Synthesis, pharmacological evaluation and molecular docking of novel R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid as dual anti-inflammatory anti-platelet aggregation agents

Article abstract of DOI:10.1007/s00210-019-01753-9

R-/S-2-(2-hydroxypropanamido) benzoic acid (R-/S-HPABA), marine-derived anti-inflammatory antiplatelet drugs, were initially synthesised in our group. However, preliminary research showed that R-/S-HPABA were eliminated rapidly because of extensive hydroxylation metabolism of phenyl ring in vivo. In order to reduce significant hydroxylation metabolism to improve pharmacological activity and bioavailability, trifluoromethyl group was incorporated into R-/S-HPABA to synthesise R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid (R-/S-HFBA), respectively. The purposes of this study were to report the synthesis of R-/S-HFBA and compare the anti-inflammatory antiplatelet effect and pharmacokinetic properties of R-/S-HFBA with those of R-/S-HPABA. Carrageenan-induced rat paw edema assay was used for the evaluation of the anti-inflammatory activity. R-/S-HFBA showed better results in inhibiting edema and were able to prolong the anti-inflammatory effect after carrageenan injection. The antiplatelet aggregation activity of R-/S-HFBA and R-/S-HPABA was studied on arachidonic acid-induced platelet aggregation of rabbit platelet-rich plasma. The aggregation inhibition rate of R-/S-HFBA was significantly (p < 0.05) higher than that of R-/S-HPABA, respectively. Molecular docking study revealed that R-/S-HFBA possess more potent binding affinity with COX-1/COX-2 than R-/S-HPABA, respectively, and that the presence of trifluoromethyl group leads to increase in activity of R-/S-HFBA. R-/S-HFBA also afford more favorable pharmacokinetic properties than R-/S-HPABA, respectively, such as higher C_max, larger AUC_0-∞, and longer t_1/2, which, as expected, are more metabolically stable.

Full text of DOI:10.1007/s00210-019-01753-9

Naunyn-Schmiedeberg's Archives of Pharmacology
https://doi.org/10.1007/s00210-019-01753-9
ORIGINAL ARTICLE
Synthesis, pharmacological evaluation and molecular docking
of novel R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic
acid as dual anti-inflammatory anti-platelet aggregation agents
1
1
1
1
1
1
Rong Rong & Rui-zhen Zhang & Xin Wang & Yu-han Dan & Yun-li Zhao & Zhi-guo Yu
Received: 10 May 2019 /Accepted: 18 October 2019
#
Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
R-/S-2-(2-hydroxypropanamido) benzoic acid (R-/S-HPABA), marine-derived anti-inflammatory antiplatelet drugs, were initially
synthesised in our group. However, preliminary research showed that R-/S-HPABAwere eliminated rapidly because of extensive
hydroxylation metabolism of phenyl ring in vivo. In order to reduce significant hydroxylation metabolism to improve pharma-
cological activity and bioavailability, trifluoromethyl group was incorporated into R-/S-HPABA to synthesise R-/S-2-(2-
hydroxypropanamido)-5-trifluoromethyl benzoic acid (R-/S-HFBA), respectively. The purposes of this study were to report
the synthesis of R-/S-HFBA and compare the anti-inflammatory antiplatelet effect and pharmacokinetic properties of R-/S-
HFBA with those of R-/S-HPABA. Carrageenan-induced rat paw edema assay was used for the evaluation of the anti-
inflammatory activity. R-/S-HFBA showed better results in inhibiting edema and were able to prolong the anti-inflammatory
effect after carrageenan injection. The antiplatelet aggregation activity of R-/S-HFBA and R-/S-HPABA was studied on arachi-
donic acid-induced platelet aggregation of rabbit platelet-rich plasma. The aggregation inhibition rate of R-/S-HFBAwas signif-
icantly (p < 0.05) higher than that of R-/S-HPABA, respectively. Molecular docking study revealed that R-/S-HFBA possess more
potent binding affinity with COX-1/COX-2 than R-/S-HPABA, respectively, and that the presence of trifluoromethyl group leads
to increase in activity of R-/S-HFBA. R-/S-HFBA also afford more favorable pharmacokinetic properties than R-/S-HPABA,
respectively, such as higher Cmax, larger AUC0-∞, and longer t , which, as expected, are more metabolically stable.
1/2
.
.
Keywords R-/S-2-(2-hydroxypropanamido) benzoic acid R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid
Anti-inflammatory Antiplatelet Molecular docking
.
.
Introduction
the inhibition of cyclooxygenase (COX) involved in the pro-
duction of pro-inflammatory prostaglandins (PGs) and throm-
boxanes (TXs). COX has two main isoforms: COX-1 is the
naturally expressed isozyme that is related with homeostatic
prostanoid biosynthesis under physiological conditions. In
particular, aspirin also can inhibit COX-1 in the platelets, sup-
press arachidonic acid (AA) metabolism, and prevent the syn-
thesis of thromboxane A (TXA ), a compound that induces
Inflammation, one of the most crucial protective mechanisms
of the body against irritation, tissue lesion or infection, is able
to induce cellular damage and release pro-inflammatory me-
diators (Newton and Dixit 2012; Maurent et al. 2017). Non-
steroidal anti-inflammatory drugs (NSAIDs) are the most
common therapeutic agents through their anti-inflammatory,
analgesic antipyretic effect (Fiorucci et al. 2001; Rao et al.
2
2
platelet aggregation. COX-2 is inducibly expressed by inflam-
matory stimulant from infections or injuries (Woo and Kwon
2
010). The anti-inflammatory activity of NSAIDs is due to
2
007; Helmersson et al. 2005; Vane and Botting 1987;
Catella-Lawson et al. 2001). However, long-term clinical use
of NSAIDs, particularly aspirin, may lead to significant side
effects, mainly gastrointestinal, cardiovascular and renal
*
*
Yun-li Zhao
yunli76@163.com
Zhi-guo Yu
zhiguo-yu@163.com
(Lazzaroni and Bianchi 2004; Ashour et al. 2013). Thus, there
is an unmet medical need for developing more effective and
safe anti-inflammatory drugs to supplement or replace the
current therapies.
1
Department of Pharmaceutical Analysis, School of Pharmacy,
Shenyang Pharmaceutical University, Shenyang 110016, China

Naunyn-Schmiedeberg's Arch Pharmacol
Fluorine continues to make great contribution to the design
and development of biologically active compounds. About
2
0% of the currently used drugs and agrochemicals are
fluorine-containing organic molecules (Wang et al. 2014;
Purser et al. 2008; Gouverneur and Müller 2012). It is well
known that incorporation of fluorine or various fluorinated
substituents into an organic molecule has had a tremendous
impact on physical and chemical properties such as solubility,
lipophilicity or biological potency (Cho et al. 2010;
Sorochinsky and Soloshonok 2010; Acena et al. 2013; Gillis
et al. 2015). In several times, the drug-receptor interactions are
also improved in the presence of fluorine moiety (Abid et al.
2
012). Among multifarious fluorine substituents,
trifluoromethyl group is one of the most important structural
fragments, because its introduction into small molecules can
enhance the effect of their interactions with cellular targets,
improve cellular membrane permeability and increase stability
by reducing oxidative metabolism (Liu et al. 2015; Ojima
Fig. 1 Chemical structures of R-HPABA (a), S-HPABA (b), R-HFBA (c)
and S-HFBA (d)
of R-/S-HFBA with those of R-/S-HPABA. The structure ac-
tivity relationship was also discussed, and the results have
been confirmed by molecular docking calculations. Results
of this investigation provide valuable scientific research data
and probably play an important role in the discovery of novel
anti-inflammatory antiplatelet aggregation drug.
2
013; Mueller et al. 2007; Hagmann 2008). The
trifluoromethyl fragment is a part of many biologically active
molecules such as highly active antiretroviral therapy agent
Sustiva, antidepressant Prozac, nonsteroidal anti-
inflammatory agent Celebrex, antimalarial agent mefloquine
and oral multikinase inhibitor sorafenib (Liu et al. 2015; Dey
et al. 2018; Russo et al. 2016; Palmer et al. 1993; Hasskarl
2
014). Incorporating a trifluoromethyl group into biologically
Material and methods
important compounds or into already known pharmaceutical
agents has been widely accepted as a powerful tool to enhance
the activity of these agents (Ismail 2002; Tressaud and Haufe
Materials
2
008; Kulikova et al. 2017).
S-2-(2-hydroxypropanamido) benzoic acid (S-HPABA;
R-/S-HPABA and R-/S-HFBA (optical purity of all com-
pounds > 99.0%) were, respectively, synthesised in our labo-
ratory. Aspirin (purity > 99.5%) was purchased from Shanghai
Aladdin biochemical technology Co., Ltd (Shanghai, China).
For in vitro study, R-/S-HPABA and R-/S-HFBA were dis-
solved and diluted by dimethyl sulfoxide (DMSO). In vivo
studies, R-/S-HPABA and R-/S-HFBA were suspended in
Fig. 1), which was initially isolated from the fermentation
broth of a marine fungus Penicillium chrysogenum by our
group, presented anti-inflammatory activity like aspirin, but
no gastrointestinal toxicity (Wang et al. 2014a; Yu et al. n.d.).
To research further on S-HPABA, a simple and efficient ste-
reospecific synthetic route was developed for S-HPABA, as
well as R-2-(2-hydroxypropanamido) benzoic acid (R-
HPABA; Fig. 1), the R-enantiomer (Wang et al. 2014b).
Moreover, our previous study showed that R-/S-HPABA also
exhibited remarkable antiplatelet aggregation and antithrom-
bosis effect like aspirin (Zhang et al. 2017). But preliminary
researches indicated that S-HPABA was eliminated rapidly
because of widely phase I and phase II metabolic reactions
in vivo. Hydroxylation metabolite of phenyl ring was the main
metabolite of S-HPABA in rats (Guan et al. 2015).
In order to reduce significant hydroxylation metabolism to
improve pharmacological activity and bioavailability,
trifluoromethyl group was incorporated into R-/S-HPABA to
synthesise R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl
benzoic acid (R-/S-HFBA; Fig. 1) in our laboratory for the first
time. Herein, we report the synthesis of R-/S-HFBA and com-
pare the pharmacodynamics and pharmacokinetics properties
0.5% aqueous solution of sodium carboxymethyl cellulose
for oral administration and dissolved in normal saline for in-
travenous administration.
Chemistry
The chemical reagents and solvents used were purchased from
commercial vendors and used without purification. Melting
points were determined on a Buchi apparatus and were uncor-
rected. IR spectra were recorded on a Bruker IFS-55 spec-
trometer. NMR spectra were determined on Brucker 600 M
AVIII spectrophotometer. The chemical shifts were given in δ
units and coupling constants (J) were measured in Hertz (Hz).
Signals are described as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), dd (double doublet). Mass spectra
were recorded on Agilent 6540 UHD Q-TOF mass

Naunyn-Schmiedeberg's Arch Pharmacol
spectrometer. TLC development was conducted on silica gel
sheets. The spots were visualised by exposure to UV-lamp at λ
times. The aqueous layer was acidised to pH = 1–4 by addition
of HCl until crystal appeared. It was then kept in freezer over-
night. The product obtained was filtered, collected and dried at
2
54 nm.
40 °C to give S-2-(2-hydroxypropanamido)-5-trifluoromethyl
Synthesis
benzoic acid. The expected compound was obtained as white
crystalline powder, pure enough to be used without further
20
Synthesis of the intermediate and target compound is illustrat-
ed in Scheme 1. In the synthetic approach, S-HFBA was pre-
pared using L-lactic acid as starting material while R-HFBA
was obtained starting with D-lactic acid in a total yield of
purification. Yield 4.57 g, 93.7%; m.p. 146–148 °C; [α] : −
D
1
−
12.2° (c = 0.5 g/100 mL, MeOH); IR (KBr, cm ): 3388,
1
3206, 1690, 1668; H NMR (MeOH-d , 600 MHz): δ 1.45
4
(d, 3H, CH ), 3.35 (s, 1H, CH-OH), 4.28 (q, 1H, CH-OH),
3
6
0.2%. Compounds 2 and 3 were synthesised according to
previously published literature procedures (Wang et al.
014b).
7.85 (d, 1H, Ar-H), 8.34 (brs, 1H, Ar-H), 8.93 (d, 1H, Ar-H);
1
3
C NMR (MeOH-d , 150 MHz): δ 21.0 (s, CH ), 69.9 (s,
4
3
2
CH-OH), 177.2 (s, -CO-NH), 169.4 (s, COOH), 125.2 (q, J =
70 Hz, CF ), 118.1(s, C-1), 144.9 (s, C-2), 121.5 (s, C-3),
2
3
Synthesis of S-methyl 2-(2-acetoxypropionyl amino)
5-trifluoromethyl benzoate compound 4
131.6 (q, J = 3 Hz, C-4), 125.6 (q, J = 30 Hz, C-5), 129.6 (q, J
1
9
-
= 3 Hz, C-6); F NMR (MeOH-d ): − 63.96 (s, 3F, CF );
4
3
−
HRMS m/z calcd for C H NF O [M-H] : 276.0489, found:
11
10
3 4
To a stirred suspension of S-methyl 2-amino-5-trifluoromethyl
benzoate (4.2 g, 19.2 mmol) in diethyl ether (300 mL), com-
pound 3 (11.56 g, 76.8 mmol) was added dropwise at − 10–0
276.0520.
°C. The reaction mixture was stirred at room temperature for 3
Pharmacology
h. After the completion of reaction (monitored by TLC), mix-
ture was washed with hydrochloric acid, water, NaHCO so-
Experimental animals
3
lution, and aqueous saturated sodium chloride then dried over
Na SO . The dried extract was filtered and concentrated under
reduced pressure to give S-methyl 2-(2-acetoxypropionyl ami-
Male Sprague-Dawley rats at age 7–8 weeks and New
Zealand white male rabbits at age 3 months were provided
by Experimental Animal Center, Shenyang Pharmaceutical
University (Shenyang, China). Animals were kept in plastic
cages in standard environmental conditions and had free
access to standard diet with water ad libitum. The ani-
mals were fasted for 12 h with only access to water prior
to experimentation. All experimental studies were per-
formed in accordance with the Guideline for Animal
Experimentation of Shenyang Pharmaceutical University
and the approval from Animal Ethics Committee of
Shenyang Pharmaceutical University.
2
4
no)-5-trifluoromethyl benzoate compound 4 (5.87 g, 92.0%).
General procedure for synthesis
of S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic
acid
To a solution of compound 4 (5.87 g, 17.6 mmol) in 40 mL
methanol and 60 mL tetrahydrofuran, 10% sodium hydroxide
was added and stirred at room temperature for 3–4 h. After
then, the reaction mixture was extracted with CH Cl three
2
2
Scheme 1 Synthesis of R-/S-
HFBA. Reagents and conditions:
a acetyl chloride, THF, 40 °C; b
2
SOCl , 50 °C; c methyl 2-amino-
5
-trifluoromethyl benzoate;
diethyl ether, room temperature; d
MeOH, THF, NaOH, room
temperature

Naunyn-Schmiedeberg's Arch Pharmacol
Anti-inflammatory activity
prepared by centrifugation of citrated blood at 1000 rpm for
15 min at room temperature. PRP samples (335 μL) with R-/S-
Carrageenan-induced hind paw edema in rats was used for
the evaluation of anti- inflammatory activity (Winter et al.
HPABA or R-/S-HFBA at a concentration of 0.64 mol/mL
(3.5 μL) were added into aggregometer cuvettes and aggrega-
tion was recorded as increased light transmission at 37 °C after
the addition of the stimulus. AA at concentration of 20 μmol/
mL (10 μL) was used as the platelet activator in PRP. PRP
samples were pre-incubated at 37 °C for 2 min before the
addition of AA. In blank and positive control experiments,
DMSO (3.5 μL) and aspirin (3.5 μL) were added instead of
the test samples, respectively. The percent inhibition rate was
evaluated and calculated according to our previous report
(Zhang et al. 2017).
1
962). Sprague-Dawley rats were randomly divided into
ten groups (five rats in each group) and administered oral-
ly in the following manner. Initially, the first group was
treated with 0.5% aqueous solution of sodium
carboxymethyl cellulose (w/v) acting as a negative con-
trol. The second group was given aspirin as a reference
standard (100 mg/kg). Rats in group 3 received 100
mg/kg of S-HPABA while rats in groups 4, 5, 6 received
5
0, 100, 200 mg/kg of S-HFBA, respectively. Rats in
group 7 were given 100 mg/kg of R-HPABA while rats
in groups 8, 9, 10 received 50, 100, 200 mg/kg of R-
HFBA, respectively. The right hind paw edema was pro-
duced by sub-plantar injection of 0.1 mL of freshly pre-
pared carrageenan (1%) in normal saline 1 h after the
administration. The paw thickness was measured using
vernier calipers and the difference between paw thickness
was recorded at hourly intervals up to 5 h. The percentage
edema (E%) and inhibition rate (I%) of each group were
calculated as follows (Sayed et al. 2018):
Molecular docking
Docking study was performed to evaluate the binding mode
and compare the binding affinity of R-/S-HFBA and R-/S-
HPABAwith COX-1/COX-2. With this purpose, crystal struc-
tures of COX-1 (PDB codes: 3N8Y) and COX-2 (PDB codes:
5IKT) were obtained from the PDB database (Sidhu et al.
2010; Orlando and Malkowski 2016). For protein prepa-
ration, water molecules, the repeating chains and other
ligands were eliminated. Hydrogen atoms were added to
the receptor and Gasteiger charges were calculated. The
Lamarckian genetic algorithm (LGA) was applied to gen-
erate the best conformation of the compounds binding in
COX-1/COX-2. The compounds were docked into the
pocket of each protein center around binding site, with
the LGA. After docking, the obtained poses with the low-
est binding energy were selected as the most probable
binding conformation. Discovery Studio 4.5 was chosen
to show the docking results.
.
Edema rate ðE%Þ ¼ ðPTt−PT0Þ
Inhibition rate ðI%Þ ¼ ðEc−EcÞ.
 100%
PT0
 100%
Ec
where PT and PT are the paw thickness (mm) before and
0
t
after carrageenan injection, respectively, and E and E are the
c
t
average percentage edema of negative control group and the
treated, respectively.
Determination of TNF-α and IL-1β levels in the rat paw
Pharmacokinetic study
Five hours after carrageenan injection, inflamed hind paws
were removed and stored at – 80 °C to be processed for
TNF-α and IL-1β determinations. The paw tissue samples
were weighed and homogenised in phosphate-buffered saline
R-/S-HFBA and R-HPABA were administered intrave-
nously (12.5 mg/kg) and intragastricly (50 mg/kg) to
conscious SD rats, respectively. Blood samples (approx-
imately 0.25 mL) were drawn from fosse orbital vein at
(
pH = 7.4). The homogenates were centrifuged at 12,000×g
0
8
0
.033, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6 and
h after intravenous administration and at 0.083, 0.25,
.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after oral
for 20 min at 4 °C, and the supernatant was obtained to be kept
at − 80 °C for cytokine assay. The TNF-α and IL-1β levels
were determined by enzyme-linked immunosorbent assay
administration. The plasma concentration of drug was
determined by HPLC method with liquid-liquid extrac-
tion. Pharmacokinetic parameters were calculated using
non-compartmental analysis of DAS 2.1 pharmacokinet-
ic program (Chinese Pharmacological Society).
(
ELISA) kits (ABclonal Biotechnology Co., Ltd, USA) ac-
cording to manufacturer’s instructions.
Anti-platelet aggregation assay
The antiplatelet aggregation effect of R-/S-HFBA and R-/S-
HPABA was studied on AA-induced platelet aggregation of
rabbit platelet-rich plasma (PRP) (Zhang et al. 2017). Blood
was collected from the carotid artery of rabbits and PRP was
Statistical analysis
Experimental results are expressed as mean ± standard devia-
tion (SD). Statistical analysis was performed using Tukey’s

Naunyn-Schmiedeberg's Arch Pharmacol
Table 1 Effects of R-/S-HFBA and R-/S-HPABA on carrageenan-induced paw edema in rats (mean ± SD, n = 5)
−1
Group
Dose (mg kg )
Edema rate (%)
Inhibition rate (%)
1
h
2 h
3 h
4 h
5 h
Control
ASP
-
40.23 ± 3.07
79.74 ± 9.42
53.37 ± 6.30
33.07
66.66 ± 8.88
45.58 ± 6.60
31.62
60.59 ± 3.86
41.98 ± 7.53
30.71
51.94 ± 4.26
39.84 ± 9.51
23.30
*
*
**
**
***
***
***
***
***
***
**
*
100
25.84 ± 7.60
5.77
22.70 ± 7.78
3.57
29.97 ± 5.98
5.50
24.62 ± 6.39
8.80
19.10 ± 5.25
2.52
20.20 ± 5.70
9.79
28.98 ± 5.97
7.96
20.98 ± 1.47
7.85
15.78 ± 5.62
0.78
3
*
*
***
***
***
S-HPABA
S-HFBA
100
50
41.25 ± 10.83
40.98 ± 4.84
38.52
39.60 ± 7.30
34.64
36.01 ± 2.30
30.67
4
48.27
*
*
**
**
54.37 ± 9.78
47.29 ± 3.33
29.06
43.48 ± 4.14
28.24
42.13 ± 8.57
18.89
2
31.82
*
*
***
***
***
***
***
*
1
00
00
42.80 ± 8.73
46.33
35.09 ± 6.63
47.36
30.96 ± 5.64
48.90
28.72 ± 6.36
44.71
3
*
**
**
*
***
***
**
***
2
34.62 ± 8.06
56.58
29.43 ± 8.19
55.85
26.90 ± 8.61
55.60
21.46 ± 7.47
58.56
5
*
***
R-HPABA
R-HFBA
100
50
44.85 ± 9.64
43.75
44.71 ± 11.12
40.74 ± 6.98
32.76
36.94 ± 9.61
28.88
4
32.93
*
**
56.95 ± 5.48
28.58
47.45 ± 2.28
28.82
39.42 ± 7.10
34.94
39.77 ± 7.21
23.43
2
*
**
**
***
***
***
***
1
2
00
00
41.20 ± 7.64
48.33
34.33 ± 6.25
31.17 ± 6.26
48.56
26.01 ± 8.05
49.92
4
48.50
*
***
***
***
***
30.03 ± 7.11
62.34
26.47 ± 3.55
60.29
23.10 ± 4.73
61.87
19.41 ± 3.91
62.63
6
*
**
***
p < 0.05, p < 0.01,
p < 0.001 vs control group
test and one-way analysis of variance by SPSS Statistics 20.0.
For a confidence interval of 95%, a value of p < 0.05 was
indicative of significant.
Assay of TNF-α and IL-1β levels
in carrageenan-injected paws
In order to observe the effects of R-/S-HFBA and R-/S-
HPABA on inflammatory cytokine secretions, TNF-α and
IL-1β levels in rat-inflamed paws were determined by
enzyme-linked immunosorbent assay. As shown in Figs. 2
and 3, all of the tested compounds and the reference drug
could significantly reduce the production of TNF-α and IL-
1β, compared to the control group (p < 0.05, p < 0.01, p <
0.001). Moreover, S-HFBA signifcantly decreased the TNF-α
and IL-1β levels compared to the S-HPABA group at the same
dose (p < 0.05). The concentrations of TNF-α and IL-1β in R-
HFBA group (100 mg/kg) were also significantly lower than
that in R-HPABA group (100 mg/kg) (p < 0.05, p < 0.01),
respectively.
Results
Anti-inflammatory activity
To evaluate the anti-inflammatory activity of R-/S-HFBA and
R-/S-HPABA, the carrageenan-induced rat paw edema model
was used (Winter et al. 1962; Sayed et al. 2018). The results of
this assay are presented in Table 1. It showed that R-/S-
HPABA (100 mg/kg) and all the doses tested for R-/S-
HFBA considerably reduced paw edema after carrageenan
administration (p < 0.05, p < 0.01, p < 0.001), compared to
control group. R-/S-HFBA decreased the paw edema in a
dose-dependent manner. Moreover, at the 100-mg/kg dose,
the paw edema inhibition rates of S-HFBA were significantly
higher (47.36%, 48.90%, 44.71%, respectively) than those of
S-HPABA (38.52%, 34.64%, 30.67%, respectively) at 3, 4
and 5 h after the administration of carrageenan. After 2 h of
administration, R-HFBA also showed a higher inhibition rate
than R-HPABA at the same dose. The results suggested that
the introduction of trifluoromethyl prolonged the anti-
inflammatory effect of R-/S-HFBA.
Anti-platelet aggregation assay
Anti-aggregation effects of R-/S-HFBA and R-/S-HPABA
were studied on AA-induced platelet aggregation. The results
are presented in Fig. 4. It showed that R-/S-HFBA and R-/S-
HPABA could significantly inhibit AA-induced platelet ag-
gregation. There was a significant (p < 0.05) increase in the
platelet aggregation inhibition by S-HFBA compared to S-
HPABA. The aggregation inhibition rate was also

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 2 Effect of R-/S-HPABA and
R-/S-HFBA on TNF-α levels in
the rat paws treated with carra-
geenan. The results are expressed
*
as mean ± SD (n = 5). p < 0.05,
*
*
*
p < 0.01, p < 0.001 vs control
#
##
group p < 0.05, p < 0.01 vs
ASP 100-mg/kg group; p <
▼
0
.05, S-HFBA 100 mg/kg vs S-
▲
HPABA 100 mg/kg; p < 0.05,
R-HFBA 100 mg/kg vs R-
HPABA 100 mg/kg
significantly (p < 0.05) higher with R-HFBA than with R-
HPABA. The results also clearly demonstrated that R-HFBA
inhibited platelet aggregation more potently than the reference
drug aspirin (p < 0.01).
compounds to evaluate their molecular docking. Docking
study had been performed from the selection of the X-ray
structure. The X-ray structures of COX-1 and COX-2 were
chosen from PDB database (PDB code 3N8Yand 5IKT). S-
HPABA and S-HFBA bind to COX-1 enzyme by releasing
free energy of − 7.67 and − 9.48 kcal/mol, respectively, and
to COX-2 releasing free energy of − 7.14 and − 9.47
kcal/mol, respectively. The binding affinity of R-HPABA
and R-HFBA to COX-1 showed score = − 7.85 and − 9.47
Molecular docking
In order to compare the binding affinity of R-/S-HFBA and
R-/S-HPABA with COX-1/COX-2, we docked all
Fig. 3 Effect of R-/S-HPABA and
R-/S-HFBA on IL-1β levels in the
rat paws treated with carrageenan.
The results are expressed as mean
*
**
±
SD (n = 5). p < 0.05, p <
*
0
.01, p < 0.001 vs control group;
#
##
p < 0.05, p < 0.01 vs ASP 100-
mg/kg group; ▼p < 0.05, S-
HFBA 100 mg/kg vs S-HPABA
00 mg/kg; ▲p < 0.05,
▲▲
p <
1
0
.01, R-HFBA 100 mg/kg vs R-
HPABA 100 mg/kg

Naunyn-Schmiedeberg's Arch Pharmacol
concentration-time curves after intragastric and intravenous
administration are shown in Figs. 9 and 10, respectively. The
main pharmacokinetic parameters based on non-
compartmental analysis are listed in Table 2 and Table 3.
The results showed that the Cmax and AUC0-∞ after the
intragastric administration of S-HFBA were nearly 2.8-fold
and 6.5-fold higher than that of S-HPABA, respectively. The
Cmax and AUC0-∞ after the intragastric administration of
R-HFBA were nearly 3.7-fold and 13.1-fold higher than that
of R-HPABA, respectively. Moreover, the elimination half-life
(
t ) of R-/S-HFBA after the intragastric administration was
1/2
significantly longer than that of R-/S-HPABA (p < 0.01, p <
.05), respectively. Meanwhile, the clearance (CL) for R-/
0
S-HFBA was found to be significantly lower than that of R-/
S-HPABA (p < 0.01). The absolute bioavailability of R-/
S-HFBA and R-/S-HPABA was 88.7%, 66.8%, 54.9% and
58.1%, respectively.
Fig. 4 The effects of R-/S-HPABA, R-/S-HFBA and aspirin (ASP) on the
platelet aggregation induced by AA. Results are given as mean ± SD (n =
†
††
*
**
3
). p < 0.05, p < 0.01 vs ASP; p < 0.05, p < 0.01 S-HPABA vs. S-
# ##
HFBA; p < 0.05, p < 0.01 R-HPABA vs. R-HFBA
kcal/mol, respectively, and to COX-2 showed score = −
Discussion
7
.67 and − 9.54 kcal/mol, respectively. Binding mode of
all compounds at COX-1/COX-2 active sites is presented
in Figs. 5, 6, 7 and 8.
R-HFBA had the same NMR, IR spectroscopy and MS data
2
0
with S-HFBA while the [α] of R-HFBA was + 13.2°. The
D
structure of S-HFBA was proven by IR spectral bands of –
−
1
−1
Pharmacokinetic study
NHCO– at 1668 cm and –NH stretching at 3206 cm .
−1
−1
Absorption peaks at 3388 cm and 1690 cm , due to car-
boxylic OH and C=O groups, respectively, were also ob-
served. In the H NMR spectra of compound, the hydrogens
Pharmacokinetic experiments were performed to compare the
pharmacokinetic behavior and absolute bioavailability of R-/
S-HPABA with those of R-/S-HFBA. Mean plasma
1
in the CH group were found as a doublet peak at 1.45 ppm.
3
Fig. 5 Docked pose of compound a R-HFBA and b R-HPABA with COX-1

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 6 Docked pose of compound a S-HFBA and b S-HPABA with COX-1
The quartet peaks at 4.28 ppm indicated the existence of linker
(3–5 h after carrageenan injection) is relative to the release of
prostaglandin, nitric oxide and cytokines, which is generated
by an inducible COX-2 isoform. The second phase is sensitive
to both steroidal and nonsteroidal anti-inflammatory agent
used in clinical therapeutics (Matsumoto et al. 2015; Ishola
et al. 2014). The edema produced in both early and late phase
involves the release of mediators (e.g. prostaglandin, bradyki-
nin), which later induces the biosynthesis of prostaglandin and
other mediators. Inhibiting the synthesis of these mediators
can effectively lead to the reduction of pain and inflammation
1
3
(
–CH–) group attach to amide group. Meanwhile, C NMR
spectra showed a singlet signal corresponding to –CONH– at
77.2 ppm and COOH group at 169.4 ppm, respectively. It
1
also revealed the CF group as quartet signals at 125.2 ppm.
3
The carrageenan-induced rat paw edema model comprises
two phases. The first phase is mediated by releasing of pro-
inflammatory mediators, such as histamine, serotonin, kinins,
and occurs up to 2 h after the injection of carrageenan (Bhukya
et al. 2009; Brooks and Day 1991). While the second phase
Fig. 7 Docked pose of compound a R-HFBA and b R-HPABA with COX-2

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 8 Docked pose of compound a S-HFBA and b S-HPABA with COX-2
(
Ueno et al. 2000). We found that R-/S-HFBA and R-/S-
inhibitory activity. Another one is likely due to the better
pharmacokinetic properties of R-/S-HFBA.
HPABA significantly inhibited rat paw edema in both phases,
suggesting that the anti-inflammatory effect of these agents is
involved with the inhibition of COX leading to the decrease of
different inflammatory mediators. Our previous study also
showed that the anti-inflammatory effect of HPABA was due
to NO, malondialdehyde and PG suppression (data are not
listed). In this study, R-/S-HFBA exhibited better results in
inhibiting the second phase of carrageenan-induced paw ede-
ma than R-/S-HPABA, respectively. One explanation for this
finding may be that R-/S-HFBA possess more potent COX
As mentioned above, carrageenan injection can induce the
release of proinflammatory cytokines. In order to investigate
the effects of those compounds on the production of inflam-
matory cytokines, TNF-α and IL-1β levels in rat paws tissue
were determined. TNF-α and IL-1β are the most important
mediators of inflammatory responses since their over expres-
sion can provoke severe proinflammatory reactions. Our re-
sults showed that R-/S-HFBA could significantly attenuate the
production of TNF-α and IL-1β in carrageenan-injected paws
Fig. 9 Mean plasma
concentration-time curves of R-/
S-HFBA and R-/S-HPABA after
intragastric administration of 50
mg/kg to rats (n = 5)

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 10 Mean plasma
concentration-time curves of R-/
S-HFBA and R-/S-HPABA after
intravenous administration of
1
2.5 mg/kg to rat (n = 5)
compared to R-/S-HPABA, respectively. These findings fur-
ther demonstrated that R-/S-HFBA had better anti-
inflammatory effect than R-/S-HPABA, respectively.
their potent anti-platelet and inhibition of COX-1. Moreover,
future studies on the antiplatelet aggregation effect of R-/S-
HFBA in vivo are needed.
In platelets, AA is released by activation of phospholipase
A and metabolised by COX and lipoxygenases finally
The purpose of the molecular docking is to evaluate the
binding affinity of the compounds with observed pharmaco-
logical effect towards COX-1/COX-2 enzyme. It is worth
mentioning that trifluoromethyl moiety imparted lipophilic
properties to R-/S-HFBA which increased its affinity with
COX-1/COX-2 active site by hydrophobic interactions. R-
HFBA could have hydrophobic interactions with side chains
of TYR355, VAL116, LEU531 and VAL349 through
trifluoromethyl group in the COX-1 binding site. Likewise,
the trifluoromethyl group of S-HFBA showed hydrophobic
contact with TYR355, VAL116, LEU531, VAL349 and
2
resulting in the generation of TXA (Hubertus et al. 2014).
2
TXA is a potent stimulant of platelet aggregation leading to
2
arterial thrombosis. Our previous study revealed that the anti-
platelet aggregation effect of R-/S-HPABA was attributed to
the inhibition of COX-1 enzyme resulting in a reduction of the
proaggregatory product TxA (Zhang et al. 2017). The results
2
in this experiment showed that R-/S-HFBA had more effect on
AA-induced platelet aggregation than R-/S-HPABA. So, the
trifluoromethyl group found in R-/S-HFBA may contribute to
Table 2 Main pharmacokinetic
parameters of R-/S-HFBA and R-/
S-HPABA after intragastric
Parameters
max (μg mL )
AUC0-t (μg mL−1 h)
AUC0-∞ (μg mL−1 h)
S-HFBA
S-HPABA
R-HFBA
R-HPABA
−1
**
##
administration of R-/S-HFBA and
C
66.55 ± 14.40
23.74 ± 3.23
41.41 ± 3.41
41.99 ± 3.75
1.73 ± 0.38
1.20 ± 0.11
0.50 ± 0.00
67.66 ± 15.56
18.34 ± 1.28
31.54 ± 2.97
32.23 ± 2.73
1.41 ± 0.27
1.56 ± 0.13
0.50 ± 0.00
−1
**
**
##
##
R-/S-HPABA (50 mg kg ) to rats
mean ± SD, n = 5)
267.01 ± 51.68
413.93 ± 55.03
(
271.90 ± 46.49
420.60 ± 56.07
##
*
t
1/2 (h)
CL (L h− kg )
2.83 ± 0.72
3.98 ± 0.47
1
−1
**
##
0.19 ± 0.04
0.12 ± 0.02
*
*
##
Tmax (h)
0.80 ± 0.11
0.90 ± 0.14
*
**
#
##
p < 0.05, p < 0.01 S-HFBA vs. S-HPABA, p < 0.05, p < 0.01 R-HFBA vs. R-HPABA
Table 3 Main pharmacokinetic
parameters of R-/S-HFBA and R-/
S-HPABA after intravenous
Parameters
max (μg mL )
AUC0-t (μg mL−1 h)
AUC0-∞ (μg mL−1 h)
S-HPABA
S-HFBA
R-HPABA
R-HFBA
−1
administration of R-/S-HFBA and
C
53.79 ± 4.20
17.81 ± 5.24
17.90 ± 5.34
0.25 ± 0.03
0.74 ± 0.19
98.01 ± 17.72
99.92 ± 13.16
101.77 ± 12.20
1.55 ± 0.32
48.43 ± 6.26
14.01 ± 1.75
14.07 ± 1.76
0.23 ± 0.05
0.90 ± 0.10
116.62 ± 17.80
129.06 ± 11.56
139.22 ± 14.88
2.27 ± 0.26
−1
R-/S-HPABA (12.5 mg kg ) to
rats (mean ± SD, n = 5)
t
1/2 (h)
CL (L h− kg )
1
−1
0.12 ± 0.02
0.09 ± 0.01

Naunyn-Schmiedeberg's Arch Pharmacol
LEU359 in the COX-1. In addition to the hydrophobic inter-
actions, R-HFBA also elicited hydrogen bonding interaction
with side chains of ARG120 in COX-2 through fluorine atom.
Also, trifluoromethyl moiety of S-HFBA showed H-bond in-
teractions with TYR385 and SER530 which proved the im-
Conflict of interest The authors declare that they have no conflict
of interest.
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