Indole-3-carbaldehyde Semicarbazone Derivatives: Synthesis, Characterization, and Antibacterial Activities

Facultad de Ingenieŕıa Industrial, Universidad de Lima, Av. Javier Prado Este 46, Lima 33, Peru Facultad de Quı́mica e Ingeneŕıa Quı́mica Universidad Nacional Mayor de San Marcos, Lima, Peru Facultad de Ciencias Naturales y Matematica, Universidad Nacional Federico Villarreal, Jr. Rı́o Chepen s/n, El Agustino, Lima, Peru Facultad de Ciencias, GIR MIOMeT, IU CINQUIMA/Quı́mica Inorgánica, Universidad de Valladolid, E-47011 Valladolid, Spain Instituto Universitario de Bio-Orgánica Antonio González, Departamento de Quı́mica Orgánica, Universidad de La Laguna, Av Astrof́ısico Fco. Sánchez 2, 38206 La Laguna, Tenerife, Spain Facultad de Ciencias Quı́micas y Farmacéuticas, Universidad de Chile, CEDENNA, Olivos 1007, 233, Independencia, 8330492 Santiago, Chile Centro de Investigación de la Biodiversidad y Recursos Genéticos de Ancash, Facultad de Ciencias, Universidad Nacional Santiago Antúnez de Mayolo, Av. Centenario 200, 02002 Independencia, Huaraz, Ancash, Peru Instituto de Quı́mica-Fı́sica “Rocasolano”, CSIC, Serrano 119, 28006 Madrid, Spain


Introduction
Diseases caused by bacteria have gained considerable attention because of their resistance to the standard antibacterial drugs [1]. Bacterial resistance is a natural process that occurs in all microorganisms and is enhanced due to the inappropriate use of drugs intended for the control of bacterial infections [2], leading to increased mortality in humans [3]. On the other hand, the World Health Organization has reported that the Gram-negative strains

Chemicals and Instrumentation.
All reagents and solvents were purchased from Merck and Sigma-Aldrich and used without further purification. e tested bacterial strains were Gram-positive (S. aureus ATTC25923 and B. subtilis ATTC11774) and Gram-negative (E. coli ATTC25922 and P. aeruginosa ATTC278533), which were obtained from the biology laboratory of the Department of Sciences, Universidad Nacional Santiago Antúnez de Mayolo. Antibacterial assays were carried out using the microdilution method according to the M07-A8 protocol followed at the Clinical & Laboratory Standards Institute [24].
Infrared (IR) spectra were recorded using a Nicolet iS10 Fourier Transform Infrared (FT-IR) spectrometer equipped with an attenuated total reflectance accessory using a diamond crystal. e measurements were obtained in absorbance mode, recorded for 32 scans at a resolution of 4 cm −1 . All the measurements were carried out with a baseline automatic correction.
NMR spectra ( 1 H and 13 C) were recorded on an Agilent instrument (500 MHz for 1 H or 126 MHz for 13 C) or a Bruker AVANCE ™ spectrometer (600 MHz for 1 H or 150 MHz for 13 C), using DMSO-d 6 or acetone-d 6 , as dissolution medium. All the 1 H NMR spectra were obtained with the instrumental settings such as number of scans (8 and 8-16), acquisition time (2.044 and 1.363 s), and resolution (0.489 and 0.734 Hz), for the Agilent and Bruker AVANCE TM spectrometers, respectively. e 13 C NMR spectra were obtained with the following parameters: number of scans (2500-10000 and 2-4), acquisition time (1.0486 s and 0.456 s), and resolution (0.953 and 2.211 Hz), for the Agilent and Bruker AVANCE TM spectrometers, respectively. e chemical shifts were measure in ppm relative to tetramethylsilane (SIMe 4 ). e coupling constant (J) is expressed in Hertz (Hz) while the splitting of proton resonances is defined as s � singlet, d � doublet, t � triplet, and m � multiplet. ESI mass spectra of the synthesized compounds were recorded on the Waters-Quattro Premier XE ™ tandem quadrupole and VG Micromass ZAB-2F mass spectrometers, using methanol as the sample dissolution medium.

General Method.
A solution of sodium acetate (164 mg, 2 mmol) in water (10 mL) was added drop by drop into a hot solution of hydrochlorinated semicarbazide (110 mg, 1 mmol) in methanol : water (100 mL, v/v, 1 : 1). To the resulting mixture was added the respective indole-3carboxaldehyde derivative (2 mmol) in ethanol (10 mL). e reaction mixture was refluxed for 3 h and stirred for 24 h at room temperature. After the slow evaporation of the solvent at room temperature, the solid product was filtered, washed several times with hot water and cold ethanol, dried in vacuo, and then recrystallized from a methanol : acetone mixture (10 mL, v/v, 1 : 1).

Antibacterial Tests.
e in vitro antibacterial activity of the semicarbazone derivatives 1-4 was investigated against the standard strains of Gram-positive (Staphylococcus aureus ATCC25923 and Bacillus subtilis ATCC11774) and Gram-negative (Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853) bacteria. In order to compare the results, tetracycline was used as standard drug. e antibacterial assays were carried out by the dilution method. To prepare the initial inoculum (IIB), two fresh bacterial colonies were suspended for 14 h in a solution of NaCl 0.8% until obtaining an optical density by 0.08-0.1 at a wavelength of 620 nm. Tetracycline (Sigma) was used as the standard antibiotic. Each assay was performed in triplicate, and reproducibility was evaluated twice. e compounds were dissolved in DMSO at 50 μg/μL as stock solution, which was diluted with Müller-Hinton broth II (MHBII, Difco) at concentrations of 12.5, 25, 50, 100, 150, and 200 μg/mL. Serial dilutions were placed inside the 96well microplates (100 μL/well) and were inoculated with 10 μL of solution of IIBd (IIB diluted in MHBII, 1 : 20). Positive growth controls were prepared using the dilutions of the compounds inoculated with bacteria-free MHBII. e microplates were incubated for 24 h at 35°C. Tetrazolium salt was used as a growth indicator [25], and 10 μL of a solution of tetrazolium violet 0.1% (TV, Sigma T0138) was added to each microplate. en, these microplates were incubated under darkness for 4 h at 35°C. e TV indicator is reduced to a violet precipitate when there is bacterial respiratory activity. Minimum inhibitory concentration (MIC) value is considered as the lowest extract concentration that inhibits bacterial growth, which is reflected by the absence of the violet precipitate.

Computational Details.
e quantum chemical calculations were carried out using the Gaussian 09 and D01 software packages [26]. e geometries of the synthesized compounds were optimized using B3LYP functional and the 6-311++G(d,p) basis set without symmetry restrictions [27][28][29]. Harmonic vibrational frequencies were obtained at the same level without scaling to verify that all the stationary points are minimal. e computed energies and enthalpies for the most stable species studied were calculated in both the gas and liquid phases (acetone and DMSO solvents), using the polarizable continuum model [29,30]. e conformational analysis of these species was also explored, and the corresponding population distribution values were determined using the Boltzmann distribution [27]. For more details, see Table S1 of Supporting Information.

Synthesis and Characterization.
Compounds 1-4 were prepared according to literature [31], as shown in Scheme 1.
e semicarbazone derivatives were obtained in satisfactory yields (56-76%) and characterized by FT-IR, ESI mass spectrometry, and NMR ( 1 H, 13 C) spectroscopy. Spectroscopic data obtained for the synthesized semicarbazones are in agreement with the proposed structures. All the synthesized compounds were recrystallized from a methanol : acetone mixture (10 mL, v/v, 1 : 1), before characterization.

Infrared Spectra.
e IR spectra of the compounds 1-4 showed absorption bands in the range of 3200-3450 cm −1 which are assigned to the N-H groups of the indole ring and terminal amine group (−NH 2 ) [27,32]. e absorption bands corresponding to the carbonyl group (C�O) appeared at 1639-1681 cm −1 [27,32,33]. is observed band indicates the presence of the keto tautomer in the solid state [34]. An intense sharp band was observed at 1536-1580 cm −1 due to vibration of the imine C�N stretching frequency [27]. For compounds 3 and 4, the presence of the OCH 3 and NO 2 substituents in the C5 and C4 positions, of the indole moiety, generated shifts of the ν(C�N) bands to lower frequencies (37-43 cm −1 ). A good linear correlation of vibrational frequencies (given as wave number σ, in cm −1 ) is obtained between these experimental IR data and the corresponding to B3LYP/6-311++G(d,p) theoretical values (see Supporting Information).

NMR Spectra.
e 1 H NMR and 13 C NMR spectra of the synthesized compounds were recorded in acetone-d 6 and DMSO-d 6 , respectively. In the 1 H NMR spectra of compounds 1-4, the signal of the HC�N proton appeared as a singlet at δ � 8.07-8.23, while the signal of the � N-NH appears as a broad singlet at δ � 9.13-10.04. ese results are similar with the chemical shifts reported for other benzaldehyde and phenoxyphenyl semicarbazone derivatives with the OH, CH 3 , Br, and NO 2 substituents at the phenyl ring [31,35]. e resonance lines of the protons corresponding to the indole ring were observed at δ � 6.83-8.37. For compounds 1 and 2, the aromatic proton signals of the indole fragment bound to the HC�N group were affected by the presence of the bromo and chloro substituents on the C5 position of the indole moiety. ese signals are shifted downfield for the protons on the C4 (0.95 and 0.79 ppm, respectively), C6 (0.20 and 0.07 ppm, respectively), and C7 (0.77 and 0.74 ppm, respectively) positions, compared to the unsubstituted indole moiety [36]. For compound 3, the presence of the methoxy substituent group on the C5 position of the indole moiety affected also the resonance signals of the aromatic protons.  [36]. ese results are in agreement with the chemical shifts observed for other acylhydrazone derivatives, ((E)-N′-((5-bromo-1Hindol-3-yl)methylene)isonicotinohydrazide, (E)-N′-((4-nitro-1H-indol-3-yl)methylene) isonicotinohydrazide, and melatonin [24,34]. e NH 2 protons of the thioamide group appeared as a broad singlet in the region δ � 5.98-6.25, in agreement with the chemical shifts found for other semicarbazone derivatives with terminal amine groups [31].
In the 13 C NMR spectra of compounds 1-4, the carbon resonance signals of the C�N group appear at δ � 136.3-138.5. ese results are similar to the chemical shifts found for other carbazone derivatives with imidazole fragment [27]. e signals observed at δ � 156.5-157.4 are characteristic for the CO carbonyl group present in all compounds. e aromatic carbons of the indole ring were observed at δ � 103.8-142.8 ppm, and these chemical shifts are in agreement with those found for other indole and carbazone derivatives [27,32,37]. On the other hand, the OCH 3 methoxy carbon signal for compound 3 appeared at 55.7 ppm [37]. e two-dimensional 1 H-1 H NOESY spectra recorded in acetone-d 6 for compounds 1 (Figure 1) and 2 show coupling between the imino proton (-CH�N) and the hydrazine proton (�N-NH). ese results are in agreement with the E configurational isomer found for other semicarbazone derivatives, whose chemical shifts of the hydrazine protons are in the range of 9.13-10.04 ppm [38,39]. In addition, in the two-dimensional 1 H-1 H NOESY spectrum recorded for the compound 1, no coupling is observed between the hydrogens of the (NH 2 ) terminal amine and �N-NH hydrazine groups, which confirms the existence of the cis conformational isomer. e synthesized compounds 1-4 present an indole ring, a side chain at the C3 position (carbaldehyde semicarbazone moiety), and substituents C5-Br (1), C5-Cl (2), C5-OCH 3 (3), and C4-NO 2 (4) (see Scheme 1). e Z/E isomerism was considered with respect to the C�N bond and cis/trans conformers with respect to the CONH amide group [40]. For each studied compound and from its isomeric-conformational analysis, the theoretical calculations have shown that the most stable conformer has a cisE geometrical configuration, where the rotation (around C3−CN bond) of side chain is characterized by the dihedral angle Φ (see Figure 2). is conformer represents more than 97% of the corresponding conformational population, in both the gas and liquid phases (acetone and DMSO) (see Table 1). us, the rest of the conformers considered (trans E, cis Z, and trans Z) display high enthalpy differences relative to the corresponding most stable one (greater than 4 kJ mol −1 ). It is important to mention that the cisE configuration obtained for the most stable 1 and 2 conformers is supported by the two-dimensional 1 H-1 H NOESY spectra.
In the most stable conformers, 1-3, the side chain and the indole moiety present an almost coplanar geometry because the dihedral angle Φ is close to 180°, while the side chain of compound 4 is out of the indole plane (Φ �156°).
is result would be related with the inductive and resonant effects of the substituent group NO 2 (strong π-withdrawing) which would reflect also the presence of a high dipolar moment (μ � 9.8 D) of the most stable conformer 4. e presence of the OCH 3 (π-donating) and Cl and Br (both π-donating/withdrawing) substituent groups for compounds 1, 2, and 3, respectively, causes a π-electron delocalization on their side chains due to the following obtained dipolar moments: μ (3) < μ (1) ≈ μ (2) < μ (4) (see Table 2).
is behavior was observed in the gas phase and in both acetone and DMSO solvents.

Antibacterial Activity.
e in vitro antibacterial activities of the compounds 1-4 were studied along that of tetracycline (standard antibacterial drug). e microorganisms used in this work included S. aureus and B. subtilis (as Grampositive bacteria) and E. coli and P. aeruginosa (as Gramnegative bacteria), and the results are presented in Table 2.
Comparing the bacterial activities of the semicarbazone derivatives and the standard drug, it became evident that compounds 1 and 2 exhibited moderate inhibitory activities against S. aureus (MIC � 100 and 150 μg/mL, respectively) and B. subtilis (MIC � 100 and 150 μg/mL, respectively) [41,42], as compared to tetracycline [43] (in this work). is same effect was obtained when comparing with the indole-2carbaldehyde-semicarbazone derivative, 2-((3-chloro-1Hindol-yl)methylene)-1-(3,5-diamine-4-propionate-2-carbonylthiophene) hydrazine, assayed against Staphylococcus aureus and Bacillus subtilis (MIC � 5 and 6 μg/mL, respectively) [44,45]. e results found in this work indicate that the position of Br and Cl atoms on the indole rings plays an important role in inhibiting bacterial growth. e presence of bromine on the fifth position of indole may have a contribution in increasing the lipophilic character of the compound 1, facilitating transport across the microorganism cell membrane and increasing antimicrobial activity [46]. is antimicrobial behavior was also observed for 3-imino-[4-benzylidene-2-phenyl-imidazole-5-one-1-(4-benzoylhydrazono)]-X-indole-    [47]. On the other hand, the compounds 3 and 4 with the OCH 3 and NO 2 substituents on the C5 and C4 positions of the indole moiety, respectively, were relatively less active (MIC � > 200 μg/mL) against the tested bacterial strains. e antibacterial results obtained for compound 4 are in agreement with those obtained for the carbohydrazide derivative, with the NO 2 substituent group on its indole ring, against S. aureus bacterial strain [48].

Conclusions
In the present study, four semicarbazone derivatives with different substituent groups on the indole moiety were synthesized and characterized by ESI-MS and standard spectroscopic techniques. e two-dimensional NMR (in acetone-d 6 ) spectral data revealed that 1 and 2 in solution exist in the cisE isomeric form.
is evidence was also confirmed by DFT calculations carried out for all the synthesized compounds. e results of the antibacterial activity showed that compounds 1 and 2 (with the Br and Cl substituents, respectively) exhibited moderate bioactivity against S. aureus and B. subtilis (as Gram-positive bacteria) while compounds 3 and 4 (with the OCH 3 and NO 2 substituents, respectively) were relatively less active against the tested bacterial strains compared with tetracycline.

Data Availability
e data used to support the findings of this study are included within the article, except for the computational results, which are found in the Supporting Information (Table S1). ese data will be available on request to bona fide researchers.

Conflicts of Interest
e authors declare that they have no conflicts of interest.