Synthesis, characterization and antimicrobial activity of nalidixic acid derivatives with spirohydantoins

Article abstract of DOI:10.1007/s13738-019-01739-7

This article presents the synthesis of a series of amides, based on the interaction of several 3-aminospirohydantoins with nalidixic acid. The target compounds were characterized by physicochemical parameters, IR, ¹H and ¹³C NMR spectral data. The antimicrobial activity of the products obtained was determined against Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Salmonella abony, the yeasts Candida albicans and Saccharomyces cerevisiae and the molds Penicillium chrysogenum and Aspergillus niger. The relationship between structure and biological activity of the products obtained was discussed. It was found that the most effective compounds are tetralin (5f) and indane (5g) derivatives, which exhibit a pronounced antimicrobial activity against both tested Gram-positive and Gram-negative bacteria.

Full text of DOI:10.1007/s13738-019-01739-7

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-019-01739-7
ORIGINAL PAPER
Synthesis, characterization and antimicrobial activity of nalidixic acid
derivatives with spirohydantoins
Marin Marinov1 · Iliana Kostova2 · Emilia Naydenova4 · Rumyana Prodanova1 · Neyko Stoyanov3
Received: 13 November 2018 / Accepted: 18 July 2019
© Iranian Chemical Society 2019
Abstract
This article presents the synthesis of a series of amides, based on the interaction of several 3-aminospirohydantoins with
nalidixic acid. The target compounds were characterized by physicochemical parameters, IR, 1H and 13C NMR spectral
data. The antimicrobial activity of the products obtained was determined against Gram-positive bacteria Staphylococcus
aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Salmonella abony, the
yeasts Candida albicans and Saccharomyces cerevisiae and the molds Penicillium chrysogenum and Aspergillus niger. The
relationship between structure and biological activity of the products obtained was discussed. It was found that the most
efective compounds are tetralin (5f) and indane (5g) derivatives, which exhibit a pronounced antimicrobial activity against
both tested Gram-positive and Gram-negative bacteria.
Keywords Synthesis · Nalidixic acid · 3-Aminospirohydantoins · Antimicrobial activity
Introduction
Over the last decade 4-quinolones that contain a fuorine
atom in a molecule called fuoroquinolones, or third-gener-
ation preparations, were introduced [1–3].
Nalidixic acid (Fig. 1) belongs to the group of quinolone
antibacterial drugs, derivatives of 4-quinolone-3-carboxylic
acid. It was introduced in the medical practice in 1963 to
treat mainly Gram-negative urinary infections. Its antibacte-
rial activity is fairly low, and most Pseudomonas strains are
resistant. It belongs to the frst generation of 4-quinolones
[1–3].
Besides pefoxacin (Fig. 4) and few others, such as ofoxa-
cin, levofoxacin, feroxacin, grepafoxacin, fuoroquinolones
are distinguished by a broad antimicrobial spectrum, bacteri-
cidal activity type and very good distribution in blood [1–3].
Fluoroquinolones are gyrase inhibitors because they
inhibit the enzyme topoisomerase (gyrase II), responsi-
ble for over-spiralization of DNA. As a result, the DNA
structure of microorganisms is disturbed and they are
terminated. The bactericidal efect of fuoroquinolones
is pronounced the most during division and growth of
microorganisms. Fluoroquinolones have a broad antimi-
crobial spectrum of action, but in Gram-positive infections
a better efect is achieved with benzylpenicillin, isoxa-
zolyl penicillin or aminoglycosides. In this respect, the
ofoxacin whose antistaphylococcal activity is higher is an
exception. Main indications of the clinical use of fuoro-
quinolones are similar to third-generation cephalosporins,
but an important advantage of them is the ability for oral
administration. Further optimization of fuoroquinolone
structure has led to preparation of a new fourth-generation
of fuoroquinolones, a representative of which is Avelox
(Fig. 5).
Later, 4-quinolones (second-generation preparations)
such as oxolinic and pipemidic acids (Fig. 2) were intro-
duced [1–3] (Fig. 3).
* Marin Marinov
m_n_marinov@abv.bg
1
Department of General Chemistry, Faculty of Plant
Protection and Agroecology, Agricultural University -
Plovdiv, 12 “Mendeleev” Blvd, 4000 Plovdiv, Bulgaria
2
Department of Biotechnologies and Food Technologies,
“Angel Kanchev, University of Ruse, Razgrad Branch, 47
“Aprilsko Vastanie” St, 7200 Razgrad, Bulgaria
3
Department of Chemistry and Chemical Technologies,
“Angel Kanchev”, University of Ruse, Razgrad Branch, 47
“Aprilsko Vastanie” St, 7200 Razgrad, Bulgaria
4
Department of Organic Chemistry, University of Chemical
Technology and Metallurgy, 8 “Kl. Ohridski” Blvd,
1756 Sofa, Bulgaria
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
CH3
H
H3C
O
H3C
N
N
O
N
H
N
N
H
O
O
F
OH
O
OH
Fig. 1 Nalidixic acid (INN)/1-ethyl-7-methyl-4-oxo-1,4-dihydro-
1,8-naphthyridine-3-carboxylic acid
Fig. 5 Avelox/1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo[4.3.0]
nonan-8-yl]-6-fuoro-8-methoxy-4-oxoquinoline-3-carboxylic acid
3-aminospirohydantoin derivatives. Furthermore, the bac-
teriostatic efect of such compounds against Gram-positive
and Gram-negative microorganisms has been previously
observed by us [4]. The purpose of the current work is to
present the synthesis of new derivatives of nalidixic acid
with 3-aminospirohydantoins, as well as the study of their
antimicrobial activity.
CH3
HN
N
N
N
O
N
O
OH
Fig. 2 Pipemidic acid (INN)/8-ethyl-5-oxo-2-(piperazin-1-yl)-5,8-
Experimental
dihydropyrido[2,3-d]pyrimidine-6-carboxylic acid
General
CH3
HN
All chemicals used were purchased from Merck and Sigma-
Aldrich. The melting points were determined with a SMP-
10 digital melting point apparatus. The elemental analysis
data were obtained with an automatic analyzer Carlo Erba
1106. The purity of the compounds was checked by thin-
layer chromatography on Kieselgel 60 F254, 0.2-mm Merck
plates, eluent system (vol. ratio): ethyl acetate:petroleum
ether = 1:2. The IR spectra were taken on PerkinElmer
FTIR-1600 spectrometer in KBr disks. The NMR spectra
were taken on a Bruker DRX-250 spectrometer, operating
at 250.13 and 62.90 MHz for 1H and 13C, respectively, using
the standard Bruker software. The chemical shifts were ref-
erenced to tetramethylsilane (TMS). The measurements in
DMSO solutions were carried out at ambient temperature
N
F
N
N
O
O
OH
Fig. 3 Enoxacin
(INN)/1-ethyl-6-fuoro-4-oxo-7-(piperazin-1-yl)-
1,4-dihydro-1,8-naphthyridine-3-carboxylic acid
H3C
N
CH3
N
F
N
N
O
1
(300 K). Typical conditions for H NMR spectra were as
follows: pulse width 30°, 1 s relaxation delay, 16 K time
domain points, zero-flled to 64 K, hard pulse with 90° pulse
width of 11.8 μs; 13C NMR spectra: pulse width 30°, 1 s
relaxation delay, 16 K time domain points, zero-flled to
32 K, hard pulse with 90° pulse width of 6.4 μs at a power
level of 3 dB below the maximum output.
O
OH
Fig. 4 Pefoxacin (INN)/1-ethyl-6-fuoro-7-(4-methylpiperazin-1-yl)-
4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid
Synthesis of 3‑aminospirohydantoin derivatives
with nalidixic acid (5a–5g, Scheme 2)
These changes in the structure of the fuoroquinolone lead
to improved activity toward Gram-positive microorganisms.
All the data above determine the research interest
in the result from a combination of nalidixic acid with
The corresponding 3-aminospirohydantoin (3a–3g)
(10 mmol) and nalidixic acid (4) (2.32 g, 10 mmol) were
dissolved in 50 mL of tetrahydrofuran (THF) with stirring
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Journal of the Iranian Chemical Society
H
N
H
N
O
O
H2NNH2.H2O
- NH4OH
NaCN / (NH4)2CO3
C2H5OH / H2O
A
O
A
A
N
NH
NH2
O
O
1
3
2
CH3
CH3
CH3
A = a)
; b)
; c)
; d)
; e)
; f)
; g)
CH3
Scheme 1 Synthesis of 3-aminospirohydantoins
at room temperature. N,N′-Dicyclohexylcarbodiimide
(DCC, 2.06 g, 10 mmol) was added to the reaction mix-
ture, and the latter was left overnight. After this interac-
tion, the N,N′-dicyclohexylcarbamide formed was filtered
off and 1 mL of glacial acetic acid was added to the fil-
trate for removing the unreacted reagent. After filtration,
the solvent was evaporated to dryness and the amides
obtained (5a–5g) were recrystallized from ethanol.
Pseudomonas aeruginosa ATCC 9027 and Salmonella
abony NTCC 6017, the yeasts Candida albicans ATCC
10231 and Saccharomyces cerevisiae ATCC 9763 and the
molds Penicillium chrysogenum and Aspergillus niger was
studied. The technique used for this investigation was the
agar well difusion method [5]. The used agar media were
as follows: tryptic soy agar (Biolife) for the tests with bac-
teria and Sabouraud dextrose agar (Biolife) for yeast and
molds. The wells (Ø=7 mm) were flled with 1% solutions
(10 mg/mL) of the compounds synthesized in ethylene gly-
col (50 µl) and, following a 30-min stay at room temperature,
the Petri dishes were placed in a thermostat at 37 °C for 24 h
for bacteria, 28 °C for 24 h for yeast and 72 h for molds.
After cultivation, the diameter of inhibition growth zones
around wells was measured in mm, and the results obtained
were evaluated as follows: Up to 15 mm, the microbial cul-
ture is weakly sensitive, from 15 to 25 mm, it is sensitive,
Antimicrobial assay
The microbial cultures were purchased from National Bank
of Industrial Microorganisms and Cell Cultures (NBIMCC),
Sofa. The antimicrobial efect of the synthesized com-
pounds (5a–5g) against Gram-positive bacteria Staphy-
lococcus aureus ATCC 6538 and Bacillus subtilis ATCC
6633, Gram-negative bacteria Escherichia coli ATCC 8739,
CH3
CH3
H
O
N
H
O
N
N
O
N
CH3
N
O
N
CH3
A
A
DCC
THF
+
H2O
N
+
N
HO
NH2
NH
O
O
O
O
4
5
3
CH3
CH3
CH3
A = a)
; b)
; c)
; d)
; e)
; f)
; g)
CH3
Scheme 2 Synthesis of 3-aminospirohydantoin derivatives with nalidixic acid
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Journal of the Iranian Chemical Society
and over 25 mm, it is very sensitive to the given synthetic
compound at tested concentration [4]. The same conditions
were applied to the nalidixic acid tests, and the data obtained
were compared with those of the corresponding derivatives
(5a–5g). The pure ethylene glycol (50 µl) was used as a
control. The data on antimicrobial activity are arithmetic
average of three measurements.
NMR spectral data. The results obtained from these analyses
are listed in Tables 1, 2 and 3, respectively.
The antimicrobial activity of the products obtained was
determined against Gram-positive bacteria Staphylococ-
cus aureus ATCC 6538 and Bacillus subtilis ATCC 6633,
Gram-negative bacteria Escherichia coli ATCC 8739, Pseu-
domonas aeruginosa ATCC 9027 and Salmonella abony
NTCC 6017, the yeasts Candida albicans ATCC 10231
and Saccharomyces cerevisiae ATCC 9763 and the molds
Penicillium chrysogenum and Aspergillus niger. The results
obtained from these analyses are listed in Table 4.
Results and discussion
The initial cycloalkanespiro-5-hydantoins (2a–2e,
Scheme 1) were synthesized from the corresponding
cycloalkanones (1a–1e) via the Bucherer–Lieb method
[6]. The 3′,4′-dihydro-2H,2′H,5H-spiro[imidazolidine-4,1′-
naphthalene]-2,5-dione (2f, Scheme 1) was obtained through
a modifcation [7] of the method reported by Sarges et al.
[8]. The 2′,3′-dihydro-2H,5H-spiro[imidazolidine-4,1′-
indene]-2,5-dione (2g, Scheme 1) was prepared in accord-
ance with Nagasawa et al. [9]. The 3-aminospirohydantoins
(3a–3g, Scheme 1) were synthesized by an interaction of
the corresponding spirohydantoins obtained (2a–2g) with
concentrated hydrazine hydrate (Scheme 1), following a
modifed procedure [4] of the previously published meth-
ods [7, 10, 11]. The analytical and the spectroscopic data of
compounds 2a–2g and 3a–3f matched those reported in the
literature [7, 10–14].
In our previous work, we have reported synthesis and
study on antimicrobial activities of amide 5a [4]. In the
current research, we investigate the efect of diferent sub-
stituents in the cyclohexane core (compounds 5a–5e), as
well as the efect of other substituents to the hydantoin ring
(compounds 5f and 5g) on the antimicrobial activity of such
products. Methyl and propyl groups of the fourth position
in the cyclohexane core (compounds 5b and 5f) do not have
any impact on the biological properties of the compounds.
The presence of a methyl group at the second position of
the cyclohexane ring (product 5c) shows less antimicro-
bial activity compared to amide 5a. Greater efficiency
against bacteria tested has been observed for compound 5d,
which contains an ethyl group at the fourth position in the
cyclohexane ring. However, the strongest biological activity
has been established for the corresponding tetralin (5f) and
indane (5g) derivatives. The latter exhibits a pronounced
antimicrobial activity against both tested Gram-positive and
Gram-negative bacteria. All amides (5a–5g), studied in this
work, do not show any activity against tested yeasts and
molds. The results presented in Table 4 show the higher
The interaction between equimolecular amounts of the
nalidixic acid (4) and the corresponding 3-aminospirohydan-
toins (3a–3g) in tetrahydrofuran medium was carried out by
the DCC method [15] in accordance with Scheme 2.
The synthesized compounds (5a–5g) were characterized
by physicochemical parameters, elemental analysis, IR and
Table 1 Physicochemical
№
Molecular formula Yield (%) M. p. (°C) Rf
Elemental analysis (%)
Calculated
parameters and elemental
analysis data of compounds
5a–5g
Found
C
C
H
N
H
N
5a* C20H23N5O4
96
88
60
72
70
71
56
206–207
204–205
168–169
187–188
210–211
214–215
172–173
0.49 60.44 5.83 17.62 60.27 5.73 17.55
0.44 61.30 6.12 17.02 61.15 6.01 16.89
0.51 61.30 6.12 17.02 61.09 5.96 16.91
0.48 62.10 6.40 16.46 61.96 6.28 16.25
0.43 62.85 6.65 15.93 62.77 6.58 15.78
0.54 64.71 5.20 15.72 64.61 5.13 15.56
0.57 64.03 4.91 16.23 63.89 4.76 16.02
/397.43/
5b C21H25N5O4
/411.45/
5c
C21H25N5O4
/411.45/
5d C22H27N5O4
/425.48/
5e
5f
C23H29N5O4
/439.51/
C24H23N5O4
/445.47/
5g C23H21N5O4
/431.44/
*Ref. [4]
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Journal of the Iranian Chemical Society
Table 2 IR spectral data of
compounds 5a–5g
№
ꢀNH
ꢀCH
ꢀ cн2
ꢀ cн2
ꢀCH
ꢀC=O
ꢀC=O
ꢀCC
ꢀCN
as
s
3
/arom./
/aliph./
/aliph./
/amide/
/arom./
5a*
3296
2948
2862
2845
2928
1775
1763
1714
1774
1751
1696
1772
1748
1700
1774
1745
1701
1773
1744
1700
1775
1746
1701
1777
1743
1699
1701
1678
1688
1691
1695
1694
1685
1599
1596
1598
1599
1598
1599
1598
1355
5b
5c
5d
5e
5f
3269
3264
3268
3263
3271
3266
2952
2946
2950
2952
2954
2957
2857
2868
2865
2866
2851
2857
2839
2929
1356
1355
1355
1356
1355
1355
2840
2919
2842
2924
2844
2923
3065
3070
2840
2915
5g
2849
2913
*Ref. [4]
Table 3 NMR spectral data of compounds 5a–5g
№
1H NMR (DMSO-d6)
13C NMR (DMSO-d6)
δ/ppm
13C DEPT 135 (DMSO-d6)
δ/ppm
δ/ppm
5a 0.98 (s, 3H, CH3), 2.43 (s, 3H, CH3, nal. ac.),
1.42–2.28 (m, 10H, CH2), 3.06 (s, 2H, CH2,
Et), 6.58 (s, 1H, CH, pyr.), 7.35 (s, 1H, NH,
sec. amide), 7.75 (s, 1H, nal. ac.), 7.83 (s, 1H,
CH, pyr.), 8.31 (s, 1H, NH, urea)
188.6 (C=O, nal. ac.), 177.3 (C=O, spirohyd. ring), 157.7 (CH), 139.4 (CH), 115.4
166.5 (C=O, 1-amide), 164.1 (C/C, pyr.), 159.4
(C=O, spirohyd. ring, urea), 157.7 (CH, nal. ac.),
139.4 (CH, pyr.), 115.4 (CH, pyr.), 62.7 (spiro
C-atom), 49.9 (CH2, Et, nal. ac.), 33.5 (CH2/CH2),
28.4 (CH2), 22.1 (CH3, nal. ac.), 20.1 (CH2/CH2),
13.8 (CH3, Et, nal. ac.)
(CH), 49.9 (CH2), 33.5 (CH2/
CH2), 28.4 (CH2), 22.1 (CH3),
20.1 (CH2/CH2), 13.8 (CH3)
5b 0.96 (s, 3H, CH3), 1.01 (s, 3H, CH3, cyclohex.), 190.2 (C=O, nal. ac.), 178.6 (C=O, spirohyd. ring), 158.2 (CH), 140.5 (CH), 115.4
1.38–1.96 (m, 8H, CH2, cyclohex.), 1.58 (s,
167.4 (C=O, 1-amide), 163.8 (C/C, pyr.), 160.3
(CH), 50.1 (CH2), 30.9 (CH2/
CH2), 29.1 (CH), 27.3 (CH2/
CH2), 21.3 (CH3), 20.4 (CH3),
1H, CH, cyclohex.), 2.63 (s, 3H, CH3, nal. ac.), (C=O, spirohyd. ring, urea), 158.2 (CH, nal. ac.),
1.42–2.28 (m, 10H, CH2), 3.15 (s, 2H, CH2,
Et), 6.62 (s, 1H, CH, pyr.), 7.20 (s, 1H, NH,
sec. amide), 7.82 (s, 1H, CH, nal. ac.), 8.01 (s,
1H, CH, pyr.), 8.42 (s, 1H, NH, urea)
140.5 (CH, pyr.), 115.4 (CH, pyr.), 62.8 (spiro
C-atom), 50.1 (CH2, Et, nal. ac.), 30.9 (CH2/CH2), 12.2 (CH3)
29.1 (CH), 27.3 (CH2/CH2), 21.3 (CH3, nal. ac.),
20.4 (CH3), 12.2 (CH3, Et, nal. ac.)
5c 0.88 (s, 3H, CH3), 1.12 (s, 3H, CH3, cyclohex.), 191.3 (C=O, nal. ac.), 179.1 (C=O, spirohyd.
158.4 (CH), 157.3 (CH), 140.6
(CH), 115.4 (CH), 50.3 (CH2),
35.5 (CH), 31.3 (CH2), 28.4
(CH2), 25.7 (CH2), 21.6 (CH2),
20.7 (CH3), 15.1 (CH3), 13.3
(CH3)
1.36–1.95 (m, 8H, CH2, cyclohex.), 2.32 (s, ring), 168.0 (C=O, 1-amide), 164.0 (C/C, pyr.),
1H, CH, cyclohex.), 2.47 (s, 3H, CH3, nal. ac.), 161.2 (C=O, spirohyd. ring, urea), 158.4 (CH,
3.21 (s, 2H, CH2, Et), 6.66 (s, 1H, CH, pyr.),
7.31 (s, 1H, NH, sec. amide), 7.82 (s, 1H, CH,
nal. ac.), 8.08 (s, 1H, CH, pyr.), 8.38 (s, 1H,
NH, urea)
nal. ac.), 157.3 (CH, ethylene), 140.6 (CH, pyr.),
115.4 (CH, pyr.), 69.4 (spiro C-atom), 50.3 (CH2,
Et, nal. ac.), 35.5 (CH), 31.3 (CH2), 28.4 (CH2),
25.7 (CH2), 21.6 (CH2), 20.7 (CH3, nal. ac.), 15.1
(CH3), 13.3 (CH3, Et, nal. ac.)
5d 0.83 (s, 3H, CH3, Et, cyclohex.), 0.98 (s, 3H,
190.8 (C=O, nal. ac.), 176.8 (C=O, spirohyd. ring), 157.5 (CH), 140.2 (CH), 115.7
CH3, Et, nal. ac.), 1.22 (s, 2H, CH2, Et,
166.4 (C=O, 1-amide), 164.3 (C/C, pyr.), 159.9
(CH), 50.5 (CH2), 35.3 (CH),
31.3 (CH2/CH2), 28.2 (CH2),
24.6 (CH2/CH2), 21.3 (CH3),
14.7 (CH3); 12.9 (CH3)
cyclohex.), 1.38–1.95 (m, 8H, CH2, cyclohex.), (C=O, spirohyd. ring, urea), 157.5 (CH, nal. ac.),
1.40 (s, 1H, CH, cyclohex.), 2.51 (s, 3H, CH3,
nal. ac.), 3.33 (s, 2H, CH2, Et), 6.68 (s, 1H,
CH, pyr.), 7.39 (s, 1H, NH, sec. amide), 7.84
(s, 1H, CH, nal. ac.), 8.11 (s, 1H, CH, pyr.),
8.44 (s, 1H, NH, urea)
140.2 (CH, pyr.), 115.7 (CH, pyr.), 63.3 (spiro
C-atom), 50.5 (CH2, Et, nal. ac.), 35.3 (CH), 31.3
(CH2/CH2), 28.2 (CH2), 24.6 (CH2/CH2), 21.3
(CH3, nal. ac.), 14.7 (CH3); 12.9 (CH3, Et, nal.
ac.)
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Journal of the Iranian Chemical Society
Table 3 (continued)
1H NMR (DMSO-d6)
№
13C NMR (DMSO-d6)
δ/ppm
13C DEPT 135 (DMSO-d6)
δ/ppm
δ/ppm
5e 0.84 (s, 3H, CH3, Pr, cyclohex.), 1.02 (s, 3H,
CH3, Et, nal. ac.), 1.21 (s, 2H, CH2, Pr,
cyclohex.), 1.30 (s, 2H, CH2, Pr), 1.39–1.96
(m, 8H, CH2, cyclohex.), 1.45 (s, 1H, CH,
cyclohex.), 2.52 (s, 3H, CH3, nal. ac.), 3.37
(s, 2H, CH2, Et), 6.68 (s, 1H, CH, pyr.), 7.13
(s, 1H, NH, sec. amide), 7.85 (s, 1H, CH, nal.
ac.), 8.13 (s, 1H, CH, pyr.), 8.54 (s, 1H, NH,
urea)
190.6 (C=O, nal. ac.), 177.1 (C=O, spirohyd. ring), 158.5 (CH), 140.2 (CH), 115.8
166.7 (C=O, 1-amide), 164.2 (C/C, pyr.), 160.2
(C=O, spirohyd. ring, urea), 158.5 (CH, nal. ac.),
140.2 (CH, pyr.), 115.8 (CH, pyr.), 63.8 (spiro
C-atom), 50.4 (CH2, Et, nal. ac.), 38.3 (CH2, Pr),
33.4 (CH), 31.5 (CH2), 31.5 (CH2), 25.3 (CH2/
CH2), 21.7 (CH2, Pr), 21.3 (CH3, nal. ac.), 15.3
(CH3), 13.6 (CH3, Et, nal. ac.)
(CH), 50.4 (CH2), 38.3 (CH2),
33.4 (CH), 31.5 (CH2), 25.3
(CH2/CH2), 21.7 (CH2), 21.3
(CH3), 15.3 (CH3), 13.6 (CH3)
5f 0.98 (s, 3H, CH3), 2.53 (s, 3H, CH3), 1.54–2.76 192.4 (C=O, nal. ac.), 179.3 (C=O, spyrohyd. ring), 158.7 (CH), 141.1 (CH), 128.8
(m, 6H, CH2, tetrahydronaphth.), 3.35 (s, 2H,
CH2, Et), 6.65 (s, 1H, CH, pyr.), 6.98–7.15 (m,
4H, CH, tetrahydronaphth.), 7.23 (s, 1H, NH,
sec. amide), 7.88 (s, 1H, CH, nal. ac.), 8.10 (s,
1H, CH, pyr.), 8.68 (s, 1H, NH, urea)
167.5 (C=O, 1-amide), 164.6 (C/C, pyr.), 160.8
(C=O, spirohyd. ring, urea), 158.7 (CH, nal. ac.),
141.1 (CH, pyr.), 139.0 (C/C, 1-benzene), 128.8
(CH/CH), 125.9 (CH/CH), 115.6 (CH, pyr.),
113.6 (CH, pyr.), 66.9 (spiro C-atom), 49.8 (CH2,
ET, nal. ac.), 41.6 (CH2), 32.3 (CH2), 28.5 (CH2),
25.3 (CH2), 22.3 (CH3), 14.6 (CH3)
(CH/CH), 125.9 (CH/CH), 115.6
(CH), 113.6 (CH), 49.8 (CH2),
41.6 (CH2), 32.3 (CH2), 28.5
(CH2), 25.3 (CH2), 22.3 (CH3),
14.6 (CH3)
5g 0.89 (s, 3H, CH3), 2.44 (s, 3H, CH3), 1.61–2.66 194.8 (C=O, nal. ac.), 180.5 (C=O, spirohyd. ring), 158.8 (CH), 141.2 (CH), 128.8
(m, 4H, CH2, ind.), 3.38 (s, 2H, CH2, Et), 6.67
(s, 1H, CH, pyr.), 6.74–7.21 (m, 4H, CH, Ph),
7.26 (s, 1H, NH, sec. amide), 7.91 (s, 1H, CH,
nal. ac.), 8.06 (s, 1H, CH, pyr.), 8.85 (s, 1H,
NH, urea)
168.7 (C=O, 1-amide), 164.2 (C/C, pyr.), 161.2
(C=O, spirohyd. ring, urea), 158.8 (CH, nal. ac.),
157.2 (C, ethylene), 141.2 (CH, pyr.), 140.0 (C/C,
benzene), 128.8 (CH/CH), 126.3 (CH/CH), 115.6
(CH, pyr.), 65.4 (spiro C-atom), 50.1 (CH2, Et,
nal. ac.), 41.8 (CH2), 32.6 (CH2), 21.8 (CH3), 17.2
(CH2), 13.5 (CH3)
(CH/CH), 126.3 (CH/CH), 115.6
(CH), 50.1 (CH2), 41.8 (CH2),
32.6 (CH2), 21.8 (CH3), 17.2
(CH2), 13.5 (CH3)
Table 4 Antimicrobial activity
Microorganism
Inhibition zone (mm)
of compounds 5a–5g
5a*
5b 5c
5d
5e 5f
5g
Nalidixic acid Eth-
ylene
glycol
Staphylococcus aureus
13.3
18.9
15.4
13.7
–
–
–
–
–
9.8
15.6
–
–
–
–
–
17.3 15.4
20.0 20.7
–
–
–
–
–
–
–
ATCC 6538
Bacillus subtilis
ATCC 6633
Escherichia coli
ATCC 8739
Pseudomonas aeruginosa
ATCC 9027
Salmonella abony NTCC 6017 13.7
10.9 19.4
23.0
10.3 17.4
21.7
–
22.7 25.4 16.2
17.5 18.8 15.6
22.3 23.2 16.7
–
*Ref. [4]
**No zone of inhibition
efcacy of compounds 5d, 5f and 5g against the tested
compounds are proven through physicochemical param-
eters, IR, 1H and 13C NMR spectral data. The antimicrobial
study of the products obtained shows that the most efec-
tive compounds are 5f and 5g, which exhibit a pronounced
antimicrobial activity against both tested Gram-positive and
Gram-negative bacteria.
microorganisms compared to the nalidixic acid.
Conclusions
The synthesis of new amides (5a–5g) based on the inter-
action of the aminospirohydantoins (3a–3g) with nalidixic
acid (4) is presented. The structures of the synthesized
Acknowledgements Financial support by the Agricultural Univer-
sity—Plovdiv, Bulgaria (Contract 06-18) is gratefully acknowledged.
1 3

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