Synthesis and Characterization of Indolyl-pyrazoline and their Evaluation of anti-inflammatory Activity

 

Minakshi Shroff1, Dr. S. J. Daharwal2, Yashwant Swarnakar3

1Deptt. of Pharmaceutical Sciences, Uttarakhand Technical University, Deheradun. (U.K.)

2University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (C.G.)

3Shri Shankaracharya Institute of  Pharmaceutical Sciences, Bhilai (C.G.)

*Corresponding Author E-mail: mahishroff@gmail.com

 

ABSTRACT:

In the present work, ten novel 3-(4,5-dihydro-3-(naphthalen-1-yl)- N1-substituted -pyrazol-5-yl)-1H-indole [1,1a,1b.1e,2] and 3-(4,5-dihydro-3-(pyridin-3-yl)-N1-substituted-pyrazol-5-yl)-1H-indole [5,5a,5b,5e.6] were synthesized by condensation between substituted pyrazoline and substituted hydrazine in the presence of glacial acetic acid. The synthesized compounds were evaluated for their anti-inflammatory activity using carrageenan-induced paw oedema method and compared to the well known NSAID indomethacin as a standard drug. The entire synthesized compounds were characterized by elemental analysis, IR, 1H NMR and mass spectroscopy.

 

KEYWORDS:  Anti-inflammatory activity, Carageenan induced paw oedema method, indole, pyrazoline, indolyl pyrazoline.

 

 


1. INTRODUCTION:

Encouraged by the diverse biological activities of pyrazoline compounds, we had decided to prepare a new series of 2- pyrazoline derivatives, having different kinds of heterocyclic moieties fused on it. 2-pyrazoline is proved to be a ‘Multiactivity Ring’. This will provide us good series of pharmacologically active indole and pyridine fused to pyrazoline. NSAIDs is continue to be a group of choice for researchers due to its wide pharmacological use and greater life expectancy ,it is not surprising that the development of newer NSAIDs continue to be in rapid pace.

 

In the view of the various activities reported for the compound possessing pyrazoline moiety. In the present study we attempted to synthesize pyrazoline along with other heterocyclic rings such as indole, naphthyl and pyridinyl, fused to form pharmacologically active derivatives, to improve the activities.

 

Chalcones were synthesized by reacting aryl aldehyde with aryl ketone by applying claisen schimdt method1. The derivatives were confirmed by determining melting point, TLC, Elemental analysis and by performing spectral studies.

 

The aforementioned numerous pharmacological activities of 2-pyrazoline prompted us to study the in vivo anti inflammatory activities of some important indolyl pyrazoline with different aromatic groups.

 

Graphical abstract:

Groups

Intermediates

Series of synthesized derivatives

If  R=

 

C1

Series 1

1,1a,1b,1e,2

 

If R=

 

C2

Series 5

5,5a,5b,5e,6

 

2. RESULT AND DISCUSSION:

The preparation of the chalcones is based on the claisen schimdt synthesis which was the first step compound of pyrazoline synthesis. The substituted 2- pyrazoline is synthesized by reaction of chalcones with hydrazine or its derivatives in different basic medium. We had taken here to simply synthesize 2-pyrazoline derivative with two more heterocyclic moieties to be attached and therefore to enhance the anti inflammatory effect. The series 1 consist of 2-pyrazoline with naphthyl and indolyl, and the series 5 consist of 2-pyrazoline with pyridinyl and indolyl moieties incorporated.

 

All the compounds simply gone through physical and spectral characterization. The IR consist of the pyrazoline ring closure characteristic to  be exhibited in IR spectra,1 series consist  v (C=N) stretching at range of 1571-1520 cm-1;v(C-N) st ranges 1361.5-1330.6 cm-1;and v(N-H) st at 3200-3130 cm-1. Indolyl group exhibits range at 3452.9-3358.43 cm-1.The series 5, contributed IR  spectra of pyrazoline ring closure ranges  at  v (C=N)st 1570-1560 cm-1; v(C-N) st 1377.22-1338.64 cm-1; v(N-H) st 1624.12-1666.55 cm-1and also indole ranges v(N-H) st 3290.67-3521.17 cm-1.The NMR (CDCl3) indolyl group at all the derivatives shows sharp doublet peak of single proton at the range δ 11.62-11.28ppm and rest of all proton peaks were showing chemical shifts at characteristic signals  satisfactory to the structures of the derivatives. . The mass spectra (EI-MS) of compounds are also in agreement with their molecular formula. The elemental analysis results were within ± 0.4% of the theoretical values.    

The pharmacological activities of both the series were showing average to good inhibitory percentage comparing with the standard indomethacin. As evaluated by Carrageenan induced rat paw oedema method. The volume was measured after 1, 2, 3, 4 and 5 hours of activity .All the synthesized compound were tested and showed inhibition of edema ranging from 25.9 to 62.04 %. However the inhibitory effects of these compounds were unpredictable during 3rd and 4th hour of measurement, but reached significant values after 5 hours; 1a showed best activity 62.04% compared to the standard drug. 5a, 5b, and 1a shows activity more than 50% and some equivalent to the standard drug indomethacin. The pyrazoline had associated two more types of heteroatoms in it, naphthyl and indolyl in 1 series and pyrazoline with pyridinyl and indolyl at 5 series.

 

3. EXPERIMENTAL:

3.1 Animals

All the heteroatoms were prone to anti-inflammatory activity if seen in the literature.  Pyrazoline having substituting phenyl ring at N1 were in general less active than substituted ones, indicating that the presence of more hetreoatoms with pyrazoline may be causing stearic hinderance in orienting the molecule to the active site, hence giving poor to average activities. But if the N1 substituted with small group like methoxy are more active than chloride substituted derivatives and also non-substituted ones. Compound 6, with phenyl substituted N1 showed least activity in both the series. Also 1e, 2, and 5e exhibited poor inhibition to oedema at 5th hour. Conclusion drawn that compounds attached with isonicotinyl moiety at N1, were not singnificant in anti inflammatory activity in comparison to the methoxy substituted N1 of pyrazolines derivatives. Therefore it deserves further attention in order to develop new leads. The anti-inflammatory activity of newly synthesized compounds was carried out on albino rat(150-200 g)  of either sex. These animals were reared with robust health by providing pellet diet and water ad libitum in the animal house under standard environment conditions of temperature, relative humidity and dark/light cycle. The rats were acclimatized for one week before initiation of experiment and its randomization into various groups. The animal experiments were previously approved by CPCSEA.

 

3.2 Material:

All the reagents and solvent used in synthesis were of laboratory grade. The solvent and reagent are of AR grade mainly. The commercially available grades of solvents and reagents are found to be of adequate purity. Melting points (m.p.) were determined in open capillary electronic apparatus and are uncorrected. The silica gel G (60-120 mesh) used for analytical chromatography (TLC) was obtained from Himedia The chemicals used for the experimental work were commercially produced from various chemicals units like s.d. fines, Himedia and Loba chemicals.

 

3.3 Scheme of synthesis:

The synthesis of the title compounds of indolyl and pyridinyl are outlined in fig.1 the required indolylaldehyde were prepared by following Vielsmeier Haack complex method.2

 

3.3.1 Synthesis of chalcones3-5 :

The synthesis of both the series of derivatives follows same kind of scheme for synthesis of their respective chalcones.  Equimolar quantities (0.01 mol) of indole-3-aldehyde  and 2-acetyl naphthelane or 2 –acetyl pyridine were taken in 100 ml conical flask and dissolved in 20 ml of ethanol to this (0.03 mol) of NaOH in minimum quantity of water was added. The mixture was stirred on a magnetic stirrer and the reaction was monitored with TLC. Reaction mixture was diluted with water and acidified with concentrated hydrochloric acid. The precipitated chalcones was filtered and recrystallized from absolute ethanol. The purity of chalcones was tested with thin layer chromatography using solvent system: petroleum ether (60-80º): ethyl acetate [70:30], colored shaded solid was obtained. 

 

3.3.2 Synthesis of pyrazoline derivatives6-8:

The solution of appropriate chalcones (0.03mol) and hydrazine derivatives (0.03 mol) was taken in mixture of ethanol (20 mL) and glacial acetic acid (60 mL) then refluxed for 4hr and kept overnight. The product was poured into ice water and the crude product was filtered and crystallized with Acetone and in some cases mixture of solvents needed like as acetone and ethanol in different ratios. Elution with solvent system ethyl acetate/petroleum ether (60-80º)] gave pure compound as yellowish shaded solid.


 

Fig 1: Scheme of reaction

 


The spectral analysis of synthesized compounds is as follows:

Compound (1): 3-(4,5-dihydro-3-(naphthalen-1-yl)-1H-pyrazol-5-yl)-1H-indole; color-light brown mp.214oC; yield-43%; IR (KBr,v,cm-1); pyrazoline ring clousure,1570 (C=N),1360.6 (C-N); 3130 (N-H)st, 1620 (N-H);indole  3452.9(N-H); 1H NMR(CDCl3) – δ indolyl N-H exhibits 11.4(1H,d); C-H exhibits 7.47-7.58(4H,t); 7.60-7.67(4H,d) and7.01-7.03(2H,d); δ of Pyrazolyl exhibits – δ 2.19-2.5(C-H,3H,t); 3.82(C-H,4H,d); 7.71(N-H,2H,d); Naphthyl, 7.85-7.90 (C-H, 4H, d); 7.92-7.96 (C-H,4H,d); 9.79 (C-H,1H,s); 8.13-8.20 (C-H,2H,d); 8.38-8.50 (C-H,2H,m); MS:m/z 309.4(M+); Anal. calcd for C21H17N3;C, 81.00; H, 5.50; N, 13.50.

 

Compound (2): 3-(4,5-dihydro-3-(naphthalen-1-yl)-1-phenyl-1H-pyrazol-5-yl)-1H-indole;color- muddy brown ;mp 190oC ;yield- 48%; IR (KBr,v,cm-1); pyrazoline ring clousure, 1551.45(C=N); 1346.07(C-N); 1659.45(N-H) st; 3159.79 (N-H); Indole:3358.43(N-H).1H NMR(CDCl3) – δ indolyl exhibits 11.28 (N-H,1H,d); 7.47-7.49 (C-H,4H,t); 7.60-7.67 (C-H,4H,d); 7.01-7.03(C-H,2H,d); Pyrazolyl: 2.49-2.53(C-H,3H,t); 1.93-1.94 (C-H,4H,d); Naphthyl : 7.94-7.85(C-H,4H,d); 7.90-7.96(C-H,4H,d); 8.13-8.20 (C-H,2H,d); 8.38-8.50 (C-H,3H,t); 9.72 (C-H,2H,d): MS:m/z 387.17(M+); Anal. calcd for C27H21N3 ; C, 83.69; H, 5.46; N, 10.90.

 

Compound (1a): (4,5-dihydro-5-(1H-indol-3-yl)-3-(naphthalen-1-yl)pyrazol-1-yl)ethanone; color-yellowish brown; mp- 193oC; yield-55%; IR (KBr,v,cm-1); pyrazoline ring clousure, 1560(C=N); 1361.5(C-N); 1600.63(N-H) st; 3200 (N-H); Indole: 3450 (N-H); Methoxy: 1750(C-H); 1023.05,2838.7(C=O). 1H NMR(CDCl3)  – δ indolyl  exhibits 11.38(N-H,1H,d); 7.58-7.67(C-H,4H,t); 7.47-7.49(C-H,4H,d); 7.01-7.3(C-H,2H,d); Pyrazolyl : 2.19-2.21(C-H,3H,d); 4.4941(C-H,4H,d); methoxy : 2.49(C-H,3H,s); Naphthyl: 7.85-7.94 (C-H,4H,d); 8.13-8.20 (C-H,4H,d); 8.44-8.50(C-H,2H,d); 8.44-8.50 (C-H,2H,d); 7.92-7.96 (C-H,3H,t); 9.79(C-H,2H,d): MS :m/z  351.3(M+); Anal.calcd C, 78.16; H, 5.42; N, 11.80; O, 4.53 for C23H19N3O.

 

Compound (1b): 4, 5-dihydro-5-(1H-indol-3-yl)-3-(naphthalen-1-yl) pyrazole-1-carbonyl chloride;color- light brown; mp-220oC ;yield-55%; IR (KBr,v,cm-1); pyrazoline ring clousure ; 1520(C=N); 1336.43(C-N); 1604.4(N-H)st; 3248.5(N-H); Indole: 3450; Acylhalide:1780(C-Cl). 1H NMR(CDCl3)  – δ indolyl  exhibits 11.500 (N-H,1H,d); 7.6-7.61(CH,4H,t); 7.56-7.58(CH,4H,d); 7.15-7.17(CH,2H,d); Pyrazolyl : 1.92-1.85(CH,3H,t); 4.63(CH,4H,d); Naphthy: 7.30-7.66(CH,4H,d); 7.89-7.91(CH,4H,d); 8.47-8.45 (CH,2H,d); 7.604-7.618(CH,3H,t); 9.66(CH,2H,d);MS :m/z 373.01(M+) ;Anal.calcd C, 70.68; H, 4.31; Cl, 9.48; N, 11.24; O, 4.28 for C22H16ClN3O.

 

Compound (1e): (4,5-dihydro-5-(1H-indol-3-yl)-3-(naphthalen-1-yl)pyrazol-1-yl)(pyridin-4-yl)methanone. Color-brownish yellow m p-187oC; yield-55%; IR (KBr,v,cm-1); pyrazoline ring clousure ;1571(C=N);1330.6(C-N); 3192.5 (N-H) st; 1628.8 (N-H); Indole:3450; Isonicotinyl aro.unsat. aldehyde: 1728.8(C=O). 1H NMR(CDCl3)  – δ indolyl  exhibits 11.40 (NH,1H,d); 7.37-7.41(CH,4H,t); 7.26(CH,4H,d); 6.80(CH,2H,d); Pyrazolyl: 1.28,1.58(CH,3H,t); 5.20 (CH,4H,d); Isonicotinyl : 7.951(CH,4H,d); 9.30(CH,4H,d); Naphthyl: 7.41-7.54(CH,4H,d); 7.50-7.54(C H,4H,d); 7.67(CH,2H,d); 7.65-7.66(CH,3H,t); 7.95(CH,2H,d) ;Anal.calcd for C27H20N4O is C, 77.87; H, 4.84; N, 13.60; O, 3.84.

 

Compound (5): 3-(4, 5-dihydro-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-1H-indole. Color –Brown; mp- 154oC; yield -91%; IR (KBr,v,cm-1); pyrazoline ring clousure; 1570(C=N); 1338.64(C-N); 1629.9(N-H); 3290.67(N-H) st; Indole: 3385.18(N-H) st; pyridine ring: 3037.9(C-H st); 1458.2( skeletal stretching). 1H NMR(CDCl3)  – δ indolyl  exhibits 11.21 (NH,1H,d); 7.58-7.60(CH,4H,t); 7.47-7.49(CH,4H,d); 6.47(CH,2H,d); Pyrazolyl 3.82(CH,3H,t); 2.1991, 2.49(CH,4H,d); 7.01-7.03(NH,2H,d); Pyridinyl: 8.44-8.38(CH,2H,d); 7.56-7.96 (CH,2H,t); 7.56-7.96(CH,2H,t); 7.67-7.71(CH,2Ht); 8.48-8.50(CH,2H,d). Anal.calcd for C16H14N4 is C, 73.26; H, 5.38; N, 21.36.

 

Compound (6): 3-(4,5-dihydro-1-phenyl-3-(pyridin-2-yl)-1H-pyrazol-5-yl)-1H-indole. Color –dark brown;mp- 210oC; yield -48%; IR (KBr,v,cm-1); pyrazoline ring clousure; 1563.36(C=N); 1377.22(C-N); 1666.55(N-H); 3445.94(N-H )st; Indole: 3521.17(N-H); Pyridine ring: 3070.78(C-H); 1463.06(Skeletal ring). 1H NMR (CDCl3) – δ indolyl exhibits -Indolyl: 11.30(NH,1H, d); 7. 31-7.33(CH, 4H, t);7. 33-7. 46 (CH,4H,d); 6.67-6.69(CH,2H,d); Pyrazolyl: 2.20, 2.24(CH,3H,t); 3.91(CH,4H,d); Phenyl : 6.67-6.71(CH,3H,d); 7.25-7.31(CH,5H,m); 7.013-7.0121(CH,3H,t);Pyridinyl : 8.33(CH,2H,d); 7.86-7.88(CH,2H,t); 7.464 -7. 746(CH,2H,t); 8. 347-8.355(CH,2H,d); MS :m/z  338.13(M+);). Anal.calcd for C20H18N4 is C, 78.08; H, 5.36; N, 16.56.

 

Compound (5a):  (4,5-dihydro-5-(1H-indol-3-yl)-3-(pyridin-2-yl)pyrazol-1-yl)ethanone. Color –Light brown; mp- 132oC;  yield-54%;   IR (KBr,v,cm-1); pyrazoline ring clousure;  1567.33(C=N); 1355.04(C-N); 1624.12(N-H )st;  3201.94(N-H);  Indole: 3450(N-H); Pyridine ring : 3003.27(C-H); 1455.34(Skeletal streching);  Methoxy group:1778.43(C-H); 2848.96(C=O).1H NMR (CDCl3)  – δ indolyl exhibits -Indolyl: 11.45(NH,1H,d); 7.457.58(CH,4H,t); 7.417.56(CH,4H,d); 6.80(CH,2H,d); Pyrazolyl:1.43, 1.450(C-H,3H,t); 4.50(C-H,4H,d); Methoxy:2.50(C-H,3H,s); Pyridinyl-8.76(CH,2H,d); 8.02-8.06(CH,2H,t); 7.60-7.68(CH,2H,t); 8.82(CH,2H,d). Ana.calcd for C18H16N4O is C, 71.04; H, 5.30; N, 18.41; O, 5.26.

 

Compound 5(b): 4,5-dihydro-5-(1H-indol-3-yl)-3-(pyridin-2-yl)pyrazole-1-carbonyl chloride. Color-light brown, yield -48%; mp- 165oC;yield-54%; IR (KBr,v,cm-1); pyrazoline ring clousure;1560(C=N); 1338.6(C-N); 1629.90(N-H)st; 3290.6(N-H); Indole: 3385.1(N-H) 2oamine; Pyridine ring: 3037.9, 1458.23 (C-H)skeletal stretching;  Isonicotinyl : 1845.9(C-H). 1H NMR (CDCl3)  – δ indolyl exhibits-Indolyl: 11.4(NH,1H,d); 7.39-7.41(CH,4H,t); 6.95-6.98(CH,4H,d); 6.98(CH,2H,d); Pyrazolyl : 2.49-2.50(CH,3H,t); 4.50(CH,3H,d); Pyridinyl: 8.00-8.02(CH,2H,d); 7.54-7.58(CH,2H,t); 8.77(CH,2H,d); Analytical calculations for C17H13ClN4O is C, 62.87; H, 4.03; Cl, 10.92; N, 17.25; O, 4.93.

 

Compound 5(e): (4,5-dihydro-5-(1H-indol-3-yl)-3-(pyridin-2-yl)pyrazol-1-yl)(pyridin-4-yl)methanone. color- dark brown; yield -59%; mp-210oC; IR (KBr,v,cm-1); pyrazoline ring clousure: 1560(C=N); 1338.64(C-N); 1629.9(N-H) st; 3290.67(N-H); Pyridine ring: 3037.9,1458.23(ring stretching ); Isonicotinyl: 1845.9(C-H);Indole :3385.18(N-H ) 2oamine. 1H NMR (CDCl3)  – δ indolyl exhibits- Indolyl:11.624(NH,1H,d); 7.03-7.47(CH,4H,t); 7.49-7.58(CH,4H,d); 7.011(CH,2H,d). Pyrazolyl: 2.19-2.21(CH,3H,t); 4.494(CH,3H,d); Isonicotinyl:7.85-7.96(CH,4H,d); 8.48-8.50(CH,4H,d); Pyridinyl:8.20-8.38(CH,2H,d); 8.13-8.20(CH,2H,t); 7.62-7.71(CH,2H,t); 8.42-8.44(CH,2H,d). Anal calcd for C22H17N5O is C, 71.92; H, 4.66; N, 19.06; O, 4.35.

 

3.4 Anti inflammatory activity screening9-10:

In vivo anti inflammatory activity was evaluated using Carrageenan induced rat paw edema model of inflammation by adopting the method of Winter et al. for the compounds listed in table:  The albino rats (150 -200 mg)of either sex were fasted with free access of water atleast 12 hour prior to experiments and were divided randomly into  12 groups of six each. Control group I received 1% sodium CMC gel (1 ml/kg) whereas group II received indomethacin (100 mg/kg). All these doses were administered orally to the test groups of synthesized compounds. The rats were dosed orally, 1h later; a sub-planter injection of 0.05ml of 1% solution of Carrageenan in sterile distilled water was administered to the left hind footpad of each animal. The paw oedema volume was measured with a digital plethysgmometer at 1, 2, 3, 4, 5 hours after Carrageenan injection. Paw edema volume was compared with vehicle control group and percentage inhibition was calculated by the following formula-

 

% Inhibition of oedema = V c -V t/V c×100

 

Where, Vc and Vt are the volume of oedema for the control and drug-treated animal groups, respectively.


 

Table 1: Paw oedema volume and %inhibition of synthesized compound against Carrageenan induced paw edema method.

Compound

3hr

% inhibition

4hr

% inhibition

5hr

% inhibition

Control

0.66

0

0.86

0

0.92

0

1

0.33±0.23

50.00

0.35±0.36

59.61

0.46±0.32

49.39

1a

0.43±0.18

35.00

0.38±0.25

55.76

0.35±0.27

62.04

1b

0.53±0.18

20.00

0.51±0.21

40.38

0.43±0.23

53.01

1e

0.58±0.18

12.50

0.56±0.20

34.61

0.68±0.23

25.90

2

0.45±0.19

32.50

0.5±0.19

42.30

0.63±0.22

31.32

5

0.43±0.16

35.00

0.4±0.19

53.84

0.46±0.21

49.39

5a

0.38±0.20

42.50

0.45±0.25

48.07

0.53±0.25

53.01

5b

0.41±0.17

37.50

0.5±0.25

42.30

0.48±0.31

56.62

5e

0.36±0.17

45.00

0.46±0.22

46.15

0.53±0.23

42.16

6

0.43±0.16

35.00

0.55±0.21

36.53

0.65±0.22

29.51

Standard

0.3±0.15

55.00

0.35±0.19

59.61

0.41±0.20

55.41

 

 

Figure 2: Effect of compounds against Carrageenan- induced hind paw oedema model.

 

Figure 3: % Inhibition of compounds against carrageeenan induced paw oedema at 5th hour of induction.

 

Figure 4: Comparative anti inflammatory profile of % inhibition of synthesized 1 series and 5 series of compounds.

 


ACKNOWLEDGEMENTS:

The authors are thankful to Dr. Amit Roy, Principal, Dr. Ram Sahu, HOD, Pharmacology, Columbia College of Pharmacy, Raipur (Chhattisgarh) for providing laboratory facilities to conduct pharmacological activities.

 

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Received on 02.08.2016             Modified on 21.12.2016

Accepted on 30.01.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(3): 677-682.

DOI: 10.5958/0974-360X.2017.00126.3