Study on the anticoagulant or procoagulant activities of type II phenolic acid derivatives

Article abstract of DOI:10.3390/molecules22122047

In this study, three type II phenolic acids (caffeic acid, p-hydroxycinnamic acid, and ferulic acid) were used to synthesize a total of 18 phenolic acid derivatives. With molecular docking for molecule design and target protein (factors) screening, in combination with the confirmation of target proteins (factors) by surface plasmon resonance, and the evaluation of haemostatic and anticoagulant activities with five blood assays (plasma recalcification time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thrombin time), the data indicated that caffeic acid derivatives showed certain anticoagulant or procoagulant activities and that two other series contained compounds with the best anticoagulant activities. Using Materials Studio analysis, particular functional groups that affect anticoagulant or procoagulant activities were revealed, and these conclusions can guide the discovery of compounds with better activities.

Full text of DOI:10.3390/molecules22122047

molecules
Article
Study on the Anticoagulant or Procoagulant Activities
of Type II Phenolic Acid Derivatives
Xuan Luo 1,2, Chuanrong Du , Hui Cheng , Jian-hua Chen and Cuiwu Lin 1,2,
1
1
3
*
1
School of Chemistry and Chemical Engineering, Guangxi University, 100 Daxue Road, Nanning 530004,
Guangxi, China; luo-xuan625@hotmail.com (X.L.); duchuanrong123@163.com (C.D.);
chui240514119@sina.com (H.C.)
Guangxi Colleges and Universities Key Laboratory of Applied Chemistry Technology and Resource
Development, Guangxi University, 100 Daxue Road, Nanning 530004, Guangxi, China
School of Resources, Environment, and Materials, Guangxi University, 100 Daxue Road, Nanning 530004,
Guangxi, China; jhchen@gxu.edu.cn
2
3
*
Correspondence: cuiwulin@163.com; Tel: +86-771-323-3718
Received: 20 September 2017; Accepted: 21 November 2017; Published: 28 November 2017
Abstract: In this study, three type II phenolic acids (caffeic acid, p-hydroxycinnamic acid, and ferulic
acid) were used to synthesize a total of 18 phenolic acid derivatives. With molecular docking for
molecule design and target protein (factors) screening, in combination with the confirmation of
target proteins (factors) by surface plasmon resonance, and the evaluation of haemostatic and
anticoagulant activities with five blood assays (plasma recalcification time, prothrombin time,
activated partial thromboplastin time, fibrinogen, and thrombin time), the data indicated that
caffeic acid derivatives showed certain anticoagulant or procoagulant activities and that two other
series contained compounds with the best anticoagulant activities. Using Materials Studio analysis,
particular functional groups that affect anticoagulant or procoagulant activities were revealed,
and these conclusions can guide the discovery of compounds with better activities.
Keywords: anticoagulant; procoagulant; haemostatic; phenolic acid; surface plasmon resonance
1
. Introduction
Phenolic acid compounds, which play essential roles in growth, development, or reproduction,
are a group of non-flavonoid polyphenols that exist in most plant tissues as secondary metabolites [
Additionally, these phenolic compounds are important components of the human diet due to their
potential antioxidant activities [ ]. With immense research on pharmacological activity, phenolic
acid compounds have shown many bioactivities, not only including familiar antioxidant [ ],
antibacterial [ ], and anti-inflammatory [ ] activities, but also haemostatic [10 11], anticancer [12 13],
and antiviral [14 15] activities. Due to their excellent physical and chemical properties, phenolic acid
1].
1,2
3–5
6
,
7
8,
9
,
,
,
compounds have aroused much attention and become one of the hotspots in drug development.
Some studies have indicated that phenolic acid compounds not only have haemostatic activity [16],
but also show better anticoagulant activity [17
anticoagulant activity components in Blumea riparia DC. and has obtained some polysaccharide,
flavonoid, and phenolic acid compounds [10 11 23 28]. Additionally, the obtained flavonoids and
phenolic acids were used as parent compounds to synthesize a series of derivatives, and their
haemostatic and anticoagulant activities were evaluated by several bioassays [29 38]. According to
–22]. Since 2002, our group has focused on studying
,
, –
–
our previous research results, phenolic acid compounds separated from Blumea riparia DC. have good
haemostatic and anticoagulant activities and could be precursors to developing new haemostatic and
anticoagulant drugs.
Molecules 2017, 22, 2047; doi:10.3390/molecules22122047
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2047
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According to their structural characteristics, all phenolic acid compounds have a carboxyl group
attached or linked to a benzene ring [39], and these compounds can be classed into two types:
type I, benzoic acid derivatives, and type II, cinnamic acid derivatives (hydroxycinnamic acids) [40].
For Type II, caffeic, p-hydroxycinnamic, ferulic, and sinapic acids are common. Notably, the structures
of most haemostatic drugs used in the clinic include phenolic hydroxyl, amide, amino, and carboxyl
groups. In the present study, based on this information, three Type II phenolic acids (caffeic acid,
p-hydroxycinnamic acid, and ferulic acid) were used as precursors to synthesize a series of derivatives.
Then, the haemostatic and anticoagulant activities of these derivatives were evaluated to guide the
design of new haemostatic and anticoagulant drugs.
2
. Materials and Methods
2
.1. Apparatus and Software
Melting points were measured on an X-4 microscopic melting point apparatus (Beijing Tech
Instruments, Beijing, China). Ultraviolet (UV) spectra were recorded on a TU-1900 UV/VIS
spectrophotometer (Shanghai Jinghua Technology Instruments, Shanghai, China). Fluorescence data
were recorded on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara,
CA, USA). Infrared (IR) spectra were determined using a Nicolet NEXUS470 spectrophotometer
with KBr pellets (Thermo Fisher Scientific, Waltham, MA, USA). Fast atom bombardment mass
spectrometry (FAB-MS) data were determined by a VGZAB-HS mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA). Surface plasmon resonance (SPR) measurements were performed
on a ProteOn XPR36 Protein Interaction Array system (Bio-Rad Laboratories, Hercules, CA, USA)
using a GLH chip. Nuclear magnetic resonance (NMR) spectra were recorded on an Advance III HD
600 MHz spectrometer (Bruker, Zurich, Swiss) with tetramethylsilane (TMS) as an internal standard
for deuterated organic solvents or 2,2,3,3-d -3-(trimethylsilyl) propionic acid sodium salt (TSP) as
4
an internal standard for deuterium oxide. Prothrombin time (PT), activated partial thromboplastin time
(
APTT), fibrinogen (FIB), and thrombin time (TT) were recorded on an LG-PABER-I semi-automated
coagulation analyser (Steellex Scientific Instrument, Beijing, China). A Milli-Q Advantage A10
(Merck Millipore, Billerica, MA, USA) supplied the pure water used in these experiments.
The molecular structures were drawn by Materials Studio (Accelrys Software, BIOVIA, San Diego,
CA, USA) to study the distances of some atoms in the structures.
2
.2. Reagents
Three material phenolic acids (caffeic acid, p-hydroxycinnamic acid, and ferulic acid) and
p-aminomethylbenzoic acid (p-AMBA), the positive control for PT, APTT, FIB, and TT, were purchased
from Zhuhai Jiaxinkang Pharmaceutical Technology Co. (Zhuhai, China). Fibrinogen was purchased
from Beijing Qinyuanhuzhi Biotechnology Co. (Beijing, China). Thrombin and deuterated solvents
for NMR analysis were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). APTT, PT,
TT, and FIB kits were supplied by HEALL Bio-Science Technology (Qingdao) Co. (Qingdao,
Shandong, China). Other chemicals and reagents were supplied by Shanghai Aladdin Biochemical
Technology Co. (Shanghai, China), Guangdong Guanghua Sci-Tech Co. (Shantou, Guangdong, China),
and Xilong Scientific Co. (Shantou, Guangdong, China).
Healthy human plasma (the platelet-poor plasma, PPP) was supplied by The First Affiliated
Hospital of Guangxi Medical University (Nanning, Guangxi, China), and all five blood assays were
conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics
Committee of First Affiliated Hospital of Guangxi Medical University (Project identification code:
2
017(KY-E-050)).

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2
.3. Preparation of Phenolic Acid Derivatives
2
.3.1. Caffeic Acid Derivatives
Preparation of diacetyl caffeic acid: Caffeic acid (50 g), 600 mL acetic anhydride and pyridine
were added to a 1000 mL round-bottom flask and reacted at room temperature for 12 h with stirring.
Subsequently, the mixture was transferred into a 5 L beaker and eight-fold iced water was added under
violent stirring. The mixture was left standing for 24 h and yielded a white precipitate. After vacuum
◦
filtration, washing, and 60 C oven drying, diacetyl caffeic acid was obtained and ready for the next
reaction without further purification.
Preparation of Cds
1
–
7
: Diacetyl caffeic acid (20 mmol) and 15 mL SOCl were added into a 50 mL
2
◦
round-bottom flask and reacted in a 60 C oil bath for 5 h with stirring. Subsequently, the solvents were
removed by a rotary evaporator, and diacetyl caffeic acyl chloride was obtained. Glycine,
β-alanine,
γ
-propalanine, 6-amidocaproic acid, taurine, tranexamic acid, or p-aminomethylbenzoic acid (40 mmol)
◦
was dissolved in 20 mL of a 2 M NaOH solution in a 50 mL round-bottom flask. At 10–15 C in a water
bath with stirring, diacetyl caffeic acyl chloride THF solution (adding 5 mL THF to dissolve obtained
diacetyl caffeic acyl chloride) was slowly added to the flask. After a 2.5 h reaction, the pH of the mixture
was adjusted to
4
–
5
by adding diluted HCl. The mixture was left standing at room temperature for
◦
2
4 h, followed by vacuum filtration, washing, and 60 C oven drying to obtain Cds.
1–
7
(The Synthetic
route of Cds. 1–7 was shown in Scheme 1).
O
O
O
OH
OH
Ac O
SOCl2
Cl
2
HO
AcO
O
AcO
OH
OAc
R
OAc
R
O
different amino acids
NaOH
NH
dilute HCl
NH
AcO
HO
OAc
OH
1 7
–
O
O
O
O
4
: R=
1
5
: R=
2
: R=
3
: R=
OH
OH
OH
OH
O
O
O
OH
OH
OH
S
7
: R=
: R=
6
: R=
O
Scheme 1. Synthetic route of Cds. 1–7.
were measured, and their structures were confirmed by using a battery
The melting points of Cds.
1–7
of spectroscopic methods, including IR, ESI-MS, and NMR. The yields, appearances, melting points,
and ESI-MS data of Cds. 1–7 were listed in Table 1 and the IR data were listed in Table 2.
Table 1. The yields, colours, and ESI-MS data of Cds. 1–7.
Cd. 1
Cd. 2
Cd. 3
Cd. 4
Cd. 5
Cd. 6
Cd. 7
Yield (%)
Color
53.2
White
powder
>244 a
238.05 b
48.3
White
powder
180–181
250.08
45.1
Yellow needle
crystal
65.3
White needle
crystal
41.3
66.3
While needle
crystal
69.6
Light yellow
powder
178–180
Yellow
powder
>200 a
◦
m.p. ( C)
229–230
164–165
162–163
c
c
c
c
c
c
ESI-MS (m/z)
264.07
292.3
286.2
318.3
312.3
a
Decomposing temperature; b [M + 1]+; c [M − 1]−.

Molecules 2017, 22, 2047
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Table 2. The IR data of Cds. 1–7 (KBr pellets, νmax, cm 1).
−
Cd. 1
Cd. 2
Cd. 3
Cd. 4
Cd. 5
Cd. 6
Cd. 7
3
3
537.51,
248.05
3629.55, 3650.56, 3496.45,
ν
3181.75
3495.45
3118.15
−
ν
ν
ν
OH(COOH)
3350.69
3409.00
1655.01
1624.10
3136.97
3363.27
1647.46
1618.62
3101.13
3307.25
1649.79
1598.80
3379.64
1646.80
1622.51
3411.44
1704.00
1654.61
3346.95
1715.59
1642.14
3274.45
1689.94
1650.91
−
NH-
−
CO−
>C=C<
1
1
577.87,
458.28
1263.63
1598.39, 1618.62, 1567.55, 1592.87, 1598.02, 1608.57,
ν
Ar
C−O(ArOH)
1456.53
1277.08
1454.79
1304.95
1520.48
1272.29
1454.86
1309.42
1460.39
1299.67
1531.97
1268.80
ν
2
.3.2. p-Hydroxycinnamic acid Derivatives
Preparation of Cd. : p-hydroxycinnamic acid (20 mmol) and 30 mL of concentrated H SO were
8
2
4
◦
added to a 100 mL round-bottom flask and reacted for 8 h in a 60 C oil bath with stirring. Subsequently,
the mixture was transferred into a 250 mL beaker, and 10 mL of distilled water was added (too viscous
to handle in the next step). With stirring, solid NaOH was added to the beaker until no more precipitate
was yielded. After vacuum filtration, washing with distilled water, and recrystallization in distilled
water, Cd.
Scheme 2).
8 was obtained, and the yield was 45.1% (The Synthetic route of Cds. 9–14 was shown in
O
O
9
8% H SO
2 4
OH
OH
HO S
3
OH
OH
OH
R
8
O
O
OH CH O, Different amines
OH
2
OH
9
–
14
9
: R=-N(CH ) 10: R'= -N(CH CH ) 11: R= -N(CH2CH2CH3)2
3 2 2 3 2
1
2: R= -N(CH CH CH CH ) 13: R= -N(CH CH OH)
2
14: R=
N
O
2
2
2
3 2
2
2
Scheme 2. Synthetic Route of Cds. 8–14.
Preparation of Cds. 9–14: p-Hydroxycinnamic acid (37 mmol), 50 mL methanol and formaldehyde–
water solution (36%, containing 49 mmol formaldehyde) was added into a 250 mL round-bottom
◦
flask and reacted for 8 h in a 60 C oil bath with reflux and stirring. After the mixture appeared
clean, 104 mmol dimethylamine, diethylamine, di-n-propylamine, di-n-butylamine, diethanolamine,
or morpholine was added to the flask, and the reaction was continued for 5 h. Large quantities of solid
◦
were precipitated in the flask. After vacuum filtration, washing with methanol, and 60 C oven drying,
Cds. 9–14 were obtained.
The melting points of Cds.
9–14 were measured, and their structures were confirmed by
using a battery of spectroscopic methods, including IR, ESI-MS, and NMR. The yields, appearances,
melting points, and ESI-MS data of Cds.
Table 4.
9–14 were listed in Table 3 and the IR data were listed in

Molecules 2017, 22, 2047
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Table 3. The yields, colours, and ESI-MS data of Cds. 8–14.
Cd. 8
Cd. 9
Cd. 10
Cd. 11
Cd.12
Cd. 13
Cd. 14
Yield (%)
Color
45.1
White
61.2
White
70.2
White
86.3
85.3
Orange lamellar
crystal
85.5
White
powder
288–289
282.2
93.5
Light yellow
lamellar crystal
237–238
Colorless
needle crystal
231–232
powder powder powder
◦
>300 a
>246 a
220.1
m.p. ( C)
172–173
250.18
209–210
c
c
b
b
b
b
c
ESI-MS (m/z)
243.00
278.3
306.3
262.2
a
Decomposing temperature; b [M + 1]+; c [M − 1]−.
Table 4. The IR data of Cds. 8–14 (KBr pellets, νmax, cm 1).
−
Cd. 8
Cd. 9
Cd. 10
Cd. 11
Cd.12
Cd. 13
Cd. 14
3
3
595.95,
230.77
3573.49, 3573.23, 3573.06, 3573.53, 3521.79, 3574.05,
ν
−
ν
OH(COOH)
3161.69
1654.02
1608.00
3463.56
1645.38
1645.38
3203.06
1700.59
1635.38
3158.49
1702.40
1637.53
3111.08
1647.02
1620.00
3202.35
1722.54
1644.43
1701.16
1634.48
−
CO−
ν
>C=C<
1
1
591.88,
517.66
1271.59
1559.81, 1603.91, 1581.23, 1581.44, 1580.19, 1578.57,
ν
Ar
C−O(ArOH)
1473.85
1268.99
1518.98
1294.21
1521.24
1262.57
1520.35
1299.67
1525.42
1299.93
1514.76
1291.03
ν
2
.3.3. Ferulic Acid Derivatives
The preparation of Cds. 15–18 was similar to the preparation of Cd. 1. The only differences were
that the amount of acetic anhydride was reduced to half in the preparation of acetyl ferulic acid, and the
amino acids were changed to 6-amidocaproic acid, taurine, tranexamic acid, or p-aminomethylbenzoic
acid (The synthetic route of Cds. 15–18 was shown in Scheme 3).
O
O
O
OH
OH
Ac O
SOCl2
Cl
2
HO
AcO
O
AcO
O
O
R
O
O
R
different amino acids
NaOH
NH
dilute HCl
NH
AcO
HO
O
O
O
–
1
5 18
O
O
OH
1
5
: R=
S
1
6
: R=
: R=
OH
O
O
17
: R=
OH
OH
1
8
Scheme 3. Synthetic route of Cds. 15–18.
The melting points of Cds. 15–18 were measured, and their structures were confirmed by
using a battery of spectroscopic methods, including IR, ESI-MS, and NMR. The yields, appearances,
melting points, and ESI-MS data of Cds. 15–18 were listed in Table 5 and the IR data were listed in
Table 6.

Molecules 2017, 22, 2047
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Table 5. The yields, colours, and ESI-MS data of Cds. 15–18.
Cd. 15
Cd. 16
Cd. 17
Cd. 18
Yield (%)
Color
m.p. ( C)
84.1
White powder
61–62
85.7
85.5
White powder
188–189
87.3
White powder
228–229
Yellow needle crystal
◦
285–286
306.3 b
300.62 b
a
326.2 b
ESI-MS (m/z)
334.4
a
M + 1]+; b [M − 1]−.
[
Table 6. The IR data of Cds. 15–18 (KBr pellets, νmax, cm 1).
−
Cd. 15
Cd. 16
Cd. 17
Cd. 18
ν
3522.27
3311.60
1693.84
3626.78, 3076.53
3313.74
3370.63
3444.83
1704.85
3464.64
3282.88
1688.55
−
OH(COOH)
ν
ν
−
NH−
1650.47
1617.38
−
CO−
ν
1650.48
1655.58
1649.58
>C=C<
ν
1583.13, 1469.73
N/A
1597.79, 1516.69
1345.22, 1155.91
1287.63
1607.74, 1514.88
N/A
1608.13, 1514.88
N/A
Ar
ν
−
SO−
ν
1299.40
1288.64
1278.45
C−O(ArOH)
2
.4. Structure Characterization
1H- and 13C-NMR data assignments of some compounds were achieved by DEPT and 2D NMR
COSY, HSQC, and HMBC). All NMR data are provided in the supplementary information.
(
2
.5. Molecular Docking
2
.5.1. Virtual Screening
The targets of the phenolic acid derivatives (PADs) were searched by PharmMapper Server,
a web-based tool designed to identify potential target candidates for the given small molecules
via ‘reverse’ pharmacophore mapping [41]. The optimized structures of PADs were prepared by
ACD/ChemSketch (Version 12.6, Advanced Chemistry Development, Inc., Toronto, ON, Canada)
and Open Babel [42] (Version 2.3.2, http://openbabel.org; accessed Oct 2014) in the mol2 format and
submitted to PharmMapper (http://59.78.96.61/pharmmapper/; access date: 12 September 2012)
for the prediction of proteins with three-dimensional structures and PAD binding sites in the Protein
Databank. In the present study, the number of reserved matched targets is defined as 300.
2
.5.2. AutoDock Docking
AutoDock is one of the most frequently used molecular docking suites and has been extensively
validated over the years [43,44]. Calculations were performed using AutoDock 4.2.1.5, which is
an automated docking suite capable of performing rigid or flexible docking [45]. The following
program settings were used in the present study: 100 runs with 2,500,000 energy evaluations and
a maximum number of 27,000 generations.
2
.6. Surface Plasmon Resonance
Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic
waves, surface plasmon polaritons, to probe interactions between an analyte in solution and
a biomolecular recognition element immobilized on the SPR sensor surface [46]. In a typical experiment,
fibrinogen and thrombin were immobilized (approximately 12,000 RU) in flow cells, with one flow cell
used as a blank. For the screening experiment, the ligands were diluted to 10
µM with running buffer
(
12 mM HEPES, 4 mM Tris, 1 mM EDTA, 1.5 mM MgCl and 0.005% Tween-20). The PAD solutions
2
were diluted to different concentrations with running buffer. The ligand was injected at a flow rate
◦
of 25
µ
L/min for 180 s during the association phase, followed by a 300 s dissociation phase at 25 C.

Molecules 2017, 22, 2047
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The GLH chip was regenerated with a short injection of 1 M NaCl between consecutive measurements.
The final graphs were obtained by subtracting blank sensorgrams from the sensorgrams for fibrinogen
or thrombin.
Kinetic and equilibrium analyses were performed using the ProteOn manager software.
The theoretical max RU of the compounds was evaluated via the following equation:
ꢀ
ꢁ
Rmax = R
r
MWprotein/MWligand
(1)
protein protein/ligand
where Rprotein is 12,000 RU, the amount of protein immobilized on the chip; rprotein/ligand is 1:1,
the ratio of the protein–ligand interaction; MWprotein is the molecular weight of fibrinogen or thrombin;
and MWligand is the molecular weight of the compounds. The kinetic SPR sensorgrams were fitted with
a 1:1 Langmuir binding mode, and the K values were achieved by the Analysis module in ProteOn
D
XPR36 software. The equation of the association process is:
d[AL]/dt = ka[A][L] − k [AL]
(2)
d
where, in the dissociation process, [A] = 0; therefore, d[AL]/dt =
−
kd[AL]. [AL] is the concentration
of the complex, t is time, and [A] and [L] are the concentrations of the analyte and the substance,
respectively. k and k are the association and dissociation rate coefficients, respectively. KD was
calculated from ka and k using the following equation:
a
d
d
K = k /ka
(3)
D
d
2
.7. Measurement of Plasma Recalcification Time
◦
For this analysis, 0.1 mL PPP (defrost and incubate at 37 C) and 0.1 mL of the different sample
◦
solutions were mixed well in test tubes (8 mm diameter). After incubation for 5 min in a 37 C water
bath, 0.1 mL CaCl solution (0.025 mM) was respectively added to each tube, and the process was
2
monitored. The period until silky fibrin appeared was recorded as the PRT. The negative control was
saline, and the positive control was p-aminomethylbenzoic acid.
2
.8. Measurement of APTT, PT, TT, and FIB
In a 2 mL centrifuge tube, 0.9 mL PPP and 0.1 different sample solution mixed well and incubated
◦
at 37 C for 5 min. APTT, PT, TT, and FIB were detected using blood coagulation factor assay kits
according to the manufacturer’s instructions. The negative control was saline, and the positive control
was p-aminomethylbenzoic acid.
3
. Results and Discussion
In vivo, haemostasis and blood circulation for removing blood stasis are two contradictory
activities. However, both activities objectively exist at the same time. In gynaecological diseases or
after surgery, wounds require stopping bleeding, but the medicines used cannot increase patient blood
viscosity, which will form thrombosis; at the same time, the medicines are needed to slowly dissipate
the congestion in blood circulation and promote the restoration of the patient’s body.
There is also a paradox in discovering and designing medicines that affect the mechanisms of
anticoagulation and procoagulation. As shown in Figure 1, the coagulation pathway is very complex,
but the situation is different near thrombin. Indeed, in that area, there is single factor control. Therefore,
in the design of medicines or the software simulation of medicine activity, thrombin or other proteins
and factors in that area are, in many cases, used as the targets. However, no matter how to design or
evaluate medicines, all obtained compounds and precursors will be used in actual blood systems to
evaluate their activities, and the activities of these compounds are a combination of their performance

Molecules 2017, 22, 2047
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on every protein and factor in the coagulation pathway. The compounds, showing good anticoagulation
activity with thrombin, may not have the same anticoagulation activity or procoagulant effect in blood.
In the present study, the anticoagulant and procoagulant activities of three phenolic acid derivative
series, 18 PADs, were investigated. Comparing the results of molecular docking and SPR with those of
five blood assays, we found that using molecular docking and SPR to assist the molecule design had
some drawbacks (one of the reasons was the paradox mentioned above). However, the experimental
data also showed that using molecular docking and SPR could generate some important information
for molecule design.
3
.1. Virtual Screening
Virtual screening of chemical libraries, as an alternative approach for the identification of new lead
compounds in drug discovery, has been widely used [47]. As the structures of biomolecules deposited
in the Protein Data Bank (PDB) have substantially increased in the past decades, searching for the
targets of a given drug or small compound (also known as inverse screening, target fishing, off-target
prediction, etc.) has become a useful approach [48]. In PharmMapper tool, 18 newly-synthesized
PADs were aligned to 2241 human protein targets, among which the top 300 targets were analysed.
From the results, three types, a total of seven protein targets, were screened out. The first type
was targets associated with coagulation, including thrombin, prothrombin, coagulation factor X,
and fibrinogen. The second type was targets associated with anticoagulant, including antithrombin-III
and plasminogen. The third type was the two-way regulatory target, vitamin K-dependent protein Z.
3
.2. AutoDock Docking
The results of the docking study for all 18 synthesized PADs with seven protein targets,
including thrombin (3RLW), prothrombin (1YPK), coagulation factor X (2J4I), fibrinogen (3GHG),
antithrombin-III (2ANT), plasminogen (1QRZ), and vitamin K-dependent protein Z (3H5C), are listed
in Table 7.
mol 1) of the 18 phenolic acid derivatives with seven protein targets.
−
Table 7. The binding energies (kJ
·
No.
3RLW
1YPK
2J4I
3GHG
2ANT
1QRZ
3H5C
Cd. 1
Cd. 2
Cd. 3
Cd. 4
Cd. 5
Cd. 6
Cd. 7
Cd. 8
Cd. 9
Cd. 10
Cd. 11
Cd. 12
Cd. 13
Cd. 14
Cd. 15
Cd. 16
Cd. 17
Cd. 18
Ave.
−22.50
−20.31
−20.60
−18.26
−18.50
−16.43
−22.75
−12.92
−18.18
−16.03
−22.11
−24.47
−19.12
−19.62
−21.47
−14.65
−20.52
−21.03
−19.42
−16.81
−17.26
−19.07
−20.75
−16.36
−22.06
−19.92
−13.87
−18.22
−21.16
−22.18
−24.77
−20.45
−20.11
−20.68
−18.73
−20.87
−21.79
−19.73
−16.02
−16.83
−17.41
−19.07
−16.48
−18.85
−18.40
−14.13
−17.20
−18.83
−19.33
−21.79
−19.28
−18.40
−19.66
−16.56
−19.87
−21.73
−18.32
−15.06
−16.23
−18.31
−19.25
−17.47
−20.40
−15.99
−15.60
−25.48
−20.94
−20.94
−23.60
−23.60
−20.58
−18.84
−17.05
−18.58
−16.37
−19.13
−17.25
−18.03
−19.92
−23.17
−17.74
−22.44
−21.11
−15.41
−20.47
−22.51
−23.08
−25.98
−21.07
−21.17
−22.91
−19.23
−23.73
−21.31
−20.92
−15.50
−16.18
−18.81
−18.31
−15.39
−19.83
−17.17
−13.58
−17.43
−18.40
−20.19
−20.29
−18.31
−18.05
−20.39
−16.57
−20.41
−17.05
−17.88
−13.13
−14.56
−15.54
−17.48
−13.44
−18.89
−17.17
−11.24
−13.69
−16.92
−19.79
−20.61
−16.44
−16.58
−19.87
−15.34
−19.68
−18.88
−16.63
For the caffeic acid derivative series (Cds.
were lower than those of the other caffeic acid derivatives. Cds.
derivatives, and the binding energy data for some compounds revealed three important findings:
The docking energies of Cd. with the seven protein targets were very low (six of the seven energy
1–
7), the binding energies of Cd.
5
with protein targets
8–14 were p-hydroxycinnamic acid
8

Molecules 2017, 22, 2047
9 of 16
values were the lowest compared with those of the other 17 compounds, and the remaining value
was the second to the lowest value). In contrast, Cd. 12 had the best docking results among the
1
8 compounds (although the binding energy with 1QRZ was not the highest value, this value was still
the second highest value). Except for Cd. , all other p-hydroxycinnamic acid derivatives had better
docking results with 3GHG and 2ANT, and binding with these two protein targets would release
8
−
1
greater than
Cd. 16, showed lower binding energies with the protein targets.
Three different series of PADs, Cds. , and 16, had worse docking results and a similar structure
−
20 kJ
·
mol of energy. For the four ferulic acid derivatives (Cds. 15–18), one derivative,
5, 8
fraction, the sulfonic acid group. These results suggest that in the docking calculation, the sulfonic
acid group could affect the binding between PADs and protein targets, particularly in connection with
the benzene ring in the PAD structure. Since the sulfonic acid group could withdraw electrons out of
the benzene ring, the low electron density generated worse docking results for Cd. 8.
A comparison of the average of each protein target with the 18 PADs revealed four coagulation
targets with better docking results, particularly 3RLW with caffeic acid derivatives and 3GHG with
p-hydroxycinnamic acid derivatives.
3
.3. Surface Plasmon Resonance Results
Based on the AutoDock docking results, thrombin and fibrinogen were the protein targets used in
SPR analysis.
Autodock can predict the combination of small molecules and proteins by theoretical calculation.
Moreover, to obtain the results, the calculation is based on the electronic distribution of the
molecule itself (such that the electron-rich regions in the electrostatic potential map of the compound
will combine with the electron deficient regions of the protein) and intermolecular interactions,
including van der Waals, electrostatic, and bond interactions. During the calculation, the effects
of the external environment are considered. Thus, the AutoDock results were not consistent with the
results of surface plasmon resonance (SPR), listed in Table 8.
In Table 8, 12 of the 18 PADs could combine with thrombin and show better results than those
obtained with fibrinogen. However, in AutoDock (Table 7), the averages of PADs with the two proteins
were very close. AutoDock predicted that Cd. 12 had the lowest binding energy with thrombin,
but in fact Cd. 13 had the smallest K , indicating that this compound had the best combination
D
with thrombin.
Table 8. The SPR results of phenolic acid derivatives with thrombin and fibrinogen.
No.
Thrombin
Fibrinogen
−
6
Cd. 1
Cd. 2
Cd. 3
Cd. 4
Cd. 5
Cd. 6
Cd. 7
Cd. 8
Cd. 9
Cd. 10
Cd. 11
Cd. 12
Cd. 13
Cd. 14
Cd. 15
Cd. 16
Cd. 17
Cd. 18
4.41 × 10
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
−
−
−
6
6
6
11.3 × 10
N/A
6.29 × 10
N/A
12.1 × 10
−
6
−6
−6
4.54× 10
10.1 × 10
−
6
44.7 × 10
14.5 × 10
N/A
N/A
N/A
N/A
N/A
N/A
N/A
−
−
6
6
1.70 × 10
12.1 × 10
N/A
−
6
N/A
16.2 × 10
−
−
6
6
43.5 × 10
N/A
7.77 × 10
N/A

Molecules 2017, 22, 2047
10 of 16
For p-hydroxycinnamic acid derivatives, AutoDock predicted that Cds.
9–
12 would have a better
combination with thrombin when the side chain was longer. Indeed, as shown in Table 8, Cd.
9 had
a smaller K due to its shorter side chain. The reason for this finding was that when the side chain
D
got increasingly longer, the hydrophobicity of the molecule increased, and because the conditions
of the assays and the environment in actual human bodies involved water as the major solvent,
increasing hydrophobicity would decrease the interactions between small molecules and proteins.
This phenomenon was very obvious in the SPR results for Cd. 13. Since the structure of Cd. 13
had two additional hydroxyl groups, which could decrease the hydrophobicity and increase its
hydrophilicity, Cd. 13 would have better combination results with proteins under the assay conditions
(water was the major solvent).
In AutoDock, p-hydroxycinnamic acid derivatives had the lowest average binding energy with
fibrinogen compared with the other two derivatives. In SPR, although only two p-hydroxycinnamic
acid derivatives had K values, p-hydroxycinnamic acid derivatives remained the best among the
D
three PADs series.
Although SPR provided a realistic combination of small molecules and proteins, the results did
not show the effects on protein activities. To examine the changes (enhance or inhibit activity) of
protein activities after combination, plasma recalcification time (PRT), PT, APTT, FIB, and thrombin
time (TT) were examined.
3
.4. PRT, PT, APTT, FIB, and TT Results
Five blood assays were used to determine the procoagulant and anticoagulation activities of
1
8 PADs, and the results are listed in Table 9.
Cds. and 13 had the best combination results with thrombin in SPR and showed significant
8
anticoagulation activities in all five assays, which might indicate that these compounds could inhibit
thrombin activity in combination and make the conversion from soluble fibrinogen to insoluble fibrin
without thrombin promotion. Cd. 16 was the second compound in the 18 PADs, which had significant
anticoagulation activity in all five blood assays. However, this compound did not combine with
thrombin and showed weak combination with fibrinogen (large K value). These results showed that
D
the anticoagulation activity of Cd. 16 might be caused by the combination with other proteins or factors
in the coagulation (clotting) cascade (See Figure 1). Cd.
9 showed combined results with thrombin and
fibrinogen in SPR, but the results with fibrinogen had a large K value, indicating that the combination
D
was extremely weak. No obvious effect in the FIB assay confirmed that Cd.
fibrinogen, and its anticoagulation activity might be caused by the combination with thrombin.
Although Cds. 13, and 16 all had the better anticoagulation activities among the 18 PADs,
these compounds belonged to two different phenolic acid derivatives: Cds. , and 13 were members
9 had little effect on
8, 9,
8, 9
of the p-hydroxycinnamic acid series and Cd. 16 was a member of the ferulic acid series, indicating
that the anticoagulation activities of these compounds affected different proteins or factors in the
coagulation cascade. Even in the same phenolic acid derivative, Cds.
anticoagulation activity, had different SPR results.
8 and 13, showing the best
Both Cds. 16 and
5 had good anticoagulation activity, and the former was the best among the
18 PADs (See Table 9). This activity difference was triggered by the only difference in their structures,
the 3-position of the benzene ring. The hydroxyl group in the 3-position of the benzene ring was
changed to a methoxy group. This change made Cd. 16 bigger and much easier to fit the receptor
(proteins or factors in the coagulation cascade).

Molecules 2017, 22, 2047
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Table 9. Plasma recalcification, prothrombin, activated partial thromboplastin, fibrinogen, and thrombin time results of the phenolic acid derivatives.
No.
PRT
APTT
PT
FIB
TT
Cd. 1
Cd. 2
Cd. 3
Anti. at High Concen.
Anti. at High Concen.
NOE
Anti. at High Concen.
Anti. at High Concen.
NOE
NOE
NOE
NOE
NOE
NOE
NOE
NOE
Anti. at Some Concen.
Sig. Procoa.
Anti. at Some Concen.
Procoa. at Some Concen.
Cd. 4
Sig. Procoa.
Sig. Procoa.
Procoa. at Some Concen.
Cd. 5
Cd. 6
Sig. Anti.
Sig. Procoa.
Sig. Anti.
Sig. Procoa.
Sig. Anti.
Sig. Procoa.
Anti. at Some Concen.
Anti. at Some Concen.
NOE
NOE
Cd. 7
Cd. 8
Sig. Procoa.
Sig. Anti.
Sig. Procoa.
Sig. Anti.
Sig. Procoa.
Sig. Anti.
NOE
Sig. Anti.
NOE
Sig. Anti.
Cd. 9
Sig. Anti.
Procoa. at High Concen.
Sig. Procoa.
Procoa. at High Concen.
Sig. Anti.
Sig. Anti.
Procoa. at High Concen.
Sig. Anti.
Procoa. at High Concen.
Procoa. at High Concen.
Sig. Anti.
Anti. at High Concen.
NOE
NOE
NOE
Sig. Anti.
Procoa. at Some Concen.
Cd. 10
Cd. 11
Cd. 12
Cd. 13
Cd. 14
Cd. 15
Cd. 16
Cd. 17
Cd. 18
Procoa. at High Concen.
Procoa. at High Concen.
Sig. Procoa.
NOE
NOE
Procoa. at High Concen.
Sig. Anti.
NOE
NOE
Sig. Anti.
Procoa. at High Concen.
Sig. Anti.
Anti. at High Concen.
Procoa. at Some Concen.
Sig. Anti.
Sig. Anti.
Sig. Anti.
NOE
Sig. Anti.
NOE
NOE
Procoa. at Some Concen.
Sig. Anti.
Procoa. at High Concen.
Sig. Anti.
Procoa. at Some Concen.
Procoa. at High Concen.
Procoa. at Some Concen.
Procoa. at Some Concen.
NOE
NOE
NOE
NOE
NOE: No obvious effect; Sig. Anti: Significant anticoagulation; Sig. Procoa: Significant procoagulant action; Anti. at High Concen.: Anticoagulation at a high concentration; Procoa.
at High Concen.: Procoagulant action at a high concentration; Anti. at Some Concen.: Anticoagulation at some concentrations; Procoa. at Some Concen.: Procoagulant action at
some concentrations.

Molecules 2017, 22, 2047
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Tissue factor
extrinsic) pathway
Contact activation
(intrinsic) pathway
Hageman factor
(
(
Factor XII)
Proconvertin
High molecular weight
kininogen, prekallikrein
(Factor VII)
Active Hageman factor
Tissue Factor
TF
Tissue factor pathway
inhibitor, TFPI
(Factor XIIa)
Christmas factor
Factor IX)
(
Active plasma
thromboplastin
antecedent (Factor XIa)
Active proconvertin,
Plasma thromboplastin
antecedent (Factor XI)
(
Factor VIIa)
Active Christmas factor
(
Factor IXa)
2+
Phospholipid, Ca
Active antihemophilic factor
Factor VIIIa)
Antihemophilic factor
(Factor VIII)
(
2
+
Phospholipid, Ca
Stuart-Prower factor
Factor X)
Active Stuart-Prower
factor (Factor Xa)
Stuart-Prower factor
(Factor X)
(
Active Proaccelerin
Factor Va)
Proaccelerin
(Factor V)
(
Prothrombin
Factor II)
Thrombin
(Factor IIa)
(
Fibrin stabilizing
factor (Factor XIII)
Common pathway
Soluble fibrin
monomer
Fibrinogen
Factor I)
(
(
Factor Ia)
Active fibrin stabilizing
factor (Factor XIIIa)
Cross-linked fibrin
Figure 1. The classical blood coagulation pathway [49].
Cd.
see Table 8) with thrombin was very weak, and no combination was observed with fibrinogen.
Cds. and had similar results compared to Cd. . These phenomena indicated that the procoagulant
activities of Cds. , and were achieved via combining with other proteins or factors in the
4 had the best procoagulant activity among the 18 PADs. However, the combination
(
6
7
4
4,
6
7
coagulation cascade rather than with thrombin and fibrinogen. This finding might be due to their
structures. All three compounds had very long R groups in their structures, and the distances
between the acylamino N and the carbonyl non-hydrogen atoms were very close (see Table 4 and
the Supplementary Information). Cd.
indicating that its R structure was suitable to combine with some proteins or factors in the coagulation
cascade and subsequently promote procoagulation. However, the three key distances of Cds. and
were shorter than those of Cd. , particularly N–hydroxyl O of COOH and N–C of COOH,
suggesting that the combinations between Cds. or and the proteins or factors were less efficient
4 showed the best procoagulant activity among the 18 PADs,
6
7
4
−
−
6
7
compared with the combination between Cd. 4 and the proteins or factors.
Mackman [50] classified anticoagulants into three main types: Vitamin K antagonists, heparins,
and direct inhibitors of factor Xa and thrombin. For the first type, Vitamin K antagonists function
by inhibiting the enzyme vitamin K epoxide reductase, which uses vitamin K to post-translationally
modify several coagulation proteins (factor VII, factor IX, factor X and prothrombin). The second
type, heparin, binds to the protein antithrombin and markedly increases the ability of this protein to
inhibit factor Xa and thrombin. The third type has two typical examples, lepirudin and desirudin.

Molecules 2017, 22, 2047
13 of 16
However, Correia-da-Silva and co-workers [51] demonstrated that some persulfated small molecules
might be a fourth type of anticoagulant, with dual anticoagulant and antiplatelet effects. The structures
of the persulfated compounds in [50] and the PADs synthesized in the present study showed some
similarities, such as benzene rings, phenolic hydroxyl groups, carbonyl groups, and sulfo groups.
This evidence suggests that the PADs might have similar functions with dual effects.
4
. Conclusions
According to the results, particularly the data from the five blood assays, the seven caffeic acid
derivatives all had certain anticoagulant or procoagulant activities; in the p-hydroxycinnamic acid
series, two derivatives, Cds. and 13, had the best anticoagulant activities among all 18 PADs.
8
Among the ferulic acid derivatives, one compound (Cd. 16) showed the best anticoagulant activity but
combined with a protein or factor that differed from the target of Cds. 8 and 13.
The anticoagulant or procoagulant activities of PADs slightly changed after structure modification.
Considering the complexities of haemostasis in vivo and the process of promoting blood circulation
for removing blood stasis, the method used in the present study provided some important data for
molecule design. In the future, the method (based on the SPR to obtain the combination data between
proteins or factors in the coagulation pathway and synthesized compounds and was assisted by
Materials Studio software to optimize the structures of the synthesized compounds and calculate the
significant structure information, such as cloud density) will be continually used to provide additional
information and data for designing molecules with in vivo activities of haemostasis and promoting
blood circulation for removing blood stasis.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: We acknowledge funding for the present study from the National Natural Science
Foundation of China (21362001), the Natural Science Foundation of Guangxi Province (2013GXNSFDA019005),
the High-Level Innovation Team and Outstanding Scholar Project of Guangxi Institutions of Higher Education
(
Guijiaoren [2014]49), and the Guangxi Postdoctoral Foundation. We also thank for Faquan Lin in the Department
of Laboratory Medicine, The First Affiliated Hospital of Guangxi Medical University, who provided all human
blood samples, the guidance for five blood assays, and other information about bio-active assays.
Author Contributions: Xuan Luo, Chuanrong Du, and Cuiwu Lin conceived and designed the experiments;
Xuan Luo and Chuanrong Du performed all experiments, molecular docking, and data analysis; Hui Cheng
assisted with molecular docking; and Jian-hua Chen guided the Materials Studio (Accelrys Software) analysis.
All authors were responsible for information processing and manuscript writing. All authors have read and
approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Because the research is continued, samples of these 18 compounds are ONLY available,
guaranteed, from the authors before December 2018. After that, not all 18 compounds are guaranteed.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(