Synthesis , Crystal Structure , and Biological Activity of cis / trans Amide Rotomers of ( Z )-N-( 2-Oxoindolin-3-ylidene ) formohydrazide

1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 2Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt 3 Department of Pharmaceutical Chemistry, College of Pharmacy, Egyptian Russian University, Badr City, Cairo 11829, Egypt 4Department of Clinical Microbiology and Immunology, College of Medicine, Mansoura University, Mansoura 35516, Egypt 5 X-Ray Crystallography Unit, School of Physics, Universiti Sains Malaysia (USM), 11800 Penang, Malaysia


Experimental
2.1.Chemistry 2.1.1.General.Melting point of 2 was measured with a Gallenkamp apparatus and was uncorrected.Infrared (IR) spectrum of 2 was recorded as KBr disk using the Perkin Elmer FT-IR Spectrum BX apparatus.The NMR spectra of 2 were recorded on a Bruker NMR spectrometer. 1 H spectrum was run at 500 MHz and 13 C spectrum was run at 125 MHz in deuterated dimethylsulfoxide (DMSO- 6 ).Chemical shifts are expressed in  values (ppm) using the solvent peak as internal standard.Mass spectrum was measured on an Agilent Triple Quadrupole 6410 QQQ LC/MS equipped with an ESI (electrospray ionization) source.

Antimicrobial Screening.
Antimicrobial activity of the synthesized compound was determined by the agar well diffusion method [29].This method was used to assess the susceptibility of the reference microorganisms to the tested compounds.Petri dishes were prepared with a base layer of Mueller-Hinton agar medium (MHA, Becton Dickinson) and Sabouraud agar.The inoculum was prepared using plate cultures of the reference microbial strains.The colonies were suspended in 0.85% saline and the turbidity was compared with the 0.5 McFarland standards, to produce a suspension of 1.5 × 10 8 CFU/mL.The suspension was loaded on a sterile cotton swab for streaking over the entire sterile agar surface to ensure a uniform distribution of inoculum.After drying, small wells (6 mm in diameter) were made in the agar plates by sterile cork borer.100 L of each compound was loaded into the different wells.Finally, compound 2 was dissolved in dimethylsulfoxide (DMSO) to a final concentration of 10 mg/mL.Ciprofloxacin (50 g/mL) and fluconazole (25 g/mL) were used as standards for antibacterial and antifungal activities, respectively, as positive controls.Negative controls were prepared using DMSO.The plates were incubated at 37 ∘ C for 24 h and 48 h for bacterial strains and yeast, respectively.The antibacterial activity was expressed as the mean of inhibition diameters (mm) produced.The experiment was carried out in triplicate and the average zone of inhibition was calculated.

Minimal Inhibitory Concentration.
The quantitative assay of the antimicrobial activity of compound 2 was determined by microplate assay (in 96-well plate) using the twofold serial dilution technique as described in (CLSI) [30].Compound 2 was prepared in DMSO and the correct volume was put in the first microplate well with Mueller-Hinton broth medium, ensuring the concentration to be 1000 g/mL in that well.The inoculum suspension was prepared in 0.85% saline, with an optical density equivalent to 0.5 McFarland standard, and diluted in Mueller-Hinton broth to obtain final concentration 6 × 10 5 CFU/mL.This suspension was inoculated in each well of a microdilution plate previously prepared with the tested compound to give concentrations from 1000 g/mL down to 1.95 g/mL.The last two wells were reserved for inoculum viability and DMSO effect.The control drug for each ATCC strain was ciprofloxacin dissolved in DMSO.The plate was covered and incubated for 24 h at 37 ∘ C. Growth was assayed by absorbance measurement at 623 nm.The MIC was defined as the lowest concentration at which the optical density (OD) was reduced to 90% of the OD in the growth control well as measured by spectrophotometer.Results were analyzed visually and spectrophotometrically.

Anticancer Activity.
HepG2 liver cancer and MCF-7 breast cancer cell lines were obtained from the National Cancer Institute (Cairo, Egypt).HepG2 cells were grown in DMEM while MCF-7 was grown in RPMI-1640.Media were supplemented with 10% heat-inactivated FBS, 50 units/mL of penicillin, and 50 g/mL of streptomycin and maintained at 37 ∘ C in a humidified atmosphere containing 5% CO 2 .The cells were maintained as "monolayer culture" by serial subculturing.Cytotoxicity was determined using the SRB method as previously described by Skehan et al. [31].Exponentially growing cells were collected using 0.25% trypsin-EDTA and seeded in 96-well plates at 1000-2000 cells/well in supplemented DMEM medium.After 24 h, cells were incubated for 72 h with various concentrations of the tested compounds as well as doxorubicin as the reference compound.Following 72 h of treatment, the cells were fixed with 10% trichloroacetic acid for 1 h at 4 ∘ C. Wells were stained for 10 min at room temperature with 0.4% SRB dissolved in 1% acetic acid.The plates were air-dried for 24 h, and the dye was solubilized with Tris-HCl for 5 min on a shaker at 1600 rpm.The optical density (OD) of each well was measured spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, FL, USA).The IC 50 values were calculated according to the equation for Boltzmann sigmoidal concentrationresponse curve using the nonlinear regression models (Graph Pad, Prism Version 5).The results reported are means of at least three separate experiments.Significant differences were analyzed by one-way ANOVA wherein the differences were considered to be significant at  < 0.05.

Results and Discussion
3.1.Chemistry.The title compound 2 was synthesized in 58% yield by the reaction of (Z)-3-hydrazonoindolin-2-one (1) with formic acid under reflux (Figure 1).Conformational isomerism occurs when the rotation about a single bond is relatively unhindered.Such isomers are known as conformational isomers, rotomers, or conformers.However, rotations about single bonds are restricted by a rotational energy barrier which must be small enough to overcome the interconversion of one rotomer to another [32].In solution phase, the compounds having arylidene-hydrazide structure (-C=N-NH-C=O) may exist, depending on the solvent, as  geometrical isomers around -C=N-bond and as cis and trans amide rotomers (conformers) around amide N-H single bond [33][34][35][36].In this study, the 1 H NMR (DMSO- 6 ) spectrum of compound 2 showed the presence of cis and trans amide rotomers of compound 2 in 25% and 75%, respectively (Figure 2).The X-ray analysis showed the  geometrical isomer of 2 around -C=N-for cis and trans amide rotomers (X-ray section, Figure 3).
The 1 H NMR spectrum of 2 showed the signals belonging to cis and trans amide rotomers (conformers) in 25% and 75%, respectively, (Figure 2).It revealed the aldehydic proton at  8.44 and 8.85 for cis and trans rotomers, respectively, whereas D 2 O exchangeable signal of hydrazone NH group appeared at  12.7 and  12.9 for trans and cis rotomers, respectively (Figure 2).
In addition, the IR spectrum of compound 2 exhibited two absorption bands in the region 3482-3412 cm −1 for 2NH groups in addition to the two absorption bands of two carbonyl groups at 1735 and 1696 cm −1 .The absorption band of aldehydic proton appeared at 2820 cm −1 in the IR of compound 2. The mass spectrum of 2 revealed a peak corresponding to the molecular ion at m/z = 190.11.
The asymmetric unit of the title compound, C 9 H 7 N 3 O 2 , contains two molecules cis and trans conformers of amide function for  geometrical isomer around the C2=N2 double bond [37] (Figure 3).
Although, the trans conformer of 2 is the kinetically favored candidate in free space, a network of intermolecular hydrogen bonds supported the formation of the cis conformer in the crystalline state.The single bond N2-N3 is clearly characterized by the distance of 1.36 Å.The double bond of C2=N2 is characterized by the distance of 1.28 Å. Selected bond distances ( Å) and bond angles ( ∘ ) of compound 2 are illustrated in Table 2.

Antimicrobial Activity.
The synthesized compound 2 is evaluated for its antimicrobial activity against five reference strains: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Candida parapsilosis using the agar well diffusion method and broth microdilution methods.Compound 2 exhibited a moderate activity against E. coli and P. aeruginosa while its inhibition zone value is close to that of the standard drug ciprofloxacin in case of S. aureus (Table 4).
According to the results obtained by the well diffusion method, the minimal inhibitory concentration (MIC) value of compound 2 determined for S. aureus was 15.6 g/mL compared to ciprofloxacin which is 0.5 g/mL.

Anticancer Activity.
Antiproliferative activity of (Z)-N  -(2-oxoindolin-3-ylidene)formohydrazide (2) was examined in two human tumor cell lines, namely, HepG2 hepatocellular carcinoma and MCF-7 breast cancer using Sulforhodamine B (SRB) colorimetric assay as described by Skehan et al. [31].Doxorubicin was included in the experiments as a reference cytotoxic drug.The results were expressed as    growth inhibitory concentration (IC 50 ) values which represent the compound concentrations required to produce a 50% inhibition of cell growth after 72 h of incubation compared to untreated controls (Table 5).From the results, it was obvious that compound 2 displayed moderate growth inhibitory activity against HepG2 hepatocellular carcinoma cell line.It was found to be that compound 2 was less potent than doxorubicin by approximately 3.5-fold with IC 50 of 105 M, while doxorubicin showed IC 50 of 29.5 M.On the other hand, compound 2 displayed weak cytotoxic activity against MCF-7 breast cancer cell line with IC 50 of 183 M comparable to doxorubicin which showed IC 50 of 3.3 M.

Conclusion
In conclusion, the title compound 2 appeared as cis and trans amide rotomers in 25% and 75%, respectively, in DMSO- 6 .X-ray single crystal analysis illustrates the interesting property of compound 2 in crystalline state as a Z geometrical isomer around -C=N-bond with cis and trans amide rotomers due to hydrogen bondings even though in free space the trans conformer is the kinetically favoured candidate.
Compound 2 showed moderate-good activity against five microbial species in its antimicrobial evaluation and a good potency against HepG2 and MCF-7 cell lines in its anticancer evaluation.

Figure 1 :
Figure 1: The synthesis of compound 2 which exists as cis and trans amide rotomers in DMSO- 6 (cis : trans = 25 : 75).The X-ray analysis showed the Z geometrical isomer of 2 around -C=N-for cis and trans amide rotomers.

Figure 4 :
Figure 4: Portion of the crystal packing of compound 2 showing hydrogen bonds as dashed lines.

Table 1 :
Experimental details of X-ray analysis for compound 2.

Table 4 :
Antibacterial activity of 2 and its mean inhibition diameters (mm).

Table 5 :
In vitro cytotoxic activities of the compound 2 against IC 50 values are the mean ± S.D. of three separate experiments. a