INTRODUCTION:

Cancer is a class of disease in which a group of cells display uncontrolled growth, invading adjacent tissues and sometimes metastasis or spreading to other locations in the body via lymph or blood. Cancer is a disease characterized by a shift in the control mechanisms that governs cell survival, proliferation and differentiation. From the crab, karkinos in Greek and cancer in Latin came the name of the disease and the name of its inducing agents, carcinogens¹. Types of Cancer classified according to organ system includes blood cancer, bone cancer, brain cancer, breast cancer,, digestive cancer, endocrine cancer, eye cancer, genitourinary cancer, gynecologic cancer etc². Cancer is a class of diseases characterized by uncontrolled cell growth. More than 100 types of cancer have been reported and each is classified by the nature of cell that is primarily affect³. Cancer pathogenesis is traceable back to DNA mutations that impact cell growth and metastasis.

The DNA mutation causing agents are termed as mutagens and further the mutagens which causes cancer is known as carcinogen. The mechanism by which cancers occur is incompletely understood. Cancer or neoplasm is thought to develop from a cell in which the normal mechanism for control of growth and proliferation are altered⁴. Four major therapy modalities exist today which can be used in attempt to bring about the requisite malignant cellular reduction as

(A) Surgery (B) Radiotherapy (C) Chemotherapy (D) Immunotherapy and other biological therapy⁵.

Thiadiazoles contains the five membered diunsaturated ring structure. This nucleus act as a “hydrogen binding domain” with “two-electron donar system”. They occur in four isomeric form as shown below⁶.

Figure 1. Different types of Thiadiazoles

Imidazothiadiazoles are formed by the fusion of 1,3,4 thiadiazole moiety with imidazole ring. The fusion of these two ring systems leads imidazothiadiazoles to have a bridgehead nitrogen atom⁷.

Figure 2. Basic Imidazo[2,1-b][1,3,4]thiadiazole nucleus

To gain an view of the mechanism of action of the novel synthesized derivatives, the active ones were subjected to molecular docking studies. Molecular docking study was performed for compounds displaying a significant inhibitory activity in order to validate their binding mode to active site. All the molecular modeling studies were carried out using Maestro 10.5 program (Schrodinger Inc. USA). The X-ray crystallographic structure of Tubulin-colchicine-soblidotin: Stathmin-like domain complex Receptor co-crystallized (PDB ID:3E22) with the Colchicine was downloaded from the protein data bank. . The protein preparation was done in three step ‘‘i.e.” preprocess, review and modify and refinement using ‘protein preparation wizard’ in Maestro 10.5. In these steps, water molecules are deleted and hydrogen atoms are added. The Energy of the structure was minimized using OPLS 2005 force field. Similarly, ligands were prepared again using force field 2005.The ligand and the default box was prepared. The ligand was docked into the grid generated from the protein using extra precision (XP). The results were evaluated by glide score (docking score) higher the docking score more is the binding affinity. The co-crystallized ligand was used to define the binding site for docking⁸^,⁹.

MATERIAL AND METHODS:

Chemical and reagents:

Chemicals used were of LR grade and purchased from Spectrochem, Sigma Aldrich, Alpha, Merck India Ltd., Loba Chemical, Central Drug House Ltd, and S.D. Fine Chemicals Ltd. All solvents were used after purification, distillation and drying. Silica gel pre-coated plates from E. Merck and Co., were used for TLC and spots were located either by UV or by dipping in potassium permanganate solution. Solvent system used for developing the chromatograms were chloroform: methanol (8:2 V/V) and Toluene: ethyl acetate: formic acid (TEF) (5:4:1 V/V/V). All the melting points were determined in open capillary tubes by heating in paraffin bath and are uncorrected. The IR spectra (KBr) were recorded on a Perkin Elmer IR spectrometer 4000-400 cm^-1 using IR grade KBr disc at SAIF, Panjab University (Chandigarh). ¹H NMR and ¹³C NMR spectra of the synthesized compounds were recorded in CDCl₃ /DMSO solution on a Bruker Avance II 400 MHz NMR spectrometer at SAIF, Panjab University (Chandigarh). Proton chemical shifts are relative to Tetramethylsilane (TMS) as internal standard. Mass spectra were recorded on MAT 120 at SAIF, Punjab University (Chandigarh).

The synthetic route of the compounds S, (3a-3h) and (S1- S8) are outlined in scheme below. Substituted 1,3,4-thiadiazole III(IIIa-IIIc) was obtained by direct cyclization of substituted naphthoic acids I(Ia-Ic) and thiosemicarbazide (II) in the presence of phosphorous oxychloride, the latter being refluxed with substituted α- haloaryl ketones IV(1-6) in dry ethanol with a drop of dimethyl formamide to yield substituted imidazo[2,1-b]]1,3,4] thiadiazoles S(1-18) in good yield as per the procedure reported by Gadad et al¹⁰. It is well established that this reaction proceeds via the intermediate iminothiadiazole which undergoes dehydrocyclisation to form the desired fused heterocycle under reflux temperature spontaneously. The ring nitrogen of the imino form of thiadiazole is involved preferably α-bromoketones forming an intermediate. It undergoes further cyclodehydration on heating with a suitable medium like ethanol and DMF to afford imidazothiadiazoles in good yields. The cyclodehydration involves intramolecular nucleophilic addition of the 2-amino group to carbonyl functions of the intermediate followed by elimination. The strongly electronegative groups impart less nucleophilic character to the nitrogen at 4th position of the 1, 3, 4- thiadiazole ¹¹.

Anticancer activity:

All of the synthesized compounds were screened against A-549 cell line to determine the growth inhibitory effect of compounds. In vitro testing was done using sulforhodamine B (SRB) assay protocol, each derivative was tested at 4 dose levels (10μg/mL, 20μg/mL, 40μg/ mL, 80μg/mL). The compounds possessed good to moderate anticancer activity. Ten of the synthesized compounds were found to possess good growth inhibition. From the in vitro screening, it has been found that the compounds substituted with electron withdrawing group, e.g. NO₂, OH, OCH₃ had better activity. The activity was directly proportional to the concentration of the compounds utilised to carry out the activity. Further the activity can be improved/ explored by incorporating substituents of different nature and studying the SAR¹².

General Procedure for the synthesis of substituted 1,3,4- thiadiazole derivatives III(IIIa-IIIc)

The mixture of substituted naphthoic acid (50mMol), thiosemicarbazide (50mMol) and POCl₃ (13mL) was stirred and heated at 75°C for 0.5 h. After cooling down to room temperature, water was added. It was refluxed for 4 h. After cooling, the mixture was alkalined to pH 8 by the dropwise addition of 50% NaOH solution under stirring. The precipitate was filtered and recrystallized from ethanol to obtain compound III(IIIa- IIIc)¹³.

Table 1. Physiochemical and Spectral properties of synthesized compounds S(1-18)

S.No	Compound	Molecular Formula	Molecular Weight	Yield %	Melting Point (˚C)	¹H NMR (DMSO, δppm)
S1		C₂₀H₁₃N₃S	327.40	51	201-203	7.4-8.3(m,12H,Ar-H) 8.4(Ar-H, Imidazole)
S2		C₂₀H₁₂N₄O₂S	372.39	56	218-220	7.8-8.3(m,7H,Ar-H) 8.3(Ar- H, Imidazole
S3		C₂₀H₁₂ClN₃S	361.84	52	206-208	7.5-8.3(m,7H,Ar-H) 8.4(Ar-H, Imidazole)
S4		C₂₁H₁₅N₃S	341.42	63	224-226	7.2-8.2(m,7H,Ar-H) 8.4(Ar-H, Imidazole) 2.3 (s, H,-CH₃)
S5		C₂₁H₁₅N₃OS	357.42	60	211-213	7.0-8.0(m,7H,Ar-H) 8.4(Ar-H, Imidazole) 3.83(s, H, -OCH₃)
S6		C₂₀H₁₂FN₃S	345.39	73	219-221	7.2-8.2(m,7H,Ar-H) 8.4(Ar-H, Imidazole)
S7		C₂₀H₁₃N₃O S	343.40	67	216-218	7. 4-8.3(m,9H,Ar-H) 8.4(Ar-H, Imidazole) 5.3 (s, H, Ar-OH)
S8		C₂₀H₁₂ClN₃OS	377.84	63	228-230	7.5-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3 (s, H, Ar-OH)
S9		C₂₁H₁₅N₃OS	357.42	66	221-223	7.2-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH) 2.3 (m, 3H, -CH₃)
S10		C₂₁H₁₅N₃O₂S	373.42	61	226-228	7.0-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.2(s,H, Ar-OH) 3.8 (m, 3H, -OCH₃)
S11		C₂₀H₁₂N₄O₃S	388.39	81	227-229	7.5-8.3(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH)
S12		C₂₀H₁₂FN₃OS	361.39	77	223-225	7.3-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH)
S13		C₂₀H₁₃N₃OS	343.40	73	210-212	7.4-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH)
S14		C₂₀H₁₂ClN₃OS	377.84	63	222-224	7.4-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.2(s,H, Ar-OH)
S15		C₂₀H₁₂N₄O₃S	388.39	83	231-233	7.4-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.2(s,H, Ar-OH)
S16		C₂₁H₁₅N₃OS	357.42	79	210-212	7.2-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH), 2.3(m, 3H,Ar-CH₃)
S17		C₂₁H₁₅N₃O₂S	373.42	74	217-219	7.0-8.2(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH), 3.8 (m, 3H, Ar-OCH₃)
S18		C₂₀H₁₂FN₃OS	361.39	71	220-222	7.3-8.3(m,8H,Ar-H) 8.4(Ar-H, Imidazole) 5.3(s,H, Ar-OH)

Molecular Docking Studies

Table 2. Molecular Docking studies of synthesized compounds S(1-18)

Cpd code	Glide XP Score	Glide XP emodel	Glide Energy	XP LowMW	XP LipophilicEvdW
S1	-4.605	-35.772	-35.589
S2	-3.596	-24.51	-37.698	-0.409	-4.754
S3	-3.987	-36.232	-37.072	-0.259	-3.36
S4	-5.556	-39.301	-38.878	-0.294	-4.146
S5	-4.001	-37.067	-41.856	-0.362	-4.682
S6	-4.537	-40.251	-39.531	-0.309	-4.338
S7	-4.313	-37.931	-43.824	-0.349	-4.636
S8	-5.462	-38.166	-33.916	-0.355	-4.565
S9	-4.124	-34.327	-39.878	-0.244	-4.399
S10	-5.158	-44.442	-43.547	-0.309	-3.874
S11	-3.976	-40.482	-40.814	-0.255	-4.553
S12	-5.232	-39.503	-42.915	-0.205	-4.227
S13	-5.959	-43.687	-39.322	-0.295	-4.315
S14	-4.177	-36.816	-36.171	-0.355	-4.952
S15	-2.882	-33.437	-44.651	-0.241	-4.397
S16	-5.703	-42.102	-38.442	-0.205	-3.068
S17	-4.907	-38.17	-44.461	-0.309	-4.933
S18	-4.57	-42.5	-39.823	-0.255	-4.42
Cholchicine	-5.574	-41.743	-48.551	-0.295	-4.754

Compound name
S4
S12
S10
S13
S16

Figure 4. Schematic diagram showing interaction made by the title compounds (docked on Tubulin-colchicine-soblidotin: Stathmin-like domain complex enzyme (PDB 3E22) using 3D and 2D representation.

Anticancer Activity profile of synthesized derivatives (S1-S18) on human lung cancer line in three experiments:

Table 3. In Vitro anticancer testing results of synthesized compounds S (1-18)

	Human Lung Cancer Cell Line A-549
	% Control Growth
	Drug Concentrations (µg/ml)
	Experiment 1				Experiment 2				Experiment 3				Average Values
	10	20	40	80	10	20	40	80	10	20	40	80	10	20	40	80
S1	66.7	81.5	68.1	6.4	67	71.5	54.6	2.4	78.6	74.5	52.4	-1.4	70.8	75.8	58.3	2.4
S2	63.1	78.8	84.1	66.4	67.9	63.9	74.9	60.6	67.6	73.4	71.3	50.6	66.2	72	76.8	59.2
S3	88.7	114.5	82.4	15.6	83.8	88.2	63.7	14.1	93.9	93.9	56.6	1.7	88.8	98.8	67.6	10.5
S4	97.8	110.2	57.2	26.7	90.4	92.3	29	14.7	92.2	87.3	26.4	9.5	93.5	96.6	37.5	17
S5	66.9	74.3	49.3	8.7	68.9	66.6	35.3	-0.7	71.7	65.3	31.8	0.4	69.2	68.7	38.8	2.8
ADR	12.6	10.1	9.5	12.7	5.3	4.9	6.3	9.2	4.4	3.5	3.3	12.1	7.5	6.2	6.4	11.3
	Human Lung Cancer Cell Line A-549
	% Control Growth
	Drug Concentrations (µg/ml)
	Experiment 1				Experiment 2				Experiment 3				Average Values
	10	20	40	80	10	20	40	80	10	20	40	80	10	20	40	80
S6	68.3	84.6	71	44.9	75.2	77.4	49.2	29.5	77.4	80.4	51.7	42.1	73.6	80.8	57.3	38.8
S7	66.4	71.5	81.4	71.7	64.2	69.7	77.4	67.2	61	81.4	76.8	50.6	63.9	74.2	78.5	63.2
S8	94.2	99.4	89.7	22.7	77.6	71.2	69.2	55.4	97.4	90.3	51.7	19.8	89.7	87	70.2	32.6
S9	99.6	101	61.2	29.3	78.5	84.3	47.2	23.6	87.5	81.4	55.7	26.7	88.5	88.9	54.7	26.5
S10	61.2	77.6	39.4	10.4	61.7	78.2	40.1	11.4	55.4	70.4	27.9	-8.7	59.4	75.4	35.8	4.4
ADR	15.7	18.6	7.8	14.8	9.6	8.8	11.2	10.7	3.4	6.5	9.9	12.7	9.6	11.3	9.6	12.7
	Human Lung Cancer Cell Line A-549
	% Control Growth
	Drug Concentrations (µg/ml)
	Experiment 1				Experiment 2				Experiment 3				Average Values
	10	20	40	80	10	20	40	80	10	20	40	80	10	20	40	80
S11	71.5	89.4	66.4	58.9	66.2	78.2	55.8	47.1	88.9	75.1	41.8	39.4	75.5	80.9	54.7	48.5
S12	66.2	56.7	47.2	13.4	60.2	59.4	51.2	20	59.4	67.9	44.4	-1.4	61.9	61.3	47.6	10.7
S13	54.2	67.9	60.2	44.2	51.9	50.2	48.7	13.5	58.8	66.6	41.2	10.4	55	61.6	50	22.7
S14	87.6	99.4	66.2	55.4	87.5	89.4	78.3	66.6	91.2	78.2	66.4	54.2	88.8	89	70.3	58.7
S15	76.4	87.2	59.2	48.9	79.5	88.3	56.4	47.9	66.7	71.2	57.6	45.2	74.2	82.2	57.7	47.3
ADR	16.4	19.4	9.8	17.5	6.3	7.4	8.4	2.3	2.1	4.6	11.4	14.9	8.3	10.5	9.9	11.6

RESULT AND DISCUSSION:

We tried to synthesized various derivatives of substituted 1,3,4-thiadiazol-2-amine III using substituted naphthoic a acid as starting material. Synthesis was carried according to reaction shown in Scheme 1. The compound III, was further condensed with various substituted bromoacetyl compounds IV in the presence of dry ethanol and refluxed for the duration of 46 h to get 2-(substituted naphtalen-2-yl)-6 (substituted) imidazo[2,1-b][1,3,4]thiadiazole derivatives S(1-18).

The reaction was monitored by thin-layer chromatography using suitable mobile phase such as toluene: ethyl acetate: formic acid (TEF) (5:4:1 V/V/V) and chloroform: methanol (8:2 V/V). The Rf values were compared and found that they were different from each other’s. After the completion of reaction the product were purified by appropriate method. The melting point of the derivatives was determined. The melting points obtained were different from each other, which confirmed the formation of new derivatives. Structure of the synthesized compounds was established on the basis of IR, 1H-NMR, 13C-NMR and Mass spectral data analyses. All the derivatives S (1-18) were further characterized by various spectral techniques. The molecular docking studies of the compounds proved that they fit appropriately into the site of the enzyme to cancerous cells. Analysis of 2D diagram indicates that various non-bonded interactions including polar hydrogen bonding interactions primarily involved between receptor and ligand molecule. The G-scores were observed in the range of 2.88- 5.95 and predicted binding energies, which are listed in (Table 2). The G-scores and binding energy for compound S4, S10, S12, S13 and S16 were observed to be comparable to the standard Colchicine. The anticancer activity of the compounds against human cancer cell line A-549 exhibited that the compounds S4, S10, S12, S13, S16 has a prominent inhibitory effect on the growth of cancerous cell when compared with the standard Adriamycin.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank UIPS Panjab Univeristy, IIT ROPAR and ACTREC Mumbai for their kind support during Spectral analysis and all other lab studies.

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Received on 07.07.2021 Modified on 13.10.2021

Research J. Pharm. and Tech 2022; 15(10):4405-4412.

DOI: 10.52711/0974-360X.2022.00738