Design, Synthesis Of Some Novel Oxadiazole Based Derivative And Evaluation For Their In-Vitro Anti-Oxidant Activity

Pharmaceutical chemistry is a science that makes use of the general laws of chemistry to study drugs, i.e. their proportion, chemical nature, composition, influence on an organism and studies of the physical and chemical proportion of drugs, the methods of quality control and the conditions of their usages. In other words, it is the chemistry of drugs. Drugs mainly exert action depending upon the biochemical pathway [1]. Pharmaceutical chemistry is a specialized science which depends on the other disciplines such as inorganic, organic, analytical, physical and colloid chemistry and also on medico-biological disciplines such as pharmacology, physiology, biological chemistry etc [2]. Pharmaceutical chemistry occupies the most important place among the related sciences such as drug technology, toxicology chemistry, pharmacognosy, the economy and the organization of pharmacy. The very breadth of knowledge required by a medicinal chemist is both a challenge and a reward. Mastering and understanding of such a breadth of subject areas is no straight forward task, but by the same token there is ample intellectual stimulation in understanding the battle against disease at the molecular level and in designing molecular ?soldiers? to win the battle [3,4]. Molecular biology and genetic engineering have produced a deluge of potential new target for drug design and have unraveled the structures and mechanisms of traditional targets while advances in computers and computer aided design have allowed medicinal chemists to take full advantage of this newly earned knowledge. Chemical modification of drug molecules to locate the number of series having optimal effects and will probably continue to be the factor necessary, to make new drugs. To establish the structure of the drug molecules the new invention in the physico-chemical directions such as X-ray crystallography different types of chromatography, spectroscopic studies like NMR, IR, Mass, U.V immensely helpful for medicinal chemist. The advances in the molecular biology, computer science, instrumentation technology gave an revolutionary turn to concept of chemotherapy leading to development or other area of drug design, QSAR studies etc [5-7].

 Oxadiazole

1,3,4-Oxadiazole is thermally stable neutral aromatic molecule. Out of its four possible isomers 1-4, 1,3,4-oxadiazole (1) is widely exploited for various applications. Other aromatic related systems are 1,3,4-oxadiazolines, 1,3,4-oxadiazolium cations [8], and the exocyclic-conjugated mesoionic 1,3,4-oxadiazole [9]. Oxadiazole moiety and its various derivatives studied frequently in the past few decades and found potent in various pharmacological and pathological conditions.[10] Among different five members heterocyclic systems pyrrole, oxadiazole, thiadiazole, triazole and their derivatives have gained importance as they constitute the structural features of many bioactive compounds. Among them 1,3,4-oxadiazoles are of significant interest in medicinal chemistry. Literature reveals that 1,3,4-oxadiazole is a highly privileged structure and its derivatives exhibit a wide range of biological activities including antibacterial, anti-tubercular, vasodialatory, antifungal, cytotoxic, anti-inflammatory and analgesic, hypolipidemic, anti-cancer and ulcerogenic activities. Furamizole is a compound which is based upon 1,3,4- oxidiazole ring and has strong antibacterial activity [11,12]. Antioxidants are the molecules that prevent cellular damage caused by oxidation of other molecules. Oxidation is a c hemical reaction that transfers electrons from one molecule to an oxidizing agent. Oxidation reactions are known to produce free radicals. These free radicals are highly reactive species which contains one or more unpaired electrons in their outermost shell. Once they are formed, the chain reaction starts. Antioxidant reacts with these free radicals and terminates this chain reaction by removing free radical intermediates and inhibits other oxidation reactions by oxidizing themselves. Though oxidation reactions are crucial for life, they can also be damaging. Plants and animals have a complex system of multiple types of antioxidants, such as vitamin C and vitamin E, as well as enzymes, such as catalase (CAT), superoxide dismutase (SOD), and various peroxidases [13]. Oxidative stress plays a key role in causing various human diseases, such as cellular necrosis, cardiovascular disease, cancer, neurological disorder, Parkinson’s dementia, Alzheimer’s disease, inflammatory disease, muscular dystrophy, liver disorder, and even aging [14]. Besides, there are some antioxidants in the form of micronutrients which cannot be manufactured by the body itself such as vitamin E, ?-carotene, and vitamin C, and hence these must be supplemented in the normal diet [15]. Antioxidants can also act as prooxidants when these are not present at the right place at the right concentration at the right time [16]. The relative importance of the antioxidant and prooxidant activities is not yet explored fully and needs further research.

Classification of Antioxidants

Antioxidants can be classified into two major types based on their source, i.e., natural and synthetic antioxidants (schematic representation of the classification of antioxidants is shown in Figure 1.

Natural Antioxidants

Natural antioxidants either are synthesized in human body through metabolic process or are supplemented from other natural sources, and their activity very much depends upon their physical and chemical properties and mechanism of action [17]. This can be further divided into two categories, i.e., enzymatic antioxidants and nonenzymatic antioxidants.

Enzymatic Antioxidants

Enzymatic antioxidants are uniquely produced in the human body and can be subdivided into primary and secondar

Primary Antioxidants

Primary antioxidants mainly include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) as described below. Superoxide Dismutase Superoxide dismutase (SOD) enzyme is found in both the dermis and the epidermis. It removes the superoxide radical (O2.?) and repairs the body cells damaged by free radical. SOD catalyzes the reduction of superoxide anions to hydrogen peroxide [18]. SOD is also known to compete with nitric oxide (NO) for superoxide anion, which inactivates NO to form peroxynitrite. Therefore, by scavenging superoxide anions, it promotes the activity of NO [19].

Nonenzymatic Antioxidants

They are a class of the antioxidants which are not found in the body naturally but are required to be supplemented for the proper metabolism [20]. Some of the known nonenzymatic antioxidants are minerals, vitamins, carotenoids, polyphenols, and other antioxidants.

Need of antioxidants

Oxidation reaction depending upon site of occurrences presents specific repercussions. If the site of occurrence is food system, then food deteriorates. When oxidation occurs in biological cell system, it causes damage or death to the cell. The oxidative deterioration of fats and oils, when present as a component in foods, is responsible for rancid odor and flavor with a consequent decrease in nutritional quality, sensory appeal and safety. This is caused by the formation of primary hydroperoxides and secondary potentially toxic compounds through auto-oxidation of unsaturated fatty acids consisting of a free radical chain mechanism [21]. The direct oxidation of unsaturated lipids with the double bond in a singlet state (no unpaired electrons, paired electrons are in the same orbital and have opposite spin) by oxygen in its ground triplet state (two free electrons in separate orbitals with same spin direction) is spin forbidden. To overcome this spin barrier, initiators or catalysts are required to start the lipid oxidation process by removing an electron from either the lipid or oxygen or by changing the electron spin of the oxygen [22]. As only trace amounts of catalysts are needed, many situations that appear to be spontaneous or uncatalyzed are actually driven by contaminants or conditions that have gone undetected or unconsidered. Indeed, in most foods, biological systems, and laboratory experiments, it is fair to say that multiple catalysts and initiators are always operative. Thus, exposure of lipids to initiators like light, metals, singlet oxygen and sensitizers (chlorophyll, hemoproteins, and riboflavin), or preformed hydroperoxide decomposition products causes generation of primary hydroperoxides. Lipoxygenase- catalyzed oxidation also produces hydroperoxides [23]. The addition of antioxidants is required to control the oxidative deterioration. In human body, about 5% of the inhaled oxygen is converted into reactive oxygen species which encompasses the hydroxyl radical, the superoxide anion radical, hydrogen peroxide, singlet oxygen, nitric oxide radical, hypochlorite radical, and various lipid peroxides. All are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes, and other small molecules, resulting in cellular damage. As a defense mechanism against reactive oxygen species, addition of antioxidants is required to food system. In human body, however, a variety of components, both endogenous (body’s immune system) and exogenous in origin, Function interactively and synergistically. As part of a healthy lifestyle and a well- balanced, wholesome diet, antioxidant supplementation is now being recognized as an important means of improving free radical protection [24].

Antioxidant in Human Health

Antioxidants in human health include:

• Nutrient-derived antioxidants like ascorbic acid (vitamin C), tocopherols and tocotrienols (vitamin E), carotenoids, and other low molecular weight compounds such as glutathione and lipoic acid.
• Antioxidant enzymes, e.g., superoxide dismutase, glutathione peroxidase, and glutathione reductase, which catalyze free radical quenching reactions.
• Metal binding proteins, such as ferritin, lactoferrin, albumin, and ceruloplasmin that sequester free iron and copper ions that are capable of catalyzing oxidative reactions.
• Numerous other antioxidant phytonutrients present in a wide variety of plant foods. For instance, oligomeric proanthocyanidins present in grape seed extracts evidenced by clinical trials revealed fifty times more potential than vitamin C in regards with bioavailability and tissue repair mechanism [25].

Toxicological Aspects

In limelight, antioxidants are popular for suppressing oxidative stress and its related diseases, however, abusive and careless application may result in toxicological effects. Researchers have marked the delusive safety of natural antioxidants with risk of toxicity and array of side effects [26]. Conceptually, every antioxidant acts as pro- oxidant after certain concentration. To assure the function of antioxidant, it is necessary to study the effect of the antioxidant with respect to dosage, pro-oxidant action, side effects, bioavailability, and interaction with other nutrients. Different studies have depicted pros and cons of antioxidants. Jakeman and Maxwell found that vitamin C supplementation prior to the exercise resulted in a faster recovery of muscle strength, however, Urso and Clarkson [27] reported that antioxidant supplements could have a negative effect on recovery from muscle damaging exercise. The pro-oxidant effects of vitamins C and E have also been reported [28]. As pro- oxidants these vitamins creates transition metal ions. The pro-oxidant effects of vitamin E supplements have been reported to cause fatal myocardial infarctions [29, 30] and inhibit glutathione S-transferase P 1-1 (GST P 1-1) [31]. Moreover, high levels of vitamin E have been reported to exacerbate impaired blood coagulation [32]. Bast and Haenen [33] reported some toxic metabolites of vitamin E like quinines which are highly cytotoxic. However, another metabolite 2,7,8-trimethyl-2-(?-carboxyethyl)-6-hydroxychroman has been reported to possess a strong nutraceutical effect [28,34]. ?-Tocopherol is an effective inhibitor of the cyclooxygenase enzyme (COX-1) [28] which is associated with significant damage to the gastrointestinal tract. ?-Carotene has been reported to act as an antioxidant at low oxidative stress, whereas under high stress conditions it stimulates lipid peroxidation [34]. Also, the unstable oxidised metabolites of ?-carotene facilitate carcinogenesis. Similar to vitamin C and E, dihydrolipoic acid, a metabolic product of lipoic acid, can also function as a pro-oxidant [35]. Caffeic acid, a widely used antioxidant, may also act as a pro-oxidant under thermal treatment. In fact, highly reactive cations were generated during the early phases of caffeic acid degradation, affecting both the oxidative status and the reaction pathway of the system [36]. The fact list of repercussions related to abusive use of antioxidants relay the need of detailed toxicological studies, and specific standards to rule out toxicological effects. It is necessary to enlighten the general public regarding toxicological as well as beneficial effects of antioxidant in a balanced manner.

 AIM & OBJECTIVE

Aim

To design, synthesis of a new substituted oxadiazole derivatives and evaluation for their anti-oxidant activity

Objectives

• The designed compounds were synthesized in the laboratory by the conventional methods.
• Synthesized compounds were characterized by IR, 1HNMR, 13C NMR, LC-MS.
• Molecular docking studies
• In vitro anti-oxidant studies

 EXPERIMENTAL SECTION

MATERIALS AND METHODS

All the chemicals were purchased from Nice chemicals. Analytical TLC was performed on Precoated sheets of silica gel G/UV-254 of 0.2mm thickness (Macherey- Nagel, Germany) using analytical grade solvent and visualized with iodine spray (10% w/w I2 in silica gel) or UV light.

Equipment and analytical instrument

Melting point was determined in capillary tubes and is uncorrected. IR spectra were taken as KBr pellets for solids on Perkin Elmer Spectrum FT-IR. 1H NMR (400MHz) and 13C NMR (100 MHz) spectra were recorded in DMSO-d6 solution with TMS as an internal standard on Bruker instrument. Spin multiplicities are given as s (singlet), d (doublet), t (triplet) and m (multiplet). Coupling constant (J) is given in hertz. Mass spectra were recorded on a thermo Finnigan LCQ Advantage MAX 6000 ESI spectrometer.

Chemistry

Synthesis of (E)-5-styryl-1,3,4-oxadiazol-2-amine (A)

A mixture of semicarbazide (50 mmol) and cinnamic acid (50 mmol) in the presence of sulphuric acid (13 mL) was heated for 35-45 minutes in a fuming cupboard in an RB flask at 65-75°C. In an ice bath, the temperature of contents was brought down to 27°C (about 1 hr) and approximately 100 mL of distilled water was added initially dropwise and later water was added slowly in 5-7 mL portions as fuming stops. Then a reflux condenser was fixed, and for about 22-24 hrs, the contents were heated. The completion of the reaction was monitored by TLC using n-hexane: ethyl acetate (5:5) as a mobile phase. The mixture was cooled and basified with the addition of 50 percent NaOH with continuous stirring. Filtered solids were washed with distilled water and the product was air-dried. Ethanol was utilized for recrystallizatio

General procedure for synthesis of N-(5-styryl-1,3,4-oxadiazol-2- yl)benzamide derivatives (1-10)

The 5-phenyl-1,3,4-oxadiazol-2-amine (0.242 g, 0.0014 mol) and hydroxybenzotriazole (HOBt) (0.450 g, 0.0029 mol) was successively added to the corresponding substituted benzoic acid drivatives (0.2 g, 0.0014 mol) in N,N- dimethylformamide (DMF) (15 mL). The mixture was cooled to 0°C in an ice bath with stirring and then 1-ethyl-3-(3-dimethylaminopropyl) carbodiimde hydrochloride (EDC. HCl) (0.563 gm, 0.00294 mol) was added. The reaction mixture was then slowly allowed to reach the room temperature over 1 hour and then stirring was further continued at this temperature till completion of reaction. Progress of reaction was monitored with TLC using n- hexane:ethylacetate (1:4) as eluent, sports are detected by using UV spectroscopy. The reaction was quenched by saturated NaHCO3 solution and then extracted with EtOAc. The organic layer was dried with anhydrous Na2SO4, filtered and concentrated under reduced pressure.

Synthesis of compound (E)-2-chloro-N-(5-styryl-1,3,4-oxadiazol-2-yl) benzamide (1)

C17H12ClN3O2; MP: 151 – 155OC; Rf: 0.99; % yield: 88%; IR (cm-1): 3280 (NH stretching amine), 2903 (CH stretching alkane), 2357 (CH stretching aromatic), 1694 (C=O stretching ketone), 1541 (C=O stretching amide), 845 (Aromatic ring), 707 (C- Halogen stretching); 1H NMR (400 MHz, CDCl3) ? 11.70 (m, 1H), 7.83 (s, 1H), 7.69 (m, 2H), 7.42 (s, 2H), 7.16 (m, 3H), 6.80 (s, 1H), 6.63 (s, 1H); 13C NMR (101 MHz, CDCl3) ? 169.67, 161.98, 137.50, 134.37, 133.51, 132.42, 131.18, 130.87, 130.34,

128.34, 127.81, 126.91, 126.57, 125.47, 124.64; Elemental Analysis: C, 62.68; H, 3.71; Cl, 10.88; N, 12.90; O, 9.82. Mass spectra: actual: 325 m/z; found:328 (M+3) m/z.

Synthesis of compound (E)-4-chloro-N-(5-styryl-1,3,4-oxadiazol-2- yl)benzamide (2)

C17H12ClN3O2; MP: 181 – 185OC; Rf: 0.95; % yield: 89%; IR (cm-1): 3373 (NH stretching amine), 2947 (CH stretching alkane), 2336 (CH stretching aromatic), 1634 (C=O stretching ketone), 1563 (C=O stretching amide), 832 (Aromatic ring), 765 (C- Halogen stretching); 1H NMR (400 MHz, CDCl3) ? 11.51 (s, 1H), 8.06 (d, J = 30.6 Hz, 2H), 7.93 (s, 4H), 7.90 (m, 2H), 7.83 (s, 2H), 7.71 (s, 1H), 6.68 (s, 1H), 6.56 (s, 1H);

13C NMR (101 MHz, CDCl3) ? 169.96, 167.91, 166.01, 134.37, 133.23, 132.58, 131.51,

130.67, 130.05, 129.45, 127.81, 126.91, 126.08, 125.47; Elemental Analysis: C, 62.68; H, 3.71; Cl, 10.88; N, 12.90; O, 9.82; Mass spectra: actual: 325 m/z; found: 328 (M+3) m/z.

Synthesis of compound (E)-3-chloro-N-(5-styryl-1,3,4-oxadiazol-2-yl) benzamide (3)

C17H12ClN3O2; Rf: 0.91; % yield: 85%; MP: 194 – 198 OC; IR (cm-1): 3391 (NH stretching amine), 2929 (CH stretching alkane), 2342 (CH stretching aromatic), 1634 (C=O stretching ketone), 1557 (C=O stretching amide), 844 (Aromatic ring), 738 (C- Halogen stretching). 1H NMR (400 MHz, CDCl3) ? 11.03 (s, 1H), 7.47 (m, 2H), 7.37 (d, J = 20.1 Hz, 2H), 7.26 (s, 4H), 7.18 (s, 1H), 7.10 (d, J = 3.1 Hz, 1H), 6.99 (s, 1H); 13C NMR (101 MHz, CDCl3) ? 159.96, 158.20, 157.62, 139.86, 139.26, 138.68, 137.00, 136.08, 135.47, 134.64, 133.82, 132.42, 128.67, 127.20, 126.57; Elemental Analysis: C, 62.68; H, 3.71; Cl, 10.88; N, 12.90; O, 9.82; Mass spectra: actual: 325 m/z; found:

325 m/z.

Synthesis of compound (E)-N-(5-styryl-1,3,4-oxadiazol-2-yl)benzamide (4)

C17H13N3O2; Rf: 0.98; % yield: 90%; MP: 147 – 149OC; IR (cm-1): 3037 (NH stretching amine), 2916 (CH stretching alkane), 2330 (CH stretching aromatic), 1652 (C=O stretching ketone), 1538 (C=O stretching amide), 835 (Aromatic ring), 764 (C- Halogen stretching). 1H NMR (400 MHz, CDCl3) ? 11.54 (s, 1H), 7.64 (s, 17H), 7.55 (s, 2H), 7.36 (s, 2H), 7.11 (s, 2H), 7.00 (s, 1H), 6.93 (s, 1H), 6.57 (s, 1H), 6.50 (s, 1H); 13C NMR (101 MHz, CDCl3) ? 171.09, 167.42, 165.08, 134.93, 134.02, 133.22, 131.24, 130.37, 129.71, 128.31, 127.78, 126.61, 125.44, 124.64; Elemental Analysis: C, 70.09; H, 4.50; N, 14.42; O, 10.98; O, 9.82; Mass spectra: actual: 291 m/z; found: 290 (M- 1)m/z.

Synthesis of compound (E)-4-methyl-N-(5-styryl-1,3,4-oxadiazol-2- yl)benzamide (5)

C18H15N3O2; Rf: 0.97; % yield: 96%; MP: 141 – 144OC; IR (cm-1): 3272 (NH stretching amine), 2917.43 (CH stretching alkane), 2336 (CH stretching aromatic), 1574 (C=O stretching ketone), 1489 (C=O stretching amide), 835 (Aromatic ring), 764 (C-Halogen stretching). 1H NMR (400 MHz, CDCl3) ? 11.05 (s, 1H), 7.83 (s, 1H), 7.69 (m, 1H), 7.42 (s, 2H), 7.16 – 7.08 (m, 3H), 6.80 (s, 1H), 6.63 (s, 1H), 2.62 (s, 3H); 13C NMR (101 MHz, CDCl3) ? 171.09, 167.42, 165.08, 134.93, 134.02, 133.22, 131.24,

130.37, 129.71, 128.31, 127.78, 126.61, 125.44, 124.64, 23.89; Elemental Analysis: C, 70.81; H, 4.95; N, 13.76; O, 10.48; Mass spectra: actual: 305 m/z; found: 306 (M+1) m/z.

Synthesis of compound (E)-2-phenyl-N-(5-styryl-1,3,4-oxadiazol-2- yl)acetamide (6)

C18H15N3O2; Rf: 0.89; % yield: 96%; MP: 151 – 153OC; IR (cm-1): 3010 (NH stretching amine), 2914 (CH stretching alkane), 2383 (CH stretching aromatic), 1581 (C=O stretching ketone), 1569 (C=O stretching amide), 835 (Aromatic ring), 764 (C- Halogen stretching). 1H NMR (400 MHz, CDCl3) ? 11.54 (s, 1H), 7.64 (s, 1H), 7.55 (s, 2H), 7.36 (s, 2H), 7.11 (s, 2H), 7.00 (s, 1H), 6.93 (s, 1H), 6.57 (s, 1H), 6.50 (s, H), 3.50 (s, 2H), 2.48 (s, H); 13C NMR (101 MHz, CDCl3) ? 170.81, 169.95, 168.52, 136.45, 134.37, 133.53, 132.39, 131.50, 131.24, 129.71, 128.65, 126.91, 126.05, 124.33, 46.78; Elemental Analysis: C, 70.81; H, 4.95; N, 13.76; O, 10.48; Mass spectra: actual: 305 m/z; found: 305 m/z.

Synthesis of compound (E)-2-(4-chlorophenyl)-N-(5-styryl-1,3,4-oxadiazol- 2-yl) acetamide (7)

C18H14ClN3O2; Rf: 0.95; % yield: 84%; MP: 145 – 148OC; IR (cm-1): 3274 (NH stretching amine), 2878 (CH stretching alkane), 2304 (CH stretching aromatic), 1647 (C=O stretching ketone), 1548 (C=O stretching amide), 845 (Aromatic ring), 745 (C- Halogen stretching). 1H NMR (400 MHz, CDCl3) ? 11.70 (m, 1H), 7.83 (s, 1H), 7.69 (m, 1H), 7.42 (s, 2H), 7.16 (m, 2H), 6.80 (s, 1H), 6.63 (s, 1H), 4.65 (m, 2H); 13C NMR (101 MHz, CDCl3) ? 171.71, 170.81, 169.95, 168.52, 136.45, 134.37, 133.53, 132.39, 131.50, 131.24, 129.71, 128.65, 126.91, 126.05, 124.33, 46.78; Elemental Analysis: C, 63.63; H, 4.15; Cl, 10.43; N, 12.37; O, 9.42; Mass spectra: actual: 339 m/z; found: 336 (M-3) m/z.

Synthesis of compound (E)-4-fluoro-N-(5-styryl-1,3,4-oxadiazol-2-yl) benzamide (8)

C17H12FN3O2; Rf: 0.90; % yield: 89%; MP: 167 – 169OC; IR (cm-1): 3090.07 (NH stretching amine), 3360.11 (OH stretching), 2935.76 (CH stretching alkane), 2348.41 (CH stretching aromatic), 1647.26 (C=O stretching ketone), 1546.00 (C=O stretching amide), 825.48 (Aromatic ring), 765.77 (C-Halogen stretching); 1H NMR (400 MHz, CDCl3) ? 11.78 (m, 1H), 7.71 (s, 2H), 7.61 (s, 2H), 7.42 (m, 1H), 7.00 (s,

1H), 6.82 (m, 1H); 13C NMR (101 MHz, CDCl3) ? 163.34, 162.21, 143.88, 135.49, 134.96, 134.06, 131.49, 131.20, 125.18, 124.35, 120.60, 120.06; Elemental Analysis: C, 66.02; H, 3.91; F, 6.14; N, 13.59; O, 10.35; Mass spectra: actual: 309 m/z; found: 309 m/z.

Synthesis of compound (E)-3-fluoro-N-(5-styryl-1,3,4-oxadiazol-2- yl)benzamide (9)

C17H12FN3O2; Rf: 0.93; % yield: 86%; MP: 177 – 180OC; IR (cm-1): 3090.07 (NH

stretching amine), 3360.11 (OH stretching), 2935.76 (CH stretching alkane), 2348.41 (CH stretching aromatic), 1647.26 (C=O stretching ketone), 1546.00 (C=O stretching amide), 825.48 (Aromatic ring), 765.77 (C-Halogen stretching); 1H NMR (400 MHz, CDCl3) ? 11.57 (s, 1H), 7.93 (s, 2H), 7.83 (s, 2H), 7.69 – 7.64 (m, 2H), 7.54 (m, 1H), 7.37 (m, 2H), 7.23 – 7.18 (m, 2H), 6.87 (s, 1H), 6.68 (s, 1H); 13C NMR (101 MHz, CDCl3) ? 165.97, 163.34, 161.08, 159.35, 157.92, 135.49, 134.96, 131.49, 128.33, 127.22, 126.10, 125.49, 124.65, 123.73; Elemental Analysis: C, 66.02; H, 3.91; F, 6.14; N, 13.59; O, 10.35; Mass spectra: actual: 309 m/z; found: 308 (M-1) m/z.

Synthesis of compound (E)-2-fluoro-N-(5-styryl-1,3,4-oxadiazol-2-yl) benzamide (10)

C17H12FN3O2; Rf: 0.97; % yield: 86%; MP: 176 – 180OC; IR (cm-1): 3387 (NH stretching amine), 2347 (CH stretching aromatic), 1622 (C=O stretching ketone), 1565 (C=O stretching amide), 820 (Aromatic ring), 651 (C-Halogen stretching); 1H NMR (400 MHz, CDCl3) ? 11.57 (s, 1H), 7.93 (s, 2H), 7.83 (s, 2H), 7.69 – 7.64 (m, 2H), 7.54

(m, 1H), 7.37 (m, 2H), 7.23 – 7.18 (m, 2H), 6.87 (s, 1H), 6.68 (s, 1H); 13C NMR (101 MHz, CDCl3) ? 165.97, 163.34, 161.08, 159.35, 157.92, 135.49, 134.96, 131.49, 128.33, 127.22, 126.10, 125.49, 124.65, 123.73; Elemental Analysis: C, 66.02; H, 3.91; F, 6.14; N, 13.59; O, 10.35; Mass spectra: actual: 309 m/z; found: 307 (M-2) m/z.

Devices and materials

In the molecular scenario in the modern drug design, the docking is commonly used to understand the interaction between the target ligand-receptor and the target lead molecule's binding orientation with its protein receptor and is quite frequently used to detect the associations between the target components. The research work was done in- silico by utilizing bioinformatics tools. Also, we utilize some of the offline programming’s like protein data bank (PDB) www.rcsb.org/pdb, PubChem database, Marvin sketch. The molecular docking studies were carried out through Discovery studio.

Preparation of protein

By utilizing the offline program protein data bank (PDB), we take the tyrosinase II (PDB ID:3NM8) was obtained from PDB website. From the protein we removed the crystal water, followed by the addition of missing hydrogens, protonation, ionization, energy minimization. The SPDBV (swiss protein data bank viewer) force field was applied for energy minimization. Prepared protein is validated by utilizing the Ramachandran plot 43.

 Identification of active sites

Identification of active amino acid present in the protein is detected by using Protein-ligand interaction profile (PLIP) https://plip-tool.biotec.tu- dresden.de/plipweb/plip/index offline tool in google. From this, I found the active amino acid present in the protein44.

Preparation of Ligands

By utilizing the Marvin sketch tool, the molecules are designed in two and three- dimensional structures. After designed molecule, the structure was optimized in 3D optimization in Marvin sketch and saved as a pdb format.

In silico ADMET prediction

A computational study was conducted to predict the pharmacokinetic properties (ADMET) of designed compounds using swiss ADME prediction. Here we calculated molecular volume (MV), molecular weight (MW), number of acceptors of hydrogen bonds (n-ON), number of donors of hydrogen bonds (n-OHNH), total CNS activity, % of oral absorption for humans, polar surface area (PSA), 1-Octyl alcohol-water distribution constant (log P o/w), and BBB penetration. The described properties help to understand the ADME properties of any drug/synthesized molecule. The drug likeness, rule-of-five and rule-of-three violations were also ascertained. For a molecule to be administered via oral route, it should possess distribution constant 5, molecular mass 500, number of donors of H-bonds 5 and number of acceptors of H-bonds 10 and only one violation of the above criteria is acceptable.

DPPH radical assay

A total antioxidant capacity assay was carried out using DPPH as radical. The experimental procedure was adapted from the literature, only with slight modification (N. Nenadis 2002 and A. Torres 2007). Briefly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in ethanol (250 mM, 2 mL) was added to 2 mL of an ethanolic solution of the test compounds. The final concentration of the test compounds in the reaction mixtures was 50 mM. Each mixture was then shaken vigorously and held for 30 min at room temperature in the dark. The decrease in absorbance of DPPH at 517 nm was then measured. Ethanol was used as a blank and a DPPH solution (2 mL) in ethanol (2 mL) as the control solution. All tests were performed in triplicate.

 RESULTS AND DISCUSSION

Synthesis

A series of novel class of design and synthesize a novel class of oxadiazole based derivatives were synthesized by the reaction between benzoic acid and semi carbazide in presence of cyclization agent it will give 5-phenyl-1,3,4-oxadiazol-2- amine. Further this compound treated with various aromatic aldehyde in presence of NaOH to give corresponding title compound. and the synthesized compounds are characterization by using IR, 1H NMR, 13C NMR, and mass spectroscopy. All the compounds and intermediates were purified by successive recrystallization from ethanol. The purity of synthesized compounds was confirmed by Melting Point and which were checked in open capillary tube and are uncorrected and TLC using Ethyl Acetate: Petroleum Ether (50:50) as solvent system. The IR spectrum of the final synthesized compounds showed absorption bands around 3300–3156 cm?1 for amide NH, while the distinguishing broad absorption peaks C=O for CONH were observed in the range 1720–1690 cm?1, 3350–3157 cm?1 for NH, 1489–1464 cm?1 for CH2, 1379– 1344 cm?1 for CH3, and 800–700 cm?1 for C=C. These compounds also exhibited appropriate peaks at corresponding ? ppm in their 1H NMR spectra and corresponding molecular ion peaks in LC–MS spectra which were in conformity with the assigned structures. The structures of synthesized compounds were confirmed by FT-IR, 1H- NMR and 13C-NMR the result was correlated with the expected structure. All the synthesized compounds were subjected for short-term in vitro anti-oxidant study.

Molecular docking studies

Based on literature studies of oxadiazole derivatives, the 10 compounds were designed for our study and these compounds were subjected to molecular docking studies. Molecular docking was carried out through discovery studio to predict the interactions model of the protein to its inhibitors. The molecular docking was performed to elucidate the binding mode competence of tyrosinase (PDB ID:3NM8) and oxadiazole analogues. The designed molecules were docked along with the native ligand and a reference standard, donepezil. The docking energy of our designed compounds ranged from 7 to 11 kcal/mol indicated good binding affinities to the target receptor, and the results are depicted in Table 1. Among the docked compounds, derivatives 1 and 2 (-10.8 kcal/mol) showed a significant binding energy towards the targeted enzyme. The compounds 2 and 3 posses 2 hydrogen bond between amino acids ASP 400 and ASP 404. The roles of certain crucial amino acids in the ligand-binding domain of the human Acetylcholinesterase inhibitors were also established. Major non- covalent interactions between the studied ligands and the ligand-binding domain of the Acetylcholinesterase inhibitors were investigated. These amino acids have been repeatedly implicated during ligand interaction with the tyrosinase inhibitors and also play important role in the inhibition of the ligand-binding domain of tyrosinase inhibitors. These non-covalent interactions, van der Waals, columbic interaction, ?–? interaction, and hydrogen interaction, are shown in Figure 2 to 11.