Novel conjugates of aspirin with phenolic acid as anti-inflammatory agents having significantly reduced gastrointestinal toxicity

Article abstract of DOI:10.1002/ardp.200900217

A series of novel conjugates of aspirin with natural phenolic acid antioxidants connected through a diol linker were designed and synthesized as potential bifunctional agents combining antioxidant and anti-inflammatory activity for reducing gastrointestinal toxicity. In general, the conjugates were found to be efficient antioxidants and many of them demonstrated much more potent anti-inflammatory activity than aspirin. Among them, 5a and 5b which bear the best anti-inflammatory activity exhibited significantly reduced ulcerogenic potency and toxicity compared to aspirin. However, it is evident that the anti-inflammatory activity of these dual-acting molecules in vivo, was not simply consistent with their antioxidant ability in vitro.

Full text of DOI:10.1002/ardp.200900217

Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
S. Jiang et al.
215
Full Paper
Novel Conjugates of Aspirin with Phenolic Acid as Anti-
inflammatory Agents Having Significantly Reduced
Gastrointestinal Toxicity
Shan Jiang1, Ning Ding2, Wei Zhang2, Yichun Zhang1, Meiyu Geng1, and Yingxia Li2
1 School of Medicine and Pharmacy, Ocean University of China, Qingdao, P.R. China
2 Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, P.R. China
A series of novel conjugates of aspirin with natural phenolic acid antioxidants connected
through a diol linker were designed and synthesized as potential bifunctional agents combining
antioxidant and anti-inflammatory activity for reducing gastrointestinal toxicity. In general, the
conjugates were found to be efficient antioxidants and many of them demonstrated much more
potent anti-inflammatory activity than aspirin. Among them, 5a and 5b which bear the best
anti-inflammatory activity exhibited significantly reduced ulcerogenic potency and toxicity
compared to aspirin. However, it is evident that the anti-inflammatory activity of these dual-act-
ing molecules in vivo, was not simply consistent with their antioxidant ability in vitro.
Keywords: Antioxidant / Aspirin / Inflammation / NSAIDs / Ulcerogenicity /
Received: September 09, 2009; Accepted: December 01, 2009
DOI 10.1002/ardp.200900217
tract, while COX-2 is inducible and short-lived; its expres-
sion is stimulated in response to a pro-inflammatory
insult [5]. COX-2 plays a major role in prostaglandin bio-
synthesis in inflammatory cells and in the central ner-
vous system [6]. Classical NSAIDs are not selective and
inhibit both COX-1 and COX-2, thus, cause peptic ulcera-
tion and dyspepsia. The development of selective COX-2
inhibitors was thought to be a reasonable target for safer
NSAIDs [6]. However, serious doubts for a substantial ben-
efit of COX-2 inhibitors have been expressed at the end of
last century [7]. Moreover, the withdrawal of several COX-
2-selective NSAIDs from the market in the beginning of
new century attracted the public concern regarding the
cardiovascular safety of the COX-2 inhibitors. To some
degree, this result eliminated the benefit of selective
inhibition of COX-2; subsequently, it made non-selective
NSAIDs more attractive than before in terms of prescrip-
tions. This was supported by the fact that there were sig-
nificant increases in non-selective NSAID prescriptions
after the withdrawal of rofecoxib and valdecoxib from
the market [8].
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the
most commonly prescribed drugs in the world due to
their anti-inflammation, anti-pyreticosis, anti-thrombo-
sis, and acesodyne properties [1]. However, gastrointesti-
nal (GI) ulceration was found to be the main and most
severe adverse effect. It has been reported that 15–35% of
all peptic ulcer complications are caused by NSAIDs [2],
and the increase of hospitalization and deaths due to GI-
related disorders parallels the increased use ofNSAIDs [3].
It is believed that the GI toxicity of NSAIDs is caused by
their “dual-insult” [4] – the suppression of cyclooxyge-
nase-1 (COX-1) activity and direct irritation of the gastric
mucosa (many NSAIDs are acids) [3]. COX-1 is a constitu-
tive isozyme and is mainly responsible for the synthesis
of cytoprotective prostaglandins in the gastrointestinal
Correspondence: Yingxia Li, Department of Medicinal Chemistry,
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai
201203, P.R. China.
E-mail: liyx417@fudan.edu.cn
Fax: +86 21 519-80127
In addition to the systemic effects of NSAIDs on gastric
mucosa due to their action on prostaglandin synthesis,
the acidic nature of NSAIDs was also considered an
important factor to induce GI ulceration, since the acid
Abbreviations: dicyclohexylcarbodiimide (DCC); 4-(dimethylamino)-pyri-
din (DMAP); 2,2-diphenyl-1-picrylhydrazyl (DPPH); gastrointestinal (GI);
intragastrically (i. g.); malondialdehyde (MDA); tetrahydropyranyl (THP)
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Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
group can produce a local action on the gastric epithe-
lium [9]. In this respect, molecular modifications to
reduce the acidic irritation of NSAIDs have been pro-
posed and proven to be a useful approach in the past
years [9].
Furthermore, reactive oxygen species (ROS) and related
free radical reactions are considered to be implicated in
numerous pathophysiological conditions, such as
inflammation, atherosclerosis, gastric ulceration, neuro-
nal degeneration, cancer, and the aging process [10]. Sev-
eral antioxidants have been shown to exhibit anti-inflam-
matory activity [11], also proving that reactive oxygen
species play a fundamental role in inflammatory condi-
tions [12].
Figure 1. Novel conjugates of aspirin with phenolic antioxidants.
Table 1. Structures and yields of the conjugates of aspirin with
phenolic antioxidants (cf. Scheme 1).
These findings indicated that the chemical derivatiza-
tion of known NSAID molecules, which combine anti-
inflammatory and antioxidant activities may lead to the
development of drugs with an improved therapeutic
index. For example, Kourounakis et al. [2] reported that
the conjugates of several widely used NSAIDs with antiox-
idant cysteamine and L-cysteine showed better anti-
inflammatory and antioxidant activities than the parent
compounds as well as the significant reduction of gastro-
intestinal toxicity [9]. Detsi et al. reported a series of novel
quinolinone 3-aminoamides and their a-lipoic acid
hybrids possess significant anti-inflammatory activity in
vivo, whereas most of them are potent 9OH scavengers
and inhibit soybean lipoxygenase in vitro [13]. The success
of this strategy has been further proven for a number of
commercially available NSAIDs, which also possess radi-
cal scavenging properties [14].
In view of the above mentioned mechanisms, in this
paper, we would like to report preliminary results on the
design, synthesis, and pharmacological evaluation of a
series of conjugates of aspirin with three natural phe-
nolic antioxidants, p-coumaric acid 1a, ferulic acid 1b,
and caffeic acid 1c, through diol linker-mediated ester
bonds (Fig. 1).
tion. With the two aspirin derivatives 3a, b in hand, six
derivatives 4a–f [16–21] of the three natural antioxidants
1a–c bearing different phenolic hydroxyl protecting
groups were coupled with 3a, b to provide 5a–i either
through their acid chlorides or promoted by DCC-DMAP
directly. Removal of the acetyl group on 5a–c using 3 M
HCl in acetone led to compounds 6a–c (Scheme 1). When
tetrahydropyranyl (THP) ether served as protecting group
for the phenolic hydroxyl 5d–i, the acetyl group on the
aspirin part was preserved during the course of THP
removal under mild acid conditions. Thus, compounds
6d–i were provided smoothly.
Results and discussion
Chemistry
The structures and synthesis of the novel aspirin conju-
gates are presented in Table 1 and Scheme 1. The synthe-
sis was started from the connection of the carboxylic acid
group of aspirin with two diol linkers, glycol ethylene 2a
and 4-(2-hydroxy-ethyl)-phenol 2b, respectively. The key
intermediate 3a [15] was generated by treating the acid
chloride of aspirin directly with excessive 2a, while com-
pound 3b was synthesized by coupling aspirin with 2c in
the presence of DCC-DMAP and followed by debenzyla-
Biological activity
Antioxidant activity in vitro
The antioxidant activity of aspirin conjugates 5a–c and
6a–i were examined through two individual investiga-
tion according to their IC50 values: the inhibition of
malondialdehyde (MDA) formation [9, 22] and the stable
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Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
Novel Conjugates of Aspirin with Phenolic Acid
217
Reagents and conditions: (i) SOCl2, reflux, 2 h; then excessive 2a, acetone, Et3N, 55.9%; (ii) 2c, DCC-DMAP, 52.8%; (iii) Pd/C, H2, 67.7%; (iv) 4a–c, SOCl2, reflux; then 3a,
Et3N; (v) 4d–f, DCC, DMAP, 3a or 3b, CH2Cl2; (vi) 5a–c, 3 M HCl, acetone, reflux; (vii) 5d–i, 5% HCl, MeOH, r. t.
Scheme 1. Synthesis of the conjugates of aspirin with phenolic antioxidants.
Table 2. Inhibition of lipid peroxidation, scavenging of 0.2 mM DPPH radical, and inhibition of croton-oil-induced mice-ear swelling by
derivatives of aspirin.
Compounds
Inhibition of MDA
formation IC50 (mM)
Elimination of DPPH
IC50 (mM)
Inhibition on croton-oil-
induced mice-ear
swelling (%)j)
Aa)
>>8c)
NTg)
22.4**
p-coumaric acid 1a series
ferulic acid 1b series
caffeic acid 1c series
A + 1ab)
5a
6a
6d
6g
>8d)
L8e)
L8f)
3.9
>125h)
NTg)
NTi)
28.3***
43.5***
27.5**
12.2
NTi)
1.8
NTi)
30.7***
A + 1bb)
5b
6b
6e
6h
4.8
2.5
3.4
2.7
0.9
0.40
NTg)
0.50
0.50
0.65
4.8
37.1***
34.4***
16.2*
14.5**
A + 1cb)
3.0
1.1
0.6
1.0
0.4
0.08
NTg)
0.10
0.12
0.11
9.5
5.8
3.0
22.8***
8.8
5c
6c
6f
6i
a): Acetylsalicylic acid (Aspirin); b): this two compounds were mixed in equal mol; c): inhibition of MDA formation was 6.6% at 8
mM; d): inhibition of MDA formation was 27.2% at 8 mM; e): inhibition of MDA formation was 44.6% at 8 mM; f): inhibition of MDA
formation was 50.7% at 8 mM; g): not tested because it had almost no elimination activity even at 125 mM; h): elimination of DPPH
was 35.8% at 125 mM; i): not tested because of whose IC50 was higher than 125 mM; j): all the tested compounds were given i. g. in 1
mmol/kg.
Each value represents the mean obtained in two independent experiments: *: P > 0.05, **: P a 0.01, ***: P a 0.001 vs. control.
free-radical scavenging ability to 2,2-diphenyl-1-picrylhy- mixtures of aspirin and their corresponding parent anti-
drazyl (DPPH) [9, 23].
oxidants. Interestingly, those conjugates comprising a
As shown in Table 2, all the aspirin conjugates inhib- caffeic acid moiety (5c, 6c, 6f, 6i) exhibited lower IC50 val-
ited MDA formation efficiently with IC50 values ranging ues than their corresponding ferulic-acid moiety-contain-
from 0.4 to 8 mM. It should be noted that all the conju- ing counterparts (5b, 6b, 6e, 6h), and, in turn, the later
gates are stronger inhibitors of MDA formation (IC50 = 8 ones’ IC50 values were lower than those of p-coumaric
mM) than aspirin (IC50 >> 8 mM) as well as the equal-molar acid series compounds (5a, 6a, 6d, 6g). These results are
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Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
Table 3. The effects of aspirin, 5a, and 5b on gastric ulcer and toxicity.
Compounds
Gastric ulcer
Toxicity
Number
of mice
Dose
(mmol/kg)
Ulcer score
(X € SD)
Number
of mice
Dose
(mmol/kg)
Mortalitya)
Toxicity
responseb)
Aspirin
5a
5b
13
12
12
4
4
4
8.62 € 7.03
4.58 € 3.64
3.83 € 4.04
16
12
12
4
4
4
3/16
0
0
16
2
1
a): Dead/total; b): including listless and sluggish behavior.
consistent with their DPPH elimination ability. It is easily Effects of compounds 5a and 5b on gastric ulcer and
to understand that the compounds without free hydrox- toxicity
yls 5a–c showed almost no free-radical scavenging ability Compounds 5a and 5b, which showed the most potent
(IC50 >> 125 mM). However, those conjugates containing anti-inflammatory activity among the test conjugates,
p-coumaric acid moiety 1a and thus bearing one or two were evaluated in vivo for their effects on gastric ulcer
free hydroxyls in total (6a, 6d, 6g) also did not possess and toxicity (Table 3). Administration of aspirin at a dose
DPPH elimination ability (IC50 >> 125 mM), whereas of 4 mmol/kg in mice resulted in a high incidence of
ferulic acid 1b and caffeic acid 1c containing conjugates ulcer (8.62 € 7.03), and this effect was accompanied by
(6b, 6e, 6h, 6c, 6f, 6i) appeared to be strong inhibitors in 3/16 mortality and 100% toxicity response (including list-
the DPPH-elimination process.
less and sluggish behavior) of the tested animals. In con-
trast, the same dose of 5a and 5b exhibited a significantly
reduced ulcer effect and all animals survived with almost
Anti-inflammatory activity
The anti-inflammatory activity of derivatives 5a–c and no toxicity response. Evidently, the designed compounds
6a–i was assessed from their ability to inhibit the croton- 5a and 5b were better than aspirin in respect of ulcero-
oil-induced mice-ear swelling (Table 2). The compounds genic potency and toxicity.
were administered intragastrically (i.g.) at a dose of 1
mmol/kg and demonstrated various degrees of inhibition
of the edema. Obviously enhanced inhibitory activities
were observed for 5a (43.5%), 6g (30.7%), 5b (37.1%), and
Conclusion
In conclusion, a series of conjugates of aspirin with natu-
ral phenolic antioxidants were designed and synthesized
to remain or enhance the analgesic activity but reduce
the risk of GI ulceration and toxicity. Among them, 5a
and 5b were found to be the best anti-inflammatory
agents. They were further confirmed to significantly
reduce ulcerogenic potency and toxicity in mice in com-
parison to aspirin. This molecular modifcation may offer
a general route to safer anti-inflammatory agents poten-
tially suitable for chronic use. However, it is evident that
the anti-inflammatory activity in vivo of these dual-acting
molecules was not simply consistent with their antioxi-
dant ability in vitro. This needs to be further explored in
the future.
6b (34.4%) in comparison to aspirin (22.4%). Co-adminis-
tration of equimolar mixtures of aspirin and phenolic
acid resulted in most cases in a lower anti-inflammatory
activity than that of the test compounds in the p-couma-
ric acid 1a and ferulic acid 1b series, with the exception
of 6a and 6d.
However, both mixtures and conjugates of caffeic acid
and aspirin (A + 1c and 5c, 6c, 6i) showed almost no effect
on croton-oil-induced mice-ear swelling except for 6f
(22.8% inhibition). This is not consistent with their anti-
oxidant ability, since caffeic acid series conjugates were
the most powerful antioxidants among the test com-
pounds. Further evidence was revealed by the fact that in
most cases conjugates bearing a p-coumaric acid 1a moi-
ety inhibited edema more efficiently than those of the
ferulic acid 1b containing counterparts, while both, their
MDA inhibition and DPPH-scavenging abilities, were evi-
dently much lower than those of the later ones.
Experimental
Chemistry
Notably, the obtained data showed the linker part had
no influence on the antioxidant and anti-inflammatory
activities of the target compounds.
Solvents were purified in the usual way. TLCs were performed on
precoated Merk silica gel 60 F254 plates (Merck, Germany). Flash
column chromatography was performed on silica gel (100–200
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Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
Novel Conjugates of Aspirin with Phenolic Acid
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mesh, Qingdao, China). Melting points were determined with a
“Yanaco” apparatus and were uncorrected. 1H-NMR and 13C-NMR
spectra were taken on a JEOL JNM-ECP 600 spectrometer (Jeol,
Japan) with tetramethylsilane (TMS) as an internal standard, and
chemical shifts are recorded in d values. Mass spectra were
obtained on a Waters Q-TOF micro mass spectrometer (Waters).
2-Acetoxy-benzoic acid 2-[3-(3,4-di-acetoxy-phenyl)-
acryloyloxy]-ethyl ester 5c
White solid, m. p.: 86–888C; Rf = 0.24 (EtOAc/petroleum ether,
4:5); 1H-NMR (CDCl3) d: 8.06 (dd, 1H, J = 1.9, 7.7 Hz), 7.66 (d, 1H, J =
15.8 Hz), 7.57 (td, 1H, J = 1.8, 8.0 Hz), 7.42 (dd, 1H, J = 2.2, 8.5 Hz),
7.37 (d, 1H, J = 1.9 Hz), 7.33 (td, 1H, J = 1.1, 7.7 Hz), 7.22 (d, 1H, J =
8.4 Hz), 7.12 (dd, 1H, J = 0.8, 8.1 Hz), 6.41 (d, 1H, J = 15.8 Hz), 4.56–
4.51 (m, 4H), 2.34 (s, 3H), 2.31 (s, 3H), 2.30 (s, 3H); 13C-NMR (CDCl3)
d: 170.0, 168.1, 168.0, 166.3, 164.1, 150.8, 143.6, 142.4 (2C),
134.2, 133.1, 131.9, 126.5, 126.1, 124.0, 123.9, 122.8 (2C), 118.6,
62.8, 62.3, 21.0, 20.7, 20.6. ESI-HRMS (m/z) calcd. for C24H22O10Na
[M + Na+]: 493.1111. Found: 493.1093.
2-Acetoxy-benzoic acid 2-(4-hydroxy-phenyl)-ethyl ester
3b
To a cold solution of aspirin (2.56 g, 14.2 mmol) and compound
2c (2.7 g, 11.8 mmol) in CH2Cl2 (300 mL) were added dicyclohex-
ylcarbodiimide (DCC; 2.93 g, 14.2 mmol) and 4-(dimethylamino)-
pyridin (DMAP; 0.15 g, 1.2 mmol). The mixture was stirred for 1
h at 08C and then overnight at r.t. After the precipitate was fil-
trated off, the solvent was removed under reduced pressure. The
oily residue was then purified by column chromatography to
give a white solid in 52.8% yield. The solid was dissolved in
EtOAc/petroleum ether (120 mL, 1:1) and hydrogenated over
10% Pd/C (0.44 g) at r.t. for 4 h. After the catalyst was filtrated off
and the solvent was removed under reduced pressure, the resi-
due was subjected to silica gel column chromatography to give
3b as a white solid in 67.7% yield; Rf = 0.1 (EtOAc/petroleum
ether, 1:4); 1H-NMR (CDCl3) d: 7.96 (dd, 1H, J = 1.6, 7.9 Hz), 7.57–
7.54 (m, 1H), 7.30 (td, 1H, J = 1.1, 7.9 Hz), 7.13–7.11 (m, 2 H), 7.10
(dd, 1H, J = 1.1, 8.2 Hz), 6.79–6.77 (m, 2H), 4.44 (t, 2H, J = 7.0 Hz),
2.97 (t, 2H, J = 7.0 Hz), 2.31 (s, 3H);13C-NMR (CDCl3) d: 169.8, 164.4,
154.3, 150.7, 133.9, 131.7, 130.1 (2C), 129.8, 126.0, 123.8, 123.3,
115.4 (2C), 65.8, 34.2, 21.0.
General procedure for the preparation of 6a–c
A solution of 5a–c (1.76 mmol) in 45 mL of acetone containing
15 mL of 3 M HCl was refluxed for 45 min, respectively, after
which the solvent was removed under reduced pressure. The
residual aqueous layer was extracted with EtOAc (80 mL). The
organic layer was separated and washed with satd. aqueous
NaHCO3 (2620 mL) and water (2620 mL), then dried over
MgSO4. After removal of the solvent under reduced pressure, the
residue was subjected to silica gel column chromatography to
give 6a, b, c in 53.5%, 48.5%, and 60.1% yield, respectively.
2-Hydroxy-benzoic acid 2-[3-(4-hydroxy-phenyl)-
acryloyloxy]-ethyl ester 6a
White solid, m.p.: 148–1508C; Rf = 0.29 (EtOAc/petroleum ether,
1:2); 1H-NMR (DMSO-d6) d: 10.42 (s, 1H, -OH), 7.78 (dd, 1H, J = 1.6,
7.7 Hz), 7.59 (d, 1H, J = 16.0 Hz), 7.56–7.51 (m, 3H), 6.99 (dd, 1H, J
= 0.8, 8.2 Hz), 6.95 (td, 1H, J = 1.1, 8.2 Hz), 6.79–6.77 (m, 2H), 6.43
(d, 1H, J = 16.0 Hz), 4.58–4.49 (m, 4H);13C-NMR (DMSO-d6) d: 168.4,
166.5, 160.0, 159.9, 145.3, 135.8, 130.4 (2 C), 130.1, 125.0, 119.4,
117.4, 115.7 (2 C), 113.6, 112.9, 63.3, 61.6. ESI-HRMS (m/z) calcd.
for C18H15O6 [M – H]: 327.0869. Found: 327.0869.
General procedure for the preparation of 5a–c
To a cold solution of 3a (10 mmol) and triethylamine (12 mmol)
in anhydrous CHCl3 (20 mL) was added 10 mmol of acid chloride
of 4a–c in 20 mL anhydrous CHCl3. The mixture was stirred for 1
h at 08C and then 1 h at r.t. After removal of the solvent, the resi-
due was subjected to silica gel column chromatography to give
5a, b, c in 67.5%, 64.9%, and 64.4% yield, respectively.
2-Hydroxy-benzoic acid 2-[3-(4-hydroxy-3-methoxy-
2-Acetoxy-benzoic acid 2-[3-(4-acetoxy-phenyl)-
acryloyloxy]-ethyl ester 5a
phenyl)-acryloyloxy]-ethyl ester 6b
Buff solid, m.p.: 121–1228C; Rf = 0.23 (CHCl3); 1H-NMR (DMSO-d6)
d: 10.44 (s, 1H, OH), 9.64 (s, 1H, OH), 7.79 (dd, 1H, J = 1.4, 7.7 Hz),
7.58 (d, 1H, J = 16.1 Hz), 7.53 (td, 1H, J = 1.8, 8.8 Hz), 7.33 (d, 1H, J =
1.8 Hz), 7.11 (dd, 1H, J = 1.9, 8.0 Hz), 7.00–6.94 (m, 2H), 6.78 (d,
1H, J = 8.0 Hz), 6.53 (d, 1H, J = 15.7 Hz), 4.59–4.48 (m, 4H), 3.81 (s,
3H); 13C-NMR (DMSO-d6) d: 168.5, 166.6, 160.1, 149.4, 147.9, 145.6,
135.8, 130.1, 125.5, 123.4, 119.4, 117.4, 115.4, 113.9, 112.9,
111.2, 63.4, 61.6, 55.7. ESI-HRMS (m/z) calcd. for C19H17O7 [M – H]:
357.0974. Found: 357.0989.
White solid, m. p.: 70–728C; Rf = 0.30 (EtOAc/petroleum ether,
1:2);1H-NMR (CDCl3) d: 8.06 (dd, 1H, J = 1.4, 7.7 Hz), 7.71 (d, 1H, J =
16.1 Hz), 7.58 (td, 1H, J = 1.8, 8.1 Hz), 7.57–7.54 (m, 2H), 7.33 (td,
1H, J = 1.1, 7.7 Hz), 7.13–7.11 (m, 3H), 6.42 (d, 1H, J = 17.7 Hz),
4.56–4.51 (m, 4H), 2.34 (s, 3H); 13C-NMR (CHCl3) d: 169.6, 169.1,
166.5, 164.1, 152.2, 150.8, 144.4, 134.1, 131.9 (2C), 129.3 (2C),
126.1, 123.9, 122.8, 122.1, 117.5, 62.8, 62.2, 21.1, 21.0 (2C). ESI-
HRMS (m/z) calcd. for C22H20O8Na [M + Na+]: 435.1056. Found:
435.1073.
2-Hydroxy-benzoic acid 2-[3-(3,4-dihydroxy-phenyl)-
acryloyloxy]-ethyl ester 6c
Buff solid, m.p.: 123–1248C; Rf = 0.13 (MeOH/CHCl3, 1:50); H-
2-Acetoxy-benzoic acid 2-[3-(4-acetoxy-3-methoxy-
phenyl)-acryloyloxy]-ethyl ester 5b
1
White solid, m. p.: 99–1008C; Rf = 0.20 (EtOAc/petroleum ether,
1:2);1H-NMR (CDCl3) d: 8.06 (dd, 1H, J = 1.9, 8.0 Hz), 7.68 (d, 1H, J =
16.1 Hz), 7.58 (td, 1H, J = 1.4, 7.3 Hz), 7.33 (td, 1H, J = 1.1, 7.7 Hz),
7.14–7.11 (m, 3H), 7.05 (d, 1H, J = 8.1 Hz), 6.41 (d, 1H, J = 16.1 Hz),
4.56–4.52 (m, 4H), 3.86 (s, 3H), 2.34 (s, 3H), 2.32 (s, 3H); 13C-NMR
(CDCl3) d: 169.7, 168.8, 166.5, 164.1, 151.4, 150.8, 144.8, 141.6,
134.2, 133.2, 131.9, 126.1, 123.9, 123.3, 122.8, 121.4, 117.6,
111.3, 62.9, 62.2, 55.9, 21.0, 20.6. ESI-HRMS (m/z) calcd. for
C23H22O9Na [M + Na+]: 465.1162. Found: 465.1171.
NMR (DMSO-d6) d: 10.43 (s, 1H, -OH), 9.64 (s, 1H, -OH), 9.15 (s, 1H, -
OH), 7.79 (dd, 1H, J = 1.4, 7.7 Hz), 7.53 (td, 1H, J = 1.4, 7.3 Hz), 7.51
(d, 1H, J = 15.8 Hz), 7.05 (d, 1H, J = 2.2 Hz), 7.00 (dd, 1H, J = 2.2, 8.4
Hz), 6.99 (dd, 1H, J = 0.7, 8.0 Hz), 6.95 (td, 1H, J = 1.1, 8.0 Hz), 6.76
(d, 1H, J = 8.0 Hz), 6.30 (d, 1H, J = 16.1 Hz), 4.58–4.48 (m, 4H); 13C-
NMR (DMSO-d6) d: 168.3, 166.4, 160.0, 148.5, 145.7, 145.5, 125.4,
121.5, 119.4, 117.4, 115.7, 114.9, 113.4, 112.9, 63.3, 61.6. ESI-
HRMS (m/z) calcd. for C18H15O7 [M – H]: 343.0818. Found:
343.0833.
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Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
General procedure for the preparation of 5d–i
2-Acetoxy-benzoic acid 2-{4-[3-(4-hydroxy-phenyl)-
To a cold mixture of 5d–i (1.2 eq.) and compound 3a (or 3b) (1.0
eq.) in CH2Cl2 were added DCC (1.2 eq.) and DMAP (0.1 eq.). The
mixture was stirred at 08C for 1 h and then at r.t. overnight.
After the precipitate was filtrated off, the solvent was removed
under reduced pressure. The residue was used for next reaction
without further purification.
acryloyloxy]-phenyl}-ethyl ester 6g
Yield over two steps: 61.3%; yellow solid, m.p.: 122–1248C; Rf =
0.12 (EtOAc/petroleum ether, 2:5); 1H-NMR (DMSO-d6) d: 10.12 (s,
1H, -OH), 7.88 (dd, 1H, J = 1.5, 7.7 Hz), 7.75 (d, 1H, J = 15.8 Hz), 7.68
(td, 1H, J = 1.4, 8.8 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.41 (td, 1H, J = 1.1,
7.3 Hz), 7.36 (d, 2H, J = 8.4 Hz), 7.23 (d, 1H, J = 8.0 Hz), 7.13 (d, 2H, J
= 8.5 Hz), 6.83 (d, 2H, J = 8.4 Hz), 6.62 (d, 1H, J = 16.1 Hz), 4.46 (t,
2H, J = 7.0 Hz), 3.03 (t, 2H, J = 7.0 Hz), 2.23 (s, 3H). ESI-HRMS (m/z)
calcd. for C26H22O7Na [M + Na+]: 469.1263. Found: 469.1282.
General procedure for the preparation of 6d–i
A solution of corresponding 5d–i in methanol (40 mL) contain-
ing five drops of 5% HCl was stirred at r.t. for 2 h. Half of the sol-
vent was removed under reduced pressure and then, 20 mL of
water and 150 mL of ether were added to the mixture. The
organic portion was separated, washed with water (3620 mL),
and dried over MgSO4. After removal of the solvent under
reduced pressure, the residue was subjected to silica gel column
chromatography to give 6d–i, respectively.
2-Acetoxy-benzoic acid 2-{4-[3-(4-hydroxy-3-methoxy-
phenyl)-acryloyloxy]-phenyl}-ethyl ester 6h
Yield over two steps: 86.7%, yellow solid, m.p.: 113–1148C; Rf =
1
0.14 (EtOAc/petroleum ether, 1:2); H-NMR (DMSO-d6) d: 9.73 (s,
1H, OH), 7.89 (dd, 1H, J = 1.8, 7.7 Hz), 7.74 (d, 1H, J = 15.7 Hz), 7.68
(td, 1H, J = 1.9, 7.7 Hz), 7.42 (d, 1H, J = 1.9 Hz), 7.41 (td, 1H, J = 1.1,
7.3 Hz), 7.39–7.36 (m, 2H), 7.24 (dd, 1H, J = 1.0, 8.0 Hz), 7.21 (dd,
1H, J = 1.8, 8.4 Hz), 7.14–7.13 (m, 2H), 6.82 (d, 1H, J = 8.0 Hz), 6.71
(d, 1H, J = 15.7 Hz), 4.46 (t, 2H, J = 7.0 Hz), 3.84 (s, 3H), 3.03 (t, 2H, J
= 7.0 Hz), 2.23 (s, 3H); 13C-NMR (DMSO-d6) d: 169.1, 165.4, 163.9,
150.5, 149.8, 149.2, 148.0, 147.0, 135.3, 134.3, 131.1, 129.9 (2C),
126.3, 125.4, 124.0, 123.7, 123.0, 121.8 (2C), 115.5, 113.4, 111.4,
65.3, 55.7, 33.6, 20.7. ESI-HRMS (m/z) calcd. for C27H24O8Na [M +
Na+]: 499.1369. Found: 499.1385.
2-Acetoxy-benzoic acid 2-[3-(4-hydroxy-phenyl)-
acryloyloxy]-ethyl ester 6d
Yield: 81.6%; white solid, m.p.: 137–1388C; Rf = 0.12 (EtOAc/petro-
leum ether, 1:2);1H-NMR (DMSO-d6) d: 7.96 (dd, 1H, J = 1.7, 7.8 Hz),
7.69 (td, 1H, J = 1.8, 7.7 Hz), 7.59 (d, 1H, J = 15.9 Hz), 7.56–7.55 (m,
2H), 7.42 (td, 1H, J = 1.3, 7.6 Hz), 7.25 (dd, 1H, J = 1.3, 8.1 Hz), 6.80–
6.78 (m, 2H), 6.42 (d, 1H, J = 17.7 Hz), 4.51–4.43 (m, 4H), 2.27 (s,
3H); 13C-NMR (DMSO-d6) d: 169.1, 166.5, 163.9, 159.9, 150.0, 145.3,
134.5, 131.3, 130.4 (2C), 126.3, 125.0, 124.0, 122.8, 115.8 (2C),
113.6, 63.1, 61.8, 20.7. ESI-HRMS (m/z) calcd. for C20H18O7Na [M +
Na+]: 393.0950. Found: 393.0932.
2-Acetoxy-benzoic acid 2-{4-[3-(3,4-di-hydroxy-phenyl)-
acryloyloxy]-phenyl}-ethyl ester 6i
Yield over two steps: 81.4%, yellow solid, m.p.: 151–1528C; Rf =
1
0.17 (EtOAc/petroleum ether, 2:3); H-NMR (DMSO-d6) d: 9.72 (s,
1H, OH), 9.21 (s, 1H, OH), 7.88 (dd, 1H, J = 1.4, 7.7 Hz), 7.68 (td, 1H,
J = 1.4, 8.1 Hz), 7.67 (d, 1H, J = 15.8 Hz), 7.41 (td, 1H, J = 1.1, 7.7
Hz), 7.36 (d, 2H, J = 8.4 Hz), 7.24 (dd, 1H, J = 1.1, 8.1 Hz), 7.14–7.12
(m, 3H), 7.09 (dd, 1H, J = 2.2, 8.5 Hz), 6.80 (d, 1H, J = 8.0 Hz), 6.50
(d, 1H, J = 15.8 Hz), 4.46 (t, 2H, J = 7.0 Hz), 3.03 (t, 2H, J = 7.0 Hz),
2.23 (s, 3H); 13C-NMR (DMSO-d6) d: 169.0, 165.2, 163.8, 149.9,
149.1, 148.8, 147.0, 145.5, 135.1, 134.2, 131.0, 129.7 (2C), 126.2,
125.3, 123.9, 123.0, 121.8, 121.7 (2C), 115.7, 115.0, 112.8, 65.2,
33.5, 20.6. ESI-HRMS (m/z) calcd. for C26H23O8 [M + H+]: 463.1393.
Found: 463.1411.
2-Acetoxy-benzoic acid 2-[3-(4-hydroxy-3-methoxy-
phenyl)-acryloyloxy] -ethyl ester 6e
Yield over two steps: 77.4%; white solid, m.p.: 77–808C; Rf = 0.20
1
(EtOAc/petroleum ether, 2:3); H-NMR (DMSO-d6) d: 9.65 (s, 1H,
OH), 7.97 (dd, 1H, J = 1.9, 7.7 Hz), 7.70 (td, 1H, J = 1.8, 7.7 Hz), 7.59
(d, 1H, J = 15.7 Hz), 7.43 (td, 1H, J = 1.1, 7.7 Hz), 7.33 (d, 1H, J = 1.8
Hz), 7.25(dd, 1H, J = 0.7, 8.0 Hz), 7.12 (dd, 1H, J = 2.2, 8.5 Hz), 6.79
(d, 1H, J = 8.1 Hz), 6.52 (d, 1H, J = 15.7 Hz), 4.51–4.43 (m, 4H), 3.81
(s, 3H), 2.27 (s, 3H); 13C-NMR (DMSO-d6) d: 169.1, 166.5, 163.9,
150.0, 149.5, 147.9, 145.6, 134.5, 131.3, 126.3, 125.5, 124.1,
123.4, 122.8, 115.5, 113.9, 111.1, 63.2, 61.8, 55.7. ESI-HRMS (m/z)
calcd. for C21H20O8Na [M + Na+]: 423.1056. Found: 423.1063.
Biology
Lipid-peroxidation assay
The anti-lipid peroxidation activity of the target compounds was
measured by determination of thiobarbituric acid (TBA) reactive
substances in 96-well plates. 10% liver homogenate from
untreated Kunming male mice (18–22 g; obtained from the
Department of Experimental Animals, Institute of Hematology,
Chinese Academy of Medical Sciences) was prepared as previ-
ously described (Sun et al., [22a]). The incubation mixture which
containing 80 lL of 10% liver homogenate, 10 lL of 25 mmol/L
FeSO4, and 10 lL of various concentrations of the test com-
pounds dissolved in 90% DMSO (90% DMSO, 10% water), was
incubated at 378C for 30 min. The reaction was quenched by
addition of 100 lL 10% CCl3COOH and then centrifuged at 3000
rpm for 10 min. 100 lL of the supernate was decanted to another
well and 100 lL TBA reagent (containing 0.67% TBA, 10%
CCl3COOH) was added to the supernatant. After incubation at
608C for 30 min, lipid peroxidation was assessed spectrophoto-
metrically (535 nm against 600 nm) (Ai). The absorbance of the
2-Acetoxy-benzoic acid 2-[3-(3,4-di-hydroxy-phenyl)-
acryloyloxy]-ethyl ester 6f
Yield: 94.0%; white solid, m.p.: 79–838C; Rf = 0.12 (EtOAc/petro-
leum ether, 2:3); 1H-NMR (DMSO-d6) d: 9.63 (s, 1H, OH), 9.15 (s, 1H,
OH), 7.96 (dd, 1H, J = 1.4, 7.7 Hz), 7.69 (td, 1H, J = 1.4, 8.1 Hz), 7.51
(d, 1H, J = 15.7 Hz), 7.42 (td, 1H, J = 1.1, 7.7 Hz), 7.25 (dd, 1H, J =
1.1, 8.0 Hz), 7.05 (d, 1H, J = 1.8 Hz), 7.00 (dd, 1H, J = 1.8, 8.0 Hz),
6.75 (d, 1H, J = 8.1 Hz), 6.29 (d, 1H, J = 15.7 Hz), 4.50–4.42 (m, 4H),
2.27 (s, 3H); 13C-NMR (DMSO-d6) d: 169.0, 166.3, 163.8, 149.9,
148.4, 145.6, 145.4, 134.3, 131.2, 126.2, 125.3, 124.0, 122.7,
121.4, 115.6, 114.8, 113.3, 63.0, 61.7, 20.6. ESI-HRMS (m/z) calcd.
for C20H18O8Na [M + Na+]: 409.0899. Found: 409.0914.
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 343, 215–221
Novel Conjugates of Aspirin with Phenolic Acid
221
control sample without test compound (A0) was measured simul-
taneously. Each experiment was performed at least twice and
the results were expressed as inhibition of malondialdehyde
(MDA) formation and calculated according to the following
equation:
uating the anti-inflammaoty activity, the effects on gastric ulcer and
toxicity of the synthesized compounds.
The authors have declared no conflict of interest.
ðA0 ꢀ AiÞ
Inhibition of MDA formation ð%Þ ¼
6100
ð1Þ
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We thank Dr. Guifang Cheng (Institute of Materia Medica, Chinese
Academy of Medical Sciences & Peking Union Medical College) for eval-
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com