Novel benzothiophene analogs are a privileged structures present in many biologically
active compounds. Benzothiophene derivatives constitute an important class of
compounds, some analogs are synthesized and evaluated for NSAIDs activity (Mahesh
et al., 2004) and many possess diverse biological activity like pesticidal
activity, antimicrobial, analgesic, antiexudative, anti-inflammatory, diuretic
and enzyme inhibition (Grazia et al., 2002).
In addition, schiff bases are also known for their antibacterial activity (Pawar
et al., 1999). In the present investigation, the strategy adapted
was keeping in view the diverse therapeutic activities of benzothiophene derivatives
and as a part of our ongoing development of efficient protocols for the preparation
of bioactive heterocycles (Rao et al., 2005;
Venugopala et al., 2004) and study of polymorphism
in heterocycles (Munshi et al., 2004). Herewith,
we describe a simple, novel, high yielding synthesis of the title compounds
by microwave method in comparison with conventional method and screened them
for their in vitro antibacterial activity by qualitative and quantitative
antibacterial activity by agar cup plate method and micro titration method,
MATERIALS AND METHODS
Melting points were determined in open capillary tubes and were found uncorrected.
Infra Red spectra were recorded on Fourier Transform IR spectrophotometer (Shimadzu
8700) using KBr (vmax in cm-1) disc method. 1H-NMR
spectra were recorded in CDCl3-d and DMSO-d6 on AMX-400
liquid state NMR spectrometer using TMS as an internal reference standard. Mass
spectra were recorded on JEOL JMS DX303 Mass spectrometer with Electron Impact
Ionization (EII) at 70ev and Elemental analysis was performed on Thermo Finnigan
FLASH EA 1112 CHNS analyzer at Indian Institute of Science, Bangalore. The purity
of the test compounds was determined by thin layer chromatography using Merck
silica gel 60 F254 coated aluminium plates using several solvent
systems of different polarity. All the chemicals used were of AR grade and were
procured from Sigma-Aldrich. Minimum inhibitory concentration of the synthesized
compounds was determined using micro-titration method where Luria broth medium
was used as medium for MIC determination.
Synthesis of Trans-3-Methoxycarbonyl-4-(2-Thienyl) But-3-Enoic Acid (1a)
and α, β-Dithenylidinesuccinic Acid (1b)
These compounds were prepared as per the procedure described by Abdel-Wahhab
and El-Rayyes (1971). Potassium metal (4.29 g, 0.11 mol) in small pieces
was dissolved in anhydrous tert. butanol (80 mL) with intermittent warming
to give potassium tert. butoxide. To this, a mixture of thiophene-2-carbaldehyde
(11.2 g, 0.1 mol), dimethyl succinate (17.5 g, 0.12 mol) and 10 mL of tert.
butanol were added gradually over 1 h with stirring at 5-10°C. The reaction
mixture was stirred at room temperature for 4 h. Then it was acidified with
4 N HCl (congo red), 50-70 mL of distilled water was added and tert.
butanol was distilled under reduced pressure. The flask was cooled to room temperature
and reddish oil separated, which was extracted with ether. The acidic portion
was extracted into sodium bicarbonate solution from ether phase. The sodium
bicarbonate layer was separated and acidified cautiously with 4 N HCl. The precipitated
orange red heavy oil was taken into ether (while extracting a yellow fine compound,
α, β-dithenylidenesuccinic acid (1b) was precipitated. The solution
was filtered to separate the compound). The ether layer was separated and dried
over anhydrous sodium sulfate. The ether was removed by distillation, the residue
(1a and b) was digested with boiling benzene to separate a further amount of
the benzene insoluble α, β-dithenylidene succinic acid (1b).
The insoluble product was treated with dilute hydrochloric acid for 30 min
and then filtered off. On crystallization from 70% acetic acid, it gave α,
β-dithenylidene succinic acid (1b) as yellow lustrous crystals, further
the compound α, β-dithenylidene succinic acid (1b) was recrystallized
with methanol to give 1.7 g (5.5%), mp. 254-256 (Lit 255-256°C). C14H10O4S2;
Elemental analysis found (expected) in %: C 54.82 (54.89), H 3.32 (3.29), S
IR (KBr, v in cm-1): 3107 (Ar-H), 1664 (C = O) and
1591 (Ar-C = C)
Evaporation of the benzene mother liquor left dark brown viscous oil which was repeatedly extracted with boiling light petroleum (b.p 60-80). Concentration and cooling (ice box) gave the half-ester trans-3-methoxycarbonyl-4-(2-thienyl) but-3-enoic acid (1a) as yellow rhombic crystals. Yield 18.99 (84.07%) mp. 116-118°C (Lit 117-118°C)
IR (KBr, v in cm-1): 3115 (Ar-H), 1710 (C = O) and
1622 (Ar-C = C)
Synthesis of Methyl 4-Acetoxybenzothiophen-6-Carboxylate (2)
This compound was prepared as per the procedure described by Abdel-Wahhab
and El-Rayyes (1971). The acid ester trans-3-methoxycarbonyl-4-(2-thienyl)
but-3-enoic acid 1a, 22.6 g (0.1 mol) was added to a mixture of sodium acetate
(8.2 g) and acetic anhydride (65 mL) and left over night at room temperature
with stirring using a magnetic stirrer. The temperature was then gradually raised
to 70-80°C over a period of 2 h and maintained for another 4 h with stirring.
Then it was allowed to cool to room temperature and the reaction mixture was
poured into warm water. The neutral portion was extracted into ether layer (200
mL) and washed with cold sodium bicarbonate solution (3x50 mL). Ether layer
was dried over anhydrous sodium sulphate and distilled off. The semisolid product
was recrystallized from light petroleum ether (bp. 60-80°C) to give methyl
4-acetoxybenzothiophen-6-carboxylate as yellow needles with yield of 23.2 g
(92.80%) mp. 84-85°C. (Lit 84-85°C)
IR (KBr, v in cm-1): 3115 (Ar-H), 1710 (-OCOCH3),
1620(-COOCH3) and 1560 (Ar-C = C)
1H-NMR (400MHz, CDCl3): δ 3.72 (s, 3H, OCOCH3),
3.81 (s, 3H,-COOCH3), 7.13 (s, 1H, Ar-H), 7.32 (d, 1H, Ar-H), 7.52
(d, 1H, Ar-H), 7.97 (s, 1H, Ar-H).
MS: m/z 250(M+), 231, 207, 185, 156 and 107.
Synthesis of 4-Hydroxy Benzothiophen-6-Carboxhydrazide (3)
Methyl 4-acetoxybenzothiophen-6-carboxylate 25.0 g (0.1 mol) and Hydrazine
hydrate 8 mL ware placed in a round bottomed flask fitted with a reflux condenser
and the mixture was heated gently under reflux for 10 min. Sufficient quantity
of absolute alcohol was added through the condenser to get a clear solution
(about 8 mL). This was refluxed for 2.5 h and ethanol was distilled off and
residue cooled. The crystals of acid hydrazide were filtered and recrystallized
from ethanol. The product was isolated as white fluffy mass with yield of 18.3
g (87.98%) and the same reaction when carried out by microwave method yield
observed was 19.4 g (93.26%), mp. 234°C.
IR (KBr, v in cm-1): 3300 (-OH), 3300 and 3211 (-NH
NH2), 1708 (C = O), 1545 (Ar C = C).
1H-NMR (400MHz, DMSO-d6): δ 6.12 (s, 2H,-NH2),
6.59 (s, 1H,-NH), 7.20 (s, 1H, Ar-H), 7.37 (d, 1H, Ar-H), 7.63 (d, 1H, Ar-H),
7.97 (s, 1H, Ar-H), 10.42 (s, 1H, phenolic OH),
MS: m/z 209(M+1), 185, 151, 135, 123 and 110.
Synthesis of Schiff Bases of 4-Hydroxy Benzothiophen-6-Carboxhydrazide (4a-p)
4-Hydroxy benzothiophen-6-carboxhydrazide 3 (0.01 mol) was refluxed with
substituted aromatic aldehydes (a-p) (0.011 mol) and acetaldehyde (0.011 mol)
in ethanol at different time intervals to get schiff bases 4a-p and 5, respectively.
Both conventional and microwave methods were employed to synthesize title compounds
using alcohol as recrystalizing solvent and the results are tabulated in Table
2. In case of microwave method different solvents were used to enhance the
products yield and the same is represented in Table 1.
|| Intermediate (3) was made to react with substituted araldehyde
in different solvents and yields
|Present method reaction condition: o-chlorobenzaldehyde
(10 m mol), intermediate 3 (10 m mol) and absolute ethanol (60 mL) were
irradiated to microwaves for 96 sec to afford product 4a
|| Reaction parameters of schiff bases of 4-hydroxy 6-carboxhydrazino
benzothiophenes (4a-p) and (5)
4-Hydroxy Benzothiophene-6-[N (3-Methylbenzaldimino)] Carboxamide
IR (KBr, v in cm-1): 3431(-OH), 3203 (-NH), 1676
(C = O), 1608 (ArC = C)
1H-NMR (400 MHZ, DMSO-d6): δ 2.34 (s, 3H,-CH3),
5.80 (s, 1H, NH), 6.97 (s, 1H, CH), 7.07-8.27 (m, 8H, Ar-H), 11.35 (s, 1H, Phenolic
MS: m/z 310(M+), 295, 268, 201, 177 and 161.
The antibacterial activity (Parmar et al., 1992)
of the test samples (4a-p) and (5) were determined by agar cup plate method
using four organisms such as B. subtilis, S. aureus, E. coli
and K. pneumoniae and two standard drugs Ampicillin and Streptomycin.
This method was based on diffusion of antibacterial component from reservoir
bore to the surrounding inoculated nutrient agar medium so that the growth of
microorganisms was inhibited as circular zone around the bore. The concentration
of test compounds was 100 μg 100 μL-1 and was prepared
in Dimethyl Sulfoxide (DMSO). The test samples and standard drugs were placed
in a bore made in petri dishes which contains different organisms and were incubated
at 37°C for 24 h. The zone of inhibitions around the bore was measured after
24 h. The antibacterial activity was classified as standards (>27 mm) highly
active (21-27 mm), moderately active (15-21 mm), least active (12-15 mm) and
less than 12 mm was taken as inactive.
||Qualitative antibacterial activity of schiff bases of 4-hydroxy
6-carboxhydrazino benzothiophene analogs (4a-p) and 5
|+: Less than 12 mm; ++: 12-15 mm (least active); +++: 15-21
mm (moderately active); ++++: 21-27 mm (highly active); +++++: >27 (standard)
-: Control; B.s: Bacillus subtilis; E.c: Escherichia
coli; S.a: Staphylococcus aureous; K.p: Klebshella pneumonia
|| Minimum inhibitory concentration of schiff bases of 4-hydroxy
6-carboxhydrazino benzothiophene analogs
All the samples were tested in triplicate. The antibacterial activity data
are recorded in Table 3.
Determination of Minimum Inhibitory Concentration (MIC)
The determination of minimum inhibitory concentration (Lowdin
et al., 1993; Kotretsou et al., 1995)
was done with four isolates of Bacillus subtilis, Staphylococcus aureus,
Escherichia coli and K. pneumoniae which were inoculated into Luria
broth medium which contains 1% tryptone, 0.5% yeast extract and 0.5% sodium
chloride. The pH of the medium was adjusted to 7.2 with sterile phosphate buffered
saline and incubated at 37°C for 24 h. The optical density of the bacteria
from mid log phase of growth was measured at 540 ηm and diluted in fresh
medium so as to get an optical density of 0.004 (corresponding to 5x105
colony forming units mL-1). To each well of the ELISA plate (Corning,
USA), 200 μL of diluted bacterial suspension was added. Graded concentrations
(0.2-500 μg/50 μL) of the synthesized promising compounds and two
standard antibiotics (Streptomycin and Ampicillin) in dimethyl sulfoxide were
added and incubated at 37°C for 24 h. At the end of incubation the effect
of the drugs on the growth of organisms were monitored by measuring the optical
density at 540 ηm using ELISA reader (Multiscan MS, Labsystems, Helsinki,
Finland). The MIC was defined as the lowest concentration of the antibiotic
or test sample allowing no visible growth. Determination of minimum inhibitory
concentration was performed in triplicate and the results are shown in Table
RESULTS AND DISCUSSION
The parent compound 4-hydroxy benzothiophen-6-carboxhydrazide was prepared
by treating methyl-4-acetoxybenzothiophene-6-carboxylate and hydrazine hydrate
in alcohol medium for 2 h. The former was prepared by cyclization of trans-3-methoxy
carbonyl-4-(2-thienyl)but-3-enoic acid in presence of sodium acetate and acetic
anhydride at room temperature and processed to obtain 4-hydroxy-benzothiophene-6-carboxhydrazide.
The intermediate trans-3-methoxy carbonyl-4-(2-thienyl)but-3-enoic acid
was prepared by Stobbe condensation method using thiophene 2-carbaldehyde and
dimethyl succinate in presence of potassium tert. butoxide as catalyst
in tert. butanol. The parent compound was treated with various aromatic
aldehydes to obtain schiff bases of substituted benzothiophene. The purified
compounds were characterized by IR, 1H-NMR, Mass spectral studies
and elemental analysis. The spectral evidences in compound 4h confirms the presence
of-OH,-NH-,-CO-and fused benzene ring, (IR at 3431, 3202, 1676 and 1608, respectively)
similarly 1H-NMR multiplet in the range of 7.07-8.27 ppm of 8H also
confirms the presence of aromatic rings. The synthetic scheme of schiff bases
of 4-hydroxy 6-carboxhydrazino benzothiophene are mentioned in scheme-1 and
the effect of solvent, reaction parameters, qualitative and quantitative antibacterial
activity are shown in Table 1-4, respectively.
Out of several solvents tried for microwave irradiated synthesis of schiff bases
of 4-hydroxy benzothiophen-6-carboxhydrazide though PEG 400 and ethylene glycol
showed satisfactory yield, maximum yield was observed with absolute ethanol.
Some novel schiff bases of 4-hydroxy 6-carboxhydrazino benzothiophene derivative were synthesized and compounds (4a-p) and (5) were tested for their qualitative antibacterial activity using agar cup plate method against two G+ve pathogenic organisms Bacillus subtilis, S. aureus and two G-ve pathogenic organisms Escherichia coli and Klebsiella pneumoniae using two standard antibiotics Ampicillin and Streptomycin. Some of the test compounds exhibited significant antibacterial activity when compared to standards (Table 3). The promising test samples were also subjected for determination of Minimum Inhibitory Concentration (MIC) using same strains of organisms and same standard drugs. Some of the compounds such as 4-hydroxy benzothiophene-6-[N (4-methylbenzaldimino)] carboxamide, 4-hydroxy benzothiophene-6-[N (4-nitrobenzaldimino)] carboxamide, 4-hydroxy benzothiophene-6-[N (4-fluorobenzaldimino)] carboxamide and 4-hydroxy benzothiophene-6-[N (4-chlorobenzaldimino)] carboxamide have shown good minimum inhibitory concentration when compared to standard Ampicillin and Streptomycin.
The authors are thankful to Prof. B.G. Shivananda, Principal, Al-Ameen College of Pharmacy, Bangalore for facilities and Dr. Chandrashekaran, Department of Organic Chemistry, Indian Institute of Science, Bangalore for 1H-NMR, Mass spectra and Elemental analysis.