INTRODUCTION:

Naturally occurring 1-indanone derivatives form a rare class of natural products generally called pterosins¹. 1-Indanone is a polynuclear hydrocarbon compound with a five-membered ring fused to benzene ring. Indanone acetic acid is a non-heterocyclic fused ring of benzene attached to cyclopentananone moiety with acetic acid side chain in 1-position. Indanones and related compounds are important bio-active molecules which possesses diverse biological activities. In recent years, these compounds have been studied for various biological activities such as anti-inflammatory^2-4, analgesic⁵, antimicrobial^6,7, antiviral⁸, anticholinergic⁹, dopaminergic¹⁰, anticancer^11-12 and antimalarial¹³, activities.

There has been a great interest to develop new non-steroidal anti-inflammatory drugs (NSAIDS) which specifically inhibit cyclooxygenase-2 (COX-2), the enzyme responsible for inflammation process. Some selective inhibitors for COX-2 have already been found and the research in this direction continues with a view to discover new drugs for inhibiting this enzyme².

MATERIALS AND METHODS:

All reagents used were of analytical reagents grade, obtained from S. D. Fine Chemicals, Spectrochem, Qualigens and Loba Chemicals. Chemicals and solvents were purified by general laboratory techniques before use. All moisture-free operations were performed in oven dried glassware. Melting points were determined using a Veego melting point apparatus having silicon oil bath and are uncorrected. IR spectra (wave numbers in cm^-1) were recorded on a Bruker Alpha FT-IR spectrophotometer using potassium bromide discs.¹H NMR spectra were recorded on Bruker Avance II 500 MHz instrument in CDCl₃with TMS as internal standard. Chemical shift values are mentioned in δ, ppm. Mass spectra were recorded on Shimadzu LC MS 2010 spectrometer. UV spectra (λ_maxin nm) were recorded on shimadzu UV-1700 UV visible spectrophotometer using methanol as a solvent. Chromatographic separations were performed on columns using silica gel 100-200 mesh and neutral alumina, activity grade I. The progress of all reactions was monitored by TLC on 2 cm X 5cm pre-coated silica gel 60 F₂₅₄ (Merck) plates of thickness 0.25 mm. The chromatograms were visualized under UV (254 nm) and/or exposure to iodine vapors. The term “dried” refers to the use of anhydrous sodium sulphate.

EXPERIMENTAL WORK:

Diethyl-3,5-diacetyl-4-phenylheptandioate (2):

A solution of benzaldehyde (30 ml, 1 mole) and ethyl acetoacetate (78 ml, 2 mole) was taken in a 250 ml round bottom flask. Diethylamine (10 ml, 0.09 mole) was added into the reaction mixture with stirring and after complete addition, reaction mixture was heated on oil bath at 80^oC for 40 min. After completion of reaction, yellow colored product was precipitated out and was recrystalized using ethanol. Finally yellow crystals of diethyl-3,5-diacetyl-4-phenylheptandioate (2) was obtained¹⁴.

Yield: 97.35%, R_f 0.68 (Ethyl acetate: Hexane, 1:1), m.p. 158-160°C

3-Phenylpentandioic acid (3):

Diethyl-3,5-diacetyl-4-phenyl heptandioate (2), (50 gm, 0.155 mole) was dissolved in a solution of potassium hydroxide (50 gm, 0.89 mole) in water (50 ml) by warming on water bath. Reaction mixture was cooled in ice bath, acidified with hydrochloric acid (60 ml) slowly until reaction mixture become acidic and the crude product was separated out as an oily globules. The biphasic solution was heated on water bath with charcoal (2 gm) for 30 min. The solution was filtered off and was kept at 0^oC in refrigerator. White colored crystals of 3-Phenylpentandioic acid (3) were separated out.

Yield: 41.94%, R_f 0.62 (Ethyl acetate: Hexane, 1:1), m.p. 142-144°C

2-(3-Oxo-2, 3-dihydro-4-inden-1-yl) acetic acid (4):

3-Phenylpentandioic acid (3), (30 gm, 0.157 mole) was dissolved in 90 ml conc. sulphuric acid in round bottom flask. Mixture was heated on water bath for 30 mins. Completion of reaction was monitored by TLC. After completion of reaction, reaction mixture was poured into the crushed ice with constant stirring, crude 2-(3-Oxo-2,3-dihydro-4-inden-1-yl) acetic acid (4) was obtained which was crystallized from methanol.

Yield: 52.70%, R_f 0.58 (Ethyl acetate: Hexane, 1:1), m.p. 136-138 °C

2-(3-Oxo-2,3-dihydro-1H–inden-1-yl)-N-phenylacetamide (5a):

2-(3-Oxo-2,3-dihydro-4-inden-1-yl) acetic acid (4) (1 gm, 0.0037mole) was taken in round bottom flask. Thionyl chloride (0.621 ml, 1mole) was added and refluxed for 40 minutes, after that aniline (0.340 ml, 0.0037 mole) was added drop wise and refluxed for 60 minutes. Then this solution was poured into crushed ice and crude 2-(3-Oxo-2,3-dihydro-1H–inden-1-yl)-N-phenylacetamide (5a) was obtained which was recrystalized from methanol.

Yield: 73.38%, R_f 0.54 (Ethyl acetate: Hexane, 1:1), UV(λ_maxin nm): 248, m.p. 152-154 °C.

2-(3-Oxo-2,3-dihydro-1H–inden-1-yl)-N-o-tolylacetamide (5b):

2-(3-Oxo-2,3-dihydro-4-inden-1-yl) acetic acid (4) (1 gm, 0.0037 mole) was taken in round bottom flask. Thionyl chloride (0.621 ml, 1mole) was added and refluxed for 40 minute. After that 2-methylaniline (1.023 ml, 0.0037 mole) was added drop wise and refluxed for 60 minute. Then this solution was added into crushed ice. So, crude 2-(3-Oxo-2,3-dihydro-1H–inden-1-yl)-N-o-tolylacetamide (5a) was obtained which was recrystalized from methanol.

Yield: 63.42%, R_f 0.52 ( Ethyl acetate: Hexane, 1:1), UV(λ_maxin nm): 295, m.p. 172-174 °C.

N-(2-Chlorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl) acetamide (5c):

2-(3-Oxo-2,3-dihydro-4-inden-1-yl) acetic acid (1 gm, 0.0037mole) (4) was taken in round bottom flask. Thionyl chloride (0.621 ml, 1 mole) was added to it and refluxed for 40 minutes. Then 2-chloroaniline (1.10 ml, 0.0037 mole) was added drop wise and refluxed for 60 minutes. Then this solution was poured into crushed ice. Crude N-(2-Chlorophenyl)-2-(3-oxo-2, 3-dihydro-1H–inden-1-yl) acetamide (5c) was obtained which was recrystalized from methanol.

Yield: 79.11%, R_f 0.56 (Ethyl acetate: Hexane, 1:1), UV(λ_maxin nm): 249, m.p. 212-214 °C.

N-(2-Methoxyphenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl) acetamide (5d):

2-(3-Oxo-2,3-dihydro-4-inden-1-yl) acetic acid (1 gm, 0.0037 mole) (4) was taken in round bottom flask. Thionyl chloride (0.621 ml, 1 mole) was added and refluxed for 40 minutes, after that 2-methoxyaniline (1.03 ml, 0.0037 mole) was added drop wise and refluxed for 60 minutes. Then this solution was added into crushed ice. Crude N-(2-Methoxyphenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl)acetamide (5d) was obtained which was recrystalized from methanol.

Yield: 56.12%, R_f 0.44 ( Ethyl acetate: Hexane, 1:1), UV(λ_maxin nm): 263.8, m.p. 202-204 °C.

N-(3-Chlorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl)acetamide (5e):

2-(3-Oxo-2,3-dihydro-4-inden-1-yl) acetic acid (1 gm, 0.0037 mole) (4) was taken in round bottom flask. Thionyl chloride (0.621 ml, 1 mole) was added and refluxed for 40 minute, after that 2-chloroaniline (1.10 ml, 0.0037 mole) was added drop wise and refluxed for 60 minutes. Then this solution was added into crushed ice. Crude N-(3-Chlorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl)acetamide (5e) was obtained which was recrystalized from methanol.

Yield: 60.75%, R_f 0.54 (Ethyl acetate: Hexane, 1:1), UV(λ_maxin nm): 242.8, m.p. 228-230 °C.

N-(4-Fluorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl)acetamide (5f):

2-(3-oxo-2,3-dihydro-4-inden-1-yl) acetic acid (1 gm, 0.0037 mole) (4) was taken in round bottom flask. Thionyl chloride (0.621 ml, 1 mole) was added and refluxed for 40 minutes, after that 4-Fluoroaniline (1.04ml, 0.0037 mole) was added drop wise and refluxed for 60 minutes. Then this solution was added into crushed ice. Crude N-(4-fluorophenyl)-2-(3-oxo-2, 3-dihydro-1H–inden-1-yl) acetamide (5f) was obtained which was recrystalized from methanol.

Yield: 82.43%, R_f 0.60 (Ethyl acetate: Hexane, 1:1), UV(λ_maxin nm) : 243.4, m.p. 156-158 °C.

RESULTS AND DISCUSSION:

The newly synthesized compounds (5a-f) were structurally conﬁrmed by analytical and spectral data. Further, substituted indanone acetic acid (5a-f) derivatives were screened for antibacterial and antifungal activity.

Compound (5a): 2-(3-Oxo-2,3-dihydro-1H–inden-1-yl)-N-phenylacetamide

IR (KBr): C=O (1718), N-H (3131), C-N (1284). ¹H NMR: 2.12 (d, 2H, CH₂), 8.32 (s,1H, NH), 7.78-7.65 (m, 4H, Ar-H). Mass: 265.8 (M+).

Compound (5b): 2-(3-Oxo-2,3-dihydro-1H–inden-1-yl)-N-o-tolylacetamide

IR (KBr): C=O (1718, 1659), N-H (3135), C-N (1258). Mass: 281.1 (M+2).

Compound (5c): N-(2-Chlorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl)

IR (KBr): C=O (1712), N-H (3125), C-N (1293), C-H (2928), C-Cl (681). Mass: 301.7 (M+2).

Compound (5d): N-(2-Methoxyphenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl) acetamide

IR (KBr): C=O (1734, 1688), N-H (3131), C-N (1288, 1157), C-O (1220). ¹H NMR: 2.55 (d, 2H, CH₂), 10.62 (s,1H, NH), 7.82-7.51 (m, 4H, Ar-H). Mass: 296.2(M+).

Compound (5e): N-(3-Chlorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl) acetamide

IR (KBr): C=O (1734, 1661), N-H (3133), C-N (1293), C-H (2927). ¹H NMR: 2.49 (d, 2H, CH₂), 9.62 (s,1H, NH), 7.95-7.82 (m, 4H, Ar-H). Mass: 301.9 (M+2).

Compound (5f): N-(4-Fluorophenyl)-2-(3-oxo-2,3-dihydro-1H–inden-1-yl) acetamide

IR (KBr): C=O (1716), N-H (3131), C-N (1293), C-F (1099). ¹H NMR: 2.09 (d, 2H, CH2), 8.25 (s,1H, NH), 7.75-7.56 (m, 4H, Ar-H). Mass: 283.8 (M+).

ANTIMICROBIAL EVALUATION:

All compounds (5a-f) were screened for in-vitro antibacterial activity against pathogenic bacteria collected from National Chemical Laboratories Pune. Two Gram-positive Bacillus subtilis National Collection of Industrial Microorganism (NCIM) No. 2063, and Staphylococcus aureus (NCIM No. 2079) and two Gram-negative Salmonella typhi (NCIM No. 2263) and Escherichia coli (NCIM No. 2065) bacterial strains were used. Antibacterial activities of novel compounds were compared using streptomycin and ampicillin as a standard drugs. Similarly antifungal studies were carried out against two fungus strains Candida albicans (NCIM No. 3102) and Aspergillus niger (NCIM No. 742) using ketoconazole as standard drug.

Antibacterial activity:

The synthesized compounds were screened for antibacterial activity against microbes using Agar well diffusion method^15,16.

In this method, Muller Hilton Agar medium was prepared and sterilized. 20 ml of media was poured in petri-plates and allowed for uniform solidification. Test bacteria were swabbed uniformly on solidified sterile Muller–Hilton agar plates using sterile cotton swab and labeled. Then aseptically, 6 mm diameter wells were made in the media of the inoculated plates with the help of a metallic borer. In each well 100µL of test sample (100 µg/ml, 50 µg/ml) in DMSO were added and labeled accordingly. Standard solution of streptomycin and ampicillin (100 µg/ml, 50 µg/ml) and DMSO control were added. These plates were incubated immediately at 37°C for 24 h in upright position and the zone of inhibition was determined by measuring the diameter of zones (mm) showing complete inhibition. The results of zone of inhibition mentioned in Table-1 were compared with the drug streptomycin and ampicillin.

Table-1: Antibacterial data of compounds 5a-f

Compounds	Conc. (μg/ml)	Zone of inhibition (mm)
Compounds	Conc. (μg/ml)	*B. subtilis*	*S. aureus*	*S. typhi*	*E. coli*
5a	100	9	7	10	11
5a	50	7	6	8	10
5b	100	10	8	13	12
5b	50	8	6	10	10
5c	100	12	11	14	14
5c	50	10	7	12	12
5d	100	11	10	15	15
5d	50	8	7	11	10
5e	100	13	12	13	14
5e	50	10	9	10	10
5f	100	17	15	16	18
5f	50	12	11	13	14
Streptomycin	100	22	20	20	23
Streptomycin	50	18	16	17	18
Ampicillin	100	20	19	20	20
Ampicillin	50	15	14	15	16
Control		0	0	0	0

Antifungal activity:

The synthesized compounds were screened for antifungal activity against fungal strains Aandida albicans and Aspergillus niger using Agar well diffusion method. The assay is a measure of test compound with standard drug which is ketoconazole¹⁷.

In this method, Sabouraud dextrose agar medium was prepared and sterilized as like Muller Hilton Agar. After solidification of media, Inoculum (0.02 ml) was introduced to molten media and poured into a petri-dish. Then aseptically, 6 mm diameter wells were made in the media of the inoculated plates with the help of a metallic borer. In each well, 50 μl of the test sample (100 µg/ml) in DMSO was introduced and labeled accordingly. Standard solution of ketoconazole (100 µg/ml) and DMSO control were added. The plates were incubated immediately at 30°C for 48 h in upright position and the zone of inhibition was determined by measuring the diameter of zones (mm) showing complete inhibition. The results of zone of inhibition mentioned in Table-2 were compared with the drug ketoconazole.

Table-2: Antifungal data of compounds 5a-f

Compounds	Zone of inhibition (mm) at 100 Conc. (μg/ml)
Compounds	*Candida albicans*	*Aspergillus niger*
5a	4	3
5b	10	8
5c	6	8
5d	11	10
5e	9	8
5f	9	7
Ketoconazole	15	12
Control	0	0

CONCLUSION:

Among all synthesized compounds, compound 5f gives a better antibacterial activity against gram positive and gram negative bacteria. It is observed that compound 5f possess para-fluorophenyl substitution on indanone acetic acid as compared to meta-chlorophenyl substitution. ortho-Methoxyphenyl derivative 5d exhibited better antifungal activity as compared to ortho-methylphenyl substitution.

ACKNOWLEDGEMENT:

We are thankful to the Director, SAIF, Punjab University, Chandigarh and Oxygen Healthcare Private Ltd. Ahmedabad for providing spectroscopic analysis of the compounds. The authors are thankful to All India Council for Technical Education, New Delhi for the financial assistance in the form of fellowship. We would like to thank University Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for providing necessary infrastructure and facility.

We would also like to thanks Late Mr. Karan Parwe, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur for his equal contribution in research and compilation of work.

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Received on 10.03.2018 Modified on 29.04.2018

Research J. Pharm. and Tech 2018; 11(7): 3013-3017.

DOI: 10.5958/0974-360X.2018.00555.3