6
R. Peraman et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
129.3, 48.2, 23.8, 15.8. Mass (ESI/MS positive mode): m/z 269 (M+Na), 247
(NH), 3048, 3015, 3011 (Ar–H), 2988, 2977, (C–H), 1685 (C@O quinoxaline), 1712
(C@O), 1611, 1624 (C@N), 1554 (C@S), 1513, 1415 (C@C), 1050 (C–N), 1212 (C–O)
864, 764 (Ar–H deformation out of plane). 1H NMR (DMSO-d6): d = 1.61 (t, 3H,
CH3), 2.43(s, H, CH3), 4.52 (q, 2H, –CH2), 6.92–7.15 (m, 2H, J = 11 Hz, quinox), 7.15–
7.39 (m, 2H, J = 10 Hz, 3 Hz quinox), 7.57–8.51 (dd, 2H, Ar, J = 9 Hz), 8.82 (m, 1H,
CH-quinolone), 11.7 (s, 1H, NH). 13C NMR (CDCl3): d = 191.1 (C@S), 164.7 (C@O
quinone), 159.5 (C@O, quinox), 158.3 (C@N, oxadiazole), 154.3, 152.8, 148.1,
147.6, 143.4, 142.9, 141.5, 133.2, 128.1, 127.7, 126.3, 124.1, 123.1, 121.1, 120.8,
49.5, 25.6, 17.3. Mass (ESI/MS positive mode): m/z 433 (M+H).
Antibacterial Screening; Disk diffusion method: 20 ml of sterile nutrient agar
medium was transferred aseptically to the sterile Petri dishes and allowed for
solidification. To this, 24 h subcultured bacteria inoculum was inoculated into
plates using sterile cotton swab dipped in the nutrient broth medium. After
inoculating, 1 mg/ml solutions of compounds in DMF solvent were prepared and
(M+H).
Synthesis of (Z)-1-(1-ethyl-7-methyl-4-oxo-1,4-dihydro-1,8-naphthyridine-3-
carbonyl)-4-(3-oxo-3,4-dihydroquinoxalin-2(1H)-ylidene)thiosemicarbazide (5):
A mixture of compound 3 (0.02 mol) and quinoxaline-2,3-dione (0.02 mol) in
absolute ethanol were heated under reflux for 22 h. The reaction mixture was
poured on to the ice and recrystallized from ethanol and then from DMF to
remove quinoxaline residues. The purity of the product was confirmed on silica
gel coated TLC plate and HPLC. Yield: 54%, mp 254–255 °C, UV (EtOH): k 369,
247 nm. IR: 3324, 3160 (NH), 3047, 3018, 3010 (Ar–H), 2989, 2972, (C–H),
1681 (C@O quinoxaline), 1710, 1716 (C@O coupled), 1612, 1619 (C@N), 1581,
1511 (C@S), 1419 (C@C), 1057 (C–N), 860, 759 (Ar–H deformation out of
plane). 1H NMR (DMSO-d6): d = 1.57 (t, 3H, CH3), 2.14 (s, H, CH3), 4.51 (q, 2H, –
CH2), 7.23 (s, 1H, NH), 6.96–7.10 (m, 2H, J = 10 Hz, quinox), 7.11–7.14 (m, 2H,
J = 14 Hz, 4 Hz, quinox), 7.76-8.51 (dd, 2H, Ar, J = 9 Hz), 8.81 (m, 2H, CH-
quinolone and NH), 11.8 (s, 1H, NH), 12.3 (s, 1H, NH). 13C NMR (CDCl3):
d = 184.3 (C@S), 168.9 (C@O), 165.5 (C@O quinone), 159.2 (C@O, quinox),
153.6, 151.8, 148.5, 146.9, 141.6, 142.8, 140.3, 131.5, 128.4, 125.5, 126.1, 123.3,
122.6, 118.1, 115.7, 48.1, 23.9, 16.1 Mass (ESI/MS positive mode): m/z 450
(M+H).
10 ll of the solution was added on blank disk using micropipette. After absorption
of solution, the disks were placed on agar plate surface and were left over for a
periodof24 h at 36 °C. The zoneinhibition inmm was measured to understandthe
antibacterial activity and statistically treated.
Method II A: Agar dilution method: MICs were determined on Mueller-Hinton agar
supplemented with 5% sheep blood. Concentrations of compounds tested were
Synthesis of (Z)-1-ethyl-7-methyl-4-oxo-N0-(3-oxo-3,4-dihydroquinoxalin-2(1H)-
doubling dilutions from 0.008 to 16 lg/ml. Plates were refrigerated and used
ylidene)-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (6):
A
mixture of
within 24 h of preparation. Bacterial suspensions were prepared in saline by
growing them overnight on blood agar plates to the density matching to 0.5
McFarland standard, then diluted at 1:10, and inoculated into test compounds-
compound (0.02 mol) and quinoxaline-2,3-dione (0.02 mol) in absolute
4
ethanol were stirred for 3 hours and after clear solution, it was refluxed for
10 h. The reaction mixture was cooled kept under refrigeration until the
formation of crystals. The crystals were separated and recrystallized from 50%
DMF in ethanol. The purity of the product was confirmed on silica gel coated
TLC plate and HPLC. Yield: 56%, mp 231–232 °C, UV (EtOH): k 350, 246 nm. IR:
3331, 3169 (NH), 3044, 3019, 3012 (Ar–H), 2991, 2975, (C–H), 1676 (C@O
quinoxaline), 1712, 1704 (C@O coupled), 1614, 1618 (C@N), 1583, 1512, 1415
(C@C), 1059 (C–N), 862, 761 (Ar–H deformation out of plane). 1H NMR (CDCl3):
d = 1.60 (t, 3H, CH3), 2.18 (s, H, CH3), 4.61 (q, 2H, –CH2), 7.32 (s, 1H, NH), 6.91–
7.08 (m, 2H, J = 13 Hz quinox), 7.14–7.18 (m, 2H, J = 12 Hz, 4 Hz, quinox), 7.79–
8.55 (dd, 2H, Ar, J = 9 Hz), 8.79 (m, 2H, CH-quinolone and NH), 10.9 (s, 1H, NH),
12.1 (s, 1H, NH). 13C NMR (CDCl3): d = 169.1 (C@O), 165.2 (C@O, quinone),
158.1 (C@O, quinox), 153.9, 152.0, 148.9, 147.3, 142.2, 141.8, 140.5, 131.8,
127.6, 125.9, 126.8, 124.3, 122.9, 120.1, 116.9, 48.9, 24.2, 15.8. Mass (ESI/MS
positive mode): m/z 408 (M+H2O), 391 (M+H).
Synthesis of 1-ethyl-7-methyl-3-(4-(3-oxo-3,4-dihydroquinoxalin-2-yl)-5-thioxo-
4,5-dihydro-1H-1,2,4-triazol-3-yl)-1,8-naphthyridin-4(1H)-one (7): The compound
5 (0.02 mol) in 10% NaOH in 90% ethanol was heated under reflux for 48 h. The
reaction mixture was neutralized with acetic acid and poured on to the ice. The
formed product was washed with water and recrystallized from ethanol. The
purityoftheproduct was confirmedonTLCplateandbyHPLC. Yield:68%, mp290–
292 °C, UV (EtOH): k 378, 249 nm. IR: 3228, 3184 (NH), 3049, 3013, 3012 (Ar–H),
2987, 2975, (C–H), 1684 (C@O, quinoxaline), 1710 (C@O), 1610, 1615 (C@N), 1523
(C@S), 1515, 1417 (C@C), 1049(C–N), 862, 760(Ar–H deformationoutofplane).1H
NMR (DMSO-d6): d = 1.56 (t, 3H, CH3), 2.55 (s, H, CH3), 4.53 (q, 2H, –CH2), 6.90 (s,
1H, NH), 6.93–7.11 (m, 2H, J = 11 Hz, quinox), 7.12–7.57 (m, 2H, J = 11 Hz, 3 Hz,
quinox), 7.74–8.49 (dd, 2H, Ar, J = 8 Hz), 8.75 (m, 1H, CH-quinolone), 12.3 (s, 1H,
NH). 13C NMR (CDCl3): d = 189.6 (C@S), 164.9 (C@O, quinone), 159.7 (C@O,
quinox), 155.4 (C@N, triazole), 153.2, 151.8, 148.7, 146.5, 142.1, 142.8, 141.8,
133.6, 128.6, 127.4, 126.7, 123.8, 122.8, 119.3, 115.7, 48.8, 24.5, 16.8. Mass (ESI/MS
positive mode): m/z 432 (M+H).
containing plates with a Steers replicator with a volume of 1 l
l (104 CFU per spot).
Plates were carefully transferred to an incubator and incubated in 5% CO2 at 36 °C
for 24 h.
Antibacterial screening; Method II B; microdilution method: Broth microdilution
testing was performed with 96-well, round-bottom microtiter plates. Each plate
included positive controls (bacteria without an antimicrobial/compounds),
negative controls (medium only), and serial dilutions of 0.008–16
lg/ml (test
compounds and standards) (G). Following the addition of a 100-
ll inoculum
(grown for 7 days) containing 2 ꢂ 106 test organisms/ml to wells, the plates were
incubated at 36 °C. The concentration was determined by changing amount of
inoculum to achieve the most consistent growth of organism. The final volume of
each well was made up to 200 ll and after 3 days of incubation period, 20 ll of 10
fold dilution of Alamar Blue was added. Alamar Blue is a ox-redox dye that turns
from blue to pink in presence of growing organisms. After 5th days of incubation,
the color intensity of each well was documented, and the MIC was recorded. The
lowest concentration used that did not result in the colour change from blue to
pink.
Antitubercular screening; Alamar Blue Assay: A stock solution of the test compounds
prepared in DMSO at 1 mg/ml was sterilized by passage through 0.22 micron
Nylonbased membranefilters. Controlsreceived 50 mlDMSO whilstIsoniazid and
rifampin were included as positive drug control. The inoculum used was 1:100
dilution to represent 1% of the mycobacterial population (102–103 CFU/ml). All the
compounds were screened for antitubercular activity at a concentration of
6.25 lg/ml. It was screened against Mycobacterium tuberculosis H37Rv (ATCC
27294) by Microplate Alamar Blue Assay (MABA). Alamar blue is an oxidation–
reduction dye that exhibit colour change from blue to red up on reduction by
mycobacteria. 50
ll 1:1 dilution of Alamar blue stock solution assay medium was
transferred to all wells for a final assay volume of 250
ll per well yielding a final
concentration of 10% alamar blue. The plates were read against an excitation
wavelengthof530 nm, and anemissionwavelengthof590 nmanditwas recorded
to determine whether any of the test compounds fluorescence at the emission
wavelength, thus interfere with the assay. Plates were returned to incubator and
the fluorescence was read at 5th day. The percent viability was determined as
fluorescence countsin thepresence oftestcompound as a percentageof that inthe
vehicle control. A compound is considered to be active only if it shows inhibition
90%. The results were shown in Table 1.
Synthesis of 1-ethyl-7-methyl-3-(4-(3-oxo-3,4-dihydroquinoxalin-2-yl)-5-thioxo-
4,5-dihydro-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (8):
A mixture of
compound 6 (0.02 mol) and carbon disulfide (0.02 mol) in dimethyl formamide
(DMF) were refluxed for 18 h. The reaction mixture was poured onto the ice and
the precipitate was washed with ice cold water. The crude was recrystallized from
90% ethanol. The purity of the product was confirmed on silica gel coated TLC plate
and HPLC. Yield: 63%, mp 281–283 °C, UV (EtOH): k 384, 248 nm. IR: 3229, 3189