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Document Type : Original Article

Authors

1 Babylon Health Directorate, Al Hillah, Iraq

2 Al-Esraa University College, Baghdad, Iraq

3 Department of Pharmaceutics, College of Pharmacy, University of Al Kafeel . Najaf. Iraq

Abstract

New 6-chloro-1,2,3,4-tetrahydrocarbazole compounds were prepared from the reaction of cyclohexanone with 4-chlorophenylhydrazine. The synthesis of heterocyclic compounds was carried out through the reaction of cyclohexanone with 4-chlorophenylhydrazine, then the produced compound was treated with two NSAIDs (indomethacin, diclofenac) by amide bond formation to form N-substituted THCZ by NSAIDs. The characterization of prepared compounds was identified using the 1H, 13C NMR and FT-IR spectroscopies. The antimicrobial activity of the synthesized compounds was tested in vitro to discover a good activity as antifungal and moderate as antibacterial, which was confirmed by the docking study of the compounds.

Graphical Abstract

Substituted Tetrahydrocarbazole Based on Indomethacin and Diclofenac with Heterocyclic Compound, Synthesis, Spectral and Antimicrobial Studies

Keywords

Main Subjects

Introduction

This research was motivated by the rise in antimicrobial resistance worldwide and the inadequate generation of novel antimicrobial drugs (1). It focused on the synthesis and antimicrobial activity of compounds produced by combining two biologically active compounds (NSAIDS and THCZ).

THCZ and Carbazole ring scaffolds are present in some alkaloids [2], they have a variety of biological effects such as antiviral (3), antitumor (4), antibacterial (5, 6) and broad-spectrum antifungal activity [7,8].

NSAIDs are considered as non-antibiotic drugs reported to display antibacterial activity [9], Some NSAIDs such as indomethacin and diclofenac have been shown to have synergistic effect with particular antimicrobial drugs [10]. Some NSAIDS like Diclofenac have an inhibitory impact on some bacteria like S. aureus [11].

The compounds 6-chloro-1,2,3,4-tetrahydrocarbazole (symbolized as 6C) and NSAID acid chloride reacted under reflux for 9 hours in the existence of triethylamine to form 6-chloro-1,2,3,4-tetrahydrocarbazole substituted at the heteroatom (N) by NSAIDs [12] as shown in Scheme 2. 6C was prepared by Borsche-Drechsel Reaction (Scheme 1) involving the addition of 4-chlorophenylhydrazine to cyclohexanone in acidic media (glacial acetic acid) with reflux for 2 hours [13]. NSAIDs (indomethacin and diclofenac) reacted with Thionyl chloride under reflux for 4 hours to produce NSAIDs acid chloride [14].

Scheme 1: Proposed mechanism of Borsche-Drechsel Reaction

Materials and Methods

Cyclohexanone and Benzene of ROMIL limited (Cambridge, UK), Indomethacin was obtained from SAFA Pharmaceutical Industries (Iraq), Diclofenac from Sama Al Fayhaa pharmaceutical industries (Iraq), 4-chlorophenylhydrazine from Hangzhou Hyper Chemicals limited (China), Thionyl chloride and methyl alcohol of CDH limited (India) and Diethyl Ether of Alpha Chemicals Private Limited (India). Other materials included the culture media (Sabouraud Dextrose Agar and Mueller-Hinton agar) of CONDA Pronadisa (Madrid, Spain), Ciprofloxacin disc of TMMEDIA (India) and DMSO of Alpha Chemicals Private Limited (India).

The Equipments used in this work included Hot plate stirrer of IKA (Germany), IR spectrometer of Shimizu (Japan), melting point system of Stuart (United Kingdom), Oven of Astell Hearso (England), Rotary evaporator of Bushi (Switzerland), 13C NMR spectrophotometer of Varian inova (USA), 1H-NMR spectrophotometer of Varian inova (USA).

Preparation of the compound 1-(6-chloro-1,2,3,4-tetrahydro-9H-carbazol-9-yl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl) ethan-1-one symbolized as (CI)

The compound (CI) was prepared by adding (0.01 mole, 3.7 g) of indomethacin acid chloride in 20 mL dry benzene and 1 mL triethylamine with stirring to (2 g, 0.01 mole) 6-chloro-1,2,3,4-tetrahydrocarbazole in 30 mL dry benzene, as shown in Scheme 2. The mixture was refluxed for 9 hours and after the distillation of the solvent the precipitate was washed with 5% sodium bicarbonate and water [12].

Acid chloride of Indomethacin was prepared (as in Scheme 2) by addition of (0.01 mole, 0.7 mL) thionyl chloride slowly to (0.025 mole, 8.9 g) indomethacin solubilized in 25 mL dry benzene, the mixture was refluxed for 4 hour, left to dry, washed with 5 mL diethyl ether and dried to produce indomethacin acid chloride [14].

The compound 6C was prepared by addition of (7.1 g, 0.05 mole) 4-chloroPhenylhydrazine during one hour to a solution of (0.05 mole, 5.2 mL) cyclohexanone in 17 mL acetic acid under reflux with stirring. The mixture was refluxed for one more hour, and filtered. The solid crude was washed by water and 75% methyl alcohol and dried to obtain 6-chloro-1,2,3,4-tetrahydrocarbazole. [15].

Preparation of the compound 1-(6-chloro-1,2,3,4-tetrahydro-9H-carbazol-9-yl)-2-(2-((2,6-dichlorophenyl) amino) phenyl) ethan-1-one Symbolized as (CD)

The compound (CD) was prepared by adding (0.01 mole, 3.1 g) of diclofenac acid chloride in 20 mL dry benzene and 1 mL triethylamine with stirring to (2 g, 0.01 mole) 6-chloro-1,2,3,4-tetrahydrocarbazole in 30 mL dry benzene. The mixture was refluxed for 9 hours. After the distillation of the solvent the precipitate was washed with 5% sodium bicarbonate and water [12].

Acid chloride of diclofenac was prepared by addition of (0.01 mole, 0.7 mL) thionyl chloride slowly to (0.025 mole, 7.4 g) diclofenac solubilized in 25 mL dry benzene. The mixture was refluxed for 4 hours, left to dry, washed with 5 mL diethyl ether and dried to produce diclofenac acid chloride [14].

Results and Discussion

The prepared compounds were summarized in the following Scheme 2.

Scheme 2:  The general rout of compound synthesis

Identification of the compounds

The synthesized compounds were identified by 1H, 13C NMR and IR spectroscopies.

The 1H NMR spectroscopy interpretation of compound 6-chloro-1,2,3,4-tetrahydrocarbazole

The 1H NMR spectrum of the compound 6C Figure 3S displays Chemical shifts. The interpretation of these spectra was summarized in Table 1.

 

Table 1: 1H NMR spectroscopy interpretation of the compound 6C

Chemical structure

Chemical shift

NO. of H

splitting

Interpretation

 

7.35

1

singlet

Proton of benzene ring

6.99

1

doublet

Proton of benzene ring

7.26

1

doublet

Proton of benzene ring

10.86

1

singlet

Proton of NH group

2.72

2

triplet

Proton of CH2 group (Allylic)

1.78

2

quintet

Proton of CH2 group (aliphatic)

1.82

2

quintet

Proton of CH2 group (aliphatic)

2.69

2

triplet

Proton of CH2 group (Allylic)

 

 

The 13C NMR spectroscopy interpretation of the compound 6C

The 13C NMR spectrum of the compound 6C Figure 4S displays Chemical shifts. The interpretation of these spectra was summarized in Table 2.

 

Table 2: 13C NMR spectroscopy interpretation of the compound 6C

Interpretation

Chemical shift

Chemical structure

Aromatic Carbon

116.85

 

Aromatic Carbon

123.13

Aromatic Carbon

120.19

Aromatic Carbon

112.33

Aromatic Carbon

134.54

Aromatic Carbon

136.96

Allylic Carbon

23.30

Aliphatic Carbon

23.18

Aliphatic Carbon

23.23

Allylic Carbon

20.90

Aromatic Carbon

108.58

Aromatic Carbon

128.92

 

The IR spectroscopy interpretation of the compound 6C

The IR spectrum of the compound 6C Figure 5S displays bands; the interpretation of these spectra was summarized in Table 3.

 

Table 3: IR spectroscopy interpretation of the compound 6C

Interpretation

Band

Chemical structure

NH band of secondary amine

3404.88

 

Aromatic -H

2938.37

Asymmetric H of cyclohexane

2905.99

Symmetric H of cyclohexane

2842.81

Aromatic C=C

1578.54

 

The 1H NMR spectroscopy interpretation of compound CI

The 1H NMR spectrum of the compound CI Figure 6S displays Chemical shifts; the interpretation of these spectra was summarized in Table 4.

 

Table 4: 1H NMR spectroscopy interpretation of the compound CI

Chemical structure

Chemical shift

NO. of H

splitting

Interpretation

 

7.25

1

s

Proton of benzene ring

7.05

1

d

Proton of benzene ring

7.34

1

d

Proton of benzene ring

2.60

2

t

Proton of CH2 group (Allylic)

1.84

2

q

Proton of CH2 group (Aliphatic)

1.85

2

q

Proton of CH2 group (Aliphatic)

2.69

2

t

Proton of CH2 group (Allylic)

3.67

2

s

Proton of CH2-CO

6.93

1

s

Proton of benzene ring

3.77

3

s

Proton of CH3-O

6.71

1

d

Proton of benzene ring

7.37

1

d

Proton of benzene ring

7.69

2

t

Proton of benzene ring

7.66

2

t

Proton of benzene ring

2.23

3

s

Proton of CH3 group(Allylic)

 

The 13C NMR spectroscopy interpretation of the compound CI

The 13C NMR spectrum of the compound CI Figure 7S displays Chemical shifts; the interpretation of these spectra was summarized in Table 5.

 

Table 5: 13C NMR spectroscopy interpretation of the compound CI

Chemical structure

Chemical shift

Interpretation

 

 

 

 

 

 

 

 

 

116.85

Aromatic Carbon

123.11

Aromatic Carbon

120.19

Aromatic Carbon

113.95

Aromatic Carbon

131.61

Aromatic Carbon

136.97

Aromatic Carbon

23.29

Allylic carbon

23.16

Cyclic carbon

23.21

Cyclic carbon

20.89

Allylic carbon

111.76

Aromatic Carbon

128.78

Aromatic Carbon

172.56

Carbonyl Carbon

30.04

Next to carbonyl

108.58

Aromatic Carbon

134.53

Aromatic Carbon

102.18

Aromatic Carbon

156.01

Aromatic next to oxygen

112.34

Aromatic Carbon

115.04

Aromatic Carbon

128.91

Aromatic Carbon

134.63

Aromatic Carbon

167.54

Carbonyl Carbon

130.68

Aromatic Carbon

131.22

Aromatic Carbon

129.53

Aromatic Carbon

138.07

Aromatic Carbon

55.87

Aliphatic next to Oxygen

13.67

Aliphatic Carbon

 

The IR spectroscopy interpretation of the compound CI

The IR spectrum of the compound CI Figure 8S displays bands; the interpretation of these spectra was summarized in Table 6.

 

Table 6: IR spectroscopy interpretation of the compound CI

Chemical structure

Band

Interpretation

 

3088.69

Aromatic C-H

3034.66

Aromatic C-H

2938.29

Asymmetric H

2842.18

Symmetric H

1579.12

Aromatic C=C

1676.64

Carbonyl

1696.90

Carbonyl

                                                                                                                                                                                                                                                                                                                                

The 1H NMR spectroscopy interpretation of the compound CD

The 1H NMR spectrum of compound CD Figure 9S displays Chemical shifts; the interpretation of these spectra was summarized in Table 7.

 

Table 7: 1H NMR spectroscopy interpretation of the compound CD

Chemical structure

Chemical shift

NO. of H

splitting

Interpretation

 

7.2

1

s

Proton of benzene ring

7.21

1

d

Proton of benzene ring

7.33

1

d

Proton of benzene ring

2.7

2

t

Proton of CH2 group (Allylic)

1.78

2

q

Proton of CH2 group (Aliphatic)

1.83

2

q

Proton of CH2 group (Aliphatic)

2.67

2

t

Proton of CH2 group (Allylic)

3.88

2

s

Proton of CH2-CO

7.1

1

d

Proton of benzene ring

6.4

1

t

Proton of benzene ring

7.25

1

t

Proton of benzene ring

7.23

1

d

Proton of benzene ring

9.85

1

s

Proton of NH group

7.37

2

q

Proton of benzene ring

7.18

1

t

Proton of benzene ring

                                                                                                                                                                                                                                                                                                                                                                                                 

 

The 13C NMR spectroscopy interpretation of the compound CD

The 13C NMR spectrum of the compound CI Figure 10S displays Chemical shifts; the interpretation of these spectra was summarized in Table 8.

 

Table 8: 13C NMR spectroscopy interpretation of the compound CD

Chemical structure

Chemical  shift

Interpretation

 

120.19

Aromatic carbon

123.30

Aromatic carbon

123.11

Aromatic carbon

112.34

Aromatic carbon

134.91

Aromatic carbon

136.97

Aromatic carbon

23.29

Allylic Carbon

23.16

Aliphatic carbon

23.22

Aliphatic carbon

20.89

Allylic Carbon

108.97

Aromatic carbon

132.38

Aromatic carbon

173.69

carbonyl group

35.55

next to carbonyl

125.45

Aromatic carbon

28.281

Aromatic carbon

116.86

Aromatic carbon

30.341

Aromatic carbon

129.85

Aromatic carbon

128.91

Aromatic carbon

143.26

Aromatic carbon

134.53

Aromatic carbon

 

The IR spectroscopy interpretation of the compound CD

The IR spectrum of the compound CD Figure 11S displays bands; the interpretation of these spectra was summarized in Table 9.

Table 9: IR spectroscopy interpretation of the compound CD

Chemical structure

Band

Interpretation

 

3404.20

NH of amine

3137.53

Aromatic H

3060.81

Aromatic H

2937.60

Asymmetric aliphatic H

2841.83

Symmetric aliphatic H

1731.81

Carbonyl group

1612.28

Aromatic C=C

 

The properties of the synthesized compounds are listed in Table (10).

 

Table 10: properties of the compounds

Colour

Melting Point

Molecular weight

Chemical Formula

Compounds

Wheat

144-147 °C

205.68

C12H12ClN

6-chloro-1,2,3,4 tetrahydrocarbazole

Brown

108-111 °C

545

C31H26Cl2N2O3

CI

Dark Red

99-103 °C

483.8

C26H21Cl3N2O

CD

 

Docking study

Molecular docking experiments were conducted to investigate the binding modes of these compounds with the target enzyme Sterol 14-demethylase (CYP51), a cytochrome P450 enzyme necessary for sterol biosynthesis in eukaryotic cells and a significant target of therapeutic medications used to treat fungal infections [16]. The receptor used was CYP51 (PDB code 5TZ1) from the RCSB protein data bank. Genetic Optimization for Ligand Docking was used to dock the identified molecules (GOLD). GOLD searches binding ligand conformational space using a genetic algorithm and assigns a score of binding residues. Poses are ranked using GOLD scores [17].

 

Table 11: Docking score with Amino Acid of Fungal candida albicans, PDB: 5TZ1

ligand

Structure

fitness score

A.A (H bond interaction

A.A (Other interaction)

REF. Ligand (Fluconazole)

 

78.58

-

THR 311, HEM 601 (3), LYS 143, HIS 468, ILE 131 (3), TYR 132 (4)

C.I.

 

88.47

TYE 132, HEM 601

HEM 601 (3), PHE 233 (2), MET 508 ( 5 ), LEU 121 (3), TYR 118 (3), LEU 376 (3), TYR 132 (2), THR 311 (7), GLY 307 (2), ILE 131, PHE 288 (7)

C.D.

 

110.7

TYR 132

HEM 601 (5), THR 311 (4), GLY 307 (2), TYR 132 (7), ILE 131 (3), MET 508 (4), LEU 121 (2)

 

Docking scores of the standard compound (fluconazole) and the synthesized compounds (6-chloro-1,2,3,4-tetrahydrocarbazole, CI and CD) with Amino Acids of Fungal candida albicans and their interactions are listed in Table 11. The interactions of the CI and CD compounds with 5TZ1 protein are shown in Figures 1 and 2, respectively.


Antimicrobial activity

The antibacterial activity of the new compounds was tested in vitro depending on well diffusion assay against 3 types of gram negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli), one type of gram positive bacteria (S. aureus) and one type of fungi (candida albicans) using agar plate (Sabouraud Dextrose Agar for the fungi and Mueller-Hinton agar for the bacteria) and 4 serial dilutions of the new compounds with DMSO as solvent. Ciprofloxacin as antibacterial and fluconazole as antifungal were used as reference drugs for the comparison.

The compound 6C showed reasonable activity against all types of bacteria except S. aureus as listed in Table 12.

 

 Table 12: Antimicrobial activity of the newly synthesized compounds based on well diffusion assay expressed as inhibition diameter zones in millimeters (mm)

 

Cipro

Flu

6C

CI

CD

 

 

 

stock

1

2

3

4

stock

1

2

3

4

stock

1

2

3

4

S. aureus.

18

 

0

0

0

0

0

15

14

12

11

7

16

14

9

7

3

P. aeruginosa

20

 

17

16

14

12

11

0

0

0

0

0

10

0

0

0

0

E.coli

23

 

20

18

0

0

0

0

0

0

0

0

0

0

0

0

0

K. pneumoniae

11

 

16

14

12

10

7

14

12

9

8

5

14

12

10

9

3

C. albicans

 

14

12

11

8

7

4

14

13

11

10

6

20

18

14

13

8

 

Conclusions

The synthesized compounds (6C, CI and CD) were identified and confirmed by 1H, 13C NMR and IR spectroscopies.

In vitro tests against fungi showed that they have good biological activity against candida albicans as expected from the docking study which show that the compounds CI and CD have higher docking scores than the control compound (fluconazole), confirming the in vitro tests.

In vitro test against bacteria showed that the compounds CI and CD exhibits no activity against P. aeruginosa and E. coli but good to moderate activity against S. aureus and K. pneumonia.

 

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

Authors' contributions

All authors contributed toward data analysis, drafting and revising the paper and agreed to responsible for all the aspects of this work.

 

Conflict of Interest

We have no conflicts of interest to disclose.

 

ORCID:

Mustafa H. Mahdi

https://www.orcid.org/0000-0002-0137-0244

HOW TO CITE THIS ARTICLE

Mustafa H. Mahdi, Ashour H. Dawood, Dhurgham Q. Shaheed. Substituted Tetrahydrocarbazole Based on Indomethacin and Diclofenac with Heterocyclic Compound, Synthesis, Spectral and Antimicrobial Studies, J. Med. Chem. Sci., 2022, 5(6) 933-942

https://doi.org/10.26655/JMCHEMSCI.2022.6.7

URL: http://www.jmchemsci.com/article_148959.html

  1. Beyer P., Moorthy V., Paulin S., Hill S.R., Sprenger M., Garner S., Simão M., Guerra R., Magrini N., Swaminathan S., Lancet, 2018, 392:264 [Crossref], [Google Scholar], [Publisher]
  2. Williams D.R., Bawel S.A., Schaugaard R.N., Lett., 2017, 19:5098 [Crossref], [Google Scholar], [Publisher]
  3. Caruso A, Ceramella J, Iacopetta D, Saturnino C, Mauro MV, Bruno R, Aquaro S., Sinicropi M.S., Molecules, 2019, 24:1912 [Crossref], [Google Scholar], [Publisher]
  4. Kulkarni M.R., Mane M.S., Ghosh U., Sharma R., Lad N.P., Srivastava A., Kulkarni-Almeida A., Kharkar P.S., Khedkar V.M., Pandit S.S., J. Med. Chem., 2017, 134:366 [Crossref], [Google Scholar], [Publisher]
  5. Su L., Li J., Zhou Z., Huang D., Zhang Y., Pei H., Guo W., Wu H., Wang X., Liu M., Yang C.G., J. Med. Chem., 2019, 162:203 [Crossref], [Google Scholar], [Publisher]
  6. Bashir M., Bano A., Ijaz A.S., Chaudhary B.A., Molecules, 2015, 20:13496 [Crossref], [Google Scholar], [Publisher]
  7. Bublitz M., Kjellerup L., Cohrt K.O.H., Gordon S., Mortensen A.L., Clausen J.D., Pallin T.D., Hansen J.B., Fuglsang A.T., Dalby-Brown W., Winther A.M., PloS one, 2018, 13:e0188620 [Crossref], [Google Scholar], [Publisher]
  8. Mohamed N.A., El-Serwy W.S., Abd El-Karim S.S., Awad G.E., Elseginy S.A., Chem. Intermed., 2016, 42:1363 [Crossref], [Google Scholar], [Publisher]
  9. Chan E.W.L., Yee Z.Y., Raja I., Yap J.K.Y., Glob. Antimicrob. Resist., 2017, 10:70 [Crossref], [Google Scholar], [Publisher]
  10. Shah P.N., Marshall-Batty K.R., Smolen J.A., Tagaev J.A., Chen Q., Rodesney C.A., Le H.H., Gordon V.D., Greenberg D.E., Cannon C.L., Agents Chemother., 2018, 62:e01574 [Crossref], [Google Scholar], [Publisher]
  11. Leão C., Borges A., Simões M., Antibiotics, 2020, 9:591 [Crossref], [Google Scholar], [Publisher]
  12. Al-Majidi S.M.H., Al-Quaz A.M.N., Al-Nahrain Sci., 2010, 13:26 [Google Scholar], [Publisher]
  13. Waldvogel S.R., Comprehensive organic name reactions and reagents.  Synthesis, 2010, 2010:892 [Google Scholar]
  14. Al-Naimi K., Alwahb H.A., Educ. Sci., 2013, 26:105 [Crossref], [Google Scholar], [Publisher]
  15. Rogers C.U., Corson B.B., Am. Chem. Soc., 1947, 69:2910 [Crossref], [Google Scholar], [Publisher]
  16. Hargrove T.Y., Friggeri L., Wawrzak Z., Qi A., Hoekstra W.J., Schotzinger R.J., York J.D., Guengerich F.P., Lepesheva G.I., Biol. Chem., 2017, 292:6728 [Crossref], [Google Scholar], [Publisher]
  17. Perveen S., Chaudhary H.S., Mag., 2015, 11:S550 [Crossref], [Google Scholar], [Publisher]