Synthesis, and in vitro antioxidant activity of novel 1, 3, 4- oxadiazole-2-thione

Rahul. R¹, Rakesh Kumar Jat¹, J. Saravanan²

1. Introduction

The living cells are disrupting by the chain reaction, that is, the attacked molecules lose its electrons and become a free radical which attacks the living cell.

Natural and synthetic are the two basic types of antioxidants structures. Various alkyl substitutions containing phenolic group are in general natural antioxidants.

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 moves from the antioxidant molecule to radical intermediate is the first step of the radical termination [1].

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.

Among the five membered aromatic compounds which are hetrocyclic1,3,4- Oxadiazoles are critical compounds. 1,3,4- oxadiazole is widely being exhibit diverse biological activities[2] like antibacterial [3], antitubercular [4], vasodilatory [5], antifungal [6], anti-inflammatory [7], anticonvulsant [8], cytotoxic [9], anaesthetic [10], analgesic [11], hypolipidimic [12], anticancer [13], antioxidant [14], and ulcerogenic [15] 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 antioxidant. With the aim of finding a promising antioxidant agents, which may have improved efficacy and fewer side effects compared with existing antioxidants, we considered it of interest to synthesize some new derivatives by incorporating thiazole moiety with mannich bases of 1,3,4-oxadiazole analogues to investigate their antioxidant property.

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. Since the electron density on the carbon atom is less electrophilic substitution at same carbon atom in the oxadiazole ring is less content but the electron releasing groups in oxadiazole can undergo association. Replacement of hydrogen atom by nucleophiles has been seen in nucleophilic substituted oxadiazoles [16].

2. Materials and Methods

Chemistry

Synthetic Procedure

Synthesis of P-Chloro Phenacyl Bromide. (A)

To a cold solution of p-nitro acetophenone (0.01 mol) in chloroform (25ml), bromine (0.012 mol) in chloroform (10ml) was gradually added for about 30minutes with continuous stirring were the temperature 0 maintained is 0-5 C. The mixture of reaction was brought to the room temperature after the addition was complete. For another 60minutes the mixture was stirred and the stirring till hydrogen gas evolution ceases. Under reduced pressure the solvent was eliminated and recrystallized the residue from ethanol to afford to get pure pchlorophencylbromide [17].

Synthesis of 4-(4′-Chlorophenacyl)-1,3-

Thiazole-2-Carboxylate. (B)

In a round bottom flask a mixture of ethyl thiooxamate (1 equivalent weight), pnitrophencylbromide (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 [18].

Synthesis of 4-(4′-Chloro Phenacyl)-1,3-

Thiazole-2-Carbohydrazide. (C)

Compound-3 was prepared by refluxing 4- (4′-nitrophenacyl)-1,3-thiazole-2- carboxylate (0.015 mol) with hydrazine hydrate (1.6mL) in ethanol (20mL) for 5 h. After cooling the mixture the product obtained was recrystallized from DMF: ethanol mixture (6:1) [19, 20].

Scheme of synthesis:

O N 2 COCH 3 S Br ,CHCl 2 3 H N 2 COOC O N 2 C H 2 COCH Br 2 (A) O N 2 N N NH O S S (D) HCHO different amines O N 2 R N ¹ N N O S S E(a-e) R = -NH(C H ) , -N(C H ) , -N(C H ) 1 6 5 6 5 2 2 5

Synthesis of 5-[4-(4′-Chloro Phenacyl)- 1,3-Thiazole-2yl]-3-(2-Methyl Substituted) -1,3,4-oxadiazole-2- thione. E(a-e)

Formaldehyde 40% (0.003mol) was added to a solution of 5-[4-(4′- nitrophenacyl)-1,3-thiazole-2yl]-3-(2- methyl substituted) -1,3,4-oxadiazole-2- thione (0.003mol) in absolute ethanol (10ml). An ethanolic solution (2ml) of the different primary or secondary amine (0.003 mol) was added portion wise to the O N 2 H 2 5 N OH COOC H 5 2 5 S (B) NH NH .H O C H OH ^{2 2 2} 2 5 O N 2 KOH N CONHNH CS ² 2 S (C) ' N , -N(CH ) O 2 3 2, 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) [20].

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 [21]. 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 (0.2 mM) is added to methanol solutions of samples or standards (0.1ml) in concentrations of (20, 40, 60, 80, 100 μg/ml). 0.1mL of methanol added to 3.9mL 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 [21].

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

Griessre action 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 -1 nitroprusside (5mmol L ) 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 [22]. 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; % 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

Melting Point

buffer without hydrogen peroxide. The reference compound is ascorbic acid, which is used for comparison [23].

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 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 R f 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 1 spectral analysis like UV, IR, H NMR and Mass were done to identify the structure of

Molecular Formula

0 Ea C¹⁹H¹⁴N⁵O³S² 169 C -172 0 Eb C²⁴H¹⁷N⁵O³S² 189 C-1 0 Ec C¹⁶H¹⁷N⁵O³S² 184 C -186 0 Ed C¹⁴H¹³N⁵O³S² 151 C -154 0 Ee C¹⁶H¹⁵N⁵O⁴S² 195 C -198 the mannich derivatives. The instrument model for UV spectra is SHIMADZU UV- 2600. IR spectra were analyzed using BRUKER VERTEX FT-IR spectrophotometer.

1 BRUKER 400Mhz is used to obtain HNMR. MASS spectra were obtained using the instrument, LCMS THERMO LCQ DECA XP.

Compound Ea

a O N 2 c j h N f l b N N d HN k O S S g e i -1 IR Spectra (cm ): 3166.00(-NHstr); 3083.32(Ar- CH); 1648.10(HC=N); 1627.6 6(-NHbent); 1499.83(Ar C=C); 1545.70(C- NO ); 1260.12(C=S); 1218.18(C-O-C); 2 1178.08(C-N); 733.74(C-S).

1 NMR Spectra: H NMR, δ(ppm)(DMSO): 8.10(d, 2H, Ar CH at a,b); 7.71(d, 2H, Ar CH at c,d); 6.91(s, 1H, CH of thiazole at e); 5.50(s, 2H, CH² at f); 3.90(s, 1H, NH at g ); 6.80(d,2H, Ar CH at h,i); 7.10(t, 2H, Ar CH at j,k); 6.41(t, 1H, Ar CH at l); solvent peaks at 2.49 and 3.31. UV-λmax (nm):361.

Solubility % yield R value

f 0 C Ethanol 59% 0.55 &DMSO 0 91 C Ethanol 61% 0.60 &DMSO 0 C Ethanol 48% 0.67 &DMSO 0 C Ethanol 52% 0.57 &DMSO 0 C Ethanol 57.5% 0.51 &DMSO

Compound Eb

a O N 2 c i g f N k b N N d N j m O S e S h o p l n -1 IR Spectra (cm ): 3106.93 (Ar-CH str); 1522.48 (Ar C=C); 1683.88 (HC=N); 758.39 (C-S); 1439.40 (NO²); 1165.12(C-O-C).

1 NMR Spectra: H NMR, δ(ppm)(DMSO): 8.30(d, 2H, Ar CH at a,b); 8.01(d, 2H, Ar CH at c,d); 7.10(s, 1H, CH of thiazole at e); 4.10(s, 2H, CH at f); 7.31(d, 4H, Ar CH at 2 l,g,m,h); 6.51(t, 4H, Ar CH at n,o,j,i); 6.91(t, 2H, Ar CH at p,k). Solvent peaks at 2.49 and 3.31. UV-λmax (nm):392.

Compound Ec

a O N 2 c N f N N b g d N i O S e S CH 3 CH h ³ J -1 IR Spectra (cm ): 3100.33 (Ar-CH str); 2924.62 (Ali-CH); 1542.16 (Ar C=C); 1686.88 (HC=N); 839.81 (C-N); 788.12 (C- S); 1445.13 (NO²); 1169.23(C-O-C).

1 NMR Spectra: H NMR, δ(ppm)(DMSO): 8.21(d, 2H, Ar CH at a,b); 7.15(d, 2H, Ar CH at c,d); 6.90(s, 1H, CH of thiazole at e); 5.46(s, 2H, CH² at f); 2.94(m, 4H, CH²-N-CH² at g,h); 1.12(t, 6H, CH³ at i,j) solvent peaks at 2.49 and 3.31. UV-λmax (nm):279.

Compound Ed

a O N 2 c N f N N b g d N CH 3 O S e S CH h ³ -1 IR Spectra (cm ): 3109.91(Ar-CH); 2998.31(Ali-CH); 1528.18(HC=N); 1528.18 (ArC=C); 1440.22(C-NO ); 1269.10(C=S); 2 1178.10(C-O-C); 819.09(C-N); 754.30(C-S).

1 NMR Spectra: HNMR, δ(ppm)(DMSO): 8.29(d, 2H, Ar CH at a,b); 7.92(d, 2H, Ar CH at c,d); 6.89(s, 1H, CH of thiazole at e); 5.45(s, 2H, CH² at f); 2.02(s, 6H, CH³-N- CH at g,h); solvent peaks at 2.49 and 3.31.

3 UV-λmax (nm): 267. Mass spectra: Molecular weight=363.

Compound Ee

a O N 2 c N f N N b d N g h O S e S i j O -1 IR spectra(cm ): 3086.77(Ar-CH); 2929.96 (Ali- CH); 1623.43 (HC=N); 1536.24 (ArC=C); 1451.70(C-NO²); 1270.17(C=S); 1245.24(C-O-C); 1170.27(C-N); 785.77(C- S).

1 NMR Spectra: H NMR, δ(ppm)(DMSO): 8.25(d, 2H, Ar CH at a,b); 7.71(d, 2H, Ar CH at c,d); 6.90(s, 1H, CH of thiazole at e); 5.49(s, 2H, CH² at f); 2.15(t, 4H, CH²-N-CH² at g,h); 3.72(t, 4H, CH -O-CH at i,j) solvent 2 2 peaks at 2.49 and 3.31.UV-λmax (nm):243.

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).

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).

Standard drug used is ascorbic acid. IC⁵⁰ Standard drug used is ascorbic acid. IC 50 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 Ec with IC⁵⁰ value of 26.7 μg/ml showed significant activity when compared to ascorbic acid with IC⁵⁰ 21.3μg/ml. In Nitric oxide and Hydrogen peroxide scavenging methods compound Ec 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 Ec 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.

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