4
MOHAMMED ET AL.
0
3
.96–3.98 (2H, m, H-5, H-6), 4.17 (1H, dd, J = 5.1, 13.0 Hz, H-6), 4.82
(C-4 ). (ESI+) m/z 416.14282 [M+Na] (C18
H
23
N NaO
3
7
requires
(
1H, t, J = 9.6 Hz, H-4), 5.06 (1H, t, J = 9.6 Hz, H-3), 5.55 (1H, d,
416.14337).
J = 8.5 Hz, H-1).
2
.2 | Biological activity
1-Ethyl-7-methyl-4-oxo-N-(1,3,4,6-tetra-O-acetyl-2-deoxy-D-
2
.2.1 | Antimicrobial activity
glucopyranose-2-yl)-[1,8]-naphthyridine-3-carboxamide (5)
Antimicrobial activities of the synthesized compounds were investi-
gated in vitro against Gram negative bacteria including food-borne
pathogens (Escherichia coli O157:H7, Salmonella enterica ATCC 13312
and Listeria monocytogenes ATCC 19115); Gram negative infectious
bacteria (Pseudomonas aeruginosa ATCC 9627; Escherichia coli
NCTC11954; Escherichia coli ATCC 8739 and resistant clinical Escheri-
chia coli isolate (not susceptible to nalidixic acid, ciprofloxacin HCl, and
Ethyl chloroformate (0.530 mL, 5.7 mmol) was added drop-wise to a
solution of TEA (0.600 mL, 4.3 mmol) and nalidixic acid (1.0 g,
ꢀ
4
.3 mmol) in CH
2
Cl
2
(100 mL) at −20 C. The mixture was left stirring
ꢀ
at −20 C until reaction completion as revealed by TLC (EtOAc:
n-hexane 10:1, R
dissolved in cold CH
mixture was stirred at −20 C for 3 h and then left stirring at RT for
4 h. Washing with H O (150 mL), aq. 1 M HCl (150 mL), aq. 5%
NaHCO (150 mL), H O (150 mL), drying over anhydrous Na SO
and evaporation yielded a gummy residue. Trituration with Et
f
= 0.70). Following, compound (4) (1.5 g, 4.3 mmol)
2
2
Cl (30 mL) was added drop-wise and the
ꢀ
®
Norfloxacin from Biolab [Amman-Jordan]); Gram positive bacteria
2
2
(
Methicillin resistant Staphylococcus aureus ATCC 33591–MRSA and
3
2
2
4
,
Methicillin sensitive Staphylococcus aureus ATCC 9253-MSSA); non
spore forming fungi (Candida albicans ATCC 10231) and spore forming
(Aspergillus flavus ATCC 9643; Fusarium solani ATCC 36031; Stachybo-
trys chartarum IBT 7711, which was provided by Professor Naresh
Magan [Cramfield University, UK] and Penicillium chrysogenum ATCC
2
O
(
100 mL) and recrystallization with n-hexane and EtOAc afforded
ꢀ
compound (5) (0.760 g, 32%) as a white solid m.p. 182.0–185.0
C
DSC: 183.5 C); IRvmax 3240.49 cm−1 (CONH), 1,746.08 (C O),
ꢀ
(
,660.77 cm−1 (C O amide); H-NMR (DMSO-d
1
1
6 H
, 500 MHz) δ
1.33
CO),
10106). All media were prepared according to manufacturer instruc-
(
3 3 3
3H, t, J = 6.7 Hz, CH ), 1.80 (3H, s, CH CO), 1.94 (6H, s, CH
tions under sterilized conditions. Bacterial broth cultures stocks were
1
.97 (3H, s, CH
3
CO), 2.65 (3H, s, CH
3
Napth), 3.95–3.97 (1H, m,
ꢀ
cultivated in the appropriate medium at 37 C for 24 h prior to testing.
H-6), 4.10–4.17 (2H, m, H-5, H-6), 4.27 (1H, dd, J = 5.0, 12.4 Hz, H-2),
.50 (2H, q, J = 6.7 Hz, CH ), 4.91 (1H, t, J = 8.8 Hz, H-4), 5.52 (1H,
0
.5 McFarland standard was used to visually approximate the con-
4
2
centration of cells in a suspension.
t, J = 9.2 Hz, H-3), 6.06 (1H, d, J = 8.0 Hz, H-1), 8.41 (1H, d,
Fungi conidial suspension was prepared from 7-day cultures
grown on malt extract agar at 30 ꢀC. Fungi conidia was attained
J = 7.4 Hz, H-6Napth), 8.46 (1H, d, J = 7.4 Hz, H-5Napth), 8.93 (1H, s,
13
H-2Napth), 9.91 (1H, d, J = 8.7 Hz, CONH); C-NMR (DMSO,
from the agar using cotton swap previously immerged in Tween
5
4
7
1
00 MHz) 15.44 (CH
2 3 3 3
CH ), 20.77–21.01 (4 × CH CO), 25.32 (CH ),
2
0 and transferred into 3 mL normal saline. The conidial suspension
6.54 (CH CH ), 52.37 (C-2), 62.07(C-6), 68.71 (C-4), 71.75 (C-3),
2
3
was vigorously vortexed for 20 s to prevent spore clumping and
then left standing at RT for 15 min to settle down. Following, the
supernatant was transferred into a sterile falcon tube and adjusted
2.71 (C-5), 92.57 (C-1), 111.68 (C-6Napth), 120.03 (C-5Napth),
21.93 (C-8aNapth), 136.34 (C-3Napth), 148.59 (C-2Napth), 148.86
(
(
(
C-4aNapth), 163.68 (C-7Napth), 164.74(CONH), 169.34–170.50
5 × CH CO), 176.12 (C-4Napth). (ESI +) m/z 584.18508 [M+Na]
NaO11 requires 584.18563).
6
to 0.5 McFarland at 530 nm to yield 8.44–18.44 × 10 sporangios-
3
6
6
C
26
H
31
N
3
pore suspensions (A. flavus, 8.44 × 10 ; F. solani, 10.63 × 10 ;
6
6
S. chartarum, 18.44 × 10 ; P. chrysogenum, 14.69 × 10 ). Mean
spores count from three trials was determined using hemocytome-
ter. The final working suspension was obtained after diluting the
stock suspension by 1:50 with malt extract broth.
1
-Ethyl-7-methyl-4-oxo-N-(2-deoxy-D-glucopyranose-2-yl)-
1,8]-naphthyridine-3-carboxamide (6)
Compound 5 (0.350 g, 0.62 mmol) was dissolved in 30 mL MeOH.
NaOCH (0.200 g, 3.7 mmol) was added gradually and left stirring at
[
Minimal inhibitory concentrations (MICs, mM) of the targeted
compounds against Gram positive, Gram negative bacteria and can-
dida spp. were determined using micro-broth dilution method. MIC
is defined as the lowest concentration of the tested compound
showing no microbial viability. Compounds were first dissolved in
DMSO at a stock solution of 2 mg mL−1. Then 100 μL was added to
first well of the 96-well plate having 100 μL of the adequate broth.
From here, the solution was serially diluted resulting in two fold
dilution of the test compound 5 (0.0070–0.8904 mM) and 6 (0.0099–
1.2710 mM) in subsequent wells. Then, 10 μL of the microbial sus-
pension (10 CFU mL−1) and 90 μL of broth were added to each well
3
room temperature for 3 h until the formation of precipitate. The
suspension was neutralized with conc. HCl. Filtration, washing with
cold MeOH and drying afforded compound (6) (0.228 g, 94%) as a
yellow solid; DSC 245 C; IRvmax 3,461.63 cm−1 (CONH), 2,829.43 cm−1
ꢀ
OH), 1,582.47 cm− (C O); H-NMR (DMSO-d
3H, t, J = 6.4 Hz, CH ), 2.62 (3H, s, CH Napth), 3.13–3.60 (7H, m,
O exchangeable), 3.83 (1H, q,
J = 7.6 Hz, H-2), 4.42–4.56 (3H, m, CH , OH, D O exchangeable),
.10 (1H, m, H-1), 7.45 (1H, d, J = 8.1 Hz, H-6Napth), 8.51 (1H, d,
J = 8.1 Hz, H-5Napth), 8.92 (1H, s, H-2Napth), 9.84 (1H, d,
1
1
(
(
6 H
, 500 MHz) δ 1.50
3
3
H-3, H-4, H-5, H-6, H-6, 3 × OH, D
2
2
2
5
6
1
3
ꢀ
J = 8.45 Hz, CONH); C-NMR (DMSO, 500 MHz) 15.49 (CH
2
CH
) 54.48 (C-2), 61.59 (C-6), 71.63 (C-4),
1.75 (C-3), 72.76 (C-5), 91.26 (C-1), 112.72 (C-6Napth), 120.16
3
),
and incubated at 37 C for 24 h. Experiments were done in triplicates.
2
7
5.32 (CH
3
), 46.36 (CH
2
CH
3
Following, absorbance of light scattering at 625 nm was measured
using Epoch spectrophotometer. Nalidixic acid (0.01682–2.1530 mM)
was used as positive control and proper sterility and negative control
(no compound) samples were prepared and evaluated.
(
C-5Napth), 121.87 (C-8’a), 136.41 (C-3Napth), 148.44 (C-2Napth),
48.59 (C-4aNapth), 163.54 (C-7Napth), 164.63 (CONH), 176.20
1