Synthesis, Characterization and Biological Activities Oxadiazole Derivatives having Thioether Linkage

 

Rajesh S. Shah and Vishal Modi*

Shri JJT University, Vidyanagari, Churu Jhunjhunu Road, Chudela, District-Jhunjhunu, Rajasthan-333001

*Corresponding Author E-mail: vishal651981@yahoo.se

 

The synthesis of novel thioether 1,3,4-oxadiazole derivatives are reported. All the synthesized compounds are characterized standard spectroscopic method and elemental analysis techniques. Synthesized compounds are screened for microbial and cytotoxic activities.

 

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INTRODUCTION:

A large number of oxadiazole derivatives have been prepared and many of these compounds have shown a wide spectrum of antimicrobial activity^1-5. The observation that some oxadiazoles with different substituents at different location on the heterocyclic ring resulted in fungicidal^6-7 and antibacterial agents^8-11 of various potencies. Since their discovery during the 20th century, antimicrobial agents (antibiotics and related antimicrobial drugs) have substantially reduced the threat posed by infectious diseases. The use of these "wonder drugs", combined with improvements in sanitation, housing, and nutrition, and the advent of widespread immunization programmes¹², and the development of numbers of antimicrobial agents for treatment of microbial infections¹³ has led to a dramatic drop in deaths from diseases that were previously widespread, untreatable, and frequently fatal. These gains are now seriously jeopardized by another recent development: the emergence and spread of microbes that are resistant to cheap and effective firstchoice, or "first-line" drugs. Worldwide emergence of multi-resistant microbial strains is a growing concern which requires a multi-pronged research strategy^{14, 15}. Some material applications of 1,3,4-oxadiazole derivatives lie in the fields of photosensitizers  and liquid crystals^16-18. Microwave activation as a non-conventional energy source has become an important method that can be used to carry out a wide range of reactions within short time and with high yields than those obtained by using conventional heating.

 

The reactions which are not possible under conventional conditions can sometimes be affected by the high energy of MWI¹⁹. Oxadiazole, a heterocyclic nucleus has attracted wide attention of the chemists in search of new therapeutic molecules. Out of its four possible isomers,1,3,4-oxadiazole is widely exploited for various applications. The therapeutic importance of these rings prompted us to develop selective molecules in which a substituent is capable of displaying higher pharmacological activities. In this paper, we have reported the synthesis of oxadiazole derivatives by the conventional method and the microwave method to find out an effective method. Synthesized compounds are also screened for microbial and cytotoxic activities.

 

MATERIALS AND METHODS:

Materials:

The requisite starting materials such as hydrazine hydrate, triethyl amine, ethanol, carbon disulfide, potassium hydroxide,1-bromo hexadecane, pyridine, 4-hydroxy phenyl benzoate, hexane, ethyl acetate etc. were procured from Aldrich Company and used without any further purification. All the solvents were purified and dried by standard methods. Analytical TLC was conducted on Merck aluminium plates with 0.2 mm of silica gel 60 F-254. Microwave synthesis was carried out by using Samsung GW71B domestic equipment.

 

Methods:

Hydrazide (II) was synthesized by condensing ethyl 4-hydroxyphenylbenzoate and hydrazine hydrate (80%).

 

 

Conventional method:

5-(4-hydroxy)phenyl-3H-1,3,4-oxadiazoline-2-thione (III)²⁰

A solution of KOH (1.6g) in water (10mL) was added dropwise to a stirred suspension of hydrazide (II) (28mmol) in ethanol (80mL) at 25°C. After all of the hydrazide has dissolved, carbon disulfide (35mmol,) was added at the same temperature. The solution was evaporated in vacuum using a rotatory evaporator. The residue was poured into a mixture of 400g ice and 100mL concentrated hydrochloride acid. The precipitate formed was filtered off, and crystallized from ethanol/water (4/1) yielding thione (III). Yield 68%             B.P. 247C

 

5-(4-hydroxy)phenyl-2-n-hexadecylthio-1,3,4-oxadiazole (IV)²⁰

Triethylamine (3.67mmol) and 1-bromo hexadecane (3.67mmol) were successively added dropwise to a stirred solution of (III) (3.67mmol) in absolute ethanol (10mL). After heating the mixture for 4h under reflux, the solvent was evaporated on a rotatory evaporator. The residue was poured into 100mL of water, the resulting precipitate was collected and crystallized from ethanol/ water (1/1) yielding compound IV. Yield 60%.

 

Ester Ia–f were synthesized by the condensation of Hydroxyoxadiazole (IV) with 4-n-alkoxybenzoyl chloride. The products were purified by column chromatography using the mixture of ethyl acetate and hexane as an eluant. Products were crystallized by mixture of ethanol/water (2/1). The following yields were obtained: Ia (40%), Ib (55%), Ic (35%), Id (37%), Ie (42%), If (50%), Ig (38%), Ih (40%).

 

Microwave method:

5-(4-hydroxy)phenyl-2-n-hexadecylthio-1,3,4-oxadiazole (IV)²⁰

Triethylamine (0.36mmol) and 1-bromo dodecane (0.36mmol) were successively added dropwise to a stirred solution of (III) (0.36mmol) in absolute ethanol (1mL). Reaction mixture was kept under microwave for 40s at 760W. The solvent was evaporated on a rotatory evaporator. The residue was poured into 100mL of water, the resulting precipitate was collected and crystallized from ethanol/ water (1/1) yielding the compound IV. Yield 90%.

Esters Ia–f were synthesized by the mixing of amino-oxadiazole (IV) (0.01mol) with 4-n-alkoxybenzoyl chloride (0.01mol) in 1mL pyridine. The resulting mixture was kept under microwave for 40 s. (1:1) cold HCl was added to the reaction mixture and filtered. The obtained products, after drying, were purified by column chromatography using the mixture of ethyl acetate and hexane as an eluent. Products were crystallized by the mixture of ethanol/water (2/1). The following yields were obtained: Ia (90%), Ib (88%), Ic (85%), Id (85%), Ie (87%), If (90%).

R= - C_nH_2n+1, Where n= 4, 5, 6, 7, 8, 10 (I a-f)

Scheme 1: Synthetic route for series I compound

 

Characterization:

The structures of the compounds were confirmed by ¹H NMR; Bruker AC-250P spectra and Fourier transform infrared (FTIR; Nicolet 550) spectra; the purity of the final products was evaluated by thin layer chromatography (TLC).

 

Spectroscopic characterization of compound (IV)

FTIR (KBr disc): cm^-1= 3300 (br, -OH), 3092 (Csp²–H); 2915 (Csp³–H); 1608 (C=C); 1521 (C=N). Elemental analysis: Calculated for C₂₄H₃₈N₂O₂S: C, 68.89; H, 9.09; N, 6.70%. Found: C, 69.89; H, 9.04; N, 6.60%.

 

Spectroscopic characterization of esters Ia–h compound (V)

Ia: ¹H NMR (CDCl₃, TMS, 250MHz): δppm=0.90 (t, 3H, CH₃); 0.95 (t, 3H, CH₃); 1.15–1.45 (m, 28H, 14xCH₂); 1.71 (m, 4H, OCH₂–CH₂ and SCH₂–CH₂); 3.20 (t, 2H, SCH₂); 3.95 (t, 2H, OCH₂); 6.90 (d, 2H, arom. H); 7.68 (m, 4H, arom. H); 7.85 (d, 2H, arom. H). FTIR (KBr disc): cm^-1= 2920 (Csp³–H); 1730 (-COO-); 1650 (C=O); 1605 (C=C). Elemental analysis: Calculated for C₃₅H₅₀N₂O₄S: C, 70.70; H, 8.41; N, 4.71%. Found: C, 70.67; H, 8.35; N, 4.69%.

 

Ib: ¹H NMR (CDCl₃, TMS, 250MHz): δppm=0.80 (t, 3H, CH₃); 0.95 (t, 3H, CH₃); 1.20–1.43 (m, 30H, 15xCH₂); 1.75 (m, 4H, OCH₂–CH₂ and SCH₂–CH₂); 3.19 (t, 2H, SCH₂); 3.95 (t, 2H, OCH₂); 6.91 (d, 2H, arom. H); 7.80 (m, 4H, arom. H); 7.85 (d, 2H, arom. H). FTIR (KBr disc): cm^-1= 2925 (Csp³–H); 1725 (-COO-); 1645 (C=O); 1605 (C=C). Elemental analysis: Calculated for C₃₆H₅₂N₂O₄S: C, 71.05; H, 8.55; N, 4.60%. Found: C, 71.01; H, 8.52; N, 4.59%.

 

Ic: ¹H NMR (CDCl₃, TMS, 250MHz): δppm= 0.85 (t, 3H, CH₃); 0.92 (t, 3H, CH₃); 1.24–1.45 (m, 32H, 16xCH₂); 1.79 (m, 4H, OCH₂–CH₂ and SCH₂–CH₂); 3.26 (t, 2H, SCH₂); 3.99 (t, 2H, OCH₂); 6.85 (d, 2H, arom. H); 7.80 (m, 4H, arom. H); 7.95 (d, 2H, arom. H). FTIR (KBr disc): cm^-1= 2930 (Csp³–H); 1750         (-COO-); 1670 (C=O); 1600 (C=C). Elemental analysis: Calculated for C₃₇H₅₄N₂O₂S: C, 71.38; H, 8.68; N, 4.50%. Found: C, 71.28; H, 8.59; N, 4.49%.

 

Id: ¹H NMR (CDCl₃, TMS, 250MHz): δppm=0.83 (t, 6H, 2xCH₃); 1.21–1.80 (m, 38H, 19xCH₂); 3.20 (t, 2H, SCH₂); 4.01 (t, 2H, OCH₂); 7.00 (d, 2H, arom. H); 7.81 (d, 2H, arom. H); 7.86 (d, 2H, arom. H); 8.00 (d, 2H, arom. H). FTIR (KBr disc): cm^-1= 2920 (Csp³–H); 1730 (-COO-); 1655 (C=O); 1610 (C=C). Elemental analysis: Calculated for C₃₈H₅₆N₂O₄S: C, 71.70; H, 8.81; N, 4.40%. Found: C, 71.68; H, 8.78; N, 4.35%.

 

Ie: ¹H NMR (CDCl₃, TMS, 250MHz): δppm=0.91 (t, 6H, 2xCH₃); 1.25–1.75 (m, 40H, 20xCH₂); 3.28 (t, 2H, SCH₂); 4.05 (t, 2H, OCH₂); 7.01 (d, 2H, arom. H); 7.90 (d, 2H, arom. H); 7.92 (d, 2H, arom. H); 7.99 (d, 2H, arom. H). FTIR (KBr disc): cm^-1= 2930 (Csp³–H); 1735 (-COO-); 1640 (C=O); 1605 (C=C). Elemental analysis: Calculated for C₃₉H₅₉N₃O₃S: C, 72.00; H, 8.92; N, 4.31%. Found: C, 71.90; H, 8.89; N, 4.28%

If: ¹H NMR (CDCl₃, TMS, 250MHz): δppm=0.90 (t, 6H, 2xCH₃); 1.25–1.80 (m, 44H, 22xCH₂); 3.26 (t, 2H, SCH₂); 4.04 (t, 2H, OCH₂); 7.00 (d, 2H, arom. H); 7.82 (d, 2H, arom. H); 7.88 (d, 2H, arom. H); 8.10 (d, 2H, arom. H). FTIR (KBr disc): cm^-1= 2910 (Csp³–H); 1740 (-COO-); 1630 (C=O); 1605 (C=C). Elemental analysis: Calculated for C₄₁H₆₃N₃O₃S: C, 72.57; H, 9.14; N, 4.13%. Found: C, 75.49; H, 9.09; N, 4.08%.

 

Biological activities:

Antibacterial and antifungal activity²¹

The compounds were tested in-vitro for their antibacterial activity against two microorganisms viz. Escherichia coli and Staphylococcus aureus, which are pathogenic in human beings by cup-plate agar diffusion method. The compounds were tested in vitro for their antifungal activity against Aspergillus oryzae and Aspergillus niger by cup-plate agar diffusion method. Procedure: All compounds were screened for antibacterial activity against E. coli and S. aureus by cup plate method¹³. For anti bacterial activity, we had taken 20g of luria broth (Hi media M-575) and 25g of agar–agar in 1000mL distilled water and heated till it dissolved. Then, the mixture was sterilized by autoclaving at 15lbs pressure and 121°C for 15min. Here, agar–agar was used to solidify the solution. After that, six Petri dishes having flat bottom were taken and filled with about 18mL of the above solution. Overlay the plate with 4mL soft agar–agar containing 0.1mL test culture. Bored four well of 8mm diameter in each plate. We had then dissolved the compound in DMF having 1000ppm concentration and added 0.1mL of testing solution into each well. This solution was allowed to diffuse at 4°C. After 20min of diffusion, the plate was incubated at 37°C overnight. After incubation, we observed the zone of inhibition and measured the diameter of the zone. For anti fungal activity, we had taken 20g Sabouraud dextrose instead of lubria broth and followed the same procedure as above. All the synthesized compounds showed good antimicrobial activity (Table 1).

 

Cytotoxicity test:

Brine shrimp lethality bioassy (BSLT). Brine shrimp lethality test has been used as a bioassay for a variety of toxic substances. This method has also been applied to plant extracts in order to facilitate the isolation of biologically active compounds. A general bioassay that appears capable of detecting a broad spectrum of bioactivity, present in crude extracts and in synthetic compounds is the brine shrimp lethality bioassay, rather than more tedious and expensive in vitro and in vivo antitumor assays. Furthermore, it does not require animal serum as is needed for cytotoxicities.

 

Procedure: Brine shrimp lethality bioassay was carried out to investigate the cytotoxicity of medicinal plants. Brine shrimps (Artemia salina) were hatched using brine shrimp eggs in a conical shaped vessel (1L), filled with sterile artificial sea water under constant aeration for 38h. After hatching, active nauplii free from egg shells were collected from brighter portion of the chamber and used for the assay. Ten nauplii were drawn through a glass capillary and placed in each vial containing 5mL of the brine solution. In each experiment, test substances whose activities are to be checked were added to the vial according to their concentrations and maintained at room temperature for 24 h under the light and surviving larvae were counted. Experiments were conducted along with control (vehicle treated), different concentrations (1-5000 µg/mL) of the test substances in a set of three tubes per dose. Replicas should be maintained to get accurate results (Table 2).

 

Table 1: Microbial activity data for synthesized series I

Sr. No.	Anti Bacterial Blank 12 mm		Anti Fungal Blank 10 mm
	E. coli	S. aureus	A. niger	A. oryzae
I-a	13.00	12.50	10.25	10.50
I-b	13.00	12.25	10.25	10.25
I-c	13.25	12.25	10.50	10.25
I-d	12.75	12.75	10.50	10.25
I-e	12.50	12.00	10.25	10.50
I-f	12.50	12.50	10.50	10.00

Furacin (As a Standard):   E. coli. : 14.75; S. aureus: 14.75; A. niger: 12.00; A. oryzae : 12.00 mm

Grieseofulvin (As a Standard): E. coli. : 12.00; S. aureus: 12.00; A. niger: 10.00; A. oryzae: 11.75 mm

 

 

Table 2: Cytotoxic activity of data for synthesized series I

Sr. No.	Solubility	ED50 μg/ml
I-a	DMSO	22.32
I-b	DMSO	43.25
I-c	DMSO	35.20
I-d	DMSO	45.39
I-e	DMSO	48.40
I-f	DMSO	45.60
Standard	Podophyllotoxin	3.88

 

RESULTS AND DISCUSSION:

The synthetic route for the preparation of series I compounds is outlined in Scheme 1. The ¹H NMR and FTIR spectra are fully consistent with the structure. Brine shrimp lethality test has been used as a bioassay for a variety of toxic substances. All the synthesized compounds (Ia–f) were tested for cytotoxic activity by the BSLT bioassay method. Among them compounds Ia, Ic showed a dose dependent cytotoxic activity at concentrations of (Ia) 22.32 µg/mL, (Ic) 35.20 µg/mL, respectively. The remaining compounds exhibited less activity when compared to the above mentioned compounds at various concentration levels. The degree of lethality is directly proportional to the concentration of the synthesized compounds. Podophyllotoxin was used as a standard drug for BSLT assay method.

 

CONCLUSION:

All the synthesized derivatives of series I were synthesized by conventional and microwave methods. Synthesis of compounds by the microwave method gives comparatively more yield and requires less time to complete the reaction. So, the microwave synthesis method is better than the conventional method. All the synthesized compounds of series I was screened for the microbial activity. In the present study, all synthesized compounds showed moderated to good microbial activities. Activity increases as the number of carbon increases in alkyl chain. All the synthesized compounds show moderate to good anti fungal activities. All the compounds were found to possess cytotoxic activity. The newly synthesized oxadiazole derivatives have good to moderate anti-bacterial and anti-fungal activities, they may be used for the development of new drugs for the treatment of bacterial and fungal diseases.

 

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Received on 08.09.2013          Modified on 25.09.2013

Accepted on 28.09.2013         © AJRC All right reserved