5e). Yield:
63.8%; mp: 193.7-194.7 °C; 1H NMR (400 MHz, CDCl3) δ 7.67 (s, 1H, Ar-H),
7.42 (d,
Hz, 1H, Ar-H), 6.86 (d,
4.44 (d, = 4.5 Hz, 2H, -CH2-), 4.17 (t,
Hz, 2H, -CH2-), 3.65 (s, 3H, -NCH3), 2.68 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.28 (d,
= 8.4 Hz, 2H, Ar-H), 6.69 (d,
= 7.5 Hz, 2H, -CH2-), 4.05 (q,
= 7.4 Hz, 2H, -CH2-), 2.10 (s,
J
= 8.5 Hz, 1H, Ar-H), 7.04 (d,
= 8.5 Hz, 3H, Ar-H),
= 7.1
J = 8.5
J
J
J
J
J
J
6H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
4.5.6.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(3, 5-dimethyl-phenyl)-amino)-propionic acid ethyl ester (5f). Yield:
43.7%; mp: 188.9-190.2 °C; 1H NMR (400 MHz, CDCl3) δ 7.67 (s, 1H, Ar-H),
13

7.42 (d,
Hz, 1H, Ar-H), 6.86 (d,
8.4 Hz, 2H, Ar-H), 4.44 (d,
-CH2-), 4.05 (q, = 7.2 Hz, 2H, -CH2-), 3.65 (s, 3H, -NCH3), 2.68 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.28 (d,
= 8.3 Hz, 2H, Ar-H), 6.85 (s, 1H, Ar-H), 6.69 (d,
= 4.4 Hz, 2H, -CH2-), 4.17 (t, = 7.4 Hz, 2H,
= 7.4
J = 8.4 Hz, 1H, Ar-H), 7.04 (d, J = 8.5
J
J =
J
J
J
J
Hz, 2H, -CH2-), 2.10 (s, 6H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
4.5.7.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(3-methoxy-phenyl)-amino)-propionic acid ethyl ester (5g). Yield:
80.9%; mp: 206.7-207.1 °C; 1H NMR (400 MHz, CDCl3) δ 7.67 (s, 1H, Ar-H),
7.42 (d,
Ar-H), 6.68 (d,
4.4 Hz, 2H, -CH2-), 4.20 (t,
J
= 8.3 Hz, 2H, Ar-H), 7.28 (d,
= 8.3 Hz, 2H), 6.60 (m, 3H, Ar-H, Ar-H, Ar-H), 4.43 (d,
= 7.3 Hz, 2H, -CH2-), 4.06 (q, = 7.2 Hz, 2H,
= 7.3 Hz, 2H, -CH2-), 1.19 (t, J
J = 8.4 Hz, 1H, Ar-H), 7.04 (m, 2H,
J
J =
J
J
-CH2-), 3.64 (s, 6H, -OCH3, -NCH3), 2.70 (t,
J
= 7.1 Hz, 3H, -CH3).
4.5.8.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(4-methoxy-phenyl)-amino)-propionic acid ethyl ester (5h). Yield:
77.4%; mp: 198.6-199.3 °C; 1H NMR (400 MHz, CDCl3) δ 7.62 (s, 1H), 7.40
(d,
1H, Ar-H), 6.93 (d,
Ar-H), 4.42 (d, = 4.5 Hz, 2H, -CH2-), 4.16 (t,
= 7.1 Hz, 2H, -CH2-), 3.66 (s, 3H, -OCH3), 3.62 (s, 3H, -NCH3), 2.67 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.22 (d,
= 8.6 Hz, 2H, Ar-H ), 6.67 (t,
= 7.3 Hz, 2H, -CH2-), 4.05 (q,
J
= 8.5 Hz, 1H, Ar-H), 7.01 (d,
J = 8.5 Hz,
J
J
= 7.8 Hz, 4H, Ar-H,
J
J
J
J =
7.3 Hz, 2H, -CH2-), 1.19 (t, J = 7.1 Hz, 3H, -CH3).
4.5.9.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(5-methyl-pyridin-2-yl)-propionic acid ethyl ester (5i). Yield: 66.6%;
mp: 188.6-189.9 °C; 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H, Py-H), 7.66 (s,
1H), 7.41 (d,
6.4 Hz, 1H, Ar-H), 7.04 (d,
Ar-H), 6.57 (d, = 8.1 Hz, 1H, Py-H), 4.46 (d,
= 7.3 Hz, 2H, -CH2-), 4.04 (q, = 7.1 Hz, 2H, -CH2-), 3.66 (s, 3H, -NCH3),
= 7.2 Hz, 2H, -CH2-), 2.19 (s, 3H, Py-CH3), 1.18 (t, = 7.1 Hz, 3H,
J
= 8.3 Hz, 2H, Ar-H), 7.23 (d,
= 8.4 Hz, 1H, Py-H), 6.69 (d,
= 4.5 Hz, 2H, -CH2-), 4.35 (t,
J
= 8.1 Hz, 1H, Ar-H), 7.11 (d,
J =
J
J
= 8.4 Hz, 2H,
J
J
J
J
2.74 (t,
-CH3).
J
J
4.5.10.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(6-methyl-pyridin-2-yl)-propionic acid ethyl ester (5j). Yield: 62.5%;
mp: 199.9-201.2 °C; 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.41 (d,
J =
8.4 Hz, 2H, Ar-H), 7.23 (d,
Py-H), 7.04 (d, = 8.4 Hz, 1H, Ar-H), 6.81 (d,
= 8.5 Hz, 2H, Ar-H), 6.40 (d, = 7.9 Hz, 1H, Py-H), 4.46 (d,
-CH2-), 4.39 (t, = 7.3 Hz, 2H, -CH2-), 4.04 (q, = 7.1 Hz, 2H, -CH2-), 3.67 (s,
= 7.3 Hz, 2H, -CH2-), 2.47 (s, 3H, Py-CH3), 1.19 (t, J =
J
= 5.8 Hz, 1H, Ar-H), 7.15 (t,
= 7.5 Hz, 1H, Py-H), 6.69 (d,
= 4.5 Hz, 2H,
J = 7.8 Hz, 1H,
J
J
J
J
J
J
J
3H, -NCH3), 2.76 (t,
7.1 Hz, 3H, -CH3).
J
14

4.5.11.
3-({2-[(4-Cyano-phenylamino)-methyl]-1-methyl-1H-benzoimidazole-5-car
bonyl}-(2-methyl-phenyl)-amino)-propionic acid ethyl ester (5k). Yield:
45.0%; mp: 202.4-203.7 °C; 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H, Ar-H),
7.42 (d,
Ar-H), 7.03 (d,
2H, Ar-H), 6.77 (d,
(d, = 4.5 Hz, 2H, -CH2-), 4.19 (t,
2H, -CH2-), 3.64 (s, 3H, -NCH3), 2.69 (t,
Ar-CH3), 1.19 (t, = 7.1 Hz, 3H, -CH3).
4.6. General procedure for preparation of compounds 6a-6j
J
= 8.6 Hz, 2H, Ar-H), 7.26 (d,
= 8.4 Hz, 1H, Ar-H), 7.00(t, 1H, Ar-H), 6.88 (d,
= 7.8 Hz, 1H, Ar-H) , 6.68 (d, = 8.6 Hz, 2H, Ar-H), 4.43
= 7.4 Hz, 2H, -CH2-), 4.05 (q, = 7.1 Hz,
= 7.4 Hz, 2H, -CH2-), 2.20 (s, 3H,
J
= 8.4 Hz, 1H, Ar-H), 7.24 (t, 1H,
J
J
= 6.9 Hz,
J
J
J
J
J
J
J
Compounds 5a-5j (10mmol), hydroxylamine hydrochloride (1.4g, 20mmol)
and trimethylamine (Et3N) (20 mmol) were mixed in 20mL ethanol. The
mixture was refluxed for 3h at 80 °C. Ethanol was then evaporated under
reduced pressure. To a acetic acid (HOAC) (20 mL) solution of the residue,
ammonium formate (1.9g, 30mmol) and 10% Pd/C (1.9g) were added with
stirring. The mixture was heated to reflux for 5h at 120 °C under a nitrogen
atmosphere. After completion of the reaction, the contents were cooled. The
solution was evaporated under vacuum, then the residue was purified by silica
gel chromatography (DCM/ MeOH=7:1), recrystallized from ethyl acetate to
obtain white solid compounds 6a-6j
.
4.6.1.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3-methyl-phenyl)-amino]-propionic acid ethyl ester (6a).
Yield: 78.3%; mp: 188.9-190.6 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d,
J
= 8.7 Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-H), 7.33 (d,
= 5.5 Hz, 1H, Ar-H), 7.16 (d, = 8.6 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 7.03
(d, = 7.9 Hz, 1H, Ar-H), 6.91 (d, = 7.7 Hz, 1H, Ar-H), 6.81 (d, = 8.7 Hz,
2H, Ar-H), 4.58 (d, = 5.4 Hz, 2H, -CH2-), 4.02 (t, = 7.2 Hz, 2H, -CH2-), 3.95
(q, = 7.1 Hz, 2H, -CH2-), 3.71 (s, 3H, -NCH3), 2.55 (t, = 7.1 Hz, 2H, -CH2-),
J = 8.3 Hz, 1H, Ar-H), 7.28 (t,
J
J
J
J
J
J
J
J
J
2.16 (s, 3H, Ar-CH3), 1.10 (t, J = 7.1 Hz, 3H, -CH3).
4.6.2.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(4-methyl-phenyl)-amino]-propionic acid ethyl ester (6b).
Yield: 72.1%; mp: 197.8-198.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.59 (d,
J
= 8.7 Hz, 2H, Ar-H), 7.44 (s, 1H, Ar-H), 7.32 (d,
(d, = 8.4 Hz, 1H, Ar-H), 7.00 (s, 4H, Ar-H), 6.81 (d,
4.58 (d, = 5.4 Hz, 2H,- CH2-), 4.01 (t, = 7.2 Hz, 2H,-CH2- ), 3.95 (q,
Hz, 2H,-CH2-), 3.71 (s, 3H, -NCH3), 2.54 (t, = 7.2 Hz, 2H, -CH2-), 2.14 (s,
= 7.1 Hz, 3H, -CH3).
J
= 8.4 Hz, 2H, Ar-H), 7.14
= 8.9 Hz, 2H, Ar-H),
= 7.1
J
J
J
J
J
J
3H, Ar-CH3), 1.67 (s, 2H, -NH2), 1.10 (t,
J
4.6.3.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(2, 4-dimethyl-phenyl)-amino]-propionic acid ethyl ester
(6c). Yield: 84.5%; mp: 155.5-156.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.62
15

(d,
7.16 (d,
Ar-H), 6.90 (d,
= 5.2 Hz, 2H, -CH2-), 4.02 (t,
-CH2-), 3.72 (s, 3H, -NCH3), 3.32 (s, 3H, Ar-CH3), 2.56 (t,
J
= 8.6 Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-H), 7.33 (d,
= 8.4 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 7.03 (d,
= 7.5 Hz, 1H, Ar-H), 6.82 (d, = 8.6 Hz, 2H, Ar-H), 4.59 (d,
= 7.1 Hz, 2H, -CH2-), 3.95 (q, = 7.1 Hz, 2H,
= 7.1 Hz, 2H,
J
= 8.4 Hz, 1H, Ar-H),
J
J
= 7.9 Hz, 1H,
J
J
J
J
J
J
-CH2-), 2.16 (s, 3H, Ar-CH3), 1.10 (t, J = 7.1 Hz, 3H, -CH3).
4.6.4.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(2, 5-dimethyl-phenyl)-amino]-propionic acid ethyl ester
1
(6d). Yield: 78.9%; mp: 165.5-166.3 °C; H NMR (400 MHz, DMSO-d6) δ
7.60 (d,
J
= 8.5 Hz, 2H, Ar-H), 7.39 (s, 1H, Ar-H), 7.32 (d,
J
J
= 7.6 Hz, 1H,
= 7.7 Hz, 1H,
Ar-H), 7.18 (d,
J
= 8.3 Hz, 1H, Ar-H), 7.07 (s, 1H), 6.94 (d,
= 7.5 Hz, 1H, Ar-H), 6.80 (d,
= 13.9, 7.0 Hz, 2H, -CH2-), 3.83 – 3.50 (m, 5H,
-NCH3, -CH2-), 2.60 (t, 2H, -CH2-), 2.15 (s, 3H, Ar-CH3), 1.99 (s, 3H,
Ar-H), 6.88 (d,
J
J = 8.4 Hz, 2H, Ar-H), 4.57 (d, J
= 4.6 Hz, 2H, -CH2-), 3.95 (d,
J
Ar-CH3), 1.10 (t, J = 6.9 Hz, 3H, -CH3).
4.6.5.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3, 4-dimethyl-phenyl)-amino]-propionic acid ethyl ester
(6e). Yield: 77.4%; mp: 226.5-227.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.57
(d,
7.16 (d,
Ar-H), 6.80 (d,
= 5.3 Hz, 2H, -CH2-), 4.00 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.46 (s, 1H, Ar-H), 7.33 (d,
= 8.3 Hz, 1H, Ar-H), 7.01 (s, 1H, Ar-H), 6.91 (d,
= 8.6 Hz, 2H, Ar-H), 6.75 (d, = 7.5 Hz, 1H, Ar-H), 4.57 (d,
= 5.3 Hz, 2H, -CH2-), 3.95 (q, J = 12.5, 5.4 Hz,
J
= 8.4 Hz, 1H, Ar-H),
J
J
= 8.0 Hz, 1H,
J
J
J
J
2H, -CH2-), 3.71 (s, 3H, -NCH3), 2.54 (t, J = 7.2 Hz, 2H, -CH2-), 2.07 (s, 3H,
Ar-CH3), 2.05 (s, 3H, Ar-CH3), 1.10 (t, J = 7.1 Hz, 3H, -CH3).
4.6.6.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3, 5-dimethyl-phenyl)-amino]-propionic acid ethyl ester
(6f). Yield: 79.5%; mp: 215.7-216.6 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.63
(d,
7.18 (d,
Ar-H), 6.74 (s, 1H, Ar-H), 4.59 (d,
7.2 Hz, 4H, -CH2-), 3.72 (s, 3H, -NCH3), 2.55 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.47 (s, 1H, Ar-H), 7.34 (d,
= 8.4 Hz, 1H, Ar-H), 6.82 (d, = 8.4 Hz, 2H, Ar-H), 6.76 (s, 2H,
= 5.3 Hz, 2H, -CH2-), 3.96 (dt, = 14.5,
= 7.1 Hz, 2H, -CH2-), 2.08
J = 8.4 Hz, 1H, Ar-H),
J
J
J
J
J
(s, 6H, Ar-CH3), 1.10 (t, J = 7.0 Hz, 3H, -CH3).
4.6.7.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3-methoxy-phenyl)-amino]-propionic acid ethyl ester (6g).
Yield: 77.9%; mp: 231.5-232.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.57 (d,
J
= 8.7 Hz, 2H, Ar-H), 7.47 (s, 1H, Ar-H), 7.34 (d,
(d, = 8.4 Hz, 1H, Ar-H), 7.07 (t, = 8.1 Hz, 1H, Ar-H), 6.81 (s, 1H, Ar-H),
6.78 (d, = 4.5 Hz, 2H, Ar-H), 6.64 (d, = 8.8 Hz, 1H, Ar-H), 4.57 (d, = 5.4
Hz, 2H, -CH2-), 4.05 (t, = 7.1 Hz, 2H, -CH2-),
J = 8.5 Hz, 1H, Ar-H), 7.18
J
J
J
J
J
J
= 7.1 Hz, 2H, -CH2-), 3.96 (q, J
16

3.71 (s, 3H, -OCH3), 2.56 (t,
(t, = 7.1 Hz, 3H, -CH3).
4.6.8.
J = 7.1 Hz, 2H, -CH2-), 1.67 (s, 3H, -NCH3), 1.10
J
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(4-methoxy-phenyl)-amino]-propionic acid ethyl ester (6h).
Yield: 80.0%; mp: 229.6-230.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.60 (d,
J
= 8.6 Hz, 2H, Ar-H), 7.44 (s, 1H, Ar-H), 7.32 (d,
(d, = 8.5 Hz, 1H, Ar-H), 7.05 (d, = 8.8 Hz, 2H, Ar-H), 6.81 (d,
2H, Ar-H), 6.74 (d, = 8.8 Hz, 2H, Ar-H), 4.58 (d, = 5.4 Hz, 2H, -CH2-), 4.00
(t, = 5.7 Hz, 2H, -CH2-), 3.96 (q, 2H, -CH2-), 3.71 (s, 3H, -OCH3), 3.61 (s,
J
= 8.4 Hz, 1H, Ar-H), 7.13
J
J
J
= 8.8 Hz,
J
J
J
3H, -NCH3), 2.54 (t, J = 7.1 Hz, 2H, -CH2-), 1.10 (t, J = 7.1 Hz, 3H, -CH3).
4.6.9.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(5-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester
1
(6i)
12.93 (s, 1H, Py-H), 12.32 (d,
12.09 (dd, = 11.5, 5.1 Hz, 2H, Ar-H), 11.86 (d,
(d, = 8.8 Hz, 2H, Ar-H), 11.51 (d, = 8.1 Hz, 1H, Py-H), 9.34 (d,
2H, -CH2-), 8.89 (t, = 7.1 Hz, 2H, -CH2-), 8.69 (q, = 7.1 Hz, 2H, -CH2-),
8.48 (s, 3H, -NCH3), 7.37 (t, = 7.1 Hz, 2H, -CH2-), 6.40 (s, 3H, Py-CH3), 5.84
(t, = 7.1 Hz, 3H, -CH3).
4.6.10.
.
Yield: 71.5%; mp: 200.5-201.4 °C; H NMR (400 MHz, DMSO-d6) δ
J
= 8.8 Hz, 2H, Ar-H), 12.19 (s, 1H, Ar-H),
= 8.4 Hz, 1H, Py-H), 11.55
= 5.4 Hz,
J
J
J
J
J
J
J
J
J
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(6-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester
(6j). Yield: 75.8%; mp: 197.7-198.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.59
(d,
7.26 (t,
Hz, 1H, Py-H), 6.81 (d,
4.60 (d, = 5.3 Hz, 2H, -CH2-), 4.17 (t,
Hz, 2H, -CH2-), 3.73 (s, 3H, -NCH3), 2.63 (t,
J
= 8.5 Hz, 2H, Ar-H), 7.44 (s, 1H, Ar-H), 7.34 (d,
= 5.4 Hz, 1H, Py-H), 7.11 (d, = 8.4 Hz, 1H, Ar-H), 6.94 (d,
= 8.6 Hz, 2H, Ar-H), 6.55 (d, = 7.9 Hz, 1H, Py-H),
= 7.0 Hz, 2H, -CH2-), 3.94 (q, = 7.1
= 7.0 Hz, 2H, -CH2-), 2.37 (s,
J
= 8.8 Hz, 1H, Ar-H),
J
J
J
= 7.6
J
J
J
J
J
J
3H, Py-CH3), 1.08 (t, J = 7.1 Hz, 3H, -CH3).
4.6.11.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(2-methyl-phenyl)-amino]-propionic acid ethyl ester (6k).
Yield: 71.4%; mp: 191.9-192.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.58 (d,
J
= 8.7 Hz, 2H, Ar-H), 7.45 (s, 1H, Ar-H), 7.33 (d,
= 5.5 Hz, 1H, Ar-H), 7.16 (d, = 8.6 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 7.03
(d, = 7.9 Hz, 1H, Ar-H), 6.91 (d, = 7.7 Hz, 1H, Ar-H), 6.81 (d, = 8.7 Hz,
2H, Ar-H), 4.58 (d, = 5.4 Hz, 2H, -CH2-), 4.02 (t, = 7.2 Hz, 2H, -CH2-), 3.95
(q, = 7.1 Hz, 2H, -CH2-), 3.71 (s, 3H, -NCH3), 2.55 (t, = 7.1 Hz, 2H, -CH2-),
2.16 (s, 3H, Ar-CH3), 1.10 (t, = 7.1 Hz, 3H, -CH3).
J = 8.3 Hz, 1H, Ar-H), 7.28 (t,
J
J
J
J
J
J
J
J
J
J
4.7. General procedure for preparation of compounds 7a-7j
The compounds 6a-6j (1.0 mmol) were dissolved in 5 mL ethanol, then a
solution of sodium hydroxide(160mg, 4mmol) in 2 mL H2O was added into this
17

system. The mixture was stirred at room temperature for 1h. After completion
of the reaction, the reaction solution was neutralized to PH=6-7 with diluted
acetic acid. The solids were filtrated and washed carefully with water and
methanol, and dried to afford compounds 7a-7j as white solid. Purities of these
target compounds have been tested.
4.7.1.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3-methyl-phenyl)-amino]-propionic acid (7a). Yield:
77.5%; mp: 246.5-247.1 °C; Purity: 96.8% ; 1H NMR (400 MHz, DMSO-d6) δ
8.91 (s, 1H, -OH), 8.66 (s, 1H, =NH), 7.66 (d,
1H, Ar-H), 7.39 (d, = 8.3 Hz, 1H, Ar-H), 7.22 (d,
(s, 1H, Ar-H), 7.07 (d, = 7.7 Hz, 1H, Ar-H), 6.95 (d,
6.87 (d, = 8.7 Hz, 3H, Ar-H), 4.65 (s, 2H, -CH2-), 4.02 (t,
-CH2-), 3.79 (d, = 14.6 Hz, 3H, -NCH3), 2.55 (t, = 7.5 Hz, 2H, -CH2-), 2.20
J
= 8.5 Hz, 2H, Ar-H), 7.51 (s,
= 8.4 Hz, 1H, Ar-H), 7.11
= 7.5 Hz, 1H, Ar-H),
= 7.2 Hz, 2H,
J
J
J
J
J
J
J
J
(s, 3H, Ar-CH3). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 172.56, 169.84,
164.33, 164.25, 164.18, 164.10, 153.07, 152.94, 143.25, 138.62, 136.40,
129.98, 129.62, 128.81, 127.99, 127.28, 125.27, 123.10, 118.94, 111.71,
109.46, 46.39, 32.11, 30.02, 20.79. ESI-HRMS: calcd for C27H28N6O3 [M+H]+ ,
485.2256, found 485.2300.
4.7.2.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(4-methyl-phenyl)-amino]-propionic acid (7b). Yield:
85.8%; mp: 253.0-254.5 °C; Purity: 97.0% ; 1H NMR (400 MHz, DMSO-d6) δ
8.93 (s, 1H, -OH), 8.65 (s, 1H, =NH), 7.67 (d,
1H, Ar-H), 7.43 (d, = 8.3 Hz, 1H, Ar-H), 7.22 (d,
(d, = 8.8 Hz, 4H, Ar-H), 6.88 (d, = 8.3 Hz, 2H, Ar-H), 4.69 (s, 2H, -CH2-),
4.02 (t, = 7.1 Hz, 2H, -CH2-), 3.79 (s, 3H, -NCH3), 2.54 (d, = 7.2 Hz, 2H,
J
= 8.3 Hz, 2H, Ar-H), 7.52 (s,
J
J
= 8.3 Hz, 1H, Ar-H), 7.04
J
J
J
J
-CH2-), 2.18 (s, 3H, Ar-CH3). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 172.53,
169.29, 164.33, 164.24, 164.17, 164.09, 153.20, 152.63, 140.34, 136.02,
135.09, 131.78, 129.74, 129.66, 127.77, 113.83, 111.93, 110.52, 46.23, 32.04,
30.61, 20.40, 18.44. ESI-HRMS: calcd for C27H28N6O3 [M+H]+ , 485.2256,
found 485.2325.
4.7.3.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(2, 4-dimethyl-phenyl)-amino]-propionic acid (7c). Yield:
83.6%; mp: 239.9-241.1 °C; Purity: 98.8% ; 1H NMR (400 MHz, DMSO-d6) δ
8.99 (s, 1H, -OH), 8.75 (s, 1H, =NH), 7.70 (d,
2H, Ar-H), 7.28 (d, = 8.4 Hz, 1H, Ar-H), 7.13 (d,
(s, 3H, Ar-H), 6.89 (d, = 8.5 Hz, 2H, Ar-H), 4.75 (s, 2H, -CH2-), 4.22(m, 2H,
-CH2-), 3.82 (s, 3H, -NCH3), 2.57 (dd, = 13.4, 6.7 Hz, 2H, -CH2-), 2.15 (s,
J
= 8.3 Hz, 2H, Ar-H), 7.50 (s,
J
J
= 7.7 Hz, 1H, Ar-H), 6.94
J
J
13
3H, Ar-CH3), 2.10 (s, 3H, Ar-CH3). C NMR (101 MHz, DMSO-d6) δ(ppm)
172.51, 168.65, 164.15, 164.08, 164.00, 153.39, 152.13, 138.17, 137.32,
134.38, 134.07, 133.29, 131.81, 129.73, 129.37, 128.58, 127.55, 125.36,
18

114.55, 113.60, 112.29, 112.22, 112.14, 45.22, 31.69, 31.55, 20.37, 17.32.
ESI-HRMS: calcd for C28H30N6O3 [M+H]+, 499.2412, found 499.2447.
4.7.4.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(2, 5-dimethyl-phenyl)-amino]-propionic acid (7d). Yield:
81.3%; mp: 247.9-249.0 °C; Purity: 98.4% ; 1H NMR (400 MHz, DMSO-d6) δ
9.07 (s, 1H, -OH), 8.82 (s, 1H, =NH), 7.73 (d,
1H, Ar-H), 7.42 (d, = 8.5 Hz, 1H, Ar-H), 7.15 (s, 1H, Ar-H), 7.00 (d,
Hz, 1H, Ar-H), 6.93 (t, = 8.4 Hz, 3H, Ar-H), 4.92 (s, 2H, -CH2-), 4.20(m, 2H,
-CH2-), 3.91 (s, 3H, -NCH3), 2.60 (t, = 7.3 Hz, 2H, -CH2-), 2.18 (s, 3H,
J
= 7.7 Hz, 3H, Ar-H), 7.60 (s,
J
J
= 7.6
J
J
Ar-CH3), 2.06 (s, 3H, Ar-CH3). 13C NMR (101 MHz, DMSO-d6) δ(ppm)
173.10, 169.69, 164.77, 164.69, 164.62, 153.77, 153.00, 141.54, 136.74,
135.34, 134.37, 132.44, 131.94, 131.49, 130.18, 129.12, 124.74, 116.56,
114.43, 113.43, 112.54, 111.24, 56.51, 46.09, 32.35, 31.31, 20.82, 17.51.
ESI-HRMS: calcd for C28H30N6O3 [M+H]+ , 499.2412, found 499.2457.
4.7.5.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3, 4-dimethyl-phenyl)-amino]-propionic acid (7e). Yield:
94.2%; mp: 255.7-256.2 °C; Purity: 96.5%; 1H NMR (400 MHz, DMSO-d6) δ
8.95 (s, 1H, -OH), 8.67 (s, 1H, =NH), 7.68 (d,
1H, Ar-H), 7.45 (d, = 8.1 Hz, 1H, Ar-H), 7.25 (d,
(s, 1H, Ar-H), 6.96 (d, = 7.8 Hz, 1H, Ar-H), 6.88 (d,
6.81 (d, = 7.7 Hz, 1H, Ar-H), 4.70 (s, 2H, -CH2-), 4.00 (t,
-CH2-), 3.80 (s, 3H, -NCH3), 2.55 (d, = 7.4 Hz, 2H, -CH2-), 2.11 (s, 3H,
J
= 8.3 Hz, 2H, Ar-H), 7.55 (s,
= 8.3 Hz, 1H, Ar-H), 7.08
= 8.3 Hz, 2H, Ar-H),
= 7.0 Hz, 2H,
J
J
J
J
J
J
J
Ar-CH3), 2.09 (s, 3H, Ar-CH3). 13C NMR (101 MHz, DMSO) δ 173.07, 169.02,
164.78, 164.70, 164.63, 153.77, 152.58, 140.33, 138.03, 135.85, 134.82,
133.70, 130.61, 130.23, 129.33, 128.94, 126.22, 126.08, 114.57, 112.82,
112.65, 56.51, 46.78, 32.45, 32.05, 19.78, 19.24, 18.71. ESI-HRMS: calcd for
C28H30N6O3 [M+H]+, 499.2412, found 499.2474.
4.7.6.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(3, 5-dimethyl-phenyl)-amino]-propionic acid (7f). Yield:
87.9%; mp: 259.5-260.2 °C; Purity: 96.2% ; 1H NMR (400 MHz, DMSO-d6) δ
8.94 (s, 1H, -OH), 8.68 (s, 1H, =NH), 7.67 (d,
1H, Ar-H), 7.45 (d, = 8.1 Hz, 1H, Ar-H), 7.27 (d,
(d, = 8.2 Hz, 2H, Ar-H), 6.82 (s, 2H, Ar-H), 6.79 (s, 1H, Ar-H), 4.69 (s, 2H,
-CH2-), 3.99 (t, = 7.0 Hz, 2H, -CH2-), 3.80 (s, 3H, -NCH3), 2.55 (d, = 7.2
J
= 8.1 Hz, 2H, Ar-H), 7.55 (s,
J
J
= 8.1 Hz, 1H, Ar-H), 6.88
J
J
J
13
Hz, 2H, -CH2-), 2.13 (s, 6H, Ar-CH3). C NMR (101 MHz, DMSO) δ 173.03,
168.94, 164.69, 164.61, 164.53, 153.73, 152.57, 142.51, 139.04, 134.73,
133.68, 130.23, 129.24, 129.19, 126.10, 115.06, 114.49, 112.81, 112.68, 56.49,
46.74, 32.47, 32.08, 21.11. ESI-HRMS: calcd for C28H30N6O3 [M+H]+,
499.2412, found 499.2474.
4.7.7.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
19

ole-5-carbonyl}-(3-methoxy-phenyl)-amino]-propionic
acid
Yield:84.6%; mp: 248.7-249.9 °C; Purity: 98.5% ; H NMR (400 MHz,
DMSO-d6) δ8.97 (s, 1H, -OH), 8.73 (s, 1H, =NH), 7.69 (d, = 8.4 Hz, 2H,
Ar-H), 7.58 (s, 1H, Ar-H), 7.53 (d, = 7.3 Hz, 1H, Ar-H), 7.31 (d, = 8.2 Hz,
1H, Ar-H), 7.11 (t, = 8.0 Hz, 1H, Ar-H), 6.90 (s, 1H, Ar-H), 6.87 (d, = 5.8
Hz, 2H, Ar-H), 6.71 (t, = 6.9 Hz, 2H, Ar-H), 4.75 (s, 2H, -CH2-), 4.05 (t,
7.3 Hz, 2H, -CH2-), 3.83 (s, 3H, -OCH3), 3.65 (s, 3H, -NCH3), 2.56 (t, = 7.2
(7g).
1
J
J
J
J
J
J
J =
J
Hz, 2H, -CH2-). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 172.60, 169.49,
164.25, 164.17, 164.10, 159.55, 153.20, 152.70, 144.18, 135.50, 129.84,
129.66, 123.76, 120.31, 117.51, 113.70, 112.21, 111.86, 110.23, 99.49, 55.14,
46.21, 32.11, 30.45. 18.41.ESI-HRMS: calcd for C27H28N6O4 [M+H]+,
501.2205, found 501.2260.
4.7.8.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(4-methoxy-phenyl)-amino]-propionic acid (7h). Yield:
83.5%; mp: 244.2-245.1 °C; Purity: 98.4% ; 1H NMR (400 MHz, DMSO-d6) δ
8.93 (s, 1H, -OH), 8.67 (s, 1H, =NH), 7.67 (d,
1H, Ar-H), 7.41 (d, = 7.6 Hz, 1H, Ar-H), 7.21 (d,
(d, = 8.2 Hz, 2H, Ar-H), 6.88 (d, = 8.5 Hz, 2H, Ar-H), 6.79 (d,
2H, Ar-H), 4.67 (s, 2H, -CH2-), 4.00 (t, = 7.1 Hz, 2H, -CH2-), 3.78 (s, 3H,
-OCH3), 3.65 (s, 3H, -NCH3), 2.54 (d,
= 7.3 Hz, 2H, -CH2-).13C NMR (101
J
= 8.2 Hz, 2H, Ar-H), 7.51 (s,
= 8.2 Hz, 1H, Ar-H), 7.11
= 8.2 Hz,
J
J
J
J
J
J
J
MHz, DMSO-d6) δ(ppm)173.16, 170.38, 164.85, 164.77, 164.70, 164.63,
157.97, 153.57, 153.32, 138.90, 136.41, 131.08, 130.14, 129.72, 123.85,
118.84, 114.78, 113.92, 113.85, 112.32, 110.21, 56.51, 55.63, 46.83, 32.57,
30.68, 18.86. ESI-HRMS: calcd for C27H28N6O4 [M+H]+, 501.2205, found
501.2272.
4.7.9.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(5-methyl-pyridin-2-yl)-amino]-propionic acid (7i). Yield:
80.4%; mp: 254.9-255.5 °C; Purity: 95.9% ; 1H NMR (400 MHz, DMSO-d6) δ
8.95 (s, 1H, -OH), 8.71 (s, 1H, =NH), 8.21 (d,
= 8.8 Hz, 2H, Ar-H), 7.52 (s, 1H, Ar-H), 7.46 (d,
8.3, 2.1 Hz, 1H, Ar-H), 7.18 (d, = 8.4 Hz, 1H, Py-H), 6.89 (d,
Ar-H), 6.87 (s, 1H, Py-H), 4.70 (s, 2H, -CH2-), 4.14 (t, = 7.5 Hz, 2H, -CH2-),
3.81 (s, 3H, -NCH3), 2.60 (t, = 7.5 Hz, 2H, -CH2-), 2.17 (d, = 9.0 Hz, 3H,
J
= 2.3 Hz, 1H, Py-H), 7.68 (d,
J
J
= 8.4 Hz, 1H), 7.41 (dd,
J
=
J
J = 7.3 Hz, 2H,
J
J
J
Py-CH3). 13C NMR (101 MHz, DMSO-d6) δ(ppm) 177.66, 174.38, 169.54,
169.47, 169.39, 169.31, 158.75, 158.34, 157.70, 153.97, 144.00, 139.98,
137.81, 136.37, 134.94, 129.92, 126.77, 121.27, 119.37, 117.32, 49.76, 37.79,
36.33, 22.46. ESI-HRMS: calcd for C26H27N7O3 [M+H]+, 486.2208, found
486.2260.
4.7.10.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(6-methyl-pyridin-2-yl)-amino]-propionic acid (7j). Yield:
85.0%; mp: 246.3-247.2 °C; Purity: 97.1%; 1H NMR (400 MHz, DMSO-d6) δ
20

8.94 (s, 1H), 8.69 (s, 1H), 7.68 (d,
7.43 (dd, = 15.8, 8.0 Hz, 2H, Ar-H, Py-H), 7.18 (d,
6.99 (d, = 7.6 Hz, 1H, Py-H), 6.88 (d, = 8.9 Hz, 2H, Ar-H), 6.67 (d, 1H,
Py-H), 4.69 (s, 2H, -CH2-), 4.17 (t, = 7.5 Hz, 2H, -CH2-), 3.80 (s, 3H,
-NCH3), 2.62 (t, = 7.5 Hz, 2H, -CH2-), 2.40 (s, 3H, Py-CH3). C NMR (101
J
= 8.7 Hz, 2H, Ar-H), 7.50 (s, 1H, Ar-H),
J
J
= 8.4 Hz, 1H, Ar-H),
J
J
J
13
J
MHz, DMSO-d6) δ(ppm) 172.97, 169.14, 164.75, 164.68, 164.61, 164.54,
157.68, 154.16, 153.71, 152.65, 140.56, 134.25, 134.16, 130.23 129.61, 126.06,
122.67, 120.42, 115.15, 114.84, 112.92, 46.48, 45.22, 32.96, 32.09, 23.34.
ESI-HRMS: calcd for C26H27N7O3 [M+H]+, 486.2208, found 486.2259.
4.7.11.
3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-methyl-1H-benzoimidaz
ole-5-carbonyl}-(2-methyl-phenyl)-amino]-propionic acid (7k). Yield:
80.3%; mp: 249.5-250.3 °C; Purity: 95.3% ; 1H NMR (400 MHz, DMSO-d6) δ
8.95 (s, 1H, -OH), 8.71 (s, 1H, =NH), 7.68 (d,
= 11.6 Hz, 2H, Ar-H), 7.25 (d, = 6.6 Hz, 2H, Ar-H), 7.12 (s, 3H, Ar-H), 6.87
(d, = 8.0 Hz, 2H, Ar-H), 4.68 (s, 2H, -CH2-), 3.78 (s, 3H, -NCH3), 3.67(m,
2H, -CH2-), 2.59 (s, 2H, -CH2-), 2.11 (d,
J = 8.1 Hz, 2H, Ar-H), 7.44 (d, J
J
J
13
J
= 19.5 Hz, 3H, Ar-CH3). C NMR
(101 MHz, DMSO-d6) δ(ppm) 173.02, 170.30, 164.86, 164.79, 164.71, 164.64,
153.61, 153.39, 142.21, 136.73, 135.23, 131.67, 130.13, 130.00, 128.11,
127.33, 123.44, 113.27, 112.26, 110.00, 45.92, 32.41, 30.59, 21.50, 18.00.
ESI-HRMS: calcd for C27H28N6O3 [M+H]+ , 485.2256, found 485.2323.
4.2 Anticoagulant assay
The thrombin inhibitory effects (IC50) of new compounds 7a-j were
determined with a commercially available chromogenic assay against human
thrombin. Lyophilized human thrombin (national standard), which was purified
from human blood, substrate and the test compounds were dissolved and
diluted to needed concentrations. Dabigatran was as the reference. Firstly,
lyophilized human thrombin (5.4ꢀg/mL) and tested compounds with different
concentrations were preincubated for 10 minutes at 37 °C in the 96-well plate.
Nextly the specificity substrate, Ac-FVR-AMC (5ꢀM), was added into the
preincubation mixture. Then the dynamic changes offluorescence intensity was
measured in a microplate detector Envision (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 inhibition rates were calculated from the ultraviolet absorption values of
inhibited wells and positive control wells. Finally, the IC50 values were gained
using a regression analysis method between the concentration and inhibition
rate. The inhibitory rate and the concentration that induced a 50% inhibition of
thrombin (IC50) were calculated by the following formulas:
Inhibition rate (%) = [VDMSO-Vsample] / VDMSO×100%
Vsample: initial velocity of compound group
VDMSO: initial velocity of blank group, treated with DMSO alone
Inhibition rate (%) =100 / [1+10(LogIC50-X)h], h is the Hill coefficient.
21

4.3 Inhibitory effects on rat arteriovenous thrombosis in vivo
32 SD rats (Shanghai Sippr Bk Laboratory Animals L.T.D.;220-260 g) were
divided into blank control (CMC-Na), positive drug (dabigatran), and two test
substances (7j and 7k) groups, equally with eight rats in each group. The
positive drug and test substances were dissolved with 0.9% CMC-Na to adjust
to the concentration of 0.5mg/mL. SD rats were administrated for 3
days(2mg/kg/day). On the fourth day, the rats were anesthetized by
intraperitoneal injection of 12% chloral hydrate and fixed in supine position.
The neck skin was incised to separate the left carotid artery and right external
jugular vein, connecting by a bypass pipe with a No. 4 surgical thread inside.
After the tested compounds were injected by intravenous administration,
making blood flow for 15 minutes, then the thread was removed and weighed,
subtracting the thread of its own weight, that is, wet weight of thrombus. The
average value and standard deviation between the experimental groups of
thrombus wet weights were calculated.
Inhibition rate of rat arteriovenous thrombosis sample (%) =
[Control group (thromboembolism weight)-sample group (thromboembolism
weight)]/control group (thromboembolism weight) × 100%.
Control group: treated with CMC-Na only.
Sample group: treated with CMC-Na and compounds.
4.4 Molecular docking
The structures of the newly synthesized compounds were constructed in the
sketch process in SYBYL-X 2.0 package running on windows workstation. The
structure was optimized using Gasteiger–Huckel method and the Tripos force
field, following by 10000 iterations and the energy gradient limit is set to 0.005
kcal/ (mol*Å). For the protein preparation, the ligand and the water were
removed from the receptor protein (PDB entry code: 1KTS). Remaining protein
was optimized using Gasteiger–Huckel charges, following by 20 iterations and
the energy gradient limit was set to 0.05 kcal/ (mol*Å).
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the Shanghai Natural Science Foundation
ꢅNo.
19ZR1455400
ꢄ.
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25

1. Ten novel methyl substituted benzimidazole derivatives as direct thrombin
inhibitors for the treatment of cardiovascular diseases were designed, synthesised and
biologically evaluated.
2. At nanomolar levels, eight compounds presented excellent in vitro inhibitory
effects on arteriovenous thrombosis.
3. Compounds 7j and 7k exhibited a potent inhibition of venous thrombosis in rats
compared with dabigatran.

Conflicts of interest
There are no conflicts to declare.