Synthesis and antithrombotic evaluation of novel dabigatran prodrugs containing a cleavable moiety with anti-platelet activity

Article abstract of DOI:10.1016/j.ejmech.2012.09.016

A novel series of prodrugs consisting of dabigatran and 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) were synthesized. The pharmacological results show that all of them possess the effect of anti-platelet aggregation induced by thrombin in vitro. Moreover, one of those compounds, Y-2 (ED₅₀ = 2.1 ± 1.3 mg/kg) shows more potent activity for inhibiting thrombosis in vivo than that of dabigatran etexilate (ED₅₀ = 4.4 ± 2.2 mg/kg).

Full text of DOI:10.1016/j.ejmech.2012.09.016

European Journal of Medicinal Chemistry 57 (2012) 21e28
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antithrombotic evaluation of novel dabigatran prodrugs containing
a cleavable moiety with anti-platelet activity
,
a
c
b,
Xiao-Zhi Yang a, Wen-Hui Yang b, Yun-Gen Xu a , Xiao-Juan Diao , Guang-Wei He , Guo-Qing Gong
*
**
a Department of Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
b Department of Pharmacology, China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China
c Hefei Yi Gong Pharmaceutical Co. Ltd., Hefei 230088, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of prodrugs consisting of dabigatran and 2-hydroxymethyl-3,5,6-trimethylpyrazine
(HTMP) were synthesized. The pharmacological results show that all of them possess the effect of
anti-platelet aggregation induced by thrombin in vitro. Moreover, one of those compounds, Y-2
(ED50 ¼ 2.1 ꢀ 1.3 mg/kg) shows more potent activity for inhibiting thrombosis in vivo than that of
dabigatran etexilate (ED50 ¼ 4.4 ꢀ 2.2 mg/kg).
Received 15 March 2012
Received in revised form
8 September 2012
Accepted 10 September 2012
Available online 18 September 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Dabigatran
HTMP
Thrombin inhibitors
Anti-platelet aggregation
Prevention and treatment of thrombosis
1. Introduction
Dabigatran etexilate (Fig. 1) is the first orally direct thrombin
inhibitor. As a double prodrug of dabigatran, it is hydrolyzed to
Thrombosis, which could occur in the arterial or the venous
circulation, has a major medical impact nowadays. Due to the
difference of pathophysiology between arterial thrombosis and
venous thrombosis, the drugs used to treat for these conditions are
different. In broad terms, arterial thrombosis is treated with anti-
platelet drugs while venous thrombosis is treated with drugs that
target factors of the coagulation cascade.
However, the activation of platelets, as the key factor of arterial
thrombosis, is closely related with the activation of thrombin,
which is an important factor of the coagulation cascade: firstly,
PAR-1, as a new target on platelet, needs thrombin to activate it [1];
secondly, the adhesion and aggregation of platelets can activate the
coagulation cascade to increase the amount of thrombin. There are
also clinical evidences for those relations: although warfarin,
dabigatran etexilate and rivaroxaban are thrombin inhibitors, they
have been used for the prevention of arterial thromboembolic
events [2]; similarly, the anti-platelet drug-aspirin can also be used
for the venous thromboembolic events [3].
generate dabigatran after oral administration. In vivo, dabigatran
(Ki ¼ 4.5 nM) can bind to human thrombin selectively and revers-
ibly to realize the strong and long-lasting anticoagulant effects [4].
On the one hand, it is used for the prevention of venous throm-
boembolic events in patients who have undergone total hip- or
knee-replacement surgery [5]. On the other hand, it is used for the
prevention of stroke and systemic embolism in patients with
nonvalvular atrial fibrillation, and this indication had been
approved by FDA on October 19, 2010.
Ligustrazine (tetramethyl pyrazine, TMP) as a major active
component of the herb medicine Chuanxiong (Ligusticum wallichii
Franchat), has long been reported to inhibit the platelet aggregation.
It was reported that 2-hydroxymethyl-3,5,6-trimethylpyrazine
(HTMP) as shown in Fig. 1 was the major metabolite of TMP
in vivo and it has a stronger inhibitory effect on platelet aggregation
than TMP [6].
In order to combine the therapeutic benefits of both dabigatran
and HTMP, we coupled dabigatran with HTMP via esterification
to afford a series of mutual prodrugs. We expected that these
ester bonds of dabigatran-HTMP could be hydrolyzed in vivo by
esterase to generate dabigatran and HTMP, which possess synerg-
istical inhibitory effect on venous or arterial thrombosis via two
routeseantiplatelet and antithrombin. The inhibitory effect of these
* Corresponding author.
** Corresponding author.
E-mail address: xu_yungen@hotmail.com (Y.-G. Xu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.016

22
X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
Fig. 1. Structures of dabigatran etexilate, HTMP and Y-2.
compounds for venous thrombosis will be discussed in this paper
and their inhibitory effects for arterial thrombosis are still under
study.
Y-1eY-7. The synthetic route for target compounds is depicted in
Scheme 1.
2.2. Pharmacological evaluation
2. Results and discussion
All synthesized compounds, along with the reference compound
dabigatran etexilate, were evaluated in rabbit thrombin-induced
platelet aggregation assay. Considering that all tested compounds
cannot inhibit thrombin directly unless hydrolyzed, they were all
incubated in rabbit liver microsomal suspensions [7] before test
in vitro. Most of tested compounds reveal similar activities (IC50 of
each compound was revealed in Table 1), meanwhile, the activity of
HTMP in the rabbit thrombin-induced platelet aggregation model is
relatively lower (IC50 ¼ 8.41 ꢀ 1.36 mM).
2.1. Chemistry
3-({2-[(4-Carbamimidoyl-phenylamino)methyl]-1-methyl-1H-
benzoimidazole-5-carbonyl}pyridin-2-yl-amino)-propionic
acid
ethyl ester hydrochloride (1) [4] reacted separately with seven
different chloroformates in the presence of potassium carbonate to
afford carbonate derivatives a1ea7. These intermediates were
converted into carboxylic acid derivatives b1eb7 by sodium
hydroxide hydrolysis. Reactions of b1eb7 with 2-hydroxymethyl-
3,5,6-trimethylpyrazine (HTMP) in the presence of 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-
Dimethylaminopyridine (DMAP) yielded the target compounds:
Compounds Y-1 (IC50 ¼ 3.13 nM) and Y-2 (IC50 ¼ 38.1 nM) were
chosen to investigate their inhibitory effects on the rat venous
thrombosis model (Reyers) [9]. The results revealed that both
compound Y-1 (ED50 ¼ 1.8 ꢀ 1.4 mg/kg) and Y-2 (ED50 ¼ 2.1 ꢀ1.3 mg/
Scheme 1. Reagents, conditions and range of yields: (a) R1eR7 chloroformate, K2CO3, THF/H2O, yields: 57.2e73.8%; (b) NaOH, EtOH/H2O, yields: 69.9e91.6%; (c) (3,5,6-
trimethylpyrazin-2-yl)methanol, EDCI, DMAP, DMF, yields: 58.8e67.6%.

X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
23
Table 1
2.3. Pharmacokinetics
Structures of target compounds Y-1eY-7 and their activities for inhibiting thrombin-
induced rabbit platelet aggregation after the incubation with rabbit liver
microsomes.
No adverse effects were found when SpragueeDawley female
rats received 20 mg/kg Y-2. Whole blood samples were collected
at different time points, and mixed with 4% formic acid. After
organic solvent treatments, plasma metabolites were determined
by liquid chromatography tandem mass spectrometry method.
Both dabigatran and HTMP were detected in the rat plasma
(Table 3).
3. Conclusion
In summary, a series of novel mutual prodrugs of dabigatran and
HTMP were designed, synthesized and evaluated. Firstly, after
preliminary pharmacological screening in vitro, further test in vivo
demonstrated that each of the compound Y-1 (ED50 ¼ 1.8 ꢀ 1.4 mg/
kg) and Y-2 (ED50 ¼ 2.1 ꢀ 1.3 mg/kg) possess a stronger activity for
inhibiting thrombosis than that of dabigatran etexilate
(ED50 ¼ 4.4 ꢀ 2.2 mg/kg). Secondly, the result of ADP-induced
platelet aggregation test shows that the moiety of HTMP might
be the cause of the stronger activity. Lastly, pharmacokinetic eval-
uation proved that Y-2 could be metabolized into dabigatran and
HTMP.
a
Compd.
R
IC50
(m
M)
Dabigatran etexilate
/
0.326 ꢀ 0.0019
0.00313 ꢀ 0.00041
0.0381 ꢀ 0.0117
0.218 ꢀ 0.103
Y-1
Y-2
Y-3
Y-4
Y-5
Y-6
Y-7
Tert-butyl
n-Hexyl
Ethyl
Benzyl
n-Pentyl
Isopropyl
Methyl
0.292 ꢀ 0.356
0.0728 ꢀ 0.0177
0.295 ꢀ 0.257
0.287 ꢀ 0.154
a
Drug concentration to achieve half-maximal inhibition of rabbit platelet
aggregation induced by thrombin. Data were expressed as mean ꢀ SD from six
independent experiments.
4. Experiment protocol
kg) (Fig. 2) possess more potent activity for inhibiting thrombosis
in vivo than that of dabigatran etexilate (ED50 ¼ 4.4 ꢀ 2.2 mg/kg), and
the ED50 value of HTMP is 42.6 ꢀ 6.4 mg/kg under the same conditions.
Based on the results obtained above, we speculated that theactivityfor
inhibiting thrombosis of Y-2 might originate from two parts: the
anticoagulant effect of dabigatran and the anti-platelet aggregation
activity of HTMP (Fig. 3).
To confirm the synergistic inhibitory effect of dabigatran and
HTMP on thrombosis, we analyzed the inhibitory effect of
compound Y-2 along with two reference compounds (dabigatran
etexilate and HTMP) on ADP-induced platelet aggregation. In this
experiment, compound Y-2 and dabigatran etexilatewereincubated
in rabbit liver microsomal suspensions before the activity assay. The
results showed that compound Y-2 (IC50 ¼ 4.03 ꢀ 2.96 mM) and
HTMP (IC50 ¼ 1.67 ꢀ 0.99 mM) displayed their activities respectively
(Table 2) while dabigatran etexilate showed no inhibitory effect in
this test.
Melting points were determined on a RDCSY-I capillary appa-
ratus and were uncorrected. The IR spectra (in KBr pellets) were
recorded on a Nicolet Impact 410 spectrophotometer. The 1H NMR
and 13C NMR spectra were recorded on a Bruker AV-300 or AV-500
NMR spectrometer using TMS as an internal standard and chemical
shifts were given in ppm with tetramethylsilane (TMS). The mass
spectra were recorded on an Agilent 1100 series LC/MSD Tarp (SL).
The HRMS spectra were acquired on a waters Micros Q-TOF appa-
ratus. All solvents were purchased from commercial sources and
used as received unless otherwise stated.
4.1. General procedure for synthesis of compounds a1da7
4.1.1. 3-({2-[(4-{Amino-[(E)-tert-butyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a1)
Compound 1 (2 g, 3.7 mmol) was dissolved in 45 mL of THF and
45 mL of water. Potassium carbonate (1.55 g, 11.2 mmol) was added,
and the mixture was stirred at room temperature for 20 min. Then,
cooled to 0 ꢁC, di-tert-butyl dicarbonate (0.98 g, 4.5 mmol) was
added drop-wise. During the addition, the temperature was kept at
0e5 ꢁC. After the addition was completed, the temperature of the
mixture was allowed to rise slowly to room temperature and stir-
ring was continued for 20 h. The organic phase was separated, dried
over anhydrous sodium sulfate, and concentrated in vacuum. The
residue was chromatographed (silica gel, dichloromethane/meth-
anol 30:1) to afford the title compound 1.63 g as white crystals:
yield 73.5%, m.p. 150e152 ꢁC,
Y-1
Y-2
10
8
6
4
ED =2.1mg/kg
50
2
1H NMR (500 MHz DMSO-d6)
d
(ppm): 1.12 (3H, t, J ¼ 7.1 Hz, e
ED50=1.8mg/kg
10
CH2CH3),1.43 (9H, s, eC(CH3)3), 2.68 (2H, t, J ¼ 7.0 Hz, >NCH2CH2e),
3.77 (3H, s, >NCH3), 3.98 (2H, q, J ¼ 7.1 Hz, eCH2CH3), 4.23(2H, t,
J ¼ 7.1 Hz, >NCH2CH2e), 4.59 (2H, d, J ¼ 4.4 Hz, eCH2NHe), 6.76 (2H,
d, J ¼ 8.8 Hz, ArH), 6.89 (1H, d, J ¼ 7.7 Hz, ArH), 7.11 (1H, t, J ¼ 7.1 Hz,
ArH), 7.16 (1H, d, J ¼ 8.4 Hz, ArH), 7.39 (1H, d, J ¼ 8.5 Hz, ArH), 7.48
(1H, s, ArH), 7.55 (1H, t, J ¼ 7.6 Hz, ArH), 7.77 (2H, d, J ¼ 8.8 Hz, ArH),
8.39 (1H, d, J ¼ 3.6 Hz, ArH), 8.80e9.70 (2H, brs, eNH2); ESI-MS(m/z):
600.3[M þ H]þ.
0
0
5
15
20
25
compound dose(mg/kg i.g.)
Fig. 2. Dose-response curve for the antithrombotic efficacy of Y-1 and Y-2 (0.1e20 mg/
kg) in clot weight in rat venous thrombosis model (Reyers). Average dry clot weight in
the vehicle-treated was 8.9 ꢀ 1.6 mg. Values are expressed as mean ꢀ SD of eight
animals/group.

24
X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
Fig. 3. Proposed metabolic route of compound Y-2.
Table 2
4.1.3. 3-({2-[(4-{Amino-[(E)-ethyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a3)
Starting from 1 and ethyl chloroformate, the title compound was
prepared following the procedure described for compound a2 to
afford a3 as a white crystals in 62.4% yield, m.p.172e174 ꢁC, 1H NMR
The inhibitory effects on ADP-induced rabbit platelet aggregation of Y-2, HTMP and
dabigatran etexilate.
Compd.
IC50a (mM)
Y-2
HTMP
Dabigatran etexilate
/
4.03 ꢀ 2.96
1.67 ꢀ 0.99
a
Drug concentration to achieve half-maximal inhibition of rabbit platelet
aggregation induced by ADP. Data were expressed as mean ꢀ SD from six inde-
pendent experiments.
(500 MHz CDCl3)
d
(ppm): 1.21 (3H, t, J ¼ 6.8 Hz, eOCH2CH3), 1.33
(3H, t, J ¼ 6.8 Hz, eNCOOCH2CH3), 2.79 (2H, t, J ¼ 6.8 Hz,
>NCH2CH2e), 3.64 (3H, s, >NCH3), 4.07 (2H, q, J ¼ 6.8 Hz, e
OCH2CH3), 4.19, (2H, q, J ¼ 6.8 Hz, eNCOOCH2CH3), 4.40e4.42
(4H, m, >NCH2CH2e, eCH2NHe), 5.52 (1H, s, eNHe), 6.57 (2H, d,
J ¼ 8.0 Hz, ArH), 6.70 (1H, d, J ¼ 7.8 Hz, ArH), 6.97e7.02 (2H, m,
ArH), 7.21 (1H, d, J ¼ 8.2 Hz, ArH), 7.33 (1H, d, J ¼ 7.6 Hz, ArH), 7.65e
7.69 (3H, m, ArH), 8.40 (1H, s, ArH), 9.30e9.70 (2H, brs, eNH2); ESI-
MS(m/z): 572.0[M þ H]þ, 594.0[M þ Na]þ.
4.1.2. 3-({2-[(4-{Amino-[(E)-hexyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a2)
Compound 1 (2 g, 3.7 mmol) was dissolved in 60 mL of THF and
12 mL of water. Potassium carbonate (1.55 g, 11.2 mmol) was added,
and the mixture was stirred at room temperature for 20 min. Then,
n-hexyl chloroformate (0.74 g, 4.5 mmol) was added drop-wise and
stirring was continued for another 5 h. The organic phase was
separated, dried over anhydrous sodium sulfate, and concentrated
in vacuum. The residue was chromatographed (silica gel,
dichloromethane/methanol 25:1) to afford the title compound
1.47 g as colorless crystals: yield 63.3%, m.p. [4] 130e132 ꢁC,
4.1.4. 3-({2-[(4-{Amino-[(E)-benzyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a4)
Starting from 1 and benzyl chloroformate, the title compound
was prepared following the procedure described for compound a2
to afford a4 as a white crystals in 73.8% yield, m.p. 165e167 ꢁC, 1H
1H NMR (300 MHz DMSO-d6)
d
(ppm): 0.87 (3H, t, J ¼ 6.9 Hz, e
NMR (500 MHz CDCl3)
d
(ppm): 1.21 (3H, t, J ¼ 7.1 Hz, eCH2CH3),
CH2CH2CH3), 1.12 (3H, t, J ¼ 7.2 Hz, eCH2CH3), 1.27e1.29 (6H, m, e
CH2CH2CH2CH2CH3), 1.57 (2H, q, J ¼ 6.6 Hz, eOCH2CH2CH2e), 2.68
(2H, t, J ¼ 6.9 Hz, >NCH2CH2e), 3.77 (3H, s, >NCH3), 3.94e4.00 (4H,
m, eOCH2CH3, eOCH2CH2CH2e), 4.22, (2H, t, J ¼ 6.9 Hz, >NCH2CH2e
), 4.59 (2H, d, J ¼ 5.4 Hz, eCH2NHe), 6.76 (2H, d, J ¼ 8.7 Hz, ArH), 6.88
(1H, d, J ¼ 8.1 Hz, ArH), 6.98 (1H, m, NH), 7.10e7.17 (2H, m, ArH), 7.39
(1H, d, J ¼ 8.4 Hz, ArH), 7.47 (1H, s, ArH), 7.54 (1H, t, J ¼ 7.7 Hz, ArH),
7.79 (2H, d, J ¼ 8.7 Hz, ArH), 8.39 (1H, d, J ¼ 4.8 Hz, ArH), 8.60e9.30
(2H, brs, eNH2); ESI-MS(m/z): 628.4[M þ H]þ.
2.80 (2H, t, J ¼ 7.2 Hz, >NCH2CH2e), 3.64 (3H, s, >NCH3),
4.07 (2H, q, J ¼ 7.1 Hz, eCH2CH3), 4.40e4.43 (4H, m, >NCH2CH2e,
eCH2NHe), 5.19 (2H, s, eOCH2Ph) 5.32 (1H, s, eNHe), 6.60 (2H, d,
J ¼ 8.4 Hz, ArH), 6.70 (1H, d, J ¼ 8.0 Hz, ArH), 6.95e6.98 (1H, m,
ArH), 7.04 (1H, d, J ¼ 8.4 Hz), 7.25e7.34 (5H, m, ArH), 7.43 (2H, d,
J ¼ 7.3 Hz, ArH), 7.67e7.72 (3H, m, ArH), 8.40 (1H, d, J ¼ 3.8 Hz, ArH),
9.20e9.70 (2H, brs, eNH2); ESI-MS(m/z): 634.0[M þ H]þ,656.0
[M þ Na]þ.
Table 3
Mean plasma concentration of HTMP or dabigatran following administration with Y-2.
Time (h)
0.167
0.25
0.5
1.0
1.5
2.0
4.0
6.0
HTMP (ng/mL)
Dabigatran (ng/mL)
423 ꢀ 105
109 ꢀ 29.6
955 ꢀ 472
245 ꢀ 138
1075 ꢀ 515
252 ꢀ 135
938 ꢀ 378
264 ꢀ 116
495 ꢀ 222
164 ꢀ 99.9
430 ꢀ 136
152 ꢀ 74.5
188 ꢀ 68.7
67.3 ꢀ 35.9
93.3 ꢀ 40.0
18.2 ꢀ 11.2

X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
25
4.1.5. 3-({2-[(4-{Amino-[(E)-pentyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a5)
Starting from 1 and n-pentyl chloroformate, the title compound
was prepared following the procedure described for compound a5
to afford a6 as a white crystals in 63.0% yield, m.p. 148e150 ꢁC, 1H
J ¼ 7.4 Hz, >NCH2CH2e), 3.76 (3H, s, >NCH3), 4.18, (2H, t, J ¼ 7.5 Hz,
>NCH2CH2e), 4.58 (2H, d, J ¼ 4.4 Hz, eCH2NHe), 6.75 (2H, d,
J ¼ 8.9 Hz, ArH), 6.94 (1H, d, J ¼ 8.1 Hz, ArH), 7.10e7.13 (1H, m, ArH),
7.16 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.3 Hz, ArH), 7.39 (1H, d, J ¼ 8.5 Hz,
ArH), 7.48 (1H, s, ArH), 7.56 (1H, td, J1 ¼7.8 Hz, J2 ¼ 1.7 Hz, ArH), 7.76
(2H, d, J ¼ 8.8 Hz, ArH), 8.37 (1H, d, J ¼ 3.6 Hz, ArH), 8.40e9.55 (2H,
brs, eNH2), 11.30e12.75 (1H, brs, eCOOH); ESI-MS(m/z): 572.2
[M þ H]þ, 570.2[M ꢂ H]ꢂ.
NMR (500 MHz CDCl3)
d
(ppm): 0.91 (3H, t, J ¼ 7.1 Hz, e
CH2CH2CH3), 1.21 (3H, t, J ¼ 7.2 Hz, eOCH2CH3), 1.33a.40 (4H, m,
eCH2CH2CH2CH3), 1.73 (2H, q, J ¼ 7.3 Hz, eOCH2CH2CH2e) 2.80
(2H, t, J ¼ 7.3 Hz, >NCH2CH2e), 3.67 (3H, s, >NCH3), 4.07 (2H, q,
J ¼ 7.1 Hz, eOCH2CH3), 4.13, (2H, t, J ¼ 6.9 Hz, eOCH2CH2CH2e),
4.41e4.43 (4H, m, >NCH2CH2e, eCH2NHe), 5.35 (1H, s, eNHe),
6.62 (2H, d, J ¼ 8.7 Hz, ArH), 6.70 (1H, d, J ¼ 8.1 Hz, ArH), 6.96e
6.99 (1H, m, ArH), 7.05 (1H, d, J ¼ 8.5 Hz, ArH), 7.25 (1H, d,
J ¼ 1.5 Hz, ArH), 7.32 (1H, td, J1 ¼ 7.9 Hz, J2 ¼ 1.9 Hz, ArH), 7.67 (1H,
s, ArH), 7.72 (2H, d, J ¼ 8.7 Hz, ArH), 8.41 (1H, d, J ¼ 3.6 Hz, ArH),
9.20e9.80 (2H, brs, eNH2); ESI-MS(m/z): 614.1[M þ H]þ, 636.0
[M þ Na]þ.
4.2.2. 3-({2-[(4-{Amino-[(E)-hexyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b2)
The title compound was prepared following the procedure
described for compound b1 from a2 to afford b2 as a white crystals
in 77.5% yield, m.p. 196e198 ꢁC, 1H NMR (500 MHz DMSO-d6)
d
(ppm): 0.87 (3H, t, J ¼ 7.0 Hz, eCH2CH2CH3), 1.28e1.34 (6H, m, e
CH2CH2CH2CH2CH3), 1.58 (2H, qui, J ¼ 7.6 Hz, eOCH2CH2CH2e),
2.61 (2H, t, J ¼ 7.5 Hz, >NCH2CH2e), 3.76 (3H, s, >NCH3), 3.98
(2H, t, J ¼ 6.7 Hz, eOCH2CH2CH2e), 4.18, (2H, t, J ¼ 7.5 Hz,
>NCH2CH2e), 4.59 (2H, d, J ¼ 4.5 Hz, eCH2NHe), 6.76 (2H, d,
J ¼ 8.9 Hz, ArH), 6.95 (1H, d, J ¼ 8.1 Hz, ArH), 7.10e7.13 (1H, m, ArH),
7.16 (1H, dd, J1 ¼ 8.5 Hz, J2 ¼ 1.5 Hz, ArH), 7.39 (1H, d, J ¼ 8.5 Hz,
ArH), 7.48 (1H, s, ArH), 7.55 (1H, td, J1 ¼ 7.8 Hz, J2 ¼ 2.0 Hz, ArH),
7.79 (2H, d, J ¼ 8.9 Hz, ArH), 8.38 (1H, d, J ¼ 4.8 Hz, ArH), 8.50e9.50
(2H, brs, eNH2), 11.30e12.55 (1H, brs, eCOOH); ESI-MS(m/z): 600.3
[M þ H]þ, 622.3[M þ Na]þ.
4.1.6. 3-({2-[(4-{Amino-[(E)-isopropyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a6)
Starting from 1 and isopropyl chloroformate, the title compound
was prepared following the procedure described for compound a2
to afford a6 as a white crystals in 62.8% yield, m.p. 128e130 ꢁC, 1H
NMR (500 MHz CDCl3)
d
(ppm): 1.21 (3H, t, J ¼ 7.2 Hz, eOCH2CH3),
1.33 (6H, d, J ¼ 6.3 Hz, eCH(CH3)2), 2.80 (2H, t, J ¼ 7.3 Hz,
>NCH2CH2e), 3.66 (3H, s, >NCH3), 4.07 (2H, q, J ¼ 7.2 Hz, e
OCH2CH3), 4.40e4.43 (4H, m, >NCH2CH2e, eCH2NHe), 4.97 (1H,
sep, J ¼ 6.3 Hz, eCH(CH3)2), 5.38 (1H, s, eNHe), 6.60 (2H, d,
J ¼ 8.6 Hz, ArH), 6.70 (1H, d, J ¼ 8.1 Hz, ArH), 6.96e6.99 (1H, m,
ArH), 7.03 (1H, d, J ¼ 8.4 Hz, ArH), 7.24 (1H, d, J ¼ 8.4 Hz, ArH), 7.32
(1H, td, J1 ¼ 7.9 Hz, J2 ¼ 1.8 Hz, ArH), 7.67 (1H, s, ArH), 7.71 (2H, d,
J ¼ 8.5 Hz, ArH), 8.40 (1H, d, J ¼ 3.6 Hz, ArH), 9.20e9.80 (2H, brs, e
NH2); ESI-MS(m/z): 586.1[M þ H]þ, 608.0[M þ Na]þ.
4.2.3. 3-({2-[(4-{Amino-[(E)-ethyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b3)
The title compound was prepared following the procedure
described for compound b1 from a3 to afford b3 as a white crystals
in 80.0% yield, m.p. 202e203 ꢁC, 1H NMR (500 MHz DMSO-d6)
d
(ppm): 1.21 (3H, t, J ¼ 6.9 Hz, eCH2CH3), 2.61 (2H, t, J ¼ 7.5 Hz,
>NCH2CH2e), 3.77 (3H, s, >NCH3), 4.02 (2H, q, J ¼ 7.1 Hz, e
CH2CH3), 4.18, (2H, t, J ¼ 7.5 Hz, >NCH2CH2e), 4.59 (2H, d,
J ¼ 5.5 Hz, eCH2NHe), 6.76 (2H, d, J ¼ 8.9 Hz, ArH), 6.94 (1H, d,
J ¼ 8.0 Hz, ArH), 7.10e7.13 (1H, m, ArH), 7.16 (1H, dd, J1 ¼ 8.5 Hz,
J2 ¼ 1.5 Hz, ArH), 7.39 (1H, d, J ¼ 8.4 Hz, ArH), 7.47 (1H, d, J ¼ 1.0 Hz,
ArH), 7.56 (1H, td, J1 ¼ 7.8 Hz, J2 ¼ 1.9 Hz, ArH), 7.79 (2H, d,
J ¼ 8.9 Hz, ArH) 8.40 (1H, d, J ¼ 3.7 Hz, ArH), 8.50e9.50 (2H, brs, e
NH2), 11.50e12.50 (1H, brs, eCOOH); ESI-MS(m/z): 544.0[M þ H]þ,
566.0[M þ Na]þ.
4.1.7. 3-({2-[(4-{Amino-[(E)-methyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid ethyl ester (a7)
Starting from 1 and methyl chloroformate, the title compound
was prepared following the procedure described for compound a2
to afford a7 as a white crystals in 57.2% yield, m.p. 156e158 ꢁC, 1H
NMR (500 MHz CDCl3)
d
(ppm): 1.21 (3H, t, J ¼ 7.2 Hz, eOCH2CH3),
2.80 (2H, t, J ¼ 7.2 Hz, >NCH2CH2e), 3.68 (3H, s, >NCH3), 3.77 (3H, s,
eOCH3), 4.07 (2H, q, J ¼ 7.2 Hz, eOCH2CH3), 4.41e4.45 (4H, m,
>NCH2CH2e, eCH2NHe), 5.37 (1H, s, eNHe), 6.64 (2H, d,
J ¼ 8.8 Hz, ArH), 6.71 (1H, d, J ¼ 8.1 Hz, ArH), 6.96e6.99 (1H, m,
ArH), 7.06 (1H, d, J ¼ 8.5 Hz, ArH), 7.25e7.28 (1H, m, ArH), 7.33 (1H,
td, J1 ¼ 7.6 Hz, J2 ¼ 2.0 Hz, ArH), 7.68 (1H, s, ArH), 7.73 (2H, d,
J ¼ 8.7 Hz, ArH), 8.40 (1H, dq, J1 ¼ 4.9 Hz, J2 ¼ 0.7 Hz ArH), 9.20e
9.70 (2H, brs, eNH2); ESI-MS(m/z): 558.2[M þ H]þ, 580.2[M þ Na]þ.
4.2.4. 3-({2-[(4-{Amino-[(E)-benzyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b4)
The title compound was prepared following the procedure
described for compound b1 from a4 to afford b4 as a white crystals
in 91.6% yield, m.p. 179e181 ꢁC, 1H NMR (500 MHz DMSO-d6)
d
(ppm): 2.60 (2H, t, J ¼ 7.5 Hz, >NCH2CH2e), 3.76 (3H, s, >NCH3),
4.17 (2H, t, J ¼ 7.5 Hz, >NCH2CH2e), 4.59 (2H, s, eCH2NHe), 5.08
(2H, s, eOCH2Ph), 6.77 (2H, d, J ¼ 8.9 Hz, ArH), 6.95 (1H, d,
J ¼ 8.1 Hz, ArH), 7.10e7.12 (1H, m, ArH), 7.15e7.17 (1H, m, ArH),
7.31e7.40 (6H, m, ArH), 7.48 (1H, s, ArH), 7.55 (1H, td, J1 ¼ 7.8 Hz,
J2 ¼ 1.8 Hz, ArH), 7.81 (2H, d, J ¼ 8.8 Hz, ArH), 8.37 (1H, d, J ¼ 3.6 Hz,
ArH), 9.00e9.70 (2H, brs, eNH2), 11.70e12.50 (1H, brs, eCOOH);
ESI-MS(m/z): 606.0[M þ H]þ, 628.0[M þ Na]þ.
4.2. General procedure for synthesis of compounds b1db7
4.2.1. 3-({2-[(4-{Amino-[(E)-tert-butyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b1)
Compound a1 (1.20 g, 2.0 mmol) was added to a solution of
sodium hydroxide (0.28 g, 7.0 mmol) in 30 mL of water and 15 mL of
ethanol and kept at room temperature for 5 h, then the solution was
concentrated in vacuum and neutralized with 10% (w/v) citric acid
under ice bath. The precipitate was collected by suction filtration
and washed with cold water to afford the desired compound as
white crystals (1.03 g, 90.1% yield), m.p. 226e228 ꢁC, 1H NMR
4.2.5. 3-({2-[(4-{Amino-[(E)-pentyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b5)
The title compound was prepared following the procedure
described for compound b1 from a5 to afford b5 as a white crystals
in 81.1% yield, m.p. 196e198 ꢁC, 1H NMR (500 MHz DMSO-d6)
(500 MHz DMSO-d6)
d (ppm): 1.43 (9H, s, eC(CH3)3), 2.61 (2H, t,

26
X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
d
(ppm): 0.88 (3H, t, J ¼ 7.0 Hz, eCH2CH2CH3), 1.29e1.32 (4H, m, e
>NCH2CH2e), 5.16 (2H, s, pyrazine-CH2Oe), 5.44 (1H, s, eCH2NHe),
6.57 (2H, d, J ¼ 8.8 Hz, ArH), 6.68 (1H, d, J ¼ 8.1 Hz, ArH), 6.95e7.00
(2H, m, ArH), 7.20 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.5 Hz, ArH), 7.28e7.31
(1H, m, ArH), 7.64e7.67 (3H, m, ArH), 8.39 (1H, dd, J1 ¼ 4.9 Hz,
J2 ¼ 1.2 Hz, ArH), 8.50e10.50 (2H, brs, eNH2); 13C NMR (75 MHz
CH2CH2CH2CH3), 1.59 (2H, qui, J ¼ 6.9 Hz, eCH2CH2CH2e), 2.58 (2H,
t, J ¼ 7.5 Hz, >NCH2CH2e), 3.76 (3H, s, >NCH3), 3.97 (2H, t,
J ¼ 6.8 Hz, eOCH2CH2CH2e), 4.17(2H, t, J ¼ 7.6 Hz, >NCH2CH2e),
4.59 (2H, d, J ¼ 5.5 Hz, eCH2NHe), 6.76 (2H, d, J ¼ 8.9 Hz, ArH),
6.94e6.96 (1H, m, ArH), 7.10e7.12 (1H, m, ArH), 7.16 (1H, dd,
J1 ¼8.4 Hz, J2 ¼ 1.5 Hz, ArH), 7.39 (1H, d, J ¼ 8.5 Hz, ArH), 7.47 (1H, d,
J ¼ 1.1 Hz, ArH), 7.55 (1H, td, J1 ¼ 7.8 Hz, J2 ¼ 2.0 Hz, ArH), 7.79
(2H, d, J ¼ 8.9 Hz, ArH), 8.37 (1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.2 Hz, ArH),
9.20e9.80 (2H, brs, eNH2), 11.30e12.50 (1H, brs, eCOOH);
ESI-MS(m/z): 586.1[M þ H]þ, 608.0[M þ Na]þ.
CDCl3)
d (ppm): 20.45, 21.32, 21.57, 28.21(3C), 29.76, 33.04, 40.69,
44.56, 65.17, 79.16, 108.71, 112.05(2C), 120.34, 120.92, 122.27, 123.27,
123.71, 128.99(2C), 129.87, 137.21, 137.23, 141.08, 144.54, 148.86(2C),
149.02, 150.39, 151.21, 152.34, 156.07, 164.11, 167.02, 170.97, 171.25;
IR(cmꢂ1): 3304.32, 2924.27, 2853.62, 1736.51, 1647.18, 1608.60,
1469.84, 1388.77, 1366.03,1321.33,1280.35, 1169.43,1142.62, 809.58,
744.75; HRMS(EIþ): m/z 706.3465 [M þ H]þ, [C38H44N9O5]þ calc. for
706.3465, found 706.3469.
4.2.6. 3-({2-[(4-{Amino-[(E)-isopropyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b6)
The title compound was prepared following the procedure
described for compound b1 from a6 to afford b6 as a white crystals
in 76.2% yield, m.p. 198e200 ꢁC, 1H NMR (500 MHz DMSO-d6)
4.3.2. 3-({2-[(4-{Amino-[(E)-hexyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid-(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-2)
d
(ppm): 1.20 (6H, d, J ¼ 6.3 Hz, eCH(CH3)2), 2.57 (2H, t, J ¼ 7.7 Hz,
The title compound was prepared following the procedure
described for compound Y-1 from b2 to afford Y-2 as a white
crystals in 66.8% yield, m.p. 140e142 ꢁC, 1H NMR (500 MHz CDCl3)
>NCH2CH2e), 3.76 (3H, s, >NCH3), 4.16 (2H, t, J ¼ 7.7 Hz,
>NCH2CH2e), 4.59 (2H, d, J ¼ 5.0 Hz, eCH2NHe), 4.80 (1H, sep,
J ¼ 6.3 Hz, eCH(CH3)2), 6.76 (2H, d, J ¼ 8.9 Hz, ArH), 6.94e6.97 (1H,
m, ArH), 7.10e7.12 (1H, m, ArH), 7.15 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.5 Hz,
ArH), 7.39 (1H, d, J ¼ 8.5 Hz, ArH), 7.48 (1H, d, J ¼ 1.0 Hz, ArH), 7.55
(1H, td, J1 ¼ 7.8 Hz, J2 ¼ 2.0 Hz, ArH), 7.79 (2H, d, J ¼ 8.9 Hz, ArH),
8.37 (1H, dq, J1 ¼ 4.9 Hz, J2 ¼ 1.3 Hz, ArH), 8.50e9.50 (2H, brs, e
NH2), 10.60e11.00 (1H, brs, eCOOH); ESI-MS(m/z): 558.0
[M þ H]þ, 580.0[M þ Na]þ.
d
(ppm): 0.89 (3H, t, J ¼ 6.17 Hz, eCH2CH3), 1.31e1.42 (6H, m, e
CH2CH2CH2CH2CH3), 1.72 (2H, qui, J ¼ 7.7 Hz, eCH2CH2CH2e),
2.48e2.51 (9H, m, pyrazine-CH3), 2.88 (2H, t, 7.3 Hz,
J
¼
>NCH2CH2e), 3.67 (3H, s, >NCH3), 4.13 (2H, t, J ¼ 6.9 Hz, e
OCH2CH2e), 4.42d.45 (4H, m, >NCH2CH2e, eCH2NHe), 5.17 (2H,
s, pyrazine-CH2Oe), 5.40 (1H, s, eCH2NHe), 6.62 (2H, d, J ¼ 8.5 Hz,
ArH), 6.69 (1H, d, J ¼ 8.0 Hz, ArH), 6.95e6.98 (1H, m, ArH), 7.03 (1H,
d, J ¼ 8.4 Hz, ArH), 7.23 (1H, d, J ¼ 7.4 Hz, ArH), 7.30 (1H, td,
J1 ¼ 7.8 Hz, J2 ¼ 1.8 Hz, ArH), 7.66e7.73 (3H, m, ArH), 8.39 (1H, d,
J ¼ 3.7 Hz, ArH), 9.00e9.90 (2H, brs, eNH2); 13C NMR (75 MHz
4.2.7. 3-({2-[(4-{Amino-[(E)-methyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid (b7)
CDCl3) d (ppm): 13.98, 20.47, 21.34, 21.59, 22.51, 25.60, 28.82, 29.81,
The title compound was prepared following the procedure
described for compound b1 from a7 to afford b7 as a white crystals
in 69.9% yield, m.p. 256e258 ꢁC, 1H NMR (500 MHz DMSO-d6)
31.50, 33.04, 40.65, 44.59, 65.19, 65.49, 108.75, 112.09(2C), 120.37,
120.96,122.30,122.61,123.75,129.12(2C),129.89,137.25(2C),141.07,
144.53, 148.88(2C), 149.05, 150.64, 151.25, 152.29, 156.06, 164.54,
167.30, 171.00, 171.27; IR(cmꢂ1): 3403.16, 3365.22, 1751.06, 1611.51,
1587.97, 1568.59, 1498.68, 1471.53, 1458.10, 1386.70, 1326.12,
1257.14, 1195.47, 1145.47, 1164.92, 1128.13, 1112.57; HRMS(EIþ): m/z
734.3788 [M þ H]þ, [C40H48N9O5]þ calc. for 734.3778, found
734.3788.
d
(ppm): 2.53 (2H, t, J ¼ 7.4 Hz, >NCH2CH2e), 3.58 (3H, s, eOCH3),
3.76 (3H, s, >NCH3), 4.15 (2H, t, J ¼ 7.8 Hz, >NCH2CH2e), 4.59 (2H,
d, J ¼ 5.4 Hz, eCH2NHe), 6.76 (2H, d, J ¼ 8.8 Hz, ArH), 6.96 (1H, d,
J ¼ 8.0 Hz, ArH), 7.10e7.12 (1H, m, ArH), 7.16 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.2 Hz, ArH), 7.39 (1H, d, J ¼ 8.5 Hz, ArH), 7.47 (1H, s, ArH), 7.55
(1H, td, J1 ¼ 7.8 Hz, J2 ¼ 1.8 Hz, ArH), 7.79 (2H, d, ¼ 8.8 Hz, ArH),
8.36 (1H, d, J ¼ 3.4 Hz, ArH), 8.50e9.70 (2H, brs, eNH2), 10.40e
10.80 (1H, brs, eCOOH); ESI-MS(m/z): 530.2[M þ H]þ, 552.2
[M þ Na]þ.
4.3.3. 3-({2-[(4-{Amino-[(E)-ethyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid-(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-3)
4.3. General procedure for synthesis of compounds Y-1dY-7
The title compound was prepared following the procedure
described for compound Y-1 from b3 to afford Y-3 as a white
crystals in 60.5% yield, m.p. 141e142 ꢁC, 1H NMR (500 MHz CDCl3)
4.3.1. 3-({2-[(4-{Amino-[(E)-tert-butyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid -(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-1)
d
(ppm): 1.34 (3H, t, J ¼ 7.1 Hz, eCH2CH3), 2.47e2.51 (9H, m, pyr-
azine-CH3), 2.88 (2H, t, J ¼ 7.2 Hz, >NCH2CH2e), 3.65 (3H, s,
>NCH3), 4.19 (2H, q, J ¼ 7.1 Hz, eCH2CH3), 4.42d.45 (4H, m,
>NCH2CH2e, eCH2NHe), 5.16 (2H, s, pyrazine-CH2Oe), 5.42 (1H, s,
eCH2NHe), 6.60 (2H, d, J ¼ 8.5 Hz, ArH), 6.69 (1H, d, J ¼ 8.0 Hz,
ArH), 6.95e6.98 (1H, m, ArH), 7.02 (1H, d, J ¼ 8.4 Hz, ArH), 7.22 (1H,
d, J ¼ 8.2 Hz, ArH), 7.29e7.32 (1H, m, ArH), 7.64e7.66 (1H, m, ArH),
7.71 (2H, d, J ¼ 8.4 Hz, ArH) 8.39 (1H, d, J ¼ 3.9 Hz, ArH), 9.00e9.90
To a solution of compound b1 (0.57 g, 1.0 mmol) in DMF(20 ml)
was added 2-hydroxymethyl-3,5,6-trimethylpyrazine (HTMP)
(0.23 g, 1.5 mmol), EDCI (0.25 g, 1.3 mmol) and DMAP (0.07 g,
0.6 mmol), and the resulting solution was stirred for 30 min at 0 ꢁC,
then slowly increasing the temperature of the reaction mixture to
room temperature. After stirring for 10 h, dichloromethane (40 mL)
was added to the reaction mixture, and the mixture was washed with
brine (20mLꢃ 5). The organiclayerwasdried overanhydrous sodium
sulfate, filtered, and concentrated. The residue was purified by
column chromatography (dichloromethane/methanol 30:1) to give
compoundY-1aswhitesolid(0.43g, 60.9%yield), m.p.126e128 ꢁC,1H
(2H, brs, eNH2); 13C NMR (75 MHz CDCl3)
d (ppm): 14.44, 20.44,
21.32, 21.57, 29.74, 33.01, 40.01, 44.56, 60.99, 65.17, 108.73,
112.02(2C), 120.28, 120.95, 122.28, 122.77, 123.69, 129.05(2C),
129.83,137.22(2C),141.03,144.50,148.86(2C),149.01,150.54,151.22,
152.28, 156.01, 164.67, 167.37, 170.98, 171.24; IR(cmꢂ1): 3411.47,
3293.48, 1737.55, 1608.79, 1468.74, 1391.08, 1364.90, 1327.43,
1264.32, 1171.98, 1141.93, 1126.00, 1099.02, 1075.87; HRMS(EIþ):
m/z 678.3151 [M þ H]þ, [C36H40N9O5]þ calc. for 678.3152, found
678.3151.
NMR (500 MHz CDCl3) d (ppm):1.53 (9H, s, eC(CH3)3), 2.47e2.50(9H,
m, pyrazine-CH3), 2.87 (2H, t, J ¼ 7.3 Hz, >NCH2CH2e), 3.61 (3H, s,
>NCH3), 4.38, (2H, d, J ¼ 4.6 Hz, eCH2NHe), 4.43 (2H, t, J ¼ 7.2 Hz,

X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
27
4.3.4. 3-({2-[(4-{Amino-[(E)-benzyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid-(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-4)
ArH), 7.23 (1H, d, J ¼ 8.5 Hz, ArH), 7.30 (1H, td, J1 ¼ 7.9 Hz,
J2 ¼ 1.9 Hz, ArH), 7.66 (1H, s, ArH), 7.72 (2H, d, J ¼ 8.6 Hz, ArH), 8.39
(1H, dd, J1 ¼ 4.8 Hz, J2 ¼ 1.2 Hz, ArH), 9.00e10.00 (2H, brs, eNH2);
13C NMR (75 MHz CDCl3)
d (ppm): 20.46, 21.34, 21.59, 22.02(2C),
The title compound was prepared following the procedure
described for compound Y-1 from b4 to afford Y-4 as a white
crystals in 67.6% yield, m.p. 149e150 ꢁC, 1H NMR (500 MHz CDCl3)
29.76, 33.03, 40.66, 44.58, 65.18, 68.11, 108.73, 112.06(2C), 120.35,
120.95, 122.30, 123.09, 123.75, 129.07, 129.88(2C), 137.23(2C),
141.05, 144.53, 148.89(3C), 150.47, 151.25, 152.28, 156.04, 164.37,
167.36, 171.00, 171.27; IR(cmꢂ1): 3395.45, 2978.24, 1735.90,
1608.90, 1470.41, 1371.88, 1330.71, 1266.07, 1168.57, 1146.73,
1109.21, 986.13, 837.88, 812.86, 749.61, 566.82; HRMS(EIþ): m/z
692.3314 [M þ H]þ, [C37H42N9O5]þ calc. for 692.3309, found
692.3314.
d
(ppm): 2.47e2.51 (9H, m, pyrazine-CH3), 2.87 (2H, t,
J ¼ 7.1 Hz, >NCH2CH2e), 3.62 (3H, s, >NCH3), 4.39e4.44 (4H, m,
>NCH2CH2e, eCH2NHe), 5.16e5.18 (4H, m, pyrazine-CH2Oe, e
OCH2Ph), 5.40 (1H, s, eCH2NHe), 6.58 (2H, d, J ¼ 8.3 Hz, ArH),
6.68 (1H, d, J ¼ 7.9 Hz, ArH), 6.94e6.96 (1H, m, ArH), 7.00 (1H, d,
J ¼ 8.4 Hz), 7.21e7.33 (5H, m, ArH), 7.42 (2H, d, J ¼ 7.3 Hz, ArH), 7.64
(1H, s, ArH), 7.69 (2H, d, J ¼ 8.3 Hz, ArH), 8.38 (1H, d, J ¼ 3.9 Hz,
ArH), 9.00e10.00 (2H, brs, eNH2); 13C NMR (75 MHz CDCl3)
4.3.7. 3-({2-[(4-{Amino-[(E)-methyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid-(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-7)
The title compound was prepared following the procedure
described for compound Y-1 from b7 to afford Y-7 as a white
crystals in 58.8% yield, m.p. 170e172 ꢁC, 1H NMR (500 MHz CDCl3)
d
(ppm): 20.45, 21.32, 21.58, 29.72, 33.02, 40.58, 44.57, 65.18, 66.88,
108.75, 112.02(2C), 120.28, 120.96, 122.29, 122.68, 123.69, 127.75,
128.11(2C), 128.27(2C), 129.10(2C), 129.83, 136.87, 137.24(2C),
141.02, 144.51, 148.87(2C), 149.02, 150.59, 151.24, 152.29, 156.02,
164.46, 167.65, 171.00, 171.26; IR(cmꢂ1): 3377.43, 2360.02, 2341.60,
1739.01, 1588.78, 1570.78, 1488.88, 1471.08, 1317.10, 1128.18,
1169.13, 1142.59, 1099.67, 1013.92, 808.79, 742.65, 698.12;
HRMS(EIþ): m/z 740.3315 [M þ H]þ, [C41H42N9O5]þ calc. for
740.3309, found 678.3315.
d
(ppm): 2.47e2.50 (9H, m, pyrazine-CH3), 2.87 (2H, t, J ¼ 7.2 Hz,
>NCH2CH2e), 3.64 (3H, s, >NCH3), 3.75 (3H, s, eOCH3), 4.39e4.44
(4H, m, >NCH2CH2e, eCH2NHe), 5.30 (2H, s, pyrazine-CH2Oe),
5.41 (1H, s, eCH2NHe), 6.59 (2H, d, J ¼ 8.6 Hz, ArH), 6.69 (1H, d,
J ¼ 8.0 Hz, ArH), 6.95e7.02 (2H, m, ArH), 7.21 (1H, d, J ¼ 8.4 Hz, ArH),
7.28e7.32 (1H, m, ArH), 7.64e7.72 (3H, m, ArH), 8.38 (1H, d,
J ¼ 4.3 Hz, ArH), 9.00e10.00 (2H, brs, eNH2); 13C NMR (75 MHz
4.3.5. 3-({2-[(4-{Amino-[(E)-pentyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid-(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-5)
The title compound was prepared following the procedure
described for compound Y-1 from b5 to afford Y-5 as a white
crystals in 63.9% yield, m.p. 134e136 ꢁC, 1H NMR (500 MHz CDCl3)
CDCl3)
d (ppm): 20.45, 21.33, 21.58, 29.75, 33.03, 40.62, 44.58,
52.30, 65.18, 108.75, 112.07(2C), 120.32, 120.37, 122.30, 122.71,
123.73, 129.04(2C), 129.88, 137.22, 137.25, 141.03, 144.52,
148.88(2C), 149.03, 150.59, 151.25, 152.26, 156.03, 165.13, 167.38,
171.00, 171.27; IR(cmꢂ1): 3415.93, 2938.05, 1736.95, 1609.03,
1587.52, 1484.35, 1470.31, 1437.88, 1393.92, 1327.32, 1268.29,
d
(ppm): 0.90 (3H, t, J ¼ 6.9 Hz, eCH2CH3), 1.34e1.37 (4H, m, e
CH2CH2CH2CH3), 1.71 (2H, qui, J ¼ 7.2 Hz, eCH2CH2CH2e), 2.47e
2.51 (9H, m, pyrazine-CH3), 2.87 (2H, t, J ¼ 7.2 Hz, >NCH2CH2e),
3.67(3H, s, >NCH3), 4.13 (2H, t, J ¼ 6.9 Hz, eOCH2CH2e), 4.42d.44
(4H, m, >NCH2CH2e, eCH2NHe), 5.16 (2H, s, pyrazine-CH2Oe),
5.52 (1H, s, eCH2NHe), 6.61 (2H, d, J ¼ 8.5 Hz, ArH), 6.69 (1H, d,
J ¼ 8.0 Hz, ArH), 6.95e6.97 (1H, m, ArH), 7.03 (1H, d, J ¼ 8.4 Hz,
ArH), 7.22 (1H, d, J ¼ 8.3 Hz, ArH), 7.31 (1H, t, J ¼ 8.2 Hz, ArH), 7.65
(1H, s, ArH), 7.69 (2H, d, J ¼ 8.4 Hz, ArH), 8.38 (1H, d, J ¼ 3.7 Hz,
ArH), 9.00e10.50 (2H, brs, eNH2); 13C NMR (75 MHz CDCl3)
1143.85, 1127.21; HRMS(EIþ): m/z 664.2993 [M
þ
H]þ,
[C35H38N9O5]þ calc. for 664.2996, found 664.2993.
4.4. Rabbit platelet aggregation induced by thrombin or ADP
4.4.1. Pretreatment of target compounds
Fresh prepared rabbit microsomal suspensions were added to
a solution of test compound (2 mg) in 40 ul DMSO [7]. The resulting
mixture was incubated for 4 h at 37 ꢁC, then diluted with saline into
four concentrations.
d
(ppm): 13.94, 20.48, 21.36, 21.61, 22.37, 28.06, 28.56, 29.81, 33.05,
40.68, 44.60, 65.21, 65.44,108.75, 112.12(2C),120.40, 120.95, 122.30,
122.84, 123.78, 129.10(2C), 129.94, 137.24(2C), 141.10, 144.55,
148.89(2C), 149.06, 150.59, 151.25, 152.26, 156.08, 164.74, 167.34,
171.00, 171.29; IR(cmꢂ1): 3393.89, 3362.83, 2954.04, 2359.88,
1737.70, 1610.81, 1588.42, 1569.76, 1470.34, 1387.38, 1326.74,
1260.33, 1195.09, 1165.52, 1145.16, 1128.23, 1112.11, 814.06, 570.90;
HRMS(EIþ): m/z 742.3446 [M þ Na]þ, [C39H45N9O5Na]þ calc. for
742.3441, found 742.3446.
4.4.2. Plasma collection and preparation
Fresh rabbit blood (25 ml) was withdrawn using 3.8% (w/v)
trisodium citrate (1 volume of citrate for 9 volumes of blood) as
anticoagulant. Samples were centrifuged at 1000 rpm for 10 min at
room temperature. The resulting platelet-rich plasma (PRP)
supernatant was removed. The residue was centrifuged at
3000 rpm for 10 min at room temperature to obtain platelet-poor
plasma (PPP). The PRP was adjusted with PPP so as to obtain
platelet counts of 350 ꢃ 109 Pl/L.
4.3.6. 3-({2-[(4-{Amino-[(E)-isopropyloxycarbonylimino]methyl}-
phenylamino)methyl]-1-methyl-1H-benzoimidazole-5-carbonyl}
pyridin-2-yl-amino)propionic acid-(3,5,6-trimethylpyrazin-2-
methylene) ester (Y-6)
The title compound was prepared following the procedure
described for compound Y-1 from b6 to afford Y-6 as a white
crystals in 62.2% yield, m.p. 160e162 ꢁC, 1H NMR (500 MHz CDCl3)
4.4.3. Platelet aggregation [8]
Platelet aggregation was measured by Success SC-2000 platelet
aggregation tester. ADP or thrombin was used to induce platelet
aggregation. Platelet aggregation was determined by Born’s
method with modifications using a four-channel SC-2000 platelet
d
(ppm): 1.32 (6H, d, J ¼ 6.3 Hz, eCH(CH3)2), 2.48e2.51 (9H, m,
aggregation tester. Drugs 10
microcuvette and incubated at 37 ꢁC. The final concentration was
10 M of ADP or 1.5 Units of thrombin. Maximum platelet aggre-
gation rates were assessed. Control studies were performed with
the drug solvent (DMSO) added at an equal volume in place of the
tested compound.
mL and PRP 200 mL were added into the
pyrazine-CH3), 2.88 (2H, t, J ¼ 7.3 Hz, >NCH2CH2e), 3.66 (3H, s,
>NCH3), 4.42d.44 (4H, m, >NCH2CH2e, eCH2NHe), 4.98 (1H, sep,
J ¼ 6.3 Hz, eCH(CH3)2), 5.17 (2H, s, pyrazine-CH2Oe), 5.36 (1H, t,
J ¼ 4.5 Hz, eCH2NHe), 6.61 (2H, d, J ¼ 8.6 Hz, ArH), 6.69 (1H, d,
J ¼ 8.1 Hz, ArH), 6.95e6.98 (1H, m, ArH), 7.03 (1H, d, J ¼ 8.5 Hz,
m

28
X.-Z. Yang et al. / European Journal of Medicinal Chemistry 57 (2012) 21e28
4.5. Venous thrombosis in rats
different time points, and mixed with 4% formic acid. After
precipitated by adding acetonitrile, blood samples were preserved
4.5.1. Preparation of test solutions and pretreatment of animals
SpragueeDawley male rats (240e260 g) received a single dose of Y-
2, dabigatran etexilate, HTMP or treatment vehicle via a gastric tube
once daily for three days. Compound Y-2 and dabigatran etexilate were
administered at doses of 1, 5,10, or 20 mg/kg body weight, HTMP was
administered at doses of 20, 40, or 60 mg/kg body weight, and control
animals received the treatment vehicle without the active agent.
at ꢂ70 ꢁC.
Acknowledgments
Financial support from the enterprise innovation drug incuba-
tion base of the Ministry of Science and Technology, China (Grant
No.2012ZX09401-006) is gratefully acknowledged. Mr. Robin Xu
(Rutgers University) is greatly acknowledged for proofreading our
manuscripts.
4.5.2. Experimentally induced venous thrombosis
An experimental venous thrombosis was described by Reyers
et al. [9], with a few changes according to the fact. Two hours after
the last administration, rats were anesthetized by intraperitoneal
injection of 20% (w/v) urethane. After the abdomen was surgically
opened, the inferior vena cava was isolated and a ligature was
applied below the left renal vein branch. Then, the abdomen was
closed. After 4 h, the abdomen was reopened under urethane
anesthesia. The inferior vena cava was clamped 2 cm below the first
ligature. This segment was emptied of blood and cut. The thrombus
was removed and placed for 20 h at 60 ꢁC for measuring dry weight.
References
[1] S. Coughlin, J. Thromb. Haemost. 3 (2005) 1800e1814.
[2] E.M. Davis, K.A. Packard, J.T. Knezevich, J.A. Campbell, Pharmacotherapy 31
(2011) 975e1016.
[3] A.R. Patel, M.K. Crist, J. Nemitz, J.L. Mayerson, J. Surg. Oncol. 102 (2010)
276e281.
[4] N.H. Hauel, H. Nar, H. Priepke, U. Ries, J.M. Stassen, W. Wienen, J. Med. Chem. 45
(2002) 1757e1766.
[5] B.I. Eriksson, H. Smith, U. Yasothan, P. Kirkpatrick, Dabigatran etexilate, Nat. Rev.
Drug Discov. 7 (2008) 557e558.
[6] S.Q. Nie, Z.C. Xie, K.C. Lin, Acta Pharmacol. Sin. 20 (1985) 689e692.
[7] C. von Bahr, C.G. Groth, H. Jansson, G. Lundgren, M. Lind, H. Glaumann, Clin.
Pharmacol. Ther. 27 (1980) 711e725.
4.6. Pharmacokinetics
[8] Wienen, J.M. Stassen, H. Priepke, J.R. Uwe, N. Hauel, Thromb. Haemost. 98
(2007) 155e162.
[9] I. Reyers, L. Mussoni, M.B. Donati, G. de Gaetano, Thromb. Res.18 (1980) 669e674.
SpragueeDawley female rats (240e260 g) were fed with 20 mg/
kg Y-2 via a gastric tube. Whole blood samples were collected at