Design, Synthesis and Antibacterial, Antifungal Activity of Some Coumarin Acetohydrazide Derivatives

 

A. R. Chabukswar¹, P.V. Adsule²*, P.B. Randhave², Manini Mantri²

¹School of Pharmacy, Dr. Vishwanath Karad MIT World Peace University, Pune-411038.

²Department of Pharmaceutical Chemistry, MAEERS Maharashtra Institute of Pharmacy, Kothrud, Pune-411038

 

ABSTRACT:

We have designed and synthesized (Z)-2-(5-methyl-2-oxo-2H-chromen-7-yl) oxy)-N’-(2-oxoindolin-3-ylidin) acetohydrazide derivatives by reacting 7-hydroxy-4-methyl-coumarin and substituted isatin to afford 12 substituted coumarin acetohydrazide derivatives. The synthesized compounds of coumarin acetohydrazide derivatives were designed and evaluated to study for their possible interactions as DNA gyrase inhibitors. All the synthesized coumarin acetohydrazide compounds have been characterized by spectral analysis IR, 1H NMR and mass spectroscopy. The compounds have been evaluated for In vitro antibacterial and antifungal activity against S. aureus, B. subtilus, E. coli, P. aeruginosa and fungi C. albicans and A. niger. In case of Gram positive bacteria and Gram negative bacteria Compound P5C, M5C, C5C exhibited significant activity. Compounds P5N, M5N, C5N shown moderate activity. Compound P5C, M5C, C5C shown significant Antifungal activity against C. albicans and A. niger. Compounds P5C, M5C, C5C are found to exert significant antibacterial as well as antifungal activity and can serve as potential compound against infectious diseases in future.

 

 

 

INTRODUCTION:

Infectious diseases due to bacteria and the fungi are common causes of death worldwide. Due to high toxicity and low resistance issues of existing drugs there is a need to design and synthesize novel compounds against infectious diseases. In recent years death caused by the infectious diseases like respiratory tract infections (3.1 million deaths), diarrhea or stomach infection (1.5 million deaths), and tuberculosis or lung infection (1.4 million deaths) worldwide. In India near about 46 percent of all deaths are due to infectious diseases. Coumarin nucleus plays important part as both natural and synthetic drug in heterocyclic chemistry. Coumarin represents very broad spectrum of biological activities such as antimicrobial¹, anti-fungal², antibacterial³, anti-oxidant⁴, antipyretic⁵, antitumor^6-9, anti-inflammatory^10,11, Anticonvulscent¹².

 

DNA gyrase B is a widely studied antibacterial and antifungal target. It is essential for processes like DNA replication, transcription, and recombination. Coumarin is found to act as DNA gyrase inhibitor. Other heterocyclic compounds reported for as DNA Gyrase inhibitors like quinolone, triazole, indazole etc. DNA gyrase is a suitable target for antibacterial and antifungal activity. However many coumarin derivatives have not been evaluated for their antibacterial and antifungal activity. Due to broad spectrum activities many scientist worked on synthesis of coumarin and its derivatives to evaluate the pharmacological activities of substituted coumarin derivatives. There are reported papers describing the synthesis of coumarin molecules. There is a huge scope for design, synthesis and evaluation of new coumarin molecules to test against anti-bacterial and anti-fungal activities. The toxicity and resistance of existing drug molecules against infectious diseases necessitates the discovery of new molecules which can be efficient and safe against infectious diseases. Infectious diseases have posed great challenge in front of human being in the post covid scenario. There is urgent need to design and synthesize novel molecules against the infectious diseases.

MATERIALS AND METHODS:

Chemicals were procured from Sigma-Aldrich Merck. The identification and characterization of synthesized compounds were done on the basis of physical along with chemical and spectral analysis data such as Melting point (M.P.), Thin layer chromatography (TLC), Infrared Spectroscopy (IR), Nuclear Magnetic Resonance Spectroscopy (¹H NMR), Mass Spectroscopy¹³. Melting point determined on Veego melting point apparatus (VMP PM, 32/1105) and was uncorrected. Thin Layer Chromatography done by using (G-60 mesh) silica gel. Reactions monitored by recoated TLC, it visualized either by iodine vapors chamber or by UV cabinet. R_f value calculation done for synthesized compounds using n-Hexane: ethyl acetate (8:2). The IR spectra of intermediate as well as final derivatives were recorded on Fourier Transform Infrared Spectrophotometer by using KBr as a standard (JASCO FTIR). Bruker spectrometer with CDCl₃ as solvent used for 1H-NMR spectra. Tetramethylsilan (TMS) as an internal standard to indicate Chemical shifts in parts per million values. GC-MS were carried out by using BRUKER Compass Data Analysis 4.2

 

Procedure for the synthesis of Coumarin Derivatives:

Substituted Coumarin (II) was synthesized from resorcinol (I) using Pechmann Condensation reaction. Prepared coumarin was subjected for esterification reaction (III). Then reacted with hydrazine hydrate to afford coumarin derivatives (IV). To this Coumarin derivative (IV) (0.002mole) substituted Isatin (0.002mole) was added in appropriate quantity of glacial acetic acid were refluxed for 8 hours at 70-80 °C, cool the reaction mixture. Reaction frequently monitored by TLC. Then it was poured off into crushed ice and separated solid collected and recrystallized from DMF.

 

Scheme-1

 

Table No.1: Physical data of Synthesized Coumarin Derivatives-

Product code	R1	R2	R3	R4	R5	Molecular Formula	Molecular Weight	% yield	M.P. (0C)	Rf value
PPI	CH3	H	H	H	H	C20H15O5N3	377.35	85	269-271	0.92
P5C	CH3	H	H	H	Cl	C20H14O5N3Cl	411.80	80	278-288	0.89
P5N	CH3	H	H	H	NO2	C20H14O7N4	422.35	85	280-284	0.90
P1M	CH3	H	H	CH3	H	C21H17O5N3	391.38	90	264-267	0.94
MPI	CH3	CH3	H	H	H	C21H17O5N3	391.38	75	261-270	0.88
M5C	CH3	CH3	H	H	Cl	C21H16O5N3Cl	425.82	85	278-280	0.90
M5N	CH3	CH3	H	H	NO2	C21H16O7N4	436.37	75	271-284	0.71
M1M	CH3	CH3	H	CH3	H	C22H19O5N3	405.40	80	295-297	0.76
CPI	CH3	H	Cl	H	H	C20H14O5N3Cl	411.80	75	192-202	0.70
C5C	CH3	H	Cl	H	Cl	C20H13O5N3Cl2	446.24	75	182-186	0.84
C5N	CH3	H	Cl	H	NO2	C20H13O7N4Cl	456.79	80	192-196	0.93
C1M	CH3	H	Cl	CH3	H	C21H16O5N3Cl	425.82	75	181-184	0.86

 

PPI

(Z)-2-(4-methyl-2-oxo-2H-chromen-7-yloxy)-N-(2-oxoindolin-3-ylidene) acetohydrazide

C₂₀H₁₅O₅N, Yield-85%, R _f=0.92, mp-269-271°C, IR(ν KBr, cm-1): 3352.64(N‑H str. Secondary amides), 3061.44(Aromatic CH-str.), 2912.95 (C‑H str. in methylene group), 1650.77 (C=O str.in ketone), 1614.13(C=O str.in amide), 1570(C=C str. aromatic), 1393.82(C‑H str.in methylene group), 1078.01(C-O str.in C-O-C), 3252.36(N‑N str.), 832.13(Aromatic C-H),1379.21(C-N amide), 2233.16(C=N); 1H NMR (400 MHz CDCl3)shows δ ppm value at- 4.78 δ (d, 2H); 7.0δ (q,1H); 6.98-7.63δ (t,3H); 9.8 δ (s,1H); 7.0-7.7 δ (q,4H); 6.24 δ (d,1H); 1.71 δ (t,3H); GC-MS (70Ev) m/z (%):378.10 (M+), 274,230,174.

 

P5C

(Z)-N-(5-chloro-2-oxoindolin-3-ylidene)-2-((4-methyl-2-oxo-2H-chromen-7-yl) oxy) acetohydrazide

C₂₀H₁₄O₅N₃Cl,Yield-80%, R_f =0.89, mp-278-288°C, IR(ν KBr, cm-1): 3329.5(N‑H str. Secondary amides), 3084(Aromatic CH-str.), 2911(C‑H str. in methylene group), 1650.77 (C=O str.in ketone),1616.06(C=O str.in amide), 1570(C=C str. aromatic), 1394.28(C‑H str.in methylene group), 1077.05(C-O str.in C-O-C), 3220.54(N‑N str.), 834.06(Aromatic C-H), 1379.82(C-N amide), 2249(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at-3.34δ (s,2H); 7.0δ (q, 1H); 6.91δ (d, 1H); 6.96 δ (d, 1H); 11.13 δ (d, 1H); 7.4-7.6 δ (q, 4H); 6.25(1H, d); 2.4 (t, 3H); GC-MS (70Ev) m/z (%): 412 (M+), 274,231,197.

 

P5N

(Z)-2-((4-methyl-2-oxo-2H-chromen-7yl) oxy)-N-(5-nitro-2-oxoindolin-3-ylidene) acetohydrazide

C20H14O7N4,Yield-85%, R_f =0.90, mp-280-284°C, IR(ν KBr, cm-1): 3446(N‑H str. Secondary amides), 3087(Aromatic CH-str.), 2917 (C‑H str. in methylene group), 1650(C=O str.in ketone),1621(C=O str.in amide), 1568(C=C str. aromatic), 1394(C‑H str.in methylene group), 1080(C-O str.in C-O-C),3235 (N‑N str.), 830(Aromatic C-H),1378(C-N amide), 2248 (C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 3.34 δ (s,2H); 7.0δ (q,1H); 11.91 δ (d,1H); 8.3δ (q,1H); 7.7 δ (d,1H); 6.25 δ (d,1H); 2.4 δ(q,3H).

 

P1M

(Z)-2-((4-methyl-2-oxo-2H-chromen-7yl) oxy)-N-(1-methyl-2-oxoindolin-3-ylidene) acetohydrazide

C21H17O5N3,Yield-90%, R_f =0.94, mp-264-267°C, IR(ν KBr, cm-1): 3341(N‑H str. Secondary amides), 3088(Aromatic CH-str.), 2915(C‑H str. in methylene group), 1650.77 (C=O str.in ketone),1618(C=O str.in amide), 1573(C=C str. aromatic), 1388(C‑H str.in methylene group), 1086(C-O str.in C-O-C), 3237(N‑N str.),833(Aromatic C-H), 1383(C-N amide), 2214(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.83 δ (s,2H,); 7.0δ (q,1H); 6.9δ (d,1H); 6.8δ (d, 1H); 2.42 δ (t,1H); 7.4-7.8 δ (q,4H);6.2 δ (d,1H,CH);1.57 δ (s,3H); GC-MS (70Ev) m/z (%):392 (M+), 274,231,197.

 

MPI

(Z)-2-((4, 8-dimethyl-2-oxo-2H-chromen-7-yl) oxy)-N-(2-oxoindolin-3-ylidene) acetohydrazide. 

C21H17O5N3,Yield-75%, R_f =0.88, mp-261-270°C, IR(ν KBr, cm-1):3369(N‑H str. Secondary amides), 3090(Aromatic CH-str.), 2917(C‑H str. in methylene group), 1637 (C=O str.in ketone), 1622(C=O str.in amide), 1383(C=C str. aromatic), 1051(C‑H str.in methylene group), 1582(C-O str.in C-O-C), 3236(N‑N str.), 858(Aromatic C-H), 1399(C-N amide), 2240(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.97 δ (s,2H); 7.1δ (q,1H); 6.9δ (d,1H); 2.4 δ (t,3H); 7.1-7.6 δ (q,4H); 6.24 δ (d,1H); 1.69 δ (s,3H); GC-MS (70Ev) m/z (%): 392 (M+), 277,231,197.

 

M5C

(Z)-N-(5-chloro-2-oxoindolin-3-ylidene)-2-((4,8-dimethyl-2-oxo-2H-chromen-7-yl)oxy) acetohydrazide

C21H16O5N3Cl,Yield-85%, R_f =0.90, mp-278-280°C, IR(ν KBr, cm-1):3356(N‑H str. Secondary amides), 3089(Aromatic CH-str.), 2916(C‑H str. in methylene group), 1687 (C=O str.in ketone), 1635(C=O str.in amide), 1580(C=C str. aromatic), 1380(C‑H str.in methylene group), 1078(C-O str.in C-O-C), 3244(N‑N str.), 835(Aromatic C-H), 1330(C-N amide), 2255(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.73 δ (s,2H); 7.2δ (s,1H); 6.6δ (d,1H), 2.38 δ (t,3H); 7.36 δ (q,4H); 6.23 δ (d,1H), 1.81 δ (s,3H).

 

M5N

(Z)-2-((4, 8-dimethyl-2-oxo-2H-chromen-7-yl) oxy)-N-(5-nitro-2-oxoindolin-3-ylidine) acetohydrazide

C21H16O7N4,Yield-75%, R_f =0.71, mp-271-284°C, IR(ν KBr, cm-1): 3356(N‑H str. Secondary amides), 3092(Aromatic CH-str.), 2916(C‑H str. in methylene group), 1643 (C=O str.in ketone),1625(C=O str.in amide), 1580(C=C str. aromatic), 1375(C‑H str.in methylene group), 1085(C-O str.in C-O-C), 3260(N‑N str.), 829(Aromatic C-H), 1392(C-N amide), 2330(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.7 δ (s,2H); 7.0δ (s,1H); 6.6δ (d,1H); 7.3 δ (d,1H); 2.34 δ (t,3H); 7.5 δ (q,4H); 6.17 δ (d,1H); 1.72 δ (s ,3H).

 

M1M

(Z)-2-((4, 8-dimethyl-2-oxo-2H-chromen-7-yl) oxy)-n-(1-methyl-2-oxoindolin-3-ylidine) acetohydrazide

C22H19O5N3,Yield-80%, R_f =0.76, mp-295-297°C, IR(ν KBr, cm-1):3340(N‑H str. Secondary amides), 3053(Aromatic CH-str.), 2916(C‑H str. in methylene group), 1687 (C=O str.in ketone), 1637(C=O str.in amide), 1584(C=C str. aromatic), 1374(C‑H str.in methylene group), 1061(C-O str.in C-O-C), 3272(N‑N str.), 835(Aromatic C-H), 1374(C-N amide), 2264(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.73 δ (s,2H); 7.0 δ (s,1H); 6.84-6.89 δ (q,4H); 7.1δ (d,1H); 2.41 δ (t,3H); 7.41-7.46 δ (q,4H); 1.57 δ (s,3H), 13.88δ (s,1 H).

 

CPI

(Z)-2-((6-chloro-4-methyl-2-oxo-2H-chromen-7-yl) oxy)-N-(2-oxoindolin-3-ylidene) acetohydrazide

C20H14O5N3Cl, Yield-75%, R_f =0.70, mp-192-202°C, IR(ν KBr, cm-1):3358(N‑H str. Secondary amides), 3045(Aromatic CH-str.), 2918(C‑H str. in methylene group), 1685 (C=O str.in ketone), 1634(C=O str.in amide), 1594(C=C str. aromatic), 1365(C‑H str.in methylene group), 1064(C-O str.in C-O-C), 3276(N‑N str.), 838(Aromatic C-H), 1370(C-N amide), 2265(C=N), 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.87 δ (s, 2H); 7.0δ (s,1H); 6.4δ (1H,d); 7.5 δ (1H,d); 7.7 δ (4H,q); 6.2 δ (1H,CH,d); 1.8 δ (3H, s); 8.0 δ (s,1H).

 

C5C

(Z)-N-(5-chloro-2-oxoindolin-ylidene)-2-((6-chloro-4-methyl-2-oxo-2H-chromen-7-yl) oxy) acetohydrazide

C20H13O5N3Cl2,Yield-75%, R_f =0.84, mp-182-186°C, IR (ν KBr, cm-1): 3342(N-H str. Secondary amides), 3065(Aromatic CH-str.), 2917(C-H str. in methylene group), 1649 (C=O str.in ketone), 1639(C=O str.in amide), 1573(C=C str. aromatic), 1390(C-H str.in methylene group), 1089(C-O str.in C-O-C), 3263(N-N str.), 839(Aromatic C-H), 1377(C-N amide), 2215(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.7 δ (s,2H); 7.2 δ (s, 1H); 6.7 δ (d,1H); 7.53 δ (d,1H); 7.5 δ (q,3H,); 6.20 δ (d,1H); 1.68 δ (s,3H).

 

C5N

(Z)-2-((6-chloro-4-methyl-2-oxo-2H-chromen-7-yl) oxy)-N-(5-nitro-2-oxoindolin-3-ylidene) acetohydrazide

C20H13O7N4Cl,Yield-80%, R_f =0.93, mp-192-196°C, IR (ν KBr, cm-1): 3329(N H str. Secondary amides), 3084(Aromatic CH-str.), 2911(C-H str. in methylene group), 1650 (C=O str.in ketone),1616(C=O str.in amide), 1570(C=C str. aromatic), 1394(C-H str.in methylene group), 1077(C-O str.in C-O-C), 3220(N-N str.), 834(Aromatic C-H), 1379(C-N amide), 2249(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.8 δ (s,2H); 7.2δ (s,1H); 6.7δ (d,1H); 7.5 δ (d,1H); 7.6 δ (q,4H); 6.2 δ (d,1H); 1.7 δ (s,3H); 8.0 δ (NH).

 

C1M

(Z)-2-((6-chloro-4-methyl-2-oxo-2H-chromen-7-yl) oxy)-N-(1-methyl-2-oxoindolin-3-ylidene) acetohydrazide.

C21H16O5N3Cl,Yield-75%, R_f =0.86, mp-181-184°C, IR (ν KBr, cm-1): 3352(N H str. Secondary amides), 3063(Aromatic CH-str.), 2917(C-H str. in methylene group), 1649 (C=O str.in ketone), 1639(C=O str.in amide), 1572(C=C str. aromatic), 1391(C-H str.in methylene group), 1088(C-O str.in C-O-C), 3251(N N str.), 839(Aromatic C-H), 1391(C-N amide), 2208(C=N); 1H NMR (400 MHz CDCl3) shows δ ppm value at- 4.5 δ (s,2H); 7.1δ (s,1H);6.9δ (d,1H); 7.4 δ (d,1H); 7.1-7.8 δ (q,4H); 6.2 δ (d,1H); 1.9 δ (s,3H); 2.4 δ (q,3H).

 

Antibacterial activity:

In vitro anti-bacterial activity was evaluated by employing the agar plate diffusion technique by estimating the zone of inhibition in mm.^14,15 In vitro anti-bacterial study of all the synthesized coumarin acetohydrazide compounds carried out against various bacterial strains like gram positive: Staphylococcus aureus (NCIM 4200), Bacillus subtilis (NCIM 3251) and Gram–ve Bacteria: Escherichia coli (NCIM 8900), Pseudomonas araginosa. Standardization of above mentioned microbial strains done by McFarland standardization method and 108cfu/ml concentration of microbial culture was used. Ciprofloxacin was used as standard drug for antibacterial activity. Compounds were prepared in DMSO (10-100µg/ml). The results of the in vitro anti-bacterial activities are summarized in Table No. 3.

 

 

Antifungal Activity:

All the synthesized coumarin acetohydrazide compounds were screened for in vitro antifungal activity against fungal strains like Candida albicans (NCIM3471), A. niger (NCIM 1196). Anti-fungal activity was evaluated as per standard reported method. Griceofulvin was used as standard drug for antifungal activity. The results of the in vitro study of anti-fungal activity are summarized in Table No. 4.

 

Molecular docking for anti-bacterial studies:

The new synthesized coumarin acetohydrazide derivatives were acquired to undergo docking in the active site of DNA gyrase enzyme. By using molecular modeling we find out the binding mode of 12 new ligands at the active site of Chlorobiocin theoretically. These studies were done to find out intermolecular ligand-receptor interactions. Chlorobiocin structurally made up of combining amino group with coumarin ring that acts as antimicrobial agent used for suppression of DNA gyrase enzyme. DNA Gyrase (PDB ID: 1kzn) 2.3 Å resolution used as main template for docking studies. In molecular docking First step is removal of water molecule and ligand from the protein. Whatever protein structure obtained now immerged in Auto dock system. Like Chlorobiocin, 12 new synthesized coumarin acetohydrazide compounds binding affinity with DNA gyrase were assessed. 3D ultra 8.0 software was used to develop three dimensional structures of all compound and were optimized by minimizing energy by Merck Molecular Force Field (MMFF), Convergence criteria (rms gradient: 0.01), systemic method (1000Kcal/mol) was selected for conformation generation and save generate conformer as a MDL Mol Files (*mol). Docking study was performed by using the routine procedure and default parameters of molecular docking AutoDock software used. For protein, polar hydrogen molecules were added and all water molecules were removed. During this procedure we have to keep ligand molecules rotating so that we will get the best conformer of the 12 ligands with the active site of the enzyme. Redocking of Chlorobiocin was done. Grid box was generated. Towards the end of the docking study cluster analysis was done. Different conformations were arranged according to root mean square deviation tolerance and according to binding free energy. After comparing all the generated conformation, selected the conformation with best scored pose and with the lowest binding energy for 12 ligands.

 

Table No. 2: Docking Score

Title	docking score
Clorobiocin	-6.191
CPI	-5.604
MPI	-5.467
P5C	-5.33
PPI	-5.325
P5N	-5.056
M5N	-5.032
C5N	-4.469
M5C	-4.391
P1M	-4.138
C1M	-4.035
M1M	-3.931
C5C	-3.529

 

 

Figure: 1-CPI Coumarin acetohydrazide with DNA Gyrase active site

 

 

Figure: 2-P5C Coumarin acetohydrazide with DNA Gyrase active site

 

 

Figure: 3-P5N Coumarin acetohydrazide with DNA Gyrase active site

 

Figure: 4- 3D representation of CPI Coumarin acetohydrazide with DNA Gyrase active site.

 

RESULTS AND DISCUSSION:

Chemistry:

Substituted Coumarin was synthesized from resorcinol using reported method. Prepared coumarins were subjected for esterification reaction. Then they were reacted with hydrazine hydrate to afford coumarin derivatives. These derivatives were then subjected for reaction with substituted isatin to give the coumarin acetohydrazide derivatives. (Scheme-1) Glacial acetic acid was found to be suitable to afford compounds in good yield. The synthesis of final coumarin acetohydrazide compounds was achieved in 8 hrs. Reaction was optimized for time duration. Substituted Coumarin derivatives were synthesized from resorcinol by using Pechmann condensation. Coumarin acetohydrazide derivatives obtained by using esterification and nucleophilic substitution reaction. Purification of compounds were done by column chromatography and dried and subjected for spectral analysis using IR, NMR and mass spectroscopy.

 

Anti-bacterial activity:

Anti-bacterial activity was performed using agar plate diffusion assay method. Gram positive as well as Gram negative each two bacterial strains were used for evaluation of anti-bacterial activity. Ciprofloxacin was used as standard in order to do comparison with activity of synthesized coumarin derivatives. The results of the antibacterial activity against all tested species are reported in Table no.3. Compounds P5C, M5C, C5C shows significant activity (Maximum inhibition: 15-20mm) against Gram positive strains S. aureus and B. subtilus and Gram negative bacteria E. coli and P. aeruginosa. Probable explanation lies in the fact that compounds P5C, M5C, C5C with halogen (chloro) group on 5^th position of Isatin contribute for the maximum activity against both Gram positive and Gram negative strains. Compounds P5N, M5N, C5N shown moderate activity (Moderate inhibition: 10-15mm) against S. aureus and B. subtilus and E. coli and P. aeruginosa due to presence of NO₂ group acts as electron withdrawing group at 5^th position of Isatin.Where as Compounds M1M, MPI and CPI shows weaker activity against both positive and negative bacterial strains (inhibition: less than 10mm) and Compounds C1M, PPI and P1M did not show any antibacterial activity against both strains. This can be due to no substitution at 5^th position of Isatin. Introduction of Chlorine or electron withdrawing Nitro group substituent on Isatin ring contributes to the Antibacterial activity.

 

Table No. 3. Zone of Inhibition (mm) for synthesized Coumarin compounds against Gram positive and Gram Negative bacteria

Sr. No.	Product Code	Zone of Inhibition of (mm) for 50µg
		Gram +ve Bacteria		Gram -ve Bacteria
		S. aureus	B. subtilus	E. coli	P. aeruginosa
1.	PPI	No zone	No zone	No zone	No zone
2.	P5C	18mm	20 mm	19 mm	20 mm
3.	P5N	15mm	15mm	14mm	15mm
4.	P1M	No zone	No zone	No zone	No zone
5.	MPI	No zone	09mm	No zone	09mm
6.	M5C	18mm	19mm	20 mm	18mm
7.	M5N	14mm	14mm	12mm	14mm
8.	M1M	8mm	9mm	8mm	8mm
9.	CPI	08mm	No zone	09mm	No zone
10.	C5C	19mm	18 mm	19mm	18mm
11.	C5N	14mm	15mm	15mm	15mm
12.	C1M	No zone	No zone	No zone	No zone
13.	Ciprofloxacin	20mm	22 mm	21mm	22mm

Maximum inhibition: 15-20mm, Moderate inhibition: 10-15mm, Poor inhibition-less than 10mm

 

Antifungal activity:

Anti-fungal activity was performed using agar plate diffusion assay. Two strains C. albicans and A. niger were used for evaluation of anti-fungal activity. Results of anti-fungal activity of synthesized coumarin acetohydrazide derivatives were compared with standard Griseofulvin. Antifungal activity results of all tested species were reported in Table no.4. Compound P5C, M5C, C5C shows excellent activity (Maximum inhibition: 15-20 mm) against both fungal strains C. albicans and A. niger. This activity can be due to introduction of Chloro group (halogen) substituent on 5^th position of Isatin ring which contributes to the Antifungal activity. Compound P5N, M5N, C5N also shown moderate activity (Maximum inhibition: 10-15 mm) against both fungal strains. Antifungal activity can be due to the presence of electron withdrawing Nitro group or Chlorine group substituent on 5^th position of Isatin ring. While M1M, CPI, and MPI shows poor inhibition (less than 10 mm) and compound PPI, P1M, C1M shown no activity. Reason for both was no halogen or nitro group substitution at 5^th position of Isatin ring decrease activity.

 

Table No. 4: Zone of Inhibition (mm) for synthesized Coumarin compounds against C. albicans and A. niger

Sr. No.	Product Code	Zone of Inhibition of (mm) for 50µg
		C. albicans	A. niger
1.	PPI	No zone	No zone
2.	P5C	20mm	19mm
3.	P5N	14mm	12mm
4.	P1M	No zone	No zone
5.	MPI	19mm	18mm
6.	M5C	19mm	20mm
7.	M5N	13mm	14mm
8.	M1M	08mm	9mm
9.	CPI	09mm	09mm
10.	C5C	20mm	19mm
11.	C5N	13mm	12mm
12.	C1M	No zone	No zone
13.	Griseofulvin	22mm	22mm

Maximum inhibition: 15-20mm, Moderate inhibition: 10-15mm, Poor inhibition-less than 10mm

 

Molecular docking:

Docking studies of Anti-bacterial activity carried out DNA gyrase (PBD Code: 1kzn). The PLP score and ΔG was obtained which shows favourable binding of these ligands to the DNA gyrase enzymes. The interactions between the ligand molecules and protein were studied. From the results of the docking studies reported in Table no.2 it was found that structure activity relationship shows substitution of Chloro and Methyl group at 6th and 8th positions (CPI, MPI, P5C) shows good activity with better binding to active site. Substitution of nitro and chloro group at Isatin ring at 5th position also shows better interaction.

 

CONCLUSION:

All the synthesized coumarin acetohydrazide derivatives exhibited interaction with the amino acid residues in active site of DNA Gyrase enzyme, it may be concluded that the coumarin derivatives may be effective Anti-bacterial agents. The synthesized derivative of coumarin acetohydrazide showed encouraging anti-bacterial activity against part B of DNA Gyrase and can be used as target molecule against bacterial infections. Designing of such compounds may be useful for development of broad spectrum anti-bacterial agents and compounds also shown Antifungal activity and can be used as Antifungal agents.

 

ACKNOWLEDGMENT:

The authors are thankful to the Management of School of Pharmacy, Dr. Vishwanath Karad MIT World Peace University, Pune and MAEER’s Maharashtra Institute of Pharmacy, Pune for providing necessary facilities to carry out this research work.

 

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Received on 18.07.2020           Modified on 23.08.2020

Accepted on 18.09.2020         © RJPT All right reserved