Study of Synthesis and Analgesic Activity of some Benzopyrone Derivatives

Sukhen Som¹* and Jayaveera K.N²

¹Department of Pharmaceutical Chemistry, M.M.U College of Pharmacy. K.K. Doddi Ramanagara- 562159. Karnataka

²Department of Chemistry, Jawaharlal Nehru Technological University Anantapur Ananthapuramu- 515002. Andhra Pradesh

*Corresponding Author E-mail: sukhen18@rediffmail.com

ABSTRACT:

Benzopyrone derivatives are associated with different pharmacological activities like analgesic, anti-inflammatory, anticancer, antihepatotoxic, antibacterial, antiviral etc. Considering the potential of this particular lead molecule, different derivatives of benzopyrone condensed with amines were synthesized and evaluated for their analgesic activity. The structures of the synthesized compounds were confirmed by their analytical as well as spectral data. Some of the compounds synthesized i.e. 4a, 4c, 4e and 4g showed significant analgesic activity.

KEYWORDS: Analgesic activity, Amines, Tail flick method.

INTRODUCTION:

The current trend in medicinal chemistry is to develop new clinically effective agents through the structural modification of a lead nucleus. The lead is a prototype compound that has the interesting biological or pharmacological activities but may have many undesirable characteristics like high toxicity, other biological activity, insolubility or metabolism problems. Such organic leads once identified, are then easy to exploit. The real test resides with the identification of such lead compounds and the optimum bioactive positions on the basic skeleton of such leads.

There is a growing interest in the pharmacological potential of natural products. In the recent years there has been a lot of attention drawn on the study of benzopyrones as inhibitors of oxidative reactions^1-5 and as anti-inflammatory agents^6-10. Compounds having chromone (4-benzopyrone) moiety are associated with interesting physiological activities such as antibacterial, antiviral, anticancer, antioxidant, antifungal, anticholesteremic, antidiabetic, antiallergic, diuretic etc¹¹.

Flavonoids, the derivatives of chromones are polyhydroxylated compounds, and they are capable of selectively reacting with free radicals or systems related to the induction of inflammatory processes. Quercetin (3, 3’, 4', 5, 7-pentahydroxy flavone) and related flavonoids are known to inhibit the growth of tumor cells and potentiates the cytotoxicity of DNA damaging anti-cancer drugs such as cis-platin¹². Few reports are also available on the influence of lipophilic substituents on the antioxidant or anti-inflammatory activities of this class of natural products¹³. Flavones inhibit CYP1A-mediated 7-ethoxy resorufin o-dethylase activity in rat and human liver microsomes¹⁴. Certain bromoflavones are found to significantly induce quinone reductase activity, which is an important mechanism of chemoprevention ^15-17. Even though natural flavonoids are highly potent, attempts have to be made to improve their stability, solubility, efficacy and kinetics. Hence, with the knowledge of semi synthetic and synthetic science, some benzopyrone derivatives were synthesized in the laboratory by convenient and cost effective methods from past few decades. Since, benzopyrone derivatives are known to exhibit a wide variety of pharmacological activities, many available literatures encouraged us to modify benzopyrone ring to explore newer activities associated with this nucleus. In view of the concerned observations therefore in our present study, we synthesized some substituted benzopyrone derivatives and evaluated their analgesic property.

MATERIALS AND METHODS:

The melting points of the synthesized compounds were determined by open capillary tube method and are uncorrected. The IR spectra were recorded in a Shimadzu 8400S FTIR spectrophotometer using KBr discs in the region of 4000-400 cm^-1. Using TMS as internal standard NMR spectra were recorded on a Brukar Spectrospin 200 spectrometer. Mass spectra were obtained by using JEOL GC mate instrument. Precoated Silica gel G plates were used to check the purity of the compounds; Benzene: ethyl acetate- 1:1 used as developing solvent; detections were carried out either in UV chamber or by using iodine vapour.

Synthesis of 7-hydroxy-2-(4-chlorophenyl)-4-benzopyrone (1a):

0.025 mol of 2, 4-dihydroxybenzoyl-4-chlorobenzoyl methane was dissolved in 40ml of glacial acetic acid and then 2ml of concentrated sulphuric acid was added with shaking. The solution was then refluxed for about 2.5 hours with intermittent shaking. The reaction mixture was then poured on crushed ice. After allowing settling the product was filtered and washed repeatedly with cold water and dried. It was recrystallised from petroleum ether (60-80⁰C). Melting point 138⁰C, yield 62%, R_f value 0.64. In a similar manner compounds 1b-l were synthesized.

Synthesis of 6-acetyl-7-hydroxy-2-(4-chlorophenyl)-4-benzopyrone (2a)

4-benzopyrone (1a) (0.01 mol) was added to 30 ml of nitrobenzene. Acetic anhydride (0.01 mol), freshly powdered 0.022 mol of anhydrous aluminium chloride were added and then the reaction mixture was heated at 150⁰C for around 4 hours. The reaction content was then cooled and to this added 75 gm of crushed ice and 4 ml of concentrated HCl. The product was filtered and recrystallised from 95% ethanol. Melting point found 186⁰C, yield 57%, R_f value 0.67. Similarly compounds 2b-l was synthesized.

Synthesis of 6-bromoacetyl-7-hydroxy-2-(4-chlorophenyl)-4-benzopyrone (3a)

Acetylated 4-benzopyrone (2a) (0.01 mol) was taken into 50 ml of glacial acetic acid. To this solution was added bromine drop wise for about 1 hour with continuous stirring. After complete addition of bromine the resulting solution was continued to stir for around 6 hours till the evolution of hydrogen bromide completes. The resulting solution was then poured on crushed ice and filtered. The product was then recrystallised from 50% alcohol. Melting point 154⁰C, yield 53%, R_f value 0.68. In a similar manner compounds 3b-l were synthesized.

Synthesis of 6-acetylamino-7-hydroxy-2-(4-chlorophenyl)-4-benzopyrone (4a)

The amine (0.01 mol) was added to a solution of bromo derivative of 4-benzopyrone (3a) (0.01 mol) in absolute alcohol (30 ml) and the mixture was heated under reflux for about 2 hours. The product was separated out on standing the above solution overnight in cold condition. The solid was filtered, washed by using cold water and dried. The compounds 4a-l was synthesized in a similar manner. Physical data are reported in table 1.

Table- 1. Physical data of synthesized derivatives:

Compound code	R1	R2	Mol. Formula	Mol. Wt.	Yield (%)	R_f value	M.P (⁰C)
4a	-Cl			399.83	49	0.59	165
4b	-Cl			383.83	70	0.72	168
4c	-Cl			413.91	53	0.71	135
4d	-Cl			357.80	61	0.69	175
4e	-NO₂			410.39	59	0.71	204
4f	-NO₂			394.39	54	0.58	175
4g	-NO₂			408.41	65	0.68	188
4h	-NO₂			368.35	57	0.72	187
4i	-Br			444.29	57	0.72	185
4j	-Br			428.29	68	0.67	179
4k	-Br			442.31	71	0.62	182
4l	-Br			402.25	63	0.68	177

4a- IR (KBr, cm^-1): 3414 O-H str, 3027 C-H str aromatic, 2930 C-H str aliphatic, 1679 C=O ketone, 1594 C=C str, 1389 C-H bending aliphatic, 1263 ether bond, 1204 OH bending, 1091 C-N vibration, 830 C-H bending aromatic, 701 C-Cl str. ¹H NMR (δ): 3.7 2H s alkyl H, 4.1-4.5 8H m alkyl H, 6.7 1H s C-3 H, 7.1-7.3 4H m Ar H, 7.7-7.8 2H m Ar H, 11.9 1H s OH. Mass (M+H): 400.83

4b- IR (KBr, cm^-1): 3413 O-H str, 3026 C-H str aromatic, 2929 C-H str aliphatic, 1676 C=O ketone, 1585 C=C str, 1388 C-H bending aliphatic, 1265 ether bond, 1198 OH bending, 1067 C-N vibration, 826 C-H bending aromatic, 751 C-Cl str. ¹H NMR (δ): 1.8-2.1 4H m alkyl H, 3.0-3.1 2H s alkyl H, 3.5-3.7 4H m alkyl H, 6.8 1H s C-3 H, 7.4-7.7 4H m Ar H, 8.0-8.1 2H m Ar H, 11.9 1H s OH. Mass (M+H): 384.82

4c- IR (KBr, cm^-1): 3416 O-H str, 3028 C-H str aromatic, 2932 C-H str aliphatic, 1679 C=O ketone, 1595 C=C str, 1387 C-H bending aliphatic, 1262 ether bond, 1206 OH bending, 1092 C-N vibration, 832 C-H bending aromatic, 705 C-Cl str. ¹H NMR (δ): 2.1-2.4 6H m alkyl H, 2.6-2.8 4H m alkyl H, 3.2 2H s alkyl H, 7.0 1H s C-3 H, 7.6-7.8 4H m Ar H, 8.0-8.1 2H m Ar H, 12.2 1H s OH. Mass (M+H): 414.90

4d- IR (KBr, cm^-1): 3413 O-H str, 3063 C-H str aromatic, 2929 C-H str aliphatic, 1676 C=O ketone, 1585 C=C str, 1388 C-H bending aliphatic, 1265 ether bond, 1199 OH bending, 1066 C-N vibration, 827 C-H bending aromatic, 751 C-Cl str. ¹H NMR (δ): 2.4-2.6 6H alkyl H, 3.0-3.1 2H s alkyl H, 6.8 1H s C-3 H, 7.4-7.6 4H m Ar H, 7.8-7.95 2H m Ar H, 12.4 1H s OH. Mass (M+H): 358.80

4e- IR (KBr, cm^-1): 3300 O-H str, 3071 C-H str aromatic, 2800 C-H str aliphatic, 1640 C=O ketone, 1568 C=C str, 1519 C-NO₂ str, 1374 C-H bending aliphatic, 1285 ether bond, 1245 OH bending, 1136 C-N vibration, 836 C-H bending aromatic. ¹H NMR (δ): 3.6-3.8 2H s alkyl H, 4.1-4.4 8H m alkyl H, 6.7 1H s C-3 H, 7.4-7.6 4H m Ar H, 7.8-8.0 2H m Ar H, 11.9 1H s OH. Mass (M+H): 411.39

4f- IR (KBr, cm^-1): 3410 O-H str, 3064 C-H str aromatic, 2929 C-H str aliphatic, 1674 C=O ketone, 1584 C=C str, 1491 C-NO₂ str, 1388 C-H bending aliphatic, 1264 ether bond, 1196 OH bending, 1075 C-N vibration, 832 C-H bending aromatic. ¹H NMR (δ): 1.8-2.1 4H m alkyl H, 3.1 2H s alkyl H, 3.5-3.8 4H m alkyl H, 6.9 1H s C-3 H, 7.6-7.8 4H m Ar H, 8.1-8.3 2H m Ar H, 12.1 1H s OH. Mass (M+H): 395.39

4g- IR (KBr, cm^-1): 3302 O-H str, 3071 C-H str aromatic, 1646 C=O ketone, 1570 C=C str, 1517 C-NO₂ str, 1375 C-H bending aliphatic, 1281 ether bond, 1246 OH bending, 1134 C-N vibration, 800 C-H bending aromatic. ¹H NMR (δ): 1.9-2.2 6H m alky H, 2.6-2.8 4H m alkyl H, 3.2 2H s alkyl H, 7.0 1H s C-3 H, 7.6-7.8 4H m Ar H, 8.0-8.1 2H m Ar H, 12.0 1H s OH. Mass (M+H): 409.41

4h- IR (KBr, cm^-1): 3301 O-H str, 3040 C-H str aromatic, 2800 C-H str aliphatic, 1642 C=O ketone, 1517 C=C str, 1478 C-NO₂ str, 1374 C-H bending aliphatic, 1281 ether bond, 1185 OH bending, 1134 C-N vibration, 833 C-H bending aromatic. ¹H NMR (δ): 2.5-2.7 6H alkyl H, 3.2 2H s alkyl H, 6.8 1H s C-3 H, 7.4-7.6 4H m Ar H, 7.8-8.0 2H m Ar H, 12.0 1H s OH. Mass (M+H): 369.35

4i- IR (KBr, cm^-1): 3419 O-H str, 3028 C-H str aromatic, 2932 C-H str aliphatic, 1679 C=O ketone, 1595 C=C str, 1387 C-H bending aliphatic, 1337 ether bond, 1206 OH bending, 1092 C-N vibration, 832 C-H bending aromatic, 610 C-Br str.. ¹H NMR (δ): 3.6-3.7 2H s alkyl H, 4.0-4.3 8H m alkyl H, 6.7 1H s C-3 H, 7.4-7.6 4H m Ar H, 7.8-7.9 2H m Ar H, 12.1 1H s OH. Mass (M+H): 445.29

4j- IR (KBr, cm^-1): 3414 O-H str, 3027 C-H str aromatic, 2929 C-H str aliphatic, 1679 C=O ketone, 1588 C=C str, 1388 C-H bending aliphatic, 1264 ether bond, 1203 OH bending, 1091 C-N vibration, 828 C-H bending aromatic, 616 C-Br str. ¹H NMR (δ): 1.9-2.1 4H m alkyl H, 3.1 2H s alkyl H, 3.5-3.8 4H m alkyl H, 7.0 1H s C-3 H, 7.6-7.8 4H m Ar H, 8.1-8.3 2H m Ar H, 11.9 1H s OH. Mass (M+H): 429.29

4k- IR (KBr, cm^-1): 3298 O-H str, 3078 C-H str aromatic, 2811 C-H str aliphatic, 1628 C=O ketone, 1529 C=C str, 1375 C-H bending aliphatic, 1240 ether bond, 1194 OH bending, 1060 C-N vibration, 834 C-H bending aromatic, 599 C-Br str. ¹H NMR (δ): 1.9-2.2 6H m alkyl H, 2.6-2.8 4H m alkyl H, 3.2 2H s alkyl H, 7.0 1H s C-3 H, 7.6-7.8 4H m Ar H, 8.0-8.1 2H m Ar H, 12.1 1H s OH. Mass (M+H): 443.31

4l- IR (KBr, cm^-1): 3415 O-H str, 3027 C-H str aromatic, 2930 C-H str aliphatic, 1679 C=O ketone, 1593 C=C str, 1388 C-H bending aliphatic, 1308 ether bond, 1204 OH bending, 1091 C-N vibration, 829 C-H bending aromatic, 601 C-Br str. ¹H NMR (δ): 2.4-2.5 6H alkyl H, 3.1 2H s alkyl H, 6.8 1H s C-3 H, 7.4-7.6 4H m Ar H, 7.8-8.0 2H m Ar H, 11.9 1H s OH. Mass (M+H): 403.25

Analgesic activity:

Analgesic activity was carried out by tail immersion method using albino rats. Here the tail of a rat is dipped up to a certain mark into the water maintained at a temperature of 55⁰C. Then the time taken for withdrawing their tail from hot water is considered as the time for creating analgesia. The animals were weighed and numbered. The basal reaction time was noted for all animals by observing the withdrawal time of the tail. The animals were divided into eight groups each consisting of six rats. To the first group injected DMSO (control), the second group with diclofenac (standard) and the other groups were injected with different derivatives of 4-benzopyrones at a dose of 68.75 mg/kg body weight. The reaction times of the animals in hot water were noted at 0, 15, 30, 60 and 120 minutes after the drug administration (Table- 2). As the reaction time increased with drugs 12 seconds was taken as cut off time.

Table-2. Analgesic activity of the synthesized compounds.

Compound code	0 min	15 min	30 min	60 min	120 min
Control	2.00 ± 0.204	2.05 ± 0.210	2.00 ± 0.210	2.1 ± 0.210	2.4 ± 0.408
Std.	2.14 ± 0.239	3.25 ± 0.144^***	4.87 ± 0.125^***	6.49 ± 0.125^***	6.6 ± 0.204^***
4a	2.01 ± 0.166	3.05 ± 0.288^***	4.61 ± 0.210^***	5.52 ± 0.288^***	5.93 ± 0.133^***
4b	1.78 ± 0.124	2.01 ± 0.166	3.21 ± 0.133^*	4.01 ± 0.125^**	4.52 ± 0.166^**
4c	2.16 ± 0.408	3.43 ± 0.108^***	4.85 ± 0.108^***	5.81 ± 0.210^***	5.93 ± 0.331^***
4d	1.75 ± 0.166	2.52 ± 0.288	3.31 ± 0.233^*	4.01 ± 0.133^*	4.81 ± 0.125^**
4e	2.22 ± 0.125	3.66 ± 0.166^***	4.15 ± 0.125^***	5.26 ± 0.133^***	5.72 ± 0.204^***
4f	2.01 ± 0.125	3.12 ± 0.144^*	3.83 ± 0.166^*	4.33 ± 0.133^*	4.92 ± 0.205^**
4g	2.11 ± 0.133	3.83 ± 0.125^***	4.12 ± 0.166^***	5.27 ± 0.114^***	5.83 ± 0.125^***
4h	2.01 ± 0.125	2.52 ± 0.133	2.94 ± 0.188	3.84 ± 0.210^**	4.91 ± 0.125^**
4i	2.01 ± 0.125	2.31 ± 0.166	2.95 ± 0.288	3.47 ± 0.204^*	4.01 ± 0.210^*
4j	2.01 ± 0.239	2.22 ± 0.125	3.21 ± 0.288	3.61 ± 0.204	4.21 ± 0.166^*
4k	1.95 ± 0.204	2.41 ± 0.144	3.30 ± 0.239	3.65 ± 0.166^*	4.13 ± 0.108^*
4l	1.88 ± 0.166	2.35 ± 0.125	3.24 ± 0.204	3.56 ± 0.288	4.01 ± 0.408^*

Statistical significance was analyzed by ANOVA followed by post-parametric Dunnet test. ***Extremely significant, **Highly significant, *Significant. n = 6

RESULTS AND DISCUSSION:

The starting material i.e. 2, 4-dihydroxybenzoyl-4-substitutedbenzoyl methane was conveniently prepared from the dibenzoyl ester of 2, 4-dihydroxy acetophenone. The title compounds (4a-l) were synthesized in a moderate to good yield after condensing bromo derivatives of substituted 4-benzopyrones (3a-l) with different amines. The structures of all the synthesized compounds were confirmed by their IR, NMR and Mass spectral analysis. The IR spectra (4a-l) showed the presence of a broad absorption band in the range of 3298 cm^-1 to 3418 cm^-1 which was confirmed to be due to -OH group. Further the appearance of a C-Br absorption band in the IR spectra of 3a-l confirmed the halogenations of 2a-l. The disappearance of the aforesaid peak and the appearance of C-N absorption band around 1060-1136 cm^-1 revealed the condensation of the suitable amines with 3a-l. The NMR spectra of the title compounds (4a-l) showed characteristic peaks for aromatic protons around δ 7-8. The appearance of sharp singlets at around δ 6.7-7.0 and 11.9-12.4 confirmed the presence of the C-3 proton and the –OH proton. Higher deshielding of these protons may be attributed to the fact that being electronegative oxygen atom deshielded the proton to a much greater extent thus appearing the signal in a downfield δ value whereas the deshielding of C-3 proton took place due to the presence of π electrons between C-2 and C-3. The protons associated with amine molecules appeared at a relatively higher δ value due to the presence of either oxygen or nitrogen or both in their structures, which are being electronegative, deshielded the protons to somewhat greater extent. The Mass spectra of the compounds gave the characteristic M+H peak from which the molecular weights of the compounds were confirmed. It was very interesting to note that some of the synthesized compounds showed significant activity when subjected for analgesic screening; compounds 4a, 4c, 4e and 4g showed very good analgesic activity. Other derivatives were weakly or moderately active.

CONCLUSION:

As the results of the analgesic screening revealed, substituted 4-benzopyrone derivatives condensed with different amines may hold the significance for future development, although thorough investigation regarding their ADME is required to optimize these compounds.

ACKNOWLEDGEMENT:

The author wish to thank the management of M.M.U College of Pharmacy, K.K. Doddi, Ramanagara- 562159, Karnataka for providing necessary facilities to carry out the research work.

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Received on 01.01.2015 Modified on 22.01.2015

Asian J. Research Chem 8(3): March 2015; Page 195-200

DOI: 10.5958/0974-4150.2015.00035.8