Design, Synthesis and Anticancer potential evaluation of Novel Naphthoic Acid linked Imidazo[2,1-b][1,3,4]thiadiazoles

 

Satvir Singh1, Divya. D. Bhandari1, Monika Gupta2, Jagdeep Singh3

1University Institute of Pharmaceutical Sciences, Chandigarh University, Gharuan, Mohali, Punjab, India.

2Department of Pharmaceutical Chemistry, Amar Shaheed Baba Ajit Singh Jujhar Singh Memorial

College of Pharmacy, Bela, Ropar, Punjab, India.

3Indian Institute of Technology, Ropar, Punjab, India.

*Corresponding Author E-mail: divya.pharma@cumail.in

 

ABSTRACT:

A total of 18 derivatives S(1-18) of naphthoic acid linked  Imidazo[2,1-b][1,3,4]thiadiazole were synthesized and tested for their anticancer potential at human cancer cell line A – 549. The interaction of the proposed derivatives with the colchicine binding domains of the receptor was also performed using Maestro 10.5 program (Schrodinger Inc. USA). Molecular docking study was performed for synthesized compounds displaying a significant inhibitory activity in order to validate their binding mode to active site. All the 18 inhibitors were docked into the active site of the receptor in two poses. The G-scores and binding energy for compound S4, S10, S12, S13 and S16 were observed to be comparable to the standard Colchicine. The compound S1, S5, S10, S12 and S16 exhibited prominent inhibition of cancer cell line.

 

KEYWORDS: Naphthoic acid, Anticancer, Docking, Colchicine, Receptor.

 

 


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.


 

Figure 3. Reaction Scheme for the synthesis of proposed derivatives (Scheme 1)

 

 


General procedure for the synthesis of 2-(substituted naphtalen-2-yl)-6 (substituted) imidazo[2,1-b][1,3,4]thiadiazole derivatives S(1-18):

A mixture of equimolar quantities of substituted 1,3,4- thiadiazole derivatives III(IIIa-IIIc) and substituted bromoacetyl compound IV(1-6) was refluxed in dry ethanol for 46 h. The excess of solvent was distilled off and the solid hydrobromide that separated was collected by filtration, suspended in water and neutralized by aqueous sodium carbonate solution to get free base S (1-18). It was filtered, washed with water, dried and recrystallized from suitable solvent like ethanol, methanol and acetone14.


 

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

Accepted on 02.12.2021           © RJPT All right reserved

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

DOI: 10.52711/0974-360X.2022.00738