Synthesis, Characterization and Pharmacological Screening of Novel Substituted Isoxazole Derivatives

 

Sudheendra and Moid A.*

Department of Pharmaceutical Chemistry, Luqman College of Pharmacy, Gulbarga – 585102, Karnataka, India.

*Corresponding Author E-mail: moid_ans55@yahoomail.com

ABSTRACT:

As per our  previous paper published (AJRC vol. 04, issue 7, 2011), here some more substituted novel isoxazole derivatives were synthesized to enhance its biological activities, so we found that the ortho and meta position has shown highest activity than para, particularly Nitro substituted groups have shown good activity than others.  The present research work was aimed to synthesize some novel substituted isoxazole derivatives and screened them for anti-microbial and analgesic activities. The prevalence of isoxazole cores in natural and biologically active molecules has stimulated the need for elegant and efficient ways to make these heterocycles as lead. In this research work the 3-substituted N-(4′-hydroxyphenyl) propanamides (B1-B10) were prepared by the reaction of N-(4-hydroxy phenyl) acetamide (A) with various substituted aryl aldehydes by refluxing in ethanol. The resultant compounds on refluxing with hydroxylamine hydrochloride gave the title compounds 4-(5′-substituted-aryl-4′, 5′-dihydro-isoxazole-3′-yl-amino) phenols (C1-C10). The structure of newly synthesized compounds has been characterized and established by spectral data viz, FT-IR, ¹HNMR and Mass. All the derivatives synthesized were subjected for their anti-bacterial and antifungal activities by cup-plate method at the concentration level of 100 mg/ml and compared with the standard Amoxicillin and clotrimazole used. Some of the compounds were subjected for their possible analgesic activity by tail-flick method at the dose of 50 mg/kg and compared with that of standard paracetamol.

 

 

 

INTRODUCTION:

This compound was first obtained in 1903. The name isoxazole was introduced by Hantzsch¹ as a modification of the term monoazole coined earlier by Claisen² to differentiate from the known isomeric oxazole. The chemistry of isoxazole has been reviewed thoroughly from time to time^3-5. Isoxazoles are a class of heterocyclic compounds having a remarkable number of applications and have been demonstrated as highly versatile building blocks for pharmaceutically active compounds^6-7Isoxazole is a five membered heterocyclic ring system containing oxygen and nitrogen in the adjacent position. Isoxazole derivatives are known to passes anti-tubercular, analgesic⁸, anti-inflammatory⁹ and anti-microbial¹⁰ activities. It has been reported that isoxazolines also possess analgesic¹¹, anti-inflammatory¹² and antimicrobial^13,14 activities. Isoxazoles have also been used as dyes, electric insulating oils, high temperature lubricants etc. while polyisoxazoles have applications as semiconductors¹⁵.

 

In view of these above fact, an attempt has been made for the synthesis of novel 4-(5′-substituted-4′, 5′-dihydro-isoxazole-3′-yl-amino) phenols possessing potent biological activities. The synthesized compounds were tested for their possible anti-microbial and analgesic activities.

 

MATERIAL AND METHODS:

The entire chemicals used were procured from SDFCL Ltd, Himedia, central drug house (p) Ltd, and Loba-chemicals. Purity of starting materials used for reaction was confirmed by checking their melting point and by thin layer chromatography. All the reactions were monitored using thin layer chromatography. Melting points were determined in open capillary tube using precision melting point apparatus and were uncorrected. The FT-IR  spectrum of the synthesized compounds has been obtained from Karnataka University, University Science Instrument Centre, Dharwad. The IR spectra were carried out by Shimadzu Perkin Elmer 8201 PC IR Spectrometer using a thin film on potassium bromide pellets. The ¹HNMR of the selected compounds has been obtained from Karnataka University, University Science Instrument Centre, Dharwad in a mixture of CDCl₃. Chemical shift values are reported as values in δ ppm relative to TMS (d=0) as internal standard. The Mass spectrum of the selected synthesized compounds has been performed in Karnataka University, University Science Instrument Centre, Dharwad. The FAB mass spectra were recorded on JEOL SX-102/DA-6000 Mass Spectrometer using Argon/Xenon (6Kv, 10Ma) as the FAB gas. Purity of the compounds was checked on “Silica Gel G” coated on laboratory micro slides prepared by dipping method or precoated plates, eluent was the mixture of different polar and non-polar solvents in varying proportions and detection was done either by observing in UV (ultra-violet) light or exposure to iodine vapors as required. The absence of TLC spots for starting materials and appearance of new TLC spot at different R_f value ensured the completion of reaction.

 

EXPERIMENTAL:

Synthesis of N-(4′-hydroxy phenyl)-acetamide (A):

The N-(4-hydroxyphenyl) acetamide (A) is prepared by the condensation of p-amino phenol ( 1.09 gm, 0.01 mol ) with acetyl chloride (0.78 ml, 0.01 mol ) by refluxing for 20 min. The product obtained is isolated dried overnight, recrystallized with ethanol.

 

Synthesis of N-(4′-hydroxyphenyl)-3- substituted-propanamides (B₁-B₁₀):

To a mixture of equimolar quantities of N-(4′-hydroxy phenyl) acetamide (A) and aromatic aldehydes in ethanol (25 ml), 2 % NaOH solution (1 ml) was added and stirred for 10.00 hrs at room temperature. Then it was refluxed for 6.00 hrs on a water bath. The excess of solvent was removed under reduced pressure. It was poured into ice-cold water. The solid thus separated was filtered, dried and recrystallized from ethanol to obtain the compound in pure form.

 

Synthesis of 4-(5′-substituted-4′, 5′-dihydro-isoxazole-3′-yl-amino) (C₁-C₁₀):

To a mixture of N-(4′-hydroxyphenyl)-3-substituted-propanamides (C₁-C₁₀) (0.01mol) and hydroxylamine hydrochloride (0.69 gm, 0.01 mol) in ethanol (25ml) was refluxed on a water bath for 6.00 hrs. The reaction mixture was concentrated under reduced pressure. It was cooled and poured into ice-cold water. The solid separated was filtered, dried and recrystallized from ethanol. The compounds thus prepared are listed in Table-1. The purity of all the compounds was established by single spot on TLC plate using silica gel G. Solvent system used: Acetone: Benzene (1:1).

SCHEME

 

R	B1-B10	C1-C10
H	B1	C1
4-Cl	B2	C2
3-Br	B3	C3
2-OH	B4	C4
2-OCH3	B5	C5
3-CH3	B6	C6
3-OCH3	B7	C7
2-NO2	B8	C8
3- NO2	B9	C9
4-NO2	B10	C10

TABLE-1:      Physical data of various substituted isoxazole derivatives (C₁-C₁₀):

 

4-[(5-substituted-4, 5-dihydro isoxazol-3-yl) amino] phenol.

 

Comp. Code	-R	Mol. Formula	Mol. weight	Solubility	Rf value	MP (OC)	% Yield
C1	-H	C15H14N2O2	254.28	Ethanol	0.54	210	77%
C2	4-Cl	C15H13ClN2O2	288.72	Ethanol	0.67	130	55%
C3	3-Br	C15H13BrN2O2	333.17	Ethanol	0.61	135	84%
C4	2-OH	C15H14N2O3	270.28	Ethanol	0.29	140	59%
C5	2-OCH3	C16H16N2O3	284.30	Ethanol	0.47	150	72%
C6	3-CH3	C16H16N2O2	268.31	Ethanol	0.34	125	69%
C7	3-OCH3	C16H16N2O3	284.30	Ethanol	0.73	115	66%
C8	2-NO2	C15H13N3O4	299.28	Ethanol	0.58	110	60%
C9	3- NO2	C15H13N3O4	299.28	Ethanol	0.46	180	63%
C10	4- NO2	C15H13N3O4	299.28	Ethanol	0.41	155	52%

 

Table-2: Spectral data of some synthesized compounds

Comp.	-R	IR (KBr, Cm-1)	1HNMR (CDCl3, δ ppm,)	Mass values
C1	H	3326(Ar-OH str.), 3163(NH str.), 3036 (C-H str.), 1655(C=N str.), 1260(C-O str.)	1.31(1H, s, CH), 2.19 (1H, s, CH2), 4.21 (1H, s, Ar-OH), 7.28(1H, s, N-H), 7.71-9.38 (m, 8H, Ar-H).	(m/z): 254 (M+)
C2	4-Cl	3420(Ar-OH str.), 3131(NH str.), 3007 (C-H str.), 2692(CH2), 1620(C=N str.), 790 (C-Cl str.).	2.22 (1H, s, CH2), 4.23 (1H, s, Ar-OH), 7.25(1H, s, N-H), 7.33(1H, d, CH) 7.40-8.62(m, 7H, Ar-H).	(m/z): 288 (M+)
C3	3-Br	3301(Ar-OH str.), 3051(NH str.), 2923 (C-H str.), 2752(CH2), 1615(C=N str.), 1210(C-O str.) 969 (C-Br str.)	2.32 (1H, s, CH2), 5.31 (1H, s, Ar-OH), 7.26(1H, s, N-H), 7.31(1H, d, CH) 7.41-8.90(m, 8H, Ar-H).	(m/z): 233 (M+)
C4	2-OH	3290(Ar-OH str.), 2955(NH str.), 2833 (C-H str.), 2853(CH2), 1617(C=N str.), 1508(C=C), 1259(C-O str.)	4.24 (2H, d, CH2), 5.31 (1H, s, Ar-OH), 6.22 (1H, s, N-H), 6.55 (1H, t, CH) 7.38-8.23(m, 8H, Ar-H).	(m/z): 270 (M+)
C5	2-OCH3	3330(Ar-OH str.), 3261(NH str.), 3057 (C-H str.), 2843(CH2), 1604(C=N str.), 1420(C=C), 1255(C-O str.) 1001 (C-O-C str.)	3.43 (3H, s, OCH3), 4.28 (2H, d, CH2), 5.21 (1H, s, Ar-OH), 6.26 (1H, s, N-H), 6.78 (1H, t, CH) 7.10-8.21(m, 8H, Ar-H).	(m/z): 284 (M+)
C8	2-NO2	3321(Ar-OH str.), 3251(NH str.), 3091 (C-H str.), 2804 (CH2), 1594(C=N str.), 1493(C- NO2 str.), 1399 (C=C str.) 1210(C-O str.)	6.28 (3H, s, NH), 4.02-4.15 (2H, d, CH2), 5.34 (1H, s, Ar-OH), 6.71 (1H, t, CH), 6.78 (1H, t, CH) 7.21-8.23(m, 8H, Ar-H).	(m/z): 299 (M+)

 

 

BIOLOGICAL EVALUATION:

Antimicrobial Screening¹⁰:

All the synthesized compounds were screened for their antimicrobial activity at the concentration level of 100 mg/ml using cup and plate method against bacterial strains Bacillus subtilis, Salmonella typhy, Escherichia coli, Staphylococcus aureus and fungal strains Candida albicans and Aspragellus fulvous. The antibacterial activities of the compounds were compared with that of standard drug Amoxicillin, where as the antifungal activities of the test compounds were compared with that of standard drug Clotrimazole.

 

Analgesic activity¹⁶:

The selected compounds among the synthesized were screened for analgesic activity by tail flick method. The compounds were administered at a dose of 50 mg/kg. Dose of The drug were fixed by toxicity study (OECD GUIDELINE- 420). Tail flick method¹⁶ was followed for the evaluation of analgesic activity using instrument analgesiometer (Inco, India). Swiss albino rats of either sex weighing between 180-250 g were randomly distributed into four groups consisting of six animals in each group. The first group served as a control group and animals ware administered with vehicle (Tween-80, 1%) orally. The second group was administered with standard drug paracetamol at a dose of 20 mg/kg body weight, orally. The animals of the other group ware treated with isoxazole derivatives at a dose of 50 mg/kg body weight, orally. The reaction time was noted at 30, 60, 90, 120 and 180 min. of time intervals. The cut off time for the reaction was 10 seconds.

Animals were randomly divided into six groups, each group containing 6 animals as follows:

1.      Group I: Served as control (Distilled water) p.o.

2.      Group II: Standard group received paracetamol (20mg/kg. p.o)

3.      Group III-VI: The animals of group 3-6 are administered with a suspention of test compounds in gum acasia (50 mg / kg p.o.)

 

RESULTS AND DISCUSSION:

The Physical and Spectral data of various substituted isoxazole derivatives  were reported in Table- 1 and 2. Biological results were reported in Tables 3 and 4, which also records the effects of standard drugs included for comparison. Series of compounds were prepared in this study, exhibited significant pharmacological properties in different biological models. The general patterns of pharmacological activity encountered with these synthesized compounds were seen mainly for their effect on pain perception. However, there was a moderate, well defined antimicrobial activity associated with many of these compounds. Considerable variation of these effects were seen with each structural changes, varying from agents that had less activity to those with high potency, and significant changes in potency resulted even from minor change in chemical structure as shown in Table 3 and 4.

 

ANTIMICROBIAL ACTIVITY:

During the synthesis of isoxazole derivatives various substituted aldehydes were used hence substitutions are changed only in the phenyl moiety of aldehydes. Among the various substituent’s particularly C₈, C₉ and C₁₀ substituted aldehyde moiety has shown highest activity and C₁, C₄, C₅ and C₇ has shown good activity against the organisms B. Subtilis, S. typhy (Gram +ve) and E.Coli, S. Aureus (Gram –ve) at the concentration lavel of 100 µg/ml compared with that of standard Amoxicillin taken, while the compounds C₂, C₃ and C₆ containing phenyl group were found to be moderately active against these organisms in comparison with the value of the standard.

 

 In case of fungus C. Albicans and A.Flavous the compounds such as C₁, C₅, C₇, C₈, C₉ and C₁₀ have shown good activity, while in case of compounds C₂, C₃, C₄ and C₆ the activity was moderate when compared with that of standard clotrimazole. Results obtained are presented in Table-3.

 

ANALGESIC ACTIVITY:

Tail flick method was followed for the evaluation of analgesic activity using instrument analgesiometer (Inco, India). The reaction time was noted at 30, 60, 90, 120 and 180 min. of time intervals. The cut off time for the reaction was 10 seconds. The compounds C₄, C₅ and C₇ among the synthesized compounds showed significant increase in reaction time while C₈ has shown moderate activity when compared to standard drug paracetamol. The results are presented in Table-4. The data were analyzed by ANOVA followed by Student‘t’ test and the level of significance was set at P<0.001.

 

TABLE-3: Antimicrobial activity of substituted isoxazole derivatives (C₁-C₁₀).

SI. No.	Compound code	Conc. (µg/ml)	Diameter of zone inhibition (mm)
			B.Subtilis	S.typhy	E.coli	S.aureus	C.albicans	A.flavous
1	C1	100	20	22	22	23	19	22
2	C2	100	18	19	20	20	21	19
3	C3	100	19	21	18	16	19	20
4	C4	100	20	21	20	23	20	21
5	C5	100	20	19	20	21	19	23
6	C6	100	17	15	20	18	20	22
7	C7	100	19	20	21	19	22	23
8	C8	100	21	23	25	22	20	24
9	C9	100	20	23	24	21	20	23
10	C10	100	19	24	22	21	23	26
11	Amoxicillin	100	23	25	27	25	-	-
12	Clotrimazole	100	-	-	-	-	25	28
13	Control	-	-	-	-	-	-	-

 

Table-4: Analgesic activity results of some synthesized isoxazole derivatives.

Group	Dose	% increase in reaction time (in sec.)
-	(mg/kg)	30 min.	1 hour	2 hour	3 hour
Control	Tween 80, 1%	3.433±0.166	3.567±0.272	3.400±0.288	3.400±0.300
Standard (paracetamol)	20	4.367±0.328	5.067±0.491	6.267±0.375	8.600±0.264**
C4	50	5.167±0.218**	6.233±0.352**	7.033±0.145***	8.133±0.578***
C5	50	4.533±0.166*	5.367±0.290*	6.400±0.360***	7.733±0.318***
C7	50	5.267±0.228**	6.313±0.413**	6.977±0.121***	7.433±0.423***
C8	50	4.167±0.142*	5.424±0.325*	5.345±0.367***	6.233±0.234**

Results are expressed in mean ± SEM (n=6) significance levels * P<0.05, ^** P < 0.01 and ^*** P < 0.001 as compared with the respective control and standard

 

Analgesic effect of substituted isoxazole derivatives by tail-flick method

 

CONCLUSION:

The purpose of the present study was to examine whether molecular modification might result in detection of new potential antimicrobial and analgesic drugs. A series of compounds were prepared and assayed in a variety of biological tests for analgesic and antimicrobial activities. All the Synthesized compounds were tested for antibacterial and antifungal activity followed by cup-plate method. Among those, compounds C₁, C₄, C₅, C₇, C₈, C₉ and C₁₀ have shown good anti-bacterial and anti-fungal activity where as compounds C₂, C₃ and C₆ have shown moderate activity on tested organisms when compared with standard drugs ciprofloxacin (antbacterial) and clotrimazole (antifungal) respectively.

 

Among the synthesized isoxazoles the compounds C₄, C₅ and C₇ were selected for analgesic activity which shown significant increase in reaction time, while the compound C₈ has shown moderate activity when compared to control as well as standard paracetamol followed by tail-flick method at the dose of 50 mg/kg. The data reported in Tables 3 and 4 showed that effect of variation in chemical structure on activity was rather unpredictable. Seldom had a particular structural modification led to uniform alteration in activity in all tests. However, some points of interest did emerge and a few generalizations can be made. The substitution which appeared to be most important for high order of activity in the greatest number of test was the ortho and meta group’s particularly Nitro substituent’s has shown highest activity than others. The substitution at 5 position of isoxazole ring resulted in compounds with potent antimicrobial and analgesic activities. Obviously, the comparative evaluation of the active compounds will require further studies. The data reported in this work may be helpful in guiding the medicinal chemists working in the area.

 

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Received on 26.09.2011         Modified on 16.10.2011

Accepted on 27.10.2011         © AJRC All right reserved