Synthesis of a series of benzothiazole amide derivatives and their biological evaluation as potent hemostatic agents

Article abstract of DOI:10.1039/c7ra13397a

A series of benzothiazole amide derivatives were synthesized through a facile and efficient method via a nucleophilic acyl substitution reaction between 2-aminobenzothiazole and various cinnamic acid compounds. The obtained products exhibited good thermal stabilities. All compounds were evaluated for their in vitro hemostatic activities using the commercially available standard drug etamsylate as a positive control. The results showed that compound Q2 had a significant partial coagulation activity, reduced capillary permeability at 5, 10 and 50 μmol L^-1, activated thrombin activity, and a more potent platelet aggregation activity than the positive control group (etamsylate, up to 1283.9 times in the nanomole range). A molecular modeling study revealed that compound Q2 was a competitive thrombin activator. Therefore, Q2 may be a potential lead for further biological screening and for the generation of drug molecules. Moreover, the structure-activity relationship of the prepared compounds is also discussed herein.

Full text of DOI:10.1039/c7ra13397a

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Synthesis of a series of benzothiazole amide
derivatives and their biological evaluation as potent
Cite this: RSC Adv., 2018, 8, 6231
hemostatic agents†
a
b
c
a
a
a
*
Anran Zhao, Jinrui Wei, Hui Cheng, Xuan Luo and Cuiwu Lin
Wenqian Nong,
A series of benzothiazole amide derivatives were synthesized through a facile and efficient method via
a nucleophilic acyl substitution reaction between 2-aminobenzothiazole and various cinnamic acid
compounds. The obtained products exhibited good thermal stabilities. All compounds were evaluated
for their in vitro hemostatic activities using the commercially available standard drug etamsylate as
a positive control. The results showed that compound Q2 had a significant partial coagulation activity,
reduced capillary permeability at 5, 10 and 50 mmol Lꢀ1, activated thrombin activity, and a more potent
platelet aggregation activity than the positive control group (etamsylate, up to 1283.9 times in the
nanomole range). A molecular modeling study revealed that compound Q2 was a competitive thrombin
activator. Therefore, Q2 may be a potential lead for further biological screening and for the generation
of drug molecules. Moreover, the structure–activity relationship of the prepared compounds is also
discussed herein.
Received 17th December 2017
Accepted 29th January 2018
DOI: 10.1039/c7ra13397a
rsc.li/rsc-advances
a key template for the development of various therapeutic
agents.8 Due to the presence of sulfur atoms in their structure,
Introduction
benzothiazoles exhibit unique properties not possessed by
other heterocyclic compounds.9 Benzothiazole amide systems
have interesting structural characteristics and strong bioactivity
proles, as well as anticancer,10 anti-inammatory,11 anti-
fungal,12 and chronic pain suppression properties.13 Further,
given their useful molecular scaffolds for the exploration and
exploitation of pharmacophore space, they have emerged as
particularly promising synthetic targets.
Chemical compounds with a low biological toxicity stem-
ming from natural products can be obtained from a wide range
of sources,14,15 and are being used as lead compounds in the
preparation of safe and low toxicity hemostatic drugs. In our
previous studies,16,17 we evaluated the activity of cinnamic acid
amide derivatives synthesized by sulfa drug 2-aminothiazole
with cinnamic acid compounds. The results showed that cin-
namic acid amide derivatives may function as hemostatic
agents. Therefore, we chose to continue our investigation on the
synthesis and properties of cinnamic acid compounds.
Excessive bleeding during surgery remains a main cause of
failure of surgical procedures.1 Homologous blood transfusion
and hemostatic drugs are commonly employed during surgery
to avoid excessive bleeding.2,3 However, homologous blood
transfusions can increase the risk of infection, leading to
increased morbidity and mortality.4 Thus, the use of hemostatic
agents is preferable. To date, commonly used hemostatic drugs,
such as p-aminomethylbenzoic acid, tranexamic acid, amino-
caproic acid, aprotinin, and etamsylate, confer adverse side
effects and safety risks despite their desirable hemostatic
effects, and are therefore being re-evaluated for clinical use.4–6
Thus, the search for safer and more effective hemostatic agents
remains crucial.
A heterocyclic ring containing nitrogen and sulfur atoms
represents a unique and universal scaffold for the design of
experimental drugs.7 Benzothiazole is an important derivative
unit of the thiazole ring, a rare heterocyclic compound found in
alkaloids, and plays an important role in drug chemistry as
According to the drug design principles of combination, the
covalent bonding of two different compounds would lead to the
synergistic effect of their properties. Thus, we designed nine
benzothiazole amide derivatives with cinnamic acid structures
and assessed their hemostatic activity. Although the synthetic
methods of compounds Q5, Q6, Q7, and Q9 have been previ-
ously reported,18–20 the synthesis of the other compounds Q1–
Q4, Q8 and the evaluation of their related biological activities
remain unreported. Thermogravimetric analysis was used to
determine the thermodynamic stability of the compounds as
aGuangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology
and Resource Development, School of Chemistry & Chemical Engineer, Guangxi
University, Nanning, 530004, China
bDepartment of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland,
OH 44115, USA
cGuangxi Scientic Research Center of Traditional Chinese Medicine, Guangxi
University of Chinese Medicine, Nanning, Guangxi 530200, PR China
† Electronic supplementary information (ESI) available. CCDC 1582748 and
1582749. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c7ra13397a
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Scheme 1 Synthesis of compounds Q1–Q4. Reagents and conditions: (a) acetic anhydride/pyridine, 120 ꢁC, 5 h, 98–100%. (b) SOCl2, DMF,
70 ꢁC, 5 min, 100%. (c) NaHCO3, pH ¼ 6–7, 0–5 ꢁC to room temperature, 3 h, 70–82%.
a proxy of drug stability. The hemostatic activity of the prepared Reaction progress was monitored by thin layer chromatography
compounds was evaluated by in vitro experiments based on the (TLC). Once the reaction was complete, the upper layer solid
hemostatic mechanism (involving normal operation of blood was collected by ltration of the mixture. The solid product was
vessels, platelets, and blood coagulation) through the platelet washed with excess distilled ethanol and water to obtain the
aggregation assay, activation of partial thromboplastin time pure products. All tested reactions proceeded smoothly under
(APTT), prothrombin time (PT), thrombin time assay (TT), optimal conditions, producing the desired products in good
human C-type natriuretic peptide determination, and thrombin yields (Table 1). However, a trend was observed wherein using
activity assay (including molecular docking of compound Q2 cinnamic acid derivatives with substituents of increasing
with thrombin). Based on the obtained results, the relationship electron-donating strength on the aromatic ring led to products
between the given structure and the corresponding hemostatic with higher yields, likely due to a decrease in electron density in
activity was discussed, providing a theoretical basis for the the aromatic ring causing an acceleration of the reaction.
screening of hemostatic drugs.
Compounds Q1–Q9 were structurally characterized by various
spectroscopic techniques, including 1H and 13C nuclear
magnetic resonance (NMR), electron ionization mass spectros-
copy, and Fourier-transform infrared spectroscopy.
Results and discussion
Chemistry
Crystal structure description
The amide bond formation reaction is an important reaction in
organic synthesis, with the amide bond being widely present in Single crystals of compounds Q7 (CCDC: 1582748) and Q9
pharmaceutical intermediates and biochemical substances.21,22 (CCDC: 1582749) were grown from dimethyl sulfoxide and the
Benzothiazole amide derivatives (Q1–Q9) were synthesized by corresponding structures were conrmed by single crystal X-ray
the reaction of cinnamic acid and its derivatives (q1–q9) with 2- diffraction analysis (Fig. 1). Furthermore, integrals in the NMR
aminobenzothiazole through the nucleophilic acyl substitution spectra conrmed the crystal structures for the two compounds
of acyl chloride (Schemes 1 and 2). NaHCO3 was used as an acid Q7 and Q9. The crystal lattice of compound Q7 belongs to the
binding agent and the reaction was performed in CH2Cl2 triclinic system. The crystal lattice of compound Q9 belongs to
ꢁ
solvent at 0–5 C. The pH value was kept between 6 and 7 by the monoclinic system, containing two N-(benzo[d]thiazol-2-yl)-
ꢀ
addition of NaHCO3 to induce precipitation. The reactants were 3-phenylpropanamide molecules. Selected bond lengths (A) and
vigorously mixed until solidication of the reaction mixture. angles (ꢁ) are provided in the ESI† section.
Scheme 2 Synthesis of compounds Q5–Q9. Reagents and conditions: (b) SOCl2, DMF, 70 ꢁC, 5 min, 100%. (c) NaHCO3, pH ¼ 6–7, 0–5 ꢁC to
room temperature, 3 h, 88–93%.
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Table 1 Synthesis of benzothiazole amide derivativesa and their biological evaluation on platelet aggregation
R1
R2
R3
R4
Yieldb (%)
EC50 (mmol Lꢀ1
)
c
Compound
2-Aminobenzothiazole
513.7
476.3
98.76
129.4
108.7
322.3
280.5
189.4
120.9
227.8
93.87
0.036
1.246
0.119
73.71
54.58
18.09
1.867
35.47
46.22
q1d
q2d
q3d
q4d
q5e
q6e
q7e
q8e
q9e
Q1
Q2
Q3
Q4
Q5
Q6
Q7
Q8
Q9
H
H
H
H
H
H
H
OCH3
—
H
H
H
H
H
H
H
OCOCH3
OCOCH3
H
H
H
OCH3
OCH3
OCH3
—
OCOCH3
OCOCH3
H
H
H
OCH3
OCH3
OCH3
—
OCH3
H
OCOCH3
H
H
H
OCH3
OCH3
—
OCH3
H
OCOCH3
H
H
H
H
H
100
99
98
OCOCH3
H
H
H
H
—
H
H
98
82
73
77
70
88
90
92
93
90
H
OCOCH3
H
H
H
H
—
H
OCH3
OCH3
—
OCH3
—
Etamsylate
a
Reaction conditions: the acid chloride of cinnamic acid compounds (1.5 mmol), 2-aminobenzothiazole (1.0 mmol) in 10.0 mL of CH2Cl2, 0–5 ꢁC to
room temperature, 3 h. b Isolated yield aer ash chromatography. c The EC50 values are expressed as the concentration required to promote ADP-
induced human platelet aggregation response by 50%; n ¼ 3. d Reaction conditions: cinnamic acid compounds (ferulic acid, p-hydroxy-cinnamic
acid, m-hydroxycinnamic acid, and o-hydroxy-cinnamic acid, 25.0 g), pyridine (5 mL) in 100 mL of acetic anhydride at 120 ꢁC for 5 h. e q5: cinnamic
acid; q6: 4-methoxycinnamic acid; q7: 3,4-dimethoxycinnamic acid; q8: 3,4,5-trimethoxycinnamic acid; q9: 3-phenylpropionic acid.
mass loss gradually increased, with the heat ow curve showing
obvious uctuations at approximately 150 ꢁC, indicating the
initiation of oxidation in air. The mass of Q4 declined consid-
erably at 180 C, with the heat ow curve showing a large uc-
tuation due to sample decomposition.
Thermogravimetric analysis
The mass loss and heat ow of the benzothiazole amide deriva-
tives upon increasing temperature are shown in Fig. 2. When the
compounds were heated to below 100 ꢁC, no signicant changes
in mass were observed. With the increase in temperature, the
ꢁ
Fig. 1 Ball-and-stick structures of compounds Q7 and Q9.
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injury as well as the action of endogenous agonists such as
adenosine diphosphate (ADP), collagen, and thrombin, fol-
lowed by the release of TXA2, forming a hemostatic plug to
obstruct the severed area and prevent bleeding.24,25 Compound
Q2 showed the highest efficacy as a platelet aggregation
promoter, with the lowest EC50 value (0.036 mmol Lꢀ1) in the
nanomole range (10 nmol Lꢀ1), demonstrating a better platelet
aggregation activity than etamsylate (positive control, EC50
¼
46.22 mmol Lꢀ1; Table 1). Compounds Q4, Q3, Q8, Q7, and Q9
(EC50 ¼ 0.119, 1.246, 1.867, 18.09, and 35.47 mmol Lꢀ1) all
showed a better platelet aggregation-promoting activity than
etamsylate. Thus, adding methoxy groups to N-(benzo[d]
thiazol-2-yl)-cinnamamide leads to a decrease in platelet
aggregation activity. However, introducing an acetoxyl group
to N-(benzo[d]thiazol-2-yl) cinnamamide would lead to an
increase in platelet aggregation activity. Thus, the substituent
type crucially affects platelet aggregation promotion.
Fig. 2 Thermogravimetric and heat flow curves of compounds Q1–
Q9 recorded at a constant heating rate. The mass loss is expressed as
the fraction M/M0 of the mass at temperature T to the initial mass of
the samples equilibrated at laboratory atmosphere.
Blood coagulation activities
Thus, the stable nature of the thermogravimetric analysis
results indicated that the prepared compounds had a suitable
thermal stability at 100 C. Therefore, these compounds were
All synthetic benzothiazole amide derivatives Q1–Q9 were
screened for in vitro blood coagulant activity, including APTT, PT,
and TT assays. APTT tests the intrinsic coagulation activity,
providing an approximate indication of coagulation factor II, V,
VII, and X activity.26 PT is an ideal and commonly used screening
assay for the detection of extrinsic coagulation pathway.27 Finally,
TT is a screening test to determine thrombin activity and the
ꢁ
further screened as hemostatic drugs.
Platelet aggregation activity
When the vascular wall is broken, platelets undergo a series of ability to convert plasma brin into brin.28 The coagulation rate
functional responses, including adhesion, diffusion, release, (Rxx) was used to evaluate the corresponding coagulation activity
and accumulation, thus promoting coagulant surface expo- of a given compound; negative values may represent partial
sure, particle formation, and clot retraction.23 The platelet coagulation activity and smaller values may indicate an
accumulation response leads to platelet adhesion at the site of improvement in the blood coagulant effects. The corresponding
Table 2
R
(%) values of the synthesized benzothiazole amide derivativesa
APTT
Concentration (mmol Lꢀ1
)
Compound
1
5
10
50
100
Etamsylate
2-Aminobenzothiazole
ꢀ7.12 ꢂ 0.23
ꢀ6.10 ꢂ 0.22
ꢀ4.52 ꢂ 0.46
ꢀ1.01 ꢂ 0.25
4.07 ꢂ 0.12
ꢀ4.87 ꢂ 0.13***
ꢀ1.72 ꢂ 0.25***
ꢀ3.97 ꢂ 0.47***
ꢀ2.19 ꢂ 0.36***
ꢀ3.47 ꢂ 0.12***
ꢀ2.18 ꢂ 0.39***
ꢀ0.96 ꢂ 0.32***
ꢀ0.73 ꢂ 0.15***
ꢀ0.48 ꢂ 0.24***
1.03 ꢂ 0.12***
0.14 ꢂ 0.37***
ꢀ5.27 ꢂ 0.35
ꢀ6.19 ꢂ 0.13***
0.86 ꢂ 0.25***
ꢀ1.92 ꢂ 0***
ꢀ4.74 ꢂ 0.23***
1.58 ꢂ 0.14***
0.01 ꢂ 0.27
ꢀ4.48 ꢂ 0.35***
1.87 ꢂ 0.38***
0.82 ꢂ 0.14***
3.15 ꢂ 0.14
q1
q2
q3
q4
q5
q6
q7
q8
q9
Q1
Q2
Q3
Q4
Q5
Q6
Q7
Q8
Q9
ꢀ0.54 ꢂ 0.16***
ꢀ3.56 ꢂ 0.24***
ꢀ1.23 ꢂ 0.27***
ꢀ1.20 ꢂ 0.24***
ꢀ0.26 ꢂ 0.38***
0.12 ꢂ 0.11***
ꢀ0.44 ꢂ 0.44***
ꢀ0.84 ꢂ 0.60***
ꢀ0.77 ꢂ 0.26***
ꢀ8.58 ꢂ 0.37***
0.55 ꢂ 0.24***
ꢀ0.96 ꢂ 0.32***
2.05 ꢂ 0.44***
0.36 ꢂ 0.35***
1.91 ꢂ 0.14***
ꢀ1.20 ꢂ 0.43***
ꢀ1.67 ꢂ 0.13***
ꢀ10.10 ꢂ 0.14***
ꢀ36.12 ꢂ 0.22***
ꢀ1.98 ꢂ 0.21***
ꢀ34.22 ꢂ 0.22***
ꢀ9.58 ꢂ 0.11***
ꢀ8.46 ꢂ 0.41***
ꢀ8.83 ꢂ 0.14***
ꢀ4.69 ꢂ 0.18
0.96 ꢂ 0.24***
0.12 ꢂ 0.24*
0.24 ꢂ 0.43***
7.57 ꢂ 0.34***
0.72 ꢂ 0.21***
2.79 ꢂ 0.15*
2.56 ꢂ 0.26***
0.60 ꢂ 0.32**
2.20 ꢂ 0.25***
ꢀ1.67 ꢂ 0.12
ꢀ2.75 ꢂ 0.24***
ꢀ2.57 ꢂ 0.22***
ꢀ5.26 ꢂ 0.37***
ꢀ20.25 ꢂ 0.30***
ꢀ0.52 ꢂ 0.10***
ꢀ4.80 ꢂ 0.14***
ꢀ7.89 ꢂ 0.11***
ꢀ7.33 ꢂ 0.34***
ꢀ4.41 ꢂ 0.24***
0.85 ꢂ 0.11***
4.51 ꢂ 0.20
ꢀ2.31 ꢂ 0.33*
ꢀ6.50 ꢂ 0.14***
ꢀ36.03 ꢂ 0.43***
ꢀ0.73 ꢂ 0.21
ꢀ38.84 ꢂ 0.08***
0.42 ꢂ 0.38***
ꢀ38.26 ꢂ 0***
0.21 ꢂ 0.21***
ꢀ34.96 ꢂ 0.30***
ꢀ10.03 ꢂ 0.34***
ꢀ8.90 ꢂ 0.11***
ꢀ6.21 ꢂ 0.14
ꢀ35.46 ꢂ 0.17***
ꢀ12.06 ꢂ 0.20***
ꢀ9.47 ꢂ 0.11***
ꢀ4.55 ꢂ 0.28***
2.24 ꢂ 0.11***
ꢀ31.90 ꢂ 0.41***
ꢀ9.47 ꢂ 0.29***
ꢀ7.90 ꢂ 0.11***
ꢀ7.31 ꢂ 0.24***
ꢀ3.52 ꢂ 0.11***
ꢀ1.01 ꢂ 0.30
ꢀ0.11 ꢂ 0.11***
ꢀ4.06 ꢂ 0.46***
ꢀ5.75 ꢂ 0.30***
ꢀ3.95 ꢂ 0.20
a
Data represent mean ꢂ SEM; n ¼ 8. *P < 0.05 vs. etamsylate; **P < 0.01 vs. etamsylate; ***P < 0.001 vs. etamsylate.
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Table 3
R
(%) values of the synthesized benzothiazole amide derivativesa
Concentration (mmol Lꢀ1
PT
)
Compound
1
5
10
50
100
Etamsylate
2-Aminobenzothiazole
ꢀ13.70 ꢂ 0.37
ꢀ10.73 ꢂ 0.11
ꢀ8.03 ꢂ 0.21
2.84 ꢂ 0.13
4.03 ꢂ 0.46
ꢀ1.05 ꢂ 0.46***
7.60 ꢂ 0.17***
ꢀ1.22 ꢂ 0.30***
4.73 ꢂ 0.45***
ꢀ3.85 ꢂ 0.27***
ꢀ1.92 ꢂ 0.26***
ꢀ2.56 ꢂ 0.30***
ꢀ2.53 ꢂ 0.39***
ꢀ3.23 ꢂ 0.56***
0.77 ꢂ 0.27***
3.21 ꢂ 0.34****
ꢀ1.51 ꢂ 0.26***
ꢀ5.22 ꢂ 0.33***
ꢀ5.91 ꢂ 0.15***
ꢀ5.36 ꢂ 0.13***
ꢀ7.24 ꢂ 0.15***
ꢀ10.22 ꢂ 0.13
ꢀ1.57 ꢂ 0.17***
2.87 ꢂ 0.59**
ꢀ4.36 ꢂ 0***
ꢀ4.48 ꢂ 0.35***
ꢀ2.20 ꢂ 0.29***
ꢀ2.93 ꢂ 0.27***
0.45 ꢂ 0.14***
0.90 ꢂ 0.40***
2.23 ꢂ 0.54**
q1
q2
q3
q4
q5
q6
q7
q8
q9
Q1
Q2
Q3
Q4
Q5
Q6
Q7
Q8
Q9
ꢀ1.35 ꢂ 0.45***
ꢀ3.39 ꢂ 0.27***
ꢀ0.30 ꢂ 0.15***
0.30 ꢂ 0.52***
ꢀ0.45 ꢂ 0.39***
ꢀ1.23 ꢂ 0.15***
1.69 ꢂ 0.46
ꢀ4.78 ꢂ 0.27***
ꢀ2.23 ꢂ 0.15***
ꢀ2.71 ꢂ 0.15***
ꢀ5.94 ꢂ 0.45***
ꢀ4.16 ꢂ 0.15***
ꢀ0.77 ꢂ 0.41***
1.52 ꢂ 0.45***
ꢀ3.70 ꢂ 0.15***
ꢀ0.89 ꢂ 0.39*
0.00 ꢂ 0.15***
ꢀ1.78 ꢂ 0.15***
ꢀ2.31 ꢂ 0.41***
1.23 ꢂ 0.27***
4.39 ꢂ 0.29***
ꢀ1.05 ꢂ 0.45***
ꢀ4.73 ꢂ 0.16***
ꢀ4.43 ꢂ 0.15***
ꢀ4.29 ꢂ 0.23***
ꢀ5.47 ꢂ 0.30***
ꢀ9.67 ꢂ 0***
ꢀ0.15 ꢂ 0.27***
1.85 ꢂ 0.31**
5.57 ꢂ 0.45***
ꢀ0.45 ꢂ 0.40***
ꢀ3.59 ꢂ 0.57***
ꢀ1.62 ꢂ 0.26***
ꢀ1.21 ꢂ 0.13***
ꢀ4.73 ꢂ 0***
6.76 ꢂ 0.34***
1.51 ꢂ 0.15***
ꢀ2.12 ꢂ 0.28***
ꢀ0.59 ꢂ 0.15***
ꢀ0.13 ꢂ 0.13***
ꢀ2.07 ꢂ 0.44***
ꢀ2.28 ꢂ 0.13***
ꢀ14.79 ꢂ 0.13***
ꢀ7.83 ꢂ 0.33***
ꢀ1.21 ꢂ 0.13***
ꢀ3.90 ꢂ 0.27***
ꢀ5.42 ꢂ 0.54***
ꢀ2.12 ꢂ 0.28***
ꢀ6.79 ꢂ 0.15***
ꢀ6.57 ꢂ 0.13***
ꢀ9.90 ꢂ 0.15***
ꢀ11.43 ꢂ 0.13***
ꢀ4.57 ꢂ 0.48***
ꢀ3.57 ꢂ 0.28***
ꢀ1.61 ꢂ 0.13***
ꢀ9.82 ꢂ 0.27***
ꢀ4.03 ꢂ 0.23***
ꢀ9.54 ꢂ 0.13***
ꢀ6.85 ꢂ 0.16***
ꢀ4.83 ꢂ 0.27***
ꢀ4.03 ꢂ 0.23***
ꢀ4.70 ꢂ 0.13***
ꢀ4.40 ꢂ 0.16***
ꢀ4.29 ꢂ 0***
ꢀ6.99 ꢂ 0.13***
ꢀ5.38 ꢂ 0.33***
ꢀ3.89 ꢂ 0.23***
ꢀ5.19 ꢂ 0.13***
ꢀ6.72 ꢂ 0.13***
a
Data represent mean ꢂ SEM; n ¼ 8. *P < 0.05 vs. etamsylate; **P < 0.01 vs. etamsylate; ***P < 0.001 vs. etamsylate.
APTT, PT, and TT values of benzothiazole amide derivatives and shortened PT compared to the positive control etamsylate. The
etamsylate (positive control) are shown in Tables 2–4, while blood coagulant mechanism is most likely due to compound Q2
a comparison with p-aminomethylbenzoic acid (another positive primarily coagulating blood through the activation of intrinsic
control) are provided in the ESI† section.
pathways and partly through thrombin-mediated promotion of
brinogen conversion to brin in plasma.
Compound Q2 exhibited the lowest APTT value (RAPTT
¼
ꢀ38.84 ꢂ 0.08% at 1 mmol Lꢀ1; Table 2), a signicantly short-
Compound Q6 exhibited the lowest PT value (RPT ¼ ꢀ14.79 ꢂ
ened TT (P < 0.001, except 1 mmol Lꢀ1; Table 4), and a somewhat 0.13% at 100 mmol Lꢀ1; Table 3), with its activity decreasing with
Table 4
R
(%) values of the synthesized benzothiazole amide derivativesa
TT
Concentration (mmol Lꢀ1
)
Compound
1
5
10
50
100
Etamsylate
2-Aminobenzothiazole
ꢀ11.75 ꢂ 0.34
ꢀ1.92 ꢂ 0.24
11.40 ꢂ 0***
ꢀ0.52 ꢂ 0.26*
ꢀ1.52 ꢂ 0.25
ꢀ0.98 ꢂ 0.24
ꢀ3.91 ꢂ 0**
ꢀ5.32 ꢂ 0.25***
ꢀ1.02 ꢂ 0.51
ꢀ1.71 ꢂ 0.24
ꢀ2.28 ꢂ 0.25
ꢀ2.06 ꢂ 0.26
5.51 ꢂ 0.44
11.51 ꢂ 0.33
16.04 ꢂ 0.17
12.57 ꢂ 0.29***
0.26 ꢂ 0.26***
ꢀ2.53 ꢂ 0.51***
0.98 ꢂ 0.42***
ꢀ4.95 ꢂ 0.26***
ꢀ6.58 ꢂ 0.44***
ꢀ2.29 ꢂ 0.43***
ꢀ4.15 ꢂ 0***
9.36 ꢂ 0.29***
3.51 ꢂ 0.31***
ꢀ3.16 ꢂ 0.45***
2.03 ꢂ 0.25***
0.58 ꢂ 0.29***
ꢀ5.47 ꢂ 0.45***
4.30 ꢂ 0.51***
q1
q2
q3
q4
q5
q6
q7
q8
q9
Q1
Q2
Q3
Q4
Q5
Q6
Q7
Q8
Q9
ꢀ1.82 ꢂ 0.26***
1.01 ꢂ 0.44***
ꢀ4.39 ꢂ 0.24***
ꢀ2.87 ꢂ 0.26***
ꢀ0.76 ꢂ 0.51***
ꢀ0.51 ꢂ 0.25***
2.44 ꢂ 0.42***
ꢀ5.61 ꢂ 0.42***
ꢀ1.82 ꢂ 0.52***
1.77 ꢂ 0.44***
ꢀ6.10 ꢂ 0.24***
ꢀ1.56 ꢂ 0.45***
3.54 ꢂ 0.25***
0.51 ꢂ 0.67***
2.04 ꢂ 0.25***
5.12 ꢂ 0.49***
8.29 ꢂ 0***
ꢀ4.30 ꢂ 0.44**
ꢀ4.12 ꢂ 0.45***
ꢀ7.38 ꢂ 0.22***
ꢀ6.67 ꢂ 0.24***
ꢀ3.11 ꢂ 0.19***
ꢀ21.91 ꢂ 0.28***
ꢀ10.56 ꢂ 0.10*
ꢀ14.46 ꢂ 0.24***
ꢀ19.31 ꢂ 0.38***
ꢀ29.13 ꢂ 0.39***
ꢀ2.61 ꢂ 0.41***
ꢀ1.27 ꢂ 0.44***
ꢀ1.55 ꢂ 0.26***
ꢀ12.15 ꢂ 0.38***
ꢀ17.14 ꢂ 0.01***
ꢀ16.70 ꢂ 0.34***
ꢀ18.10 ꢂ 0.24***
ꢀ13.62 ꢂ 0.23***
ꢀ5.21 ꢂ 0.47***
ꢀ17.14 ꢂ 0.22***
ꢀ27.96 ꢂ 0.19***
ꢀ11.61 ꢂ 0.24***
2.03 ꢂ 0.51***
3.04 ꢂ 0.25***
0.01 ꢂ 0.52***
1.03 ꢂ 0.25***
ꢀ11.50 ꢂ 0.38***
ꢀ11.43 ꢂ 0.41***
ꢀ4.47 ꢂ 0***
ꢀ12.80 ꢂ 0.01***
ꢀ24.52 ꢂ 0.24***
ꢀ14.18 ꢂ 0.19***
ꢀ15.00 ꢂ 0.41***
ꢀ20.19 ꢂ 0.23***
ꢀ4.98 ꢂ 0.47***
ꢀ16.05 ꢂ 0.38***
ꢀ16.31 ꢂ 0.39***
ꢀ17.54 ꢂ 0.41***
ꢀ14.53 ꢂ 0.22***
ꢀ16.43 ꢂ 0.41***
ꢀ9.13 ꢂ 0.34***
ꢀ13.33 ꢂ 0.24***
ꢀ20.66 ꢂ 0.47***
ꢀ0.95 ꢂ 0.24***
ꢀ13.23 ꢂ 0.22***
ꢀ13.20 ꢂ 0.34***
ꢀ18.48 ꢂ 0.24***
ꢀ20.24 ꢂ 0.24***
ꢀ11.97 ꢂ 0.41***
ꢀ10.9 ꢂ 0.24***
ꢀ17.57 ꢂ 0.22***
ꢀ28.35 ꢂ 0.01***
ꢀ3.79 ꢂ 0.24***
a
Data represent mean ꢂ SEM; n ¼ 8. *P < 0.05 vs. etamsylate; **P < 0.01 vs. etamsylate; ***P < 0.001 vs. etamsylate.
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Fig. 3 The EC50 values for the activation of thrombin activity by
compounds Q2 and Q8. aThe EC50 values are expressed as the
concentration required for a thrombin activity response of 50%; n ¼ 3.
Fig. 5 Compound Q2 docking into the thrombin binding pocket.
compound Q8 mainly achieved a blood coagulation effect
through stimulation of brinogen or an increase of brin levels.
However, coagulation was not observed to be due to intrinsic or
extrinsic pathways.
Combined with blood coagulant activity, N-(benzo[d]thiazol-
2-yl)-cinnamamide with a methoxy group substitution lead to
a decrease in APTT and PT. Conversely, introducing an acetoxyl
group to N-(benzo[d]thiazol-2-yl) cinnamamide lead to an
increase in APTT, but a decrease in PT. Moreover, the position
of the acetoxyl group was also observed to inuence the relevant
hemostatic activity, where APTT and PT were increased when
decreasing concentration. Nevertheless, Q6 showed a lower
activity than etamsylate at 1, 5 and 10 mmol Lꢀ1, and a higher
activity at 50 and 100 mmol Lꢀ1. Further, it showed the lowest
RPT value at 1 mmol Lꢀ1 (ꢀ13.70 ꢂ 0.37%). The PT values of
other compounds were higher than ꢀ10%, indicating that the
extrinsic pathways were less affected.
Compound Q8 exhibited the lowest TT value (RTT ¼ ꢀ29.13 ꢂ
0.39%; Table 4). However, a rare effect on APTT and PT was
observed, wherein the RAPTT and RPT values of Q8 were all lower
than ꢀ5% (which can be regarded as no activity), indicating that
Fig. 4 The classical systems for blood coagulation including extrinsic, intrinsic, and/or the common coagulation pathways.30
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and Q2 and facilitating Q2 anchoring within the thrombin
binding site. The above molecular simulations elucidated the
underlying interactions between Q2 and thrombin, providing
valuable information for the further development of thrombin
activators.
In vitro determination of human C-type natriuretic peptide
(CNP)
CNP, a local vascular regulator, is a member of the natriuretic
family,32 with potent vasodilator properties.33 The level of CNP
in plasma and tissue has been shown to increase upon vascular
injury, with a decrease in CNP content suggesting that vascular
injury has improved.34 At low concentrations (5, 10, and 50 mmol
Lꢀ1), the plasma CNP level of Q2 was signicantly lower than
that of etamsylate (P < 0.05 or P < 0.01; Fig. 6). Yet, at high
concentrations (100 and 200 mmol Lꢀ1), the reverse was
observed. Etamsylate is able to reduce capillary perme-
ability.35–38 Thus, we could infer that Q2 may lead to a reduction
in capillary permeability at low concentrations (at 5, 10, and 50
mmol Lꢀ1).
Fig.
6 Effects of compounds Q2 and etamsylate on capillary
permeability. The results were expressed as the concentration of
CNP (pg mLꢀ1). Values are reported as the mean ꢂ SEM of three
independent experiments.
Conclusions
the acetoxyl group was located in the ortho or para positions in
the benzene ring compared to the meta position.
We have developed a facile and efficient method for the
synthesis of benzothiazole amide derivatives via a nucleophilic
acyl substitution reaction of 2-aminobenzothiazole with cin-
namic acid compounds. Products bearing methoxy groups were
easily obtained in high yields (up to 93%) and had suitable
thermal stabilities as demonstrated by thermogravimetric
analysis. Compound Q2 showed good hemostatic activity, with
a signicantly shortened APTT and TT, reduced CNP level,
activated thrombin activity, and more potent platelet aggrega-
tion activity compared to etamsylate (up to 1283.9 times),
indicating that these benzothiazole amide derivatives, and
particularly Q2, should be further screened for the possible
generation of potential hemostatic drug molecules.
Thrombin activity assay
Thrombin is a key component in blood coagulation, positioned
in the coagulation cascade to cleave brinogen into brin,
resulting in blood clot formation.29 Q2 and Q8, which had
a good coagulation activity, were selected for thrombin activity
assays (Fig. 3). The EC50 of thrombin activity of Q2 (135.9 mmol
Lꢀ1) and Q8 (232.1 mmol Lꢀ1) were both lower than for etam-
sylate (126.5 mmol Lꢀ1). In combination with blood coagulant
activity results and the fact that Q2 signicantly shortened APTT
and TT compared to etamsylate, we can infer that the coagula-
tion activity mechanism of Q2 functioned through its interac-
tion with thrombin as well as with other proteins or factors in
the blood coagulation cascade (Fig. 4).
Experimental
Materials and methods
A series of cinnamic acid derivatives of analytical grade were
purchased from Shengshun Trading Co., Ltd. (Zhuhai, China).
Docking of compound Q2 with thrombin
To further assess the interaction mode of compound Q2 with 2-Aminobenzothiazole was purchased from Aladdin reagents
thrombin, a molecular docking study was performed using (Shanghai, China). Etamsylate was purchased from Hubei Tia-
Autodock vina 1.1.2 soware.31 The theoretical binding mode of nyao Pharmaceutical Co., Ltd. APTT, PT, and TT test kits were
Q2 in the thrombin binding site is illustrated in Fig. 5. Q2 purchased from Heall Bio-Science Technology, Qingdao, China.
adopted a compact conformation to bind inside the thrombin ADP was purchased from Sigma-Aldrich (Shanghai, China).
pocket. The benzothiazolyl group of Q2 was located within the Human CNP ELISA Kit were purchased from Shanghai mlbio
hydrophobic pocket, surrounded by Tyr-60A, Leu-99, Ile-174, Biotechnology Co. (Shanghai, China). Thrombin ($1000 NIH U
and Trp-215 residues, forming stable hydrophobic bonding. mgꢀ1 protein) prepared from human plasma was purchased
Detailed analysis showed that the phenyl group of Q2 formed from Sigma-Aldrich (USA). Chromozym-TH was purchased from
anion–p interactions with Asp-189 and Glu-192 residues. Roche (Swiss). All other reagents were commercial materials of
Importantly, three key polar interactions were observed analytical purity and were used without further purication.
between the carbonyl “O” of Q2 and Asp-189 (bond distance: 3.1
1H and 13C NMR spectra were performed in dimethylsulf-
A), Ala-190 (bond distance: 2.5 A), and Gly-219 (bond distance: oxide-d6 on a Bruker Ascend 600 at 600 MHz for 1H and 150
ꢀ
ꢀ
3.0 A), representing the main interactions between thrombin MHz for 13C, respectively. X-ray crystallographic studies were
ꢀ
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performed using a Nonius Kappa CCD diffractometer equipped 8.18 Hz), 7.38 (t, 1H, J ¼ 7.55 Hz), 7.30 (t, 1H, J ¼ 7.86 Hz),
with a graphite monochromator and an Oxford cryostream, 7.19 (d, 1H, J ¼ 7.75 Hz), 7.09 (d, 1H, J ¼ 9.53 Hz), 7.03 (t, 1H,
using Mo-Ka radiation (l ¼ 0.7107 A). Infrared spectra were J ¼ 1.63 Hz), 6.67 (d, 1H, J ¼ 15.59 Hz), 2.30 (s, 3H); 13C NMR
ꢀ
performed as KBr sheets on a Tensor 27 FTIR spectrometer (151 MHz, CDCl3) d 169.27, 164.24, 161.16, 151.11, 147.64,
(Bruker, Germany). ESI-MS were collected using a LC-MS 2010A 144.24, 135.59, 132.28, 129.96, 126.95, 126.42, 124.17,
instrument (Shimadzu, Japan).
124.11, 122.07, 121.07, 120.21, 119.36, 21.22; ESI-MS m/z
Plasma coagulation assays and platelet aggregation deter- 336.8 (M ꢀ H)ꢀ.
mination were performed using a blood coagulation analyzer
(E)-2-(3-(Benzo[d]thiazol-2-ylamino)-3-oxoprop-1-en-1-yl)
(LG-PABER-I, GTM-STEELLEX Instrument Co., Ltd., Beijing, phenyl acetate (Q4). White powder, yield 70%, mp 197.2–
1
China). The enzyme–drug reaction was monitored using a BIO- 197.8 C; IR (KBr) n (cmꢀ1): 1762, 1679, 1602; H NMR (600
ꢁ
RAD iMark microplate reader.
MHz, CDCl3) d 12.61 (s, 1H), 8.04 (d, 1H, J ¼ 15.64 Hz), 7.95
(d, 1H, J ¼ 7.33 Hz), 7.87 (d, 1H, J ¼ 7.85 Hz), 7.42 (m, 3H),
7.30 (t, 1H, J ¼ 7.84 Hz), 7.13 (d, 1H, J ¼ 8.09 Hz), 7.09 (t, 1H,
J ¼ 7.58 Hz), 6.69 (d, 1H, J ¼ 15.62 Hz), 2.37 (s, 3H); 13C NMR
Synthesis of compound Q1–Q4
As shown in Scheme 1, cinnamic acid compounds (25.0 g; (151 MHz, CDCl3) d 169.38, 164.14, 160.87, 149.77, 147.76,
compound 1) and pyridine (5 mL) were added to a solution of 138.35, 132.28, 131.69, 127.40, 126.86, 126.71, 126.28,
acetic anhydride (100 mL). Aer the addition, the solution was 124.33, 123.33, 122.06, 120.43, 120.11, 21.07; ESI-MS m/z
stirred at 120 ꢁC for 5 h using an oil bath. Deionized water (800 336.8 (M ꢀ H)ꢀ.
mL) was added and the crude acetoxy intermediate (compound
2) was precipitated and separated.39 Compound 2 (1.5 mmol)
Synthesis of compound Q5–Q9
was dissolved in N,N-dimethylformamide (1 mL) and thionyl
chloride (10 mL), and stirred at 70 ꢁC for 5 min. Through Cinnamic acid compounds (1.5 mmol) and N,N-dime-
distillation under reduced pressure, the liquid residue was thylformamide (1 mL) were added to 10 mL of thionyl chloride,
ꢁ
collected and dispersed in 20 mL of CH2Cl2, to which 2-ami- and allowed to react at 70 C for 5 min. The liquid residue ob-
ꢁ
nobenzothiazole (1.0 mmol) was added, and stirred at 0–5 C. tained following distillation under reduced pressure was
The pH value was kept between 6 and 7 by addition of NaHCO3 retained. A solution of the liquid residue in CH2Cl2 was added
to induce precipitation. The upper layer solid was then collected dropwise to a CH2Cl2 solution of 2-aminobenzothiazole (1.0
by ltration of the mixture, and washed with excess distilled mmol) under ice cooling. The mixture was stirred at 0–5 ꢁC for 3
ethanol and water to obtain the pure products. Data for hours. TLC was used to monitor whether the reaction had
compounds Q1–Q4 are given below.
completed. The pH value was kept between 6 and 7 by addition
(E)-4-(3-(Benzo[d]thiazol-2-ylamino)-3-oxoprop-1-en-1-yl)- of NaHCO3 to induce precipitation. Aer the reaction, the
2-methoxyphenyl acetate (Q1). White powder, yield 82%, mp amides were isolated using ltration of the mixture and then
183.0–184.9 ꢁC; IR (KBr) n (cmꢀ1): 1765, 1633, 1618, 1198; 1H washed with excess distilled ethanol and water to obtain the
NMR (600 MHz, DMSO-d6) d 12.57 (s, 1H), 8.00 (d, 1H, J ¼ pure products. The synthesis route is shown in Scheme 2 and
7.70 Hz), 7.80 (d, 1H, J ¼ 15.58 Hz), 7.77 (d, 1H, J ¼ 8.00 Hz), the data for compounds Q5–Q9 are indicated below.
7.44 (m, 2H), 7.32 (m, 1H), 7.28 (dd, 1H, J ¼ 1.76 and 8.19
N-(Benzo[d]thiazol-2-yl)cinnamamide (Q5). White powder,
Hz), 7.20 (d, 1H, J ¼ 8.12 Hz), 6.97 (d, 1H, J ¼ 15.80 Hz), 3.85 yield 88%, mp 187.3–189.8 ꢁC; IR (KBr) n (cmꢀ1): 1698, 1634,
(s, 3H), 2.28 (s, 3H); 13C NMR (151 MHz, DMSO-d6) d 168.37, 1601; 1H NMR (600 MHz, DMSO-d6) d 12.60 (s, 1H), 8.00 (d, 1H, J
164.00, 158.02, 151.17, 148.68, 142.47, 140.96, 133.20, ¼ 7.74 Hz), 7.80 (d, 1H, J ¼ 15.82 Hz), 7.77 (d, 1H, J ¼ 8.01 Hz),
131.69, 126.13, 123.57, 121.72, 120.57, 119.71, 112.35, 55.86, 7.67 (m, 2H), 7.46 (m, 4H), 7.32 (d, 1H, J ¼ 7.52 Hz), 6.97 (d, 1H, J
20.39; ESI-MS m/z 366.7 (M ꢀ H)ꢀ.
¼ 15.82 Hz); 13C NMR (151 MHz, DMSO-d6) d 164.02, 158.05,
(E)-4-(3-(Benzo[d]thiazol-2-ylamino)-3-oxoprop-1-en-1-yl) 148.68, 143.11, 134.19, 131.69, 130.52, 129.11, 128.12, 126.13,
phenyl acetate (Q2). White powder, yield 73%, mp 183.0– 123.56, 121.72, 120.54, 119.40; ESI-MS m/z 280.8 (M + H)+.
1
184.9 C; IR (KBr) n (cmꢀ1): 1756, 1641, 1596; H NMR (600
(E)-N-(Benzo[d]thiazol-2-yl)-3-(4-methoxyphenyꢁl)acrylamide
ꢁ
MHz, DMSO-d6) d 12.64 (s, 1H), 8.00 (d, 1H, J ¼ 7.90 Hz), 7.81 (Q6). White powder, yield 90%, mp 201.8–202.3 C; IR (KBr) n
(d, 1H, J ¼ 15.80 Hz), 7.77 (d, 1H, J ¼ 7.96 Hz), 7.71 (d, 2H, J (cmꢀ1): 1681, 1624, 1162; 1H NMR (600 MHz, DMSO-d6) d 12.49
¼ 8.59 Hz), 7.45 (t, 1H, J ¼ 8.25 Hz), 7.32 (t, 1H, J ¼ 7.57 Hz), (s, 1H), 7.98 (d, 1H, J ¼ 7.80 Hz), 7.75 (m, 2H), 7.52 (d, 2H, J ¼
7.25 (d, 2H, J ¼ 8.59 Hz), 6.94 (d, 1H, J ¼ 15.82 Hz), 2.29 (s, 8.55 Hz), 7.44 (t, 1H, J ¼ 7.55 Hz), 7.30 (t, 1H, J ¼ 7.47 Hz), 7.03
3H); 13C NMR (151 MHz, DMSO-d6) d 169.00, 164.00, 158.07, (d, 2H, J ¼ 8.55 Hz), 6.82 (d, 1H, J ¼ 15.75 Hz), 3.81 (s, 3H); 13
C
151.97, 148.67, 142.15, 131.84, 131.78, 129.35, 126.13, NMR (151 MHz, DMSO-d6) d 164.36, 161.19, 158.27, 148.72,
123.56, 122.60, 121.72, 120.59, 119.55, 20.86; ESI-MS m/z 142.87, 131.70, 129.90, 126.80, 126.06, 123.43, 121.66, 120.43,
336.8 (M ꢀ H)ꢀ.
116.78, 114.58, 55.36; ESI-MS m/z 309.2 (M ꢀ H)ꢀ.
(E)-3-(3-(Benzo[d]thiazol-2-ylamino)-3-oxoprop-1-en-1-yl)
(E)-N-(Benzo[d]thiazol-2-yl)-3-(3,4-dimethoxyphenyl)acrylamide
phenyl acetate (Q3). White powder, yield 77%, mp 197.2– (Q7). White powder, yield 92%, mp 204.4–206.8 ꢁC; IR (KBr) n
1
197.8 C; IR (KBr) n (cmꢀ1): 1754, 1694, 1578; H NMR (600 (cmꢀ1): 1701, 1639, 1159; 1H NMR (600 MHz, DMSO-d6), d 12.44 (s,
ꢁ
MHz, CDCl3) d 12.76 (s, 1H), 7.93 (d, 1H, J ¼ 7.43 Hz), 7.87 1H), 7.99 (d, 1H, J ¼ 7.69 Hz), 7.75 (t, 2H, J ¼ 11.59 Hz), 7.44 (d, 1H,
(d, 1H, J ¼ 15.58 Hz), 7.85 (d, 1H, J ¼ 7.97 Hz) 7.44 (t, 1H, J ¼ J ¼ 7.65 Hz), 7.31 (d, 1H, J ¼ 7.54 Hz), 7.25 (m, 2H), 7.05 (d, 1H, J ¼
6238 | RSC Adv., 2018, 8, 6231–6241
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8.83 Hz), 6.85 (d, 1H, J ¼ 15.73 Hz), 3.83 (s, 3H), 3.81 (s, 3H); 13
C
Live subject statement. All experiments concerning healthy
human plasma were performed in compliance with accordance
with the WHO guidelines on blood drawing (WHO Publication
ISBN-13: 978-92-4-159922-1, 2010), and approved by the ethics
committee at the Ethics Committee of First Affiliated Hospital
of Guangxi Medical University. All experiments concerning
human volunteers were performed aer obtaining informed
consent.
NMR (151 MHz, DMSO-d6) d 164.36, 158.17, 151.06, 148.96, 148.69,
143.26, 131.70, 127.02, 126.10, 123.48, 122.31, 121.68, 120.47,
116.98, 111.84, 110.62, 55.68, 55.50; ESI-MS m/z 340.9 (M + H)+.
(E)-N-(Benzo[d]thiazol-2-yl)-3-(3,4,5-trimethoxyphenyl)acrylamide
(Q8). Yellow powder, yield 93%, mp 137.4–138.8 ꢁC; IR (KBr) n
(cmꢀ1): 1703, 1633, 1153; 1H NMR (600 MHz, CDCl3) d 12.95 (s, 1H),
7.91 (d, 1H, J ¼ 7.83 Hz), 7.85 (dd, 2H, J ¼ 11.88 and 14.24 Hz), 7.42
(t, 1H, J ¼ 7.35 Hz), 7.35 (t, 1H, J ¼ 7.53 Hz), 6.60 (m, 3H), 3.87 (s,
3H), 3.65 (s, 6H); 13C NMR (151 MHz, CDCl3) d 164.58, 161.28,
153.55, 147.58, 145.55, 140.73, 132.23, 129.47, 126.93, 124.23,
122.05, 120.10, 117.45, 105.72, 61.12, 56.16; ESI-MS m/z 368.8 (M ꢀ
H)ꢀ.
Platelet aggregation determination in vitro
Functional promotion activity of the nine synthesized
compounds was evaluated by measuring the promotion of ADP-
induced platelet aggregation in human platelet-rich plasma
(PRP) by Born's method.44 PRP was prepared from collected
blood by low speed centrifugation at 800 rpm for 10 min at
24 ꢁC, whereas platelet-poor plasma (PPP) was prepared by
centrifuging PRP at 3000 rpm for a further 10 min. ADP was
selected as an agonist for platelet aggregation test.
Light transmission of 100% was calibrated by 280 mL PPP
and 20 mL sample solution (samples were dissolved and diluted
with 80% macrogol 400), and then platelet aggregation was
induced by the addition of ADP (nal concentration: 5 mmol
Lꢀ1) to 280 mL PRP and 20 mL sample solution. Platelet aggre-
gation kinetics were measured and platelet aggregation was
expressed as a percentage of the PPP transmission value. The
changes in light transmission were recorded. Experiments were
completed within 3 h aer blood collection.
N-(Benzo[d]thiazol-2-yl)-3-phenylpropanamide (Q9). White
powder, yield 90%, mp 197.3–199.9 ꢁC; IR (KBr) n (cmꢀ1): 1756,
1
1641, 1596; H NMR (600 MHz, DMSO-d6) d 12.36 (s, 1H), 7.96
(d, 1H, J ¼ 7.58 Hz), 7.72 (d, 1H, J ¼ 8.01 Hz), 7.42 (t, 1H, J ¼ 7.66
Hz), 7.28 (m, 5H), 7.18 (t, 1H, J ¼ 7.11 Hz), 2.95 (t, 2H, J ¼ 7.72
Hz), 2.82 (t, 2H, J ¼ 7.74 Hz); 13C NMR (151 MHz, DMSO-d6)
d 171.46, 157.82, 148.52, 140.65, 131.42, 128.36, 128.26, 126.08,
126.05, 123.45, 121.65, 120.46, 36.81, 30.24; ESI-MS m/z 280.8
(M ꢀ H)ꢀ.
Crystallographic data collection and structure determination
Single-crystal X-ray diffraction data of compounds Q7 and Q9
was collected on a CCD area detector diffractometer with
graphite monochromated Mo/Ka radiation (l ¼ 0.071073 nm).
The structures were solved by direct methods using Olex2
soware40 and rened with full-matrix least squares on F2 using
the ShelXL41 program package. All non-hydrogen atoms were
rened anisotropically. The hydrogen atoms attached to all
carbon and nitrogen atoms were geometrically xed and the
positional and temperature factors were rened isotropically.
Structural illustrations were generated using Diamond 3.1 for
Windows.
Blood coagulation assay in vitro
The activation of APTT, PT, and TT assays were examined by
commercial kits following the manufacturer's instructions
using a hematology analyzer. Samples were dissolved in and
diluted with 80% macrogol 400. A 100 mL aliquot of the sample
solution was then mixed with 900 mL of plasma and incubated
at 37 ꢁC for 5 min. APTT was determined by incubating 50 mL of
the plasma solution with 50 mL of APTT activating agent for
3 min at 37 ꢁC, followed by addition of 50 mL of CaCl2 (25 mM).
PT was determined by incubating 50 mL of the plasma solution
Thermogravimetric analysis
The thermogravimetric experiments were performed using
a NETZSCH STA 409 PC/PG thermogravimetric analyzer under
air atmosphere at a ow rate of 20 mL minꢀ1 for the sample.
The ꢁexplored temperature interval ranged between 30 ꢁC and
ꢁ
for 3 min at 37 C, followed by addition of 100 mL of thrombo-
plastic agent. TT was determined by incubating 50 mL of the
plasma solution for 3 min at 37 ꢁC, followed by addition of 100
mL of APTT activating agent.
The coagulation activity was assessed by assaying the plasma
clotting time of TT and APTT, and the decrease in International
Normalized Ratio (INR) of PT.
250 C, at a heating rate of 0.5 C minꢀ1. The decomposition
ꢁ
temperature (Td) was taken at the maximum of the heat ow
peaks42 and the maximum heat ux changes occurred at the
same point.
The RAPTT, RPT, or RTT were calculated as follows:
Human plasma samples
APTTC ꢀ APTT0
RAPTT
¼
ꢃ 100%
(1)
(2)
(3)
Healthy volunteers (aged 20–25) were recruited for this study.
Whole blood was drawn from the volunteer's hand vein. The
fresh vein blood samples were collected in the morning with
overnight fasting using routine procedures. Blood was collected
in plastic tubes with 3.8% sodium citrate (citrate/blood: 1/9, v/v)
to ensure plasma anticoagulation.43 Samples were centrifuged
for 15 minutes at 1000 ꢃ g at 2–8 ꢁC within 30 minutes of
collection.
APTT0
PTC ꢀ PT0
RPT
¼
¼
ꢃ 100%
PT0
TTC ꢀ TT0
RTT
ꢃ 100%
TT0
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In this set of equations, APTTC, PTC, and TTC correspond to
the APTT, PT, and TT at determined sample concentrations. The
factors APTT0, PT0, and TT0 correspond to the control assays in
which the concentration of the sample was 0 mM. All the data
were obtained as mean (n ¼ 8) values.
Statistical analysis
Signicant differences between the groups were determined
using a one-way analysis of variance (ANOVA) in SPSS 22.0
(SPSS Inc., Chicago, Illinois, USA), followed by Dunnett's test
with P < 0.05. The EC50 values were determined using linear
regression in SPSS 22.0.
Determination of thrombin activity
Conflicts of interest
The thrombin activation assay was conducted using the
chromogenic substrate method. The substrate Tos–Gly–Pro–
Arg–pNA (chromozym-TH) can be hydrolyzed by thrombin and
cleaved into two parts, a residual peptide and a yellow
substance, p-nitroaniline, which can be measured with
a microplate reader. A 20 mL aliquot of the thrombin solution
(1 U mLꢀ1, in 0.1% bovine serum albumin, pH 6.5) was mixed
with 20 mL of the test compound solution (dissolved in 50 mM
There are no conicts to declare.
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (21362001), and the Guangxi Key
Laboratory of Traditional Chinese Medicine Quality Standards
(guizhongzhongkai201104). We acknowledge MogoEdit (a
professional English editing company) for English revision of
the manuscript.
ꢁ
Tris buffer, pH 8.3). The mixture was incubated at 37 C for
10 min before the addition of 20 mL of chromozym-TH solution
(2 mM). Aer a further 10 min of incubation at 37 ꢁC, 50 mL of
acetic acid (50% w/v) and 40 mL of water were successively
added to terminate the reaction. For each concentration, three
replicate wells were used. The absorbance was monitored at
405 nm. The Tris-treated control was considered the blank
control. The EC50 values represent the concentration that
caused a 50% activation relative to the Tris-treated control.
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