Synthesis and biological evaluation of novel active arylidene derivatives of 5,6-dihydro-4H-cyclopenta[b]- and 4,5,6,7-tetrahydrobenzo[b]-thiophene-2-carboxylic acid

Article abstract of DOI:10.1007/s11164-016-2763-9

A series of novel arylidene derivatives of 5,6-dihydro-4H-cyclopenta[b]-thiophene-2-carboxylic acid and 4,5,6,7-tetrahydrobenzo[b]-thiophene-2-carboxylic acid were synthesized by reacting benzylidene derivatives of chloro aldehyde with 2-mercaptoacetic acid. Benzylidene derivatives of chloro aldehyde were prepared from Vilsmeier reaction of 2-benzylidenecyclopentanone and 2-benzylidenecyclohexanone derivatives, obtained from condensation of various aromatic aldehydes with cyclopentanone and cyclohexanone. All synthesized compounds were characterized by nuclear magnetic resonance (NMR), infrared (IR), and mass spectroscopy and X-ray single-crystal analysis. The synthesized compounds were screened for their in?vitro antimicrobial and antifungal activity. Good antimicrobial activity, especially against methicillin-resistant Staphylococcus aureus, was observed for most of the compounds tested. In particular, compound 9f emerged as an effective antibacterial agent and may be a potential candidate for future drug discovery and development.

Full text of DOI:10.1007/s11164-016-2763-9

Res Chem Intermed
DOI 10.1007/s11164-016-2763-9
Synthesis and biological evaluation of novel active
arylidene derivatives of 5,6-dihydro-4H-cyclopenta[b]-
and 4,5,6,7-tetrahydrobenzo[b]-thiophene-2-carboxylic
acid
P. R. Kathiravan1,2
S. Muthukumaran2
M. Venugopal3
•
•
Received: 21 July 2016 / Accepted: 4 October 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A series of novel arylidene derivatives of 5,6-dihydro-4H-cyclopenta[b]-
thiophene-2-carboxylic acid and 4,5,6,7-tetrahydrobenzo[b]-thiophene-2-carboxylic
acid were synthesized by reacting benzylidene derivatives of chloro aldehyde with
2-mercaptoacetic acid. Benzylidene derivatives of chloro aldehyde were prepared
from Vilsmeier reaction of 2-benzylidenecyclopentanone and 2-benzylidenecyclo-
hexanone derivatives, obtained from condensation of various aromatic aldehydes
with cyclopentanone and cyclohexanone. All synthesized compounds were char-
acterized by nuclear magnetic resonance (NMR), infrared (IR), and mass spec-
troscopy and X-ray single-crystal analysis. The synthesized compounds were
screened for their in vitro antimicrobial and antifungal activity. Good antimicrobial
activity, especially against methicillin-resistant Staphylococcus aureus, was
observed for most of the compounds tested. In particular, compound 9f emerged as
an effective antibacterial agent and may be a potential candidate for future drug
discovery and development.
Keywords Vilsmeier reaction Á Benzylidene derivatives of chloro aldehyde Á
5,6-Dihydro-4H-cyclopenta[b]-thiophene-2-carboxylic acid derivatives Á
4,5,6,7-Tetrahydrobenzo[b]-thiophene-2-carboxylic acid derivatives Á
Antimicrobial activity
&
P. R. Kathiravan
prkathir@yahoo.com
1
2
Orchid Pharma Ltd, R&D Centre, Chennai 600119, India
Department of Chemistry, Ramakrishna Mission Vivekananda College,
Mylapore, Chennai 600004, India
3
Ven Biotech Private Limited, Chennai, India
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P. R. Kathiravan et al.
Introduction
Antimicrobial resistance has been considered to be one of the greatest threats to
global health for several years. As a consequence of mutations in microbes and
continued wide use of antibiotics in hospitals, the community, and agriculture, the
rate of resistance to common antibiotics has increased in recent years [1]. Discovery
of new antimicrobials with completely new scaffolds or modification of the
structure of well-known antimicrobials are the two main approaches to address this
problem [2]. Another strategy involves combining two or more pharmacophores
from different antimicrobials to obtain molecules exhibiting powerful synergistic
effects [3]. Heterocycles containing nitrogen and sulfur have been the subject of
chemical and biological studies due to their versatile synthetic applicability and
interesting pharmacological properties.
Thiophenes are an important class of heterocyclic compounds, being widely used
as building blocks in many agrochemicals and pharmaceuticals, including
antibacterial, antifungal, analgesic, antiinflammatory, antioxidant, local anesthetic,
and antitumor agents; For example, ticarcillin, cefoxitin, cephalothin, and
cephaloridine are thiophene-containing b-lactam antibiotics that are widely used
for treating microbial infections, while tioconazole and sertaconazole are antifungal
agents with thiophene moiety in their core structure (Fig. 1) [4]. Raloxifene is an
oral selective estrogen receptor modulator (SERM) containing a thiophene ring,
being used to treat osteoporosis in postmenopausal women [5].
Literature survey reveals that tetrahydrobenzothiophene derivatives have
attracted continuous interest because of their various biological properties.
Tetrahydrobenzothiophene derivatives were reported to have low micromolar
inhibition of hepatitis C virus (HCV) nonstructural (NS)5B 4a full-length
polymerase with selectivity against human DNA polymerase and calf thymus
polymerase a [6]. They were also found to possess antiinflammatory and analgesic
activities [7, 8]. A variety of tetrahydrobenzothiophene derivatives have been
studied for their antibacterial [9, 10] and antitumor activity [11]. Replacement of
aryl group with heterocyclic tetrahydrobenzothiophene has been proved to improve
the potency and selectivity towards inhibition of Bruton’s tyrosine kinase, which
plays a significant role in the development of immunological disorders such as
rheumatoid arthritis, lupus, multiple sclerosis, and B-cell malignancies [12].
In view of this biological importance and in continuation of our studies on the
chemistry of such compounds, we designed and synthesized new tetrahydroben-
zothiophene-2-carboxylic acid derivatives with benzylidene moiety via aldol
condensation of different para-substituted aromatic aldehydes and cyclic ketones
in presence of base. Subsequently, the condensed product was subjected to
Vilsmeier reaction, resulting in formation of chloro aldehyde derivatives. These
chloro aldehydes were reacted with 2-mercapto acetic acid in presence of base to
give our expected compounds. The synthesized compounds were characterized and
evaluated for their antimicrobial activity against various microorganisms such as
Staphylococcus aureus (methicillin-sensitive and methicillin-resistant strains,
MSSA and MRSA, respectively), Escherichia coli, Pseudomonas aeruginosa, and
123

Synthesis and biological evaluation of novel active…
HO
O
O
H
S
HN
O
S
NH
H
N
O
NH2
O
S
O
S
O
N
O
O
OH
O
Ticarcillin
H
Cefoxitin
HN
S
S
HN
O
N
O
O
N
O
N
O
O
S
S
O
O
OH
OH
Cephalotin
Cephaloridine
N
N
Cl
Cl
Cl
N
N
O
Cl
O
Cl
S
S
Cl
Sertaconazole
Fig. 1 Example thiophene-containing b-lactam antibiotics and antifungal agents
Tioconazole
fungal strain Candida albicans, and the results were interpreted to determine their
effect as antimicrobial agents.
Experimental
General considerations
All chemicals and reagents used were of laboratory grade and procured from Sigma
Aldrich. All solvents used were purchased from commercial suppliers and used
123

P. R. Kathiravan et al.
without further purification. Melting points were determined using a Bu¨chi
apparatus by open capillary tube method and are uncorrected. IR spectra were
recorded using a PerkinElmer series 2000 Fourier-transform infrared (FTIR)
1
spectrophotometer from KBr pellets. H and 13C NMR spectra were recorded on
a Bruker Advance 400 spectrometer. Chemical shifts (d) are reported in ppm
relative to tetramethylsilane as internal standard. Coupling constants (J) are in hertz
(Hz). Proton and carbon spectra were typically obtained at room temperature. Mass
spectra were recorded on an PerkinElmer Sciex API 3000 ESI mass spectrometer.
Precoated silica gel GF254 plates from Merck were used for thin-layer chromatog-
raphy. Elemental analyses were carried out using a Thermo Finnigan Flash EA 1112
elemental analyzer.
Synthesis of 2-benzylidenecyclopentanone and 2-benzylidenecyclohexanone
derivatives
These compounds were synthesized according to previously reported procedure
[13–18].
General procedure for synthesis of compounds 6a–c and 7a–g
To dimethylformamide (45 ml), phosphorus oxychloride (0.12 mol) was added
dropwise over a period of 15–30 min with stirring at 0–5 °C. Compound 4a–c and
5a–g (0.05 mol) dissolved in dimethylformamide (DMF, 70 ml) was added
dropwise, followed by warming to room temperature, heating to temperature of
55–60 °C maintained for 4 h, cooling, and pouring slowly into 50 % solution of
sodium acetate in water (100 ml) at 0–5 °C. The product was filtered, washed with
water, followed by slurry washing with ethanol (100 ml) to obtain compounds 6a–
c and 7a–g.
(E)-2-Chloro-3-(benzylidene)cyclopent-1-enecarbaldehyde (6a)
Pale-yellow powder; yield: 60 %; m.p. 99–101 °C; IR (KBr) cm-1: 3435, 2942,
1
2845, 1659, 1566, 1448, 1246, 999, 880; H NMR (400 MHz, DMSO-d6): d 10.09
(s, 1H), 7.58–7.56 (d, J = 7.6 Hz, 2H), 7.46 (t, J = 8.0 Hz, 2H), 7.37 (t,
J = 7.6 Hz, 1H), 7.03 (s, 1H), 3.03–2.99 (m, 2H), 2.69–2.68 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): d 188.2, 147.6, 143.5, 140.8, 136.2, 131.1, 129.4, 129.3,
128.8, 126.9, 27.5, 27.3; ESI–MS m/z calcd. 218.05; found: 219.1 [M ? H]?; anal.
calcd. for C13H11ClO (%): C, 71.40; H, 5.07; found (%): C, 71.38; H, 5.12.
(E)-2-Chloro-3-(4-fluorobenzylidene)cyclopent-1-enecarbaldehyde (6b)
Pale-yellow powder; yield: 63 %; m.p. 146–148 °C; IR (KBr) cm-1: 3435, 2942,
1
2845, 1659, 1566, 1448, 1246, 999, 880; H NMR (400 MHz, CDCl3): d 10.19 (s,
1H), 7.59–7.46 (m, 2H), 7.13–7.09 (m, 2H), 7.02 (s, 1H), 3.09–3.01 (m, 2H),
2.82–2.81 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 188.2, 163.4, 160.9
(1J19F–13C = 246 Hz), 147.5, 143.2, 143.2 (4J19F–13C = 2.4 Hz), 140.8, 132.8,
123

Synthesis and biological evaluation of novel active…
132.0, 131.9 (3J19F–13C = 8.3 Hz), 125.7, 116.5, 116.3, 116.1 (2J19F–13C = 21.4 Hz),
27.5, 27.1; ESI–MS m/z calcd. 236.04; found: 237.1 [M ? H]?; anal. calcd. for
C13H10ClFO (%): C, 65.97; H, 4.26; found (%): C, 65.92; H, 4.28.
(E)-2-Chloro-3-(4-chlorobenzylidene)cyclopent-1-enecarbaldehyde (6c)
Pale-yellow powder; yield: 65 %; m.p. 183–185 °C; IR (KBr) cm-1: 3434, 3047,
2831, 1656, 1570, 1491, 1247, 1008, 888; 1H NMR (400 MHz, CDCl3): d 10.19 (s,
1H), 7.44–7.37 (m, 4H), 7.01 (s, 1H), 3.02–3.00 (m, 2H), 2.82–2.79 (m, 2H); ESI–
MS m/z calcd. 252.01; found: 253.1 [M ? H]?; anal. calcd. for C13H10Cl2O (%): C,
61.68; H, 3.98; found (%): C, 61.74; H, 3.94.
(E)-3-Benzylidene-2-chlorocyclohex-1-enecarbaldehyde (7a)
Pale-yellow powder; yield: 70 %; m.p. 87–90 °C; IR (KBr) cm-1: 3434, 2949,
2875, 1663, 1560, 1440, 1255, 1001, 882; 1H NMR (400 MHz, DMSO-d6): d 10.28
(s, 1H), 7.49 (s, 1H), 7.45–7.41 (m, 4H), 7.38–7.36 (m, 1H), 2.77–2.74 (m, 2H),
2.40–2.37 (m, 2H), 1.65–1.62 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 191.6,
146.0, 136.1, 134.7, 134.6, 133.3, 130.3, 128.9, 128.8, 28.3, 24.8, 21.2; ESI–MS m/
z calcd. 232.07; found: 233.1 [M ? H]?; anal. calcd. for C14H13ClO,(%) C, 72.26;
H, 5.63; found (%): C, 72.22; H, 5.68.
(E)-2-Chloro-3-(4-fluorobenzylidene)cyclohex-1-enecarbaldehyde (7b)
Yellow powder; yield: 68 %; m.p. 96–98 °C; IR (KBr) cm-1: 3435, 2943, 2879,
1657, 1560, 1447, 1252, 999, 887; 1H NMR (400 MHz, DMSO-d6): d 10.26 (s, 1H),
7.51–7.49 (m, 2H), 7.46 (s, 1H), 7.28–7.23 (m, 1H), 2.74–2.71 (m, 2H), 2.39–2.36
(m, 2H), 1.66–1.60 (m, 2H); ESI–MS m/z calcd. 250.06; found: 251.1 [M ? H]?;
anal. calcd. for C14H12FO (%): C, 67.07; H, 4.82; found (%): C, 67.02; H, 4.78.
(E)-2-Chloro-3-(4-chlorobenzylidene)cyclohex-1-enecarbaldehyde (7c)
Greenish-yellow powder; yield: 66 %; m.p. 76–79 °C; IR (KBr) cm-1: 3435, 2954,
2861, 1659, 1558, 1488, 1250, 1012, 884; 1H NMR (400 MHz, DMSO-d6): d 10.28
(s, 1H), 7.55–7.49 (m, 4H), 7.46 (s, 1H), 2.75–2.72 (m, 2H), 2.40–2.37 (m, 2H),
1.67–1.61 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 191.6, 145.7, 135.3, 135.0,
134.9, 133.4, 132.0, 131.9, 128.9, 28.2, 24.8, 21.2; ESI–MS m/z calcd. 266.03;
found: 267.0 [M ? H]?; anal. calcd. for C14H12Cl2O (%): C, 62.94; H, 4.53; found
(%): C, 62.88; H, 4.58.
(E)-3-(4-Bromobenzylidene)-2-chlorocyclohex-1-enecarbaldehyde (7d)
Pale-yellow powder; yield: 66 %; m.p. 82–85 °C; IR (KBr) cm-1: 3435, 2953,
2860, 1658, 1557, 1485, 1252, 1004, 889; 1H NMR (400 MHz, DMSO-d6): d 10.29
(s, 1H), 7.65–7.61 (m, 2H), 7.43–7.41 (m, 3H), 2.80–2.73 (m, 2H), 2.40–2.37 (m,
2H), 1.66–1.63 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 191.1, 145.2, 134.8,
123

P. R. Kathiravan et al.
134.6, 131.8, 131.5, 131.4, 121.6, 27.8, 24.3, 20.7; ESI–MS m/z calcd. 309.98;
found: 311.0 [M ? H]?; anal. calcd. for C14H12BrClO (%): C, 53.96; H, 3.88;
found (%): C, 53.92; H, 3.86.
(E)-2-Chloro-3-(4-methylbenzylidene)cyclohex-1-enecarbaldehyde (7e)
Yellow powder; yield: 65 %; m.p. 72–75 °C; IR (KBr) cm-1: 3435, 2947, 2862,
1664, 1559, 1442, 1249, 996, 883; 1H NMR (400 MHz, DMSO-d6): d 10.28 (s, 1H),
7.46 (s, 1H), 7.38–7.36 (m, 2H), 7.26–7.24 (m, 2H), 2.78–2.75 (m, 2H), 2.39–2.37
(m, 2H), 2.33 (s, 3H), 1.67–1.61 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 191.1,
145.7, 138.0, 133.9, 133.3, 132.9, 132.8, 129.9, 129.1, 27.9, 24.3, 20.9, 20.7; ESI–
MS m/z calcd. 246.08; found: 247.1 [M ? H]?; anal. calcd. for C15H15ClO (%): C,
73.02; H, 6.13; found (%): C, 73.12; H, 6.08.
(E)-2-Chloro-3-(4-isopropylbenzylidene)cyclohex-1-enecarbaldehyde (7f)
Yellow powder; yield: 64 %; m.p. 72–75 °C; IR (KBr) cm-1: 3433, 2921, 2862,
1657, 1559, 1441, 1248, 996, 883; 1H NMR (400 MHz, DMSO-d6): d 10.28 (s, 1H),
7.46 (s, 1H), 7.37–7.35 (m, 2H), 7.25–7.19 (m, 2H), 3.09 (m, 1H), 2.76–2.71 (m,
2H), 2.38–2.36 (m, 2H), 1.64–1.62 (m, 2H), 1.28–1.27 (d, J = 6.1 Hz, 6H); 13C
NMR (100 MHz, DMSO-d6): d 191.0, 145.8, 138.0, 133.8 133.3, 132.9, 129.9,
129.1, 128.9, 27.9, 24.3, 20.9, 20.8, 20.7; ESI–MS m/z calcd. 274.11; found: 275
[M ? H]?; anal. calcd. for C17H19ClO (%): C, 74.31; H, 6.97; found (%): C, 4.26;
H, 6.98.
(E)-2-Chloro-3-(4-methoxybenzylidene)cyclohex-1-enecarbaldehyde (7g)
Pale-yellow powder; yield: 68 %; m.p. 77–79 °C; IR (KBr) cm-1: 3435, 2935,
1
2839, 1657, 1557, 1440, 1255, 995, 893; H NMR (400 MHz, DMSO-d6): d 10.27
(s, 1H), 7.46–7.44 (m, 3H), 7.02–6.99 (m, 2H), 3.80 (s, 3H), 2.78–2.75 (m, 2H),
2.38–2.37 (m, 2H), 1.66–1.63 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 191.4,
159.8, 146.5, 133.9 133.3, 132.5, 132.2, 128.5, 114.8, 55.7, 28.5, 24.7, 21.2; ESI–
MS m/z calcd. 262.08; found: 263.1 [M ? H]?; anal. calcd. for C15H15ClO2,(%); C,
68.57; H, 5.75; found (%): C, 68.52; H, 5.72.
General procedure for synthesis of compounds 8a–c and 9a–g
Compound 6a–c and 7a–g (0.0042 mol) was added at room temperature to solution
of potassium hydroxide (0.017 mol) and 2-mercapto acetic acid (0.0064 mol) in
methanol:water (40:10 ml). The mixture was refluxed for 4 h and cooled to room
temperature. The reaction mixture was slowly acidified with conc. HCl over
30–45 min at 25–30 °C. The product was filtered, washed with water, and dried.
The pure product was isolated by column chromatography over silica gel (60–120
mesh) using ethyl acetate/hexane mixture.
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Synthesis and biological evaluation of novel active…
(E)-6-Benzylidene-5,6-dihydro-4H-cyclopenta[b]-thiophene-2-carboxylic
acid (8a)
Pale-brown powder; yield: 65 %; m.p. 204–206 °C; IR (KBr) cm-1: 3429, 2925,
2853, 1668, 1530, 1447, 1280, 1168, 1039, 863; 1H NMR (400 MHz, DMSO-d6): d
7.57 (s, 1H), 7.49–7.48 (m, 2H), 7.39–7.38 (m, 2H), 7.27–7.23 (m, 1H), 6.74 (s,
1H), 2.97–2.94 (m, 2H), 0.81–0.78 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d
163.5, 151.9, 151.3, 139.7, 137.6, 129.6, 129.1, 128.6, 127.2, 120.5, 34.3, 27.6;
ESI–MS m/z calcd. 256.01; found: 255.1 [M - H]-; anal. calcd. for C15H12O2S
(%): C, 70.29; H, 4.72; found (%): C, 70.34; H, 4.76.
(E)-6-(4-Fluorobenzylidene)-5,6-dihydro-4H-cyclopenta[b]-thiophene-2-
carboxylic acid (8b)
Brown powder; yield: 67 %; m.p. 222–224 °C; IR (KBr) cm-1: 3436, 2918, 2852,
1
1649, 1531, 1438, 1222, 1171, 1072, 868; H NMR (400 MHz, DMSO-d6): d 7.56
(s, 1H), 7.53–7.50 (m, 2H), 7.23–7.19 (m, 2H), 6.74 (s, 1H), 3.32–3.29 (m, 2H),
2.96–2.93 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.5, 162.5, 160.0, 151.8,
151.2, 139.4, 139.3, 137.6, 134.2, 134.1, 130.6, 130.5, 129.5, 119.4, 116.0, 115.8,
34.2, 27.5; ESI–MS m/z calcd. 274.05; found: 273.1 [M - H]-; anal. calcd. for
C15H11FO2S (%): C, 65.68; H, 4.04; found (%): C, 65.66; H, 4.08.
(E)-6-(4-Chlorobenzylidene)-5,6-dihydro-4H-cyclopenta[b]-thiophene-2-
carboxylic acid (8c)
Brown powder; yield: 66 %; m.p. 250–252 °C; IR (KBr) cm-1: 3436, 2921, 2854,
1
1667, 1532, 1449, 1285, 1168, 1009, 864; H NMR (400 MHz, DMSO-d6): d 7.56
(s, 1H), 7.50–7.48 (m, 2H), 7.43–7.41 (m, 2H), 6.73 (s, 1H), 3.27 (m, 2H),
2.96–2.93 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.5, 151.7, 151.6, 140.6,
137.9, 136.5, 131.5, 130.2, 129.5, 129.0, 119.2, 34.4, 27.6; ESI–MS m/z calcd.
290.02; found: 289.1 [M - H]-; anal. calcd. for C15H11ClO2S (%): C, 61.96; H,
3.81; found (%): C, 61.94; H, 3.84.
(E)-7-Benzylidene-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-carboxylic acid
(9a)
Yellow powder; yield: 68 %; m.p. 191–194 °C; IR (KBr) cm-1: 3434, 2936, 2857,
1680, 1539, 1450, 1264, 1166, 764; 1H NMR (400 MHz, DMSO-d6): d 7.46 (s, 1H),
7.42–7.37 (m, 4H), 7.29–7.26 (m, 1H), 6.88 (s, 1H), 2.75 (m, 2H), 2.51 (m, 2H),
1.78 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.7, 144.0, 140.1, 136.7, 134.4,
133.2, 128.9, 128.8, 127.4, 123.8, 27.3, 25.8, 23.5; ESI–MS m/z calcd. 270.07;
found: 269.1 [M - H]-; anal. calcd. for C16H14O2S (%): C, 71.08; H, 5.22; found
(%): C, 71.14; H, 5.24.
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P. R. Kathiravan et al.
(E)-7-(4-Fluorobenzylidene)-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-
carboxylic acid (9b)
Yellow powder; yield: 66 %; m.p. 222–225 °C; IR (KBr) cm-1: 3435, 2944, 2829,
1637, 1545, 1448, 1271, 1161, 1014, 870; 1H NMR (400 MHz, DMSO-d6): d
7.48–7.43 (m, 3H), 7.23–7.19 (m, 2H), 6.87 (s, 1H), 2.73–2.67 (m, 4H), 1.79–1.76
(m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.4, 162.7, 160.3, 144.3, 140.2,
134.8, 133.2, 133.1, 132.9, 131.7, 131.6, 131.3, 122.9, 115.8, 115.6, 27.2, 25.7,
23.5; ESI–MS m/z calcd. 288.06; found: 287.1 [M - H] -; anal. calcd. for
C16H13FO2S (%): C, 66.65; H, 4.54; found (%): C, 66.70; H, 4.58.
(E)-7-(4-Chlorobenzylidene)-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-
carboxylic acid (9c)
Yellow powder; yield: 65 %; m.p. 213–216 °C. IR (KBr) cm-1: 3435, 2938, 2859,
1
1666, 1540, 1449, 1265, 1165, 1012, 867; H NMR (400 MHz, DMSO-d6): d 7.48
(s, 1H), 7.42 (m, 4H), 6.86 (s, 1H), 2.71–2.68 (m, 4H), 1.78–1.76 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): d 163.4, 144.1, 140.5, 135.6, 134.7, 133.8, 131.9, 131.6,
131.4 128.8, 122.7, 27.2, 25.7, 23.5; ESI–MS m/z calcd. 304.03; found: 303.1
[M - H]-; anal. calcd. for C16H13ClO2S (%): C, 63.05; H, 4.30; found (%): C,
63.12; H, 4.26.
(E)-7-(4-Bromobenzylidene)-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-
carboxylic acid (9d)
Yellow powder; yield: 65 %; m.p. 205–208 °C; IR (KBr) cm-1: 3435, 2940, 2841,
1668, 1541, 1450, 1266, 1167, 1008, 872; 1H NMR (400 MHz, DMSO-d6): d
7.60–7.56 (m, 2H), 7.47 (s, 1H), 7.38–7.34 (m, 4H), 6.84 (s, 1H), 2.74–2.68 (m,
4H), 1.80–1.77 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.1, 143.4, 140.1
135.5, 134.0, 133.5, 131.3, 131.2, 122.2, 120.1, 26.8, 25.3 23.0; ESI–MS m/z calcd.
347.98; found: 347.0 [79MBr - H]-; 349.0 [81MBr - H]-; anal. calcd. for
C16H13BrO2S (%): C, 55.03; H, 3.75; found (%): C, 55.16; H, 3.70.
(E)-7-(4-Methylbenzylidene)-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-
carboxylic acid (9e)
Brown powder; yield: 66 %; m.p. 178–180 °C; IR (KBr) cm-1: 3435, 2942, 2860,
1
1675, 1542, 1449, 1268, 1170, 1020, 875; H NMR (400 MHz, DMSO-d6): d 7.59
(s, 1H), 7.31–7.29 (m, 2H), 7.21–7.19 (m, 4H), 6.84 (s, 1H), 2.73 (m, 2H), 2.68 (m,
2H), 2.31 (s, 3H), 1.77 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.0, 144.0,
139.4, 136.4, 134.1, 133.4, 131.9, 129.2, 128.9, 123.5, 26.9, 25.3, 23.1, 20.8; ESI–
MS m/z calcd. 284.09; found: 283.2 [M - H]-; anal. calcd. for C17H16O2S (%): C,
71.80; H, 5.67; found (%): C, 71.78; H, 5.64.
123

Synthesis and biological evaluation of novel active…
(E)-7-(4-Isopropylbenzylidene)-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-
carboxylic acid (9f)
Yellow powder; yield: 65 %; m.p. 215–217 °C; IR (KBr) cm-1: 3434, 2925, 2854,
1
1673, 1541, 1447, 1266, 1168, 1019, 874; H NMR (400 MHz, CDCl3): d 7.58 (s,
1H), 7.28–7.26 (m, 1H), 7.19–7.17 (m, 3H), 6.93 (s, 1H), 2.80–2.74 (m, 4H), 2.36
(m, 4H), 1.87 (m, 2H), 1.25 (m, 3H); 13C NMR (100 MHz, DMSO-d6): d 162.9,
144.2, 139.4, 136.4, 134.3, 133.4, 131.9, 130.7, 129.2, 128.9, 123.5, 26.9, 25.3,
23.1, 20.8; ESI–MS m/z calcd. 312.12; found: 311.0 [M - H]-; anal. calcd. for
C19H20O2S (%): C, 73.04; H, 6.45; found (%): C, 73.10; H, 6.48.
(E)-7-(4-Methoxybenzylidene)-4,5,6,7-tetrahydrobenzo[b]-thiophene-2-
carboxylic acid (9g)
Pale-yellow powder; yield: 70 %; m.p. 219–221 °C; IR (KBr) cm-1: 3435, 2935,
2835, 1669, 1539, 1445, 1254, 1167, 1030, 872; 1H NMR (400 MHz, DMSO-d6): d
7.47 (s, 1H), 7.37–7.35 (m, 2H), 6.96–6.94 (m, 2H), 6.83 (s, 1H), 3.77 (s, 3H),
2.73–2.66 (m, 4H), 1.78–1.75 (m, 2H); 13C NMR (100 MHz, DMSO-d6): d 163.4,
158.8, 144.9, 139.5, 134.8, 131.2, 131.1, 130.7, 129.2, 123.8, 114.3, 55.6, 27.3,
25.7, 23.5; ESI–MS m/z calcd. 300.08; found: 299.1 [M - H]-; anal. calcd. for
C17H16O3S (%): C, 67.98; H, 5.37; found (%): C, 67.92; H, 5.34.
Biology
Antimicrobial activity
The synthesized compounds were screened for antimicrobial activity by agar well
diffusion method [19] using bacterial strains Staphylococcus aureus (ATCC 29213),
Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853) and
fungal strain Candida albicans (ATCC 90028). Compounds which showed activity
against S. aureus ATCC 29213 were further tested against methicillin-resistant
S. aureus strains ATCC 33591 and NRS 100.
Ampicillin and ciprofloxacin, and fluconazole were used as standard antibacterial
and antifungal substances, respectively. Dimethyl sulfoxide (DMSO) was used as
negative control. Mu¨ller–Hinton agar (MHA) medium was used for bacteria, and
Sabouraud dextrose agar medium for fungal strains.
Mu¨ller–Hinton agar plates were inoculated with overnight-grown bacterial
cultures previously adjusted to 0.5 McFarland standard turbidity and diluted to 1:10
using sterile saline solution by following Clinical and Laboratory Standards Institute
(CLSI) recommendations. Sabouraud dextrose agar plates were inoculated with
overnight-grown fungal culture previously adjusted to 0.8 McFarland standard
turbidity and diluted to 1:10 using sterile saline solution by following CLSI
recommendations. For agar well diffusion, wells were formed on Mu¨ller–Hinton
agar plates for bacterial cultures and Sabouraud dextrose agar plates for fungal
cultures, and the test compounds were added to the wells. Standard antibiotics used
for quality control were also added to respective wells. All plates were incubated at
123

P. R. Kathiravan et al.
37° C for 18–24 h, and the zone of inhibition was measured to determine the
antimicrobial activity.
The test compounds were dissolved in dimethylsulfoxide (DMSO), then the
antimicrobial effect of the synthesized compounds was evaluated. The wells were
filled with 50 ll of test compound at concentration of 200 lg/ml. The plates were
incubated at 37 °C for 48 h. Antimicrobial activity was evaluated by measuring the
zone of growth inhibition of bacteria surrounding the wells after 24 and 48 h.
Ampicillin (10 lg/ml), ciprofloxacin (5 lg/ml), and fluconazole (25 lg/ml) served
as antimicrobial controls. DMSO was taken as negative control, which did not
produce any significant zone of inhibition.
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of the compounds against the tested
bacteria was determined by broth microdilution method [20]. The MIC is the lowest
concentration of an antimicrobial compound that inhibits visible growth of a
microorganism after overnight incubation. Stock solutions of the compounds were
prepared in DMSO at concentration of 2.56 mg/ml. Stock solution was diluted 1:10
in cation-adjusted Mu¨ller–Hinton broth, and 100 ll of the diluted solution was
added to the first well of a 96-well microplate. The compounds were then diluted in
doubling dilutions to obtain descending concentrations (128, 64, 32, 16, 8, 4, 2, and
1 lg/ml) of the compound. The references, linezolid and vancomycin, were
included in the assay as positive controls. MIC determinations were performed in
triplicate, and the results averaged (Table 2).
Results and discussion
Chemistry
We synthesized a novel series of benzylidene derivatives of dihydro-cyclopenta[b]-
thiophene-2-carboxylic acid and tetrahydrobenzothiophene-2-carboxylic acid as
shown in Scheme 1. In an effort to prepare benzylidene derivatives of dihydro-
cyclopenta[b]-thiophene-2-carboxylic acid and tetrahydrobenzothiophene-2-car-
boxylic acid, our initial experiments were directed towards reaction of aldehyde
moiety with dihydro-cyclopenta[b]-thiophene-2-carboxylic acid and tetrahydroben-
zothiophene-2-carboxylic acid, which failed to afford the desired product. A recent
study revealed formation of 7a and 7b as byproducts of vinylogous aldol reaction
[21] when a cyclic b-chloro enal was reacted with an aldehyde in presence of
tetrabutylammonium fluoride in tetrahydrofuran at 25 °C.
In this work we synthesized benzylidene derivatives of chloro aldehyde through
Vilsmeier reaction. Initially, cyclohexanone was condensed with aldehydes in
presence of sodium hydroxide in water at 90–95 °C through reported procedure to
get 4a–c and 5a–g [13–18]. These condensation products were subjected to
Vilsmeier reaction using dimethylformamide (DMF) and phosphorus oxychloride
(POCl3) to yield chloro aldehydes 6a–c and 7a–g in 60–70 % yield [22, 23]. The
123

Synthesis and biological evaluation of novel active…
O
O
O
ii
i
n
n
X
X
4 a-c (X= H, F, Cl) n = 1
5 a-g [X= H, F, Cl, Br, CH3, CH(CH3)2,
1
2 n = 1
3 n = 2
X= H, F, Cl, Br, CH3,
CH(CH3)2, OCH3
OCH3] n = 2
O
OH
Cl
O
S
iii
n
n
X
X
8 a-c (X= H, F, Cl) n = 1
9 a-g [X= H, F, Cl, Br, CH3, CH(CH3)2,
6 a-c (X= H, F, Cl) n = 1
7 a-g [X= H, F, Cl, Br, CH3, CH(CH3)2,
OCH3] n = 2
OCH3] n = 2
Scheme 1 Reagents: (i) sodium hydroxide, (ii) DMF, POCl3, (iii) 2-mercapto acetic acid, KOH
chloro aldehyde was then reacted and cyclized with mercapto acetic acid in
presence of potassium hydroxide in methanol under reflux condition to give
benzylidene derivatives of tetrahydrobenzothiophene-2-carboxylic acid in 65–70 %
yield [24] and further purified by using column chromatography. A possible
synthetic mechanism of the thiophene carboxylic acid derivatives is given in Fig. 2.
The structure of the synthesized intermediate compounds 6a–c and 7a–g was
confirmed by various spectral techniques such as NMR, mass, and IR. The IR
spectra of the compounds revealed a band at around *1658 cm-1, corresponding to
carbonyl group of aldehyde group in cyclopentyl moiety, and at *1663 cm-1 for
1
cyclohexyl moiety. In the H NMR spectra, singlets at around d 10.19 ppm and d
10.28 ppm correspond to –CH proton of aldehyde group of cyclopentyl and
cyclohexyl moiety, respectively. Singlets at around d 7.02 and 7.49 ppm correspond
to benzylidene proton of cyclopentyl and cyclohexyl moiety. The multiplet at
around d 3.03–2.99 ppm and d 2.82–2.79 ppm corresponds to methylene protons of
cyclopentyl group. The multiplet at around d 2.75–2.71 ppm, d 2.39–2.31 ppm, and
d 1.65–1.60 ppm corresponds to methylene protons of cyclohexyl group. The 13C
NMR spectra recorded in DMSO-d6 showed a signal at d 188.2 ppm corresponding
to carbonyl carbon of aldehyde group in cyclopentyl moiety. Signals at around d
27.4 ppm and d 27.3 ppm confirm methylene carbons of cyclopentyl group. The 13
C
123

P. R. Kathiravan et al.
Cl
O
O
O
R
SH
H
KOH
SH
H
KO
n
HO
KO
O
O
KO
-OH
O
-Cl
-H
S
O
S
Cl
R
H
R
H
n
n
OK
H
O
O
H
S
OK
-H
S
OH
-OH
R
R
O
n
n
O
O
OH
OK
S
S
HCl
R
R
n
n
R = Aryl; n=1,2
Fig. 2 Plausible reaction mechanism for synthesis of thiophene 2-carboxylic acid derivatives
NMR spectra recorded in DMSO-d6 showed a signal at d 191.4 ppm, corresponding
to carbonyl carbon of aldehyde group in cyclohexyl moiety. Signals at around d
28.3 ppm, d 24.3 ppm, and d 20.9 ppm confirmed methylene carbons of cyclohexyl
group. Mass spectra acquired in positive and negative electrospray ionization (ESI)
mode confirmed the molecular mass of the compounds. In addition to the above
spectral evidence, the structure of synthesized compound 7a was further confirmed
by single-crystal X-ray diffraction unequivocally. The crystal data were deposited at
the Cambridge Crystallographic Data Centre (CCDC) with no. 1449251. Similarly,
the structures of 7b and 7e were assigned by single-crystal X-ray diffraction results,
deposited at CCDC with nos. 1405840 and 1405845, respectively. Based on the
single-crystal data, the structure of the obtained compounds was confirmed to be the
E-isomer. Oak Ridge thermal ellipsoid plot (ORTEP) diagrams for the above
crystalline compounds are shown in Fig. 3a–c.
123

Synthesis and biological evaluation of novel active…
Fig. 3 ORTEP diagrams of a (E)-3-benzylidene-2-chlorocyclohex-1-enecarbaldehyde (compound 7a,
CCDC 1449251), b (E)-2-chloro-3-(4-fluorobenzylidene)cyclohex-1-enecarbaldehyde (compound 7b,
CCDC 1405840), and c (E)-2-chloro-3-(4-methylbenzylidene)cyclohex-1-enecarbaldehyde (compound
7e, CCDC 1405845)
The structure of synthesized compounds 8a–c and 9a–g was confirmed by
various spectral techniques such as NMR, mass, and IR data. The IR spectra
revealed a band at around 3400–3450 cm-1, corresponding to –OH stretching of the
acid group. The band at around *1669 cm-1 corresponds to carbonyl group of
123

P. R. Kathiravan et al.
1
carboxylic acid group. In the H NMR spectra, the singlets at around d 7.56 and d
7.48 ppm correspond to benzylidene proton of cyclopentyl and cyclohexyl moiety.
Singlets observed at around d 6.74 and 6.84 ppm correspond to thiophene ring
proton of cyclopentyl and cyclohexyl moiety, respectively. The multiplet at around
d 3.32–3.29 ppm and d 2.96–2.93 ppm corresponds to methylene protons of
cyclopentyl group, and that at d 1.78–1.76 ppm and d 2.73–2.68 to methylene
protons of cyclohexyl group. The 13C NMR spectra recorded in DMSO-d6 showed a
signal at d 163.5 and d 163.4 ppm, corresponding to carbonyl carbon of the acid
group. The signals at d 34.3 and 27.3 ppm confirmed methylene carbons of
cyclopentyl group. The signals at d 27.2, 25.3, and 23.1 ppm confirmed methylene
carbons of cyclohexyl group. Mass spectra acquired in positive and negative ESI
mode confirmed the molecular mass of the compounds.
Biology
Antimicrobial activity
Synthesized compounds 8a–c and 9a–g were tested for their antimicrobial activity
by agar well diffusion method against S. aureus (ATCC 29213), methicillin-
resistant S. aureus (ATCC 33591, NRS 100), E. coli (ATCC 25922), and
P. aeruginosa (ATCC 27853), and C. albicans (ATCC 90028) for testing antifungal
activity. All synthesized compounds 8a–c and 9a–g displayed variable degree of
antimicrobial activity against S. aureus (ATCC 29213) strain, as indicated in Fig. 4.
None of these compounds were active against Gram-negative bacterial strains such
as E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) or fungal strain
9a 9b 9c 9d 9e 9f 9g 8a 8b 8c Ampicillin Ciprofloxacin
50
45
40
35
30
25
20
15
10
5
0
Staphylococcus aureus
ATCC 29213
Escherichia coli
ATCC 25922
Pseudomonas aeruginosa
ATCC 27853
Fig. 4 Sensitivity pattern of bacterial strains to benzylidene derivatives of thiophene-2-carboxylic acid
123

Synthesis and biological evaluation of novel active…
C. albicans (ATCC 90028). The zones of inhibition (in mm) of the synthesized
compounds are summarized in Table 1.
As the synthesized compounds showed marked activity against the Staphylo-
coccus strain, further experiments were planned to determine the minimum
inhibitory concentration (MIC) for these compounds against methicillin-sensitive
and methicillin-resistant S. aureus strains. The MIC value of the compounds against
the tested bacteria was determined by broth microdilution method, and the values
are given in Table 2. The most active compound against ATCC 29213 S. aureus
strain was the –CH(CH3)2 derivative (9f) with MIC of 16 lg/ml, while the lowest
activity was observed for the –CH3 and –OCH3 derivatives (9e, g) with MIC of
128 lg/ml. Among the halogen substituents, chloro and bromo substitution (8c, 9b–
d) had beneficial effect in inhibiting growth of ATCC 29213 S. aureus strain, with
MIC values falling below 64 lg/ml, whereas fluoro substitution of the cyclopentyl
derivative (compound 8b) abolished the antibacterial potency (MIC = 128 lg/ml).
The MICs of the compounds against the methicillin-resistant strains (ATCC 33591
and NRS 100) of S. aureus were determined according to the same procedure
employed for the microbiological assay against the sensitive standard strain (ATCC
29213). The halo-substituted cyclohexyl and cyclopentyl derivatives showed
activity against the multidrug-resistant strains of S. aureus in the order
Table 1 Zone of inhibition (mm) of arylidene derivatives of 5,6-dihydro-4H-cyclopenta[b]- and 4,5,6,7-
tetrahydrobenzo[b]-thiophene-2-carboxylic acid 8a–c and 9a–g (lg/ml)
Compound
Staphylococcus
aureus ATCC 29213
Escherichia coli
ATCC 25922
Pseudomonas
aeruginosa ATCC
27853
C. albicans
ATCC 90028
Zone diameter (mm)
9a
18
19
23
22
12
20
13
16
16
20
28
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
19
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
–
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NZ
NA
NA
25
9b
9c
9d
9e
9f
9g
8a
8b
8c
Ampicillin
Ciprofloxacin 32
43
35
Fluconazole
DMSO
NA
NZ
NA
NZ
NA
NZ
NZ
NZ no zone; controls: ampicillin and ciprofloxacin (standard antibacterial agents), and fluconazole (s-
tandard antifungal agent)
123

P. R. Kathiravan et al.
Table 2 Antibacterial activity (MIC, lg/ml) of benzylidene derivatives of thiophene-2-carboxylic acid
tested in broth microdilution assay
Compound
MSSA
MRSA
Staphylococcus
aureus ATCC 29213
Staphylococcus
aureus ATCC 33591
Staphylococcus
aureus NRS 100
9a
64
64
64
64
64
64
9b
9c
64
32
32
9d
32
32
32
9e
128
16
128
32
128
16
9f
9g
128
[128
128
32
128
[128
128
32
128
[128
128
32
8a
8b
8c
Linezolid
Vancomycin
2
2
1
1
2
1
Br [ Cl [ F. It was observed that the most active compound against ATCC 33591
and NRS 100 S. aureus strains was 9f with MIC = 16–32 lg/ml. These results are
in conformity with those previously determined for the ATCC 29213 strain.
In general, benzylidene substituents chosen in this work increased the antibac-
terial property of the thiophene carboxylic acid to a larger extent. The presence of
the weakly electron-donating isopropyl group attached to the benzylidene moiety
(9f) exerted a positive influence on the antibacterial activity. However, this
influence was not seen with the smaller alkyl group substituent (9e). Substitution by
the strong electron-donating methoxy group (9g) resulted in an analogue that was
inactive against the tested Staphylococcus aureus strains. On the other hand, weakly
deactivating halogens on the benzylidene moiety also had positive influence on the
antibacterial activity, with potency decreasing in the order Br [ Cl [ F. It is clear
that electronic and steric effects probably play the most important role in
determining the antibacterial activity.
Conclusions
A new series of arylidene derivatives of 5,6-dihydro-4H-cyclopenta[b]- and 4,5,6,7-
tetrahydrobenzo[b]-thiophene-2-carboxylic acid (8a–c, 9a–g) were synthesized and
characterized by using 1H and 13C NMR, IR, and mass spectroscopy. Antimicrobial
evaluation of all the compounds revealed that compounds 9a–d, f and 8c showed
comparable inhibitory active against S. aureus of both methicillin-susceptible and
methicillin-resistant strains. Thus, the results of this study identify 9f as the most
bioactive compound of this series, since it exhibited excellent bactericidal activities.
More importantly, the similar antibacterial spectra seen for both methicillin-
123

Synthesis and biological evaluation of novel active…
sensitive and methicillin-resistant S. aureus strains indicate that this microorganism
does not yet have mechanisms of resistance to this class of compounds. Such novel
thiophene carboxylic acid analogues could represent an alternative approach for
development of drugs for treatment of infections caused by methicillin-resistant
S. aureus strains.
Acknowledgments The authors thank the management of Orchid Pharma Limited, Chennai-600119,
India and Ramakrishna Mission Vivekananda College, Chennai-600004, India for providing the required
facilities. We also thank Mr. T. Shanmuganathan for helping carry out the reactions and Mr. T. G.
Rajaraman, Dr. K. Parathasarathy, Mr. N. Dhatchanamoorthy, and Mrs. B. Jeyaseeli for helping with
manuscript preparation.
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