Design, synthesis and biological evaluation of 4-methoxy diaryl isophthalates as antiplatelet agents

Article abstract of DOI:10.1007/s00044-017-2075-7

A series of 4-methoxy diphenyl isophthalates, which are the isosturctural analogs of picotamide, were designed and synthesized using the concept of isosterism. The structures of these new analogs were characterized by all means of spectroscopy, including 1H NMR, 13C NMR, and MS spectra. In vitro antiplatelet aggregation activities of these compounds were investigated by using Born’s test method. Among the 19 compounds tested for both ADP and collagen inducers, six of them (P216–P219 and P220–P221) were found to exhibit higher activity in vitro antiplatelet aggregation than Picotamide. In particular, the compound P220 bearing a nitrooxyl group showed the highest acitivity 72.1% (induced by collagen) and 72.5% (induced by ADP), with IC50 values of 0.30 μM/L induced by ADP (1.3 μM/L) and LD50 CloseSPigtSPi 2500 mg/kg, could have dual mechanism of action. Evaluation of cytotoxic activity of the compounds against L929 cell line revealed that none of the compounds have significant cytotoxicity. Through the careful analysis of in vitro activity data, the SAR of these compounds was preliminarily deduced. The results of this study showed that 4-methoxy diaryl isophthalates derivatives are potential to become an antiplatelet aggregation agents.

Full text of DOI:10.1007/s00044-017-2075-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2018) 27:488–496
DOI 10.1007/s00044-017-2075-7
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of 4-methoxy diaryl
isophthalates as antiplatelet agents
1
,2 ●
1,2 ●
1 ●
1 ●
1 ●
Liu Xiu-jie
Liu Xu-guang
Wang Chao-qing
Meng Jie Shi xin-xin Yan ya-nan
1
Received: 6 May 2017 / Accepted: 14 September 2017 / Published online: 26 September 2017
Springer Science+Business Media, LLC 2017
©
●
Abstract A series of 4-methoxy diphenyl isophthalates,
which are the isosturctural analogs of picotamide, were
designed and synthesized using the concept of isosterism.
The structures of these new analogs were characterized by
all means of spectroscopy, including 1H NMR, 13C NMR,
and MS spectra. In vitro antiplatelet aggregation activities
of these compounds were investigated by using Born’s test
method. Among the 19 compounds tested for both ADP and
collagen inducers, six of them (P216–P219 and P220–P221)
were found to exhibit higher activity in vitro antiplatelet
aggregation than Picotamide. In particular, the compound
P220 bearing a nitrooxyl group showed the highest acitivity
Keywords 4-Methoxy diphenyl isophthalates Antiplatelet
aggregation SAR, Organic synthesis
●
Introduction
Thrombotic disorders are important cause of morbidity and
mortality in the world (Reddy et al. 2011). Platelets play a
crucial role in hemostasis and thrombosis (Brito et al. 2010;
Mashayekhi et al. 2013). Antiplatelet aggregation drugs can
prevent thrombosis through the inhibition of platelet adhe-
sion, aggregation, and release. Antiplatelet aggregation
drugs employed in therapy can be divided to several drug
categories, such as nonsteroidal anti-inflammatory drugs
(NSAIDS), glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists,
and adenosine diphosphate receptor antagoinsts (Butwick
and Carvalho 2010).
Despite the documented utility of all these drugs, their
efficacy and selectivity are not sufficiently high and there
are substantial rooms to improve for the antiplatelet drugs.
Of all the antiplatelet agents in current widespread use,
Pictoamide may be the unique one. Picotamide (Fig. 1, left),
N,N′-bis(3-picolyl)-4-methoxyisophthalamide, is a deriva-
tive of 4-methoxyliso-phtalic acid and a antiplatelet aggre-
gating drug, which inhibits both thromboxane A (TXA )
7
2.1% (induced by collagen) and 72.5% (induced by ADP),
with IC50 values of 0.30 μM/L induced by ADP (1.3 μM/L)
and LD50 > 2500 mg/kg, could have dual mechanism of
action. Evaluation of cytotoxic activity of the compounds
against L929 cell line revealed that none of the compounds
have significant cytotoxicity. Through the careful analysis
of in vitro activity data, the SAR of these compounds was
preliminarily deduced. The results of this study showed that
4
-methoxy diaryl isophthalates derivatives are potential to
become an antiplatelet aggregation agents.
*
*
Liu Xiu-jie
liuxiujie@hotmail.com
2
2
receptors and TXA synthesis (Yip and Benavente 2011;
Violi et al. 1988) and does not interfere with endothelial
prostacyclin(PGI ) production, in contrast to Aspirin (Gre-
2
Liu Xu-guang
liuchembc@163.com
2
1
2
sele et al. 1989; Celestini and Violi 2007; Sartori et al.
The College of Chemistry and Chemical Engineering, Tianjin
University of Technology, Tianjin 300384, China
1993).
Driven by the well documented pictoamide, our group
has been carrying a project on the synthesis and study of the
antiplatelet acivity of some pictoamide analogs (Liu et al.
Tianjin Key Laboratory of Organic Solar Cells and Photochemical
Conversion, School of Chemistry and Chemical Engineering
Tianjin University of Technology, Tianjin 300384, China

Med Chem Res (2018) 27:488–496
489
2
006, 2011, 2012). Initially, we focused our attention on the
Instruments
compounds to the original structure of pictoamide (Fig. 1,
Series 1). We have carried out the antiplatelet aggregating
activity studies of a series of 4-methoxyisophthalamides,
which are the structural analogs of picotamide. Screening of
the series 1 highlighted several relatively potent com-
pounds. Specifically, compound P113 with a benzyl group
attached to the phenyl ring in the parent compound showed
the highest activity, with IC50 value is as low as 0.02 μM/L
and the LD50 is over 5000 mg/kg. It is 36 times as Pictoa-
mide’s (IC50 = 0.76 μM/L, LD50 = 2500 mg/kg).
Melting points were determined with a Kofler micro melting
point apparatus without corrected; IR spectra were recorded
on Fourier transfrom infrared (FTIR) 1700 infrared spec-
trophotometer and expressed in cm using KBr pelets; 1H-
NMR spectra are determined in deuterated solvents
−1
(DMSO/CDCl ), using tetramethylsilane as an internal
3
standard (chemical shift was measured in δ ppm) with a
Bruker-400 MHz spectrometer. Splitting patterns were as
follows: s, singlet; d, doublet; dd, double doublet; t, triplet;
q, quartet; qt, quintet; m, multiplet; Mass spectra were
measured on Xevo GZQ-Tof VHRS mass spectrometer.
It is well-known that isosterism are wildly utilized in the
medicinal chemistry. We envisioned that replacing the two
amide components in the P1 series with two ester compo-
nents might open another door for the pictoamide-like drugs
(
Fig. 2, right). After extensively literature search, It is sur-
Methods and results
prising for us the P2 series are underexplored as antiplatelet
drugs before our work. Indeed, our preliminary results
showed that the compound P202 bearing a bromide at the
para position of the phenyl ring exhibited higher antiplatelet
activity than Pictoamide (Fig. 2, P202).
Structural design and synthetic routes
19 4-Methoxyl isophthalates were designed and synthesized
based on the use of acyloxy subunits. With the aim of
guaranteeing acceptable levels of diversity for the series P2
library. Single or multiple substituents on the phenyls rings
are designed (Scheme 1). It can be divided into four several
subset. Subset P203–P208 are aimed to study the halogen
atoms (F, Cl, and Br) attached to the ortho or meta-position
on the phenyl ring, since the halogen atoms on the para-
positions has been published previously (Meng et al. 2011).
The second subset P209–P216 are focused on the investi-
gation on the antiplatelet activity influenced by introducing
Intriguing by the preliminary result and in order to
release the full potential of series P2 as antiplatelet drugs,
we carried out greater detailed studies on the effect on the
substituent on the phenyl ring in series P2. The chemical
structures of each new compounds have been confirmed by
1
13
H nuclear magnetic resonance (NMR), C NMR, mass
spectrometry (MS), and infrared (IR) spectra, and in vitro
antiplatelet aggregation activities of series P2 were inves-
tigated by using Born’s test for both ADP and collagen
inducers. The compounds with higher inhibition ratio were
selected to determine acute toxicity in mice and the L-929
cell lines of cytotoxicity.
electron withdrawing (NO , COOMe, and NHCOMe) and
2
donating group (OMe) to the phenyl ring. In order to study
the mechanism of antiplatelet activities of the prodrug is
Fig. 1 Structures of picotamide,
4
-methoxylisophthalamides and
P113
Fig. 2 Structures relationship between series P1 and P2

4
90
Med Chem Res (2018) 27:488–496
Scheme 1 Synthetic routes to the 4-methoxy diphenyl isophthalates (P203–P219)
Scheme 2 Synthetic routes to the P220 and P221
another reason to design P215 and P216, envisioned that
aspirin and acetaminophen have the similar methyl salicy-
late and acetamino subunit (Seymour et al. 1984). Besides
the single substituent, multiple substituent derivatives
P217–P219 are also included to compare the activity with
mono-substituted ones. Taking into account that NO group
is an important messenger group in the regulation of many
physiological functions (Ellis et al. 2005; Zhao et al. 2012),
P219 bearing a NO group and two chloride groups was
designed.
followed by nucleophilic substitution with silver nitrate.
Experimental details are depicted in the synthetic pathways
shown in Scheme 2.
Experimental details
The synthesis of 4-methoxyl-isophthaloyl dichloride fol-
lowed the literature procedure (Liu et al. 2006).
Typical procedures for condensation of 4 with phenols.
The target compounds P203–P219 were accessible from
the universal building block 4 (Scheme 1). Blank chlor-
omethylation of anisole gave 2,4-bis(chloromethyl)-1-
methoxybenzene 2, subsequently 2 was oxidized to produce
4-Methoxydi(4-acetamidophenyl)isophthalate (P216)
A three-necked flask (100 mL) was charged with acet-
aminophen (10.0 mmol, 1.51 g) in anhydrous tetra-
hydrofuran (15 mL), 4-methoxyl-isophthaloyl dichloride 4
(5.0 mmol, 1.17 g) in dry tetrahydrofuran (10 mL) was
added portionwise to the above solution at room tempera-
4
-methoxyisophthalic acid 3. Treatment of 3 with thionyl
chloride delivered the universal building block 4-
methoxyisophthaloyl dichloride 4. Condensation of 4 with
various phenols produced P203–P219.
ture under N . Ten minutes after addition of compound 4,
2
The design of P220 and P221 derivatives was supported
dry pyridine (1 mL) was added dropwise to the reaction
mixture. The mixture was stirred for additional 8 h at room
by the presence of a similar ONO fragment in the the non-
2
steroidal anti-inflammatory drug (NSAID) naproxcinod
temperature under N . After the reaction completed as
2
(
Scheme 2), the nitroxybutyl ester is also act as a nitric
monitored by thin layer chromatography (TLC), the bulky
solvent was removed under reduced pressure. The con-
centrate was cooled in a refrigerator. The precipitate was
filtered and washed with water and ethanol successively.
The crude product was purified by recrystallization from
oxide (NO) donor (Ellis et al. 2005; Nagy et al. 2006). P220
was prepared by reaction of compound 4 with silver nitrate
in a single step. P221 can be accessed from 4 in two steps,
condensation of 4 with 2-chloroethan-1-ol afforded 5,

Med Chem Res (2018) 27:488–496
491
acetone to yield the desired product P216 as white solid.
Yield: 60.3%, M.p: 237–239 °C.
The other compounds P203–P215 and P217–P219 were
prepared in the similar manner.
MHz, CDCl , TMS) δ (ppm): 4.054 (s, 3H, OCH ), 7.149
3
3
(d, 1H, 5-H), 7.168–7.390 (m, 8H, 2 × C H ), 8.375 (dd,
6
4
1
3
1H, 6H), 8.812 (d, 1H, 2-H). C NMR (101 MHz, DMSO-
d6) δ (ppm): 170.1 (C=O), 166.2 (C=O), 162.8, 149.9,
148.3, 135.5, 133.3, 132.6, 126.7, 123.2, 121.5, 120.3,
P203 Yield = 54.00%; M.p: 125–126 °C; MS (m/z): 790.8
115.3, 109.3, 108.6, 54.8 (OCH3).
+
−1
[
2M + Na] ; IR (KBr) cm : 1747.39, 1732.14 (υC=O),
1
610.34 (υC=C), 1500.25 (βAr–H), 1258.25, 1175.65,
P208 Yield = 51.76 %; M.p: 134–135 °C; MS (m/z):
1034.2 [2M + Na] ; IR (KBr) cm : 1725.83 (υC=O),
1616.06, 1584.90 (υC=C), 1498.05, 1472.22 (βArH),
1
+
−1
1
044.32 (υC–O–C), 753.35 (γAr–H); H NMR (400 MHz,
CDCl , tetramethylsilane (TMS)) δ (ppm): 4.054 (s, 3H,
3
OCH ), 7.164 (d, 1H, 5-H), 7.192–7.295 (m, 8H, 2 ×
1280.17, 1218.99, 1190.40, 1086.98 (υC–O–C), 852.32,
3
1
3
1
C H ), 8.415 (dd, 1H, 6-H), 8.925 (d, 1H, 2-H); C NMR
768.91 (γAr–H); H NMR (400 MHz, CDCl , TMS) δ (ppm):
6
4
3
(
(
1
101 MHz, DMSO-d6) δ (ppm): 169.14 (C=O), 166.1
C=O), 162.8, 148.3, 136.5, 132.6, 127.8, 122.6, 121.5,
15.8, 55.7 (OCH3).
4.052 (s, 3H, OCH ), 7.154 (d, 1H, 5-H), 7.180–7.448 (m,
3
8H, 2 × C H ), 8.371 (dd, 1H, 6-H), 8.807 (d, 1H, 2-H).
6
4
1
3
C NMR (101 MHz, DMSO-d6) δ (ppm): 170.1 (C=O),
66.2 (C=O), 164.4, 149.9, 148.3, 135.5, 133.3, 132.6,
1
P204 Yield = 62.58 %; M.p: 115–116 °C; MS (m/z):
126.7, 123.2, 121.5, 120.3, 119.3, 117.8, 54.8 (OCH3).
+
−1
8
56.5 [2M + Na] ; IR (KBr) cm : 1755.82,1742.02
(
1
υC=O), 1609.76 (υC=C), 1476.93 (βAr–H), 1280.56,
199.04, 1071.11 (υC–O–C), 756.97 (γAr–H); H NMR (400
P209 Yield = 41.02 %; M.p: 137–138 °C; MS (m/z): 461
1
+
−1
[M + Na] ; IR (KBr) cm : 1750.19 (υC=O), 1603.28
(υC=C), 1527.73, 1348.95 (υNO2), 1279.78, 1199.88,
MHz, CDCl , TMS) δ (ppm): 4.056 (s, 3H, OCH ), 7.172
(
1
3
3
1
d, 1H, 5H), 7.237–7.504 (m, 8H, 2 × C H ), 8.438 (dd,
1089.29 (υC–O–C), 734.75 (γArH); H NMR (400 MHz,
6
4
1
3
H, 6-H), 8.994 (d, 1H, 2-H); C NMR (101 MHz, DMSO-
CDCl , TMS) δ (ppm): 4.061 (s, 3H, OCH ), 7.184 (d, 1H,
3
3
d6) δ (ppm): 169.3 (C=O), 165.2 (C=O), 163.4, 147.9,
5-H), 7.397–8.171 (m, 8H, 2 × C H ), 8.422 (dd, 1H, 6-H),
6 4
8.963 (d, 1H, 2-H). C NMR (101 MHz, DMSO-d6) δ
1
3
1
48.3, 136.5, 133.3, 132.6, 127.8, 126.7, 123.2, 121.5,
1
20.3, 116.1, 55.8 (OCH3).
(ppm): 169.1 (C=O), 167.2 (C=O), 164.7, 148.9, 148.3,
136.5, 133.3, 132.6, 132.3, 119.3, 117.8, 55.8 (OCH3).
P205 Yield = 43.44 %; M.p: 139–140 °C; MS (m/z):
+
−1
5
(
1
07.1 [2M + Na] ; IR (KBr) cm : 1756.84, 1741.88
P210 Yield = 35.23 %; M.p: 181–182 °C; MS (m/z): 461
-1
υC=O), 1609.68 (υC=C), 1471.94 (βArH), 1279.55, 1196.82,
069.00, 1047.27 (υC–O–C), 756.37 (γAr–H); H NMR (400
MHz, CDCl , TMS) δ (ppm): 4.064 (s, 3H, OCH ), 7.169
[M + Na]+; IR (KBr) cm : 1728.98, 1709.76 (υC=O),
1539.89, 1349.62 (υNO2), 1276.76, 1236.84, 1071.74
(υC–O–C), 812.35, 732.47 (γAr–H); 1H NMR (400 MHz,
1
3
3
(
1
d, 1H, 5-H), 7.192–7.672 (m, 8H, 2 × C H ), 8.455 (dd,
H, 6-H), 9.053 (d, 1H, 2-H). C NMR (101 MHz, DMSO-
CDCl3, TMS) δ (ppm): 4.090 (s, 3H, OCH ), 7.210 (d, 1H,
6
4
3
1
3
5-H), 7.624–8.194 (m, 8H, 2 × C H ), 8.430 (dd, 1H, 6-H),
6
4
d6) δ (ppm): 169.3 (C=O), 165.2 (C=O), 163.7, 147.9,
8.879 (d, 1H, 2-H). 13C NMR (101 MHz, DMSO-d6) δ
(ppm): 172.1 (C=O), 167.2 (C=O), 164.5, 153.3, 148.7,
136.8, 127.6, 125.6, 120.7, 116.3, 55.8 (OCH3).
1
48.3, 136.5, 133.3, 132.6, 127.8, 126.7, 123.2, 121.5,
1
20.3, 115.3, 54.8 (OCH3).
P206 Yield = 49.76%; M.p: 117–118 °C; MS (m/z): 385.2
P211 Yield = 39.27 %; M.p: 210–212 °C; MS (m/z): 461
[M + Na] ; IR (KBr) cm : 1739.32 (υC=O), 1610.49
+
−1
+
−1
[
M + H] ; IR (KBr) cm : 1743.73, 1708.04 (υC=O),
1
1
608.32 (υC=C), 1487.09 (βArH), 1281.63, 1248.90,
(υC=C), 1522.28, 1348.09 (υNO2), 1280.62, 1215.19,
1
1
225.64, 1125.84 (υC–O–C), 770.09, 675.45 (γAr–H);
H
1162.06, 1089.21 (υC–O–C), 866.73, 746.48 (γAr–H);
H
NMR (400 MHz, CDCl , TMS) δ (ppm): 4.054 (s, 3H,
NMR (400 MHz, CDCl , TMS) δ (ppm): 4.083 (s, 3H,
3
3
OCH ), 7.158 (d, 1H, 5-H), 7.364–7.428 (m, 8H, 2 ×
OCH ), 7.204 (d, 1H, 5H), 7.424–7.454, 8.326–8.355 (m,
3
3
1
3
C H ), 8.381 (dd, 1H, 6H), 8.823 (d, 1H, 2-H). C NMR
8H, 2 × C H ), 8.423 (dd, 1H, 6-H), 8.856 (d, 1H, 2-H).
6
4
6 4
1
3
(
(
101 MHz, DMSO-d6) δ (ppm): 170.1 (C=O), 166.2
C=O), 162.8, 149.9, 148.3, 135.5, 133.3, 132.6, 126.7,
C NMR (101 MHz, DMSO-d6) δ (ppm): 170.2 (C=O),
165.6 (C=O), 160.5, 155.6, 154.3, 144.6, 143.2, 136.5,
1
23.2, 121.5, 120.3, 115.3, 109.3, 54.8.
131.6, 125.3, 122.5, 122.3, 117.6, 55.9 (OCH3).
P207 Yield = 53.64 %; M.p: 132–133 °C; MS (m/z):
P212 Yield = 73.94 %; M.p: 157–159 °C; MS (m/z):
838.9 [2M + Na] ; IR (KBr) cm : 1747.18, 1727.14
+
−1
+
−1
8
1
1
56.5 [2M + Na] ; IR (KBr) cm : 1725.93 (υC=O),
591.78 (υC=C), 1498.30 (βAr–H), 1282.56, 1219.33,
(υC=O), 1610.87 (υC=C), 1497.17 (β ), 1168.22, 1109.68,
ArH
1
1
194.02 (υC–O–C), 770.66, 680.20 (γAr–H); H NMR (400
1048.03 (υC–O–C), 757.54 (γArH); H NMR (400 MHz,

4
92
Med Chem Res (2018) 27:488–496
CDCl , TMS) δ (ppm): 3.837 (d, 6H, 2 × C H OCH ),
(m, 8H, 2 × C H ), 8.352 (dd, 1H, 6-H), 8.594 (d, 1H, 2-H),
3
6
4
3
6 4
1
3
4
.033 (s, 3H, OCH ), 7.130 (d, 1H, 5-H), 6.969–7.262 (m,
10.028 (s, 2H, 2 × CONH). C NMR (101 MHz, DMSO-
d6) δ (ppm): 169.8 (C=O), 168.9 (NHC=O), 167.8
(NHC=O), 165.4 (C=O), 163.7, 145.0, 143.0, 136.5,
3
8
H, 2 × C H ), 8.395 (dd, 1H, 6-H), 8.942 (d, 1H, 2-H).
6
4
1
3
C NMR (101 MHz, DMSO-d6) δ (ppm): 171.1 (C=O),
1
1
66.3 (C=O), 162.8, 151.7, 150.2, 138.5, 138.4, 136.5,
135.3, 122.2, 121.2, 121.8, 117.4, 113.5, 55.9 (OCH ), 26.3
3
31.8, 126.5, 125.4, 121.0, 117.6, 115.3, 56.5 (OCH ), 55.3
(CH ), 24.1 (CH ).
3
3
3
(
OCH ), 54.6 (OCH ).
3 3
P217 Yield = 53.33 %; M.p: 134–137 °C; MS (m/z):
+
−1
P213 Yield = 49.48 %; M.p: 103–104 °C; MS (m/z):
487.12 [M + H] ; IR (KBr) cm : 1740, 1615, 1466, 1275,
+
−1
1
8
1
1
38.9 [2M + Na] ; IR (KBr) cm : 1731.74 (υC=O),
1196, 1122; H NMR (400 MHz, CDCl , TMS) δ (ppm):
3
607.91 (υC=C), 1491.37 (βAr–H), 1270.25, 1207.51,
4.077 (s, 3H, OCH ), 7.191–7.537 (m, 6H, 2 × C H ), 7.16
3
6 3
1
13
146.23 (υC–O–C), 773.19, 757.89, 686.96 (γAr–H); H NMR
(d, 1H, 5-H), 8.373 (dd, 1H, 6-H), 8.801 (d, 1H, 2-H).
C
(
400 MHz, CDCl , TMS) δ (ppm): 3.827 (s, 6H, 2 ×
NMR (101 MHz, DMSO-d6) δ (ppm): 170.1 (C=O), 166.2
(C=O), 163.7, 151.6, 150.8, 136.5, 133.8, 131.6,131.7,
130.2,122.8, 122.4, 121.2, 117.4, 113.5, 55.7 (OCH3).
3
C H OCH ), 4.047 (s, 3H, OCH ), 7.145 (d, 1H, 5H),
6
1
6
4
3
3
.783–6.852, 7.307–7.355 (m, 8H, 2 × C H ), 8.379 (dd,
6 4
1
3
H, 6-H), 8.845 (d, 1H, 2-H). C NMR (101 MHz, DMSO-
d6) δ (ppm): 170.1 (C=O), 166.7 (C=O), 164.2, 162.3,
P218 Yield = 50.57 %; M.p: 100–102 °C; MS (m/z):
+
−1
1
61.4, 152.3, 151.6, 136.5, 131.7, 130.4, 117.4, 113.9,
405.2 [M + H] ; IR (KBr) cm : 1729.92 (ʋC=O), 1604.96,
1500.83 (ʋC=C), 1268.76, 1176.53, 1120.00, 1036.69
1
11.1, 107.6, 106.5, 56.2 (OCH ), 54.1 (OCH ), 52.4
3
3
1
(
OCH3).
(ʋC–O–C), 834.38, 757.02, 664.79; H NMR (400 MHz,
CDCl , TMS) δ (ppm): 2.19 (s, 3H, CH ), 2.24 (s, 3H,
3
3
P214 Yield = 56.87 %; M.p: 119–120 °C; MS (m/z):
CH ), 2.33 (s, 6H, 2 × CH ), 4.03 (s, 3H, OCH ), 7.02–7.08
3
3
3
+
−1
8
38.9 [2M + Na] ; IR (KBr) cm : 1733.66, 1707.49
(m, 8H, 2 × –C H ), 7.13 (d, 1H, J = 8.8 Hz, 5-H), 8.39
6 3
(
8
υC=O), 1508.85 (υC=C), 1183.57, 1086.73 (υC–O–C),
(dd, 1H, J = 8.8 Hz, J = 2.0 Hz, 6-H), 8.85 (d, 1H, J =
1 2
1
13
47.59, 825.46 (γAr–H); H NMR (400 MHz, CDCl , TMS)
2.0 Hz, 2-H). C NMR (101 MHz, DMSO-d6) δ (ppm):
173.6 (Ar–NO), 172.9 (Ar–NO), 170.0 (C=O), 165.2
(C=O), 169.3, 168.1,163.7, 138.5, 137.2, 132.1, 131.6,
119.4, 117.8, 117.6, 55.6 (OCH3).
3
δ (ppm): 3.828 (d, 6H, 2 × C H -OCH ), 4.038 (s, 3H,
6
4
3
OCH ), 7.160 (d, 1H, 5-H), 6.927–7.144 (m, 8H, 2 ×
3
1
3
C H ), 8.366 (dd, 1H, 6-H), 8.828 (d, 1H, 2-H); C NMR
6
4
(
(
101 MHz, DMSO-d6) δ (ppm): 169.9 (C=O), 165.3
C=O), 164.7, 157.6, 157.3, 141.7, 140.6, 136.5, 131.6,
P219 Yield = 70.37 %; M.p: 139–141 °C; MS (m/z):
545.12 [M + H] ; IR (KBr) cm : 1752.07 (ʋC=O),
+
−1
1
22.4, 122.6, 117.8, 114.7, 113.6, 56.7 (OCH ), 55.3
3
(
OCH ), 54.4 (OCH ).
3 3
1606.91, 1449.31 (ʋC=C), 1270.97, 1179.72, 1076.04,
1
1
017.97 (ʋC–O–C), 852.07, 769.1, 541.01; H NMR (400
P215 Yield = 68.59 %; M.p: 95–97 °C; MS (m/z): 487.3
MHz, CDCl , TMS) δ (ppm): 4.088 (s, 3H, OCH ), 7.444
3
3
+
−1
[
M + Na] ; IR (KBr) cm : 1729.14 (υC=O), 1606.61
(m, 4H, 2 × C H ), 7.228 (d, 1H, 5-H), 8.511 (dd, 1H, 6-H),
6
2
1
3
(
υC=C), 1276.85, 1188.86, 1083.51, 1046.77 (υC–O–C),
9.070 (d, 1H, 2-H);
C NMR (101 MHz, DMSO-d6) δ
1
7
50.60, 693.87 (γAr–H); H NMR (400 MHz, CDCl , TMS)
3
(ppm): 166.3 (C=O), 165.8 (C=O), 163.6, 136.5, 132.2,
122.7, 117.4, 113.5, 55.7 (OCH3).
δ (ppm): 3.836 (d, 6H, 2 × C H -COOCH ), 4.049 (s, 3H,
6
4
3
OCH ), 7.170 (d, 1H, 5-H), 7.263–8.090 (m, 8H, 2 ×
3
1
3
C H ), 8.425 (dd, 1H, 6-H), 9.009 (d, 1H, 2-H). C NMR
6
4
(
(
101 MHz, DMSO-d6) δ (ppm): 170.1 (C=OOCH ), 169.9
Experimental procedures for preparation of P220 and
P221
3
C=O), 168.8 (C=OOCH ), 165.8 (C=O), 163.7, 153.6,
3
1
53.4, 136.4, 133.2, 130.0, 130.3, 129.8, 125.6, 124.8,
1
23.1, 122.4, 117.4, 119.6, 56.1 (OCH ), 51.3 (CH ), 50.2
4-Methoxy-1,3-nitric acid anhydride (P220)
3
3
(
CH3).
A three-necked flask (100 mL) was charged with compound
4 (13 mmol, 3.0 g) in acetonitrile (10 mL), silver nitrate (13
mmol, 3.0 g) dissolved in acetonitrile (5 mL) was slowely
P216 Yield = 60.40 %; M.p: 237–239 °C; MS (m/z):
+
−1
4
1
1
1
63.4 [M + H] ; IR (KBr) cm : 3369.48 (υNH), 1740.50,
716.27 (υO=C–O), 1686.39 (υO=C–NH), 1608.66 (υC=C),
533.47 (βNH), 1506.65 (βAr–H), 1405.41 (υC–N), 1189.32,
added to stirred solution at room temperature under N in
2
dark. The mixture continued stirring for 24 h at 50 °C in a
water bath with a mechanical stirrer. After the reaction is
complete, the mixture was cooled to room temperature and
the solid was filtered. Filtrate was concentrated under
1
048.64 (υCOC), 843.69 (γArH); H NMR (400 MHz,
CDCl , TMS) δ (ppm): 2.055 (s, 6H, 2 × COCH ), 4.007 (s,
3
3
3
H, OCH ), 7.440 (d, 1H, 5-H), 7.197–7.228, 7.634–7.656
3

Med Chem Res (2018) 27:488–496
493
Table 1 In vitro antiplatelet aggregation activities & acute toxicity of
compounds P201, 203–221
solution (10 mL) and cold water (25 mL) for twice. The oil
layer was collected and dried over anhydrous magnesium
sulfate. After filtration, the filtration was dried over desic-
cant; the mixture was concentrated under reduced pressure
to obtain 6 as a white solid. Yield: 64.3%.
Groups
Dose
μmol/L)
Inhibition (%)
IC50
(μM/L)
LD50
(mg/kg)
(
Collagen ADP ADP
Solvent
P201
P203
P204
P205
P206
P207
P208
P209
P210
P211
P212
P213
P214
P215
P216
P217
P218
P219
P220
P221
Aspirin
–
–
–
–
–
–
–
–
Bis(2-(nitrooxy)ethyl)4-methoxyisophthalate (P221)
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
1.3
46.5
49.1
40.3
46.2
48.0
48.4
50.2
52.3
52.9
44.8
47.3
43.6
50.6
43.7
64.1
48.9
69.2
59.8
72.1
61.6
68.3
54.2
47.4 0.81
49.5 0.79
A three-necked flask (100 mL) was charged with a solution
of compound 6 (1.0 g, 3.0 mmol) in acetonitrile (10 mL).
And silver nitrate (6.0 mmol, 1.05 g) was added slowly to
the stirred solution. The mixture was stirred for 10 h in a
41.8
–
47.8 0.85
48.8 0.91
48.5 0.90
50.2 0.80
52.0 0.92
53.1 0.83
–
–
50°C water bath with a mechanical stirrer. The mixture was
–
cooled to room temperature and concentrated under reduced
pressure to obtain a light yellow liquid. The liquid was
dissolved with acetic ether (25 mL) and washed with cold
saturated sodium chloride solution (25 mL) and cold water
–
–
45.7
48.5 0.94
44.4
51.5 0.85
44.4
–
–
–
(30 mL) successively. Separating the oil phase, dried over
–
–
anhydrous magnesium sulfate (MgSO ). After filtering the
4
–
desiccant, the mixture was concentrated under reduced
pressure to obtain P221 as yellow liquid. 0.46 g, yield:
–
–
64.3 0.54
66.3 0.78
68.0 0.70
59.7 0.77
72.5 0.30
62.4 0.61
69.6 0.33
56.5 0.76
1486
>2500
>2500
>2500
>2500
2102
–
44.5%.
P221 Yield = 44.5 %; M.p: liquid; MS (m/z): 375.2 [M +
+
−1
H] ; IR (KBr) cm : 2962.30 (ʋ–CH3), 1716.02 (ʋ–C=O),
627.65 (ʋ–C=O), 1502.96 (ʋNO2), 1125.13 (ʋNO2), 1022.12
1
(ʋ–O–C–), 1459.41 (benzene), 767.35 (benzene), 1273.6,
1
4678.66; H NMR (400 MHz, CDCl , TMS) δ (ppm): 8.42
3
Picotamide 1.3
1250
(d, 1H, benzene-H), 7.28 (s, 1H, benzene-H), 7.07 (d, 1H,
benzene-H), 4.60 (t, 4H, –CH –), 4.00 (t, 4H, –CH –), 3.83
2
2
1
3
(
1
1
s, 3H, –OCH3). C NMR (101 MHz, DMSO-d6) δ (ppm):
reduced pressure and cooled. The precipitate was filtered to
obtain a yellow solid. The product was dried in a vacuum
oven to give P220. 3.62 g. Yield: 97.2 %, Mp: 212 °C
65.9 (C=O), 163.2 (C=O), 162.1, 135.4, 130.7, 122.5,
13.4, 62.2, 72.3, 56.1 (OCH3).
(
decomposition).
Biological evaluation
P220 Yield = 97.2 %; M.p: over 212 °C; MS (m/z): 287.2
+
−1
[
(
(
(
M + H] ; IR (KBr) cm : 1652.52 (ʋC=O), 1384.87
In vitro antiplatelet aggregation activity
ʋNO2), 1273.77 (ʋNO2), 1087.27 (ʋ–C–O–N), 1003.94
ʋC–O–N); H NMR (400 MHz, CDCl , TMS) δ (ppm): 8.53
d, 1H, benzene-H), 8.18 (s, 1H, benzene-H), 7.15 (d, 1H,
benzene-H), 3.84 (s, 3H, –OCH3). C NMR (101 MHz,
DMSO-d6) δ (ppm): 166.3 (C=O), 165.8 (C=O), 163.6,
1
3
The in vitro antiplatelet aggregation activities of 19 4-
methoxyl isophthalates were evaluated by their ability to
inhibit platelet aggregation or rabbit platelet-rich plasma
(PRP) using the Born test (Barbosa et al. 2006): Venous
blood from the rat eye socket vein was collected into tubes
containing 3.8% sodium citrate (1:9, v/v). Platelet aggre-
gation was assessed in platelet-rich plasma (PRP), obtained
by centrifugation of citrated whole blood at room tem-
perature for 10 min (500–800 rpm). The aggregation rate
was measured by platelet aggregation analyzer after sti-
mulation with collagen (5 mM) and ADP using platelet-
poor plasma (PPP) set to zero. The PPP was obtained by
centrifugation of PRP at room temperature for 15 min
1
3
1
36.5, 132.2, 122.7, 117.4, 113.5, 55.7 (OCH3).
Bis(2-(chclorly)ethyl)4-methoxyisophthalate (6)
A three-necked flask (100 mL) was charged with a solution
of compound 4 (8.0 mmol, 2.0 g) in toluene (15 ml). 2-
chloroethanol (17 mmol, 1.38 g) was added dropwise. The
mixture was stirred and refluxed for 6 h. Then the mixture
was cooled and washed with cold 5% NaOH aqueous
−
1
(3000 rpm). A solution of the compounds (1.3μmol L ) in

4
94
Med Chem Res (2018) 27:488–496
Table 2 Cytotoxicity effect of P216, P217, P218, P219 and P220,
P221 on L-929 cells
DMSO or chloroform (5 μL) was added into PRP (200 μL),
and the same volume of DMSO or chloroform without test
compound was added to a reference sample (according to a
pre-experiment, 5 μL of DMSO or chloroform appears to
have no significant effect on the platelet aggregation). After
incubating for 2 min, the platelet aggregation was assessed
and the percentage inhibition of platelet aggregation was
calculated using the corresponding ADP. Platelet aggrega-
tion inhibition (%) was calculated by the following formula:
Compd.
Dose (μmol/L)
Absorbance
Survival rate (%)
Blank group
Control group
P220
–
0.184
0.767
0.740
0.503
0.701
0.503
0.711
0.610
0.651
0.574
0.672
0.556
0.572
0.44
–
–
–
10
95.37
54.72
93.24
54.72
93.85
60.21
86.13
66.57
88.41
65.21
66.55
40.29
90.92
85.26
1
00
P216
10
1
00
P217
10
Inhibition % ¼ ð1 À D=SÞ Â 100%;
1
00
P218
10
where D is the platelet aggregation in the presence of test
compounds, and S is the platelet aggregation in the presence
of solvent. The primary screening data for all compounds
1
00
P219
10
1
00
(
1.3 μM) in vitro activities on antiplatelet aggregation of the
synthesized compounds are given in Table 1 and the IC50
values were calculated for compounds with a inhibition rate
of more than 47%.
P221
10
100
10
Picomaide
0.885
0.735
1
00
Acute toxicity test
was calculated as follows. (Xiong et al. 2007).
With the IC50 values (0.78 μM/L) of new compounds less
than or close to the Picotamides, six compounds P216,
P217, P218, P219, P220, and P221 were selected to con-
tinue the acute toxicity test in mice. One hundred and eight
mice (weight 18–20 g) were randomly divided into eighteen
groups by weight and sex. Doses were 625,1250, and 2500
mg/kg, formulated with 0.5% CMC–Na. The acute toxicity
test included continuous observation for 2 weeks after
intragastric administration (Xu et al. 1991, Table 1).
Abs ðtest cellÞ
Cell viability ð%Þ ¼
 100%
Abs ðcontrolled cellÞ
Discussion
Biological evaluation
Cytotoxicity effect on L-929 cells
In total nineteen 4-methoxy diphenyl isophthalates were
designed and synthesized and the chemical structures were
1
13
Mouse fibroblast cells (L929) was chosen to evaluate the
in vitro cytotoxicity of the materials and the drugs via Cell
Counting Kit-8 (CCK-8) assays. L929 was cultivated in a
humidified 5% carbon dioxide atmosphere at 37 °C on 96-
confirmed by H NMR, C NMR, and MS spectroscopy.
The primary screening data for all compounds (1.3 μM) and
the acute toxicity are shown in Table 1, and the IC50 values
were calculated for compounds with a inhibition rate of
more than 47%. Te results, in Table 1, demonstrate that
compound P220 bearing a nitrooxyl group and with IC50
values of 0.30 induced by ADP and LD50 > 2500 mg/kg
and more potential than both Picotamide and Aspirin, show
highest acitivities 72.1% (induced by collagen) and 72.5%
(induced by ADP) and could have dual mechanism of
action, as the ADP and collagen induced the aggregation by
4
well microplates, with 1 × 10 cells per well immersed in
complete growth medium. The cells with 100 mL of RPMI-
1
640 per well were allowed to attach for 24 h. Subse-
quently, the cells were then exposed to target compounds at
a range of concentrations at 37 °C for 48 h. Target com-
pounds concentration from 100 to 1000 mg mL were added
1
to L929 cells. After incubation for 48 h, the medium was
removed and replaced with 100 mL of fresh complete
medium of RPMI-1640. Then, CCK-8 solution was added
to the 96-well plates at 10 mL per well and incubated for a
further 2 h, and the absorbance at 450 nm was measured on
a microplate reader (Bio-Tek FLx800 fluorescence micro-
plate reader) (Xu et al. 1991, Table 2). This process was
repeated eight times in parallel. The results are expressed as
the relative cell viability (%) with respect to control wells, it
thromboxane (TXA ) formation. The other five compounds
2
(P216, P217, P218, P219, and P221) were able to inhibit
the platelet aggregation induced by ADP in 64.3, 66.3, 68.0,
59.7, and 62.4%, superior to Picotamide (56.5%) and four
compounds had a higher inhibitory rate at 1.3 μM induced
by collagen in 64.1%, 69.2, 59.8, and 61.6% than Picota-
mide (54.2%). As shown in Table 1, with the same LD50,
compounds P217, P218, P219, and P220 showed very low

Med Chem Res (2018) 27:488–496
495
acute toxicity at quite high LD50 (2500 mg/kg). In the test of
cytotoxicity effect on L-929 cells, as shown in Table 2,
among the six compounds tested at the dose of 10 μmol/L,
the cell survival rate of three compounds (P220, P216, and
P217) was higher than that of Picotamide (90.92%) and the
cell survival rate of compound P220 (95.37%) was the
highest, however, when the concentration increased by ten
times, the cell survival rate of six compounds was lower
than that of Picotamide (85.26%).
72.1% (induced by collagen) and 72.5% (induced by ADP).
Further study needed to be explored.
Conclusion
Nineteen novel analogs of Picotamide were prepared and
evaluated as potential antiplatelet aggregation agents. The
strongest antiplatelet activity to all tested compounds was
observed for compound P220, followed by P218, P217,
P216, P221, and P219. The preliminary SAR study shows
that the antiplatelet aggregation activity can be fine changed
via the introduction of substituents in various positions of
the phenyl group. In general, the analogs with an electron-
withdrawing group in para-position turned out to be more
potent than the others. It has been identified that compound
P220 with a nitric acid ester exhibited the most activity,
lower acute toxicity and cytotoxicity. Introduction of an
additional nitroso group in ortho-position modified the
activity only slightly while nitric acid ester and nitroox-
yethyl ester with no phenyl showed good activity. In con-
clusion, 4-methoxy diphenyl isophthalate derivatives are
potential to be studied and might help in developing new
antiplatelet drugs with elevated activity. In the test of
cytotoxicity effect on L-929 cells, as shown in Table 2,
among the six compounds tested at the dose of 10 μmol/L,
the cell survival rate of three compounds (P220, P216,
P217) was higher than that of Picotamide (90.92%) and the
cell survival rate of compound P220 (95.37%)was the
highest, however, when the concentration increased by ten
times, the cell survival rate of six compounds was lower
than that of Picotamide (85.26%). And further work is
needed to better understand the role of these specific factors
in the future.
Preliminary structure–activity relationship (SAR)
exploration
For ADP-induced platelet aggregation, with IC50 as a jud-
ging basis values, there are 12 compounds with a score of
0
.85 or less. The order of inhibition of platelets is:
P220 (0.30) > Aspirin (0.33) > P216 (0.54) > P221
(
(
(
0.61) > PN218
0.76) > P217 (0.78) > P203 (0.79) > P208 (0.80) > P210
0.83) > P214 (0.85).
(0.70)) > PN219
(0.77) > Picotamide
With different mono-halogen substituted phenyl rings in
the same position, the order of inhibition of platelets is: in
ortho-position of the phenyl: F(P203) > Br(P205) > Cl
(
P204); in meta-position: Br(P208) > F(P206) > Cl(P207).
no matter in given high or low dose and in para-position is
Br(P202) > Cl. Thus, in general, fluorine- and bromine-
substituted compounds are more potent than the chlorine-
substituted analogs.
The activity order of inhibition of platelets of the same
halogen at different positions is: ortho-F(P203) > ortho-F
(
P206) and ortho-Cl(P204) < meta-Cl(P207) < para-Cl and
ortho-Br(P205) < meta-Br(P208) < para-Br(P202). That is,
ortho-position < meta-position < para-position. Para-
substitution leads to stronger antiplatelet aggregation
activity. As to P204, P207 the activity is higher than P217
and P218, respectively, that means the activity can be
enhanced when another chlorine-substitutes in any positions
added.
The activity order of the target compounds with different
positions (ortho-, meta- and para-) of methoxy and nitro
mono-substituted phenyl groups is: para-(P214) > ortho-
Acknowledgements The authors grate to the National Science
Foundation of China (11341014) & the Committee of Science and
Technology of Tianjin of China(15JCZDJC33100) for the financial
supports and Shenyang Pharmaceutical University of China for run-
ning platelet aggregation assays.
Compliance with ethical standards
(
P212) > meta-(P213) and meta-(P210) > ortho-(P209) >
Conflict of interest The authors declare that they have no competing
para-(P211), respectively. It seems that nitro groups are
better than methoxy groups. In contrast to P216, compound
P215 does not exhibit good activity and has much lower
inhibition of platelets than Picotamide. The activity of the
former is higher than that of Picotamide, but lower than that
of Aspirin. Introduction of an additional nitroso group could
increase the antiplatelet activity and especially compound
P220, bearing a nitrooxyl group and with IC50 values of
interests.
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