Synthesis and biological evaluation of a new series of cinnamic acid amide derivatives as potent haemostatic agents containing a 2-aminothiazole substructure

Article abstract of DOI:10.1016/j.bmcl.2017.07.058

Ten new cinnamic acid derivatives containing a 2-aminothiazole substructure were designed and synthesized. This series of compounds exhibited good thermostabilities as demonstrated by thermogravimetric analysis. In coagulation assays (prothrombin time, activated partial thromboplastin time and thrombin time) in vitro, most compounds demonstrated excellent activities to promote blood coagulation. Among the studied series, compounds N1, N4, N5 and W5 exhibited a significant coagulation activity. Further studies indicated that compound N5 (IC₅₀ = 1.87 μmol/L) displayed the most suitable efficacy of promoting platelet aggregation than the clinically used haemostatic drug etamsylate (IC₅₀ = 46.22 μmol/L). Furthermore, the relationship between the functional groups of the compounds and the corresponding blood coagulant activity was explored in this study.

Full text of DOI:10.1016/j.bmcl.2017.07.058

Accepted Manuscript
Synthesis and Biological Evaluation of a New Series of Cinnamic Acid Amide
Derivatives as Potent Haemostatic Agents Containing a 2-Aminothiazole Sub-
structure
Wenqian Nong, Anran Zhao, Jinrui Wei, Xiao Lin, Lisheng Wang, Cuiwu Lin
PII:
S0960-894X(17)30765-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.058
BMCL 25171
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
15 June 2017
15 July 2017
21 July 2017
Please cite this article as: Nong, W., Zhao, A., Wei, J., Lin, X., Wang, L., Lin, C., Synthesis and Biological Evaluation
of a New Series of Cinnamic Acid Amide Derivatives as Potent Haemostatic Agents Containing a 2-Aminothiazole
Substructure, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.058
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Synthesis and Biological Evaluation of a New Series of
Cinnamic Acid Amide Derivatives as Potent Haemostatic
Agents Containing a 2-Aminothiazole Substructure
Wenqian Nonga, Anran Zhaob, Jinrui Weic, Xiao Lind, Lisheng Wanga and Cuiwu Lina, *
a
Guangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology and Resource Development, School of
chemistry& chemical engineer in Guangxi University, Nanning, 530004, China
b Department of Chemistry, Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, United States
c Guangxi Scientific Research Center of Traditional Chinese Medicine, Guangxi University of Chinese Medicine, Nanning, Guangxi
530200, PR China
d
Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Traditional Medical and
Pharmaceutical Sciences, Nanning 530022, China
* Corresponding author. Tel.: +86 13878162266; e-mail: cuiwulin@163.com
1

Graphical Abstract
This study demonstrates the use of new compound N5 with potential characteristics for the
development
of
drugs
combining
coagulant
and
platelet
activities.
2

ABSTRACT
Ten new cinnamic acid derivatives containing a 2-aminothiazole substructure were designed
and synthesized. This series of compounds exhibited good thermostabilities as demonstrated by
thermogravimetric analysis. In coagulation assays (prothrombin time, activated partial
thromboplastin time and thrombin time) in vitro, most compounds demonstrated excellent
activities to promote blood coagulation. Among the studied series, compounds N1, N4, N5 and
W5 exhibited a significant coagulation activity. Further studies indicated that compound N5 (IC50
= 1.87 μmol/L) displayed the most suitable efficacy of promoting platelet aggregation than the
clinically used haemostatic drug etamsylate (IC50 = 46.22 μmol/L). Furthermore, the relationship
between the functional groups of the compounds and the corresponding blood coagulant activity
was explored in this study.
Keywords: Cinnamic acid derivatives; 2-aminothiazole; Crystal structure; Platelet aggregation;
Coagulant activity
To date, excessive blood loss during surgery still remains a critical operative
complication in patients undergoing major surgery, potentially increasing the risk of
postoperative morbidity and mortality.1 Excessive blood loss generally results in
prolonged hospital stays, delayed rehabilitation and increased mortality.2 In surgical
procedures, transfusion of homologous blood components is often associated with
infectious and immunologic risks as well as increased mortality. Therefore,
blood-saving strategies are essential to reduce the amount of homologous blood used
in transfusion.3 In 1988, a Lancet editorial article described that the use of drugs can
reduce surgical blood loss.4 For this reason, improving the efficacy of haemostatic
agents represents one of the direct routes to reduce bleeding during surgery. A variety
of medications have been studied as haemostatic agents. As such, newer but
expensive to produce medications such as aprotinin, etamsylate and hemostatic acid
have been studied in recent years.5 However, studies6 by Mangno et al. have shown
that drugs such as aprotinin exhibit considerable safety risks with the clinical usage to
be reevaluated. Therefore, the search for safer and more efficient hemostatic drugs
still represents a crucial, albeit unmet, scientific goal.
Normal haemostatic mechanisms involve normal functions of blood vessels,
platelets and blood coagulation. Blood coagulation represents an important part of
haemostasis in which the wall of damaged blood vessels is covered by a platelet and
fibrin-containing clot to reduce bleeding and begin damage repair of the vessel.7, 8
Simultaneously, the extrinsic coagulation pathway is activated. Hence, an inexpensive
drug with high efficiency of haemostatic activity through more than two pathways as
mentioned above would offer significant advantages. Natural products and/or
structures stemming from natural product are highly significant for the development
process of new drugs.9, 10 Compounds based on cinnamic acid represent natural
extracts with a variety of biological activities and can be found in a large number of
Chinese herbal medicines.11 Cinnamic acid derivatives are reported to possess various
biologically relevant activities such as antioxidant,12 anti-inflammatory,13
anti-malarial,14 anti-tumor,15 anti-diabetic,16 anti-tubercular17 and anti-cancer18
3

activities. Moreover, the coagulation activity of this compound species was evaluated
in our previously reported study.19 Using cinnamic acid as a lead compound, we
carried out further structural modifications and studied the effects. According to a
patent publication20 by Shiota et al., 2-aminothiazole was used to synthesize a drug
composition that promotes thrombocytosis.
According to the combination principle of drug design, two different compounds
were combined via a covalent bond in anticipation of a synergistic effect of each
individual compound. Hence, we designed ten new cinnamic acid derivatives with an
amide group and 2-aminothiazole structure to evaluate the blood coagulation activity.
We determined the thermodynamic stability of the compounds using
thermogravimetry for the evaluation of drug stability. Based on the haemostatic
mechanism,8 the blood clotting activities of the above referenced compounds were
evaluated by in vitro experiments of a plasma coagulation assay (prothrombin time,
activated partial thromboplastin time and thrombin time) and platelet aggregation
determination. The relationship between structure and the corresponding coagulant
activity was also discussed based on the obtained test results.
The new cinnamic acid derivatives N1-N5 (cf. Scheme 1), W1-W5 (cf. Scheme
2 and 3) were synthesized and further purified by reverse phase C18 silica gel column
chromatography (mobile phase A: methanol; mobile phase B: water. The isocratic
system was A:B = 55%:45%). The detailed description of the experimental
information, corresponding yields, melting points, characteristic IR, NMR, MS and
HPLC purity data are provided in the Supporting information section.
Scheme 1 Reagents and conditions: (a) AA/Py, 120 °C, 99-100%. (b) SOCl2, 70 °C, 100%. (c) NaOAc, pH = 6-7,
0-5°C to rt, 80-95%.
Scheme 2 Reagents and conditions: (d) SOCl2/DMF, 70 °C, 100%. (e) NaOAc, pH = 5-6, 0 °C to rt, 72-95%.
4

Scheme 3 Reagents and conditions: (d) SOCl2/DMF, 70 °C, 100%. (e) NaOAc, pH = 5-6, 0 °C to rt, 91.2%.
Single crystals of compounds N1, N3, W1 and W2 (CCDC1534821-1534824) 21
were grown from N,N-dimethylformamide or dimethyl sulfoxide and the
corresponding structures were confirmed by single crystal X-ray diffraction analysis
(cf. Figure 1). Coupled with the integrals in the NMR spectra, X-ray diffraction
analysis provided the crystal structures for the four compounds N1, N3, W1 and W2.
The crystal lattices of compounds N1, W1 and W2 belong to the monoclinic system
but exhibit different space groups. The crystal lattices of compound N3 belong to the
orthorhombic
system,
containing
two
(E)-4-(3-oxo-3-(thiazol-2-ylamino)prop-1-en-1-yl)phenyl acetate molecules. Selected
bond lengths (Å) and angles (°) are listed in Tables 1 and 2, a detailed description of
the method is provided in the Supporting information section.
Fig. 1 Ball-and-stick structures of compounds N1, N3, W1 and W2.
Table 1 Selected bond lengths (Å) for compounds N1, N3, W1 and W2
Compound N1
N(2)-C(4)
Compound N3
N(009)-C(4A)
Compound W1
Compound W2
N(2)-C(1)
1.3762(18)
1.2279(17)
1.4395(19)
1.3670(17)
1.1962(19)
1.3945(17)
1.3586(18)
1.3749(19)
1.2277(19)
1.3995(18)
1.3674(19)
1.190(2)
N(2)-C(2)
1.3756(17)
1.3745(17)
1.2178(17)
1.474(2)
1.394(3)
1.397(2)
1.221(3)
1.510(4)
1.470(4)
O(1)-C(4)
O(2)-C(15)
O(3)-C(13)
O(4)-C(13)
O(3)-C(10)
O(2)-C(9)
O(003)-C(4A)
O(007)-C(10A)
O(007)-C(13A)
O(008)-C(13A)
N(2)-C(4)
O(1)-C(4)
C(4)-C(5)
C(5)-C(6)
N(2)-C(9)
O(1)-C(9)
C(4)-C(9)
C(4)-C(14)
1.3265(19)
5

Table 2 Selected bond lengths (Å) and angles (°) for compounds N1, N3, W1 and W2
Compound N1 Compound N3 Compound W1
O(003)-C(4A)-N(009) C(4)-N(2)-C(2)
Compound W2
C(9)-N(2)-C(1)
O(1)-C(4)-N(2)
121.37(13)
124.71(13)
117.17(12)
121.11(14)
124.23(14)
122.94(12)
124.18(15)
126.16(15)
123.09(12)
121.20(13)
124.23(13)
121.30(12)
123.34(10)
120.72(13)
122.5(2)
122.0(3)
123.4(2)
114.6(2)
113.2(2)
O(1)-C(4)-C(5)
C(9)-O(2)-C(15)
C(13)-O(3)-C(10)
O(4)-C(13)-O(3)
O(003)-C(4A)-C(5A)
C(13A)-O(007)-C10A
O(1)-C(4)-N(2)
O(1)-C(4)-C(5)
N(1)-C(2)-N(2)
N(2)-C(2)-S(1)
C(6)-C(5)-C(4)
O(1)-C(9)-N(2)
O(1)-C(9)-C(4)
N(2)-C(9)-C(4)
C(14)-C(4)-C(9)
117.04(11) O(008)-C(13A)-O(007)
122.53(13) O(008)-C(13A)-C(14A)
O(4)-C(13)-C(14) 127.62(13)
The mass losses and the heat flux of the cinnamic acid amide derivatives upon
changing the temperatures are shown in Figures 2 and 3 and a detailed description of
the method is provided in the Supporting information section. Compound W2 started
to decompose at 157 °C while others decomposed above 200 °C as determined by
decreasing quality characteristics. It can be inferred that the thermal stability of
compound W2 decreases with the decrease of the degree of unsaturation.
Thermogravimetric analysis indicated that these compounds exhibit a suitable thermal
stability at 150 °C. Therefore, these compounds are appropriate for further screening
as coagulant drugs.
Fig. 2 Thermogravimetric curves and heatflow curves of N1-N5 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 equlibrated at
laboratory atmosphere.
6

Fig. 3 Thermogravimetric curves and heatflow curves of W1-W5 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 equlibrated at
laboratory atmosphere.
To investigate the coagulant activity effects of the 10 compounds (N1-N5,
W1-W5), we conducted assays of PT (prothrombin time), APTT (activated partial
thromboplastin time), TT (thrombin time) and platelet aggregation activities. PT is an
ideal and commonly used screening assay for the detection of extrinsic coagulation
systems,22 whereas APTT is a test of the intrinsic coagulation activity. The latter may
be a rough detection of coagulation factor II, V, VII and X activity.22, 23 Note that a
prolonged PT may suggest the inhibition of the extrinsic and/or the common
coagulation pathway, whereas a prolonged APTT may suggest the inhibition of the
intrinsic and/or the common pathway.24 TT is a screening test for determining the
thrombin activity and the ability of converting plasma fibrin into fibrin.25 In
particular, TT reflects the conversion time of fibrinogen to fibrin. A shortened TT
may represent an increase in coagulatable fibrinogen or an increase in its activity.26
The coagulation rate (Rxx) was used to evaluate the haemostasis activity. Negative
values of drugs may represent haemostasis activity and smaller values may indicate
that improved clotting effects of the compounds are demonstrated. Overall, platelet
aggregation represents a vastly complex process. Its activation is mediated primarily
through platelet adhesion at the site of injury as well as the action of endogenous
agonists such as ADP (adenosine diphosphate), collage, and thrombin, followed by
the release of TXA2, which acts as an amplifying factor.27 Activation is enhanced by
the formation and liberation of TXA2, the secretion of ADP and serotonin from the
platelet dense granules, and the exposure of a procoagulant surface that promoted the
generation of thrombin.27 These agonists stimulate platelet aggregation via specific
receptors on the platelet membrane.28 All results of PT, APTT, TT assay can be found
listed in Table 3-5, and a detailed description of the method is provided in the
Supporting information section. The results of the ADP induced platelet aggregation
assay in human platelet-rich plasma (PRP) using the Born’s method29 are listed in
Table 6.
7

Table 3 RPT (%) values of the synthesized compounds
Concentration (μmol/L)
Compound
1
5
10
50
100
1.15 ± 0.28
1.05 ± 0.18
0.73 ± 0.48
0.63 ± 0.28
0.73 ± 0.18
Haemostatic acid
-6.63 ± 0.23***
-1.98 ± 0.23***
-4.30 ± 0.18***
-3.20 ± 0.62***
-0.10 ± 0.58
-1.68 ± 0.10**
-5.69 ± 0.61***
-0.56 ± 0.52
-1.51 ± 0.57**
-1.70 ± 0.25**
0.56 ± 0.34
-4.91 ± 0.65***
-2.24 ± 0.31***
-4.09 ± 0.38***
-4.40 ± 0.17***
-1.05 ± 0.10*
-3.25 ± 0.28***
-7.09 ± 0.35***
-2.44 ± 0.49***
-2.27 ± 0.09***
0.28 ± 0.41
-2.84 ± 0.39**
-4.39 ± 0.41***
-1.36 ± 0.69
-5.29 ± 0.17***
-0.73 ± 0.10
-4.62 ± 0.46***
-5.59 ± 0.17***
-3.19 ± 0.34***
0.00 ± 0.49
-5.17 ± 0.52***
-5.77 ± 0.22***
-1.68 ± 0.46**
-4.30 ± 0.66***
-0.42 ± 0.28
-2.20 ± 0.56***
-4.40 ± 0.17***
-2.81 ± 0.25***
-0.75 ± 0.50
-1.49 ± 0.25**
0.55 ± 0.25
-5.43 ± 0.65***
-7.32 ± 0.45***
-3.88 ± 0.36***
1.20 ± 0.40
2-aminothiazole
N1
N2
N3
-0.84 ± 0.31
-1.78 ± 0.55*
-3.70 ± 0.20***
0.00 ± 0.50
N4
N5
W1
W2
W3
W4
W5
-6.75 ± 0.19***
-2.99 ± 0.34***
2.55 ± 0.25
-0.22 ± 0.16
3.22 ± 0.25*
-0.95 ± 0.57*
Data represent mean ± SEM. n = 4.
* P < 0.05 vs. haemostatic acid.
** P < 0.01 vs. haemostatic acid.
*** P < 0.001 vs. haemostatic acid.
Table 4 RAPTT (%) values of the synthesized compounds
Concentration (μmol/L)
Compound
1
5
10
50
100
-6.37 ± 0.23
-3.71 ± 0.49***
-1.14 ± 0.25***
-2.94 ± 0.11***
-1.68 ± 0.52***
-4.64 ± 0.41*
-5.55 ± 0.00
-4.78 ± 0.52
1.00 ± 0.38***
-1.28 ± 0.28***
-0.14 ± 0.37***
0.56 ± 0.37***
-7.74 ± 0.11
-2.00 ± 0.25***
-2.57 ± 0.38***
-6.00 ± 0.33
-3.10 ± 0.22***
-6.57 ± 0.20
-9.51 ± 0.30
-5.81 ± 0.22*
-2.42 ± 0.14***
-2.70 ± 0.34***
-6.47 ± 0.48
1.55 ± 0.51***
-7.28 ± 0.30
-6.83 ± 0.39
0.00 ± 0.51***
-6.28 ± 0.25
-2.04 ± 0.60***
-2.71 ± 0.22***
-9.06 ± 0.49
-8.15 ± 0.34
-6.72 ± 0.47
-1.56 ± 0.57***
0.28 ± 0.65***
-0.56 ± 0.38***
1.96 ± 0.37***
-5.23 ± 0.46
2.53 ± 0.29***
-1.85 ± 0.39**
0.00 ± 0.57***
-2.45 ± 0.52*
-8.61 ± 0.20**
-6.12 ± 0.11
-2.71 ± 0.38
0.28 ± 0.65***
-0.71 ± 0.33***
1.97 ± 0.37***
1.69 ± 0.24***
Haemostatic acid
-0.43 ± 0.29***
-3.42 ± 0.38***
-2.94 ± 0.23***
-3.88 ± 0.22***
-10.19 ± 0.60***
-8.61 ± 0.20
2-aminothiazole
N1
N2
N3
N4
N5
-9.95 ± 0.47**
-1.85 ± 0.25***
0.14 ± 0.28***
-1.41 ± 0.32***
2.25 ± 0.14***
W1
W2
W3
W4
W5
Data represent mean ± SEM. n = 4.
* P < 0.05 vs. haemostatic acid.
** P < 0.01 vs. haemostatic acid.
*** P < 0.001 vs. haemostatic acid.
8

Table 5 RTT (%) values of the synthesized compounds
Concentration (μmol/L)
Compound
1
5
10
50
100
-7.13 ± 0.63
-3.53 ± 0.23
-5.76 ± 0.33
-13.94 ± 0.31***
-11.45 ± 0.38*
-5.05 ± 0.24
-2.40 ± 0.24*
-7.93 ± 0.44
-3.15 ± 0.48
-6.54 ± 0.64
-14.62 ± 0.24***
-29.01 ± 0.24***
-4.99 ± 0.63
-4.75 ± 0.48
-4.28 ± 0.36
-4.04 ± 0.24
Haemostatic acid
-5.06 ± 0.22
-6.47 ± 0.61
-5.41 ± 0.20
-3.53 ± 0.62
2-aminothiazole
-7.88 ± 0.20**
-30.77 ± 0.33***
-14.54 ± 0.22***
-7.21 ± 0.24*
-13.46 ± 0.43***
-10.79 ± 0.38***
-4.60 ± 0.48
-13.06 ± 0.31***
-28.13 ± 0.64***
-22.03 ± 0.38***
-15.39 ± 0.24***
-21.39 ± 0.42***
-11.23 ± 0.22***
-5..33 ± 0.64
-12.35 ± 0.42***
-27.89 ± 0.42***
-21.15 ± 0.58***
-19.95 ± 0.00***
-26.20 ± 0.24***
-13.66 ± 0.39***
-7.02 ± 0.84*
-11.06 ± 0.35***
-15.63 ± 0.00***
-6.39 ± 0.22
N1
N2
N3
-21.64 ± 0.24***
-21.88 ± 0.24***
-9.69 ± 0.48*
-7.93 ± 0.44***
-16.95 ± 0.24***
-14.62 ± 0.47***
-50.24 ± 0.62***
N4
N5
W1
W2
W3
W4
W5
-7.51 ± 0.24*
-16.04 ± 0.24***
-22.17 ± 0.41***
-8.23 ± 0.24***
-22.41 ± 0.62***
-10.14 ± 0.41***
-13.80 ± 0.24***
-23.82 ± 0.47***
-21.70 ± 0.45***
Data represent mean ± SEM. n = 4.
* P < 0.05 vs. haemostatic acid.
** P < 0.01 vs. haemostatic acid.
*** P < 0.001 vs. haemostatic acid.
Table 6 In vitro activities on platelet aggregation of the synthesized compounds
Compound
IC50a (μmol/L)
Compound
IC50a (μmol/L)
Etamsylate
46.22
2-Aminothiazole
201.96
36.57
45.11
73.71
54.54
18.09
25.46
N1
N2
N3
N4
N5
W1
W2
W3
W4
W5
75.63
58.12
35.47
1.87
a Concentration required reducing ADP-induced human platelet aggregation response by 50%. The IC50 values are
expressed as the average of at least three determinations.
Compound N5 showed the highest efficacy as a platelet aggregation promoter
and the highest IC50 value (1.87 μmol/L) in the micromolar range (1 μmol/L).
Compounds W5, N1, N4, W1 and W2 (IC50 = 18.09, 25.46, 35.47, 36.57 and 45.11
μmol/L) all demonstrated a better haemostatic activity than etamsylate (positive
control, IC50 = 46.22 μmol/L, Table 6). Simultaneously, compound N5 significantly
shortened the TT (p <0.05 or p < 0.001, Table 5), while shortening the APTT and PT
to a certain extent. Compound N1 exhibited the lowest PT value (RPT = -7.32 ± 0.45
at 100 μmol/L, Table 3), a significantly shortened TT (p <0.01 or p < 0.001, Table 5)
and exhibited an excellent platelet aggregation activity compared to the positive
control. Compound N1 also shortened APTT and PT to some extent. The haemostatic
mechanism is most likely due to compounds N1 and N5 promoting the fibrinogen
conversion to fibrin in the plasma while promoting platelet adhesion, stimulating the
formation and the release of agonist ADP-activated TXA2 to aggregate platelets.
9

However, the intrinsic and extrinsic pathways were found to be less affected. The
results of compound N5 further highlight the important effect of the chemical group
in 1-position of the N-(thiazol-2-yl)cinnamamide scaffold on the platelet activity.
Generally, the mechanisms of stimulation-induced platelet aggregation are different at
the beginning of the aggregation process: one correlates to the activation of a specific
receptor, and another one deals with the massive entry of external calcium into the
platelet (in the case of ionomycin).30 Both pathways have some common features: an
increase in the level of cytosolic calcium, the binding of fibrinogen to its receptor and
the phosphorylation of certain proteins.31 These points lead us to believe that the
mode of action of compound N5 focused on one of these key steps in the process of
platelet aggregation. The significantly reduced TT values of compound N5 indicate
that the compound does not feature the required structural elements to block the
fibrinogen receptor. However, in this study the effects of cytoplasmic calcium levels
and phosphorylation status on platelet protein have not been studied but the
mechanism of this action is subject to continuous research in our laboratory.
Although compound N4 exhibited a minimum APTT value (RAPTT = -10.19 ±
0.60, Table 4), it also significantly shortened the TT value (p <0.05 or p < 0.001, cf.
Table 5) and promoted platelet aggregation compared to the positive control
etamsylate. However, a rare effect on PT could be observed. This in turn suggests that
compound N4 achieved the desired haemostatic results primarily through
thrombin-mediated fibrin formation and partly by activating intrinsic pathways of
blood coagulation and promoting platelet aggregation. However, hardly any
haemostatic effects were found to be due to activating extrinsic pathways of blood
coagulation.
Compound W5 exhibited the lowest TT value (RTT = -50.24 ± 0.62, Table 5) and
was found to be effective in promoting platelet aggregation relative to the positive
control etamsylate while increasing PT and APTT to a certain extent. This in turn
indicates that compound W5 mainly achieved a haemostatic effect through
stimulating fibrinogen or increasing fibrin and activation of platelet aggregation.
However, coagulation was not found to be caused by intrinsic and extrinsic pathways.
In combination with the experimental studies of the 10 compounds, preliminary
SARs analysis showed that the condensation of acids and amine to form amides could
shorten PT, APTT and TT, and promote platelet aggregation. The addition of a
methoxy functional group could shorten the TT and promote platelet aggregation. The
formyloxy group in ortho- or para-position of the phenyl functionality could shorten
not only the TT but also the PT to a certain extend. Changing the double bonds to a
single bond on the branch could extend PT, APTT and TT and inhibit platelet
aggregation. However, the number of methoxy groups or the position of the formyl
group on the benzene ring has been found to have little effect on APTT.
In conclusion, the structure-activity relationships of a series of studied potent
haemostatic agents demonstrated that the introduction of carboxyl groups to amino
groups decreased PT, APTT, TT and promoted platelet aggregation; the position and
10

the quantity of methoxy and formyloxy groups also influenced the PT, APTT, TT and
platelet aggregation activities of the cinnamic acid amide derivatives containing
thiazoles. In addition, the introduction of methoxy and formyloxy groups resulted in
an improved platelet aggregation effect compared to the mere conversion of carboxyl
groups into amide groups. However, further work is necessary to better define the
relationship between the functional groups of the compounds and the blood
coagulation activity. This understanding may facilitate the design of chemical
compounds exhibiting a higher potency to serve as procoagulant and will further
provide information on the exploitation and utilization of amide compounds
containing thiazole as a procoagulant for bleeding treatments during surgery. Finally,
this work demonstrated that compound N5 features potential characteristics for the
development of drugs combining both coagulant and platelet effects.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (21362001), the Guangxi Key Laboratory of Traditional Chinese Medicine
Quality Standards (guizhongzhongkai201104) and the high level innovation team and
outstanding scholar project of Guangxi institutions of higher education (guijiaoren
[2014] No. 49). We acknowledge MogoEdit (a professional English editing company)
for English revising of the manuscript.
References and notes
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