synthesis, characterization, molecular docking studies and...

Supplementary Information

Synthesis, characterization, molecular docking studies and biological activity

of coumarin linked 2-pyridone heterocycles

N C Desai*a, Tushar J Karkar

b, Rajesh H Vekariya

c, Surbhi B Joshi

a, Krunalsinh A Jadeja

a & Darshita V Vaja

a

a Division of Medicinal Chemistry, Department of Chemistry, Mahatma Gandhi Campus

Maharaja Krishnakumarsinhji Bhavnagar University, Bhavnagar 364 002, India

(DST-FIST Sponsored & UGC NON-SAP) b C B Patel Computer College and JNM Patel Science College (DRB), New City light Road, Bharthana (Vesu), Surat 395 007,

India c Research Scientist, Piramal Pharma Solutions, Plot No. 18, Pharmez, Matoda Village, Ahmedabad 382 220, India

E-mail: [email protected]

Received 30 August 2019; accepted (revised) 16 December 2019

Captions:

Materials and method

Biological assay 1H NMR of compound 4a

1H NMR of compound 4f

1H NMR of compound 4k

1H NMR of compound 4m

IR Spectra of compound 4f

IR Spectra of compound 4m

Mass Spectra of compound 4f 13

C NMR of compound 4f

Materials and method

Melting points were noted on Gallenkamp apparatus and were left uncorrected. The completion

of reaction and the purity of all compounds were checked on aluminum-coated TLC plates

60F245 (E. Merck) using various solvent systems as mobile phase and visualized under ultraviolet

(UV) light, or iodine vapor. Perkin-Elmer 2400 CHN analyzer was used for elemental analysis

(% C, H, N). IR spectra were recorded on Perkin Elmer FT-IR spectrophotometer. 1H NMR

spectra were recorded on a Bruker Avance II 500 MHz while 13

C NMR spectra on Varian

Mercury-400, 100 MHz in DMSO-d6 as a solvent and tetramethylsilane (TMS) as an internal

standard using 5 mm tube. Chemical shifts were reported in ppm units with use of δ scale. Mass

spectra were also scanned on a Shimadzu LCMS 2010 spectrometer. The essential chemicals

were purchased from E. Merck. Buchi Rota vapor instrument was used for distillation purpose.

Physical and characterization data of 4-(4-chlorophenyl)-6-((2-hydroxybenzylidene)amino)-2-

oxo-1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitrile

(4b)

Yield: 73 %, m.p. 152-156 °C; IR (KBr, cm-1

): 3415 (O-H, Ar-OH), 2930 (C-H, CH3), 2205 (-

C≡N stretching, nitrile group), 1730 (>C=O stretching, coumarin ring), 1570, 1594 (>C=N-,

>C=C< stretching, aromatic ring), 1583, 1405 (C-H bending, -CH=N linkage), 1259 (C-O-C

coumarin ring), 736 (-C-Cl stretching);1H NMR (500 MHz, DMSO-d6, δ ppm): 9.84 (s, 1H, Ar-

OH), 9.24 (s, 1H, Ar-CH=N-), 8.92 (s, 1H, C4 proton of coumarin), 6.90-7.88 (m, 12H, Ar-H of

coumarin ring and phenyl ring), 2.09 (s, 3H, -C(CH3)=N-); 13

C NMR (100 MHz, DMSO-d6, δ

ppm): 14.4, 114.6, 115.7, 115.8 (2), 116.2, 117.5, 118.1, 118.6, 121.3, 123.7, 125.5, 127.5,

128.5, 128.9 (2), 130.6 (2), 130.4, 132.4, 132.6,133.7 (2), 153.4, 153.5, 155.6, 159.7, 160.0,

161.5, 163.5, 169.9; LCMS: m/z 559.10 (M+). Anal. calcd. For C31H18ClN5O4, C, 66.49; H, 3.24;

N, 12.51; Found: C, 66.44; H, 3.21; N, 12.46.

Physical and characterization data of 4-(4-chlorophenyl)-6-((3-hydroxybenzylidene)amino)-2-


(4c)


): 3405 (O-H, Ar-OH), 2937 (C-H, CH3), 2209 (-

C≡N stretching, nitrile group), 1737 (>C=O stretching, coumarin ring), 1577, 1590 (>C=N-,

>C=CC=O stretching, coumarin ring), 1574, 1593 (>C=N-,

>C=CC=O stretching, coumarin ring), 1572, 1591 (>C=N-, >C=C< stretching,

aromatic ring), 1587, 1404 (C-H bending, -CH=N linkage), 1490, 1348 (-N=O stretching, -NO2)

1264 (C-O-C coumarin ring), 731 (-C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δ ppm):

9.24 (s, 1H, Ar-CH=N-), 8.91 (s, 1H, C4 proton of coumarin), 6.85-7.90 (m, 12H, Ar-H of



ppm): 14.4, 114.7, 115.3, 115.6 (2), 116.1, 118.4, 123.3, 124.2, 125.6, 127.6, 128.1, 128.5, 128.6

(2), 130.3 (2), 130.4, 130.8, 131.7, 133.7 (2), 134.8, 147.6, 153.3, 153.5, 155.6, 159.5, 160.2,

163.4, 169.6; LCMS: m/z 588.09 (M+). Anal. calcd. For C31H17ClN6O5, C, 63.22; H, 2.91; N,

14.27; Found: C, 63.28; H, 2.94; N, 14.31.

Physical and characterization data of 4-(4-chlorophenyl)-6-((3-nitrobenzylidene)amino)-2-oxo-

1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitrile (4f)

Yield: 71 %, m.p. 224-228 °C; IR (KBr, cm-1): 2980 (C-H, CH3), 2208 (-C≡N stretching, nitrile

group), 1699 (>C=O stretching, coumarin ring), 1597, 1557 (>C=N-, >C=C< stretching,

aromatic ring), 1589, 1408 (C-H bending, -CH=N linkage), 1489, 1348 (-N=O stretching, -NO2),



coumarin ring and phenyl ring), 1.25 (s, 3H, -C(CH3)=N-); 13C NMR (100 MHz, DMSO-d6, δ

ppm): 14.4, 114.5, 115.1, 115.5 (2), 116.4, 118.4, 121.3, 123.5, 125.7, 126.6, 127.9, 128.6, 128.9

(2), 129.8, 130.4 (2), 130.9, 133.7 (2), 134.9, 135.5, 148.4, 153.6, 153.9, 155.7, 159.8, 160.2,


14.27; Found: C, 63.25; H, 2.96; N, 14.21.

Physical and characterization data of 4-(4-chlorophenyl)-6-((4-nitrobenzylidene)amino)-2-oxo-

1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitrile (4g)


): 2941 (C-H, CH3), 2221 (-C≡N stretching, nitrile


aromatic ring), 1582, 1404 (C-H bending, -CH=N linkage), 1491, 1340 (-N=O stretching, -NO2)





ppm): 14.4, 114.6, 115.5, 115.7 (2), 116.5, 118.6, 123.3, 124.5 (2), 125.6, 127.6 (2), 127.7,

128.5, 128.6 (2), 130.3 (2), 130.7, 133.8 (2), 139.9, 150.5, 153.2, 153.6, 155.4, 159.7, 160.3,


14.27; Found: C, 63.29; H, 2.92; N, 14.24.

Physical and characterization data of 6-((2-chlorobenzylidene)amino)-4-(4-chlorophenyl)-2-


(4h)

Yield: 65 %, m.p. 256-260 °C; IR (KBr, cm-1): 2945 (C-H, CH3), 2218 (-C≡N stretching, nitrile


aromatic ring), 1581, 1407 (C-H bending, -CH=N linkage), 1263 (C-O-C coumarin ring), 733 (-

C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δ ppm): 9.20 (s, 1H, Ar-CH=N-), 8.92 (s, 1H,

C4 proton of coumarin), 6.67-8.15 (m, 12H, Ar-H of coumarin ring and phenyl ring), 1.30 (s, 3H,

-C(CH3)=N-); 13C NMR (100 MHz, DMSO-d6, δ ppm): 14.4, 84.5, 88.4, 115.5, 116.3, 116.7,

118.4, 123.2, 125.7, 126.6, 127.4, 127.6, 128.4, 128.8 (2), 130.3 (2), 130.5, 130.6, 132.6, 133.6,

133.7 (2), 133.9, 153.1, 153.5, 155.6, 159.3, 160.8, 163.8, 163.9; LCMS: m/z 577.07 (M+). Anal.

calcd. For C31H17Cl2N5O3, C, 64.37; H, 2.96; N, 12.11; Found: C, 64.30; H, 2.99; N, 12.19.



(4i)







-C(CH3)=N-); 13

C NMR (100 MHz, DMSO-d6, δ ppm): 14.4, 114.5, 115.5, 115.8, 116.4, 118.3,

123.5, 125.6, 127.3, 127.5, 127.8, 128.2, 128.5 (2), 130.1 (2), 130.4, 130.7, 131.4, 133.8 (2),

134.6, 135.3, 153.4, 153.7, 155.9, 159.1, 160.0, 163.5, 169.6; LCMS: m/z 577.07 (M+). Anal.

calcd. For C31H17Cl2N5O3, C, 64.37; H, 2.96; N, 12.11; Found: C, 64.39; H, 2.91; N, 12.16.



(4j)







-C(CH3)=N-); 13

C NMR (100 MHz, DMSO-d6, δ ppm): 14.4, 114.7, 115.5, 115.7 (2), 116.2,

118.5, 123.1, 125.5, 127.7, 128.1, 128.7 (2), 128.9 (2), 130.3 (2), 130.5 (3), 131.6, 133.4 (2),

136.7, 153.2, 155.9, 159.3, 160.3, 163.9, 169.7; LCMS: m/z 577.07 (M+). Anal. calcd. For

C31H17Cl2N5O3, C, 64.37; H, 2.96; N, 12.11; Found: C, 64.44; H, 2.99; N, 12.18.

Physical and characterization data of 4-(4-chlorophenyl)-6-((4-fluorobenzylidene)amino)-2-oxo-

1-((1-(2-oxo-2H-chromen-3-yl)ethylidene)amino)-1,2-dihydropyridine-3,5-dicarbonitrile (4k)





C-F stretching), 730 (-C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δ ppm): 9.20 (s, 1H, Ar-

CH=N-), 8.65 (s, 1H, C4 proton of coumarin), 6.82-8.18 (m, 12H, Ar-H of coumarin ring and

phenyl ring), 1.30 (s, 3H, -C(CH3)=N-); 13

C NMR (100 MHz, DMSO-d6, δ ppm): 14.4, 114.5,

115.1, 115.4 (2), 115.8 (2), 116.5, 118.3, 123.7, 125.7, 127.6, 128.5, 128.8 (2), 129.5, 130.4 (2),

130.7, 130.9 (2), 133.7 (2), 153.3, 155.5, 155.6, 159.3, 160.5, 163.8, 165.4, 169.3; LCMS: m/z

561.10 (M+). Anal. calcd. For C31H17ClFN5O3, C, 66.26; H, 3.05; N, 12.46; Found: C, 66.21; H,

3.09; N, 12.51.

Physical and characterization data of 4-(4-chlorophenyl)-6-((3-methoxybenzylidene)amino)-2-


(4l)




aromatic ring), 1591, 1409 (C-H bending, -CH=N linkage), 1270 (C-O-C coumarin ring), 1157,

1035 (-Ar-O-CH3 stretching), 730 (-C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δ ppm):


coumarin ring and phenyl ring), 3.80 (s, 3H, Ar-OCH3), 1.32 (s, 3H, -C(CH3)=N-); 13

C NMR

(100 MHz, DMSO-d6, δ ppm): 14.4, 55.5, 111.5, 114.9, 115.5, 115.8 (2), 116.3, 116.6, 118.4,

121.7, 123.1, 125.5, 127.9, 128.5, 128.8 (2), 129.9, 130.3 (2), 130.8, 133.7 (2), 134.7, 153.5,

153.7, 155.4, 159.6, 160.0, 160.8, 163.3, 169.5; LCMS: m/z 573.12 (M+). Anal. calcd. For

C32H20ClN5O4, C, 66.96; H, 3.51; N, 12.20; Found: C, 66.90; H, 3.59; N, 12.27.

Physical and characterization data of 4-(4-chlorophenyl)-6-((4-methoxybenzylidene)amino)-2-


(4m)








C NMR

(100 MHz, DMSO-d6, δ ppm): 14.4, 55.7, 114.5 (2), 114.8, 115.1, 115.6 (2), 116.4, 118.6, 123.5,

125.4, 126.3, 127.5, 128.3, 128.9 (2), 130.1 (2), 130.3 (2), 130.7, 133.8 (2), 153.2, 153.6, 155.8,

159.2, 160.4, 162.6, 163.5, 169.6; LCMS: m/z 573.12 (M+). Anal. calcd. For C32H20ClN5O4, C,

66.96; H, 3.51; N, 12.20; Found: C, 66.99; H, 3.46; N, 12.12.

Physical and characterization data of 4-(4-chlorophenyl)-2-oxo-1-((1-(2-oxo-2H-chromen-3-

yl)ethylidene)amino)-6-((3,4,5-trimethoxybenzylidene)amino)-1,2-dihydropyridine-3,5-

dicarbonitrile (4n)








C NMR

(100 MHz, DMSO-d6, δ ppm): 14.4, 56.3 (2), 60.6, 104.3 (2), 114.5, 115.4, 115.7 (2), 116.3,

118.5, 123.6, 125.6, 127.6, 128.1, 128.5, 128.9 (2), 130.3 (2), 130.9, 133.7 (2), 141.3, 153.1,

153.4 (3), 155.4, 159.6, 160.5, 163.9, 169.7; LCMS: m/z 633.14 (M+). Anal. calcd. For

C34H24ClN5O6, C, 64.41; H, 3.82; N, 11.05; Found: C, 64.47; H, 3.88; N, 11.01.

Physical and characterization data of 6-((4-bromobenzylidene)amino)-4-(4-chlorophenyl)-2-


(4o)





1015 (C-Br), 733 (-C-Cl stretching); 1H NMR (500 MHz, DMSO-d6, δ ppm): 9.25 (s, 1H, Ar-

CH=N-), 8.97 (s, 1H, C4 proton of coumarin), 7.03-7.96 (m, 12H, Ar-H of coumarin ring and

phenyl ring), 1.30 (s, 3H, -C(CH3)=N-); 13

C NMR (100 MHz, DMSO-d6, δ ppm): 14.4, 114.9,

115.2, 115.7, 116.4, 118.3, 123.6, 125.3 (2), 127.7, 128.4, 128.6 (2), 128.8 (2), 130.2 (2), 130.5,

131.7 (2), 132.7, 133.5 (2), 153.1, 153.5, 155.5, 159.6, 160.2, 163.6, 169.6; LCMS: m/z 621.02

(M+). Anal. calcd. For C31H17BrClN5O3, C, 59.78; H, 2.75; N, 11.24; Found: C, 59.71; H, 2.79;

N, 11.29.

Biological assay

Antibacterial bioassay

The newly synthesized compounds 4a-o were screened for their antibacterial activity against

Gram positive [Staphylococcus aureus (MTCC-96), Streptococcus pyogenes (MTCC-443)], and

Gram-negative bacteria [Escherichia coli (MTCC-442), Pseudomonas aeruginosa (MTCC-441)]

at different concentrations of 1000, 500, 200, 100, 50, 25, 12.5 µg ml-1

. Several of the newly

synthesized compounds 4a-o was found to exhibit moderate to excellent antimicrobial activity.

Antibacterial activity was carried out as per National Committee for Clinical Laboratory

Standards (NCCLS) protocol using serial broth dilution method and all the standard strains used

for the antimicrobial activity were procured from Institute of Microbial Technology (IMTECH),

Chandigarh. “The drugs which were found to be active in primary screening were similarly

diluted to obtain 100, 50, 25 and 12.5 µg ml-1

concentrations for secondary screening to test in a

second set of dilution against all microorganisms. Inoculum size for test strain was adjusted to

106 CFU/mL (Colony Forming Unit per milliliter) by likening the turbidity. Mueller-Hinton

Broth was used as a nutrient medium to grow and dilute the synthesized compound suspension

for test organisms. 2 % DMSO was used as a diluents/vehicle to acquire the desired

concentration of synthesized compounds and standard drugs to test upon standard microbial

strains. Diluted 1000 µg/mL concentrated solution of synthesized compounds were used as stock

solution. The control tube containing not any antibiotic was instantly subcultured [before

inoculation] by spreading a loopful evenly over quarter of a plate of medium suitable for the

growth of test organisms. The culture tubes were then incubated for 24 h at 37 ºC, and the

growth was observed visually and spectrophotometrically. After that, 10 µg ml-1

suspensions

were further inoculated on appropriate media and development was noted after 24 and 48 h. The

lowest concentration preventing appearance of turbidity was considered as minimum inhibitory

concentration (MIC, µg ml-1

) i.e., the amount of growth from the control tube before incubation

(which represents the original inoculum) was compared. Solvent had no influence on strain

growth and the result of this was greatly affected by the size of inoculum. DMSO and sterilized

distilled water were used as negative control while ‘chloramphenicol’ antibiotic (1 U strength)

was used as positive control. Standard drug ‘chloramphenicol’ was used in this study for

evaluating antibacterial activity which showed 100, 100, 250 and 100 µg ml-1

MIC against E.

coli, P. aeruginosa, S. aureus, and S. pyogene, respectively as shown in Table 1.

Antifungal bioassay

The same newly synthesized compounds 4a-o were screened for their antifungal activity against

in six sets against C. albicans (MTCC-227), A. niger (MTCC-282) and A. clavatus (MTCC-

1323) at various primary concentrations of 1000, 500 and 250 µg ml-1

. The primary screen active

compounds were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25 and 12.5 µg ml-1

concentrations for secondary screening to test in a second set of dilution against all

microorganisms. ‘Nystatin’ was used as a standard drug for antifungal activity, which showed

500, 100 and 100 µg ml-1

MIC against C. albicans, A. niger and A. clavatus, respectively (Table

1). For growth of fungi, in the present procedure, we have used Sabourauds dextrose broth at 28

ºC in aerobic condition for 48 h. DMSO and sterilized distilled water used as negative controls

while ‘Nystatin’ (1 U strength) was used as a positive control.

MTT assay for cytotoxic activity

In vitro cytotoxicity activity of compounds 4a-o was measured by means of the IC50 using the

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay process. “The IC50

determination was achieved according to the National Committee for Clinical Laboratory

Standards (NCCLS) recommendations. All synthesized compounds were dissolved in 0.1 %

DMSO with the stock concentration of 10 g/L and diluted with medium freshly before drug

administration. Cell lines were sowed into 96-well plates at density of 8,000 cells/ well. After

seeding it for 24 h, each compound dilution was added in duplicate, and cultivation continued at

37 ºC in a moistened atmosphere containing 10 % FBS, 1 % glutamine, and 50 µM/mL

gentamicin sulfate in a 5 % CO2 and 95 % air. After 24 h, 20 µL MTT reagent at 5 mg/mL in

PBS (filter sterilized, light protected and stored at 4 ºC) per well was added, and after 4 h of

incubation at 37 ºC, MTT is changed to a blue formazan product by mitochondrial succinate

dehydrogenase. This product was eluted from cells by addition of 150 mL of DMSO. The

absorbance at 570 nm was determined by an ELISA using ELX800 micro plate

spectrophotometer”.

1H NMR of compound 4a

1H NMR of compound 4f

1H NMR of compound 4k

1H NMR of compound 4m

IR Spectra of compound 4f

IR Spectra of compound 4m

Mass Spectra of compound 4f

13C NMR of compound 4f

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