Synthesis, Characterization and Anticancer Evaluation of Novel Analogues of Pyrazoles

 

Babli Khatun¹, Venkatesh Kamath², Muddukrishna B. S.³, Aravinda Pai¹*

¹Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal - 576104, Karnataka, India.

²Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal - 576104, Karnataka, India.

³Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical Sciences,

Manipal Academy of Higher Education, Manipal - 576104, Karnataka, India.

 

ABSTRACT:

In the present study, a series of novel substituted pyrazole chalcones and pyrazole oximes (CF 1-15) were synthesized and characterized. Logp values were determined to assess their hydrophobicity. MTT (methyl tetrazolium) assay was performed on A-549 (lung cancer) cell lines. The MTT assay results showed the effect of various substituents on the pyrazole template that could influence their cytotoxic effect. Out of the 15 compounds screened against A-549 cell lines, the compound CF-6 showed appreciable cytotoxicity against the standard doxorubicin.

 

 

 

1. INTRODUCTION:

“Cancer, is known as a malignant neoplasm, is a wide group of diseases where all are involved in unregulated cell growth. Here, cells split and proliferate in an uncontrollable manner by developing malignant tumors, and spread to close by parts of the body”. The cancer may even escalate to parts of the body that are distant from the original site through the lymphatic system or bloodstream. Further it originates from a single abnormal cell having an altered DNA sequence caused by mutation and uncontrolled proliferation of these cells occur which leads to clinically significant stages. There are more than 200 different known cancers that exasperate human bodies.¹

 

In the past few decades several hallmarks of cancer have been identified. According to a review by “Hanahan D and Weinberg RA, the hallmarks of cancer” consists six biological capabilities which encompass sustaining “proliferative signaling”, “evading growth suppressors”, “resisting cell death”, “enabling replicative immortality”, “inducing angiogenesis”, and “activating invasion” and “metastasis” which are possessed during the multistep evolution of human tumors².

 

There is a concern regarding the major public health in the world owing to the emergence of cancer due to ecological imbalance set. In the past few decades much surveillance has drawn towards Pyrazole and its derivatives because of its extensive range of biological activities. Thousands of researchers utilize molecular modelling as a tool for designing novel pyrazole analogs that may target those receptors which are responsible for cancer management that including protein kinase, tyrosine kinase, vascular endothelial growth factor (VEGF), BRAF gene, cyclin dependent kinase (CDK) and tumor growth factor (TGF) ³.

 

Pyrazoles are the important members of heterocyclic family with two neighbouring nitrogens among which one is basic and the other is neutral in a five-membered ring system. These are aromatic molecules for their requisite planar conjugated ring structures having six delocalized π-electrons. Pyrazoles possess antimicrobial, analgesic, anticancer, anti-tubercular, anti-inflammatory, antidepressant, anticonvulsant, antihyperglycemic, antipyretic, anthelmintic, antioxidant and herbicidal properties. Pyrazole is a multifunctional lead compound that has evolved into therapeutically active agents. Various synthetic pathways are conceded for the evolution of pyrazole restraining reactions to impart novel and potent molecule having extensive opportunity in the area of medicinal chemistry. ⁴

 

Fig. 1: Biological activities of Pyrazole moiety

 

The pyrazole moiety is present as the core in a variety of leading anticancer drugs such as Ruxolitinib (blood cancer), axitinib (renal cancer), crizotinib (lung cancer) etc.

 

MATERIALS AND METHODS:

Chemistry:

Instruments, chemicals and reagents:

All the chemicals and regents were purchased from Sigma chemicals, SRL Ranbaxy and cisco chemicals. UV spectrum was recorded using Shimadzu UV-1800, IR spectrum was recorded using IR affinity from Shimadzu, and Mass spectrum was recorded using LCMS utilizing APCI as ionization method, NMR spectrum was recorded using Bruker-400 and redox potential was determined using CH-electrochemical analyzer USA.

 

Synthetic Procedure:

Step-1(Preparation of Chalcone):

Acetophenone (0.01mol) and corresponding aromatic aldehyde (0.01mol) were dissolved in 15ml ethanol, 0.5 ml solution of sodium hydroxide (0.03mol) was added slowly and stirred the mixture for 24 hours. The reaction was monitored by TLC. After completion of the reaction, the reaction mixture was poured in 10ml of ice water and kept for stirring and was neutralized by adding HCl dropwise. It was observed that solid precipitation was forming. Then the solid product was filtered, dried and purified by recrystallization using ethanol. (Product I) ⁵

 

Step-2(Cyclization of chalcone in presence of Hydrazine hydrate and Glacial Acetic acid):

In a round bottomed flask chalcone (0.001mol), Hydrazine hydrate (0.001mol) and glacial acetic acid (15ml) was taken and then refluxed and after four hours few drops of concentrated hydrochloric acid was added and continued refluxing. The reaction was monitored by taking TLC. After completion of the reaction the reaction mixture was poured into ice water. Then it was kept overnight for cooling and solid precipitation of product was observed. The product was filtered. (Product II)

 

Step-3a (Preparation of Oxime):

A mixture of product II (0.001mol), hydroxylamine hydrochloride (0.001mol), ethanol (10ml) and water was taken in a round bottom flask and sodium hydroxide was added with shaking in that mixture and refluxed for 12 hours. The reaction mixture was monitored by taking TLC. After completion the reaction mixture was cooled and ice cold diluted HCl was added dropwise for neutralization. After neutralization the solid product was precipitated and the product was filtered (Product IIIa). ⁶

 

Step-3b (preparation of chalcone):

Product II (0.01mol) and aromatic aldehyde (0.01mol) were dissolved in (5ml, 95%) ethanol, 0.5ml, solution of sodium hydroxide (0.03mol) was added slowly and stirred the mixture for 24 hours. The reaction was monitored using TLC. The reaction mixture was poured in 10ml of ice water and kept for stirring and was neutralised by adding HCl dropwise. It was observed that solid precipitation was forming. Then solid product was filtered, dried and purified through recrystallization with the help of ethanol. (product IIIb) ⁷

 

The melting point, partition coefficient, molar absorptivity and Rf values were recorded using standard procedures.

 

Biological activity:

MTT Assay:

Principle:

MTT assay is a colorimetric assay which quantifies depletion of yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)⁸ by mitochondrial succinate dehydrogenase. The MTT undertakes cells followed by proceeding to the mitochondria where this gets diminished to formazan which is an insoluble, dark purple product. After that cells are solubilized using acidic isopropanol. Then solubilized formazan is then is quantified by spectrometric means.

 

 

The MTT Assay quantifies the cell multiplication and contrarily when metabolic events conducts sudden cell death, the depletion in cell viability. In deprivation of cells the MTT reagent produces lower absorbance values. For the accurate measurement of changes in the deprivation of cells, the linear relationship between cell number and signal produced is inveterate for every cell types.

 

MTT Assay Protocol:

MTT assay was performed to check the cytotoxic effect of chalcones on two cell lines. The cancer cells were grown in specified media (DMEM) consisting of with 8-10% phosphate buffered saline and 50µg mL^-1 erythromycin phosphate, maintained at ambient temperature with 5% CO₂. Specified number of cells were seeded in a 96-well plate and were incubated for a period of 24 hours. Specified concentration of sample solutions were prepared in DMSO. Quercetin was used as a standard drug. The absorbance was measured at 540 nm. The toxicity of the compounds on each cell lines were expressed as the IC50 value

 

Fig. 2: General Scheme for Synthesis of Pyrazole Derivatives

 

 

RESULTS AND DISCUSSIONS:

Synthesis and characterization:

The synthesis of all the final test compound was achieved in 3 steps. In the first step, nucleophilic attack on carbonyl carbon was performed. The reaction was monitored through TLC using solvent system comprising of chloroform: methanol in the ratio 9:1. Purification of the compound from step one, was done by recrystallization from ethanol and the purification required at both, step 2 and step 3 was done using ethanol; using gradient flow (up till 4%). The formation of the above product was confirmed by melting point, IR, Mass spectroscopy and NMR.

 

The second step involved by forming an acid catalyzed imine in which the hydrazine hydrate attacks the carbonyl carbon and this diamine compounds are deprotonated to form the acid catalyst and gives the pyrazole product and was confirmed by melting point, UV, IR, Mass spectroscopy and NMR.

 

In Step 3 a oxime is formed by reacting with an aromatic ketone and hydroxylamine hydrochloride with sodium hydroxide providing alkaline environment. Nucleophilic addition of hydroxylamine to the aromatic ketone forms the basis of this reaction. The product was purified by recrystallization using ethanol and confirmed by melting point, UV, IR, Mass spectroscopy and NMR. In step 3b follows the same mechanism of step 1 to get final product was purified by recrystallization using ethanol. The formation of the above product was confirmed by melting point, IR and Mass spectroscopy. Physical characterization is presented in Table 1

 

 

Table 1: Physico chemical characterization of synthesized test compounds

Sr. No.	Com. Code	Structure	MF	MW	MP	% yield	Rf	Log P	λmax
1	CF-1		C25H20Cl2N2O2	450	240	70	0.29	6.1	259
2	CF-2		C18H18ClN3O2	343	340	52	0.35	3.7	263
3	CF-3		C25H20BrClN2O2	494	330	68	0.30	6.3	256
4	CF-4		C18H18BrN3O2	387	331	50	0.36	3.9	268
5	CF-5		C18H18ClN3O	327	380	53	0.31	4.32	246
6	CF-6		C18H18ClN3O	327	380	53	0.31	4.32	246
7	CF-7		C24H18BrClN2O2	481	345	73	0.61	6.12	214
8	CF-8		C17H16ClN3O2	329	349	53	0.52	3.44	205
9	CF-9		C27H22ClN3O2	455	375	71	0.37	5.10	261
10	CF-10		C20H23N3O3	353	335	51	0.41	3.36	301
11	CF-11		C26H26ClN3O2	441	320	69	0.26	5.59	261
12	CF-12		C26H26N4O4	452	262	65	0.21	4.57	266
13	CF-13		C25H19Cl3N2O2	485	260	72	0.28	6.67	294
14	CF-14		C25H19Cl2N3O4	495	253	62	0.32	6.31	256
15	CF-15		C26H19Cl2N3O2	476	295	76	0.23	6.15	257

 

 

In vitro anticancer activity:

The anticancer activity⁹ of the test compounds were performed by MTT assay protocol. The effect of synthesized compounds on cell viability is expressed in terms of their IC50 value and were compared with a standard drug doxorubicin as shown in the Table. In the present study, Pyrazole derivative like CF-6 (Chloro methyl substituted pyrazole oxime) has shown an appreciable cytotoxicity against A-549 (lung cancer cell lines) with an IC 50 value of 12.5 micromolar which is comparable to that of the standard doxorubicin with an IC 50 value of 0.3 micromolar against the same. Anticancer activity of the synthesized test compounds were given in Table 2.

 

Table 2: Anticancer activity for the synthesized test compounds

S. No.	NAME	IC 50- A549 (μM)
1.	Doxorubicin	0.3
2.	CF-1	37.5
3.	CF-3	27.5
4.	CF-6	12.65
5.	CF-9	25
6.	CF-2	47.5
7.	CF-4	37.5
8.	CF-5	35
9.	C-13	47.5

 

CONCLUSIONS:

In the present study, we have performed the anticancer screening for different analogues of pyrazole substituted chalcones and pyrazole oximes against A-549 lung cancer cell lines. Out of the 15 pyrazole derivatives synthesized and screened, the oxime derivative CF-6 emerged out as a potent cytotoxic agent with appreciable cytotoxicity against A-549 (lung cancer) cell lines. The lipophilicity value was in line with its anticancer activity. The present study provides insight for the better understanding of mechanistic studies of novel pyrazole analogues against anticancer targets such as Hydrolase identified in lung cancer.

 

ACKNOWLEDGEMENTS:

Authors acknowledge Manipal Academy of Higher Education for the support provided to carry out the present research.

 

REFERENCES:

1.            Rehman K, Tariq M, Akash MS, Gillani Z, Qazi MH. Effect of HA 14‐1 on Apoptosis‐ Regulating Proteins in HeLa Cells. Chemical Biology and Drug Design. 2014 Mar;83(3): 317-23.

2.            Hejmadi M. Introduction to Cancer Biology. Bookboon; 2009.

3.            Hanahan D, Weinberg RA. The Hallmarks of Cancer. Cell. 2000 Jan 7;100(1): 57-70.

4.            Kumar H, Saini D, Jain S, Jain N. Pyrazole scaffold: a remarkable tool in the development of anticancer agents. European Journal of Medicinal Chemistry. 2013 Dec 1;70: 248-58.

5.            Zheng Y, Zheng M, Ling X, Liu Y, Xue Y, An L, Gu N, Jin M. Design, synthesis, quantum chemical studies and biological activity evaluation of pyrazole–benzimidazole derivatives as potent Aurora A/B kinase inhibitors. Bioorganic and Medicinal Chemistry Letters. 2013 Jun 15;23(12): 3523-30.

6.            Li X, Wang L, Long L, Xiao J, Hu Y, Li S. Synthesis and biological evaluation of 1, 2, 4-trisubstituted imidazoles and 1, 3, 5-trisubstituted pyrazoles as inhibitors of transforming growth factor β type 1 receptor (ALK5). Bioorganic and Medicinal Chemistry Letters. 2009 Aug 15;19(16): 4868-72.

7.            Kupcewicz B, Ciolkowski M, Karwowski BT, Rozalski M, Krajewska U, Lorenz IP, Mayer P, Budzisz E. Copper (II) complexes with pyrazole derivatives–synthesis, crystal structure, DFT calculations and cytotoxic activity. Journal of Molecular Structure. 2013 Nov 25;1052: 32-7.

8.            Koca I, Özgür A, Coşkun KA, Tutar Y. Synthesis and anticancer activity of acyl thioureas bearing pyrazole moiety. Bioorganic and Medicinal Chemistry. 2013 Jul 1;21(13): 3859-65.

9.            Fan C, Su H, Zhao J, Zhao B, Zhang S, Miao J. A novel copper complex of salicylaldehyde pyrazole hydrazone induces apoptosis through up-regulating integrin β4 in H322 lung carcinoma cells. European Journal of Medicinal Chemistry. 2010 Apr 1;45(4): 1438-46.

 

 

 

 

Received on 29.04.2020            Modified on 04.07.2020

Accepted on 09.08.2020         © RJPT All right reserved