Biometals (2018) 31:571–584

https://doi.org/10.1007/s10534-018-0106-6 (0123456789().,-volV)
(0123456789().,-volV)

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 .
João F. G. Ferreira . Sofia M. P. S. Bachi . Nivaldo L. Speziali .
Luiz M. Farias . Paula P. Magalhães . Heloisa Beraldo

Received: 27 February 2018 / Accepted: 23 April 2018 / Published online: 7 May 2018
Ó Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract In the present work a family of novel the compounds under study are suitable for in vivo
secnidazole-derived Schiff base compounds and their evaluation and the microorganisms should be classi-
copper(II) complexes were synthesized. The antimi- fied as susceptible to them. Electrochemical studies on
crobial activities of the compounds were evaluated the reduction of the nitro group revealed that the
against clinically important anaerobic bacterial compounds show comparable reduction potentials,
strains. The compounds exhibited in vitro antibacterial which are in the same range of the bio-reducible drugs
activity against Bacteroides fragilis, Bacteroides secnidazole and benznidazole. The nitro group reduc-
thetaiotaomicron, Bacteroides vulgatus, Bacteroides tion potential is more favorable for the copper(II)
ovatus, Parabacteroides distasonis and Fusubac- complexes than for the starting ligands. Hence, the
terium nucleatum pathogenic anaerobic bacteria. antimicrobial activities of the compounds under study
Upon coordination to copper(II) the antibacterial might in part be related to intracellular bio-reduction
activity significantly increased in several cases. Some activation. Considering the increasing resistance rates
derivatives were even more active than the antimicro- of anaerobic bacteria against a wide range of antimi-
bial drugs secnidazole and metronidazole. Therefore, 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 Keywords 5-Nitroimidazole  Copper(II)
users. complexes  Schiff bases  Anaerobic bacteria 
Antimicrobial activity  Bio-reduction
A. A. Oliveira  A. P. A. Oliveira  L. L. Franco 
M. O. Ferencs  S. M. P. S. Bachi  H. Beraldo (&)
Departamento de Quı́mica, Universidade Federal de
Minas Gerais, Belo Horizonte, MG 31270-901, Brazil
e-mail: hberaldo@ufmg.br; Introduction
heloisaberaldoufmg@gmail.com

J. F. G. Ferreira  L. M. Farias  P. P. Magalhães The importance of nitro compounds in medicinal

Departamento de Microbiologia, Universidade Federal de chemistry emerged with the discovery of chloram-
Minas Gerais, Belo Horizonte, MG 31270-901, Brazil phenicol, an antibacterial agent containing a nitro-
N. L. Speziali
phenyl group, first isolated in 1947 from a culture of
Departamento de Fı́sica, Universidade Federal de Minas Streptomyces venezuelae (Ehrlich et al. 1947). Since
Gerais, Belo Horizonte, MG 31270-901, Brazil

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then, nitro-heterocyclic compounds have been exten- half-life, which allows the administration of lower
sively investigated as antimicrobial agents (Patterson doses and makes single-dose applications possible
and Wyllie 2014; O’Shea et al. 2016). (Gillis and Wiseman 1996; De Backer et al. 2010; Ang
2-Nitroimidazole (azomycin, Fig. 1a), isolated in et al. 2017).
1953 from the bacterium Nocardia mesenterica, was Anaerobic bacteria are the most predominant
found to be active against Trichomonas vaginalis, the components of the human indigenous microbiota and
causative parasite of trichomoniasis (Anderson et al. the most frequent cause of endogenous bacterial
2012). After this finding, the synthesis and biological infections. Anaerobic infections are often considered
evaluation of nitroimidazole derivatives led to the as life threatening and may occur at the central
discovery of metronidazole in 1957 (Fig. 1b), a potent nervous system, oral cavity, head and neck, chest,
antiprotozoal agent with greater activity than azomy- abdomen, pelvis, skin and soft tissues. The most
cin and acceptable toxicity (Anderson et al. 2012; relevant anaerobes associated with the etiopathogen-
Wright et al. 2014). esis of approximately 95% of anaerobic infections are
In 1960 metronidazole became the first drug to be Gram-negative rods, among them Bacteroides, Pre-
introduced into human therapy for the treatment of votella, Porphyromonas, Fusobacterium, Bilophila
trichomoniasis and later, for treating infections caused and Sutterella (Brook 2016).
by a variety of anaerobic bacteria (Wright et al. 2014). The polymicrobial etiology of anaerobic infections,
Metronidazole is classified as an essential medicine by mainly an association between anaerobes and facul-
the World Health Organization (WHO 2018). It is tative bacteria, results in difficulties in treating these
widely used for the treatment of infections caused by infections (Brook 2016). Another obstacle for the
Giardia lamblia, Trichomonas vaginalis, Entamoeba effective treatment is the increasing resistance rates of
histolytica and Blastocystis sp. protozoa, as well as anaerobic bacteria against a wide range of antimicro-
against Clostridium difficile and Bacteroides fragilis bial agents, including metronidazole, frequently
anaerobic bacteria and the microaerophilic species employed in clinics (Akhi et al. 2015; Tan et al.
Helicobacter pylori (Dunn et al. 2010). 2017; Hastey et al. 2016).
Afterwards, different derivatives of nitroimidazoles 5-Nitroimidazoles are prodrugs that require bioac-
were synthetized and a second-generation of 5-ni- tivation of the nitro group in the intracellular envi-
troimidazole antimicrobial agents has been developed, ronment in order to exert their biological effect. After
including secnidazole (Fig. 1c), tinidazole (Fig. 1d) these compounds enter the cells the nitro group is
and ornidazole (Fig. 1e) (Nagata et al. 2012; Kapoor reduced to a short-lived nitro anion radical (R-NO 2 ),
et al. 2003). the critical step in the activation of nitroimidazoles
Secnidazole proved to be a very promising alter- (Ang et al. 2017).
native for metronidazole due to its broad spectrum of While the nitro group is crucial for the activity of
activity and potency against anaerobic microorgan- 5-nitroimidazoles against anaerobic bacteria, different
isms. Secnidazole presents appropriate pharmacoki- substituents in the imidazole side chains may result in
netics, being rapidly and completely absorbed after different spectra of activity and pharmacokinetic
oral administration. It has a long terminal elimination properties.

Fig. 1 Chemical structures of nitroimidazole derivatives a 2-nitroimidazole, b metronidazole, c secnidazole, d tinidazole and
e ornidazole

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Biometals (2018) 31:571–584 573

Hydrazones and thiosemicarbazones are Schiff Materials and methods

base compounds which show potent antimicrobial
activity (Divar et al. 2017; Abdelrahman et al. 2017; Equipment and reagents
Popiolek 2017). In several cases the antimicrobial
activity of these compounds increases upon metal All reagents were purchased from Aldrich and Merck
coordination (Ferreira et al. 2016). In particular, and used as received. Partial elemental analyses were
complexation to copper(II) often results in antibacte- performed on a Perkin Elmer CHN 2400 analyzer.
rial activity enhancement (Recio-Despaigne et al. Melting points were determined with a Mettler
2012; Mendes et al. 2007). MQAPF-302 apparatus. Magnetic susceptibility mea-
In this way, in the present work novel secnidazole- surements were performed with a Johnson Matthey
derived compounds functionalized with different MSB/AUTO balance. An YSI model 31 conductivity
Schiff base groups were obtained (Fig. 2) as well as bridge was employed for molar conductivity mea-
their copper(II) complexes. The antimicrobial effects surements. Infrared spectra were recorded on a Perkin
of the compounds were evaluated against Bacteroides Elmer Spectrum One employing the attenuated total
fragilis, Bacteroides thetaiotaomicron, Bacteroides reflectance (ATR) method (4000–400 cm-1). NMR
vulgatus, Bacteroides ovatus, Parabacteroides dista- spectra were obtained at 25 °C with a Bruker DPX-
sonis and Fusubacterium nucleatum pathogenic anaer- 400 Advance (400 MHz) spectrometer using DMSO-
obic bacteria. Electrochemistry studies on the d6 as solvent. Mass spectra were recorded with a
reduction of the nitro group were performed as well, Shimadzu LCMS-IT-TOF instrument working at
aiming to investigate the ability of the compounds to high-resolution.
undergo bio-reduction activation.

Fig. 2 Syntheses of
secnidazole-derived
compounds 2, (HL3–HL8)
and copper(II) complexes
(Ca–Cf). Reagents and
conditions: (a) PDC,
CH2Cl2, r.t. 48 h; (b) HL3–
HL5: NH2NHCOR, AcOH,
MeOH, reflux, 4 h; HL6–
HL8: NH2NH2CSNHR,
AcOH, MeOH, reflux, 4 h;
(c, d) CuCl2, MeOH, reflux,
8h

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Electrochemical studies magnetic susceptibilities and by means of their

infrared spectra. All data that support the chemical
Cyclic voltammetry was carried out at room temper- characterization of the compounds are reported in
ature with a conventional three-electrode cell (with Supplementary Information (SI).
volumetric capacity of 10 mL) in a lAutolab type II 1-(2-Methyl-5-nitro-1H-imidazol-1-yl)propan-2-one
potentiostat (Echo Chemie, Utrecht, the Netherlands) (2) White solid. Yield: 77% (oxidant: PCC) and 84%
using the GPES 4.9 software. The working electrode (oxidant: PDC). Anal. Calc. for C7H9N3O3 (%): C,
was a Metrohm glassy carbon electrode, the auxiliary 45.90; H, 4.95; N, 22.94. Found: C, 46.22; H, 4.83; N,
electrode was a platinum wire and Ag/AgCl, Cl- 22.96. FW: 183.17 g mol-1. IR (ATR, cm-1): 3110 (m
(3.0 M) was used as the reference electrode. The C2-H), 1726 (m C=O), 1529 (m C=N), 1456/1361 (mass/ms
glassy carbon electrode was previously fine-polished NO2). 1H NMR [400.13 MHz, DMSO-d6, d(ppm)]:
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, C1CH3],
5 min. Solutions for analysis were prepared in spec- 2.27 [s, 3H, C5CH3]. 13C{1H} NMR [100.61 MHz,
troscopic dimethylformamide (DMF) containing DMSO-d6, d(ppm)]: 200.8 [C5], 151.3 [C1], 138.3
1 mM of analyte and 0.1 M of tetrabutylammonium [C3], 132.3 [C2], 54.8 [C4], 27.0 [C5CH3)], 13.5
perchlorate (TBAP) as supporting electrolyte. Before [C1CH3]. HRMS [ESI(?), IT-TOF] calculated for
recording the voltammograms, the test solution was C7H10N3O3, [M ? H]?: 184.0722, found: 184.0722.
thoroughly purged with high purity nitrogen gas to (E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
remove any traces of dissolved oxygen. Cyclic pan-2-ylidene)hydrazinecarboxamide (HL3) White-
voltammograms were recorded in the 1.50 to yellowish solid. Yield: 45%. Melting point:
- 2.50 V potential range using 250 mV s-1 scan rate. 211.8–213.2 °C. Anal. Calc. for C8H12N6O3 (%):
C, 40.00; H, 5.04; N, 34.98. Found: C, 39.75; H, 4.64;
Syntheses of the 5-nitroimidazole-derived Schiff N, 34.08. FW: 240.22 g mol-1. IR (ATR, cm-1):
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
Compounds (HL3–HL8) were synthetized from the NO2). 1H NMR [400.13 MHz, DMSO-d6, d(ppm)]:
intermediate (2), prepared by oxidation of secnidazole 9.39 [s, 1H, N4H], 8.40 [s, 1H, H2], 6.13 [sl, 2H,
(1) with pyridinium chlorochromate (PCC) or pyri- N5H], 5.13 [s, 2H, H4], 2.50 [s, 3H, C1CH3], 1.92 [s,
dinium dichromate (PDC), according to procedures 3H, C5CH3]. 13C{1H} NMR [100.61 MHz, DMSO-
described in the literature (Fig. 2) (Corey and Schmidt d6, d(ppm)]: 156.8 [C6], 150.7 [C1], 142.3 [C5],
1979; Breuning et al. 2009). HL3–HL8 were obtained 138.3 [C3], 128.8 [C2], 50.3 [C4], 14.5 [C5CH3)],
by stirring under reflux a methanol solution (5 mL) of 12.8 [C1CH3]. HRMS [ESI(?), IT-TOF] calculated
2 with an equimolar amount of the desired hydrazine for C8H12N6NaO3, [M ? Na]?: 263.0869, found:
derivative (0.55 mmol) for 4 h, with addition of few 263.0885.
drops of acetic acid as catalyst. After cooling to room (E)-N0 -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
temperature, 10 mL of ethyl ether and 10 mL of pan-2-ylidene)acetohydrazide (HL4) White-yellow-
isopropanol were added and the obtained precipitate ish solid. Yield: 81%. Melting point: 165.4–167.8 °C.
was filtered off, washed with ethyl ether and dried in a Anal. Calc. for C9H13N5O3 (%): C, 45.18; H, 5.48; N,
desiccator under reduced pressure. 29.27. Found: C, 44,10; H, 5,19; N, 28,34. FW:
Copper(II) complexes (Ca–Cf) were obtained by 239.24 g mol-1. IR (ATR, cm-1): 3264 (m N4-H),
reacting equimolar amounts (0.5 mmol) of the desired 3142 (m C2-H), 1674 (m C=O), 1653 (m C=C), 1530 (m
thiosemicarbazone or hydrazone with CuCl22H2O in C=N), 1471/1362 (mass/ms NO2). 1H NMR
methanol (10 mL) under reflux for 8 h. The resulting [400.13 MHz, DMSO-d6, d(ppm)]: 10.32 [s, 1H,
solids were filtered off and washed with methanol and N4H], 8.04 [s, 1H, H2], 5.10 [s, 2H, H4], 2.37 [s,
ethyl ether, and dried under reduced pressure. 3H, C1CH3], 1.93 [s, 3H, C5CH3], 1.66 [s, 3H, H7].
13
The Schiff base ligands were characterized by C{1H} NMR [100.61 MHz, DMSO-d6, d(ppm)]:
elemental analysis and by means of their infrared, 172.2 [C6], 151.5 [C1], 144.9 [C5], 138.6 [C3], 132.4
NMR and HRMS spectra. Complexes (Ca–Cf) were [C2], 49.9 [C4], 19.9 [C7], 14.4 [C5CH3)], 13.5
characterized by microanalyses, molar conductivities,

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Biometals (2018) 31:571–584 575

[C1CH3]. HRMS [ESI(?), IT-TOF] calculated for [C5CH3)], 13.8 [C1CH3]. HRMS [ESI(?), IT-TOF]
C9H14N5O3, [M ? H]?: 240.1097, found: 240.1103. calculated for C9H14N6NaO2S, [M ? Na]?: 293.0797,
(E)-N0 -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro- found: 293.0796.
pan-2-ylidene)benzohydrazide (HL5) Yellow solid. (E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
Yield: 66%. Melting point: 205.1–206.2 °C. Anal. pan-2-ylidene)-N-phenylhydrazinecarbothioamide
Calc. for C14H15N5O3 (%): C, 55.81; H, 5.02; N, (HL8) White-yellowish solid. Yield: 84%. Melting
23.24. Found: C, 56.43; H, 4.88; N, 23.49. FW: point: 116.2–118.0 °C. Anal. Calc. for C14H16N6O2S
301.31 g mol-1. IR (ATR, cm-1): 3185 (m N4-H), (%): C, 50.59; H, 4.85; N, 25.28. Found: C, 48.40; H,
3123 (m C2-H), 1670 (m C=O), 1585 (m C=C), 4.92; N, 24.17. FW: 332.38 g mol-1. IR (ATR,
1578/1526 (m C=N), 1461/1355 (mass/ms NO2). 1H cm-1): 3296 (m N4-H), 3138 (m C2-H), 1593 (m
NMR [400.13 MHz, DMSO-d6, d(ppm)]: 10.70 [s, C=C), 1554/1530 (m C=N), 1471/1362 (mass/ms NO2),
1H, N4H], 8.06 [s, 1H, H2], 7.78 [sl, 2H, H8, H12], 772 (m C=S). 1H NMR [400.13 MHz, DMSO-d6,
7.58–7.17 [m, 3H, H9, H10, H11], 5.22 [s, 2H, H4], d(ppm)]: 10.87 [s, 1H, N4H], 8.83 [s, 1H, N5H], 8.15
2.42 [s, 3H, C1CH3], 2.04 [s, 3H, C5CH3]. 13C{1H} [s, 1H, H2], 7.51 [d, 2H, H8, H12], 7.35 [t, 2H, H9,
NMR [100.61 MHz, DMSO-d6, d(ppm)]: 163.1 [C6], H11], 7.16 [t, 1H, H10], 5.23 [s, 2H, H4], 2.43 [s, 3H,
157.0 [C1], 151.7 [C5], 138.5 [C3], 133.7 [C7], 132.7 C1CH3], 2.10 [s, 3H, C5CH3].13C{1H} NMR
[C2], 131.5 [C10], 128.3 [C9, C11], 127.7 [C8, C12], [100.61 MHz, DMSO-d6, d(ppm)]: 175.8 [C6],
50.1 [C4], 15.8 [C5CH3)], 13.7 [C1CH3]. HRMS 152.1 [C1], 148.1 [C5], 138.7 [C3], 138.2 [C7],
[ESI(?), IT-TOF] calculated for C14H15N5NaO3, 132.6 [C2], 128,5 [C9, C11], 125.0 [C10], 122.7 [C8,
[M ? Na]?: 324.1073, found: 324.1072. C12], 50.2 [C4], 15.3 [C5CH3)], 13.7 [C1CH3].
(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan- HRMS [ESI(?), IT-TOF] calculated for C14H16N6-
2-ylidene)hydrazinecarbothioamide (HL6) White solid. NaO2S, [M ? Na]?: 355.0953, found: 355.0940.
Yield: 74%. Melting point: 168.4–171.2 °C. Anal. Calc. Bis[(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)
for C8H12N6O2S (%): C, 37.49; H, 4.72; N, 32.79. propan-2-ylidene)hydrazinecarboxamide)dichloro-
Found: C, 38.26; H, 4.79; N, 31.32. FW: copper(II)], [Cu(HL3)2Cl2] (Ca) Dark blue solid.
256.28 g mol-1. IR (ATR, cm-1): 3170 (m N4-H), Yield: 50%. Melting point: 183.0–184.5 °C. Anal.
3083 (m C2-H), 1644 (m C=C), 1611/1530 (m C=N), Calc. for C16H24Cl2CuN12O6 (%): C, 31.25; H,
1463/1360 (mass/ms NO2), 804 (m C=S). 1H NMR 3.93; N, 27.34. Found: C, 31.36; H, 3.70; N,
[400.13 MHz, DMSO-d6, d(ppm)]: 10.42 [s, 1H, 27.25. FW: 614.89 g mol-1. Molar conductivity
N4H], 8.16 [s, 1H, N5H], 8.07 [s, 1H, H2], 6.41 [s, (DMSO, 1 mM): 18.83 cm2 X-1 mol-1. IR (ATR,
1H, N5H], 5.13 [s, 2H, H4], 2.38 [s, 3H, C1CH3], 2.01 [s, cm-1): 3434 (m N4-H), 1712 (m C=O), 1683
3H, C5CH3]. 13C{1H} NMR [100.61 MHz, DMSO-d6, m(C=C), 1566/1520 m(C=N), 1484/1390 (mass/ms
d(ppm)]: 178.9 [C6], 151.8 [C1], 147.4 [C5], 138.5 [C3], NO2). Magnetic moment: 2.00 lB.
132.6 [C2], 50.1 [C4], 15.1 [C5CH3)], 13.6 [C1CH3]. [(E)-N 0 -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)
HRMS [ESI(?), IT-TOF] calculated for C8H13N6O2S, propan-2-ylidene)acetohydrazidedichlorocopper
[M ? H]?: 257.0820, found: 257.0796. (II)] [Cu(HL4)Cl 2 ]‘CH 3OH (Cb) Green solid.
(E)-N-methyl-2-(1-(2-methyl-5-nitro-1H-imidazol- Yield: 51%. Melting point: decomposition at
1-yl)propan-2-ylidene)hydrazinecarbothioamide (HL7) 195 °C. Anal. Calc. for C 9.5 H 15 Cl 2 CuN 5 O 3.5 (%):
Yellow solid. Yield: 62%. Melting point: 194.2– C, 29.28; H, 3.88; N, 17.97. Found: C, 30.33; H,
196.9 °C. Anal. Calc. for C9H14N6O2S (%): C, 39.99; 3.58; N, 17.76. FW: 389.70 g mol-1 . Molar con-
H, 5.22; N, 31.09. Found: C, 39.86; H, 5.41; N, 30.45. ductivity (DMSO, 1 mM): 28.82 cm 2 X -1 mol -1 .
FW: 270.31 g mol-1. IR (ATR, cm-1): 3183 (m N4-H), IR (ATR, cm -1 ): 3126 (m N4-H), 1736 (m C=O),
1555/1531 (m C=N), 1471/1362 (mass/ms NO2), 786 (m 1634 m(C=C), 1550 m(C=N), 1476/1388 (m ass /m s
C=S). 1H NMR [400.13 MHz, DMSO-d6, d(ppm)]: NO 2). Magnetic moment: 1.60 l B .
10.31 [s, 1H, N4H], 8.07 [s, 1H, H2], 7.24 [s, 1H, N5H], [(E)-N0 -(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro-
5.12 [s, 2H, H4], 2.89 [d, 3H, H7], 2.39 [s, 3H, C1CH3], pan-2-ylidene)benzohydrazidedichlorocopper(II)] [Cu
1.96 [s, 3H, C5CH3]. 13C{1H} NMR [100.61 MHz, (HL5)Cl2] (Cc) Green solid. Yield: 50%. Melting point:
DMSO-d6, d(ppm)]: 178.9 [C6], 151.9 [C1], 147.0 [C5], decomposition at 210 °C. Anal. Calc. for C14H15Cl2-
138.6 [C3], 132.7 [C2], 50.3 [C4], 30.7 [C7], 14.8 CuN5O3 (%): C, 38.59; H, 3.47; N, 16.07. Found: C,

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38.09; H, 3.21; N, 16.39. FW: 435.75 g mol-1. Molar SHELXS-2013/1 (Sheldrick 2013, 2015). Full-matrix
conductivity (DMSO, 1 mM): 33.18 cm2 X-1 mol-1. least-squares refinement procedure on F2 with aniso-
IR (ATR, cm-1): 3126 (m N4-H), 1736 (m C=O), 1585 tropic thermal parameters was carried on using
m(C=C), 1556/1526 m(C=N), 1480/1386 (mass/ms NO2). SHELXL-2014/7 (Sheldrick 2014). Positional and
Magnetic moment: 1.63 lB. anisotropic atomic displacement parameters were
[(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro- refined for all non-hydrogen atoms. Hydrogen atoms
pan-2-ylidene)hydrazinecarbothioamidedichlorocop- were placed geometrically and the positional param-
per(II)] [Cu(HL6)Cl2]‘H2O (Cd) Dark green solid. eters were refined using a riding model.
Yield: 75%. Melting point: decomposition at 160 °C. A suitable single crystal of HL6 was obtained at
Anal. Calc. for C8H13Cl2CuN6O2.5S (%): C, 24.04; H, room temperature from slow evaporation of the
3.28; N, 21.02. Found: C, 23.91; H, 3.20; N, 21.28. mother liquor. A summary of the crystal data, data
FW: 399.74 g mol-1. Molar conductivity (DMSO, collection details and refinement results is listed in
1 mM): 43.57 cm2 X-1 mol-1. IR (ATR, cm-1): 3224 Table 1. Molecular graphics were plotted using
(m N4-H), 788 (m C=S), 1640 m(C=C), 1594/1526 (m PLATON (Spek 2009).
C=N), 1474/1390 (mass/ms NO2). Magnetic moment:
1.60 lB. Antimicrobial activity
[(E)-N-methyl-2-(1-(2-methyl-5-nitro-1H-imidazol-
1-yl)propan-2-ylidene)hydrazinecarbothioamidedichloro- In vitro susceptibility to the compounds under study
copper(II)] [Cu(HL7)Cl2] (Ce) Dark green solid. Yield: was evaluated according to Clinical & Laboratory
65%. Melting point: decomposition at 180 °C. Anal. Calc. Standards Institute (CLSI) (2015). Six reference strains
for C9H14Cl2CuN6O2S (%): C, 26.71; H, 3.49; N, 20.76. of Gram-negative anaerobic bacteria were tested:
Found: C, 26.81; H, 3.73; N, 20.20. FW: 404.76 g mol-1. Bacteroides fragilis (ATCC 25285), Bacteroides
Molar conductivity (DMSO, 1 mM): 26.75 cm2 X-1 thetaiotaomicron (ATCC 29741), Bacteroides vulga-
mol-1. IR (ATR, cm-1): 3374 (m N4-H), 784 (m C=S), tus (ATCC 8482), Bacteroides ovatus (ATCC 8483),
1562/1530 m(C=N), 1474/1370 (mass/ms NO2). Magnetic Parabacteroides distasonis (ATCC 1295) and Fusub-
moment: 1.63 lB. acterium nucleatum (ATCC 25586).
[(E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)pro- In order to evaluate the minimum inhibitory
pan-2-ylidene)-N-phenylhydrazinecarbothioamidedich- concentration of each compound, bacterial strains
lorocopper(II)] [Cu(HL8)Cl2]‘H2O (Cf) Dark green were cultured in Brucella Agar supplemented with
solid. Yield: 61%. Melting point: decomposition at hemin (5 lg/mL), vitamin K1 (1 lg/mL) and horse
205 °C. Anal. Calc. for C14H17Cl2CuN6O2.5S (%): C, blood (5% v/v) (BA-S), at 37 °C for 48 h in an
35.34; H, 3.45; N, 17.66. Found: C, 35.18; H, 3.25; N, anaerobic chamber (85% N2, 10% H2 and 5% CO2).
17.22. FW: 475.84 g mol-1. Molar conductivity The inoculum was prepared in sterile saline solution
(DMSO, 1 mM): 46.82 cm2 X-1 mol-1. IR (ATR, 0.9% w/v and standardized to obtain visual turbidity
cm-1): 3300 (m N4-H), 760 (m C=S), 1596 m(C=C), 1552 comparable to that of the standard 0.5 of the McFar-
m(C=N), 1496/1368 (mass/ms NO2). Magnetic moment: land scale, which provides a concentration of approx-
1.63 lB. imately 1.5 9 108 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
of approximately 1.5 9 107 CFU/mL. The adjusted
Single crystal X-ray diffraction measurements were suspensions were used in the final inoculation up to
carried out on an Oxford-Diffraction GEMINI-Ultra 30 min after preparation.
diffractometer (LabCri-UFMG) using graphite-En- The compounds under evaluation were tested for
hance Source Mo Ka radiation (k = 0.71073 Å) at determination of minimum inhibitory concentration
293(2) K. Data collection, cell refinements and data (MIC) by the agar dilution method. Briefly, the
reduction were performed using the CrysAlisPro technique consisted of adding 4.0 mL of working
software package (Oxford 2010). An absorption solution of the compounds containing nine different
correction based on a multi-scan method was applied. concentrations (ranging from 320 to 1.25 lg/mL) in
The structure was solved by direct methods using flasks containing 36 mL of the culture medium

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Biometals (2018) 31:571–584 577

Table 1 Crystal data and Compound HL6

structure refinement results
for HL6 Empirical formula C8H12N6O2S
Formula weight (g mol-1) 256.30
Crystal system Triclinic
Space group P-1
Wavelength (Å) 0.71073
Temperature (K) 293 (2)
Unit cell dimensions
a (Å) 7.2881 (3)
b (Å) 8.5774 (4)
c (Å) 10.1348 (4)
a/b/c (°) a = 90.017(4)/b = 103.727(4)/c = 104.761(4)
V (Å3) 593.91 (5)
Z 2
Density calculated (mg m-3) 1.433
Crystal size (mm3) 0.02 9 0.02 9 0.02
F(000) 268
Absorption coefficient (mm-1) 0.274
Index ranges - 10 B h B 10
- 12 B k B 12
- 14 B l B 14
2h range for data collection (°) 4.146 to 62.07
Reflections collected 31726
Independent reflections [Rint, Rsigma] 3532 [0.0621, 0.0413]
Completeness to h = 31.035o (%) 93.1
Data/restraints/parameters 3532/0/154
Final R indexes [I [ 2r(I)] R1 = 0.0555, wR2 = 0.1407
Final R indexes (all data) R1 = 0.0994, wR2 = 0.1684
Goodness-of-fit on F2 1.019
Dqmáx. and Dqmin. 0.40 and - 0.36

followed by plating. In the preparation of the working control) as well as the control to assure the non-
solutions of the compounds, DMSO was used as the interference of DMSO in cell viability. The tests were
solvent and sterile distilled water as the diluent, so that performed in duplicate with absolute agreement of the
the final concentration of DMSO was less than 0.5% in results and the MIC values were expressed in lM.
all tests. Inoculation was performed by adding
approximately 105 CFU/spot of each bacterial strain
using the Steers replicator and subsequent anaerobic Results and discussion
incubation at 37 °C for 48 h.
The minimum inhibitory concentration (MIC) was Characterization
expressed as the lowest concentration that resulted in
visual inhibition of the microorganism growth com- Microanalyses and high-resolution mass spectra were
pared with the growth in the culture medium free of compatible with the formation of 2 and HL3–HL8.
the tested compound (positive control). In addition, Microanalyses and molar conductivity data were in
incubation in the absence of inoculum was performed accordance with the formation of [Cu(HL3)2Cl2] (Ca).
to confirm the sterility of the culture medium (negative In all other cases (Cb–Cf) the data were compatible

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578 Biometals (2018) 31:571–584

with formation of [Cu(HL)Cl2] complexes. Com- NMR spectra the signal at 200.8 ppm associated to the
plexes (Ca–Cf) showed magnetic moments in the C5 nucleus in 2 is not present in the spectra of HL3–
1.63–2.00 BM range, in accordance with d9 electronic HL8, while a signal close to 150 ppm was assigned to
configuration. The presence of crystallization solvent the iminic carbon.
molecules in complexes Cb, Cd and Cf was confirmed In order to define the stereochemistry of the
by their thermogravimetry curves, which showed compounds in solution, two-dimensional Nuclear
weight losses of 4.09% (calcd. 4.10%), 2.29% (calcd. Overhauser Effect Spectroscopy (NOESY) was
2.25%) and 1.93% (calcd.1.89%), respectively. employed. Contour maps showed intramolecular
1
In the infrared spectra of HL3–HL8 three common H-1H coupling involving C5CH3 and N4H, in accor-
features associated with the imidazole nucleus were dance with E stereochemistry around the azomethine
observed: a vibration attributed to m(C2-H) in the double bond.
3083–3142 cm-1 range, a vibration attributed to
m(C=N) near 1520–1540 cm-1 and two bands near X-ray crystallography
1470 cm-1 and 1360 cm-1 corresponding to the
asymmetric and symmetric stretching modes of the The atom arrangements and atom numbering
NO2 group, respectively (Rodrigues et al. 2010). Upon scheme for HL6 are shown in Fig. 3. Selected
coordination to copper(II) the m(C2-H) vibration shifts intramolecular bond lengths and angles are listed in
to 3098–3282 cm-1. The m(C=N) absorption shifts to Table 2. The thiosemicarbazone HL6 crystallizes in
1500–1525 cm-1 and the asymmetric and symmetric the triclinic system, space group P-1, with two
stretching modes of NO2 shift to 1496–1368 cm-1. molecules per unit cell. The C6–S and N3–C5 bond
In addition, in all compounds absorptions at distances are 1.690(2) and 1.275(3) Å, as expected for
1600–1670 cm-1 were attributed to the m(C=C) C=S and C=N double bonds (Allen et al. 1987). The
vibration and absorptions at 1550–1580 cm-1 were compound crystallized in the EE conformation in
attributed to m(C=N3). The vibrations attributed to relation to the N3–C5 and C6–N4 bonds.
m(C=O) and m (C=S), observed at 1661–1714 cm-1 In the crystal packing N5-HO2 and N5-HN1
(HL3-HL5) (Recio-Despaigne et al. 2010) and inter-molecular hydrogen bonds were observed (see
804–772 cm-1 (HL6-HL8) (Mendes et al. 2001), Table S1 and Fig. S35).
respectively, are observed at 1712–1736 cm-1 (Ca–
Cc) and 788–760 cm-1 (Cd–Cf), respectively. Antimicrobial activity
The NMR spectra were recorded in DMSO-d6. The
1
H resonances were doubtless assigned on the basis of Secnidazole (1), the intermediate ketone (2), Schiff
chemical shifts, multiplicities and by using 2D bases (HL3–HL8) and the copper(II) complexes (Ca–
homonuclear 1H–1H correlation spectroscopy Cf) were evaluated in vitro by the agar dilution method
(COSY). The carbon type (C, CH) was determined for their ability to inhibit the growth of six strains of
by using distortionless enhancement by polarization Gram-negative anaerobic bacterial species. For com-
transfer (DEPT-135) experiments and the assignments parison, metronidazole was also investigated. The
were made by 2D heteronuclear multiple quantum lowest concentrations of the studied compounds that
coherence (HMQC) and heteronuclear multiple bond resulted in visual inhibition of the microorganism
coherence (HMBC) experiments. growth (MIC values) are listed in Table 3.
In the 1H NMR spectra of 2 and HL3–HL8 two The bacterial strains were quite susceptible to the
signals characteristic of the imidazole nucleus were antimicrobial action of 2 and HL3–HL8. Indeed, low
observed: a singlet near to 2.40 ppm and another near micromolar MIC values were obtained. In some cases
to 8.00 ppm, corresponding to C1CH3 and H2, the compounds under study proved to be more potent
respectively. In addition, a singlet around 2.00 ppm than secnidazole (1) and metronidazole. Although
attributed to C5CH3, and a singlet corresponding to the CuCl2 per se was devoid of activity against the tested
methylene H4 near to 5.10 ppm were observed. bacterial strains, upon coordination to copper(II) the
Formation of the Schiff bases was evidenced by the antibacterial activity significantly increased in several
presence of a signal corresponding to N4H in the cases. In fact, MIC = 29.78 lM (HL4) and MIC =
region between 9.08 and 11.01 ppm. In the 13C{1H} 25.36 lM (HL6), while MIC = 3.32 lM (Cb) and

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Biometals (2018) 31:571–584 579

Fig. 3 Molecular plot of

HL6 showing the labeling
scheme of the non-H atoms
and their displacement
ellipsoids at the 50%
probability level

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

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580 Biometals (2018) 31:571–584

Table 3 Minimum inhibitory concentration (MIC) values for Bacteroides vulgatus (ATCC 8482), Bacteroides ovatus
the 5-nitroimidazole-derived compounds, their copper(II) (ATCC 8483), Parabacteroides distasonis (ATCC 1295) and
complexes and CuCl2 against Bacteroides fragilis (ATCC Fusubacterium nucleatum (ATCC 25586)
25285), Bacteroides thetaiotaomicron (ATCC 29741),
Compound MIC (lM)
Bacteroides Bacteroides Bacteroides Bacteroides Parabacteroides Fusubacterium
fragilis thetaiotaomicron vulgatus ovatus distasonis nucleatum

1 2.64 5.27 2.64 5.27 5.27 2.64

2 5.46 5.46 2.73 5.46 43.68 –
HL3 14.18 14.18 7.09 7.09 14.18 4.16
Ca 3.32 6.64 3.32 3.32 C 13.26 3.32
HL4 29.78 7.45 3.72 3.72 7.45 8.36
Cb 3.32 6.64 3.32 3.32 C 13.27 3.32
HL5 13.28 13.28 13.28 6.64 13.28 6.64
Cc 13.28 13.28 13.28 6.64 13.28 6.64
HL6 25.36 25.36 12.68 3.17 6.34 3.90
Cd 7.80 7.80 3.90 3.90 C 31.22 1.95
HL7 3.24 6.47 3.24 3.24 6.47 3.24
Ce 3.70 3.70 3.70 1.85 7.40 3.70
HL8 4.84 4.84 4.84 4.84 2.42 12.03
Cf C 6.02 C 6.02 6.02 3.01 C 6.02 6.02
CuCl2 C 187.77 C 187.77 C 187.77 C 187.77 187.77 187.77
Metronidazole 3.17 3.17 3.17 3.17 6.34 1.46

Fig. 4 Bio-reductive
processes of nitroimidazole
compounds

Taking into consideration that the biological assays stable only at pH [ 7 or by adding an aprotic co-
were performed in anaerobic conditions, electrochem- solvent (Carbajo et al. 2002). Therefore, in order to
istry studies on the reduction of the nitro group of the achieve the best experimental conditions that ensure
nitroimidazole derivatives were performed. The redox the nitro radical anion stability, a medium formed by

potentials E8 (RNO2 R-NO 2 ) of nitroimidazole
DMF and tetrabutylammonium perchlorate (TBAP) as
derivatives and of complexes (Ca–Cf) were deter- supporting electrolyte was employed in the cyclic
mined aiming to evaluate the ability of the compounds voltammetry studies.
to be reduced in anaerobic media. The complete set of Secnidazole (1), 2 and (HL3–HL8) displayed a
cyclic voltammograms are shown in Supplementary similar electrochemical reduction process, typical of
Information (SI). nitroimidazoles in aprotic medium (Cavalcanti et al.
Using the non-usual pulse radiolysis technique, the 2004). As shown in Fig. 5, in general the compounds

monoelectronic transfer process RNO2 R-NO exhibited a stable well-defined couple (system Ic/Ia)
2 can
be observed and measured in aqueous solution. with EpIc near -1100 mV, corresponding to a mono-
However, usual electrochemical techniques are not electronic transfer process attributed to the formation,
very useful due to the limited stability of the produced in the timescale of the experiment, of a stable nitro
R-NO radical anion (R-NO 2 ).
2 radical. This radical may be sufficiently

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Biometals (2018) 31:571–584 581

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-1. Dotted line shows a short sweep with the isolated RNO2 R-NO 2 couple

The voltammograms also exhibited a second wave DEI (EpIa - EpIc) (Table 4) (Brett and Brett 1993).
near - 2000 mV, assigned to the generation of IpIa/IpIc values higher than 0.82 in most of the cases
hydroxylamine (R-NHOH) species and, in some cases, and DE values for the first wave in the 74–181 mV
an irreversible oxidation near to - 500 mV, related to range at 250 mV s-1 suggest a behavior typical of
formation of nitroso (R-NO) species was observed. quasi-reversible systems.
The electrochemical processes related to the nitroim- The voltammograms of the copper(II) complexes
idazole moiety of the structures are represented by (Fig. 5c) show two quasi-reversible processes which

Eqs. 1–3. The remaining processes in HL3–HL8 are were attributed to the RNO2 R-NO 2 (peaks Ic and Ia)
related to the Schiff base moiety (Table 4). and CuI/CuII (peaks IVc and IVa) couples [see
Peaks Ic and Ia R-NO2 þ e R-NO ð1Þ Eq. (4)].
2
 II   1
Cu ðHLÞx Cl2 þ e ! CuI ðHLÞx Cl2 ð4Þ
Peak IIc
ð2Þ x ¼ 1 or 2
R-NO  þ
2 þ 3e þ 4H ! R-NHOH þ H2 O
As shown in Table 4, the obtained EpIc values for
Peak IIIa R-NHOH R-NO þ 2e þ 2Hþ ð3Þ the intermediate ketone (2) and the novel nitroimida-
The characterization of the voltammetric first wave zole-derivatives (HL3–HL8) show that the electro-
was performed by using the parameters IpIa/IpIc and chemical behaviors of the compounds within the nitro-
imidazole moiety are very similar, meaning a similar

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582 Biometals (2018) 31:571–584

Table 4 Cyclic 
Compoundsb RNO2 R-NO
2
CuII/CuI
voltammetry parametersa
corresponding
 to the EpIc (mV) DEI (mV) IpIa/IpIc EpIc (mV) DEI (mV) IpIa/IpIc
RNO2 R-NO 2 and Cu /
II
I 1 - 1090 146 0.83 – – –
Cu couples for 1, 2, HL3–
HL8 and complexes Ca–Cf 2 - 1105 87 0.93 – – –
versus Ag/AgCl, Cl- HL3 - 1135 181 0.87 – – –
(3.0 M) reference electrode
Ca - 1033 74 0.89 515 152 1.35
HL4 - 1101 122 0.87 – – –
Cb - 985 78 0.95 478 185 1.36
HL5 - 1115 78 0.82 – – –
Cc - 880 89 0.50 622 152 1.32
HL6 - 1120 83 0.83 – – –
Cd - 1025 98 0.75 435 225 0.94
a HL7 - 1125 141 0.92 – – –
Scan rate of 250 mV s-1;
b
compounds at 1 mM Ce - 1000 97 0.76 560 129 1.31
dissolved in DMF using HL8 - 1130 98 0.85 – – –
0.1 M of TBAP as Cf - 994 83 0.58 466 190 0.90
supporting electrolyte

tendency for nitro radical anion formation. Upon MIC values of the compounds under study are
coordination to copper(II) the EpIc values become comparable to each other and to those of secnidazole
more favorable, suggesting a higher tendency to the and metronidazole.
formation of the nitro anion radical in the complexes. Upon coordination to copper(II) the antibacterial
Furthermore, the EpIc values of complexes (Ca–Cf) activity increased in several cases. Interestingly, the
did not differ much from those of the well-known reduction potentials of the nitro group were in general
enzymatic reducible drugs secnidazole (compound 1, more favorable for the complexes than for the starting
EpIc = - 1076 mV) and benznidazole (EpIc- ligands, suggesting that this might be one of the
= - 1019 mV) (Bollo et al. 2001). reasons for their improved antimicrobial action.
Previous works by other authors indicated that
Schiff base derivatives of metronidazole (Zhang et al.
Conclusions 2014) and secnidazole (Li et al. 2013) present
antibacterial effects against Gram-positive and
In conclusion, compounds HL3–HL8 exhibited Gram-negative aerobic bacteria. The novel derivatives
in vitro antibacterial activity against the six reference investigated in the present work showed potent
strains evaluated in this study. In some cases, the activity against Gram-negative pathogenic anaerobic
compounds were even more active than secnidazole bacterial strains. Therefore, we may consider these
and metronidazole, presenting lower minimum inhi- compounds suitable for in vivo evaluation and the
bitory concentrations than those of the reference microorganisms should be classified as susceptible to
drugs. them.
Since the compounds under study showed reduction The literature reports decreased clinical suscepti-
potentials in the same range as the bio-reducible drugs bility to metronidazole and other antimicrobials
secnidazole and benznidazole, their biological activ- among Bacteroides species (Löfmark et al. 2010;
ities might also be related to intracellular bio-reduc- Liu et al. 2008). Considering the increasing resistance
tion activation. to antimicrobial agents among anaerobic pathogens,
Although according to Lipinki’s rules, an absolute the present work constitutes an important contribution
correlation of a single parameter with the biological to the development of new antibacterial drug
activity is not expected, the similar electrochemical candidates.
behaviors at least partially explain the reason why the

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Biometals (2018) 31:571–584 583

Acknowledgements The authors gratefully acknowledge Corey EJ, Schmidt G (1979) Useful procedures for the oxidation
CNPq, CAPES-PNPD, FAPEMIG and INCT-INOFAR (Proc. of alcohols involving pyridinium dichromate in aprotic
CNPq 573.364/2008-6) for financial support and student grants. media. Tetrahedron Lett 5:399–402
Funding was provided by CNPq (Grant Numbers 421902/2016- De Backer E, Dubreuil L, Brauman M, Acar J, Vaneechoutte M
7 and 303746/2013-0). (2010) In vitro activity of secnidazole against Atopobium
vaginae, an anaerobic pathogen involved in bacterial
vaginosis. Clin Microbiol Infect 16:470–472
Divar M, Khalafi-Nezhad A, Zomorodian K, Sabet R, Faghih Z,
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