Design, synthesis, anticoagulant activity evaluation and molecular docking studies of a class of N -ethyl dabigatran derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.05.020

A class of N-ethyl dabigatran derivatives was designed based on pharmacological strategies for inhibition of thrombin activity and the structure-activity relationship studies of the previous dabigatran derivatives. Activities of these novel compounds were predicted based on CoMFA model, and most of the compounds had comparable predicted activity with dabigatran. All of screened compounds were synthesized and characterized by 1^HNMR13C NMR and HRMS. Subsequently, these compounds were evaluated inhibitory activity on thrombin. Among these compounds, 9a-9e, 9h, 9l-9n and 9p exhibited comparable inhibitory activity to dabigatran (IC₅₀ Combining double low line 1.20 nM), additionally, compound 9p (IC₅₀ Combining double low line 0.96 nM) exhibited better inhibitory activity than dabigatran. Moreover, compound 9p also exhibited a fairly good inhibitory activity for arteriovenous thrombosis with inhibition rate of (85.35 ± 0.72) %, which was comparable to that of dabigatran (85.07 ± 0.61) %. These results, along with related molecular docking studies, could provide an important basis for further development of compound 9p as a potent thrombin inhibitor.

Full text of DOI:10.1016/j.ejmech.2016.05.020

Accepted Manuscript
Design, synthesis, anticoagulant activity evaluation and molecular docking studies of
a class of N-ethyl dabigatran derivatives
Weixin Ren, Yujie Ren, Shuai Wang
PII:
S0223-5234(16)30400-7
10.1016/j.ejmech.2016.05.020
EJMECH 8612
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 January 2016
Revised Date: 5 May 2016
Accepted Date: 6 May 2016
Please cite this article as: W. Ren, Y. Ren, S. Wang, Design, synthesis, anticoagulant activity evaluation
and molecular docking studies of a class of N-ethyl dabigatran derivatives, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.020.
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ACCEPTED MANUSCRIPT
A class of N-ethyl dabigatran derivatives was designed, predicted, synthesized and
evaluated anticoagulant activity. Compound 9p which exhibited better inhibitory
activity than dabigatran could as a potential thrombin inhibitor.

ACCEPTED MANUSCRIPT
Design, synthesis, anticoagulant activity evaluation and molecular
docking studies of a class of N-ethyl dabigatran derivatives
Weixin Ren, Yujie Ren* and Shuai Wang
College of Chemical and Environmental Engineering, Shanghai Institute of
Technology, Shanghai, China
Correspondence: Dr. Yujie Ren, College of Chemical and Environmental
Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai 201418,
China.
E-mail: clab@sit.edu.cn
Abstract A class of N-ethyl dabigatran derivatives was designed based on
pharmacological strategies for inhibition of thrombin activity and the
structure-activity relationship studies of the previous dabigatran derivatives. Activities
of these novel compounds were predicted based on CoMFA model, and most of the
compounds had comparable predicted activity with dabigatran. All of screened
1
13
compounds were synthesized and characterized by H NMR, C NMR and HRMS.
Subsequently, these compounds were evaluated inhibitory activity on thrombin.
Among these compounds, 9a–9e, 9h, 9l–9n and 9p exhibited comparable inhibitory
activity to dabigatran (IC50 = 1.20 nM), additionally, compound 9p (IC50 = 0.96 nM)
exhibited better inhibitory activity than dabigatran. Moreover, compound 9p also
exhibited a fairly good inhibitory activity for arteriovenous thrombosis with inhibition
rate of (85.35 ± 0.72) %, which was comparable to that of dabigatran (85.07 ±
0.61) %. These results, along with related molecular docking studies, could provide an
important basis for further development of compound 9p as a potent thrombin
inhibitor.
Key Words: N-methyl dabigatran derivatives / synthesis / anticoagulant activity /
molecular docking
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1. Introduction
Atrial fibrillation (AF) increases the risks of stroke and death [1, 2], patients with
AF have a nearly 5-fold increase in stroke risk [3]. The prevalence of AF is increasing
significantly worldwide, and AF affects approximately 1%–2% of the world
population [4–7]. Improving treatment for AF is therefore a key health care focus. The
main clinical impact of AF is increased the risk of stroke, accordingly, anticoagulant
is employed in the prevention of stroke or systemic embolism (SE) for patients with
AF and co-existing heart failure (HF) [8, 9].
Traditionally, prophylactic treatment has been based on vitamin K antagonists
(VKAs), and it had been used for more than 60 years for their known effectiveness in
preventing thromboembolic events [10, 11]. Managing AF by using VKA therapy has
several disadvantages, such as monitoring required and the high risk of hemorrhages,
thus, VKA therapy is infrequently used to treat AF cases [12]. Recently, novel oral
anticoagulants (NOACs) have been developed for stroke prevention. Compared with
traditionally anticoagulants, NOACs have better specificity, more rapid onset of action,
and shorter half-lives [13], Examples of NOACs are dabigatran, rivaroxaban, and
apixaban; these have potential for use as stroke prevention treatments for patients with
nonvalvular AF [14,15].
Particularly, dabigatran is a novel synthetic anticoagulant drug belonging to the
class of reversible direct thrombin inhibitors (DTIs). It is the prodrug of dabigatran
etexilate, and has a high specificity for thrombin [16]. This drug is superior to other
anticoagulants because of the following practical advantages: predictable
anticoagulant effect, oral administration, no need for monitoring, and few drug
interactions [17, 18]. Dabigatran etexilate has recently been approved in the United
State and Europe Union for stroke prevention in patients with AF [19–22]. Safety and
efficiency are the major concerns in using anticoagulant drugs in clinical practice.
However, the bioavailability after oral administration of dabigatran etexilate is 7.2%
[23], and it also has a risk of life-threatening hemorrhage. In the RE-LY trial, the
major bleeding incidence of life-threatening hemorrhage increased from 2.71% to
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3.11% when dosage for patients treated with from 110mg to 150 mg [24]. A new
dabigatran derivative that has better efficacy may contribute to decrease side effects
because drug dosage would be reduced, and has high-hydrophobicity may contribute
to enhance the banding force between drugs and target enzyme [25].
A previous study shows that dabigatran enhances clot lysis mainly via two
mechanisms, as follows: reduction of plasmin generation and alteration of the
viscoelastic properties of the clot. Both effects are depended on the ability of the drug
to reduce thrombin generation and activity [17]. Effects are based on the combination
of drugs and target enzyme. We studied the pharmacological strategies for inhibition
of thrombin activity and the structure-activity relationship of the previous dabigatran
derivatives (Fig.1) [25–30]. The benzamidine moiety binds to Asp 189 in the
specificity pocket, which is necessary for the inhibition of thrombin activity. The
benzimidazole ring as scaffold forms p-p stacking interactions with the Trp60D,
which is crucial for the thrombin protein binding. Carboxylate group is needed for
pharmacokinetics, and it does not interfere with the thrombin binding site. The
N-methyl group occupies the P-pocket and the pyridine ring occupies the D-pocket.
The two sections interact with the protein mainly due to the hydrophobic
characteristics of the groups. Therefore, N-ethyl-substituted N-methyl occupies the
P-pocket and may favor activity. Aryl-substituted pyridine or methyl substituted on
these ring could increase hydrophobicity, which may also be favor activity.
We designed a class of N-ethyl dabigatran derivatives. All compounds’ scores were
calculated via molecular docking. Activities were predicted based on a molecular
model, and these compounds were synthesized and evaluated for inhibitory activities.
We expected to find dabigatran derivatives that have higher inhibition activities than
dabigatran, and such derivatives are worth studying in the future.
2. Results and discussion
2.1 Docking scores and activity prediction
Molecular models, such as comparative molecular field analysis (CoMFA), were
established on the analysis of three-dimensional quantitative structure-activity
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relationship (3D-QSAR) of dabigatran in our previous work [28]. All designed
compounds were scored via molecular docking. Based on the CoMFA model,
activities of these novel compounds were predicted. The docking result and the
predicted result showed that all designed compounds obtained higher scores than
dabigatran, and most of the compounds had comparable activity with dabigatran.
Additionally, predicted activity values of those p-position methyl substituted
compounds were lower than the others.
2.2 Chemistry
The synthetic route of N-ethyl dabigatran derivatives is shown in Scheme 1.
Compounds 9a–9p were synthesized using corresponding amines (1a–1p) according
to the literature [31]. In the synthesis of compound 5, the author used Zn/NH4Cl as
reductant, and THF/H2O as solvent in this reaction. This method not only needed a
long duration for the reaction but also had high cost. Moreover, purification was done
by column chromatography. Zinc powder is an effective and cheap reductant and is
widely used in the nitroreduction reaction. Justin J. Maresh et al. used Zn/HCl for
nitroreduction [32]. Takehito Tsukinoki et al. used zinc to reduce nitroarenes in
various conditions, such as NH4Cl/MeOH [33]. After a number of attempts, we
adopted Zn as a reducing agent and AcOH/H2O as a solvent in this paper. This method
needed only 1 hour. Using this method not only saved energy but also reduced cost.
We studied the reaction process, and compounds 4a–4pꢀAcOH and H2O could
constitute a homogeneous system. Zinc powder can be ionized well in acetic acid and
has high reducibility in an acidic environment. The reaction is exothermic. The
temperature of the reaction system can reach up to 60 °C–70 °C, and this temperature
range was favorable to the reaction. Therefore, the reaction was performed well, and
thereby contributing to the subsequent purification. Many experiments were
performed. Furthermore, we found that compounds 5a–5p could be purified through
hyperacoustic-aided crystal by using ethyl acetate and petroleum ether solvents. We
obtained high purity (97%–99%) products with this method. The purification of later
products benefited from this method, and it might be an excellent method for
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application in other syntheses.
2.3 Inhibition activity evaluation
2.3.1 Inhibition rate tests
To confirm the activity of these synthesized compounds, we first conducted
inhibition rate tests. The thrombin inhibition rates of compounds 9a–9p were tested at
a concentration of 1 ꢀg/mL and are exhibited in Fig. 2.
Fig. 2 shows that all the compounds had high inhibition rates (>90%), thereby
demonstrating that these compounds inhibited thrombin. Additionally, we studied
dates of the inhibition rate. We found that there was little difference between 9a, 9b,
9h, 9l, 9m, and 9p (o-position methyl substituted) and 9d, 9f, 9i, 9k, and 9o
(p-position methyl substituted). The inhibition rates of the former were a little higher
than those of the later.
2.3.2 Inhibitory activity in vitro
To save resources and improve efficiency, combining this result with the result of
the predicted IC50, we selected 11 compounds as representatives to evaluate their
inhibitory activity in vitro, and the IC50 values were exhibited in Table 1.
As shown in Table 1, compounds 9a–9e, 9h, 9l–9n, and 9p had comparable
inhibition activities with dabigatran. Furthermore, compound 9p had better inhibitory
activity compared with dabigatran, but compound 9f exhibited worse inhibitory
activity than the others. Additionally, inhibitory activities of compounds 9b, 9h, and
9p (o-position methyl substituted) were better than compounds 9d, 9f, and 9h
(p-position methyl substituted). These results were consistent with the predicted IC50
and the inhibition rate tests in Fig. 2, possibly because the hydrophobic and
electron-donating group at o-position is favorable for activity, but not favorable for
activity at p-position.
2.3.3 Inhibitory activity in vivo
According to the analysis of inhibitory activity in vitro, compounds 9b and 9p were
selected to evaluate their effects on blood clotting in rats. The results of inhibitory
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activity in vivo were exhibited in Table 2.
As shown in Table 2, compounds 9b and 9p could inhibit the embolus weight of
arteriovenous thrombosis effectively. Moreover, compound 9p exhibited a fairly good
inhibitory activity for arteriovenous thrombosis with inhibition rate of (85.35 ±
0.72) %, which was comparable to that of dabigatran (85.07 ± 0.61) %.
2.4 Molecular docking study
Molecular docking could simulate the bonding form between molecule and target
enzymes [34–37]. To understand the recognition processes of these derivatives, we
studied the molecular modeling between dabigatran and the promising compound 9p
and the thrombin protein, and performed it in Surflex-Dock (Sybyl 2.0). The X-ray
crystal structure of dabigatran (PDB code: 1KTS) was retrieved from the RCSB
Protein Data Bank. The docking results are exhibited in Figs. 3 and 4.
As shown in Fig. 3, the original ligand was redocked to validate the docking
reliability. Their binding modes are basically similar. The benzimidazole ring of the
two compounds is located in the same pocket. The pyridine ring of the redocked
ligand is also positioned in D-pocket, except slight rotation of bonds. After validating
the docking reliability, the compound 9p with highest activity was selected for
exploring the probable binding conformation. It bonded with the protein compactly,
and the binding modes were basically similar with dabigatran.
As shown in Fig. 4, compared with dabigatran, compound 9p also formed hydrogen
bonds with Asp189 (the most important specific binding sites). The benzimidazole
ring forms p-p stacking interactions with the Trp60D residue. N-ethyl group occupies
the P-pocket, and the 6-position methyl substituted pyridine ring occupies the
D-pocket tightly. Van der Waals forces were formed between the target compound and
important residues (such as: Asp221, Cys220, Ala190, Cys191, Glu217, Arg173,
Ile174, Arg175, Glu97A, Asn98, Asp100, Leu99, Leu97A, Tyr60A, Lys60F, His57,
Phe227, Trp215, Val213, Ser195 and Glu192). Additionally, compound 9p has a high
amount of residues around the N-ethyl group (Trp215 and Lys60F) and around the
6-methyl substituted pyridine ring (Arg175 and Asp100). Moreover, the distance
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between the two groups and residues is nearer than that between the two groups and
dabigatran. Compound 9p has stronger binding affinity than dabigatran.
3. Conclusion
Activities of 16 novel designed compounds were predicted via molecular models.
1
All compounds were synthesized and characterized by H NMR, 13C NMR, and
HRMS. Zinc powder was used as reductant, and AcOH/H2O was used as solvent for
the synthesis of 5a–5p. Furthermore, compounds 5a–5p were purified through
hyperacoustic-aided crystal by using ethyl acetate and petroleum ether. These not only
improved efficiency but also reduced the cost. Subsequently, the synthesized
compounds were evaluated for inhibitory activity on thrombin. Compounds 9a–9e, 9h,
9l–9n and 9p had comparable inhibition activity to dabigatran, and compound 9p
exhibited better inhibitory activity than dabigatran in vitro. Additionally, compound
9p also exhibited a fairly good inhibitory activity for arteriovenous thrombosis in vivo,
which was comparable to that of dabigatran. These results, along with related
molecular docking studies, demonstrated that the promising compound 9p could be a
potential thrombin inhibitor for further study.
4. Experimental
4.1 Materials and methods
All reagents were A.R. grade and purchased from Shanghai Chemical Reagent
Company in China. Melting points were measured on WRS-1B and were uncorrected.
NMR spectra were carried out using a Brucker Avance 500 or 400MHz MHz NMR
spectrometer using TMS as internal reference. The HRMS spectra were recorded on a
SolariX-70FT-MS Bruker spectrometer. The reactions were monitored by thin-layer
chromatography (TLC). TLC was performed on silica gel plates (GF254) with
visualisation of components by UV light (254 nm) or exposure to I2.
The intermediates 3–5 were purified through hyperacoustic forced crystal by
SY7200-D ultrasonic cleaner. Purity were measured on HPLC (P230ꢁ).
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4.2 Synthesis of the compounds 9a–9p
4.2.1 General procedure for the synthesis of compounds 2a–2p
Aromatic amine derivatives (1a–1p) (5.0g, 45mmol), ethyl acrylate (68mmol) and
TfOH as catalyst (10 mol% with respect to amine) were mixed, and the reaction
mixture was heated under reflux temperature overnight. The reaction was detected by
TLC. The solvent was concentrated via reduced-pressure distillation. The residue was
purified by column chromatography on silica gel eluted with PE/EtOAc = (10:1–5:1).
Compounds 2a–2p were obtained. Yield: 75%–85%.
4.2.2 General procedure for the synthesis of compounds 4a–4p
A mixture of 4-chloro-3-nitrobenzoic acid (25 mmol) and ethylamine solution
(0.1mol) was stirred at 70 ꢂ for 5h. cooled to room temperature, the resulting
solution was poured into water and acidized to pH 4–5 with AcOH . Filtering and
drying, we obtained yellow solid 4-substituted-3-nitrobenzoic acid .Yield 99%.
In a mixture of 4-substituted-3-nitrobenzoic acid (24mmol) and DCM (50 mL),
containing N,N-dimethylforma- mide (DMF),thionyl chloride (36 mmol) was slowly
dropping added at reflux temperature for 2 h. Cooled to room temperature, the
reaction mixture was concentrated to near dryness by vacuum distillation. It obtained
oily residue 3. Compounds 2a–2p (28 mmol) were dissolved in DCM (30 mL)
containing triethylamine (Et3N) (35 mmol). Subsequently, it was dropping added in a
DCM (40 mL) solution of the oily residue 3 at room temperature, and then the
mixture was stirred for 1–3h. After the completion of reaction, the reaction mixture
was extracted with H2O/DCM in the separatory funnel. The DCM layer was
concentrated by vacuum distillation, it obtained yellow oily residue (4a–4p).
4.2.3 General procedure for the synthesis of compounds 5a–5p
Compounds 4a–4p (20 mmol) were dissolved by a mixture solvent of AcOH/H2O
(25/25mL). Subsequently, Zn (80mmol) was added and stired at room temperature for
1h. Then the mixture was filtered, extracted (DCM 3*50 mL) and concentrated
by vacuum distillation. The residue was dissolved in EtOAc(1–3ml) and were forced
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crystal by adding PE (1~3ml) in ultrasonic. Filtered and dried, white and high-purity
solid (5a–5p) were obtained.
4.2.3.1 3-[(3-Amino-4-ethylamino-benzoyl)-phenyl-amino]-propionic acid ethyl ester
(5a). Yield: 85.2%; m.p. 195–196 ; HPLC: 99.3% 1H NMR (500 MHz, CDCl3) δ
(ppm) 7.28 (d, J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.15 (t, J = 7.3 Hz, 1H), 7.08 (d, J = 7.8
Hz, 2H), 6.86 (d, J = 1.4 Hz, 1H), 6.73 (dd, J = 8.2, 1.3 Hz, 1H), 6.31 (d, J = 8.3 Hz,
1H), 4.20 (t, J = 7.4 Hz, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.09 (q, J = 7.1 Hz, 2H), 2.71
(t, J = 7.4 Hz, 2H), 1.25 (t, J = 7.1 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H).
4.2.3.2 3-[(3-Amino-4-ethylamino-benzoyl)-o-tolyl-amino]-propionic acid ethyl ester
(5b).Yield: 86.3%; m.p. 133–135 ; HPLC: 97.3% 1H NMR (500 MHz, CDCl3) δ
(ppm) 7.14 (d, J = 9.1 Hz, 3H), 7.07 (d, J = 6.0 Hz, 1H), 6.86 (s, 1H), 6.67 (d, J = 7.3
Hz, 1H), 6.28 (d, J = 7.6 Hz, 1H), 4.38 – 4.30 (m, 1H), 4.07 (q, J = 7.1 Hz, 2H), 3.88
– 3.81 (m, 1H), 3.08 (q, J = 6.4 Hz, 2H), 2.76 – 2.71 (m, 2H), 2.20 (s, 3H), 1.27
–1.21(m, 6H).
4.2.3.3 3-[(3-Amino-4-ethylamino-benzoyl)-m-tolyl-amino]-propionic acid ethyl ester
(5c).Yield: 83.7%; m.p. 148–149 ; HPLC: 97.0% 1H NMR (500 MHz, CDCl3) δ
(ppm) 7.11 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.5 Hz, 1H), 6.93 (s, 1H), 6.89 (s, 1H),
6.83 (d, J = 7.7 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H), 6.31 (d, J = 8.3 Hz, 1H), 4.17 (t, J =
7.4 Hz, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.09 (q, J = 7.1 Hz, 2H), 2.71 (t, J = 7.4 Hz,
2H), 2.29 (s, 3H), 1.24 (dt, J = 17.1, 7.1 Hz, 6H).
4.2.3.4 3-[(3-Amino-4-ethylamino-benzoyl)-p-tolyl-amino]-propionic acid ethyl ester
(5d). Yield: 86.5%; m.p. 133–136 ; HPLC: 98.6% 1H NMR (400 MHz, CDCl3) δ
(ppm) 7.02 (d, J = 8.1 Hz, 2H), 6.93 (d, J = 8.2 Hz, 2H), 6.84 (d, J = 1.8 Hz, 1H),
6.71 (dd, J = 8.3, 1.8 Hz, 1H), 6.29 (d, J = 8.3 Hz, 1H), 4.14 (t, J = 7.4 Hz, 2H), 4.04
(q, J = 7.1 Hz, 2H), 3.06 (q, J = 7.1 Hz, 2H), 2.67 (t, J = 7.4 Hz, 2H), 2.27 (s, 3H),
1.21 (dt, J = 14.6, 7.1 Hz, 6H).
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4.2.3.5 3-[(3-Amino-4-ethylamino-benzoyl)-(2,3-dimethyl-phenyl)-amino]-propionic
acid ethyl ester (5e). Yield: 83.3%; m.p. 119–120 ; HPLC: 97.7% 1H NMR (400
MHz, CDCl3) δ (ppm) 6.99 (d, J = 10.8 Hz, 2H), 6.86 (d, J = 7.1 Hz, 2H), 6.60 (d, J
= 8.1 Hz, 1H), 6.24 (d, J = 8.0 Hz, 1H), 4.39 – 4.29 (m, 1H), 4.04 (q, J = 7.1 Hz, 2H),
3.78 – 3.68 (m, 1H), 3.05 (q, J = 7.1 Hz, 2H), 2.78 – 2.61 (m, 2H), 2.22 (s, 3H), 2.09
(s, 3H), 1.24 – 1.21 (m, 3H), 1.19 (t, J = 5.3 Hz, 3H).
4.2.3.6 3-[(3-Amino-4-ethylamino-benzoyl)-(2,4-dimethyl-phenyl)-amino]-propionic
acid ethyl ester (5f). Yield: 86.1%; m.p. 121–122 ; HPLC: 99.0% 1H NMR (400
MHz, CDCl3) δ (ppm) 6.97 (d, J = 8.1 Hz, 3H), 6.92 (s, 1H), 6.68 (d, J = 8.1 Hz,
1H), 6.29 (d, J = 8.2 Hz, 1H), 4.37 – 4.29 (m, 1H), 4.09 (q, J = 7.1 Hz, 2H), 3.86 –
3.79 (m, 1H), 3.08 (q, J = 6.8 Hz, 2H), 2.76 – 2.71 (m, 2H), 2.29 (s, 3H), 2.16 (s, 3H),
2.09 (s, 3H), 1.31 – 1.18 (m, 6H).
4.2.3.7 3-[(3-Amino-4-ethylamino-benzoyl)-(2,5-dimethyl-phenyl)-amino]-propionic
acid ethyl ester (5g). Yield: 81.2%; m.p. 124–126 ; HPLC: 98.8% 1H NMR (400
MHz, CDCl3) δ (ppm) 6.99 (d, J = 7.2 Hz, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.87 (d, J
= 5.2 Hz, 2H), 6.64 (d, J = 7.1 Hz, 1H), 6.24 (d, J = 8.0 Hz, 1H), 4.30 – 4.19 (m, 1H),
4.05 (q, J = 7.1 Hz, 2H), 3.89 – 3.78 (m, 1H), 3.05 (q, J = 6.9 Hz, 2H), 2.70 (t, J = 7.4
Hz, 2H), 2.24 (s, 3H), 2.08 (s, 3H), 1.21 (dt, J = 14.4, 7.3 Hz, 6H).
4.2.3.8 3-[(3-Amino-4-ethylamino-benzoyl)-(2,6-dimethyl-phenyl)-amino]-propionic
acid ethyl ester (5h). Yield: 78.4%; m.p. 151–153 ; HPLC: 97.2% 1H NMR (400
MHz, CDCl3) δ (ppm) 7.07 – 7.03 (m, 1H), 6.99 (d, J = 7.2 Hz, 2H), 6.83 (d, J = 1.8
Hz, 1H), 6.57 (dd, J = 8.3, 1.9 Hz, 1H), 6.22 (s, 1H), 4.05 (q, J = 7.1 Hz, 2H), 4.00 –
3.94 (m, 2H), 3.03 (q, J = 7.1 Hz, 2H), 2.80 – 2.73 (m, 2H), 2.17 (s, 6H), 1.20 (dt, J =
7.1, 5.4 Hz, 6H).
4.2.3.9 3-[(3-Amino-4-ethylamino-benzoyl)-(3,4-dimethyl-phenyl)-amino]-propionic
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acid ethyl ester(5i). Yield: 81.7%; m.p. 126–129 ; HPLC: 97.1% 1H NMR (400
MHz, CDCl3) δ (ppm) 6.95 (d, J = 7.9 Hz, 1H), 6.89 – 6.83 (m, 2H), 6.75 – 6.70 (m,
2H), 6.29 (d, J = 8.3 Hz, 1H), 4.12 (t, J = 7.5 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.07
(q, J = 7.1 Hz, 2H), 2.66 (t, J = 7.5 Hz, 2H), 2.17 (s, 3H), 2.16 (s, 3H), 1.21 (dt, J =
14.4, 7.1 Hz, 6H).
4.2.3.10 3-[(3-Amino-4-ethylamino-benzoyl)-(3,5-dimethyl-phenyl)-amino]-ropionic
acid ethyl ester (5j). Yield: 88.2%; m.p. 134–136 ; HPLC: 96.8% 1H NMR (400
MHz, CDCl3) δ (ppm) 6.89 (d, J = 1.8 Hz, 1H), 6.77 (s, 1H), 6.72 (dd, J = 8.3, 1.8 Hz,
1H), 6.66 (s, 2H), 6.29 (d, J = 8.3 Hz, 1H), 4.14 – 4.09 (m, 2H), 4.05 (q, J = 7.1 Hz,
2H), 3.07 (q, J = 7.1 Hz, 2H), 2.70 – 2.64 (m, 2H), 2.20 (s, 6H), 1.22 (dt, J = 14.2, 7.1
Hz, 6H).
4.2.3.11
3-[(3-Amino-4-ethylamino-benzoyl)-(3-fluoro-4-methyl-phenyl)-amino]-
propionic acid ethyl ester (5k). Yield: 80.3%; m.p. 135–136 ; HPLC: 97.7% 1H
NMR (400 MHz, CDCl3) δ (ppm) 7.00 (t, J = 8.4 Hz, 1H), 6.85 (d, J = 1.9 Hz, 1H),
6.77 (dd, J = 10.5, 1.8 Hz, 1H), 6.75 – 6.70 (m, 2H), 6.32 (d, J = 8.3 Hz, 1H), 4.12 (t,
J = 7.3 Hz, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.09 (q, J = 7.1 Hz, 2H), 2.67 (t, J = 7.3 Hz,
2H), 2.20 (s, 3H), 1.23 (dt, J = 16.5, 7.1 Hz, 6H).
4.2.3.12
3-[(3-Amino-4-ethylamino-benzoyl)-(4-fluoro-3-methyl-phenyl)-amino]-
propionic acid ethyl ester(5l). Yield: 81.5%; m.p. 144–145 ; HPLC: 97.3% 1H
NMR (400 MHz, CDCl3) δ (ppm) 6.92 (dd, J = 6.6, 1.8 Hz, 1H), 6.86 (d, J = 1.8 Hz,
1H), 6.84 (s, 1H), 6.83 – 6.79 (m, 1H), 6.67 (dd, J = 8.2, 1.7 Hz, 1H), 6.30 (d, J = 8.3
Hz, 1H), 4.11 (t, J = 7.4 Hz, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.08 (q, J = 7.2 Hz, 2H),
2.67 (t, J = 7.4 Hz, 2H), 2.19 (s, 3H), 1.22 (dt, J = 15.5, 7.1 Hz, 6H).
4.2.3.13 3-[(3-Amino-4-ethylamino-benzoyl)-(2,4-difluoro-phenyl)-amino]- propionic
acid ethyl ester(5m). Yield: 84.6%; m.p. 113–114 ; HPLC: 98.0% 1H NMR (400
MHz, CDCl3) δ (ppm) 7.17–7.12 (m5, 1H), 6.88 (d, J = 1.9 Hz, 1H), 6.81 (t, J = 8.2
11

ACCEPTED MANUSCRIPT
Hz, 2H), 6.72 (dd, J = 8.2, 1.4 Hz, 1H), 6.34 (d, J = 8.3 Hz, 1H), 4.10 (q, J = 7.1 Hz,
4H), 3.11 (q, J = 7.1 Hz, 2H), 2.73 (s, 2H), 2.09 (s, 1H), 1.28 (t, J = 5.2 Hz, 3H), 1.24
(t, J = 5.2 Hz, 3H).
4.2.3.14 3-[(3-Amino-4-ethylamino-benzoyl)-(4-methyl-pyridin-2-yl)-amino]-ropionic
acid ethyl ester (5n). Yield: 82.2%; m.p. 112–113 ; HPLC: 97.3% 1H NMR (400
MHz, CDCl3) δ (ppm) 8.13 (d, J = 5.1 Hz, 1H), 6.74 (d, J = 6.7 Hz, 2H), 6.51 (d, J
= 11.2 Hz, 2H), 6.12 (d, J = 8.4 Hz, 1H), 4.15 (t, J = 7.2 Hz, 2H), 3.89 (q, J = 7.1 Hz,
2H), 2.89 (q, J = 7.0 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H), 1.99 (s, 3H), 1.03 (t, J = 7.1 Hz,
6H).
4.2.3.15 3-[(3-Amino-4-ethylamino-benzoyl)-(5-methyl-pyridin-2-yl)-amino]-ropionic
acid ethyl ester (5o). Yield: 84.6%; m.p. 114–115 ; HPLC: 97.1% 1H NMR (500
MHz, CDCl3) δ (ppm) 8.27 (s, 1H), 7.22 (dd, J = 8.2, 1.8 Hz, 1H), 6.86 (d, J = 1.8
Hz, 1H), 6.74 (dd, J = 8.2, 1.8 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 6.35 (d, J = 8.3 Hz,
1H), 4.35 (t, J = 7.4 Hz, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.12 (q, J = 7.1 Hz, 2H), 2.75
(t, J = 7.4 Hz, 2H), 2.28 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H).
4.2.3.16 3-[(3-Amino-4-ethylamino-benzoyl)-(6-methyl-pyridin-2-yl)-amino]-ropionic
acid ethyl ester (5p). Yield: 80.2%; m.p. 119–120 ; HPLC: 98.2% 1H NMR (400
MHz, CDCl3) δ (ppm) 7.32 (d, J = 2.3 Hz, 1H), 6.91 (d, J = 7.1 Hz, 2H), 6.77 (dd, J =
8.2, 1.9 Hz, 1H), 6.52 (d, J = 7.9 Hz, 1H), 6.37 (d, J = 8.3 Hz, 1H), 4.41 (t, J = 7.3 Hz,
2H), 4.10 (q, J = 7.1 Hz, 2H), 3.14 (q, J = 7.1 Hz, 2H), 2.79 (t, J = 7.3 Hz, 2H), 2.58
(s, 3H), 2.14 (s, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz, 3H).
4.2.4 Procedure for the synthesis of common compound 6
A mixture of aminobenzyl cyanide (0.1mol) and bromoacetic acid (0.15mol) was
stirred at 100 ꢂ in water for 5 h. cooled to room temperature, solid separated. We
obtained white solid (4-cyano-2-substituted-phenylamino)-acetic acid (6), yield
80–87%.
12

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4.2.5 General procedure for the synthesis of compounds 7a–7p
A mixture of (4-cyano-2-substituted-phenylamino)-acetic acid (6) (15 mmol),
HOBt (15 mmol), EDCI (15 mmol) and 4 mL DMF was dissolved in 30 mL
tetrahydrofuran (THF), and the mixture was stirred under ice-cooling for 30 min, then,
a THF (40 mL) solution of the 5a–5p (13mmol) was slowly added at room
temperature, the mixture was stirred for 10 h. After the completion of reaction, the
solvent THF was removed by vacuum distillation. Then the mixture was extracted
with DCM (3*50 mL) and concentrated by vacuum distillation. The residue was
added in AcOH (50 mL) and heated to reflux for 2.5 h. After the completion of
reaction, the solvent AcOH was removed by vacuum distillation. Then, ammonia
(NH3·H2O) was added and let pH to 8~9, the mixture was extracted with DCM (3*50
mL) and the DCM layer was dried over anhydrous Na2SO4, and concentrated by
vacuum distillation.
The
residue
was
chromatographed
(silicagel,
dichloromethane/methanol=100:1) to obtain compounds. Finally, the compounds were
dissolved in EtOAc (5ml) and were forced crystal by adding PE (1–3ml) in
ultrasonic cleaner. Filtered and dried, white and high-purity solid (7a–7p) were
obtained.
4.2.5.1
3-({2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-phenyl-amino)-propionic acid ethyl ester (7a). Yield: 81.5%; m.p.
195–196 ; 1H NMR (501 MHz, CDCl3) δ (ppm) 7.70 (s, 1H), 7.48 (d, J = 8.6 Hz,
2H), 7.36 (dd, J = 8.5, 1.1 Hz, 1H), 7.22 (t, J = 7.7 Hz, 2H), 7.16 (d, J = 8.5 Hz, 1H),
7.13 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H), 5.47 (t, J =
3.9 Hz, 1H), 4.50 (d, J = 4.5 Hz, 2H), 4.27 (t, J = 7.3 Hz, 2H), 4.15 (q, J = 7.3 Hz,
2H), 4.09 (q, J = 7.2 Hz, 2H), 2.75 (t, J = 7.3 Hz, 2H), 1.40 (t, J = 7.3 Hz, 3H), 1.23 (t,
J = 7.1 Hz, 3H).
4.2.5.2
3-({2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-o-tolyl-amino)-propionic acid ethyl ester (7b). Yield: 83.7%; m.p. 175 –
13

ACCEPTED MANUSCRIPT
177 ; 1H NMR (500 MHz, CDCl3) δ (ppm)7.63 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H),
7.37 (d, J = 8.6 Hz, 1H), 7.17 – 7.07 (m, 5H), 6.73 (d, J = 8.4 Hz, 2H), 5.44 (s, 1H),
4.48 (d, J = 4.0 Hz, 2H), 4.17 – 4.12 (m, 2H), 4.09 (q, J = 7.0 Hz, 2H), 2.85 – 2.71 (m,
2H), 2.26 (s, 3H), 1.68 (s, 1H), 1.40 (t, J = 7.1 Hz, 3H), 1.24 (t, J = 7.0 Hz, 3H).
4.2.5.3
3-({2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-m-tolyl-amino)-propionic acid ethyl ester (7c). Yield:83.1%; m.p.
187–188 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.72 (s, 1H), 7.48 (d, J = 8.4 Hz,
2H), 7.37 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 7.7 Hz, 1H), 6.96
(s, 1H), 6.94 (d, J = 7.7 Hz, 1H), 6.83 (d, J = 7.9 Hz, 1H), 6.73 (d, J = 8.5 Hz, 2H),
5.48 (s, 1H), 4.50 (d, J = 4.4 Hz, 2H), 4.25 (t, J = 7.4 Hz, 2H), 4.15 (q, J = 7.2 Hz,
2H), 4.10 (q, J = 7.2 Hz, 2H), 2.74 (t, J = 7.4 Hz, 2H), 2.26 (s, 3H), 1.40 (t, J = 7.3 Hz,
3H), 1.24 (t, J = 7.1 Hz, 3H).
4.2.5.4
3-({2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-p-tolyl-amino)-propionic acid ethyl ester (7d).Yield: 82.8%; m.p.
158–160 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69 (s, 1H), 7.47 (d, J = 8.5 Hz,
2H), 7.35 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 8.0 Hz, 2H), 6.97
(d, J = 7.9 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H), 5.51 (s, 1H), 4.49 (d, J = 4.3 Hz, 2H),
4.23 (t, J = 7.3 Hz, 2H), 4.15 (q, J = 7.2 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.73 (t, J =
7.3 Hz, 2H), 2.25 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
4.2.5.5
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(2,3-dimethyl-phenyl)-amino]-propionic acid ethyl ester (7e). Yield: 87.6%;
m.p. 150–151 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.63 (s, 1H), 7.46 (d, J = 8.5
Hz, 2H), 7.32 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.99 (d, J = 7.2 Hz, 1H),
6.95 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 6.72 (d, J = 8.5 Hz, 2H), 5.51 (s, 1H),
4.47 (d, J = 4.1 Hz, 2H), 4.11–4.15 (m, 2H), 4.10 – 4.05 (m, 2H), 2.67–2.84 (m, 2H),
2.20 (s, 3H), 2.18 (s, 3H), 1.38 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
14

ACCEPTED MANUSCRIPT
4.2.5.6
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(2,4-dimethyl-phenyl)-amino]-propionic acid ethyl ester (7f). Yield: 85.4%;
m.p. 155–157 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.64 (s, 1H), 7.46 (d, J = 6.8
Hz, 2H), 7.35 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H),
6.92 (s, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 8.3 Hz, 2H), 5.50 (s, 1H), 4.48 (d, J
= 3.5 Hz, 2H), 4.46 – 4.40 (m, 1H), 4.17 – 4.11 (m, 2H), 4.11 – 4.06 (m, 2H),
3.79–3.85 (m, J = 1H), 2.84 – 2.68 (m, 2H), 2.22 (s, 3H), 2.20 (s, 3H), 1.39 (t, J = 5.8
Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
4.2.5.7
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(2,5-dimethyl-phenyl)-amino]-propionic acid ethyl ester (7g). Yield: 84.5%;
m.p. 163–165 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.65 (s, 1H), 7.47 (d, J = 8.3
Hz, 2H), 7.36 (d, J = 9.0 Hz, 1H), 7.14 (d, J = 8.9 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H),
6.91 (d, J = 5.1 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H), 5.49 (s, 1H), 4.48 (d, J = 4.1 Hz,
2H), 4.43 – 4.34 (m, 1H), 4.14 (q, J = 6.4 Hz, 2H), 4.11 – 4.06 (m, 2H), 3.94 – 3.86
(m, 1H), 2.78 (t, J = 6.3 Hz, 2H), 2.23 (s, 3H), 2.16 (s, 3H), 1.39 (t, J = 7.3 Hz, 3H),
1.24 (t, J = 7.0 Hz, 3H).
4.2.5.8
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(2,6-dimethyl-phenyl)-amino]-propionic acid ethyl ester (7h). Yield: 83.3%;
m.p. 184–185 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.58 (s, 1H), 7.46 (d, J = 8.2
Hz, 2H), 7.37 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 7.08 – 7.03 (m, 1H), 6.99
(d, J = 7.5 Hz, 2H), 6.72 (d, J = 8.4 Hz, 2H), 5.50 (s, 1H), 4.47 (d, J = 4.5 Hz, 2H),
4.17 – 4.12 (m, 2H), 4.11 – 4.06 (m, 4H), 2.84 (t, J = 7.5 Hz,2H), 2.25 (s, 6H), 1.39 (t,
J = 7.3 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H).
4.2.5.9
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(3,4-dimethyl-phenyl)-amino]-propionic acid ethyl ester (7i). Yield: 85.5%;
m.p. 149–151 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.71 (s, 1H), 7.46 (d, J = 8.6
Hz, 2H), 7.37 (dd, J = 8.6, 0.7 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 7.9 Hz,
15

ACCEPTED MANUSCRIPT
2H), 6.72 – 6.75 (m,3H), 5.51 (s, 1H), 4.49 (d, J = 4.4 Hz, 2H), 4.21 (t, J = 7.5 Hz,
2H), 4.15 (q, J = 7.2 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.72 (t, J = 7.4 Hz, 2H), 2.15
(s, 6H), 1.40 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
4.2.5.10
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(3,5-dimethyl-phenyl)-amino]-propionic acid ethyl ester (7j). Yield: 86.1%;
m.p. 163–166 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.74 (s, 1H), 7.49 (d, J = 8.6
Hz, 2H), 7.38 (dd, J = 8.5, 1.2 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 4.9 Hz,
2H), 6.73 (s, 1H), 6.70 (s, 2H), 5.47 (t, J = 4.3 Hz, 1H), 4.51 (d, J = 4.4 Hz, 2H), 4.22
(t, J = 7.5 Hz, 2H), 4.17 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 2.74 (t, J = 7.5
Hz, 2H), 2.18 (s, 6H), 1.68 (s, 3H), 1.41 (t, J = 7.3 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H).
4.2.5.11
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(3-fluoro-4-methyl-phenyl)-amino]-propionic acid ethyl ester (7k). Yield:
85.8%; m.p. 167–168 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.69 (s, 1H), 7.48 (d,
J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.00 (t, J = 8.2 Hz,
1H), 6.81 – 6.76 (m, 2H), 6.74 (d, J = 8.4 Hz, 2H), 5.48 (t, J = 3.7 Hz, 1H), 4.51 (d, J
= 4.5 Hz, 2H), 4.22 (t, J = 7.2 Hz, 2H), 4.17 (q, J = 6.9 Hz, 2H), 4.11 (q, J = 7.0 Hz,
2H), 2.73 (t, J = 7.2 Hz, 2H), 2.17 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H), 1.24 (t, J = 7.2 Hz,
3H).
4.2.5.12
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(4-fluoro-3-methyl-phenyl)-amino]-propionic acid ethyl ester (7l). Yield:
87.6%; m.p. 185–186 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.68 (s, 1H), 7.47 (d,
J = 8.5 Hz, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 6.98 (d, J = 5.8 Hz,
1H), 6.82 (s, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.74 (d, J = 8.6 Hz, 2H), 5.50 (t, J = 4.4
Hz, 1H), 4.51 (d, J = 4.4 Hz, 2H), 4.21 (t, J = 7.3 Hz, 2H), 4.17 (q, J = 7.5 Hz, 2H),
4.10 (q, J = 7.1 Hz, 2H), 2.72 (t, J = 7.2 Hz, 2H), 2.17 (s, 3H), 1.41 (t, J = 7.2 Hz, 3H),
1.24 (t, J = 7.1 Hz, 3H).
16

ACCEPTED MANUSCRIPT
4.2.5.13
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(2,4-difluoro-phenyl)-amino]-propionic acid ethyl ester (7m). Yield: 82.2%;
m.p. 148–149 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.67 (s, 1H), 7.50 (d, J = 8.7
Hz, 2H), 7.38 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.20 – 7.16 (m, 1H), 6.75
(d, J = 8.6 Hz, 4H), 4.52 (d, J = 4.5 Hz, 2H), 4.16 – 4.20 (m, 4H), 4.11 (q, J = 7.2 Hz,
2H), 2.77 (d, J = 40.7 Hz, 2H), 1.65 (s, 2H), 1.43 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.1
Hz, 3H).
4.2.5.14
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(4-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester (7n). Yield: 76%;
m.p. 167–169 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 8.27 (d, J = 5.0 Hz, 1H), 7.75
(s, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 7.8 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H),
6.85 (d, J = 5.1 Hz, 1H), 6.75 (d, J = 8.7 Hz, 2H), 6.66 (s, 1H), 5.52 (t, J = 4.6 Hz,
1H), 4.52 (d, J = 4.4 Hz, 2H), 4.40 (t, J = 7.3 Hz, 2H), 4.18 (q, J = 7.1 Hz, 2H), 4.10
(q, J = 7.1 Hz, 2H), 2.81 (t, J = 7.3 Hz, 2H), 2.11 (s, 3H), 1.75 (s, 1H), 1.42 (t, J = 7.2
Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
4.2.5.15
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(5-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester (7o). Yield: 71%;
m.p. 172–174 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 8.25 (s, 1H), 7.70 (s, 1H),
7.45 (d, J = 8.7 Hz, 2H), 7.33 (dd, J = 8.3, 1.2 Hz, 1H), 7.19 – 7.14 (m, 2H), 6.73 (d, J
= 8.6 Hz, 2H), 6.64 (d, J = 8.0 Hz, 1H), 4.51 (d, J = 4.5 Hz, 2H), 4.39 (t, J = 7.4 Hz,
2H), 4.16 (q, J = 7.2 Hz, 2H), 4.08 (q, J = 7.2 Hz, 2H), 2.79 (t, J = 7.3 Hz, 2H), 2.24
(s, 3H), 1.40 (t, J = 7.2 Hz, 3H), 1.22 (t, J = 7.1 Hz, 3H).
4.2.5.16
3-[{2-[(4-Cyano-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole-5-
carbonyl}-(6-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester (7p). Yield:
70.8%; m.p. 157–158 ; 1H NMR (500 MHz, CDCl3) δ (ppm) 7.72 (s, 1H), 7.46 (d,
J = 8.6 Hz, 2H), 7.33 (d, J = 8.5 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.4 Hz,
1H), 6.86 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 8.6 Hz, 2H), 6.47 (d, J = 7.8 Hz, 1H), 5.55
17

ACCEPTED MANUSCRIPT
(t, J = 4.0 Hz, 1H), 4.51 (d, J = 4.5 Hz, 2H), 4.44 (t, J = 7.3 Hz, 2H), 4.16 (q, J = 7.2
Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.81 (t, J = 7.3 Hz, 2H), 2.52 (s, 3H), 1.83 (s, 1H),
1.40 (t, J = 7.2 Hz, 3H), 1.23 (t, J = 7.1 Hz, 3H).
4.2.6 General procedure for the synthesis of compounds 8a–8p
A mixture of compounds (7a–7p) (5.7 mmol), hydroxylamine hydrochloride (24
mmol) and triethylamine (24mmol) were dissolved in 30 mL ethanol (EtOH), and the
mixture was heated at reflux temperature for 3 h. The solvent was distilled off. In a
acetic acid (AcOH) (20 mL) solution of the residue, 10% Pd/C (24mmol) and
ammonium formate (34 mmol) were added to, and the mixture was stirred at reflux
temperature under nitrogen atmosphere for 5 h. The reaction mixture was filtered and
the filtrate was concentrated through vacuum distillation. The residue was
chromatographed (silica gel, dichloromethane/methanol = 7:1) to obtain solid or oil
compounds. Finally, the compounds were dissolved in EtOAc (5ml) and were forced
crystal by adding PE (1~3ml) in ultrasonic. Filtered and dried, white and high-purity
solid (8a–8p) were obtained.
4.2.6.1 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-phenyl-amino)-propionic acid ethyl ester (8a). Yield: 71.6%, m.p.
183–185 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.62 (d, J = 8.7 Hz, 2H), 7.47
(s, 1H), 7.39 (d, J = 8.5 Hz, 1H), 7.25 (t, J = 7.6 Hz, 2H), 7.19 (t, J = 9.8 Hz, 3H),
7.14 (t, J = 7.4 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 4.62 (d, J = 5.4 Hz, 2H), 4.25 (q, J =
7.0 Hz, 2H), 4.10 (t, J = 7.1 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H),
1.71 (s, 2H), 1.24 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H).
4.2.6.2 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-o-tolyl-amino)-propionic acid ethyl ester (8b). Yield: 77.3%; m.p.
200–201 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm)7.61 (d, J = 8.4 Hz, 2H), 7.39
(d, J = 11.4 Hz, 2H), 7.29 (s, 1H), 7.23 (t, J = 7.4 Hz, 2H), 7.13 (d, J = 8.7 Hz, 2H),
6.83 (d, J = 8.5 Hz, 2H), 4.61 (d, J = 4.4 Hz, 2H), 4.23 (d, J = 6.8 Hz, 2H), 3.99 (q, J
18

ACCEPTED MANUSCRIPT
= 7.0 Hz, 2H), 3.74 – 3.66 (m, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.14 (s, 3H), 1.79 (s, 3H),
1.23 (t, J = 6.7 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H), 0.94 (t, J = 7.2 Hz, 1H).
4.2.6.3 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-m-tolyl-amino)-propionic acid ethyl ester (8c). Yield: 71.5%; m.p.
199–200 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.61 (d, J = 8.6 Hz, 2H), 7.50
(s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 7.08 (d, J =
7.8 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.84 (d, J = 8.6 Hz,
2H), 4.62 (d, J = 5.3 Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H), 4.07 (t, J = 7.2 Hz, 2H), 4.00
(q, J = 14.2, 7.2 Hz, 2H), 2.60 (t, J = 7.2 Hz, 2H), 2.21 (s, 3H), 1.24 (t, J = 7.1 Hz,
3H), 1.14 (t, J = 7.1 Hz, 3H).
4.2.6.4 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-p-tolyl-amino)-propionic acid ethyl ester (8d). Yield: 76.6%; m.p.
200–201 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 7.62 (d, J = 8.7 Hz, 2H), 7.48
(s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.08 – 7.03 (m, 4H), 6.85
(d, J = 8.7 Hz, 2H), 4.62 (d, J = 5.3 Hz, 2H), 4.25 (q, J = 7.2 Hz, 2H), 4.05 (t, J = 7.1
Hz, 2H), 4.00 (q, J = 7.1 Hz, 2H), 2.58 (t, J = 7.1 Hz, 2H), 2.19 (s, 3H), 1.24 (t, J =
7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H).
4.2.6.5 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,3-dimethyl-phenyl)-amino]-propionic acid ethyl ester (8e). Yield:
74.8%; m.p. 200–202 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.60 (d, J = 8.5
Hz, 2H), 7.41 (s, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 8.7 Hz, 1H), 7.03 – 6.96
(m, 3H), 6.83 (d, J = 8.3 Hz, 2H), 4.61 (d, J = 5.1 Hz, 2H), 4.23 (q, J = 7.4 Hz, 2H),
3.99 (q, J = 7.0 Hz, 2H), 2.70 – 2.53 (m, 2H), 2.15 (s, 3H), 2.10 (s, 3H), 1.23 (t, J =
6.8 Hz, 3H), 1.14 (t, J = 7.0 Hz, 3H), 0.94 (t, J = 7.1 Hz, 1H).
4.2.6.6 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,4-dimethyl-phenyl)-amino]-propionic acid ethyl ester (8f). Yield:
19

ACCEPTED MANUSCRIPT
78.1%; m.p. 200–202 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.62 (d, J = 8.5
Hz, 2H), 7.41 (s, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.23 (d, J = 7.4 Hz, 1H), 7.11 (d, J =
7.8 Hz, 1H), 6.95 (s, 0H), 6.93 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 8.2 Hz, 2H), 4.61 (d, J
= 4.7 Hz, 1H), 4.27 – 4.22 (m, 1H), 4.00 (q, J = 7.0 Hz, 1H), 3.67 – 3.63 (m, 1H),
2.62 (t, J = 7.7 Hz, 1H), 2.16 (s, 1H), 2.10 (s, 1H), 1.24 (t, J = 6.5 Hz, 2H), 1.14 (t, J =
6.8 Hz, 2H).
4.2.6.7 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,5-dimethyl-phenyl)-amino]-propionic acid ethyl ester (8g). Yield:
73.3%; m.p. 183–184 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 7.61 (d, J = 7.6 Hz,
2H), 7.43 (s, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 7.12 (s, 1H), 7.00
(d, J = 6.4 Hz, 1H), 6.93 (d, J = 7.3 Hz, 1H), 6.84 (d, J = 8.3 Hz, 2H), 4.61 (d, J = 3.8
Hz, 2H), 4.25 – 4.21 (m, 2H), 4.00 (q, J = 6.7Hz, 2H), 3.74 (m, 2H), 2.64 (t, J = 8.9
Hz, 2H), 2.20 (s, 3H), 2.04 (s, 3H), 1.23 (t, J = 5.7 Hz, 3H), 1.14 (t, J = 6.9 Hz, 3H),
0.94 (t, J = 7.1 Hz, 1H).
4.2.6.8 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,6-dimethyl-phenyl)-amino]-propionic acid ethyl ester (8h). Yield:
72.6%; m.p. 196–199 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 7.61 (d, J = 8.7 Hz,
2H), 7.40 (d, J = 8.5 Hz, 1H), 7.31 (s, 1H), 7.24 (d, J = 8.5 Hz, 1H), 7.07 – 7.04 (m,
1H), 7.03 (d, J = 5.5 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H), 4.61 (d, J = 5.1 Hz, 2H), 4.24
(q, J = 6.9 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.92 (t, J = 7.3 Hz, 2H), 2.70 (t, J = 7.4
Hz, 2H), 2.18 (s, 6H), 1.23 (t, J = 7.0 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H).
4.2.6.9 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(3,4-dimethyl-phenyl)-amino]-propionic acid ethyl ester (8i). Yield:
75%; m.p. 198–199 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 7.61 (d, J = 8.6 Hz,
2H), 7.51 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.07 (s, 1H), 6.96
(d, J = 7.9 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 7.8 Hz, 1H), 4.62 (d, J = 5.2
Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H), 4.06 – 4.02 (m, 2H), 4.01 – 3.97 (m, 2H), 2.58 (t, J
20

ACCEPTED MANUSCRIPT
= 7.2 Hz, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 1.24 (t, J = 7.0 Hz, 3H), 1.14 (t, J = 7.1 Hz,
3H).
4.2.6.10 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(3,5-dimethyl-phenyl)-amino]-propionic acid ethyl ester (8j). Yield:
76.7%; m.p. 200–204 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 7.63 (d, J = 8.7 Hz,
2H), 7.52 (s, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 8.8
Hz, 2H), 6.81 (s, 2H), 6.79 (s, 1H), 4.64 (d, J = 5.5 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H),
4.06 – 4.01 (m, 4H), 4.01 – 3.97 (m, 2H), 2.60 (q, J = 7.2 Hz, 6H), 2.13 (s, 6H), 1.24
(t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H).
4.2.6.11 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(3-fluoro-4-methyl-phenyl)-amino]-propionic acid ethyl ester (8k). Yield:
76.3%; m.p. 213–216 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 7.64 (d, J = 8.7 Hz,
2H), 7.52 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 7.11
(d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 4.64 (d, J = 5.3 Hz, 2H), 4.27 (q, J = 6.7
Hz, 2H), 4.07 (t, J = 7.1 Hz, 2H), 4.00 (q, J = 7.2 Hz, 2H), 2.62 – 2.58 (m, 2H), 2.11
(s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H).
4.2.6.12 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(4-fluoro-3-methyl-phenyl)-amino]-propionic acid ethyl ester (8l).
Yield: 72.1%; m.p. 205–206 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.61 (d, J =
8.7 Hz, 2H), 7.51 (s, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 5.8 Hz, 1H), 7.21 (d, J
= 8.4 Hz, 1H), 7.00 (d, J = 8.6 Hz, 1H), 6.97 (s, 1H), 6.85 (d, J = 8.6 Hz, 2H), 4.63 (d,
J = 5.3 Hz, 2H), 4.26 (q, J = 6.8 Hz, 2H), 4.05 (t, J = 7.0 Hz, 2H), 4.00 (q, J = 7.0 Hz,
2H), 2.60 (t, J = 7.0 Hz, 2H), 2.13 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz,
3H).
4.2.6.13 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,4-difluoro-phenyl)-amino]-propionic acid ethyl ester (8m). Yield:
21

ACCEPTED MANUSCRIPT
77.8%; m.p. 223–225 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm)7.62 (d, J = 8.5 Hz,
2H), 7.57 (d, J = 8.2 Hz, 1H), 7.49 (s, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.32 (s, 1H), 7.21
(d, J = 6.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 4.64 (d, J = 4.9 Hz, 2H), 4.26 (q, J = 7.0
Hz, 2H), 4.02 (t, J = 6.3 Hz, 2H), 4.00 – 3.95 (m, 2H), 2.63 (d, J = 10.0 Hz, 2H), 1.25
(t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H).
4.2.6.14 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(4-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester (8n). Yield:
70.5%; m.p. 202–205 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm) 8.20 (d, J = 5.0 Hz,
1H), 7.62 (d, J = 8.7 Hz, 2H), 7.50 (s, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 8.4
Hz, 1H), 6.98 (d, J = 4.9 Hz, 1H), 6.91 (s, 1H), 6.85 (d, J = 8.7 Hz, 2H), 4.64 (d, J =
5.4 Hz, 2H), 4.28 (q, J = 7.0 Hz, 3H), 4.17 (t, J = 7.1 Hz, 2H), 3.98 (q, J = 7.1 Hz,
2H), 2.67 (t, J = 7.2 Hz, 2H), 2.11 (s, 3H), 1.24 (t, J = 7.0 Hz, 3H), 1.13 (t, J = 7.1 Hz,
3H).
4.2.6.15 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(5-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester (8o). Yield:
71.3%; m.p. 221–224 ; 1H NMR (500 MHz, DMSO-d6) δ(ppm)8.23 (s, 1H), 7.62
(d, J = 8.7 Hz, 2H), 7.48 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.40 (dd, J = 8.3, 1.2 Hz,
1H), 7.18 (d, J = 8.4 Hz, 1H), 6.84 (t, J = 9.3 Hz, 3H), 4.64 (d, J = 5.3 Hz, 2H), 4.27
(q, J = 6.7 Hz, 2H), 4.18 (t, J = 7.0 Hz, 2H), 3.98 (q, J = 7.1 Hz, 2H), 2.66 (t, J = 7.1
Hz, 2H), 2.19 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H).
4.2.6.16 3-[{2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(6-methyl-pyridin-2-yl)-amino]-propionic acid ethyl ester (8p). Yield:
68.2%; m.p. 200–202 ; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.64 (d, J = 8.7
Hz, 2H), 7.48 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.40 (d, J = 4.6 Hz, 1H), 7.16 (d, J =
8.2 Hz, 1H), 6.99 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 6.62 (d, J = 8.0 Hz,
1H), 4.65 (d, J = 5.4 Hz, 2H), 4.28 (q, J = 7.0Hz, 2H), 4.21 (t, J = 7.0 Hz, 2H), 3.98 (q,
J=7.0Hz, 2H), 2.67 (t, J = 7.1 Hz, 2H), 2.42 (s, 3H), 1.25 (t, J = 7.0 Hz, 3H), 1.12 (t, J
22

ACCEPTED MANUSCRIPT
= 7.1 Hz, 3H).
4.2.7 General procedure for the synthesis of compounds 9a–9p
Compounds 8a–8p (2 mmol) were dissolved in ethanol/H2O (5/5mL), and a
solution of sodium hydroxide (6 mmol) were added, and keeping stirred at room
temperature for 30 min. The mixture was acidized with acetic acid to pH = 6–7 and it
precipitated white solid. Filtered, washed with water and methanol and dried,
compounds 9a–9p were obtained.
4.2.7.1 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-phenyl-amino)-propionic acid (9a). White solid, yield: 90.2%; m.p.
244–246 ; HPLC: 98.3% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.96 (s,
1H), 8.71 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 11.1 Hz, 2H), 7.31 (d, J = 8.7
Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.22 (m, 3H), 7.15 (t, J = 7.0 Hz, 1H), 6.88 (d, J =
8.9 Hz, 2H), 4.79 (s, 2H), 4.35 (q, J = 6.7 Hz, 2H), 4.06 (t, J = 7.5 Hz, 2H), 3.16 (s,
2H), 2.56 (t, J = 7.5 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). 13C NMR (126 MHz,
DMSO-d6+2HCl) δ (ppm) 173.03, 170.29, 164.74, 153.36, 152.99, 143.76, 135.71,
130.69, 130.18, 129.69, 128.40, 127.13, 123.88, 119.46, 113.87, 113.80, 113.30,
112.21, 110.23, 48.99, 46.86, 39.06, 32.64, 15.20. HRMS (ESI) m/z Calcd for
C27H28O6N3 (M+H): 485.22229, Fond: 458.23193 [M+H]+.
4.2.7.2 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoImidazole
-5-carbonyl}-o-tolyl-amino)-propionic acid (9b). Yield: 90.3%; m.p. 259–260 ;
HPLC: 98.6% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.91 (s, 1H), 8.66 (s,
1H), 7.66 (d, J = 8.7 Hz, 2H), 7.47 (s, 1H), 7.40 (d, J = 11.7 Hz, 2H), 7.25 (t, J = 9.6
Hz, 2H), 7.13 (d, J = 5.9 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.63 (s, 2H), 4.23 (t, J =
12.9 Hz, 2H), 3.82 – 3.56 (m, 2H), 2.59 (dd, J = 11.8, 7.6 Hz, 2H), 2.14 (s, 3H), 1.23
(t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6+2HCl) δ (ppm) 173.05, 170.34,
164.71, 153.37, 152.97, 142.24, 135.88, 135.22, 131.70, 130.40, 130.18, 129.99,
128.14, 127.37, 123.46, 119.11, 113.72, 113.27, 112.15, 110.04, 56.46, 45.97, 38.99,
23

ACCEPTED MANUSCRIPT
32.38, 18.01, 15.23. HRMS (ESI) m/z Calcd for C28H30O6N3 (M+H): 499.23794,
Fond: 499.24786 [M+H]+.
4.2.7.3 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoImidazole
-5-carbonyl}-m-tolyl-amino)-propionic acid (9c). Yield: 93.3%; m.p. 252–253 ;
HPLC: 99.2% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.93 (s, 1H), 8.69 (s,
1H), 7.67 (d, J = 8.6 Hz, 2H), 7.53 (s, 1H), 7.46 (d, J = 7.3 Hz, 1H), 7.25 (d, J = 8.4
Hz, 1H), 7.13 (s, 1H), 7.09 (t, J = 7.7 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 6.88 (t, J =
9.0 Hz, 3H), 4.69 (s, 2H), 4.29 (q, J = 6.6 Hz, 2H), 4.03 (t, J = 7.5 Hz, 2H), 2.55 (t, J
= 7.6 Hz, 2H), 2.21 (s, 3H), 1.25 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6+2HCl) δ (ppm) 172.96, 169.30, 164.62, 153.01, 152.64, 141.31, 138.84,
134.11, 133.09, 130.92, 130.28, 129.79, 127.81, 126.59, 125.51, 115.06, 114.87,
113.70, 112.62, 112.43, 56.43, 45.93, 32.03, 20.44, 14.80, 14.55. HRMS (ESI) m/z
Calcd for C28H30O6N3 (M+H): 499.23794, Fond: 499.24750 [M+H]+.
4.2.7.4 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-p-tolyl-amino)-propionic acid (9d). Yield: 88.7%; m.p. 246–247 ;
HPLC: 97.6% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.91 (s, 1H), 8.65 (s,
1H), 7.66 (d, J = 8.8 Hz, 2H), 7.50 (s, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.4
Hz, 1H), 7.11 – 7.02 (m, 4H), 6.87 (d, J = 8.9 Hz, 2H), 4.66 (s, 2H), 4.27 (q, J = 6.8
Hz, 2H), 4.02 (t, J = 7.5 Hz, 2H), 2.57 – 2.52 (m, 2H), 2.19 (s, 3H), 1.24 (t, J = 7.1 Hz,
3H). 13C NMR (100 MHz, DMSO-d6+2HCl) δ (ppm) 173.08, 170.26, 164.72, 154.88,
153.26, 152.97, 141.05, 139.17, 136.43, 135.45, 131.11, 130.32, 130.19, 128.21,
124.04, 119.04, 113.88, 113.32, 112.23, 110.43, 46.87, 32.57, 21.50, 20.89, 15.53,
15.16. HRMS (ESI) m/z Calcd for C28H30O6N3 (M+H): 499.23794, Fond: 499.24792
[M+H]+.
4.2.7.5 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,3-dimethyl-phenyl)-amino)-propionic acid (9e). Yield: 87.6%; m.p.
255–256 ; HPLC: 97.7% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.97 (s, 1H),
24

ACCEPTED MANUSCRIPT
8.74 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.52 (d, J = 8.3 Hz, 1H), 7.49 (s, 1H), 7.26 (d, J
= 8.5 Hz, 1H), 7.00 (d, J = 6.7 Hz, 2H), 6.97 (d, J = 7.6 Hz, 1H), 6.87 (d, J = 8.8 Hz,
2H), 4.74 (s, 2H), 4.35 – 4.28 (m, 2H), 2.63 (m, 2H), 2.13 (d, J = 13.0 Hz, 6H), 1.26 (t,
13
J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d6+2HCl) δ (ppm) 173.05, 170.02,
164.71, 153.19, 152.99, 143.49, 139.23, 135.19, 131.43, 130.20, 129.38, 128.53,
127.96, 125.80, 124.29, 118.62, 114.02, 113.31, 112.27, 110.67, 56.46, 46.91, 32.59,
21.27, 18.90, 15.08. HRMS (ESI) m/z Calcd for C29H32O6N3 (M+H): 513.25359,
Fond: 513.26341[M+H]+.
4.2.7.6 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,4-dimethyl-phenyl)-amino)-propionic acid (9f). Yield: 91.7%; m.p.
250–251 ; HPLC: 97.3% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.94 (s, 1H),
8.71 (s, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 12.0 Hz, 2H), 7.27 (d, J = 8.3 Hz,
1H), 7.14 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 10.9 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 4.70
(s, 2H), 4.36 – 4.25 (m, 2H), 3.71 – 3.56 (m, 2H), 2.54–2.59 (m, 2H), 2.13 (d, J =
23.1 Hz, 6H), 1.26 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6+2HCl) δ (ppm)
173.09, 170.36, 164.78, 153.27, 152.96, 139.57, 137.34, 135.56, 134.84, 132.23,
130.94, 130.18, 129.77, 127.99, 123.68, 118.61, 113.93, 113.31, 112.23, 110.31, 45.98,
32.34, 21.50, 20.88, 17.91, 15.16. HRMS (ESI) m/z Calcd for C29H32O6N3 (M+H):
513.25359, Fond: 513.26259 [M+H]+.
4.2.7.7 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,5-dimethyl-phenyl)-amino)-propionic acid (9g). Yield: 89.3%; m.p.
248–249 ; HPLC: 98.0% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.89 (s,
1H), 8.70 (s, 1H), 7.65 (d, J = 8.7 Hz, 2H), 7.44 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.25
(d, J = 8.4 Hz, 1H), 7.15 (s, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H),
6.86 (d, J = 8.7 Hz, 2H), 4.62 (s, 2H), 4.34 – 4.20 (m, 2H), 4.20 – 4.10 (m, 1H), 3.75
– 3.66 (m, 1H), 2.59 (t, J = 7.0 Hz, 2H), 2.20 (s, 3H), 2.03 (s, 3H), 1.23 (t, J = 7.0 Hz,
3H). 13C NMR (100 MHz, DMSO-d6+2HCl) δ (ppm) 173.10, 170.32, 164.72, 153.44,
152.96, 142.17, 140.79, 136.62, 136.14, 131.94, 131.44, 130.17, 128.85, 123.42,
25

ACCEPTED MANUSCRIPT
119.50, 113.62, 113.29, 112.13, 109.83, 46.31, 38.88, 32.40, 20.85, 17.60, 15.29.
HRMS (ESI) m/z Calcd for C29H32O6N3 (M+H): 513.25359, Fond: 513.26344
[M+H]+.
4.2.7.8 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
5-carbonyl}-(2,6-dimethyl-phenyl)-amino)-propionic acid (9h). Yield: 81.3%; m.p.
250–253 ; HPLC: 96.6% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.90 (s, 1H),
8.64 (s, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.41 (s, 1H), 7.31 (d, J = 1.0 Hz, 1H), 7.25 (dd,
J = 8.5, 1.5 Hz, 1H), 7.04 (q, J = 4.7 Hz, 3H), 6.85 (d, J = 8.9 Hz, 2H), 4.63 (s, 2H),
4.25 (q, J = 6.9 Hz, 2H), 3.87 (t, J =8.0Hz,2H), 2.65 (t, J =7.8Hz, 2H), 2.18 (s, 6H),
13
1.23 (t, J = 7.1 Hz, 3H). C NMR (126 MHz, DMSO-d6+2HCl) δ (ppm) 172.98,
169.92, 164.80, 164.73, 153.24, 153.13, 141.33, 135.68, 135.58, 130.70, 130.33,
130.20, 129.53, 128.28, 123.63, 117.82, 113.97, 113.32, 112.25, 110.47, 46.28, 32.36,
18.77, 18.34, 15.46, 15.10. HRMS (ESI) m/z Calcd for C29H32O6N3 (M+H):
513.25359, Fond: 513.26362 [M+H]+.
4.2.7.9 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(3,4-dimethyl-phenyl)-amino)-propionic acid (9i). Yield: 88.4%; m.p.
247–248 ; HPLC: 97.2% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.97 (s,
1H), 8.73 (s, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.60 (s, 2H), 7.34 (d, J = 8.6 Hz, 1H), 7.11
(d, J = 1.8 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 8.9 Hz, 2H), 6.83 (dd, J =
7.9, 1.6 Hz, 1H), 4.80 (s, 2H), 4.36 (q, J = 6.8 Hz, 2H), 4.00 (t, J = 7.5 Hz, 2H), 2.54
(d, J = 7.7 Hz, 2H), 2.12 (s, 3H), 2.10 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H). 13C NMR (126
MHz, DMSO-d6+2HCl) δ (ppm) 172.97, 169.20, 164.82, 164.74, 153.01, 152.73,
140.69, 137.94, 135.62, 133.94, 133.45, 130.59, 130.27, 128.94, 126.10, 125.71,
116.00, 114.85, 113.54, 112.57, 112.14, 56.45, 46.89, 32.53, 19.81, 19.26, 18.87,
14.68. HRMS (ESI) m/z Calcd for C29H32O6N3 (M+H): 513.25359, Fond: 513.25989
[M+H]+.
4.2.7.10 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
26

ACCEPTED MANUSCRIPT
-5-carbonyl}-(3,5-dimethyl-phenyl)-amino)-propionic acid (9j). Yield: 92.1%; m.p.
256–257 ; HPLC: 98.1% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.96 (s, 1H),
8.73 (s, 1H), 7.68 (d, J = 8.8 Hz, 2H), 7.59 (m, 2H), 7.33 (d, J = 8.4 Hz, 1H), 6.89 (d,
J = 8.9 Hz, 2H), 6.85 (s, 2H), 6.80 (s, 1H), 4.77 (s, 2H), 4.35 (q, J =6.5 Hz, 2H),3.98(t,
13
J =7.4 Hz, 2H), 2.54 (t, J =7.6 Hz,2H), 2.13 (s, 6H), 1.28 (t, J = 7.1 Hz, 3H). C
NMR (126 MHz, DMSO-d6+2HCl) δ (ppm) 173.03, 170.23, 164.76, 153.38, 152.92,
143.64, 138.77, 135.79, 130.76, 130.32, 130.18, 128.71, 125.98, 123.79, 119.42,
113.27, 112.20, 110.19, 56.49, 47.05, 39.04, 32.64, 21.17, 19.11, 15.21. HRMS (ESI)
m/z Calcd for C29H32O6N3 (M+H): 513.25359, Fond: 513.26371 [M+H]+.
4.2.7.11 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(3-fluoro-4-dimethyl-phenyl)-amino)-propionic acid (9k). Yield: 84.7%;
m.p. 235–236 ; HPLC: 96.4% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 9.00
(s, 1H), 8.76 (s, 1H), 7.69 (t, J = 7.1 Hz, 3H), 7.64 (s, 1H), 7.38 (d, J = 8.5 Hz, 1H),
7.19 (dd, J = 10.8, 1.9 Hz, 1H), 7.14 (t, J = 8.5 Hz, 1H), 6.94 (d, J = 1.8 Hz, 1H), 6.90
(d, J = 9.0 Hz, 2H), 4.86 (s, 2H), 4.40 (q, J = 6.9 Hz, 2H), 4.04 (t, J = 7.4 Hz, 2H),
13
2.56 (t, J = 7.4 Hz, 2H), 2.11 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H). C NMR (100 MHz,
DMSO-d6+2HCl) δ (ppm) 173.10, 170.35, 164.73, 161.85, 159.42, 153.33, 153.10,
142.97, 135.88, 132.14, 130.51, 130.32, 130.18, 124.51, 123.73, 123.19, 123.02,
119.41, 115.15, 114.92, 112.21, 110.35, 46.83, 32.62, 18.97, 15.22, 14.13. HRMS
(ESI) m/z Calcd for C28H29FO6N3 (M+H): 517.22852, Fond: 517.23569 [M+H]+.
4.2.7.12 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(4-fluoro-3-dimethyl-phenyl)-amino)-propionic acid (9l). Yield: 87.6%;
m.p. 250–251 ; HPLC: 96.5% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.92 (s,
1H), 8.68 (s, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.53 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.28
(d, J = 5.7 Hz, 1H), 7.22 (dd, J = 8.4, 0.9 Hz, 1H), 6.98 (d, J = 7.9 Hz, 2H), 6.87 (d, J
= 8.9 Hz, 2H), 4.67 (s, 2H), 4.28 (q, J = 6.9 Hz, 2H), 4.01 (t, J = 7.5 Hz, 2H), 2.55 (t,
J = 7.5 Hz, 2H), 2.13 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6+2HCl) δ (ppm) 173.05, 170.05, 164.72, 160.53, 158.11, 153.19, 153.02,
27

ACCEPTED MANUSCRIPT
139.49, 135.22, 131.25, 130.20, 128.09, 125.78, 125.60, 124.18, 118.63, 115.98,
115.75, 114.02, 112.28, 110.71, 56.45, 46.90, 32.56, 18.88, 15.08, 14.57. HRMS (ESI)
m/z Calcd for C28H29FO6N3 (M+H): 517.22852, Fond: 517.23790 [M+H]+.
4.2.7.13 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(2,4-difluoro-phenyl)-amino)-propionic acid (9m). Yield: 83%; m.p.
254–257 ; HPLC: 97.7% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.93 (s,
1H), 8.68 (s, 1H), 7.67 (d, J = 8.9 Hz, 2H), 7.62 (dd, J = 8.9, 2.8 Hz, 1H), 7.51 (s, 2H),
7.22 (dd, J = 22.0, 8.6 Hz, 2H), 6.88 (d, J = 8.9 Hz, 2H), 4.70 (s, 2H), 4.30 (q, J = 6.7
13
Hz,2H), 3.98 (t, J = 6.9 Hz, 2H), 2.72 – 2.52 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H). C
NMR (100 MHz, DMSO-d6+2HCl) δ (ppm) 173.09, 171.02, 164.75, 162.57, 162.45,
160.11, 160.00, 153.44, 153.18, 141.16, 136.37, 132.04, 130.15, 129.33, 122.88,
119.36, 112.84, 112.62, 112.15, 110.09, 105.43, 105.17, 104.92, 46.29, 38.88, 32.78,
21.52, 15.28. HRMS (ESI) m/z Calcd for C27H26F2O6N3 (M+H): 521.20344, Fond:
521.20994 [M+H]+.
4.2.7.14 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(4-methyl-pyridin-2-yl)-amino)-propionic acid (9n).Yield: 82.7%; m.p.
236–238 ; HPLC: 96.8% 1H NMR (400 MHz, DMSO-d6+2HCl) δ (ppm) 8.91 (s, 1H),
8.68 (s, 1H), 8.18 (d, J = 5.6 Hz, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.52 (d, J = 1.1 Hz,
1H), 7.46 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.5, 1.4 Hz, 1H), 7.00 (s, 2H), 6.88 (d, J =
8.9 Hz, 2H), 4.68 (s, 2H), 4.30 (q, J = 6.9 Hz, 2H), 4.13 (t, J = 7.5 Hz, 2H), 2.65 –
2.56 (m, 2H), 2.13 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6+2HCl) δ (ppm) 172.93, 169.92, 164.70, 156.00, 153.28, 152.92, 150.00,
148.65, 134.54, 132.81, 130.24, 125.01, 123.30, 122.57, 117.05, 114.52, 113.37,
112.44, 111.75, 56.43, 45.26, 33.03, 20.83, 18.91, 14.83. HRMS (ESI) m/z Calcd for
C27H29O7N3 (M+H): 500.23319, Fond: 500.24048 [M+H]+.
4.2.7.15 3-({2-[(4-Carbamimidoyl-phenylamino)-methyl]-1-ethyl-1H-benzoimidazole
-5-carbonyl}-(5-methyl-pyridin-2-yl)-amino)-propionic acid (9o). Yield: 86.5%; m.p.
28