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S. Emami et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 405–411
12.8, 1.8 Hz, 1H, H-2a), 4.52 (dd, J = 12.8, 1.8 Hz, 1H, H-2b), 5.48 (t,
J = 1.8 Hz, 1H, H-3), 6.80–7.10 (m, 2H, H-6 and H-8), 7.31 (dt, J =
6.4, 2.4 Hz, 1H, H-7), 7.83 (dd, J = 8.4, 2.4 Hz, 1H, H-5).
7.62 Hz, 1H, H-6 chroman), 7.31 (t, J = 7.25 Hz, 1H, H-7 chroman),
7.54 (d, J = 7.17 Hz, 1H, H-8 quinoline), 7.89 (d, J = 13.13 Hz, 1H, H-
5 quinoline), 8.51 (d, J = 7.35 Hz, 1H, H-5 chroman), 8.65 (s, 1H, H-
2 quinoline), 11.56 (s, 1H, OH oxime), 15.20 (s, 1H, COOH). Anal.
calcd. for C26H25FN4O5: C, 63.41; H, 5.12; N, 11.38. Found: C,
63.35; H, 5.19; N, 11.22.
General procedure for the synthesis of N-(2,3-dihydro-4-
hydroxyimino-4H-1-benzopyran-3-yl)-
piperazinylquinolones 9a–d
2,3-Dihydro-9-fluoro-3-methyl-10-[4-(4-hydroxyimino-2,3-
dihydro-4H-1-benzopyran-3-yl)piperazin-1-yl]-7-oxo-7H-
A mixture of 3-bromo-2,3-dihydro-4H-1-benzopyran-4-one oxime
13 (0.5 mmol), piperazinylquinolone (1–3 or N-desmethyl levo-
floxacin 14, 0.5 mmol) and NaHCO3 (0.5 mmol) in DMF (5 mL),
was stirred at room temperature for 24–72 h. After consump-
tion of piperazinylquinolone (monitored by TLC), water (10 mL)
was added and the precipitate was filtered, washed with water
and crystallized from DMF / water to give compound 9a–d as (E)-
oximes.
pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid 9d
(E)-Isomer; yield: 95%; m.p.: 216–2178C; 1H-NMR (500 MHz,
DMSO-d6) d: 1.43 (d, J = 5.98 Hz, 3H, CH3), 2.55–2.81 (m, 4H, piper-
azine), 3.05 (s, 1H, H-3 chroman), 3.20–3.29 (m, 4H, piperazine),
4.15 (d, J = 12.25 Hz, 1H, H-2a chroman), 4.34 (d, J = 9.68 Hz, 1H,
H-2a pyridobenzoxazine), 4.55 (d, J = 10.57 Hz, 1H, H-2b pyrido-
benzoxazine), 4.81 (d, J = 11.0 Hz, 1H, H-2b chroman), 4.82–4.99
(m, 1H, H-3 benzoxazine), 6.91 (d, J = 8.22 Hz, 1H, H-8 chroman),
6.95 (t, J = 7.55 Hz, 1H, H-6 chroman), 7.31 (t, J = 7.63 Hz, 1H, H-7
chroman), 7.55 (d, J = 12.07 Hz, 1H, H-8 pyridobenzoxazine), 8.50
(d, J = 7.97 Hz, 1H, H-5 chroman), 8.94 (s, 1H, H-5 pyridobenzoxa-
zine), 11.53 (s, 1H, OH oxime), 15.17 (s, 1H, COOH). 13C-NMR (125
MHz, DMSO-d6) d: 18.74, 51.02, 51.63, 55.67, 62.05, 67.54, 68.91,
104.21, 115.92, 117.34, 120.83, 125.61, 131.84, 131.88, 141.09,
145.08, 147.05, 155.96, 166.92, 177.22. Anal. calcd. for
C26H25FN4O6: C, 61.41; H, 4.96; N, 11.02. Found: C, 61.57; H, 5.11;
N, 10.88.
1,4-Dihydro-1-ethyl-6-fluoro-7-[4-(4-hydroxyimino-2,3-
dihydro-4H-1-benzopyran-3-yl)piperazin-1-yl]-4-oxo-3-
quinoline carboxylic acid 9a
(E)-Isomer; yield: 93%; m.p.: 238–2398C; 1H-NMR (500 MHz,
DMSO-d6) d: 1.38 (t, J = 6.97 Hz, 3H, -CH3 ethyl), 2.65–2.83 (m, 4H,
piperazine), 3.06 (s, 1H, H-3 chroman), 3.20–3.28 (m, 4H, pipera-
zine), 4.16 (d, J = 12.20 Hz, 1H, H-2a chroman), 4.54 (q, J = 6.86 Hz,
2H, -CH2-ethyl), 4.84 (d, J = 12.27 Hz, 1H, H-2b chroman), 6.90 (d, J
= 8.26 Hz, 1H, H-8 chroman), 6.95 (t, J = 7.57 Hz, 1H, H-6 chro-
man), 7.15 (d, J = 6.94 Hz, 1H, H-8 quinoline), 7.30 (t, J = 7.71 Hz,
1H, H-7 chroman), 7.90 (d, J = 13.10 Hz, 1H, H-5 quinoline), 8.49
(d, J = 7.89 Hz, 1H, H-5 chroman), 8.93 (s, 1H, H-2 quinoline),
11.54 (s, 1H, OH oxime), 15.31 (s, 1H, COOH). Anal. calcd. for
C25H25FN4O5: C, 62.49; H, 5.24; N, 11.66. Found: C, 62.63; H, 5.11;
N, 11.83.
Antibacterial activity
Compounds 9a–d were evaluated for their in-vitro antibacterial
activity against selected strains using conventional agar-dilu-
tion method in comparison to the reference drugs [24]. Drugs
(10.0 mg) were dissolved in DMSO (1 mL) and the solution was
diluted with water (9 mL). Further progressive twofold serial
dilution with melted Mueller–Hinton agar was performed to
obtain the required concentrations of 100, 50, 25, 12.5, 6.25,
3.13, 1.56, 0.78, 0.39, 0.19, 0.098, 0.049, 0.024, 0.012, 0.006,
0.003, and 0.0015 lg/mL. The bacteria inocula were prepared by
suspending overnight colonies from Mueller–Hinton agar
media in 0.85% saline. The inocula were adjusted photometri-
cally at 600 nm to a cell-density equivalent of approximately 0.5
McFarland standard (1.56108 CFU/mL). The suspensions were
then diluted in 0.85% saline to give 107 CFU/mL. Petri dishes
were spot-inoculated with 1 lL of each prepared bacterial sus-
pension (104 CFU/spot) and incubated at 35–378C for 18 h. The
minimum inhibitory concentration (MIC) was the lowest con-
centration of the test compound, which resulted in no visible
growth on the plate. To ensure that the solvent had no effect on
bacterial growth, a control test was performed with test medium
supplemented with DMSO at the same dilutions as used in the
experiment.
1,4-Dihydro-1-ethyl-6-fluoro-7-[4-(4-hydroxyimino-2,3-
dihydro-4H-1-benzopyran-3-yl)piperazin-1-yl]-4-oxo-1,8-
naphthyridine-3-carboxylic acid 9b
(E)-Isomer; yield: 96%; m.p.: 206–2078C; 1H-NMR (500 MHz,
DMSO-d6) d: 1.36 (t, J = 7.00 Hz, 3H, -CH3 ethyl), 2.60–2.82 (m, 4H,
piperazine), 3.04 (s, 1H, H-3 chroman), 3.69–3.82 (m, 4H, pipera-
zine), 4.15 (d, J = 11.94 Hz, 1H, H-2a chroman), 4.46 (q, J = 6.99 Hz,
2H, -CH2- ethyl), 4.82 (d, J = 12.29 Hz, 1H, H-2b chroman), 6.91 (d, J
= 8.26 Hz, 1H, H-8 chroman), 6.95 (t, J = 7.65 Hz, 1H, H-6 chro-
man), 7.31 (t, J = 7.24 Hz, 1H, H-7 chroman), 8.05 (d, J = 13.49 Hz,
1H, H-5 naphthyridine), 8.50 (d, J = 8.00 Hz, 1H, H-5 chroman),
8.95 (s, 1H, H-2 naphthyridine), 11.56 (s, 1H, OH oxime), 15.30 (s,
1H, COOH). 13C-NMR (125 MHz, DMSO-d6) d: 15.53, 47.34, 47.40,
48.06, 50.79, 61.82, 67.37, 108.90, 113.43, 115.83, 117.40, 120.16,
120.33, 120.89, 131.88, 131.93, 144.91, 145.68, 147.05 148.55,
150.03, 155.918, 166.72, 177.17. Anal. calcd. for C24H24FN5O5: C,
59.87; H, 5.02; N, 14.55. Found: C, 60.01; H, 4.89; N, 14.50.
MTT colorimetric assay
1-Cyclopropyl-1,4-dihydro-6-fluoro-7-[4-(4-hydroxyimino-
2,3-dihydro-4H-1-benzopyran-3-yl)piperazin-1-yl]-4-oxo-
The cytotoxic activity of target compounds were assessed using
MTT colorimetric assay [25] against normal mouse fibroblasts
(NIH/3T3). Briefly, cultures in the exponential growth phase
were trypsinized and diluted in complete growth medium to
give a total cell count of 56104 cells/mL. 100 lL of suspension
was added to the wells of sterile 96-well plates. After plating,
50 lL of a serial dilution of every agent was added. Each com-
pound dilution was assessed in triplicate. Three wells containing
only normal mouse fibroblast (NIH/3T3) cells suspended in
3-quinoline carboxylic acid 9c
(E)-Isomer; yield: 61%; m.p.: 245–2468C; 1H-NMR (500 MHz,
DMSO-d6) d: 1.10–1.20 (m, 2H, cyclopropyl), 1.26–1.32 (m, 2H,
cyclopropyl), 2.66–2.86 (m, 4H, piperazine), 3.07 (s, 1H, H-3 chro-
man), 3.20–3.28 (m, 4H, piperazine), 3.76 (br s, 1H, cyclopropyl),
4.16 (d, J = 12.10 Hz, 1H, H-2a chroman), 4.84 (d, J = 12.10 Hz, 1H,
H-2b chroman), 6.91 (d, J = 8.24 Hz, 1H, H-8 chroman), 6.95 (t, J =
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