Bioactivity Study of Thiophene and Pyrazole Containing Heterocycles

Chalcones 3a-f were prepared by reacting thiophene containing pyrazolyl aldehyde (2) with different 2-hydroxy acetophenones 1a-f. The compounds 3a-f were transformed into different Pyrazolines 4a-f. The formation of chromene derivatives 5a-f occurred from the cyclization of 3a-f, which were then transformed into pyrazole derivatives 6a-f. Newly synthesized compounds have promising antibacterial activity against S. typhii and S. aureus, while weak activity against B. subtilis and E. coli. Compounds 5d and 6d had significant antifungal action towards A. niger, while most of the compounds were moderately active towards T. viride. Some of the synthesized compounds showed promising α-amylase inhibitory activity at 1 mg/mL concentration.


INTRODUCTION
In the field of medicinal chemistry, most drugs have different heterocyclic scaffolds that show potential biological activities. Microbes are responsible for different human diseases. As these microorganisms develop resistance towards many of the present drug molecules, there is a need for continuous research on developing new potential medicinal agents. The presence of oxygen, sulphur and nitrogen containing heterocyclic nucleus imparts very effective pharmacological properties to therapeutic agents. In these scaffolds, the presence of Fluorine increases bioactivity of molecules several times 1,2 .
Thiophene derivatives have varied therapeutic applications. Thiophene containing heterocyclic compounds have created interest among the researchers owing to their vast spectrum of biological functions including antimicrobial 3,4 , antiparasitic 5,6 , antiviral 7 , anticancer 8,9 , enzyme inhibitors 10 , anti-inflammatory and analgesic 11 properties. Some of the commercially available drugs that contain thiophene as an integral component are Suprophen and Tiaprofenic acid as an anti-inflammatory, Rolaxifene and OSI-930 as an anticancer, Methapyrilene as anti-histamine, Tienilic acid as an antihypertensive, Ticlopidine as antiplatelet, Olanzapine as antisychotic, Etizolam as anti-anxiety and Tigabine as anticonvulsant agents.
Chromones belong to the flavonoid family and are widely used in folk medicine because of their interesting biological activities 47, 48 . The large number of bioactive molecules have chromone moiety as an essential pharmacophore 49,50 . Many of the biologically active chromones show anti-bacterial and antifungal 51,52 , anti-inflammatory 53,54 , antioxidant 55,56 , anticancer 57-59 , anti-HIV 60 , anti-obesity 61 , antiviral 62 , antidiabetic 48 , anticonvulsant 63 and anti-tubercular activities 64 . In light of the significance of thiophene, pyrazole, chalcone and chromone in numerous areas, particularly in medicinal chemistry (biological activity), the present study focuses on synthesis of these scaffolds and their biological activities.
Inhibition of the digestive enzymes is one of the approaches to manage diabetis. These enzymes catalyse the hydrolysis of starch into smaller carbohydrates like glucose 65 . This is one of the important steps in maintaining blood sugar level. In diabetics, hypoglycaemia can be achieved by inhibiting the enzyme alpha-amylase and the inhibitor can prove a potential anti-diabetic agent.

RESULT AND DISCUSSION
A well-known literature approach was used to synthesize various acetophenones (1a-f) and arylaldehyde (2) derivatives, as illustrated in Scheme-I and Scheme-II. The synthesis of chalcones, 3a-f was carried out by Claisen-Schmidt condensation by using 1a-f and 2. Different spectral methods have verified the formation of 3a-f. I.R. spectra of compound 3d shows band at 3439, 1639 cm -1 . HRMS shows a molecular ion peak at 459.0135 support 3d formation. 1 HNMR signal at 5.88 indicate olefinic proton and also the signal at δ 8.85 shows proton of pyrazole ring. In ethanol, the molecule 3a-f interacts with hydrazine hydrate to produce bipyrazolyl phenols, 4a-f. This formation of pyrazolines 4a-f was confirmed by spectral technique. The I.R. Spectrum 3334 cm -1 and Molecular ion peak at 473.0410 in HRMS validated formation of 4d. The most important confirmation of 4d formation is in 1 HNMR spectra which shows two doublets at δ 3.17 and δ 3.71 confirm the presence of diastereotopic protons of methylene group of pyrazoline ring while the δ 5.09 triplet of methine in pyrazoline ring. These signals strongly support the formation of 4a-f. Refluxing chalcone 3a-f in DMSO with a catalytic quantity of iodine yielded 2-substituted chromone 5a-f. The I.R. spectrum at 1653 cm -1 and the molecular ion peak at 456.9967 verify 5d. 1 HNMR spectra validate chromone formation as there is absence of downfield signal above δ 10.0 implies absence of O-H and also singlet at δ 6.80 is due to 3-position proton chromone. Chromones 5a-f when refluxed in ethanol and hydrazine hydrate were transformed into pyrazoles 6a-f. The I.R. Spectrum at 3404 cm -1 and In HRMS molecular ion peaks at 471.0243 supports 6d formation. The most significant confirmation is the presence of a singlet at δ 12.70 in 1 HNMR spectra of O-H proton.

Amylase Inhibitory Activity
In a test tube 500 μL of test sample, 500 μL solution of α-amylase whose concentration is 0.5 mg/ mL and phosphate buffer of 0.2 mM concentration were mixed and kept undisturbed 10 min at room temperature. Then the contents of the test tube were allowed to react 1% starch solution in phosphate buffer having pH 6.9. Then dinitrosalicylic acid was used to extinguish reaction. After incubation of 5 min test tubes were cooled and the contents were diluted with distilled water. The absorbance of resulting solutions was recorded at the wavelength 540nm. Then the results were compared with well-known inhibitor of the acarbose.
The results are recorded as % inhibition of enzyme activity. %Inhibition = (Ac-As)/Ac x 100 Where AC -Absorbance for Control and AS -Absorbance for Sample

EXPERIMENTAL
Open capillary technique was used for melting points which are uncorrected. IR Affinity-I Fourier transform infrared spectrophotometer ( S h i m a d z u ) , B r u k e r Av a n c e I I 5 0 0 M H z spectrophotometer and Waters SYNAPT G2 HDMS were used to record the IR, 1 H NMR and Mass spectra. Samples for NMR were prepared in DMSO-d 6 and TMS was internal reference. Absorption frequencies in terms of chemical shift were expressed in δ ppm. Mass spectra were recorded on.

5-yl]phenol, 4a
A mixture of 2 mL hydrazine hydrate, 15 mL ethanol and substituted pyrazolyl chalcone 3a (0.0015 mol) was taken in R.B. flask and refluxed for 4 hours. Then by adding 2 mL of glacial acetic acid, heated for further 4 hours. Once the reaction was finished the contents were taken into crushed ice. The resulting product had been filtered. On crystallization from ethanol, pure compound 4a was obtained. The compound 4b-4f were prepared using the same procedure.

1H-pyrazol-4-yl]-4H-chromen-4-one, 5a
Substituted pyrazolyl propenone 3a (0.001 mol) was refluxed in 12 mL of DMSO containing 0.2 g Iodine at 135-145 o C for 3-4 h and kept aside for 24 hours. Then the mixture was transferred to smashed ice and filtered solid was treated with 15% sodium thiosulphate for removal of unreacted Iodine. The compound 5a was purified by recrystallizing it from ethanol. The compound 5b-5f were prepared using the same procedure.

CONCLUSION
Flourine and thiophene containing different pyrazolyl compounds were prepared in this present work and spectroscopic evidence strongly supports the suggested compounds. Compound 6a and 6b are promising alpha-amylase inhibitory activity in comparison with reference compound Acarbose. These compounds can be considered as lead compounds as anti-diabetic agents. Results of the antimicrobial study show that all the synthesized compounds can be modified structurally to improve their antimicrobial profile.