Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives of 4, 5-diaryl thiophene-2-carboxylic acid and their biological evaluation

Article abstract of DOI:10.1007/s12039-017-1274-6

Abstract: In our earlier studies, we have shown that the introduction of amino moieties at carboxylic acid of 4,5-diarylthiophene-2-carboxylic acid significantly improved the anti-inflammatory activity of the compound against the standard drug diclofenac

Full text of DOI:10.1007/s12039-017-1274-6

J. Chem. Sci. © Indian Academy of Sciences.
DOI 10.1007/s12039-017-1274-6
REGULAR ARTICLE
Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
of 4, 5-diaryl thiophene-2-carboxylic acid and their biological
evaluation
T SHANMUGANATHANa,b,∗, M VENUGOPALc, K PARTHASARATHYd,
N DHATCHANAMOORTHYa, Y ARUNe and A A M PRINCEb
aOrchid Pharma Ltd, R&D Centre, Chennai, Tamilnadu 600 119, India
bDepartment of chemistry, Ramakrishna Mission Vivekananda College, Mylapore, Chennai, Tamilnadu
600 004, India
cVen Biotech Private Limited, Chennai, Tamilnadu 600 095, India
dDepartment of Chemistry, Siddha Central Research Institute, Central Council for Research in Siddha, Chennai,
Tamilnadu 600 106, India
eOrganic Chemistry Division, Central Leather Research Institute (CSIR), Adyar, Chennai, Tamilnadu
600 020, India
Email: t_shanmuganathan09@yahoo.com
MS received 5 January 2017; revised 1 April 2017; accepted 2 April 2017
Abstract. In our earlier studies, we have shown that the introduction of amino moieties at carboxylic acid of
4,5-diarylthiophene-2-carboxylic acid significantly improved the anti-inflammatory activity of the compound
against the standard drug diclofenac sodium. In the present study, we have synthesized new derivatives of 4,5-
diarylthiophene-2-carboxylic acid by modifying the hydroxyl group of the phenyl ring and carboxylic acid group
of the thiophene ring. A series of novel 4,5-diarylthiophene-2-carboxylic acid derivatives containing bis-allyloxy
and hydroxypropoxy with methyl or ethyl ester moieties were synthesized, characterized and subsequently
evaluated for anti-inflammatory and antioxidant property. Among the novel compounds, the inhibition of bovine
serum albumin denaturation assay revealed that the compound 4,5-bis(4-(3-hydroxypropoxy)phenyl)thiophene-
2-carboxylic acid (15) and ethyl ester (13) having anti-inflammatory activity better than the standard drug
diclofenac sodium. The antioxidant screening showing 4,5-bis(4-(allyloxy)phenyl)thiophene-2-carboxylic acid
(10), 4,5-bis(4-(3-hydroxypropoxy)phenyl)thiophene-2-carboxylic acid methyl ester (11) and 4,5-bis(4-(3-
hydroxypropoxy)phenyl)thiophene-2-carboxylic acid ethyl ester (13) exhibited a slightly moderate antioxidant
activity than standard ascorbic acid. Molecular docking analysis was performed for the synthesized compounds
with the cyclooxygenase-2 (COX-2) receptor (PDB 1D: 1PXX). Docking studies revealed that all the synthesised
compounds exhibit greater binding affinity than the standard drug. Particularly, the compound ethyl 4,5-bis(4-
(allyloxy)phenyl)thiophene-2-carboxylate (8) and allyl 4,5-bis(4-(allyloxy)phenyl)thiophene-2-carboxylate (9)
having high free energy binding of −10.40 and −10.48 Kcal/mol, respectively.
Keywords. Bis-allyloxy derivatives; hydroxypropoxy derivatives; 4,5-diarylthiophene-2-carboxylic acid;
anti-inflammatory; antioxidant; molecular docking.
1. Introduction
inhibitors, have been designed, synthesized and clin-
ically introduced as gastrointestinal sparing NSAIDs.
Hence, the beneficial effect of selective inhibition of
COX-2 over COX-1 has triggered therapeutic interest
on the former, resulting in the evolution of drugs with
reduced side effect to the patients.
The general structure of COX-2 selective inhibitors
consist of two aryl groups attached to neighboring
atoms of a central ring, one of the aryl groups is para-
substituted with either a methyl sulfonyl (SO2CH3)
group, e.g., etoricoxib and rofecoxib or a sulfonamide
(SO2NH2), e.g., celecoxib and parecoxib.2 The central
Non-steroidal anti-inflammatory drugs (NSAIDs) are
therapeutic agents commonly used in the treatment of
inflammation, pain and fever. The therapeutic effect
of these substances involves inhibition of cyclooxyge-
nases (COX) thereby preventing prostaglandin (PGs)
and thromboxane A2 formation from arachidonic acid.1
There are two isoforms, COX-1 and COX-2, where
inhibition of COX-1 is responsible, in part, for gastroin-
testinal and renal side effects, whereas selective COX-2
*
For correspondence

T Shanmuganathan et al.
Figure 1. Some of the non-steroidal anti-inflammatory drugs.
rings of compounds are thiophene, pyrazole, furanone, lumiracoxib was reported with its carboxylate group
isoxazole, and cyclopentene. There are also exam- forming hydrogen bonding interactions with Ser530 and
ples of cycloalkyl, alkoxy or phenoxy moieties in Tyr385 at the top of the active site.15 These findings have
the non-sulfonyl containing ‘aryl’ ring.3–5 One of the encouraged us to replace the amide moiety with car-
important heterocyclic systems which attract progres- boxylic acid group of the previously reported potent 4,5-
sive interest of many researchers is thiophene, because diarylthiophene-2-carboxamide derivatives.16 In view
of plethora of biological, pharmacological and indus- of these observations and in continuance of our effort
trial importance. Thiophene containing drugs exhibit a to develop novel thiophene analogues,16 it was thought
variety of biological activities which include antimi- worthwhile to synthesize new allyloxy and hydrox-
crobials, antipsychotics,6 sedatives,7–9 antidepressants ypropoxyderivativesof4,5-diarylthiophene-2-carboxylic
(e.g., Duloxetine and Trazodone) and antihypertensive acidandtoevaluatethemfortheiranti-inflammatoryand
(e.g., Tiamenidine) drugs. Thiophene derivatives are antioxidant properties.
also well known for their pronounced anti-inflammatory
activities as evident from the approval of Lornoxicam,
Tenoxicam, Tiaprofenic acid and Tenidap by the US
2. Experimental
Food and Drug Administration (FDA). Recently, several
new Thiophene-2-carboxylic acid derivatives have been
reported as potent HCV NS5B polymerase inhibitors,10
5-LOX/COX-inhibitor,11 and with anti-inflammatory
activities.12 The introduction of carboxylic acid into a
biologicallyactivecompoundnotonlyimpactsthewater
solubility of the compound but also establishes rela-
tively strong electrostatic interactions or hydrogen-bond
bridges with the COX enzymes, conferring both binding
affinity and specificity to the drug–target as in the case
of flurbiprofen.13 Also, carboxylic acid moiety is criti-
cal for their biological activity of several NSAID’s like
aspirin, ibuprofen, naproxen, indomethacin, diclofenac,
and lumiracoxib (Figure 1). In addition, esters and
amides of indomethacin indeed are more COX2 selec-
tive and possess potent anti-inflammatory effects with-
out being ulcerogenic.14 Another analogue of diclofenac
with a carboxylic moiety and a selective COX2 inhibitor
2.1 Materials and methods
All the chemicals and reagents used were lab grade material
procured from Alfa aesar, India. All the solvents used were
purchased from commercial suppliers and were used with-
out further purification. The melting points were determined
using Buchi apparatus by the open capillary tube method and
were uncorrected. The IR spectra were recorded in Perkin-
Elmer series 2000 FTIR spectrophotometer using KBr pellet.
1H NMR and 13C NMR spectra were obtained in CDCl3,
DMSO-d6 on a Bruker spectrometer at 400 and 100 MHz,
respectively. The chemical shifts are reported in ppm (δ)
relative to tetramethylsilane as internal standard, coupling
constants (J) are in hertz (Hz). Spin multiplicities are given
as s (singlet), d (doublet), t (triplet), dd (doublet of doublet),
bs (broad signal) and m (multiplet). Residual proton and car-
bon solvent signal for CDCl3, δH7.26 ppm, δC77.0 ppm, δ6-
DMSO, δH2.50 ppm, δC40.0 ppm. Proton and carbon spectra

Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
were typically obtained at room temperature. Mass spectra in chlorobenzene (60 mL) at 25–30◦C. The reaction mix-
were recorded on ESI—Perkin Elmer Sciex, API 3000 mass ture was heated to 95–105◦C and maintained for 3 h. The
spectrometer. Pre-coated silica gel GF254 plates from Merck progress of the reaction was monitored using TLC (hex-
were used for thin layer chromatography (TLC). The elemen- ane:ethyl acetate 3:7). After completion of the reaction,
tal analyses wererecorded inThermo FinniganFlashEA1112 aqueous HCl (1:1, 50 mL) was added to the mass at 25−30◦C.
elemental analyser.
The product was extracted with ethyl acetate (50 mL × 2)
and the organic layer was washed with water. Ethyl acetate
was evaporated under vacuum and the residue was triturated
with dichloromethane (100 mL), filtered to get compound 4.
4.85 g (88% yield). Light green powder, M.p.: 240−243◦C.
2.2 Synthesis
2.2a Synthesis of 2,3-bis(4-methoxy phenyl)-3-chloro- IR(KBr)cm−1: 3428, 3318, 1639, 1545, 1442, 1255, 1069,
1
2-prop-2-ene aldehyde (2): Phosphorous oxychloride 829. H NMR (400 MHz, CDCl3 + DMSO-d6): δ 8.48 (bs,
(POCl3) (7.66 g, 0.05 mole) was added drop wise over a 1H), 8.19 (bs, 1H), 7.75 (s, 1H), 7.17 (d, J = 8.5 Hz, 2H),
period of 15–30 min with stirring at 0−5◦C to 20 mL of 7.11 (d, J = 8.4 Hz, 2H), 6.77–6.75 (m, 4H). 13C NMR
dimethylformamide. The mass was maintained at 0−5◦C over (100 MHz, CDCl3+DMSO-d6):δ 164.3, 157.5, 156.4, 144.9,
30 min and a solution of desoxyanisoin (10 g, 0.04 mole) 137.8, 136.2, 130.9, 130.4, 130.0, 127.1, 124.9, 115.6, 115.5.
in dimethylformamide (50 mL) at 0−5◦C was added under ESI-MS m/z Calculated 312.0. Found: 313.0 [M + H]+.
stirring. The reaction mass was heated to 70−75◦C and main-
tained for 4 h. The progress of the reaction was monitored
using TLC (toluene). After completion, the reaction mix-
ture was cooled and poured slowly into 25% solution of
2.2d Synthesis of methyl 4,5-bis(4-hydroxyphenyl)
thiophene-2-carboxylate (5): 4,5-Bis(4-hydroxyphenyl)
thiophene-2-carboxylic acid (3.12 g, 0.01 mol) dissolved in
sodium acetate in water (100 mL). The product was filtered
methanol (78 mL) and concentrated sulphuric acid (1.0 g,
and washed with water, followed by slurry wash with ethanol
0.01 mol) was added slowly and refluxed for 8 h. The reaction
(100 mL) which after drying gave 9.1 g (78% yield) of the
wasmonitoredbyusingTLC(hexane:ethylacetate3:7). After
the reaction was completed, the reaction mixture was cooled
and concentrated. To this residue 20 mL of water added, the
title compound 2. White powder, M.p.: 145−147 ◦C. [Lit.12;
158◦C]. IR(KBr) cm−1: 2932, 1680, 1513, 1442, 1080, 813,
772. 1H NMR (400 MHz, CDCl3): δ 9.66 (s, 1H), 7.52
product was extracted with ethyl acetate (2 × 50 mL). The
organic phase was separated, washed with water, 10% sodium
carbonate solution followed by water and evaporated under
vacuum. The residue was triturated with heptane (20 mL),
filtered to get compound 5. 2.8 g (86% yield). The product
obtained did not require any further purification for next step.
(d, J = 7.8 Hz, 2H), 7.25–7.22 (m, 2H), 6.99–6.91 (m, 4H),
3.89 (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
190.5, 162.1, 159.7, 155.1, 139.8, 132.4, 132.1, 131.1, 130.6,
128.4, 126.7, 114.2, 113.9, 55.8, 55.5. ESI-MS m/z Calculated
302.1. Found: 303.1 [M + H]+.
Off-white powder, M.p.: 211−213◦C. IR (KBr)cm−1
:
2.2b Synthesisof4,5-bis(4-methoxyphenyl)thiophene-
2-carboxylic acid (3): 2,3-Bis(4-methoxy phenyl)-3-
chloro-2-prop-2-ene aldehyde 2 (5 g, 0.0165 mol) was added
to a solution of potassium hydroxide (4 g, 0.714 mol) and
2-mercapto acetic acid (3.1 g, 0.034 mol) in methanol: water
(40mL:10mL)mixtureatroomtemperature. Themixturewas
refluxed for 4 h, and reaction was monitored using TLC (hex-
ane: ethyl acetate 3:7). After completion, the reaction mixture
was cooled to room temperature and slowly acidified with
concentrated HCl over 30–45 min at 25−30◦C. The product
3 was filtered, washed with water and dried to get compound
3. 4 g (71% yield). Yellow powder, M.p.: 211−213◦C. [Lit.12;
215◦C]. IR(KBr) cm−1: 2933, 2542, 1668, 1546, 1449, 1247,
1033, 827. 1H NMR (400MHz, CDCl3): δ 10.7 (bs, 1H), 7.87
(s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.6 Hz, 2H),
6.85–6.81 (m, 4H), 3.83 (s, 3H), 3.81 (s, 3H). 13C NMR
(100 MHz, CDCl3): δ 168.0, 159.9, 159.1, 147.0, 138.4,
137.9, 130.6, 130.3, 129.5 128.1, 125.9, 114.3, 114.1, 55.5,
55.4. ESI-MS m/z Calculated 340.1. Found: 339.1 [M−H]−.
1
3325, 2949, 1664, 1547, 1441, 1227, 1079, 834, 756. H
NMR (400 MHz, CDCl3 +DMSO-d6) δ 8.12 (s, 1H), 7.76 (s,
2H), 7.17 (d, 2H, J = 8.44 Hz), 7.10 (d, 2H, J = 8.4 Hz),
6.77–6.75 (m, 4H), 3.89 (s, 3H). 13C NMR (100 MHz,
CDCl3 + DMSO-d6) δ 162.9, 157.5, 156.4, 145.4, 137.9,
136.5, 130.4, 130.1, 129.5, 127.0, 124.8, 115.7, 115.6, 52.12.
ESI-MS m/z Calculated 326.1. Found: 327.1 [M+H]+. Anal.
Calculated for C18H14O4S : C, 66.24; H, 4.32%. Found: C,
66.28; H, 4.30%.
2.2e Synthesis of ethyl 4,5-bis(4-hydroxyphenyl)
thiophene-2-carboxylate (6): 4,5-Bis(4-hydroxyphenyl)
thiophene-2-carboxylic acid (3.12 g, 0.01 mol) dissolved in
ethanol (78 mL) and concentrated sulphuric acid (1.0 g,
0.01 mol) was added slowly and refluxed for 8 h. The reaction
was monitored by using TLC (hexane:ethyl acetate 3:7). After
the reaction was completed, the reaction mixture was cooled
and concentrated. To this residue water (20 mL) was added
and the product was extracted with ethyl acetate (2×50 mL).
2.2c Synthesis of 4,5-bis(4-hydroxyphenyl)thiophene- The organic phase was separated, washed with water, 10%
2-carboxylic acid (4):
4,5-Bis(4-methoxyphenyl) sodium carbonate solution followed by water and evapo-
thiophene-2-carboxylic acid 3 (6 g, 0.0176 mol) was added rated under vacuum. The residue was triturated with heptane
to the mixture of aluminium chloride (9.38 g, 0.0704 mol) (20 mL), filtered to get compound 6. 3.0 g (88% yield). The

T Shanmuganathan et al.
product obtained did not require any further purification for compound 8. 2.94 g (88% yield). The product obtained was
next step.
used for next step without further purification.
Yellow powder, M.p.: 202−204◦C. IR (KBr) cm−1: 3317,
Yellow powder, M.p.: 65−67◦C IR (KBr) cm−1: 3429,
1
2937, 1656, 1547, 1442, 1228, 1077, 833, 756. H NMR 2925, 2862, 1696, 1650, 1546, 1441, 1247, 1073, 827,
(400 MHz, CDCl3 + DMSO-d6) δ 9.19(s, 1H), 8.99 (s, 1H), 755. 1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.24
7.71(s, 1H), 7.14 (d, 2H, J = 8.56 Hz), 7.08 (d, 2H, J = (d, 2H, J = 8.76 Hz), 7.18 (d, 2H, J = 8.68 Hz), 6.85–6.82
8.52 Hz), 6.76–6.73 (m, 4H), 4.37 (q, 2H, J = 7.08 Hz), (m, 4H), 6.11–6.00 (m, 2H), 5.44–5.39 (m, 2H), 5.31–
1.39 (t, 3H, J = 7.12 Hz). 13C NMR (100 MHz, CDCl3 + 5.28 (m, 2H), 4.54–4.52 (d, 4H, J = 5.32 Hz), 4.39–4.33
DMSO-d6) δ 160.9, 156.8, 155.7, 143.8, 136.8, 134.9, 129.2, (q, 2H, J = 7.12 Hz), 1.40 (t, 3H, J = 7.12 Hz). 13C
128.9, 125.4, 123.4, 123.2, 114.6, 114.5, 59.86, 13.36. ESI- NMR (100 MHz, CDCl3) δ 162.4, 158.7, 157.9, 144.8, 137.8,
MS m/z Calculated 340.1. Found: 341.1 [M + H]+. Anal. 136.1, 133.2, 133.1, 133.0, 132.9, 130.7, 130.4, 130.1, 128.3,
Calculated for C19H16O4S: C, 67.04; H, 4.74%. Found: C, 126.1, 117.9, 117.8, 114.8, 114.7, 114.7, 114.6, 68.8, 68.7,
67.08; H, 4.72%.
61.2, 14.4. ESI-MS m/z Calculated 420.1. Found: 421.3
[M + H]+. Anal. Calculated for C25H24O4S: C, 71.40; H,
5.75%. Found: C, 71.44; H, 5.78%.
2.2f Synthesis of methyl 4,5-bis(4-(allyloxy)phenyl)
thiophene-2-carboxylate (7): Allyl bromide (2.5 g,
0.02 mol) was added to the mixture of methyl 4,5-bis(4-
hydroxyphenyl)thiophene-2-carboxylate (2.5 g, 0.008 mol),
potassiumcarbonate(2.77g, 0.02mol)inacetonitrile(50mL).
The reaction mixture was refluxed for 4 h. The reaction was
monitored by using TLC (hexane: ethyl acetate 1:1). After the
completion of the reaction, the reaction mixture was cooled
and filtered to remove insolubles. The organic mass was
evaporated under vacuum and to this residue water (20 mL)
was added and the product was extracted with ethyl acetate
(2 × 50 mL). The organic phase was separated, washed with
water, and evaporated under vacuum. The residue was tritu-
rated with hexane (20 mL), cooled to 10–15◦C and filtered
to get compound 7. 2.7 g (87% yield). The product obtained
was used for next step without further purification. Pale brown
powder, M.p.: 66−68◦C. IR (KBr) cm−1: 3390, 2946, 2862,
2.2h Synthesis of allyl 4,5-bis(4-(allyloxy)phenyl)
thiophene-2-carboxylate (9): Allyl bromide (4.2 g,
0.035 mol) was added to the mixture of ethyl 4,5-bis(4-
hydroxyphenyl)thiophene-2-carboxylic acid (3 g, 0.01 mol),
potassiumcarbonate(4.8g, 0.035mol)inacetonitrile(75mL).
The reaction mixture was refluxed for 4 h. The reaction was
monitored by using TLC (hexane:ethyl acetate 1:1). After
the reaction was completed, the reaction mixture was cooled
and filtered to remove insolubles. The organic mass was
evaporated under vacuum and to this residue water (20 mL)
was added and the product was extracted with ethyl acetate
(2 × 50 mL). The organic phase was separated, washed with
water, and evaporated under vacuum to get compound 9.
3.4 g (82% yield). Brown liquid, IR (KBr) cm−1: 2924, 2862,
1709, 1547, 1443, 1243, 1069, 831, 753. 1H NMR (400 MHz,
CDCl3) δ 7.72 (s, 1H), 7.16 (d, 2H, J = 8.4 Hz), 7.10 (d, 2H,
J = 8.4 Hz), 6.77–6.74 (m, 4H), 6.01–5.91 (m, 3H), 5.36–
5.31 (m, 3H), 5.22–5.20 (m, 3H), 4.73 (d, 2H, J = 5.32 Hz),
4.45 (d, 4H, J = 5 Hz). 13C NMR (100 MHz, CDCl3) δ
201.7, 162.1, 158.9, 158.0, 145.3, 138.0, 136.5, 133.3, 133.1,
132.2, 130.6, 130.4, 130.2, 128.4, 126.2, 118.5, 118.0, 117.9,
114.9, 114.8, 68.9, 65.7, 31.1. ESI-MS m/z Calculated 432.1.
Found: 433.2 [M + H]+. Anal. Calculated for C26H24O4S :
C, 72.20; H, 5.59%. Found: C, 72.26; H, 5.56%.
1
1704, 1651, 1543, 1440, 1242, 1073, 833, 754. H NMR
(400Mz, CDCl3) δ 7.78 (s, 1H), 7.24 (d, 2H, J = 6.76 Hz),
7.18 (d, 2H, J = 6.76 Hz), 6.85–6.82 (m, 4H), 6.07–6.02
(m, 2H), 5.44–5.39 (m, 2H), 5.31–5.28 (m, 2H), 4.54–4.52
(m, 4H), 3.89 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 162.9,
158.8, 157.9, 145.0, 137.9, 136.4, 133.3, 133.1, 130.5, 130.3,
130.2, 128.3, 126.1, 118.0, 117.87, 114.9, 114.8, 68.9, 68.8,
52.25. ESI-MS m/z Calculated 406.1. Found: 407.1 [M+H]+.
Anal. Calculated for C24H22O4S: C, 70.91; H, 5.46%. Found:
C, 70.95; H, 5.48%.
2.2i 4,5-Bis(4-(allyloxy)phenyl)thiophene-2-carboxylic
acid (10): Ethyl 4,5-bis(4-(allyloxy)phenyl)thiophene-2-
2.2g Synthesis of ethyl 4,5-bis(4-(allyloxy)phenyl) carboxylate (0.6 g, 0.0014 mol) was added at room tempera-
thiophene-2-carboxylate (8): Allyl bromide (2.5 g ture to a solution of sodium hydroxide (0.54 g, 0.0135 mol)
0.02 mol) was added to the mixture of ethyl 4,5-bis(4- in ethanol:water (8 mL:2 mL). The mixture was refluxed for
hydroxyphenyl)thiophene-2-carboxylate (2.7 g, 0.008 mol), 2 h. The reaction was monitored by using TLC (hexane: ethyl
potassiumcarbonate(2.77g, 0.02mol)inacetonitrile(50mL). acetate 2:8). After completion of the reaction, the mixture
The reaction mixture was refluxed for 4 h. The reaction was was cooled and the impurities were extracted with toluene.
monitored by using TLC (hexane:ethyl acetate 1:1). After The aqueous phase was acidified with hydrochloric acid and
the reaction was completed, the reaction mixture was cooled the product was extracted with ethyl acetate (20 mL). The
and filtered to remove insolubles. The organic mass was organic phase was washed with water, dried and evaporated
evaporated under vacuum and to this residue water (20 mL) to get compound 10. (0.42 g, 90% yield). Off-white pow-
was added and the product was extracted with ethyl acetate der, M.p.: 110−111◦C. IR (KBr) cm−1: 2919, 2856, 2537,
(2 × 50 mL). The organic phase was separated, washed with 1672, 1544, 1444, 1247, 1074, 830, 757. 1H NMR (400 MHz,
water, and evaporated under vacuum. The residue was tritu- DMSO-d6) δ 7.71 (s, 1H), 7.22–7.17 (m, 4H), 6.95–6.89 (m,
rated with hexane (20 mL), cooled to 10–15◦C filtered to get 4H), 6.05–6.01 (m, 2H), 5.42–5.38 (m, 2H), 5.28-5.25 (m,

Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
2H), 4.56 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 162.8, 2.2l Ethyl 4,5-bis(4-(3-hydroxypropoxy)phenyl)thio
158.4, 157.5, 143.6, 137.6, 135.5, 133.7, 133.5, 131.8, 130.2, phene-2-carboxylate (13): Ethyl 4,5-bis(4-(allyloxy)
129.9, 127.5, 125.4, 117.8, 117.6, 115.1, 114.8, 68.3, 68.2, phenyl)thiophene-2-carboxylate (2.1 g, 0.005 mol) was dis-
59.8, 20.8, 14.1. ESI-MS m/z Calculated 392.1. Found: 391.4 solved in tetrahydrofuran (50 mL) and cooled to 0◦C. Borane
[M-H]−. 393.2 [M + H]+, Anal. Calculated for C23H20O4S: 1 M solution in THF (11.8 mL) was added and stirred at
C, 70.39; H, 5.14%. Found: C, 70.42; H, 5.16%.
0◦C for 1 h. The reaction mixture was slowly warmed to
room temperature and maintained for 4 h. The reaction mix-
ture was quenched with water (1 mL) followed by addition
of 2 N sodium hydroxide solution (2 mL), hydrogen perox-
ide (40% w/w, 2 mL) solution and stirred for 1 h at room
temperature. The product was extracted with ethyl acetate
(100 mL) and the organic phase was separated, washed with
20% w/w sodium chloride solution followed by water, dried
and evaporated under vacuum. The reaction produced a mix-
tureofcompounds, themajor(80%)componentofthemixture
being ethyl 4,5-bis(4-(3-hydroxypropoxy)phenyl)thiophene-
2-carboxylate (13) (R f = 0.25, SiO2, 30% ethyl acetate in
hexanes), and the minor (20%) ethyl 5(4)-
(4-(2-hydroxypropoxy)phenyl)-4(5)-(4-(3-hydroxypropoxy)
phenyl)thiophene-2-carboxylate (14) (Rf = 0.4, SiO2, 30%
ethyl acetate in hexanes). The product was isolated by col-
umn chromatography by eluting with 6:4 v/v hexane–ethyl
acetate to get compound 13 (1.3 g, 57% yield) and 14
(0.4 g, 17% yield). Yellow liquid, IR (KBr) cm−1: 3413,
2.2j Methyl 4,5-bis(4-(3-hydroxypropoxy)phenyl)thio
phene-2-carboxylate (11): Methyl 4,5-bis(4-(allyloxy)
phenyl)thiophene-2-carboxylate (2 g, 0.005 mol) dissolved
in tetrahydrofuran (50 mL) and cooled to 0◦C. Borane 1 M
solution in THF (11.8 mL) was added and stirred at 0◦C
for 1 h. The reaction mixture was slowly warmed to room
temperature and maintained for 4 h. The reaction mixture
was quenched with water (1 mL) followed by addition of
2 N sodium hydroxide solution (2 mL), hydrogen perox-
ide (40% w/w, 2 mL) solution and stirred for 1 h at room
temperature. The product was extracted with ethyl acetate
(100 mL). The organic phase was separated and washed
with 20% w/w sodium chloride solution followed by water,
dried and evaporated under vacuum. The reaction produced
a mixture of compounds, the major (80%) component of the
mixture being methyl 4,5-bis(4-(3-hydroxypropoxy)phenyl)
thiophene-2-carboxylate (11) (Rf = 0.25, SiO2, 30% ethyl
acetate in hexanes), and the minor (20%) methyl 5(4)-
(4-(2-hydroxypropoxy)phenyl)-4(5)-(4-(3-hydroxypropoxy)
phenyl) thiophene-2-carboxylate (12) (Rf = 0.4, SiO2, 30%
ethyl acetate in hexanes). The product was isolated by col-
umn chromatography by eluting with 6:4 v/v hexane–ethyl
acetate to get compound 11 (1.2 g, 55% yield) and 12
(0.3 g, 14% yield). Yellow liquid, IR (KBr) cm−1: 3423,
1
2926, 2874, 1704, 1547, 1443, 1246, 1061, 830, 754. H
NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.25 (d, 2H, J =
8.8 Hz), 7.18 (d, 2H, J = 8.72 Hz), 6.85–6.82 (m, 4H), 4.40
(q, 2H, J = 7.12 Hz), 3.89 (m, 4H), 2.85 (s, 2H), 2.08–1.98
(m, 4H), 1.44–1.38(m, 3H). 13C NMR (100 MHz, CDCl3) δ
162.6, 159.1, 158.2, 144.9, 137.9, 136.3, 130.9, 130.6, 130.3,
128.5, 126.2, 114.7, 114.6, 65.8, 65.7, 61.3, 60.4, 60.3, 32.2,
32.1, 14.5. ESI-MS m/z 501.3 [M + HCOO]−. ESI-MS m/z
Calculated 456.2. Found: 501.3 [M + HCOO]−. Anal. Calcu-
lated for C25H28O6S: C, 65.77; H, 6.18%. Found: C, 65.74;
H, 6.16%.
1
2928, 2877, 1712, 1547, 1446, 1247, 1063, 832, 755. H
NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.23 (d, 2H, J =
8.76 Hz), 7.17 (d, 2H, J = 8.72 Hz), 6.83–6.80 (m, 4H), 4.12
(t, 4H, J = 5.88 Hz), 3.89 (s, 3H), 3.87–3.84 (m, 4H), 2.06-
*1.97 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 163.0, 159.1,
158.2, 145.2, 138.0, 136.5, 130.6, 130.3, 130.2, 128.4, 126.2, 2.2m Ethyl 5(4)-(4-(2-hydroxypropoxy)phenyl)-4(5)-
114.7, 114.6, 65.7, 65.6, 60.5, 60.3, 52.3, 32.1, 29.8. ESI-MS (4-(3-hydroxypropoxy)phenyl)thiophene-2-carboxylate
m/z Calculated 442.2. Found: 487.2 [M + HCOO]−. Anal. (14): Yellow liquid, IR (KBr) cm−1: 3421, 2926, 2873,
Calculated for C24H26O6S: C, 65.14; H, 5.92%. Found: C, 1705, 1547, 1443, 1247, 1072, 830, 754. 1H NMR (400 MHz,
65.16; H, 5.96%.
CDCl3)δ 7.70 (s, 1H), 7.17–7.14 (m, 2H), 7.10–7.08 (m,
2H), 6.77–6.73 (m, 4H), 4.31–4.27 (m, 2H), 4.14–4.10
(m, 1H), 4.06 (t, 2H, J = 5.28 Hz), 3.88–3.84 (m, 1H),
3.81–3.77 (m, 2 H), 3.74 (t, 1H, J = 8.28 Hz), 1.99–
1.96 (m, 2H), 1.35–1.33(m, 3H), 1.22–1.20 (m, 3H). 13C
NMR (100 MHz, CDCl3) δ 161.4, 157.9, 157.0, 143.9, 136.9,
135.0, 129.8, 129.5, 129.2, 127.6, 125.4, 113.7, 113.5, 72.2,
65.2, 64.6, 60.2, 59.4, 30.9, 17.6, 13.4. ESI-MS m/z Calcu-
lated 456.2. Found: 501.2 [M + HCOO]−. Anal. Calculated
for C25H28O6S: C, 65.77; H, 6.18%. Found: C, 65.72; H,
6.14%.
2.2k Methyl5(4)-(4-(2-hydroxypropoxy)phenyl)-4(5)-
(4-(3-hydroxypropoxy)phenyl)thiophene-2-carboxylate
(12): Yellow liquid, IR (KBr) cm−1: 3400, 2925, 2872,
1710, 1546, 1446, 1246, 1076, 830, 754. 1H NMR (400 MHz,
CDCl3) δ 7.78 (s, 1H), 7.25–7.22 (m, 2H), 7.19–7.16 (m, 2H),
6.84–6.82 (m, 4H), 4.20 (brs, 1H), 4.14 (t, 2H, J = 8.76 Hz),
3.96–3.94 (m, 1H), 3.90 (s, 3H), 3.89–3.87 (m, 2 H), 3.82–
3.79 (t, 1H, J = 8.32 Hz), 2.28–2.07 (m, 1H), 2.06–2.04 (m,
2H), 1.70–1.65 (m, 1H), 1.30 (d, 3H) J = 6.36 Hz). 13C
NMR (100 MHz, CDCl3)δ 163.0, 158.8, 158.2, 145.2, 138.1,
136.6, 130.7, 130.5, 130.4, 130.3, 128.4, 126.2, 114.8, 114.7, 2.2n 4,5-Bis(4-(3-hydroxypropoxy)phenyl)thiophene-
73.4, 66.4, 65.8, 60.6, 52.4, 32.1, 18.92. ESI-MS m/z Calcu- 2-carboxylic
acid
(15):
Methyl
4,5-bis(4-
lated 442.2. Found: 487.3 [M + HCOO]−. Anal. Calculated (3-hydroxypropoxy)phenyl)thiophene-2-carboxylate (0.5 g,
for C24H26O6S: C, 65.14; H, 5.92%. Found: C, 65.18; H, 0.001 mol) was added at room temperature to a solution
5.94%.
of sodium hydroxide (0.5 g, 0.0125 mol) in ethanol:water

T Shanmuganathan et al.
(8 mL:2 mL). The mixture was refluxed for 2 h. The addition of phosphate buffered saline. The control represents
reaction was monitored by TLC using hexane:ethyl acetate 100% protein denaturation. The results were compared with
(2 mL:8 mL) as a solvent system. After the reaction was com- reference drug diclofenac sodium. The percentage inhibition
pleted, the reaction mixture was cooled and the impurities of protein denaturation was calculated by using the following
were extracted with toluene. The aqueous phase was acid- formula:
ified with hydrochloric acid and the product was extracted
with ethyl acetate (20 mL). The organic phase was washed
with water, dried and evaporated to get compound 15. (0.44 g,
Percentage Inhibition = 100
− [(optical density of test solution
91% yield). Pale brown powder, M.p.: 136−138◦C. IR (KBr)
cm−1: 3385, 2925, 2854, 2602, 1686, 1547, 1442, 1248, 1086,
832, 757. 1H NMR (400MHz, DMSO-d6) δ 7.71 (s, 1H),
7.23 (d, 2H, J = 8.64 Hz), 7.17 (d, 2H, J = 8.6 Hz), 6.93
(d, 2H, J = 8.76 Hz), 6.90 (d, 2H, J = 8.72 Hz), 4.05 (quar-
tet, 4H, J = 6.0 Hz), 3.58(t, 4H, J = 6.08 Hz), 1.90 (quintet,
4H, J = 6.12 Hz). 13C NMR (100 MHz, DMSO-d6) δ 162.8,
158.9, 158.0, 143.6, 137.6, 131.7, 130.2, 129.9, 127.3, 125.1,
114.8, 114.5, 114.3, 64.6, 64.5, 57.2, 32.1, 32.0. ESI-MS m/z
Calculated 428.1. Found: 427.2 [M-H]−. Anal. Calculated for
C23H24O6S: C, 64.47; H, 5.65%. Found: C, 64.49; H, 5.62%.
− optical density of product control)
÷ (optical density of test control)] × 100.
2.4 In vitro anti-oxidant activity
The antioxidant activity of the test drug was determined using
the 1,1-diphenyl-2 picrylhydrazyl (DPPH) free radical scav-
enging assay.19 The test drug was mixed with 95% methanol
to prepare the stock solution in required concentration
(100 μg/mL). From the stock solution, 10 μg/mL, 20 μg/mL,
40 μg/mL, 60 μg/mL and 100 μg/mL concentration of test
drug was prepared. Ascorbic acid was used as standard and
was prepared in same concentration as that of the test drug
by using methanol as solvent. Final reaction mixture contain-
ing 1 mL of 0.3 mmol DPPH methanol solution was added
to 2.5 mL of sample solution of different concentrations and
allowed to react at room temperature after 15 min incuba-
tion period at 37◦C. Absorbance was read out at 517 nm.
Control reading was observed without adding test drug. %
scavenging = [(Absorbance of control − Absorbance of test
sample) ÷ (Absorbance of control)] × 100.
2.2o 5(4)-(4-(2-Hydroxypropoxy)phenyl)-4(5)-(4-(3-
hydroxypropoxy)phenyl)thiophene-2-carboxylic acid
(16): Methyl 5(4)-(4-(2-hydroxypropoxy)phenyl)-4(5)-(4-
(3-hydroxypropoxy)phenyl)thiophene-2-carboxylate (0.5 g,
0.001 mol) was added at room temperature to a solution
of sodium hydroxide (0.5 g, 0.0125 mol) in ethanol:water
(8 mL:2 mL). The mixture was refluxed for 2 h. The reaction
was monitored by using TLC (hexane:ethyl acetate 2:8). After
the reaction was completed, the reaction mixture was cooled
and the impurities were extracted with toluene. The aqueous
phase was acidified with hydrochloric acid and the product
was extracted with ethyl acetate (20 mL). The organic phase
was washed with water, dried and evaporated to get compound
16. (0.42 g, 87% yield). Yellow powder, M.p.: 150−152◦C.
IR (KBr) cm−1: 3394, 2923, 2853, 1682, 1545, 1445, 1246,
1060, 830, 754. 1H NMR (400 MHz, DMSO-d6) δ 12.9 (brs,
1H), 7.71 (s, 1H), 7.22–7.17 (m, 4H), 6.93–6.87 (m, 4H), 4.90
(s, 1H), 4.58 (2,1H), 4.05–4.01 (m, 2H), 3.97–3.93 (m, 1H),
3.85–3.76 (m, 2H), 3.57–3.55 (m, 2 H), 1.89–1.83 (m, 2H),
1.16 (t, 3H, J = 6.24 Hz). 13C NMR (100 MHz, DMSO-d6) δ
162.8, 158.9, 158.0, 143.6, 137.6, 131.8, 130.2, 129.9, 127.3,
127.2, 125.2, 125.1, 115.1, 114.9, 114.8, 114.5, 114.2, 64.8,
64.5, 57.2, 32.1, 20.1. ESI-MS m/z Calculated 428.1. Found:
427.2 [M-H]−. Anal. Calculated for C23H24O6S: C, 64.47;
H, 5.65%. Found: C, 64.45; H, 5.68%.
2.5 Molecular docking studies
Molecular docking was performed for the synthesised com-
pounds in order to investigate their possible binding mode in
the cyclooxygenase-2 (COX-2) receptor. Docking was per-
formed using the Auto Dock Tools (ADT) version 1.5.6 and
Auto Dock version 4.2.5.1 docking program.20,21 The struc-
ture of COX-2 was downloaded from the Protein Data Bank
(PDB ID: 1PXX).22 The co-crystallized ligand and water
molecules present in the 1PXX structure were removed. Then,
polar hydrogen atoms were added, and lower occupancy
residue structures were deleted. ADT was used to replace
any incomplete side chains. Gasteiger charges were added to
each atom and merged the non-polar hydrogen atoms to the
protein structure. The hydrogen bond distance between donor
and acceptor atoms were defined as 1.9 Å with a tolerance of
0.5 Å and the threshold for acceptor-hydrogen-donor angle
was set to not less than 120◦. For further studies in ADT,
the structures were saved in PDBQT file format. A grid box
2.3 In vitro anti-inflammatory activity
(anti-denaturation assay)
The in vitro anti-inflammatory activity of synthesized com- centred on 27.131, 24.348 and 14.747 with the dimension of
pounds were studied using bovine serum albumin denatura- 60 × 60 × 60 Å3 and 0.375 Å spacing was created around
tion method.17,18 In brief, increasing concentrations of the the binding site of co-crystallised ligand using ADT. Grid
test or reference compound were incubated with 0.5% w/v of energy calculations were carried out with the centre of the
bovine serum albumin at 37◦C for 20 min and the temperature box was set at co-crystallised ligand centre. Default docking
was increased to keep the samples at 57◦C for 30 min. After parameters were used and twenty docked conformations for
cooling to room temperature, the turbidity was measured each compound were generated. Genetic algorithm was used
using UV-Visible spectrophotometer at 660 nm following to estimate the energy of the binding interactions. PyMOL

Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
was used to visualise the binding modes and interactions of hydroxide in aqueous ethanol under reflux condition
the docked compounds with amino acid residues in the active
site of COX-2 receptor.
to get compound 15 in 88% yields. Similarly compound
12 and 14 was hydrolysed with sodium hydroxide in
aqueous ethanol under reflux condition to get compound
16 in 88% yield.
3. Results and Discussion
3.1 Chemistry
3.2 Characterization
In the present work, we synthesized a new series of The structure of the synthesized intermediate compound
allyloxy and hydroxypropoxy derivatives of 4,5-diaryl 7 was confirmed by various spectral techniques such
thiophene-2-carboxylic acid, as shown in Scheme 1. as NMR, Mass and IR data. IR spectrum of the com-
The first step in the Scheme 1 is the Vilsmeier reac- pound 7 revealed that the bands between ∼2950 cm−1
tion23,24 of desoxyanisoin 1 with dimethylformamide and ∼ 2850 cm−1 correspond to the asymmetric and
(DMF) and phosphorous oxychloride (POCl3) to give symmetric stretching of methylene groups and the band
compound 2 in 78% yield after recrystallization from at around ∼ 1704 cm−1 corresponds to the carbonyl
1
ethanol.12 Compound 2 was condensed and cyclized group of the ester moiety. The H NMR spectrum
with 2-mercaptoacetic acid (thioglycolic acid) in the recorded in CDCl3 showed twenty-two protons. The
presence of potassium hydroxide to afford compound singlet observed at δ 7.78ppm corresponds to the aryl
325 in 71% yield. Treatment of compound 3 with alu- proton of the thiophene moiety. The multiplet at around
minium chloride in chlorobenzene16,26 at 95–105◦C over δ 4.54–4.52ppm corresponds to the methylene protons
3 h under stirring is vital for the demethylation of com- attached to phenoxy ring system. The singlet observed
pound 3 to get compound 4 in good yield (88%). The at δ 3.89ppm corresponds to the methyl proton of
obtained compound 4 did not require any further purifi- the ester group. The 13C NMR spectrum recorded in
cation.
CDCl3 showed twenty-four signals, in which the signal
The compound 4 obtained was converted to the cor- observed at δ 162.9ppm correspond to carbonyl carbon
responding ester 5 and 6 by using alcohol in presence of the ester moiety. The two signals at δ 114.9ppm and
of sulphuric acid. Esterification of compound 4 with at δ 114.8ppm correspond to the four aryl carbons at
methanol or ethanol and sulphuric acid under reflux con- the ortho position to hydroxyl derivative of the phenyl
dition over 8 h gave 86% and 88% yield of compound rings. The signals at δ 68.9 and δ 68.8ppm confirmed
5 and6, respectively. The allyation of compound 4, 5 the methylene carbons of allyloxy group. The signal
and 6 with ally bromide and potassium carbonate27 in at δ 52.3ppm confirmed the methyl carbon of ester
acetonitrile under reflux condition gave 87%, 88% and group. Mass spectrum acquired in positive ionization
82%, yield of compound 7, 8 and 9, respectively.
ESI mode, showed a signal at 407.1Da corresponding
The allyl carboxylic acid ester 7 or 8 or 9 was hydrol- to [M + H]+, which confirmed the molecular mass of the
ysed with sodium hydroxide to get compound 10 in compound. In addition to the above spectral evidences
90% yield. Similarly compound 14 was hydrolysed with discussed, the structure of synthesized compound 7 was
sodium hydroxide to get carboxylic acid compound 16 further confirmed unequivocally with single-crystal X-
in 88% yield.
ray diffraction data. The crystal data was deposited at
The allyl carboxylic acid ester 7 was converted CCDC, and the CCDC No. is 1406560. The ORTEP dia-
to alcohol by hydroboration followed by oxidation gram of above crystal compound is shown in Figure 2.
with hydrogen peroxide and sodium hydroxide solu-
The structure of the synthesized compound 15 was
tion.27 The reaction produced a mixture of compounds, confirmed by various spectral techniques such as NMR,
the major (80%) component of the mixture being Mass and IR data. IR spectrum of the compound
methyl4,5-bis(4-(3-hydroxypropoxy)phenyl)thiophene- 15 revealed that the broad band observed at around
2-carboxylate compound 11 and the minor (20%) 3360–3300 cm−1 corresponds to the carboxylic acid
methyl 5(4)-(4-(2-hydroxypropoxy)phenyl)-4(5)-(4-(3- stretching coupled with the hydroxyl stretching of the
hydroxypropoxy)phenyl)thiophene-2-carboxylatecom- propyl group. The bands observed at ∼ 2925 cm−1 and
pound 12. The product was isolated by column chro- ∼ 2854 cm−1 correspond to the asymmetric and sym-
matography eluting with 6:4 v/v hexane–ethyl acetate to metric stretching of methylene groups. The band at
afford the product 11 (55% yield) and 12 (14% yield).
around ∼ 1686 cm−1 corresponds to the carbonyl group
1
Similarly compound 8 was converted to alcohol of the acid moiety. The H NMR spectrum recorded
compound 13 (57% yield) and 14 (17% yield). The car- in DMSO-d6 showed twenty-four protons in which the
boxylic acid ester 11 or 13 was hydrolysed with sodium protons of hydroxyl and carboxylic groups weren’t

T Shanmuganathan et al.
Scheme 1. Synthesis of 4,5-diarylthiophene-2-carboxylic acid derivatives. Reagents and conditions: (i)
DMF and POCl3, at 70−75◦C for 4h; (ii) 2-mercapto acetic acid, KOH, methanol and water, at reflux for
4h; (iii) AlCl3 and chlorobenzene, at 95−105◦C for 3h; (iv) methanol or ethanol, sulphuric acid, at reflux
for 8h; (v) allyl bromide, K2CO3, acetonitrile reflux for 4h; (vi) NaOH, ethanol and water, at reflux for 2h;
(vii) Borane in THF at 0◦C 1h, H2O2/NaOH at RT 1h.
observed. The singlet observed at δ 7.71ppm corre- carbonyl carbon of the acid group. The four signals at
sponds to the aryl proton of the thiophene ring system. δ 114.9, δ 114.8, δ 114.7 and δ114.5ppm correspond
The quartet at δ 4.05ppm corresponds to four methy- to the four aryl carbons of the phenoxy ring. The signal
lene protons attached to two hydroxy groups, the triplet at δ 57.5 ppm corresponds to the methylene groups of
at δ 3.58ppm corresponds to four methylene protons the hydroxy propyl side chain. Mass spectrum acquired
attached to two phenoxy group. The 13C NMR spectrum in negative ionization ESI mode showed the signal at
recorded in DMSO-d6 showed twenty-three signals, in 427.2Da which corresponds to [M − H] confirmed the
which the signal observed at δ 162.9ppm correspond to molecular mass of the compound.

Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
acid analogues. In the present study, we developed
and synthesized some newer derivatives of compound
4 by introducing hydroxyl substituents to the phenyl
ring and esterification of carboxylic acid of thiophene
ring. The anti-inflammatory activity of novel substi-
tuted 4,5-diarylthiophene-2-carboxylic acid derivatives
were evaluated using inhibition of bovine serum albu-
min denaturation method and compared with standard
drug diclofenac sodium. The results are summarized in
Table 1.
Most of the derivatives exhibited anti-inflammatory
activity that is comparable to diclofenac sodium. The
free acid 4 is less potent; however, derivatisation of
the hydroxy group to allyl with bisallyloxy substituted
phenyl moiety (10) resulted in analogues having anti-
inflammatory activity comparable to the standard drug.
Although, bisallyloxy substituted phenyl moiety with
methyl (7), ethyl (8) and allyl (9) esters of acid group
resulted in less active analogues. In contrast, the com-
pound4,5-bis(4-(3-hydroxypropoxy)phenyl)thiophene-
2-carboxylic acid (15) and its methyl (11), or ethyl
ester (13) exhibited better anti-inflammatory activity in
comparison to the standard drug whereas their byprod-
ucts 5(4)-(4-(2-hydroxypropoxy)phenyl)-4(5)-(4-(3-
hydroxypropoxy)phenyl)thiophene-2-carboxylic acid
(16) and its methyl (12) or ethyl ester (14) showed mod-
erate activity.
Taken together, these results clearly suggest that
allyloxy/hydroxypropoxy substitution on the phenyl
group of 4,5-diarylthiophene-2-carboxylic acid deriva-
tives is advantageous in improving or retaining the
anti-inflammatory spectrum. Further, an important SAR
observation relevant to the nature of substituents to the
diaryl ring indicates that symmetricity of substituents is
Figure 2. ORTEP diagram of compound 7 (methyl
4,5-bis(4-(allyloxy)phenyl)thiophene-2-carboxylate, CCDC
1406560).
3.3 Biological evaluation: structure activity
relationship (SAR)
3.3a In vitro anti-inflammatory activity: In our ear-
lier studies, we have identified that the introduction
of amino moieties at carboxylic acid of thiophene
ring16 significantly enhanced the anti-inflammatory
activity spectrum of 4,5-diarylthiophene-2-carboxylic
Table 1. In vitro anti-inflammatory activity of compounds 4, 7–16 by inhibition of
protein denaturation method (Bovine serum albumin).
Entry Compounds Activity (% inhibition of protein denaturation)
25μg/mL 50μg/mL 100μg/mL 200μg/mL 400μg/mL
1
2
3
4
5
6
7
8
4
7
8
9
10
11
12
13
14
15
16
Std
4.86
10.42
5.21
9.84
18.85
12.5
16.47
26.04
17.71
29.17
51.05
45.83
38.54
50.00
31.25
43.75
22.92
51.04
28.13
35.41
21.88
35.42
62.5
55.21
46.88
62.50
46.88
57.3
30.33
43.75
29.17
41.67
70.83
60.42
52.08
75.00
54.17
70.63
45.83
66.66
14.58
34.38
22.92
19.79
35.42
14.58
21.88
8.33
20.83
42.71
31.25
29.17
40.63
23.96
34.38
15.63
40.65
9
10
11
12
31.29
60.41
32.29
Std: Diclofenac sodium

T Shanmuganathan et al.
Table 2. Antioxidant activity of compounds 4, 7–16 by using 1,1-diphenyl-2-picrylhydrazyl
(DPPH) free radical scavenging assay.
Entry Compounds
% Inhibition
Antioxidant
IC50 value
10 μg/mL 20 μg/mL 40 μg/mL 60 μg/mL 100 μg/mL
1
2
3
4
5
6
7
8
4
7
8
3.59
2.78
6.76
1.27
24.1
21.25
9.62
27.27
13.95
9.73
12.05
41.01
7.61
3.17
15.01
8.77
30.87
28.86
15.12
39.32
19.45
13.11
24.63
61.84
14.16
9.83
19.34
12.68
27.17
18.39
46.93
43.66
27.91
52.75
28.86
32.03
35.41
86.89
23.78
19.45
30.97
23.78
53.28
50.63
34.88
65.22
37.53
37.42
41.23
92.39
208.64
255.54
164.96
204.97
80.95
89.48
148.87
56.16
146.81
129.19
120.77
5.754
17.34
13.53
39.22
37.42
20.82
45.77
24.84
23.57
29.07
73.25
9
10
11
12
13
14
15
16
Std
9
10
11
12
Std: Ascorbic acid
required for optimal anti-inflammatory as in case of 11 radical scavenging assay. The free radical scavenging
and 15 whereas when the symmetricity is lost (12, 14 activity of the synthesized compounds was assessed
and 16) a reduction in activity was observed. This indi- through their ability to quench the DPPH using ascor-
cates that lengthier substituents to the diaryl ring are bic acid as a standard. The potencies for the antioxidant
well tolerated resulting in substantial increase of anti- activity of compounds 4, 7–16 to the reference drug are
inflammatory potency.
shown in Table 2. In general, all the synthesized com-
The 4,5-diarylthiophene-2-carboxylic acid 4 or its pounds were less potent than the reference compound.
carboxyamide analogue reported in our earlier work16 Among the synthesized compounds, compound 10, 11
possess very little anti-inflammatory property in com- and 13 exhibited slightly moderate antioxidant activity
parison to diclofenac. On the other hand, derivatization when compared to the standard.
of the acid to ester or the amide with p-halo substi-
tuted phenyl moiety resulted in analogues possessing
3.4a Molecular docking studies: Molecular docking
activity comparable to diclofenac. The lipophilic sub-
analysis was performed for the synthesised compounds
stituent, namely the hydroxypropyl moiety to the diaryl
with the cyclooxygenase-2 (COX-2) receptor, which is
ring as in 13 and 15 appears to be the most important
substituent with regard to enhancing the activity which
was not seen with substitution to the diaryl ring with
a methoxy group.16 Taken together, the SAR analysis
with the carboxylic acid derivatives mentioned herein
and with the carboxamide derivatives reported earlier16
indicates that a bulkier lipophilic substitution to the
diaryl ring and with simple esters of the carboxylate
group might provide a lipophilic-hydrophilic balance
to the 4,5-diarylthiophene skeleton which led to an
increase in anti-inflammatory spectrum. In conclusion,
this study has identified some important substituents and
its arrangement on the diaryl ring which is crucial for
the anti-inflammatory property of 4,5-diarylthiophene-
2-carboxylic acid.
an important target for the anti-inflammatory activity.28
COX-2 is an attractive target for medicinal chemists
because of its very high expression in inflamed tissues as
well as many tumors.29,30 The compounds were docked
using Auto Dock Tools (ADT) version 1.5.6 and Auto
Dock version 4.2.5.1 docking program20,21 into the crys-
tal structure of COX-2 receptor (PDB ID: 1PXX).22
To verify the reproducibility of the docking calcula-
tion, the co-crystallised ligand was extracted from the
complex and submitted for one-ligand run calculation.
Dockingofco-crystallisedligandwithboundX-raycon-
formation for 1PXX exhibits root-mean-square devia-
tion (RMSD) value of 0.58 Å. This result signifies that
this method is valid enough to be used for docking stud-
ies of other compounds (Figure 3A).
Docking simulation of all the synthesised compounds
was performed using the same protocol of validation
study. For each of the test molecules, dockings were
3.4 Antioxidant activity
The antioxidant activity of the test drug was determined achieved by taken into 2.5 million energy evaluations.
using the 1,1-diphenyl-2 picrylhydrazyl (DPPH) free The conformation of docked ligand with COX-2 recep-

Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
Figure 3. Molecular docking simulation with COX-2 receptor (1PXX). (A) Method val-
idation using crystallised and docked ligand diclofenac; (B) docking simulation of all the
compounds in the active site; and (C) docking simulation of the most binding energy com-
pound 9.
tor was analysed in terms of energy, hydrogen bonding, PyMol software. The free energy of binding (FEB)
π
π
interaction. The final coor- of all compounds were estimated and given in the
hydrophobic and
−
dinates of the ligand and receptor were saved after Table 3.
the clear analysis of ligand–receptor interactions. Lig-
The docking studies demonstrate that the synthe-
and and receptor interactions were investigated using sised compounds have good free energy of binding with

T Shanmuganathan et al.
Table 3. Free energy of binding (FEB) of all the synthesised
compounds.
Sl. No.
Compound
Free Energy of Binding (kcal/mol)a
COX-2 (PDB ID: 1PXX)
1
2
3
4
5
6
7
8
4
7
8
9
10
11
12
13
−8.97
−9.95
−10.40
−10.48
−9.80
−9.45
−9.25
−8.90
−8.85
−8.98
−8.75
−8.15
9
14
15
16
10
11
12
Diclofenac
aCalculated using Autodock4
COX-2 receptor. Compounds display free energy of 4. Conclusions
binding value from −8.75 to −10.48kcal/mol. When
In summary, a new series of bishydroxypropoxy sub-
compared with the standard drug diclofenac sodium,
all the synthesised compounds exhibit greater binding
affinity. Docking revealed that all the synthesised com-
pounds shows various interactions such as hydrophobic,
stituted 4, 5-diarylthiophene-2-carboxylic acid deriva-
tives were synthesized, characterized and evaluated for
their in vitro anti-inflammatory activity. The in vitro
anti-inflammatory activity revealed that the compound
π
π
interaction and hydrogen bond-
hydrophilic,
−
4,5-bis(4-(3-hydroxypropoxy)
phenyl)thiophene-2-
ing with 25 binding site amino acids namely HIS-90,
VAL-116, ARG-120, GLN-192, VAL-344, TYR-348,
VAL-349, LEU-352, SER-353, TYR-355, LEU-359,
PHE-381, LEU-384, TYR-385, TRP-387, ARG-513,
ILE-517, PHE-518, MET-522, VAL-523, GLY-526,
ALA-527, SER-530, LEU-531andLEU-534. Thedock-
ing conformation of all the compounds with COX-2
receptor is shown in Figure 3B.
carboxylic acid (15) and ethyl ester (13) having anti-
inflammatory activity better than the standard drug
diclofenac sodium. The antioxidant screening showed
4,5-bis(4-(allyloxy)phenyl)thiophene-2-carboxylicacid
(10), 4,5-bis(4-(3-hydroxypropoxy)phenyl)thiophene-
2-carboxylic acid methyl ester (11) and 4,5-bis(4-(3-
hydroxypropoxy)phenyl)thiophene-2-carboxylic acid
ethyl ester (13) exhibited a slightly moderate antioxidant
activity than the standard ascorbic acid. Docking stud-
ies with COX-2 enzyme revealed that all the synthesised
compoundsexhibitgreaterbindingaffinitythanthestan-
dard drug. In particular, the compounds ethyl 4,5-bis(4-
(allyloxy)phenyl)thiophene-2-carboxylate (8) and allyl
4,5-bis(4-(allyloxy)phenyl)thiophene-2-carboxylate(9)
have high free energy binding of −10.40 and
−10.48 Kcal/mol, respectively. Further studies are
in progress to improve the biological activities of
bishydroxypropoxy substituted 4,5-diarylthiophene-2-
carboxylic acid derivatives.
Among all the compounds docked, compounds 8 and
9 exhibit very high binding with COX-2 receptor. Com-
pound 9 shows the binding affinity of −10.48kcal/mol
with three hydrogen bonds with two active site amino
acids, namely ARG-120 and TYR-385. In Compound 9,
estercarbonylandoxygeninteractwiththeO-HofTYR-
385 and forms two hydrogen bonds with the bond length
of 2.3Å and 2.4Å, respectively. One of the phenoxy
oxygen interacts with the N-H of ARG-120 and forms
a hydrogen bond with the bond length of 2.7Å. In addi-
tion to the polar interactions, hydrophobic interaction
was observed with the VAL-116, VAL-349, LEU-352,
LEU-356, LEU-384, VAL-523, ILE-517, GLY-526 and
ALA-527 amino acids. Furthermore, phenyl rings of the
Supplementary information (SI)
π
π
interaction with the phenyl
compound 9 display
−
rings of the TYR-355 and PHE-518. The docking con-
formation of compound 9 with COX-2 receptor is shown
in Figure 3C.
The characterization of the compounds 2–16 using 1H NMR,
13C NMR, IR and Mass spectral data (Figures S1–S60) are
given in the Supplementary Information, which is available
at www.ias.ac.in/chemsci.

Synthesis of novel bis-allyloxy and hydroxypropoxy derivatives
11. Radwan M A A, Shehab M A and El-Shenawy
Acknowledgements
S M 2008 Synthesis and biological evaluation of
5-substituted benzo[b]thiophene derivatives as anti-
inflammatory agents Monatsh. Chem. 140 445
Theauthors are thankful tothemanagement of OrchidPharma
Limited, Chennai 600 119, India and Ramakrishna Mission
Vivekananda College, Chennai 600 004, India for providing
the required facilities.
12. Wierzbicki M, Sauveur F, Bonnet J and Tordjman C 1998
Thiophene compounds U.S. Patent 5705525
13. Lindner M, Sippl W and Radwan A A 2010
Pharmacophore elucidation and molecular docking
studies on 5-phenyl-1-(3-pyridyl)-1h-1,2,4-triazole-3-
carboxylic acid derivatives as COX-2 inhibitors Sci.
Pharm. 78 195
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