Synthesis and in vitro Antimicrobial Activity of Nalidixic Acid Hydrazones

Article abstract of DOI:10.1002/jhet.2468

A series of nalidixic acid-based hydrazones have been synthesized and evaluated for their in vitro antimicrobial activity using the broth microdilution method against a panel of reference strains of microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and fungi belonging to yeasts Candida spp. and molds Aspergillus spp., Penicillium spp., and Rhizopus spp. Nalidixic acid derivatives were obtained by condensation reaction of nalidixic acid hydrazide with substituted (hetero)aromatic aldehydes. All compounds have been characterized by¹H NMR and¹³C NMR spectra. The antimicrobial activity indicated that compound with indole substituent could be a promising lead for future development of active antifungal agents.

Full text of DOI:10.1002/jhet.2468

Month 2015
Synthesis and in vitro Antimicrobial Activity of Nalidixic
Acid Hydrazones
a
b
b
*
Łukasz Popiołek, Anna Biernasiuk, and Anna Malm
aDepartment of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin, 4A Chodźki Street, 20-093,
Lublin, Poland
bDepartment of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, 1 Chodźki Street,
20-093, Lublin, Poland
*
E-mail: lukasz.popiolek@umlub.pl
Received March 1, 2015
DOI 10.1002/jhet.2468
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
A series of nalidixic acid-based hydrazones have been synthesized and evaluated for their in vitro antimi-
crobial activity using the broth microdilution method against a panel of reference strains of microorganisms,
including Gram-positive bacteria, Gram-negative bacteria, and fungi belonging to yeasts Candida spp. and
molds Aspergillus spp., Penicillium spp., and Rhizopus spp. Nalidixic acid derivatives were obtained by con-
densation reaction of nalidixic acid hydrazide with substituted (hetero)aromatic aldehydes. All compounds
1
have been characterized by H NMR and 13C NMR spectra. The antimicrobial activity indicated that com-
pound with indole substituent could be a promising lead for future development of active antifungal agents.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
RESULTS AND DISCUSSION
Nalidixic acid (1,8-naphthyridine derivative) was the
first clinically useful synthetic quinolone antibiotic intro-
duced in the 1960s. It failed to achieve adequate concentra-
tions in the treatment of systemic infections following oral
or potential administration but got concentrated in the urine
where it was effective for eradicating urinary tract infec-
tions [1]. It acts against bacteria by selectively inhibiting
the type II topoisomerase DNA gyrase and topoisomerase
IV, enzymes that play a critical role in bacterial cell growth
and division [2]. Structure activity relationship of com-
pounds based on nalidixic acid [3–5] has led to a large
group of synthetic antibacterial agents collectively known
as the quinolones [6].
Hydrazones are attractive target compounds for new
drug development because of their synthetic and biological
versatility including antimicrobial activities. Furthermore,
a number of hydrazone derivatives have been claimed to
possess other interesting activities like anticancer [7,8],
anti-inflammatory [9,10], antibacterial [11,12], antifungal
[13], and antitubercular activity [14,15].
Chemistry. The aim of this study was the synthesis,
spectral analysis, and in vitro antimicrobial evaluation of
nalidixic acid-based hydrazones (3–16). In the first step
of synthesis, methyl ester of nalidixic acid (1) was
synthesized by the reaction of nalidixic acid chloride with
methanol. Then in the second step, the obtained ester (1)
was converted to appropriate hydrazide by the reaction
with hydrazine hydrate. Finally in the third step of
synthesis, the desired hydrazones of nalidixic acid (3–16)
were synthesized by a condensation reaction of nalidixic
acid hydrazide (2) with various substituted (hetero)aromatic
aldehydes. Using the aforementioned reactions, 12 new
nalidixic acid-based hydrazones were obtained (3,4 and
7–16). Compounds 5 and 6 were synthesized earlier [16].
In the 1H NMR spectra of hydrazones (3–16), one typical
proton singlet signal for the N¼CH group was observed in
the range of δ 8.67–9.14 ppm and for the CONH group at δ
12.42–13.32 ppm. Whereas in the 13C NMR of compounds
3–16, three signals for N¼CH group [17] and two C¼O
groups were observed at δ 145.1–155.1ppm and δ 161.1–
180.2ppm, respectively, which successfully confirmed the
formation of expected products. All other aliphatic and aro-
matic signals were found at the usual regions.
Encouraged by the aforementioned facts and in continu-
ation of our synthetic work on searching for microbiologi-
cally active molecules, in this current research, we design,
synthesized, and evaluated for in vitro antimicrobial activity
new nalidixic acid-based hydrazones.
The target new nalidixic acid-based hydrazones (3–16)
were synthesized via the route outlined in Scheme 1. The
© 2015 HeteroCorporation

Ł. Popiołek, A. Biernasiuk, and A. Malm
Vol 000
Scheme 1. Synthetic pathway to nalidixic acid based hydrazones (3–16).
synthesized compounds (3–16) were in vitro screened for
antibacterial and antifungal activities.
Germany) and used without further purification. Melting
points were determined in Fisher-Johns blocks (Fisher
Scientific, Germany) and presented without any corrections.
Microbiology. The results of our study indicated, that
among the newly synthesized nalidixic acid derivatives,
compounds 3, 6, 8, and 16 had no inhibitory effect on the
growth of reference strains of Gram-positive or Gram-
negative bacteria. According to the data presented in
Table 1, the other compounds showed different bacteriostatic
or bactericidal activity against tested bacteria [minimal
inhibitory concentration (MIC)= 125–1000 μg/mL; minimal
bactericidal concentration (MBC) = 125– >1000 μg/mL]
or were inactive against these microorganisms.
These derivatives of nalidixic acid indicated also some ac-
tivity against fungi, belonging to Candida spp. The com-
pounds 4, 8, 11, 12, 13, and 15 inhibited their growth with
MIC =500–1000 μg/mL [minimal fungicidal concentration
(MFC) ≥ 1000μg/mL] or exhibited no activity. The molds,
that is, Aspergillus niger ATCC 16404, Aspergillus fumigatus,
Penicillium spp. and Rhizopus spp. were insensitive to them.
The compound 15, as the only, indicated high fungicidal
or fungistatic activity against fungi belonging to yeasts
(MIC =15.62–500μg/mL and MFC =15.62–1000 μg/mL)
and molds (MIC = 31.25–125μg/mL and MFC > 1000 μg/mL)
(Table 2).
1
The H NMR and 13C NMR spectra were recorded on a
Bruker Avance 300 apparatus (Bruker BioSpin GmbH,
Germany) in DMSO-d6 or CDCl3 with TMS as an internal
standard. Chemical shifts are given in ppm (δ-scale). The
purity of obtained compounds was checked by TLC on
aluminium oxide 60 F254 plates (Merck Co. USA), in a
CHCl3/EtOH (10:1, v/v) solvent system. The spots were
detected by exposure to a UV lamp at 254nm. Elemental
analyses of the obtained compounds were performed on an
AMZ 851 CHX analyser (PG, Gdańsk, Poland). The
maximum percentage differences between calculated and
found values for each element were within the error and
amounted to 0.4%.
Synthesis of nalidixic acid methyl ester (1). The compound
was prepared according to a literature procedure [18].
Analytical and spectral data consisted of those reported in
the literature.
Synthesis of nalidixic acid hydrazide (2). To the methanolic
solution of 1 (2 mL of 1 in 5 mL of methanol), 2 mL of
hydrazine monohydrate (98%) was added dropwise and
refluxed for 4 h. The reaction mixture was poured into a
beaker containing ice cold water. The precipitate was
filtered off, dried, and recrystallized form ethanol.
1
Yield: 71%; m.p.: 186–188°C. H NMR (DMSO-d6)
CONCLUSIONS
δ (ppm)=1.36–1.40 (t, 3H, CH3, J=6Hz), 2.65 (s, 3H, CH3),
2.70 (br s, 2H, NH2), 4.53–4.60 (q, 2H, CH2, J = 9 Hz,
J = 6 Hz), 7.44–7.47 (d, 1H, H6-naphthyridine, J = 9 Hz),
8.53–8.54 (d, 1H, H5-naphthyridine, J = 12 Hz), 8.96
(s, 1H, H2-naphthyridine), 10.51 (br s, 1H, CONH);
13C NMR (DMSO) δ (ppm) = 12.4, 24.3, 45.8, 102.9,
113.3, 121.9, 137.3, 145.6, 159.9, 160.7, 167.4 (C¼O),
180.6 (C¼O). Analysis for C12H14N4O2 (246.26)
Calculated: C: 58.53%, H: 5.73%, N: 22.75%; Found:
In this research, we design, synthesized, and analyzed 12
new nalidixic acid-based hydrazones (3,4 and 7–16) and
subjected them to in vitro antimicrobial assays. Our
antimicrobial activity results indicate that a few of the
obtained compounds showed some antimicrobial activity.
Particularly noteworthy is compound 15 with antifungal
effect against yeasts (MIC = 15.62–500μg/mL and
MFC = 15.62–1000 μg/mL) and molds (MIC = 31.25–
125μg/mL and MFC > 1000μg/mL).
C: 58.59%, H: 5.70%, N: 22.78%.
Synthesis of nalidixic acid hydrazones (3–16)
General procedure.
A 2.46 g (10 mM) of 2 was
EXPERIMENTAL
dissolved in 15mL of methanol. The mixture was stirred
till a clear solution was obtained. After that, appropriate
(hetero)aromatic aldehydes were added (11 mM), and
Chemistry. All reagents were purchased from Sigma-
Aldrich (Munich, Germany) and Merck Co. (Darmstadt,
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DOI 10.1002/jhet

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Synthesis and in vitro Antimicrobial Activity of Nalidixic Acid Hydrazones
the solution was stirred at rt for 3h. The precipitate
obtained was filtered off, dried, and recrystallized from
ethanol.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-
3-carboxylic acid (3-bromo-4-hydroxy-benzylidene)-hydrazide
1
(3). Yield: 81%; m.p.: 307–309°C. H NMR (DMSO-d6)
δ (ppm)= 1.41–1.44 (t, 3H, CH3, J=3Hz), 2.70 (s, 3H,
CH3), 4.62–4.65 (q, 2H, CH2, J= 3 Hz), 7.03–7.04
(d, 1H, Ph, J = 3 Hz), 7.53–7.55 (d, 1H, H6-
naphthyridine, J = 6 Hz), 7.60–7.61 (m, 1H, ArH),
7.89–7.90 (d, 1H, ArH, J = 3 Hz), 8.31 (s, 1H, H2-
naphthyridine), 8.65–8.67 (d, 1H, H5-naphthyridine,
J = 6 Hz), 8.99 (s, 1H, OH), 9.11 (s, 1H, N¼CH),
13.01 (s, 1H, CONH); 13C NMR (DMSO) δ (ppm)
= 13.1, 24.7, 45.9, 104.1, 109.9, 116.3, 119.3,
121.9, 127.2, 128.4, 130.4, 137.3, 148.6 (N¼CH),
153.3, 153.7, 159.4, 160.7, 161.9 (C¼O), 179.8
(C¼O). Analysis for C19H17BrN4O3 (429.27)
Calculated: C: 53.16%, H: 3.99%, N: 13.05%;
Found: C: 53.21%, H: 3.96%, N: 13.08%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-
3-carboxylic acid (5-bromo-2-hydroxy-benzylidene)-hydrazide
(4). Yield: 73%; m.p.: 296–298°C. 1H NMR (CDCl3)
δ (ppm)= 1.55–1.57 (t, 3H, CH3, J=3 Hz), 2.74 (s, 3H,
CH3), 4.61–4.65 (q, 2H, CH2, J=6Hz, J= 3Hz), 6.93–6.97
(m, 1H, ArH), 7.33–7.35 (d, 1H, ArH, J= 6 Hz), 7.37–7.39
(d, 1H, H6-naphthyridine, J = 6 Hz), 7.49–7.50 (d, 1H,
ArH, J = 3 Hz), 8.30 (s, 1H, H2-naphthyridine), 8.66
(s, 1H, OH), 8.67–8.69 (d, 1H, H5-naphthyridine,
J = 6 Hz), 9.01 (s, 1H, N¼CH), 13.25 (s, 1H, CONH);
13C NMR (CDCl3) δ (ppm) = 12.7, 24.1, 45.1, 103.8,
114.7, 118.9, 119.1, 119.4, 121.9, 130.4, 130.6,
137.3, 150.4 (N¼CH), 153.3, 156.3, 159.1, 160.3,
161.1 (C¼O), 180.1 (C¼O). Analysis for
C19H17BrN4O3 (429.27) Calculated: C: 53.16%,
H: 3.99%, N: 13.05%; Found: C: 53.19%, H: 4.02%,
N: 13.01%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-
3-carboxylic acid (4-hydroxy-3-ethoxy-benzylidene)-hydrazide
(5).
Yield: 51%; m.p.: 226–228°C (lit. 239°C). 1H
NMR (CDCl3) δ (ppm) =1.44–1.50 (t, 3H, CH3, J=9Hz),
1.51–1.53 (t, 3H, CH3, J= 3 Hz), 2.72 (s, 3H, CH3),
4.21–4.28 (q, 2H, CH2, J=9Hz), 4.56–4.65 (q, 2H, CH2,
J=9 Hz), 5.97 (s, 1H, OH), 6.91–6.94 (m, 1H, ArH),
7.05–7.25 (m, 1H, ArH), 7.32–7.35 (d, 1H,
H6-naphthyridine, J = 9 Hz), 7.56–7.57 (d, 1H, ArH,
J = 3Hz), 8.14 (s, 1H, H2-naphthyridine), 8.65–8.68 (d,
1H, H5-naphthyridine, J = 9 Hz), 9.03 (s, 1H, N¼CH),
13.05 (s, 1H, CONH); 13C NMR (CDCl3) δ (ppm)
= 13.8, 15.2, 24.3, 46.8, 64.3, 103.7, 111.8, 116.2,
119.3, 121.4, 122.9, 123.8, 125.9, 137.3, 147.6, 149.1
(N¼CH), 153.3, 159.9, 160.8, 161.1 (C¼O), 178.9
(C¼O). Analysis for C21H22N4O4 (394.42) Calculated:
C: 63.95%, H: 5.62%, N: 14.20%; Found: C: 63.98%,
H: 5.59%, N: 14.25%.
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Ł. Popiołek, A. Biernasiuk, and A. Malm
Vol 000
Table 2
The activity data expressed as minimal inhibitory concentration (minimal fungicidal concentration) [μg/mL] against the strains of fungi.
Candida
albicans
ATCC 2091
Candida
albicans
TCC 10231
Candida
parapsilosis
ATCC 22019
Aspergillus
niger ATCC
16404
Aspergillus
fumigatus
Species
Penicillium spp. Rhizopus spp.
MIC (MFC)
[μg/ml] of
the tested
4
8
11
12
13
1000 (>1000)
500 (>1000)
—
1000 (>1000)
1000 (>1000)
500 (1000)
0.244
—
—
1000 (>1000)
500 (>1000)
1000 (>1000)
—
500 (1000)
15.62 (15.62)
1.953
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1000 (1000)
500 (500)
1000 (>1000)
31.25 (250)
0.976
compounds
—
31.25 (>1000)
—
—
125 (>1000)
—
—
125 (>1000)
—
15
FLU
The standard antibiotic–fluconazole– used as positive control.
ATCC, American Type Culture Collection.
N¼CH), 13.12 (s, 1H, CONH); 13C NMR (CDCl3) δ
(ppm) = 12.4, 24.3, 45.8, 56.8, 103.9, 114.3, 114.4, 119.3,
121.9, 126.9, 129.1, 132.3, 137.3, 148.6 (N¼CH), 153.5,
157.2, 159.8, 160.7, 161.8 (C¼O), 180.2 (C¼O).
Analysis for C20H19BrN4O3 (443.29) Calculated: C:
54.19%, H: 4.32%, N: 12.64%; Found: C: 54.23%, H:
4.27%, N: 12.68%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (4-methoxy-benzylidene)-hydrazide (6). Yield:
1
89%; m.p.: 244–246°C (lit. 260°C). H NMR (DMSO-d6)
δ (ppm)=1.40–1.44 (t, 3H, CH3, J=6Hz), 2.69 (s, 3H,
CH3), 3.82 (s, 3H, CH3), 4.60–4.66 (q, 2H, CH2,
J=6Hz), 7.02–7.05 (d, 2H, ArH, J=9Hz), 7.52–7.55 (d,
1H, H6-naphthyridine, J=9Hz), 7.71–7.73 (d, 2H, ArH,
J=6Hz), 8.36 (s, 1H, H2-naphthyridine), 8.59–8.61 (d,
1H, H5-naphthyridine, J=6Hz), 9.10 (s, 1H, N¼CH),
13.00 (s, 1H, CONH); 13C NMR (DMSO) δ (ppm)
=12.4, 24.3, 45.7, 103.8, 114.3, 114.4, 119.3, 121.9,
127.1, 129.1, 129.2, 137.3, 149.3 (N¼CH), 153.3, 159.9,
160.1, 160.7, 161.8 (C¼O), 179.6 (C¼O). Analysis
for C20H20N4O3 (364.40) Calculated: C: 65.92%,
H: 5.53%, N: 15.38%; Found: C: 65.88%, H: 5.55%,
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (2-chloro-3-methoxy-benzylidene)-hydrazide
1
(9). Yield: 53%; m.p.: 222–224°C. H NMR (CDCl3)
δ (ppm) = 1.53–1.57 (t, 3H, CH3, J = 6 Hz), 2.73 (s, 3H,
CH3), 3.96 (s, 3H, CH3), 4.59–4.66 (q, 2H, CH2, J=6Hz),
6.96–7.07 (m, 1H, ArH), 7.30–7.32 (m, 1H, ArH),
7.35–7.38 (d, 1H, H6-naphthyridine, J=9Hz), 7.82–7.92
(m, 1H, ArH), 8.67–8.70 (d, 1H, H5-naphthyridine,
J = 9 Hz), 9.04 (s, 1H, H2-naphthyridine), 9.14 (s, 1H,
N¼CH), 13.28 (s, 1H, CONH); 13C NMR (CDCl3) δ
(ppm) = 15.4, 25.3, 47.2, 56.3, 111.9, 113.0, 119.9,
120.2, 121.7, 127.1, 132.7, 136.3, 145.1, 148.0, 155.1
(N¼CH), 155.4, 159.4, 161.7, 163.7 (C¼O), 176.7
(C¼O). Analysis for C20H19ClN4O3 (398.84) Calculated:
C: 60.23%, H: 4.80%, N: 14.05%; Found: C: 60.17%, H:
4.82%, N: 14.09%.
N: 15.42%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (2,3-dimethoxy-benzylidene)-hydrazide (7). Yield:
82%; m.p.: 252–254°C. 1H NMR (CDCl3) δ (ppm)
=1.53–1.57 (t, 3H, CH3, J = 6 Hz), 2.73 (s, 3H, CH3),
3.90 (s, 6H, 2xOCH3), 4.59–4.66 (q, 2H, CH2, J=6Hz,
J=9 Hz), 6.94–6.98 (dd, 1H, ArH, J= 3 Hz), 7.07–7.13 (t,
1H, ArH, J= 9Hz), 7.35–7.37 (d, 1H, H6-naphthyridine,
J=6 Hz), 6.79–7.83 (dd, 1H, ArH, J= 3 Hz), 8.61 (s, 1H,
H2-naphthyridine), 8.67–8.70 (d, 1H, H5-naphthyridine,
J = 9 Hz), 9.05 (s, 1H, N¼CH), 13.15 (s, 1H, CONH);
13C NMR (CDCl3) δ (ppm) = 13.5, 23.4, 45.3, 54.0,
59.9, 110.4, 112.1, 113.8, 117.1, 118.4, 119.8, 122.3,
126.1, 134.4, 142.4, 146.1, 146.8, 150.8 (N¼CH),
159.7, 161.8 (C¼O), 174.9 (C¼O). Analysis for
C21H22N4O4 (394.42) Calculated: C: 63.95%, H: 5.62%,
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (2-bromo-5-fluoro-benzylidene)-hydrazide
1
(10). Yield: 71%; m.p.: 244–246°C. H NMR (DMSO-d6)
δ (ppm)= 1.41–1.45 (t, 3H, CH3, J=6 Hz), 2.52 (s, 3H,
CH3), 4.61–4.67 (q, 2H, CH2, J=6Hz), 7.43–7.54 (m, 1H,
ArH), 7.63–7.84 (m, 1H, ArH), 7.90–7.93 (d, 1H,
H6-naphthyridine, J=9Hz), 8.60–8.63 (d, 1H, ArH, J=9Hz),
8.65 (s, 1H, H2-naphthyridine), 8.87–8.89 (s, 1H,
H5-naphthyridine, J = 6 Hz), 9.14 (s, 1H, N¼CH), 13.27
(s, 1H, CONH); 13C NMR (DMSO) δ (ppm)=12.4, 24.5,
45.8, 103.2, 116.3, 118.1, 118.4, 119.3, 121.9, 132.5,
137.3, 137.5, 145.1 (N¼CH), 153.3, 159.4, 160.8, 161.8
(C¼O), 162.1, 179.1 (C¼O). Analysis for C19H16BrFN4O2
(431.26) Calculated: C: 52.92%, H: 3.74%, N: 12.99%;
Found: C: 52.96%, H: 3.77%, N: 12.95%.
N: 14.20%; Found: C: 63.93%, H: 5.68%, N: 14.17%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (3-bromo-4-methoxy-benzylidene)-hydrazide
1
(8). Yield: 74%; m.p.: 254–256°C. H NMR (CDCl3)
δ (ppm) = 1.54–1.57 (t, 3H, CH3, J = 6 Hz), 2.73 (s, 3H,
CH3), 3.96 (s, 3H, CH3), 4.61–4.64 (q, 2H, CH2, J=3Hz),
6.93–6.94 (d, 1H, ArH, J=3Hz), 7.35–7.36 (d, 1H, ArH,
J=3Hz), 7.71–7.73 (d, 1H, H6-naphthyridine, J=6Hz),
8.09 (s, 1H, ArH), 8.14 (s, 1H, H2-naphthyridine), 8.67–
8.69 (d, 1H, H5-naphthyridine, J =6 Hz), 9.04 (s, 1H,
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (2-chloro-6-fluoro-benzylidene)-hydrazide
1
(11). Yield: 90%; m.p.: 242–244°C. H NMR (CDCl3)
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Synthesis and in vitro Antimicrobial Activity of Nalidixic Acid Hydrazones
δ (ppm)=1.53–1.56 (t, 3H, CH3, J= 3Hz), 2.72 (s, 3H, CH3),
4.58–4.65 (q, 2H, CH2, J=6Hz, J=9Hz), 7.05–7.11 (m, 1H,
ArH), 7.24–7.25 (m, 2H, ArH), 7.34–7.37 (d, 1H, H6-
naphthyridine, J=9Hz), 8.49 (s, 1H, H2-naphthyridine),
8.66–8.68 (d, 1H, H5-naphthyridine, J = 6 Hz), 9.05 (s,
1H, N¼CH), 13.26 (s, 1H, CONH); 13C NMR (CDCl3) δ
(ppm) = 15.3, 25.3, 47.1, 111.9, 115.3, 120.9, 121.7,
125.6, 130.7, 135.2, 136.3, 141.3, 148.5 (N¼CH), 159.5,
161.6, 162.9, 163.7 (C¼O), 176.7 (C¼O). Analysis for
C19H16ClFN4O2 (386.81) Calculated: C: 59.00%, H:
4.17%, N: 14.48%; Found: C: 59.03%, H: 4.15%, N:
56.91%, H: 4.21%, N: 15.62%; Found: C: 56.87%, H:
4.19%, N: 15.64%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid [(1H-indol-3-yl)methylidene]-hydrazide
1
(15). Yield: 63%; m.p.: 206–208°C. H NMR (CDCl3)
δ (ppm)=1.49–1.51 (t, 3H, CH3, J=6Hz, J=9Hz), 2.68
(s, 3H, CH3), 4.47–4.54 (q, 2H, CH2, J=6Hz, J=9Hz),
7.20–7.26 (m, 5H, indole), 7.34–7.37 (d, 1H, H6-naphthyridine,
J = 9 Hz), 8.52 (s, 1H, H2-naphthyridine), 8.66–8.69
(d, 1H, H5-naphthyridine, J=9Hz), 8.67 (s, 1H, N¼CH),
9.07 (s, 1H, NH), 12.99 (s, 1H, CONH); 13C NMR
(CDCl3) δ (ppm)=15.2, 25.1, 46.4, 103.6, 105.7, 111.6,
113.2, 118.4, 119.3, 120.7, 121.2, 121.9, 127.12, 136.9,
137.3, 141.2, 146.6, 148.9 (N¼CH), 162.7, 166.3 (C¼O),
174.7 (C¼O). Analysis for C21H19N5O2 (373.41) Calculated:
C: 67.55%, H: 5.13%, N: 18.76%; Found: C: 67.64%, H:
14.51%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid (2,3-difluoro-benzylidene)-hydrazide (12). Yield:
71%; m.p.: 258–260°C. 1H NMR (CDCl3) δ (ppm)
=1.52–1.57 (t, 3H, CH3, J=9Hz, J=6 Hz), 2.71 (s, 3H,
CH3), 4.58–4.65 (q, 2H, CH2, J=6Hz), 7.10–7.19 (m, 1H,
ArH), 7.34–7.37 (d, 1H, H6-naphthyridine, J = 9 Hz),
7.85–7.90 (m, 1H, ArH), 7.93–7.99 (m, 1H, ArH), 8.48
(s, 1H, H2-naphthyridine), 8.65–8.68 (d, 1H, H5-
naphthyridine, J = 9 Hz), 9.02 (s, 1H, N¼CH), 13.32 (s,
1H, CONH); 13C NMR (CDCl3) δ (ppm) = 15.3, 25.3,
47.2, 111.8, 118.5, 120.1, 121.7, 122.2, 122.5, 124.1,
124.2, 136.3, 140.1, 147.1, 148.5 (N¼CH), 155.1, 161.8,
163.8 (C¼O), 176.7 (C¼O). Analysis for C19H16F2N4O2
(370.35) Calculated: C: 61.62%, H: 4.35%, N: 15.13%;
5.10%, N: 18.73%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid [(6-chloro-benzo[1,3]dioxol-5-yl)methylidene]-
1
hydrazide (16). Yield: 69%; m.p.: 254–256°C. H NMR
(CDCl3) δ (ppm)=1.54–1.58 (t, 3H, CH3, J=6Hz), 2.72
(s, 3H, CH3), 4.38–4.44 (q, 2H, CH2, J = 6 Hz), 4.68
(s, 2H, CH2), 6.84 (s, 1H, ArH), 6.92 (s, 1H, ArH),
7.36–7.39 (d, 1H, H6-naphthyridine, J = 9 Hz), 7.75
(s, 1H, H2-naphthyridine), 8.67–8.70 (d, 1H, H5-
naphthyridine, J=9Hz), 8.95 (s, 1H, N¼CH), 12.90 (s,
1H, CONH); 13C NMR (CDCl3) δ (ppm)=12.4, 24.3,
45.8, 87.4, 103.2, 107.4, 108.6, 119.3, 121.9, 127.1, 128.1,
137.3, 146.6, 149.4 (N¼CH), 149.9, 153.3, 159.9, 160.7,
161.8 (C¼O), 180.2 (C¼O). Analysis for C20H17ClN4O4
(412.83) Calculated: C: 58.19%, H: 4.15%, N: 13.57%;
Found: C: 58.23%, H: 4.11%, N: 13.52%.
Found: C: 61.66%, H: 4.31%, N: 15.11%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid [(1H-pyrrol-2-yl)methylidene]-hydrazide (13).
Yield: 23%; m.p.: 252–254°C. 1H NMR (CDCl3) δ (ppm)
=1.54–1.56 (t, 3H, CH3, J=3Hz), 2.73 (s, 3H, CH3),
4.60–4.64 (q, 2H, CH2, J=3Hz, J=6Hz), 6.26–6.28 (m,
1H, pyrrole), 6.51–6.52 (m, 1H, pyrrole), 6.96 (s, 1H,
pyrrole), 7.35–7.36 (d, 1H, H6-naphthyridine, J=3Hz),
8.07 (s, 1H, H2-naphthyridine), 8.67–8.69 (d, 1H, H5-
naphthyridine, J=6Hz), 9.01 (s, 1H, N¼CH), 12.42 (s,
1H, CONH); 13C NMR (CDCl3) δ (ppm)=12.4, 24.3,
45.8, 103.8, 110.8, 116.2, 119.3, 119.5, 121.9, 124.7,
136.9 (N¼CH), 137.3, 153.3, 159.9, 160.7, 161.8 (C¼O),
180.1 (C¼O). Analysis for C17H17N5O2 (323.35)
Calculated: C: 63.15%, H: 5.30%, N: 21.66%; Found: C:
Microbiology
In vitro antimicrobial assay. The examined compounds
3–16 were in vitro screened for antibacterial and antifungal
activities using the broth microdilution method according
to both the European Committee on Antimicrobial
Susceptibility Testing [19] and the Clinical and Laboratory
Standards Institute guidelines [20] against a panel of reference
and clinical or saprophytic strains of microorganisms,
including Gram-positive bacteria (Staphylococcus aureus
ATCC 25923, S. aureus ATCC 43300, S. aureus ATCC
6538, Staphylococcus epidermidis ATCC 12228, Bacillus
subtilis ATCC 6633, Bacillus cereus ATCC 10876,
Micrococcus luteus ATCC 10240), Gram-negative bacteria
(Escherichia coli ATCC 25922, Klebsiella pneumoniae
ATCC 13883, Proteus mirabilis ATCC 12453, Bordetella
bronchiseptica ATCC 4617, Salmonella typhimurium
ATCC 14028, Pseudomonas aeruginosa ATCC 9027) and
fungi belonging to yeasts (Candida albicans ATCC 2091,
C.albicans ATCC 10231, Candida parapsilosis ATCC
22019) and molds (Aspergillus niger ATCC 16404,
Aspergillus fumigatus, Penicillium spp., and Rhizopus
spp.). The microorganisms belonging to ATCC came from
the American Type Culture Collection, routinely used for
63.18%, H: 5.25%, N: 21.69%.
1-Ethyl-7-methyl-4-oxo-1,4-dihydro[1,8]naphthyridine-3-
carboxylic acid [(5-chlorofuran-2-yl)methylidene]-hydrazide (14).
1
Yield: 72%; m.p.: 202–204°C. H NMR (CDCl3) δ (ppm)
=1.50–1.55 (t, 3H, CH3, J=9Hz, J=6Hz), 2.70 (s, 3H,
CH3), 4.56–4.63 (q, 2H, CH2, J=6Hz), 6.27–6.28 (d, 1H,
furan, J=3Hz), 6.83–6.84 (d, 1H, furan, J=3Hz), 7.32–
7.34 (d, 1H, H6-naphthyridine, J = 6 Hz), 8.41 (s, 1H,
H2-naphthyridine), 8.65–8.69 (d, 1H, H5-naphthyridine,
J=12Hz), 9.02 (s, 1H, N¼CH), 13.17 (s, 1H, CONH); 13
C
NMR (CDCl3) δ (ppm)=15.3, 25.3, 47.2, 108.9, 109.4,
121.5, 131.3, 136.8, 139.1, 141.2, 146.2, 148.7, 149.1
(N¼CH), 161.6, 163.7, 163.7 (C¼O), 176.6 (C¼O).
Analysis for C17H15ClN4O3 (358.78) Calculated: C:
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Ł. Popiołek, A. Biernasiuk, and A. Malm
Vol 000
the evaluation of antimicrobials. All the used microbial
cultures were first subcultured on nutrient agar or
Sabouraud agar at 35°C for 18–24 h or 30°C for 24–48 h
for bacteria and fungi, respectively.
The surface of Mueller-Hinton agar or Mueller-Hinton
agar with 5% sheep blood (for bacteria) and RPMI 1640
with MOPS (for fungi) were inoculated with the suspen-
sions of bacterial or fungal species. Microbial suspensions
were prepared in sterile saline (0.85% NaCl) with an
optical density of McFarland standard scale 0.5 – approxi-
mately 1.5 ×108 CFU (colony forming units)/mL for bacte-
ria and 0.5 McFarland standard scale – approximately
5 × 105 CFU/mL for fungi.
In this study, no bioactivity was defined as an
MIC >1000 μg/mL, mild bioactivity as an MIC in the
range 501–1000μg/mL, moderate bioactivity with MIC
from 126 to 500 μg/mL, good bioactivity as an MIC in
the range 26–125 μg/mL, strong bioactivity with MIC
between 10 and 25 μg/mL and very strong bioactivity as
a MIC <10 μg/mL [22].
The MBC/MIC or MFC/MIC ratios were calculated in
order to determine bactericidal/fungicidal (MBC/MIC ≤ 4,
MFC/MIC ≤ 4) or bacteriostatic/fungistatic (MBC/MIC > 4,
MFC/MIC > 4) effect of the tested compounds.
REFERENCES AND NOTES
Samples containing examined compounds were dis-
solved in 1mL dimethyl sulfoxide (DMSO). Furthermore,
bacterial and fungal suspensions were put onto petri dishes
with solid media containing 2 mg/mL of the tested com-
pounds, followed with incubation at 37°C for 24h and
30°C for 48h for bacteria and fungi, respectively. The
inhibition of microbial growth was judged by comparison
with that of a control culture prepared without any
sample tested. Ciprofloxacin or fluconazole (Sigma) was
used as a reference antibacterial or antifungal compound,
respectively.
Subsequently, MIC of the compounds was examined by
the microdilution broth method, using their twofold dilu-
tions in Mueller-Hinton broth or Mueller-Hinton broth
with 5% sheep blood (for bacteria) and RPMI 1640 broth
with MOPS (for fungi) prepared in 96-well polystyrene
plates. Final concentrations of the compounds ranged from
1000 to 0.488μg/mL. Microbial suspensions were pre-
pared in sterile saline (0.85% NaCl) with an optical density
of 0.5 McFarland standard. Next, 2 μL of each bacterial or
fungal suspension was added per well containing 200 μL
broth and various concentrations of the examined com-
pounds. After incubation (37°C for 24 h for bacteria and
yeasts and 30°C for 48 h for molds), the MIC was assessed
spectrophotometrically as the lowest concentration of the
samples showing complete bacterial or fungal growth inhi-
bition. Appropriate DMSO, growth, and sterile controls
were carried out. The medium with no tested substances
was used as control.
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The MBC or MFC are defined as the lowest concentra-
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by removing 20 μL of the culture used for MIC determina-
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observed were assessed as
a bactericidal/fungicidal
concentration. All the experiments were repeated three
times, and representative data are presented [21].
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet