Synthesis, characterization & biological evaluation of 1, 3, 4- oxadiazoles as antioxidant agents

Rahul. R¹, Rakesh Kumar Jat¹, J. Saravanan²

1. Introduction

Among the five membered aromatic compounds which are hetrocyclic, 1,3,4- Oxadiazoles are critical compounds. There are four oxadiazoles that exist in different isomeric forms such as 1,2,4, 1,2,5, 1,2,3 and 1,3,4-oxadiazoles. Out of this 1,3,4-oxadiazole is widely being exhibit diverse biological activities [1] like antibacterial [2], antitubercular [3], vasodilatory [4], antifungal [5], antiinflammatory [6], anticonvulsant [7], cytotoxic [8], anaesthetic [9], analgesic [10], hypolipidimic [11], anticancer [12], © antioxidant [13], and ulcerogenic [14] activities. 1,3,4-oxadiazole derivatives and their mannich bases were reported to possess anticancer and antioxidant activity. Furthermore, certain thiazole derivatives are well known for their Due to presence of an extra heteroatom there is inductive effect which makes oxadiazole a weak base. There are two pyridine like nitrogen (-N=) present in oxadiazole. The conjugated diene character in oxadiazole is mainly due to reduction in aromaticity of oxadiazole ring. Replacement of hydrogen atom by nucleophiles has been seen in nucleophilic substituted oxadiazoles[15].

Free radicals are not stable, short lived and very reactive due to presence of odd number of electrons. So to gain stability it captures the odd electrons so that it reacts with other compounds free radicals generally capture the electrons of the nearest stable molecule. The living cells are disrupted by the chain reaction, that is, the attacked molecule lose it’s electrons and become a free radical which attack the living cell. Nitrogen compounds, phenolic compounds are categorized as synthetic antioxidants. The sterically hindered phenols and secondary aromatic amines form the primary antioxidants compounds. The hydrogen atom migrate from the antioxidant molecule to radical intermediate is the first step of the radical termination [16].

Free radical scavenging is one of the best known mechanisms by which antioxidant inhibit lipid oxidation. The in vitro antioxidant activity can be performed by three methods, DPPH assay, Nitric oxide scavenging assay and Hydrogen peroxide radical scavenging assay. Some new 2- amino methyl thiazole derivatives (Figure 1) were prepared [17] and posses

In view of the above studies, we considered it of interest to synthesize some new derivatives by incorporating thiazole moiety with mannich bases of 1,3,4-oxadiazole analogues and investigated their antioxidant property.

2. Materials and Methods

Chemistry

Synthetic Procedure

Step-1: Synthesis of P-Chloro Phenacyl Bromide. (1)

To a cold solution of p-chloroacetophenones (0.01mol) in chloroform (25ml), bromine (0.012mol) in chloroform (10ml) was gradually added for about 30 minutes with continuous stirring were the 0 temperature maintained is 0-5 C. The mixture of reaction was brought to the room temperature after the addition was complete. For another 60 minutes the mixture was stirred till hydrogen gas evolution ceases. Under reduced pressure the solvent was eliminated and recrystallized the residue from ethanol to afford to get pure p-chlorophencylbromide [18].

Step-2: Synthesis of 4-(4′-Chloro- phenacyl)-1,3-Thiazole-2-Carboxylate.

(2) In a round bottom flask a mixture of ethyl thiooxamate (1 equivalent weight), pchloro phencylbromide (1.1 equivalent weight) and ethanol 10-15ml were taken, and refluxed for 2 hr, the ethanol was distilled off under vacuum and it was neutralized with sodium bicarbonate.

Ethyl acetate is used to extract the mixture, and then washed with water. Under vacuum the solvent was eliminated. Crude product obtained was recrystallized from ethanol [l9].

different amines

Scheme of synthesis:

Cl Br ,CHCl 2 COCH 3 Cl NH NH .H 2 2 N CONHNH² C H OH 2 5 S (3) CS KOH 2 Cl N N NH HCHO O S S (4) R¹ = -NH(C⁶H⁵) , -N(C⁶H⁵)² , -N(C²H⁵)

Step-3: Synthesis of 4-(4′-Chloro Phenacyl)-1,3-Thiazole-2-Carbo hydrazide. (3)

Compound-3 was prepared by refluxing 4- (4′-chlorophenacyl)-1,3-thiazole-2- carboxylate (0.015 mol) with hydrazine hydrate (1.6 mL) in ethanol (20 mL) for 5 h. After cooling the mixture the product obtained was recrystallized from DMF: ethanol mixture (6:1) [20, 21].

Step-4: Synthesis of 5-[4-(4′-Chloro Phenacyl)-1,3-Thiazole-2yl] -1,3,4- Oxadiazole-2-Thione. (4)

Potassium hydroxide (0.04mol), ethanol (25ml), 4-(4′-chlorophenyl) thiazole-2- Cl 3 COCH Br 2 (1) S C H OH ^{2 5} H N 2 COOC H 2 5 Cl O 2 N COOC H 2 5 S (2) Cl R N ¹ N N O S S 5(a-e) ' N O ² , -N(CH³)^2, carbohydrazide (0.02mol) were mixed together. With stirring carbon disulfide (14ml) was added. For 8 hours the reaction mixture was heated. Under reduced pressure solvent was eliminated. The residue was filtered after washing with water. Filtrate was cooled and using dilute hydrochloric acid it is neutralized to pH 6. The final product obtained was filtered, washed with water, dried and recrystallized using ethanol [20, 21].

Step-5: Synthesis of 5-[4-(4′-Chloro Phenacyl)-1,3-Thiazole-2yl]-3-(2-

Methyl Substituted) -1,3,4-Oxadiazole- 2-Thione. (5a-e)

Formaldehyde 40% (0.003mol) was added to a solution of 5-[4-(4′- chlorophenacyl)-1,3-thiazole-2yl]-3-(2- methyl substituted) -1,3,4-oxadiazole-2- thione (0.003 mol) in absolute ethanol (10ml). An ethanolic solution (2ml) of the different primary or secondary amine (0.003 mol) was added portion wise to the above reaction mixture, stirred for 3 hours at room temperature, and left overnight in a refrigerator. The precipitate formed was filtered, dried, and crystallized from DMF: Water (5:2) [21].

Pharmacological Study Results

Antioxidant activity results: DPPH assay (2, 2-diphenyl -1- picrylhydrazyl)

The DPPH assay was used to study radical scavenging activity of test samples according to Scherer and Godoy [22]. The measurement is taken at 517 nm. After the addition of an antioxidant the decrease in the absorption of the DPPH solution was taken. Standard taken is Ascorbic acid.

Procedure

Using 2,2-diphenyl-1-picrylhydrazyl radical(DPPH) antioxidant potency of the test samples and standard were evaluated. 3.9mL of DPPH in methanol solution (0.2mM) is added to methanol solutions of samples or standards (0.1ml) in concentrations of (20, 40, 60, 80, 100 microgram/ml). 0.1mL of methanol added to 3.9 mL of DPPH solution is the control. Triplicate of the procedure were done. In the dark, 90-min incubation is done at room temperature, then absorbance was measured at 517 nm. Without test compound, but an equivalent amount of methanol taken is the control [22].

Ascorbic acid was used as standard. The results obtained from DPPH assay is shown in table 2. IC⁵⁰ values for standard and test samples were determined using ED50 plus V 1.0 software. Percentage inhibition is calculated using the formula; % scavenging activity =

Nitric Oxide Scavenging Activity

Nitric oxide scavenging activity can be estimated by the use of Griessre action (Garrat, 1964). The compound sodium nitroprusside produce NO by decomposing in aqueous solution at pH 7.2. In aerobic condition, NO reacts with oxygen to produce stable products (nitrate and nitrite). The quantities of which can be determined using Griess reagent. The completion between scavengers of nitric oxide and oxygen leads to decrease in production of nitrite ions.

Procedure

Griess reaction is used to measured Nitric oxide scavenging activity, spectrophotometrically. Different concentration of test -1 sample (20, 40, 60, 80, 100 μg mL ) in methanol is mixed with sodium nitro -1 prusside (5mmolL ) in phosphate buffered saline (pH-7.4) and incubated for 30 minutes at 25°C. Instead of test sample equivalent amount of methanol is taken and used as control. 1.5mL of Griess reagent is used to dilute 1.5mL of the incubated solution after 30 minutes. Absorbance was evaluated at 517nm with reference to standard and percentagescavenging property was evaluated [23]. Ascorbic acid was used as standard. The results obtained from nitric oxide scavenging assay is shown in table 3. IC⁵⁰ values for standard and test samples were determined using ED50 plus V 1.0 software. Percentage inhibition is calculated using the formula;

Melting Point

% scavenging activity =

Hydrogen Peroxide Radical Scavenging Assay

By the action of oxidase enzymes, in vivo hydrogen peroxide is generated. Through the reduction product called hydroxyl radical (OH•) hydrogen peroxide is scavenged.

Here hydrogen peroxide is scavenged by the test sample, which is the basis of this method. Using phosphate buffer solution (pH 7.4), hydrogen peroxide solution (2mmol/l) was prepared. To hydrogen peroxide solution (0.6 ml) test samples (20, 40, 60, 80, 100 μg/ml) were added.

After 10 min absorbance of hydrogen peroxide was evaluated against blank at 230 nm which containing phosphate buffer without hydrogen peroxide. The reference compound is ascorbic acid, which is used for comparison [24].

Ascorbic acid was used as standard. The results obtained from hydrogen peroxide assay is shown in table 4. IC⁵⁰ values for standard and test samples were determined using ED50 plus V 1.0

Molecular Formula

0 0 5a C¹⁸H¹³ClN⁴OS² 97 C -99 0 5b C H ClN OS 197 C -199 24 17 4 2 0 5c C¹⁶H¹⁷ClN⁴OS² 105 C -108 0 0 5d C H ClN OS 86 C -90 14 13 4 2 0 5e C¹⁶H¹⁵ClN⁴O²S² 124 C -126 software. Percentage inhibition is calculated using the formula; % scavenging activity = Data are the mean of three or more experiments and reported as mean ± standard error of the mean (SEM)

3. Result and Discussion

Physical data: Physical data like Molecular formula, Melting point, Solubility and Rf value were evaluated for the mannich derivatives and is shown in table 1.

Spectral Analysis:

1 The spectral analysis like UV, IR, H NMR and Mass were done to identify the structure of the mannich derivatives. The instrument model for UV spectra is SHIMADZU UV-2600. IR spectra were analyzed using BRUKER VERTEX FT-IR spectrophotometer. BRUKER 400Mhz is 1 used to obtain HNMR. MASS spectra were obtained using the instrument, LCMS THERMO LCQ DECA XP.

Solubility % yield R value

f C Ethanol 59% 0.69 &DMSO 0 C Ethanol 49.5% 0.75 &DMSO 0 C Ethanol 44% 0.81 &DMSO C Ethanol 53% 0.65 &DMSO 0 C Ethanol 51% 0.89 &DMSO

Spectral data of compound 5a

a Cl c j h N f l N N b d HN k O S g e S i -1 IR Spectra (cm ): 3286.64(- NH structure); 3983.76 (Ar- CH); 1648.10(HC=N); 1627.6 6 (-NHbent);1499.83(Ar C=C);1260.12(C=S); 1218.18(C-O-C);1178.08(C-N);733.74(C-Cl); 814.20(C-S).

1

NMR Spectra: H NMR, δ(ppm)(DMSO):

7.22(d, 2H, Ar CH at a,b); 7.39(d, 2H, Ar CH at c, d); 8.11(s, 1H, CH of thiazole at e); 5.11(s, 2H, CH at f); 3.81(s, 1H, NH at g ); 2 6.51(d,2H, Ar CH at h,i); 6.90 (t, 2H, Ar CH at j,k); 6.71(t, 1H, Ar CH at l); solvent peaks at 2.49 and 3.31.

UV-λmax (nm): 347.

Mass spectra: Molecular weight=400

Spectral data of compound 5b

-1 IR SPECTRA (cm ): 3079.82(Ar-CH str); 2918.98(Ali- CH); 1588.12 & 1508(Ar C=C); 1658.46(C=N); 1221.34(C-O-C); 1266.12 (C=S); 828.40(C -S); 778.75(C-Cl).

a Cl c i g N f k b N N d N j m O S e S h o l p n 1

NMR Spectra: H NMR, δ(ppm)(DMSO):

7.29(d, 2H, Ar CH at a, b); 7.38(d, 2H, Ar CH at c, d); 8.12(s, 1H, CH of thiazole at e); 5.17(s, 2H, CH² at f); 7.11(d, 4H, Ar CH at l, g, m, h); 6.91(t, 4H, Ar CH at n, o, j, i); 6.82(t, 2H, Ar CH at p, k). Solvent peaks at 2.49 and 3.31.

UV-λmax (nm): 397.

Mass spectra: Molecular weight=477

Spectral data of compound 5c

a Cl c N f N N b g d N i O S e S CH 3 CH h ³ J -1 IR Spectra (cm ): 3065.79(Ar-CH structure); 2916.36(Ali- CH); 1524.65 & 1584.40(Ar C=C); 1679.33(C=N); 1270.18(C=S); 1207.15(C-O-C); 751.89(C- S); 721.65(C-Cl).

1

NMR Spectra: H NMR, δ(ppm) (DMSO):

7.32(d, 2H, Ar CH at a, b); 7.12(d, 2H, Ar CH at c, d); 6.90(s, 1H, CH of thiazole at e); 5.47(s, 2H, CH at f); 2.95(m, 4H, CH -N- 2 2 CH² at g, h); 1.11(t, 6H, CH³ at i, j) solvent peaks at 2.49 and 3.31.

UV-λmax (nm): 270 Mass spectra: Molecular weight=380

Spectral data of compound 5d

a Cl c N f N N b g d N CH 3 O S e S CH h ³ -1 IR Spectra (cm ): 3076.74(Ar-CH str); 2 926(Ali- CH); 1516.24 & 1581.11(Ar C=C); 1648.25(C=N); 1160.27(C-O-C); 1251.70 (C=S); 1040(C-N); 754.08(C-S); 695.15(C- Cl).

1

NMR Spectra: H NMR, δ(ppm) (DMSO):

7.31(d, 2H, Ar CH at a, b); 7.22(d, 2H, Ar CH

values in μg/ml for samples were determined using ED50 plus V 1.0 software. Data are the mean of three or more experiments and reported as mean ± standard error of the mean(SEM).

values in μg/ml for samples were determined using ED50 plus V 1.0 software. Data are the mean of three or more experiments and reported as mean ± standard error of the mean(SEM).

at c, d); 6.89(s, 1H, CH of thiazole at e); 5.46(s, 2H, CH at f); 2.01(s, 6H, CH -N-CH 2 3 3 at g, h); solvent peaks at 2.49 and 3.31.

UV-λmax (nm): 266 Mass spectra: Molecular weight=352

C Spectral data of compound 5e

a Cl c N f N N b d N g h O S e S i j O -1 IR spectra (cm ): 3052.46(Ar-CH str); 2900.76(Ali- CH); 1513.60 & 1600.48(Ar C=C); 1122.24(C-N); 1228.20(C-O-C); Standard drug used is ascorbic acid. IC⁵⁰ Standard drug used is ascorbic acid. IC⁵⁰ 1663.48(C=N); 1270.14(C =S); 817.26(C-S); 764.88(C-Cl).

1 NMR Spectra: H NMR,δ(ppm)(DMSO): 8.24(d, 2H, Ar CH at a, b); 7.70(d, 2H, ArCH at c, d); 6.91(s, 1H, CH of thiazole at e); 5.49(s, 2H, CH at f); 2.16(t, 4H, CH -N-CH 2 2 2 at g, h); 3.71(t, 4H, CH²-O-CH² at i, j) solvent peaks at 2.49 and 3.31.

UV-λmax (nm): 281 Mass spectra: Molecular weight=394

Antioxidant activity results:

The derivatives prepared were evaluated for antioxidant activity by three methods like DPPH assay, Nitric oxide scavenging assay and Hydrogen peroxide scavenging assay. The compounds were tested in five different concentrations.

values in μg/ml for samples were determined using ED50 plus V 1.0 software.Data are the mean of three or more experiments and reported as mean ± standard error of the mean(SEM) Standard drug used is ascorbic acid. IC 50

Conclusion

The physical and spectral analysis of the mannich derivatives were carried out. In vitro antioxidant activity was carried out by three methods. In DPPH assay compound 5c with IC⁵⁰ value of 26.7μg/ml showed significant activity when compared to ascorbic acid with IC⁵⁰ 21.3μg/ml. In Nitric oxide and Hydrogen peroxide scavenging methods compound 5c showed maximum activity when compared to other derivatives but was not significant to the result obtained in DPPH method. It is well established that organic molecules incorporating an electron donating group can act as free radical trapping agents and are capable of opposing oxidative challenges. Compound 5c showed maximum antioxidant activity in all the three methods. This may be due to the presence of chloro substitution at 4ˈ position of phenyl ring and the diethyl group attached to amine. Both chloro and diethyl group are electron releasing which are important in radical scavenging activity.

References

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