5-Nitroimidazole-derived Schiff bases and their copper(II) complexes exhibit potent antimicrobial activity against pathogenic anaerobic bacteria

Article abstract of DOI:10.1007/s10534-018-0106-6

In the present work a family of novel secnidazole-derived Schiff base compounds and their copper(II) complexes were synthesized. The antimicrobial activities of the compounds were evaluated against clinically important anaerobic bacterial strains. The compounds exhibited in vitro antibacterial activity against Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Parabacteroides distasonis and Fusubacterium nucleatum pathogenic anaerobic bacteria. Upon coordination to copper(II) the antibacterial activity significantly increased in several cases. Some derivatives were even more active than the antimicrobial drugs secnidazole and metronidazole. Therefore, the compounds under study are suitable for in vivo evaluation and the microorganisms should be classified as susceptible to them. Electrochemical studies on the reduction of the nitro group revealed that the compounds show comparable reduction potentials, which are in the same range of the bio-reducible drugs secnidazole and benznidazole. The nitro group reduction potential is more favorable for the copper(II) complexes than for the starting ligands. Hence, the antimicrobial activities of the compounds under study might in part be related to intracellular bio-reduction activation. Considering the increasing resistance rates of anaerobic bacteria against a wide range of antimicrobial drugs, the present work constitutes an important contribution to the development of new antibacterial drug candidates.

Full text of DOI:10.1007/s10534-018-0106-6

Biometals
https://doi.org/10.1007/s10534-018-0106-6
5-Nitroimidazole-derived Schiff bases and their copper(II)
complexes exhibit potent antimicrobial activity against
pathogenic anaerobic bacteria
.
Alexandre A. Oliveira Ana P. A. Oliveira Lucas L. Franco Micael O. Ferencs
.
.
.
.
.
.
Jo a˜ o F. G. Ferreira Sofia M. P. S. Bachi Nivaldo L. Speziali
.
.
Luiz M. Farias Paula P. Magalh a˜ es Heloisa Beraldo
Received: 27 February 2018 / Accepted: 23 April 2018
Ó Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract In the present work a family of novel
secnidazole-derived Schiff base compounds and their
copper(II) complexes were synthesized. The antimi-
crobial activities of the compounds were evaluated
against clinically important anaerobic bacterial
strains. The compounds exhibited in vitro antibacterial
activity against Bacteroides fragilis, Bacteroides
thetaiotaomicron, Bacteroides vulgatus, Bacteroides
ovatus, Parabacteroides distasonis and Fusubac-
terium nucleatum pathogenic anaerobic bacteria.
Upon coordination to copper(II) the antibacterial
activity significantly increased in several cases. Some
derivatives were even more active than the antimicro-
bial drugs secnidazole and metronidazole. Therefore,
the compounds under study are suitable for in vivo
evaluation and the microorganisms should be classi-
fied as susceptible to them. Electrochemical studies on
the reduction of the nitro group revealed that the
compounds show comparable reduction potentials,
which are in the same range of the bio-reducible drugs
secnidazole and benznidazole. The nitro group reduc-
tion potential is more favorable for the copper(II)
complexes than for the starting ligands. Hence, the
antimicrobial activities of the compounds under study
might in part be related to intracellular bio-reduction
activation. Considering the increasing resistance rates
of anaerobic bacteria against a wide range of antimi-
crobial drugs, the present work constitutes an impor-
tant contribution to the development of new
antibacterial drug candidates.
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10534-018-0106-6) con-
tains supplementary material, which is available to authorized
users.
Keywords 5-Nitroimidazole Á Copper(II)
complexes Á Schiff bases Á Anaerobic bacteria Á
Antimicrobial activity Á Bio-reduction
A. A. Oliveira Á A. P. A. Oliveira Á L. L. Franco Á
M. O. Ferencs Á S. M. P. S. Bachi Á H. Beraldo (&)
Departamento de Qu ´ı mica, Universidade Federal de
Minas Gerais, Belo Horizonte, MG 31270-901, Brazil
e-mail: hberaldo@ufmg.br;
Introduction
heloisaberaldoufmg@gmail.com
The importance of nitro compounds in medicinal
chemistry emerged with the discovery of chloram-
phenicol, an antibacterial agent containing a nitro-
phenyl group, first isolated in 1947 from a culture of
Streptomyces venezuelae (Ehrlich et al. 1947). Since
J. F. G. Ferreira Á L. M. Farias Á P. P. Magalh a˜ es
Departamento de Microbiologia, Universidade Federal de
Minas Gerais, Belo Horizonte, MG 31270-901, Brazil
N. L. Speziali
Departamento de F ´ı sica, Universidade Federal de Minas
Gerais, Belo Horizonte, MG 31270-901, Brazil
123

Biometals
then, nitro-heterocyclic compounds have been exten-
sively investigated as antimicrobial agents (Patterson
and Wyllie 2014; O’Shea et al. 2016).
half-life, which allows the administration of lower
doses and makes single-dose applications possible
(Gillis and Wiseman 1996; De Backer et al. 2010; Ang
et al. 2017).
2
-Nitroimidazole (azomycin, Fig. 1a), isolated in
1
953 from the bacterium Nocardia mesenterica, was
Anaerobic bacteria are the most predominant
components of the human indigenous microbiota and
the most frequent cause of endogenous bacterial
infections. Anaerobic infections are often considered
as life threatening and may occur at the central
nervous system, oral cavity, head and neck, chest,
abdomen, pelvis, skin and soft tissues. The most
relevant anaerobes associated with the etiopathogen-
esis of approximately 95% of anaerobic infections are
Gram-negative rods, among them Bacteroides, Pre-
votella, Porphyromonas, Fusobacterium, Bilophila
and Sutterella (Brook 2016).
found to be active against Trichomonas vaginalis, the
causative parasite of trichomoniasis (Anderson et al.
2
012). After this finding, the synthesis and biological
evaluation of nitroimidazole derivatives led to the
discovery of metronidazole in 1957 (Fig. 1b), a potent
antiprotozoal agent with greater activity than azomy-
cin and acceptable toxicity (Anderson et al. 2012;
Wright et al. 2014).
In 1960 metronidazole became the first drug to be
introduced into human therapy for the treatment of
trichomoniasis and later, for treating infections caused
by a variety of anaerobic bacteria (Wright et al. 2014).
Metronidazole is classified as an essential medicine by
the World Health Organization (WHO 2018). It is
widely used for the treatment of infections caused by
Giardia lamblia, Trichomonas vaginalis, Entamoeba
histolytica and Blastocystis sp. protozoa, as well as
against Clostridium difficile and Bacteroides fragilis
anaerobic bacteria and the microaerophilic species
Helicobacter pylori (Dunn et al. 2010).
The polymicrobial etiology of anaerobic infections,
mainly an association between anaerobes and facul-
tative bacteria, results in difficulties in treating these
infections (Brook 2016). Another obstacle for the
effective treatment is the increasing resistance rates of
anaerobic bacteria against a wide range of antimicro-
bial agents, including metronidazole, frequently
employed in clinics (Akhi et al. 2015; Tan et al.
2017; Hastey et al. 2016).
Afterwards, different derivatives of nitroimidazoles
were synthetized and a second-generation of 5-ni-
troimidazole antimicrobial agents has been developed,
including secnidazole (Fig. 1c), tinidazole (Fig. 1d)
and ornidazole (Fig. 1e) (Nagata et al. 2012; Kapoor
et al. 2003).
5-Nitroimidazoles are prodrugs that require bioac-
tivation of the nitro group in the intracellular envi-
ronment in order to exert their biological effect. After
these compounds enter the cells the nitro group is
ÁÀ
reduced to a short-lived nitro anion radical (R-NO ),
2
the critical step in the activation of nitroimidazoles
(Ang et al. 2017).
Secnidazole proved to be a very promising alter-
native for metronidazole due to its broad spectrum of
activity and potency against anaerobic microorgan-
isms. Secnidazole presents appropriate pharmacoki-
netics, being rapidly and completely absorbed after
oral administration. It has a long terminal elimination
While the nitro group is crucial for the activity of
5-nitroimidazoles against anaerobic bacteria, different
substituents in the imidazole side chains may result in
different spectra of activity and pharmacokinetic
properties.
Fig. 1 Chemical structures of nitroimidazole derivatives a 2-nitroimidazole, b metronidazole, c secnidazole, d tinidazole and
e ornidazole
1
23

Biometals
Hydrazones and thiosemicarbazones are Schiff
Materials and methods
base compounds which show potent antimicrobial
activity (Divar et al. 2017; Abdelrahman et al. 2017;
Popiolek 2017). In several cases the antimicrobial
activity of these compounds increases upon metal
coordination (Ferreira et al. 2016). In particular,
complexation to copper(II) often results in antibacte-
rial activity enhancement (Recio-Despaigne et al.
Equipment and reagents
All reagents were purchased from Aldrich and Merck
and used as received. Partial elemental analyses were
performed on a Perkin Elmer CHN 2400 analyzer.
Melting points were determined with a Mettler
MQAPF-302 apparatus. Magnetic susceptibility mea-
surements were performed with a Johnson Matthey
MSB/AUTO balance. An YSI model 31 conductivity
bridge was employed for molar conductivity mea-
surements. Infrared spectra were recorded on a Perkin
Elmer Spectrum One employing the attenuated total
2
012; Mendes et al. 2007).
In this way, in the present work novel secnidazole-
derived compounds functionalized with different
Schiff base groups were obtained (Fig. 2) as well as
their copper(II) complexes. The antimicrobial effects
of the compounds were evaluated against Bacteroides
fragilis, Bacteroides thetaiotaomicron, Bacteroides
vulgatus, Bacteroides ovatus, Parabacteroides dista-
sonis and Fusubacterium nucleatum pathogenic anaer-
obic bacteria. Electrochemistry studies on the
reduction of the nitro group were performed as well,
aiming to investigate the ability of the compounds to
undergo bio-reduction activation.
-
1
reflectance (ATR) method (4000–400 cm ). NMR
spectra were obtained at 25 °C with a Bruker DPX-
400 Advance (400 MHz) spectrometer using DMSO-
d as solvent. Mass spectra were recorded with a
6
Shimadzu LCMS-IT-TOF instrument working at
high-resolution.
Fig. 2 Syntheses of
secnidazole-derived
compounds 2, (HL3–HL8)
and copper(II) complexes
(
Ca–Cf). Reagents and
conditions: (a) PDC,
CH Cl , r.t. 48 h; (b) HL3–
HL5: NH NHCOR, AcOH,
MeOH, reflux, 4 h; HL6–
HL8: NH NH CSNHR,
AcOH, MeOH, reflux, 4 h;
c, d) CuCl , MeOH, reflux,
2
2
2
2
2
(
2
8
h
123

Biometals
Electrochemical studies
magnetic susceptibilities and by means of their
infrared spectra. All data that support the chemical
characterization of the compounds are reported in
Supplementary Information (SI).
Cyclic voltammetry was carried out at room temper-
ature with a conventional three-electrode cell (with
volumetric capacity of 10 mL) in a lAutolab type II
potentiostat (Echo Chemie, Utrecht, the Netherlands)
using the GPES 4.9 software. The working electrode
was a Metrohm glassy carbon electrode, the auxiliary
1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-one
(2) White solid. Yield: 77% (oxidant: PCC) and 84%
(oxidant: PDC). Anal. Calc. for C H N O (%): C,
7
9 3 3
45.90; H, 4.95; N, 22.94. Found: C, 46.22; H, 4.83; N,
-1
-
-1
22.96. FW: 183.17 g mol . IR (ATR, cm ): 3110 (m
C2-H), 1726 (m C=O), 1529 (m C=N), 1456/1361 (m /m
electrode was a platinum wire and Ag/AgCl, Cl
(
3.0 M) was used as the reference electrode. The
ass
s
1
glassy carbon electrode was previously fine-polished
NO ). H NMR [400.13 MHz, DMSO-d , d(ppm)]:
2
6
with 0.3 lm alumina slurry on a polishing felt during
8.05 [s, 1H, H2], 5.31 [s, 2H, H4], 2.35 [s, 3H, C1CH ],
3
1
2.27 [s, 3H, C5CH3]. C{ H} NMR [100.61 MHz,
3
1
5
min. Solutions for analysis were prepared in spec-
troscopic dimethylformamide (DMF) containing
mM of analyte and 0.1 M of tetrabutylammonium
DMSO-d , d(ppm)]: 200.8 [C5], 151.3 [C1], 138.3
6
1
[C3], 132.3 [C2], 54.8 [C4], 27.0 [C5CH )], 13.5
3
perchlorate (TBAP) as supporting electrolyte. Before
recording the voltammograms, the test solution was
thoroughly purged with high purity nitrogen gas to
remove any traces of dissolved oxygen. Cyclic
voltammograms were recorded in the 1.50 to
[C1CH ]. HRMS [ESI(?), IT-TOF] calculated for
3
?
C H N O , [M ? H] : 184.0722, found: 184.0722.
7
10 3 3
(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
pan-2-ylidene)hydrazinecarboxamide (HL3) White-
yellowish solid. Yield: 45%. Melting point:
211.8–213.2 °C. Anal. Calc. for C H N O (%):
-
1
-
2.50 V potential range using 250 mV s scan rate.
8
12 6 3
C, 40.00; H, 5.04; N, 34.98. Found: C, 39.75; H, 4.64;
-
N, 34.08. FW: 240.22 g mol . IR (ATR, cm ):
1
-1
Syntheses of the 5-nitroimidazole-derived Schiff
bases and their copper(II) complexes
3180 (m N4-H), 3123 (m C2-H), 1698 (m C=O), 1670
(
m C=C), 1576, 1540 (m C=N), 1461/1361 (mass/ms
1
Compounds (HL3–HL8) were synthetized from the
intermediate (2), prepared by oxidation of secnidazole
NO ). H NMR [400.13 MHz, DMSO-d , d(ppm)]:
2
6
9.39 [s, 1H, N4H], 8.40 [s, 1H, H2], 6.13 [sl, 2H,
(
1) with pyridinium chlorochromate (PCC) or pyri-
dinium dichromate (PDC), according to procedures
described in the literature (Fig. 2) (Corey and Schmidt
N5H], 5.13 [s, 2H, H4], 2.50 [s, 3H, C1CH ], 1.92 [s,
3
1
3H, C5CH3]. C{ H} NMR [100.61 MHz, DMSO-
3
1
d6, d(ppm)]: 156.8 [C6], 150.7 [C1], 142.3 [C5],
138.3 [C3], 128.8 [C2], 50.3 [C4], 14.5 [C5CH )],
1
979; Breuning et al. 2009). HL3–HL8 were obtained
by stirring under reflux a methanol solution (5 mL) of
with an equimolar amount of the desired hydrazine
3
12.8 [C1CH ]. HRMS [ESI(?), IT-TOF] calculated
3
?
for C H N NaO , [M ? Na] : 263.0869, found:
2
8
12
6
3
derivative (0.55 mmol) for 4 h, with addition of few
drops of acetic acid as catalyst. After cooling to room
temperature, 10 mL of ethyl ether and 10 mL of
isopropanol were added and the obtained precipitate
was filtered off, washed with ethyl ether and dried in a
desiccator under reduced pressure.
263.0885.
0
(E)-N -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
pan-2-ylidene)acetohydrazide (HL4) White-yellow-
ish solid. Yield: 81%. Melting point: 165.4–167.8 °C.
Anal. Calc. for C H N O (%): C, 45.18; H, 5.48; N,
9
13 5 3
29.27. Found: C, 44,10; H, 5,19; N, 28,34. FW:
-1
239.24 g mol . IR (ATR, cm ): 3264 (m N4-H),
3142 (m C2-H), 1674 (m C=O), 1653 (m C=C), 1530 (m
C=N), 1471/1362 (m /m
[400.13 MHz, DMSO-d , d(ppm)]: 10.32 [s, 1H,
N4H], 8.04 [s, 1H, H2], 5.10 [s, 2H, H4], 2.37 [s,
-
1
Copper(II) complexes (Ca–Cf) were obtained by
reacting equimolar amounts (0.5 mmol) of the desired
thiosemicarbazone or hydrazone with CuCl Á2H O in
1
2
2
ass
s
NO2).
H
NMR
methanol (10 mL) under reflux for 8 h. The resulting
solids were filtered off and washed with methanol and
ethyl ether, and dried under reduced pressure.
6
3H, C1CH ], 1.93 [s, 3H, C5CH ], 1.66 [s, 3H, H7].
3
3
1
3
1
The Schiff base ligands were characterized by
elemental analysis and by means of their infrared,
NMR and HRMS spectra. Complexes (Ca–Cf) were
characterized by microanalyses, molar conductivities,
C{ H} NMR [100.61 MHz, DMSO-d , d(ppm)]:
6
172.2 [C6], 151.5 [C1], 144.9 [C5], 138.6 [C3], 132.4
[C2], 49.9 [C4], 19.9 [C7], 14.4 [C5CH )], 13.5
3
1
23

Biometals
[
C1CH ]. HRMS [ESI(?), IT-TOF] calculated for
3
[C5CH )], 13.8 [C1CH ]. HRMS [ESI(?), IT-TOF]
3 3
?
C H N O , [M ? H] : 240.1097, found: 240.1103.
?
calculated for C H N NaO S, [M ? Na] : 293.0797,
9
14
5
3
9
14
6
2
0
(
E)-N -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
found: 293.0796.
pan-2-ylidene)benzohydrazide (HL5) Yellow solid.
Yield: 66%. Melting point: 205.1–206.2 °C. Anal.
Calc. for C H N O (%): C, 55.81; H, 5.02; N,
(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
pan-2-ylidene)-N-phenylhydrazinecarbothioamide
(HL8) White-yellowish solid. Yield: 84%. Melting
point: 116.2–118.0 °C. Anal. Calc. for C H N O S
1
4 15 5 3
2
3
3
1
3.24. Found: C, 56.43; H, 4.88; N, 23.49. FW:
-
1
4 16 6 2
1
-1
01.31 g mol . IR (ATR, cm ): 3185 (m N4-H),
(%): C, 50.59; H, 4.85; N, 25.28. Found: C, 48.40; H,
-
4.92; N, 24.17. FW: 332.38 g mol . IR (ATR,
1
123 (m C2-H), 1670 (m C=O), 1585 (m C=C),
1
-1
cm ): 3296 (m N4-H), 3138 (m C2-H), 1593 (m
578/1526 (m C=N), 1461/1355 (m /m NO ).
H
ass
s
2
NMR [400.13 MHz, DMSO-d , d(ppm)]: 10.70 [s,
C=C), 1554/1530 (m C=N), 1471/1362 (m /m NO ),
6
ass
s
2
1
1
7
2
H, N4H], 8.06 [s, 1H, H2], 7.78 [sl, 2H, H8, H12],
772 (m C=S). H NMR [400.13 MHz, DMSO-d6,
d(ppm)]: 10.87 [s, 1H, N4H], 8.83 [s, 1H, N5H], 8.15
[s, 1H, H2], 7.51 [d, 2H, H8, H12], 7.35 [t, 2H, H9,
H11], 7.16 [t, 1H, H10], 5.23 [s, 2H, H4], 2.43 [s, 3H,
.58–7.17 [m, 3H, H9, H10, H11], 5.22 [s, 2H, H4],
1
.42 [s, 3H, C1CH ], 2.04 [s, 3H, C5CH ]. C{ H}
3
1
3
3
NMR [100.61 MHz, DMSO-d , d(ppm)]: 163.1 [C6],
6
1
3
1
1
57.0 [C1], 151.7 [C5], 138.5 [C3], 133.7 [C7], 132.7
C2], 131.5 [C10], 128.3 [C9, C11], 127.7 [C8, C12],
0.1 [C4], 15.8 [C5CH )], 13.7 [C1CH ]. HRMS
C1CH3], 2.10 [s, 3H, C5CH ]. C{ H} NMR
3
[
[100.61 MHz, DMSO-d , d(ppm)]: 175.8 [C6],
6
5
152.1 [C1], 148.1 [C5], 138.7 [C3], 138.2 [C7],
132.6 [C2], 128,5 [C9, C11], 125.0 [C10], 122.7 [C8,
C12], 50.2 [C4], 15.3 [C5CH )], 13.7 [C1CH ].
3
3
[
ESI(?), IT-TOF] calculated for C H N NaO ,
14 15 5 3
?
[
M ? Na] : 324.1073, found: 324.1072.
E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-
-ylidene)hydrazinecarbothioamide (HL6) White solid.
3
3
(
HRMS [ESI(?), IT-TOF] calculated for C H N
14 16 6-
?
NaO S, [M ? Na] : 355.0953, found: 355.0940.
2
2
Yield: 74%. Melting point: 168.4–171.2 °C. Anal. Calc.
Bis[(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)
propan-2-ylidene)hydrazinecarboxamide)dichloro-
copper(II)], [Cu(HL3) Cl ] (Ca) Dark blue solid.
for C H N O S (%): C, 37.49; H, 4.72; N, 32.79.
8
12 6 2
Found: C, 38.26; H, 4.79; N, 31.32. FW:
2
2
-
1
-1
2
3
1
56.28 g mol . IR (ATR, cm ): 3170 (m N4-H),
Yield: 50%. Melting point: 183.0–184.5 °C. Anal.
083 (m C2-H), 1644 (m C=C), 1611/1530 (m C=N),
Calc. for C H Cl CuN O (%): C, 31.25; H,
1
6
24
2
12 6
1
463/1360 (m /ms NO ), 804 (m C=S). H NMR
3.93; N, 27.34. Found: C, 31.36; H, 3.70; N,
ass
2
-
27.25. FW: 614.89 g mol . Molar conductivity
1
[
400.13 MHz, DMSO-d , d(ppm)]: 10.42 [s, 1H,
6
2
-1
-1
N4H], 8.16 [s, 1H, N5H], 8.07 [s, 1H, H2], 6.41 [s,
(DMSO, 1 mM): 18.83 cm X mol . IR (ATR,
-
1
1
H, N5H], 5.13 [s, 2H, H4], 2.38 [s, 3H, C1CH ], 2.01 [s,
3
cm ): 3434 (m N4-H), 1712 (m C=O), 1683
m(C=C), 1566/1520 m(C=N), 1484/1390 (mass/ms
NO ). Magnetic moment: 2.00 l .
1
3
1
H, C5CH3]. C{ H} NMR [100.61 MHz, DMSO-d ,
3
6
d(ppm)]: 178.9 [C6], 151.8 [C1], 147.4 [C5], 138.5 [C3],
32.6 [C2], 50.1 [C4], 15.1 [C5CH )], 13.6 [C1CH ].
2
B
0
1
[(E)-N -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)
propan-2-ylidene)acetohydrazidedichlorocopper
(II)] [Cu(HL4)Cl ]Á‘CH OH (Cb) Green solid.
3
3
HRMS [ESI(?), IT-TOF] calculated for C H N O S,
8
13 6 2
?
[
M ? H] : 257.0820, found: 257.0796.
E)-N-methyl-2-(1-(2-methyl-5-nitro-1H-imidazol-
-yl)propan-2-ylidene)hydrazinecarbothioamide (HL7)
Yellow solid. Yield: 62%. Melting point: 194.2–
96.9 °C. Anal. Calc. for C H N O S (%): C, 39.99;
2
3
(
Yield: 51%. Melting point: decomposition at
1
195 °C. Anal. Calc. for C9.5H Cl CuN O (%):
15
2
5
3.5
C, 29.28; H, 3.88; N, 17.97. Found: C, 30.33; H,
-
3.58; N, 17.76. FW: 389.70 g mol . Molar con-
1
1
9
14 6 2
2
-1
-1
H, 5.22; N, 31.09. Found: C, 39.86; H, 5.41; N, 30.45.
ductivity (DMSO, 1 mM): 28.82 cm X mol
.
-1
-1
-1
FW: 270.31 g mol . IR (ATR, cm ): 3183 (m N4-H),
555/1531 (m C=N), 1471/1362 (m /m NO ), 786 (m
IR (ATR, cm ): 3126 (m N4-H), 1736 (m C=O),
1634 m(C=C), 1550 m(C=N), 1476/1388 (mass/ms
NO ). Magnetic moment: 1.60 l .
1
ass
s
2
1
C=S). H NMR [400.13 MHz, DMSO-d , d(ppm)]:
6
2
B
0
1
5
1
0.31 [s, 1H, N4H], 8.07 [s, 1H, H2], 7.24 [s, 1H, N5H],
[(E)-N -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
.12 [s, 2H, H4], 2.89 [d, 3H, H7], 2.39 [s, 3H, C1CH ],
3
pan-2-ylidene)benzohydrazidedichlorocopper(II)] [Cu
13
1
.96 [s, 3H, C5CH3]. C{ H} NMR [100.61 MHz,
(HL5)Cl ] (Cc) Green solid. Yield: 50%. Melting point:
2
DMSO-d , d(ppm)]: 178.9 [C6], 151.9 [C1], 147.0 [C5],
decomposition at 210 °C. Anal. Calc. for C14H15Cl2-
6
138.6 [C3], 132.7 [C2], 50.3 [C4], 30.7 [C7], 14.8
CuN O (%): C, 38.59; H, 3.47; N, 16.07. Found: C,
5
3
123

Biometals
-
1
3
8.09; H, 3.21; N, 16.39. FW: 435.75 g mol . Molar
2
SHELXS-2013/1 (Sheldrick 2013, 2015). Full-matrix
2
least-squares refinement procedure on F with aniso-
-1
-1
.
conductivity (DMSO, 1 mM): 33.18 cm X mol
-1
IR (ATR, cm ): 3126 (m N4-H), 1736 (m C=O), 1585
m(C=C), 1556/1526 m(C=N), 1480/1386 (m /m NO ).
tropic thermal parameters was carried on using
SHELXL-2014/7 (Sheldrick 2014). Positional and
anisotropic atomic displacement parameters were
refined for all non-hydrogen atoms. Hydrogen atoms
were placed geometrically and the positional param-
eters were refined using a riding model.
ass
s
2
Magnetic moment: 1.63 lB.
(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
[
pan-2-ylidene)hydrazinecarbothioamidedichlorocop-
per(II)] [Cu(HL6)Cl ]Á‘H O (Cd) Dark green solid.
2
2
Yield: 75%. Melting point: decomposition at 160 °C.
A suitable single crystal of HL6 was obtained at
room temperature from slow evaporation of the
mother liquor. A summary of the crystal data, data
collection details and refinement results is listed in
Table 1. Molecular graphics were plotted using
PLATON (Spek 2009).
Anal. Calc. for C H Cl CuN O2.5S (%): C, 24.04; H,
8
13
2
6
3
.28; N, 21.02. Found: C, 23.91; H, 3.20; N, 21.28.
-
FW: 399.74 g mol . Molar conductivity (DMSO,
1
2
-1
-1
-1
1
mM): 43.57 cm X mol . IR (ATR, cm ): 3224
m N4-H), 788 (m C=S), 1640 m(C=C), 1594/1526 (m
C=N), 1474/1390 (m /m NO ). Magnetic moment:
(
ass
s
2
1
.60 lB.
(E)-N-methyl-2-(1-(2-methyl-5-nitro-1H-imidazol-
-yl)propan-2-ylidene)hydrazinecarbothioamidedichloro-
copper(II)] [Cu(HL7)Cl ] (Ce) Dark green solid. Yield:
Antimicrobial activity
[
1
In vitro susceptibility to the compounds under study
was evaluated according to Clinical & Laboratory
Standards Institute (CLSI) (2015). Six reference strains
of Gram-negative anaerobic bacteria were tested:
Bacteroides fragilis (ATCC 25285), Bacteroides
thetaiotaomicron (ATCC 29741), Bacteroides vulga-
tus (ATCC 8482), Bacteroides ovatus (ATCC 8483),
Parabacteroides distasonis (ATCC 1295) and Fusub-
acterium nucleatum (ATCC 25586).
2
6
5%. Melting point: decomposition at 180 °C. Anal. Calc.
for C H Cl CuN O S (%): C, 26.71; H, 3.49; N, 20.76.
9
14
2
6 2
-1
Found: C, 26.81; H, 3.73; N, 20.20. FW: 404.76 g mol .
-1
Molar conductivity (DMSO, 1 mM): 26.75 cm X
mol . IR (ATR, cm ): 3374 (m N4-H), 784 (m C=S),
2
-1
-1
1562/1530 m(C=N), 1474/1370 (m /m NO ). Magnetic
ass
s
2
moment: 1.63 lB.
[
(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
In order to evaluate the minimum inhibitory
concentration of each compound, bacterial strains
were cultured in Brucella Agar supplemented with
hemin (5 lg/mL), vitamin K1 (1 lg/mL) and horse
blood (5% v/v) (BA-S), at 37 °C for 48 h in an
anaerobic chamber (85% N , 10% H and 5% CO ).
pan-2-ylidene)-N-phenylhydrazinecarbothioamidedich-
lorocopper(II)] [Cu(HL8)Cl ]Á‘H O (Cf) Dark green
2
2
solid. Yield: 61%. Melting point: decomposition at
2
3
1
05 °C. Anal. Calc. for C H Cl CuN O S (%): C,
5.34; H, 3.45; N, 17.66. Found: C, 35.18; H, 3.25; N,
1
4
17
2
6 2.5
2
2
2
-1
7.22. FW: 475.84 g mol . Molar conductivity
2
The inoculum was prepared in sterile saline solution
0.9% w/v and standardized to obtain visual turbidity
comparable to that of the standard 0.5 of the McFar-
land scale, which provides a concentration of approx-
-1
-1
(
DMSO, 1 mM): 46.82 cm X mol . IR (ATR,
-
1
cm ): 3300 (m N4-H), 760 (m C=S), 1596 m(C=C), 1552
m(C=N), 1496/1368 (m /m NO ). Magnetic moment:
ass
s
2
8
1
.63 lB.
imately 1.5 9 10 CFU (colony forming units)/mL. A
1:10 dilution of the inoculum was prepared in sterile
Crystal structure determination
saline solution 0.9% w/v, resulting in a concentration
7
of approximately 1.5 9 10 CFU/mL. The adjusted
Single crystal X-ray diffraction measurements were
carried out on an Oxford-Diffraction GEMINI-Ultra
diffractometer (LabCri-UFMG) using graphite-En-
suspensions were used in the final inoculation up to
30 min after preparation.
The compounds under evaluation were tested for
determination of minimum inhibitory concentration
(MIC) by the agar dilution method. Briefly, the
technique consisted of adding 4.0 mL of working
solution of the compounds containing nine different
concentrations (ranging from 320 to 1.25 lg/mL) in
flasks containing 36 mL of the culture medium
˚
hance Source Mo Ka radiation (k = 0.71073 A) at
2
93(2) K. Data collection, cell refinements and data
reduction were performed using the CrysAlisPro
software package (Oxford 2010). An absorption
correction based on a multi-scan method was applied.
The structure was solved by direct methods using
1
23

Biometals
Table 1 Crystal data and
structure refinement results
for HL6
Compound
HL6
Empirical formula
Formula weight (g mol
Crystal system
8 12 6 2
C H N O S
-
1
)
256.30
Triclinic
P-1
Space group
˚
Wavelength (A)
0.71073
293 (2)
Temperature (K)
Unit cell dimensions
˚
a (A)
7.2881 (3)
8.5774 (4)
˚
b (A)
˚
c (A)
10.1348 (4)
a/b/c (°)
V (A )
a = 90.017(4)/b = 103.727(4)/c = 104.761(4)
3
˚
593.91 (5)
Z
2
-3
)
Density calculated (mg m
3
Crystal size (mm )
1.433
0.02 9 0.02 9 0.02
268
F(000)
-
1
)
Absorption coefficient (mm
Index ranges
0.274
- 10 B h B 10
-
-
12 B k B 12
14 B l B 14
2
h range for data collection (°)
4.146 to 62.07
31726
Reflections collected
Independent reflections [Rint, Rsigma
o
Completeness to h = 31.035 (%)
]
3532 [0.0621, 0.0413]
93.1
Data/restraints/parameters
Final R indexes [I [ 2r(I)]
Final R indexes (all data)
3532/0/154
R
1
1
= 0.0555, wR
= 0.0994, wR
2
2
= 0.1407
= 0.1684
R
2
Goodness-of-fit on F
1.019
Dqm a´ x. and Dqmin.
0.40 and - 0.36
followed by plating. In the preparation of the working
solutions of the compounds, DMSO was used as the
solvent and sterile distilled water as the diluent, so that
the final concentration of DMSO was less than 0.5% in
all tests. Inoculation was performed by adding
control) as well as the control to assure the non-
interference of DMSO in cell viability. The tests were
performed in duplicate with absolute agreement of the
results and the MIC values were expressed in lM.
5
approximately 10 CFU/spot of each bacterial strain
using the Steers replicator and subsequent anaerobic
incubation at 37 °C for 48 h.
Results and discussion
The minimum inhibitory concentration (MIC) was
expressed as the lowest concentration that resulted in
visual inhibition of the microorganism growth com-
pared with the growth in the culture medium free of
the tested compound (positive control). In addition,
incubation in the absence of inoculum was performed
to confirm the sterility of the culture medium (negative
Characterization
Microanalyses and high-resolution mass spectra were
compatible with the formation of 2 and HL3–HL8.
Microanalyses and molar conductivity data were in
accordance with the formation of [Cu(HL3) Cl ] (Ca).
2
2
In all other cases (Cb–Cf) the data were compatible
123

Biometals
with formation of [Cu(HL)Cl ] complexes. Com-
2
NMR spectra the signal at 200.8 ppm associated to the
C5 nucleus in 2 is not present in the spectra of HL3–
HL8, while a signal close to 150 ppm was assigned to
the iminic carbon.
plexes (Ca–Cf) showed magnetic moments in the
1
9
.63–2.00 BM range, in accordance with d electronic
configuration. The presence of crystallization solvent
molecules in complexes Cb, Cd and Cf was confirmed
by their thermogravimetry curves, which showed
weight losses of 4.09% (calcd. 4.10%), 2.29% (calcd.
In order to define the stereochemistry of the
compounds in solution, two-dimensional Nuclear
Overhauser Effect Spectroscopy (NOESY) was
employed. Contour maps showed intramolecular
2
.25%) and 1.93% (calcd.1.89%), respectively.
1
1
In the infrared spectra of HL3–HL8 three common
features associated with the imidazole nucleus were
H- H coupling involving C5CH and N4H, in accor-
3
dance with E stereochemistry around the azomethine
observed: a vibration attributed to m(C2-H) in the
double bond.
-
1
3
083–3142 cm
range, a vibration attributed to
-
1
m(C=N) near 1520–1540 cm and two bands near
X-ray crystallography
-
1
-1
1
470 cm
and 1360 cm
corresponding to the
asymmetric and symmetric stretching modes of the
The atom arrangements and atom numbering
scheme for HL6 are shown in Fig. 3. Selected
intramolecular bond lengths and angles are listed in
Table 2. The thiosemicarbazone HL6 crystallizes in
the triclinic system, space group P-1, with two
molecules per unit cell. The C6–S and N3–C5 bond
NO group, respectively (Rodrigues et al. 2010). Upon
2
coordination to copper(II) the m(C2-H) vibration shifts
-
1
to 3098–3282 cm . The m(C=N) absorption shifts to
-
500–1525 cm and the asymmetric and symmetric
1
1
-
1
stretching modes of NO shift to 1496–1368 cm
2
.
˚
distances are 1.690(2) and 1.275(3) A, as expected for
In addition, in all compounds absorptions at
1
-
1
600–1670 cm
were attributed to the m(C=C)
vibration and absorptions at 1550–1580 cm were
C=S and C=N double bonds (Allen et al. 1987). The
compound crystallized in the EE conformation in
relation to the N3–C5 and C6–N4 bonds.
-
1
attributed to m(C=N3). The vibrations attributed to
-
1
m(C=O) and m (C=S), observed at 1661–1714 cm
In the crystal packing N5-HÁÁÁO2 and N5-HÁÁÁN1
inter-molecular hydrogen bonds were observed (see
Table S1 and Fig. S35).
(
HL3-HL5) (Recio-Despaigne et al. 2010) and
-
1
8
04–772 cm
(HL6-HL8) (Mendes et al. 2001),
-
respectively, are observed at 1712–1736 cm (Ca–
-
Cc) and 788–760 cm (Cd–Cf), respectively.
1
1
Antimicrobial activity
The NMR spectra were recorded in DMSO-d . The
6
1
H resonances were doubtless assigned on the basis of
Secnidazole (1), the intermediate ketone (2), Schiff
bases (HL3–HL8) and the copper(II) complexes (Ca–
Cf) were evaluated in vitro by the agar dilution method
for their ability to inhibit the growth of six strains of
Gram-negative anaerobic bacterial species. For com-
parison, metronidazole was also investigated. The
lowest concentrations of the studied compounds that
resulted in visual inhibition of the microorganism
growth (MIC values) are listed in Table 3.
chemical shifts, multiplicities and by using 2D
1
homonuclear
1
H– H correlation spectroscopy
(
COSY). The carbon type (C, CH) was determined
by using distortionless enhancement by polarization
transfer (DEPT-135) experiments and the assignments
were made by 2D heteronuclear multiple quantum
coherence (HMQC) and heteronuclear multiple bond
coherence (HMBC) experiments.
1
In the H NMR spectra of 2 and HL3–HL8 two
The bacterial strains were quite susceptible to the
antimicrobial action of 2 and HL3–HL8. Indeed, low
micromolar MIC values were obtained. In some cases
the compounds under study proved to be more potent
than secnidazole (1) and metronidazole. Although
signals characteristic of the imidazole nucleus were
observed: a singlet near to 2.40 ppm and another near
to 8.00 ppm, corresponding to C1CH3 and H2,
respectively. In addition, a singlet around 2.00 ppm
attributed to C5CH , and a singlet corresponding to the
3
CuCl per se was devoid of activity against the tested
2
methylene H4 near to 5.10 ppm were observed.
Formation of the Schiff bases was evidenced by the
presence of a signal corresponding to N4H in the
bacterial strains, upon coordination to copper(II) the
antibacterial activity significantly increased in several
cases. In fact, MIC = 29.78 lM (HL4) and MIC =
25.36 lM (HL6), while MIC = 3.32 lM (Cb) and
1
3
1
region between 9.08 and 11.01 ppm. In the C{ H}
1
23

Biometals
Fig. 3 Molecular plot of
HL6 showing the labeling
scheme of the non-H atoms
and their displacement
ellipsoids at the 50%
probability level
˚
in the bio-reductive activation of 5-nitroimidazoles
Table 2 Selected bond lengths (A) and angles (°) for HL6
(
Ang et al. 2017; Leitsch et al. 2007).
Bonds
Angles
The formation of the nitro radical anion is pre-
C6–S
1.690 (2)
1.317 (3)
1.348 (3)
1.387 (2)
1.276 (3)
1.490 (3)
1.507 (3)
1.460 (3)
N5–C6–N4
N5–C6–S
118.2 (2)
sumed to trigger a series of reduction steps, leading to
the production of nitroso, hydroxylamine and amine
species, thought to contribute to the pharmacological
action of nitroimidazole derivatives (Fig. 4). Although
the metabolites can form adducts with different
biomolecules, including DNA, proteins and lipids,
the exact molecular target of action of nitroimidazole
compounds has not so far been completely elucidated
N5–C6
C6–N4
N4–N3
N3–C5
C7–C5
C5–C4
C4–N2
122.68 (17)
119.13 (16)
118.41 (17)
116.69 (18)
127.4 (2)
N4–C6–S
C6–N4–N3
C5–N3–N4
N3–C5–C7
N3–C5–C4
C7–C5–C4
N2–C4–C5
117.49 (19)
115.07 (19)
113.72 (18)
(Ang et al. 2017; Miyamoto et al. 2013).
In aerobic conditions, the bio-reductive activation
of 5-nitroimidazoles becomes reversible and the
radical anion is reoxidized, in a process resembling a
‘
‘futile’’ cycle which results in decreased biological
effects of the nitroimidazoles (Ang et al. 2017).
Indeed, 5-nitroimidazole derivatives have
MIC = 7.80 lM (Cd) against Bacteroides fragilis.
Similarly, MIC = 12.68 lM (HL6), while MIC =
3
.90 lM (Cd) against Bacteroides vulgatus.
E8 & - 0.47 V versus normal hydrogen electrode
in water at pH 7, significantly lower than the one-
electron reduction potential exhibited by molecular
oxygen, E8 & - 0.15 V, which explains why effec-
tive doses of activated 5-nitroimidazole are only
achieved in environments with low oxygen concen-
tration, as it occurs in some microaerophilic or
anaerobic microorganisms (Kizaka-Kondoh and
Konse-Nagasawa 2009).
Electrochemistry studies on the reduction
of the nitro group
5
-Nitroimidazoles are prodrugs that require bioacti-
vation by reduction of the nitro group in the intracel-
lular environment in order to exert their biological
effect. Electron donors, including ferredoxins and
reductase enzymes have been suggested to participate
123

Biometals
Table 3 Minimum inhibitory concentration (MIC) values for
the 5-nitroimidazole-derived compounds, their copper(II)
Bacteroides vulgatus (ATCC 8482), Bacteroides ovatus
(ATCC 8483), Parabacteroides distasonis (ATCC 1295) and
Fusubacterium nucleatum (ATCC 25586)
complexes and CuCl
2
2
against Bacteroides fragilis (ATCC
5285), Bacteroides thetaiotaomicron (ATCC 29741),
Compound
MIC (lM)
Bacteroides
fragilis
Bacteroides
thetaiotaomicron
Bacteroides
vulgatus
Bacteroides
ovatus
Parabacteroides
distasonis
Fusubacterium
nucleatum
1
2.64
5.46
5.27
5.46
2.64
2.73
5.27
5.46
5.27
43.68
14.18
C 13.26
7.45
2.64
–
2
HL3
Ca
14.18
3.32
14.18
6.64
7.09
7.09
4.16
3.32
8.36
3.32
6.64
6.64
3.90
1.95
3.24
3.70
12.03
6.02
187.77
1.46
3.32
3.32
HL4
Cb
29.78
3.32
7.45
3.72
3.72
6.64
3.32
3.32
C 13.27
13.28
13.28
6.34
HL5
Cc
13.28
13.28
25.36
7.80
13.28
13.28
25.36
7.80
13.28
13.28
12.68
3.90
6.64
6.64
HL6
Cd
3.17
3.90
C 31.22
6.47
HL7
Ce
3.24
6.47
3.24
3.24
3.70
3.70
3.70
1.85
7.40
HL8
Cf
4.84
4.84
4.84
4.84
2.42
C 6.02
C 187.77
3.17
C 6.02
C 187.77
3.17
6.02
3.01
C 6.02
187.77
6.34
CuCl
2
C 187.77
3.17
C 187.77
3.17
Metronidazole
Fig. 4 Bio-reductive
processes of nitroimidazole
compounds
Taking into consideration that the biological assays
were performed in anaerobic conditions, electrochem-
istry studies on the reduction of the nitro group of the
stable only at pH [ 7 or by adding an aprotic co-
solvent (Carbajo et al. 2002). Therefore, in order to
achieve the best experimental conditions that ensure
the nitro radical anion stability, a medium formed by
DMF and tetrabutylammonium perchlorate (TBAP) as
supporting electrolyte was employed in the cyclic
voltammetry studies.
nitroimidazole derivatives were performed. The redox
ÁÀ
ꢀ
potentials E8 (RNO2 R-NO ) of nitroimidazole
2
derivatives and of complexes (Ca–Cf) were deter-
mined aiming to evaluate the ability of the compounds
to be reduced in anaerobic media. The complete set of
cyclic voltammograms are shown in Supplementary
Information (SI).
Secnidazole (1), 2 and (HL3–HL8) displayed a
similar electrochemical reduction process, typical of
nitroimidazoles in aprotic medium (Cavalcanti et al.
2
004). As shown in Fig. 5, in general the compounds
Using the non-usual pulse radiolysis technique, the
ÁÀ
ꢀ
exhibited a stable well-defined couple (system Ic/Ia)
with EpIc near -1100 mV, corresponding to a mono-
electronic transfer process attributed to the formation,
monoelectronic transfer process RNO2 R-NO2 can
be observed and measured in aqueous solution.
However, usual electrochemical techniques are not
very useful due to the limited stability of the produced
in the timescale of the experiment, of a stable nitro
ÁÀ
ÁÀ
radical anion (R-NO ).
R-NO radical. This radical may be sufficiently
2
2
1
23

Biometals
Fig. 5 Cyclic voltammogram of (a) secnidazole (1), (b) HL3 and (c) complex (Ca) in aprotic media (TBAP 0.1 M in DMF) versus Ag/
ꢀ
-
AgCl, Cl (3 M); scan rate of 250 mV s . Dotted line shows a short sweep with the isolated RNO2 R-NO2 couple
-1
ÁÀ
The voltammograms also exhibited a second wave
near - 2000 mV, assigned to the generation of
hydroxylamine (R-NHOH) species and, in some cases,
an irreversible oxidation near to - 500 mV, related to
formation of nitroso (R-NO) species was observed.
The electrochemical processes related to the nitroim-
idazole moiety of the structures are represented by
Eqs. 1–3. The remaining processes in HL3–HL8 are
related to the Schiff base moiety (Table 4).
DE (Ep - Ep ) (Table 4) (Brett and Brett 1993).
I
Ia
Ic
Ip /Ip values higher than 0.82 in most of the cases
Ia Ic
and DE values for the first wave in the 74–181 mV
range at 250 mV s suggest a behavior typical of
quasi-reversible systems.
-
1
The voltammograms of the copper(II) complexes
(Fig. 5c) show two quasi-reversible processes which
ÁÀ
ꢀ
were attributed to the RNO2 R-NO (peaks Ic and Ia)
I
2
II
and Cu /Cu (peaks IVc and IVa) couples [see
Eq. (4)].
À
ÁÀ
2
Peaks Ic and Ia
R-NO þ e ꢀ R-NO
ð1Þ
ð2Þ
ð3Þ
2
Â
Ã
As shown in Table 4, the obtained Ep values for
Â
ÃÀ1
II
À
I
Cu ðHLÞ Cl2 þ e ! Cu ðHLÞ Cl2
x
x
Peak IIc
ð4Þ
x ¼ 1 or 2
ÁÀ
À
þ
R-NO þ 3e þ 4H ! R-NHOH þ H O
2
2
Ic
À
þ
Peak IIIa
R-NHOH ꢀ R-NO þ 2e þ 2H
the intermediate ketone (2) and the novel nitroimida-
zole-derivatives (HL3–HL8) show that the electro-
chemical behaviors of the compounds within the nitro-
imidazole moiety are very similar, meaning a similar
The characterization of the voltammetric first wave
was performed by using the parameters Ip /Ip and
Ia Ic
123

Biometals
ꢀ
Table 4 Cyclic
voltammetry parameters
corresponding to the
b
ÁÀ
II
Cu /Cu
I
Compounds
RNO2 R-NO2
EpIc (mV)
a
DE (mV)
I
Ip /Ip
Ia Ic
EpIc (mV)
DE (mV)
I
Ip /Ip
Ia Ic
ꢀ
ÁÀ
II
RNO2 R-NO and Cu /
2
I
Cu couples for 1, 2, HL3–
1
- 1090
- 1105
- 1135
- 1033
- 1101
- 985
146
87
0.83
0.93
0.87
0.89
0.87
0.95
0.82
0.50
0.83
0.75
0.92
0.76
0.85
0.58
–
–
–
HL8 and complexes Ca–Cf
-
2
–
–
–
versus Ag/AgCl, Cl
(
HL3
Ca
181
74
–
–
–
3.0 M) reference electrode
515
–
152
–
1.35
–
HL4
Cb
122
78
478
–
185
–
1.36
–
HL5
Cc
- 1115
- 880
78
89
622
–
152
–
1.32
–
HL6
Cd
- 1120
- 1025
- 1125
- 1000
- 1130
- 994
83
98
435
–
225
–
0.94
–
a
-1
HL7
Ce
141
97
Scan rate of 250 mV s
;
b
560
–
129
–
1.31
–
compounds at 1 mM
dissolved in DMF using
.1 M of TBAP as
supporting electrolyte
HL8
Cf
98
0
83
466
190
0.90
tendency for nitro radical anion formation. Upon
coordination to copper(II) the EpIc values become
more favorable, suggesting a higher tendency to the
formation of the nitro anion radical in the complexes.
Furthermore, the EpIc values of complexes (Ca–Cf)
did not differ much from those of the well-known
enzymatic reducible drugs secnidazole (compound 1,
MIC values of the compounds under study are
comparable to each other and to those of secnidazole
and metronidazole.
Upon coordination to copper(II) the antibacterial
activity increased in several cases. Interestingly, the
reduction potentials of the nitro group were in general
more favorable for the complexes than for the starting
ligands, suggesting that this might be one of the
reasons for their improved antimicrobial action.
Previous works by other authors indicated that
Schiff base derivatives of metronidazole (Zhang et al.
2014) and secnidazole (Li et al. 2013) present
antibacterial effects against Gram-positive and
Gram-negative aerobic bacteria. The novel derivatives
investigated in the present work showed potent
activity against Gram-negative pathogenic anaerobic
bacterial strains. Therefore, we may consider these
compounds suitable for in vivo evaluation and the
microorganisms should be classified as susceptible to
them.
Ep = - 1076 mV) and benznidazole (EpIc-
Ic
=
- 1019 mV) (Bollo et al. 2001).
Conclusions
In conclusion, compounds HL3–HL8 exhibited
in vitro antibacterial activity against the six reference
strains evaluated in this study. In some cases, the
compounds were even more active than secnidazole
and metronidazole, presenting lower minimum inhi-
bitory concentrations than those of the reference
drugs.
Since the compounds under study showed reduction
potentials in the same range as the bio-reducible drugs
secnidazole and benznidazole, their biological activ-
ities might also be related to intracellular bio-reduc-
tion activation.
The literature reports decreased clinical suscepti-
bility to metronidazole and other antimicrobials
among Bacteroides species (L o¨ fmark et al. 2010;
Liu et al. 2008). Considering the increasing resistance
to antimicrobial agents among anaerobic pathogens,
the present work constitutes an important contribution
to the development of new antibacterial drug
candidates.
Although according to Lipinki’s rules, an absolute
correlation of a single parameter with the biological
activity is not expected, the similar electrochemical
behaviors at least partially explain the reason why the
1
23

Biometals
Acknowledgements The authors gratefully acknowledge
CNPq, CAPES-PNPD, FAPEMIG and INCT-INOFAR (Proc.
CNPq 573.364/2008-6) for financial support and student grants.
Funding was provided by CNPq (Grant Numbers 421902/2016-
Corey EJ, Schmidt G (1979) Useful procedures for the oxidation
of alcohols involving pyridinium dichromate in aprotic
media. Tetrahedron Lett 5:399–402
De Backer E, Dubreuil L, Brauman M, Acar J, Vaneechoutte M
(2010) In vitro activity of secnidazole against Atopobium
vaginae, an anaerobic pathogen involved in bacterial
vaginosis. Clin Microbiol Infect 16:470–472
7
and 303746/2013-0).
Divar M, Khalafi-Nezhad A, Zomorodian K, Sabet R, Faghih Z,
Jamali M, Pournaghz H, Khabnadideh S (2017) Synthesis
of some novel semicarbazone and thiosemicarbazone
derivatives of isatin as possible biologically active agents.
J Pharm Res Int 17:1–13
Dunn LA, Burgess AG, Krauer KG, Erkmann L, Vanelle P,
Crozet MD, Gillin FD, Upcroft P, Upcroft JA (2010) A
new-generation 5-nitroimidazole can induce highly
metronidazole-resistant Giardia lamblia in vitro. Int J
Antimicrob Agents 36:37–42
References
Abdelrahman MA, Salama I, Gomaa MS, Elaasser MM, Abdel-
Aziz MM, Soliman DH (2017) Design, synthesis and 2D
QSAR study of novel pyridine and quinolone hydrazone
derivatives as potential antimicrobial and antitubercular
agents. Eur J Med Chem 138:698–714
Akhi MT, Ghotaslou R, Beheshtirouy S, Asgharzadeh M, Pir-
zadeh T, Asghari B, Alizadeh N, Ostadgavahi AT, Some-
saraei VS, Memar MY (2015) Antibiotic susceptibility
pattern of aerobic and anaerobic bacteria isolated from
surgical site infection of hospitalized patients. Jundishapur
J Microbiol 8:e20309
Allen FH, Kennard O, Watson DG, Brammer L, Orpen AG,
Taylor R (1987) Tables of bond lengths determined by
X-ray and neutron diffraction. Part 1. Bond lengths in
organic compounds. J Chem Soc Perkin Trans 2:S1–S19
Anderson RJ, Groundwater PW, Todd A, Worsley AJ (2012)
Nitroimidazole antibacterial agents. Antibacterial agents:
chemistry, mode of action, mechanisms of resistance and
clinical applications. Wiley, Chichester, pp 85–101
Ang CW, Jarrad AM, Cooper MA, Blaskovich MAT (2017)
Nitroimidazoles—molecular fireworks that combat a broad
Ehrlich J, Bartz QR, Smith RM, Joslyn DA, Burkholder PR
(1947) Chloromycetin, a new antibiotic from a soil acti-
nomycete. Science 106:417
Ferreira IP, Pil o´ EDL, Recio-Despaigne AA, Da Silva JG,
Ramos JP, Marques LB, Prazeres PHDM, Takahashi JA,
Souza-Fagundes EM, Rocha W, Beraldo H (2016) Bis-
muth(III) complexes with 2-acetylpyridine- and 2-ben-
zoylpyridine-derived hydrazones: antimicrobial and
cytotoxic activities and effects on the clonogenic survival
of human solid tumor cells. Bioorg Med Chem
24:2988–2998
Gillis JC, Wiseman LR (1996) Secnidazole. Drugs 51:621–638
Hastey CJ, Boyd H, Schuetz AN, Anderson K, Citron DM,
Dzink-Fox J, Hackel M, Hecht DW, Jacobus NV, Jenkins
SG, Karlsson M, Knapp CC, Koeth LM, Wexler H, Roe-
Carpenter DE (2016) Changes in the antibiotic suscepti-
bility of anaerobic bacteria from 2007-2009 to 2010-2012
based on the CLSI methodology. Anaerobe 42:27–30
Kapoor VK, Chadha R, Venisetty PK, Prasanth S (2003)
Medicinal significance of nitroimidazoles—some recent
advance. J Sci Ind Res 62:659–665
spectrum of infectious diseases.
0:7636–7657
J
Med Chem
6
Bollo S, N u´ n˜ ez-Vergara LJ, Bont a´ M, Chauviere G, P e´ ri e´ J,
Squella JA (2001) Cyclic voltammetric studies on nitro
radical anion formation from megazol and some related
nitroimidazole derivatives. J Electroanal Chem 511:46–54
Brett CMA, Brett AMO (1993) Electrochemistry—principles,
methods and applications, 1st edn. Oxford University Press
Inc., New York
Breuning M, H a¨ user T, Mehler C, D a¨ schlein C, Strohmann C,
Oechsner A, Braunschweig H (2009) Enantioselective
synthesis of tricyclic amino acid derivatives based on a
Kizaka-Kondoh S, Konse-Nagasawa H (2009) Significance of
nitroimidazole compounds and hypoxia-inducible factor-1
for imaging tumor hypoxia. Cancer Sci 100:1366–1373
Leitsch D, Kolarich D, Wilson IBH, Altmann F, Duch eˆ ne M
(2007) Nitroimidazole action in Entamoeba histolytica: a
central role for thioredoxin reductase. PLoS Biol
5:1820–1834
2
,6
rigid 4-azatricyclo[5.2.1.0 ]decane skeleton. Beilstein J
Org Chem 5:81
Brook I (2016) Spectrum and treatment of anaerobic infections.
J Infect Chemother 22:1–13
Carbajo J, Bollo S, N u´ n˜ ez-Vergara LJ, Campero A, Squella JA
Li Y, Zhao CP, Ma HP, Zhao MY, Xue YR, Wang XM, Zhu HL
(2013) Design, synthesis and antimicrobial activities
evaluation of Schiff base derived from secnidazole
derivatives as potential FabH inhibitors. Bioorg Med Chem
21:3120–3126
Liu CY, Huang YT, Liao CH, Yen LC, Lin HY, Hsueh PR
(2008) Increasing trends in antimicrobial resistance among
clinically important anaerobes and bacteroides fragilis
isolates causing nosocomial infections: emerging resis-
tance to carbapenems. Antimicrob Agents Chemother
52:3161–3168
L o¨ fmark S, Edlund C, Nord CE (2010) Metronidazole is still the
drug of choice for treatment of anaerobic infections. Clin
Infect Dis 50:S16–S23
Mendes IC, Teixeira LR, Lima R, Beraldo H, Speziali NL, West
DX (2001) Structural and spectral studies of
(2002) Cyclic voltammetric study of the disproportionation
reaction of the nitro radical anion from 4-nitroimidazole in
protic media. J Electroanal Chem 53:187–194
Cavalcanti JCM, Abreu FC, Oliveira NV, Moura MABF,
Chaves JG, Alves RJ, Bertinaria M, Fruttero R, Goulart
MOF (2004) Effect of the leaving group on the electrodic
reduction mechanism of anti-Helicobacter pylori metron-
idazole derivatives, in aprotic and protic media. Bioelec-
trochemistry 63:353–357
Clinical and Laboratory Standards Institute (CLSI) (2015)
Performance standards for antimicrobial susceptibility
testing; twenty-fifth informational supplement. CLSI doc-
ument M100-S25, Pennsylvania, USA
123

Biometals
thiosemicarbazones derived from 3- and 4-formylpyridine
and 3- and 4-acetylpyridine. J Mol Struct 559:355–360
Mendes IC, Moreira JP, Mangrich AS, Balena SP, Rodrigues
BL, Beraldo H (2007) Coordination to copper(II) strongly
enhances the in vitro antimicrobial activity of pyridine-
derived N(4)-tolyl thiosemicarbazones. Polyhedron
Recio-Despaigne AA, da Costa FB, Piro OE, Castellano EE,
Louro SRW, Beraldo H (2012) Complexation of 2-acet-
ylpyridine- and 2-benzoylpyridine-derived hydrazones to
copper(II) as an effective strategy for antimicrobial activity
improvement. Polyhedron 38:285–290
Rodrigues C, Batista AA, Ellena J, Castellano EE, Ben ´ı tez D,
Hugo Cerecetto, Gonz a´ lez M, Teixeira LR, Beraldo H
(2010) Coordination of nitro-thiosemicarbazones to
ruthenium(II) as a strategy for anti-trypanosomal activity
improvement. Eur J Med Chem 45:2847–2853
Sheldrick GM (2013) SHELXS-2013/1: program for crystal
structure solution. University of G o¨ ttingen, G o¨ ttingen
Sheldrick GM (2014) SHELXL-2014/7: program for the solu-
tion of crystal structures. University of G o¨ ttingen,
G o¨ ttingen
Sheldrick GM (2015) Crystal structure refinement with
SHELXL. Acta Cryst C71:3–8
Spek AL (2009) Structure validation in chemical crystallogra-
phy. Acta Crystallogr Sect D D65:148–155
Tan TY, Ng LS, Kwang LL, Rao S, Eng LC (2017) Clinical
characteristics and antimicrobial susceptibilities of anaer-
obic bacteremia in an acute care hospital. Anaerobe
43:69–74
2
6:3263–3270
Miyamoto Y, Kalisiak J, Korthals K, Lauwaet T, Cheung DY,
Lozano R, Cobo ER, Upcroft P, Upcroft JA, Berg DE,
Gillin FD, Fokin VV, Sharpless KB, Eckmann L (2013)
Expanded therapeutic potential in activity space of next-
generation 5-nitroimidazole antimicrobials with broad
structural diversity. Proc Nat Acad Sci 110:17564–17569
Nagata N, Marriott D, Harkness J, Ellis JT, Stark D (2012)
Current treatment options for Dientamoeba fragilis infec-
tions. Int. J Parasitol Drugs Drug Resist 2:204–215
O’Shea IP, Shahed M, Aguilera-Venegas B, Wilkinson SR
(2016) Evaluating 5-nitrothiazoles as trypanocidal agents.
Antimicrob Agents Chemother 60:1137–1140
Oxford Diffraction (2010) CrysAlisPro CCD and CrysAlisPro
RED: Version 1.171.35.21 (Release 20-01-2012 CrysA-
lis171.NET), Oxford Diffraction Ltd, Yarnton, Oxford-
shire, England
Patterson S, Wyllie S (2014) Nitro drugs for the treatment of
trypanosomatid diseases: past, present, and future pro-
spects. Trends Parasitol 30:289–298
WHO Model Lists of Essential Medicines (2018) http://www.
who.int/medicines/publications/essentialmedicines/en/.
Accessed 28 Jan 2018
Popiolek L (2017) Hydrazide–hydrazones as potential antimi-
crobial agents: overview of the literature since 2010. Med
Chem Res 26:287–301
Wright PM, Seiple IB, Myers AG (2014) The evolving role of
chemical synthesis in antibacterial drug discovery. Angew
Chem Int Ed Engl 53:8840–8869
Recio-Despaigne AA, Vieira LF, Mendes IC, da Costa FB,
Speziali NL, Beraldo H (2010) Organotin(IV) complexes
with 2-acetylpyridine benzoyl hydrazones: antimicrobial
activity. J Braz Chem Soc 21(7):1247–1257
Zhang X, Sangani CB, Jia LX, Gong PX, Wang F, Wang JF, Zhu
HL (2014) Synthesis and antibacterial evaluation of novel
Schiff’s base derivatives of nitroimidazole nuclei as potent
E. coli FabH inhibitors. RSC Adv 4:54217–54225
1
23