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

Authors: Hatem A. Abdel-Aziz; Hazem A. Ghabbour; Wagdy M. Eldehna; Maha M. Qabeel; Hoong-Kun Fun
Journal: Journal of Chemistry
 (2014)

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## Abstract

(Z)-N′-(2-Oxoindolin-3-ylidene)formohydrazide (2) was  synthesized by the reaction of (Z)-3-hydrazonoindolin-2-one (1) with formic acid under reflux. The structure of 2 was characterized by IR, Mass, 1H NMR, and X-ray crystal structure determination. Interestingly, compound 2 appeared in DMSO-d6 as cis and trans amide rotomers in 25% and 75%, respectively. The X-ray analysis showed the Z geometrical isomer of 2 around –C=N– for cis and trans amide rotomers. The crystal of 2 belongs to monoclinic, space group P21/c, with a=4.5206 (1) Å, b=22.4747 (7) Å, c=17.3637 (5) Å, β=103.752 (1)°, Z=8, V=1713.57 (8) Å3, Dc=1.467 Mg m−3, μ=0.11 mm−1, F(000)=784, R=0.047, and wR=0.123 for 3798 observed reflections with I>2σ(I). Compound 2 exhibited a moderate activity in its antimicrobial evaluation against E. coli and P. aeruginosa and a good activity against S. aureus close to that of the standard drug ciprofloxacin. The in vitro anticancer activity of 2 was evaluated against two human tumor cell lines, namely, HepG2 hepatocellular carcinoma and MCF-7 breast cancer. HepG2 cancer cell line was more susceptible to compound 2 than MCF-7.


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## Body

## 1. Introduction

Indoline-2,3-dione or indole-1H-2,3-dione, commonly known as isatin, is a well-known natural product found in plants of genusIsatis andCouroupita guianensisAubl. It is also isolated as a metabolite of adrenaline in humans [1]. Due to the importance of isatin, it has received extensive investigations [2]. Isatin is a versatile precursor in a large number of pharmacologically active agents [3] with antifungal [4], antiviral [5], anti-HIV [6], antiprotozoal [7], antitubercular [8], antimalarial [9], antileishmanial [10], and antiepileptic inhibition activities [11]. Antibacterial [12–15], antitumor, and antineoplastic properties of isatin derivatives have also been reported [16, 17]. The isatin derivative, sunitinib, is an anticancer drug for the treatment of gastrointestinal stromal cancers, metastatic renal cell carcinoma, and pancreatic neuroendocrine tumors [18–20]. In addition, isatin-based derivative SU9516 was reported as a potential inhibitor of cyclin-dependent kinases (CDKs) with apoptotic activity against colon carcinoma cells [21]. Furthermore, we have reported the synthesis ofN,N′-hydrazono-bis-isatins as active agents against multidrug-resistant cancer cells [22] and the synthesis of isatin-based chromene hydrazones with good cytotoxic activity against leukemia K562, breast MDA-MB-468, and colon HT-29 cell lines [23]. In view of our findings and in continuation of our interest in chemistry and biological activity of isatins [22–24], we report herein the synthesis, crystal structure, antimicrobial activity, and cytotoxic properties of the title compound.


## 2. Experimental

### 2.1. Chemistry

#### 2.1.1. General

Melting point of2 was measured with a Gallenkamp apparatus and was uncorrected. Infrared (IR) spectrum of2 was recorded as KBr disk using the Perkin Elmer FT-IR Spectrum BX apparatus. The NMR spectra of2 were recorded on a Bruker NMR spectrometer. 1H spectrum was run at 500 MHz and 13C spectrum was run at 125 MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in δ 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.


#### 2.1.2. Synthesis of (Z)-N′-(2-Oxoindolin-3-ylidene)formohydrazide (2)

A stirred solution of formic acid (20 mL) and (Z)-3-hydrazonoindolin-2-one (1) [25] (1.61 g, 10 mmol) was refluxed for 1 h. The reaction mixture was then left to cool. Then formic acid was removed by distillation. The solid obtained was recrystallized from acetic acid to give compound2 in 58% yield, mp 190–192°C; IRν 3482–3412 (2NH), 2820 (CH aldehydic), 1735, 1696 (2C=O), 1617 (C=N) cm−1; 1H NMR (DSMO-d6) (the ratio ofcis/trans = 25/75) δ 6.94–6.97 (m, 1H, ArH), 7.08–7.11 (m, 1H, ArH), 7.38–7.41 (m, 1H, ArH), 7.53–7.55 (m, 1H, ArH), 8.44 (s, 1H, –CH=Ocis rotomer), 8.85 (s, 1H, –CH=Otrans rotomer), 11.30 (br. s, 1H, D2O exchangable, NH isatin), 12.70 (s, 1H, NH, D2O exchangable,trans rotomer), 12.90 (s, 1H, NH, D2O exchangable,cis rotomer); 13C NMR (DSMO-d6) δ 111.69, 119.97, 121.11, 123.15, 132.21, 137.02, 143.07, 162.62, 168.23; MS (ESI)m/z 190.11 (M+ + 1).


### 2.2. X-Ray Crystallography

X-ray data was collected on a Bruker D8 Venture area diffractometer equipped with graphite monochromatic MoK∖α radiation (λ = 0.71073 Å) at 293 (2) K.Single crystal of2, which is suitable for X-ray analysis, was grown by slow evaporation from acetic acid. Computing details: APEX II [26]; cell refinement: SAINT; data reduction: SAINT; program(s) used to solve structure: SHELXTL [27]; program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON [28].


### 2.3. Antimicrobial Activity

#### 2.3.1. Microorganisms and Media

In this study, we used five reference strains:Escherichia coli ATCC 10536,Pseudomonas aeruginosa ATCC 15442,Staphylococcus aureus ATCC 6538,Candida albicans ATCC 90029, andCandida parapsilosis ATCC 22019. The bacterial strains were grown on Mueller-Hinton agar medium (MHA, Becton Dickinson) at 37°C for 24 h and the yeast on Sabouraud agar medium for 48 h.


#### 2.3.2. 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 × 108 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 μL of each compound was loaded into the different wells. Finally, compound2 was dissolved in dimethylsulfoxide (DMSO) to a final concentration of 10 mg/mL. Ciprofloxacin (50 μg/mL) and fluconazole (25 μg/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.


#### 2.3.3. Minimal Inhibitory Concentration

The quantitative assay of the antimicrobial activity of compound2 was determined by microplate assay (in 96-well plate) using the twofold serial dilution technique as described in (CLSI) [30]. Compound2 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 μg/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 × 105 CFU/mL. This suspension was inoculated in each well of a microdilution plate previously prepared with the tested compound to give concentrations from 1000 μg/mL down to 1.95 μg/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.


### 2.4. 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% CO2. 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 IC50 values were calculated according to the equation for Boltzmann sigmoidal concentration-response 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 P<0.05.


## 2.1. Chemistry

### 2.1.1. General

Melting point of2 was measured with a Gallenkamp apparatus and was uncorrected. Infrared (IR) spectrum of2 was recorded as KBr disk using the Perkin Elmer FT-IR Spectrum BX apparatus. The NMR spectra of2 were recorded on a Bruker NMR spectrometer. 1H spectrum was run at 500 MHz and 13C spectrum was run at 125 MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in δ 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.


### 2.1.2. Synthesis of (Z)-N′-(2-Oxoindolin-3-ylidene)formohydrazide (2)

A stirred solution of formic acid (20 mL) and (Z)-3-hydrazonoindolin-2-one (1) [25] (1.61 g, 10 mmol) was refluxed for 1 h. The reaction mixture was then left to cool. Then formic acid was removed by distillation. The solid obtained was recrystallized from acetic acid to give compound2 in 58% yield, mp 190–192°C; IRν 3482–3412 (2NH), 2820 (CH aldehydic), 1735, 1696 (2C=O), 1617 (C=N) cm−1; 1H NMR (DSMO-d6) (the ratio ofcis/trans = 25/75) δ 6.94–6.97 (m, 1H, ArH), 7.08–7.11 (m, 1H, ArH), 7.38–7.41 (m, 1H, ArH), 7.53–7.55 (m, 1H, ArH), 8.44 (s, 1H, –CH=Ocis rotomer), 8.85 (s, 1H, –CH=Otrans rotomer), 11.30 (br. s, 1H, D2O exchangable, NH isatin), 12.70 (s, 1H, NH, D2O exchangable,trans rotomer), 12.90 (s, 1H, NH, D2O exchangable,cis rotomer); 13C NMR (DSMO-d6) δ 111.69, 119.97, 121.11, 123.15, 132.21, 137.02, 143.07, 162.62, 168.23; MS (ESI)m/z 190.11 (M+ + 1).


## 2.1.1. General

Melting point of2 was measured with a Gallenkamp apparatus and was uncorrected. Infrared (IR) spectrum of2 was recorded as KBr disk using the Perkin Elmer FT-IR Spectrum BX apparatus. The NMR spectra of2 were recorded on a Bruker NMR spectrometer. 1H spectrum was run at 500 MHz and 13C spectrum was run at 125 MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in δ 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.


## 2.1.2. Synthesis of (Z)-N′-(2-Oxoindolin-3-ylidene)formohydrazide (2)

A stirred solution of formic acid (20 mL) and (Z)-3-hydrazonoindolin-2-one (1) [25] (1.61 g, 10 mmol) was refluxed for 1 h. The reaction mixture was then left to cool. Then formic acid was removed by distillation. The solid obtained was recrystallized from acetic acid to give compound2 in 58% yield, mp 190–192°C; IRν 3482–3412 (2NH), 2820 (CH aldehydic), 1735, 1696 (2C=O), 1617 (C=N) cm−1; 1H NMR (DSMO-d6) (the ratio ofcis/trans = 25/75) δ 6.94–6.97 (m, 1H, ArH), 7.08–7.11 (m, 1H, ArH), 7.38–7.41 (m, 1H, ArH), 7.53–7.55 (m, 1H, ArH), 8.44 (s, 1H, –CH=Ocis rotomer), 8.85 (s, 1H, –CH=Otrans rotomer), 11.30 (br. s, 1H, D2O exchangable, NH isatin), 12.70 (s, 1H, NH, D2O exchangable,trans rotomer), 12.90 (s, 1H, NH, D2O exchangable,cis rotomer); 13C NMR (DSMO-d6) δ 111.69, 119.97, 121.11, 123.15, 132.21, 137.02, 143.07, 162.62, 168.23; MS (ESI)m/z 190.11 (M+ + 1).


## 2.2. X-Ray Crystallography

X-ray data was collected on a Bruker D8 Venture area diffractometer equipped with graphite monochromatic MoK∖α radiation (λ = 0.71073 Å) at 293 (2) K.Single crystal of2, which is suitable for X-ray analysis, was grown by slow evaporation from acetic acid. Computing details: APEX II [26]; cell refinement: SAINT; data reduction: SAINT; program(s) used to solve structure: SHELXTL [27]; program(s) used to refine structure: SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL and PLATON [28].


## 2.3. Antimicrobial Activity

### 2.3.1. Microorganisms and Media

In this study, we used five reference strains:Escherichia coli ATCC 10536,Pseudomonas aeruginosa ATCC 15442,Staphylococcus aureus ATCC 6538,Candida albicans ATCC 90029, andCandida parapsilosis ATCC 22019. The bacterial strains were grown on Mueller-Hinton agar medium (MHA, Becton Dickinson) at 37°C for 24 h and the yeast on Sabouraud agar medium for 48 h.


### 2.3.2. 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 × 108 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 μL of each compound was loaded into the different wells. Finally, compound2 was dissolved in dimethylsulfoxide (DMSO) to a final concentration of 10 mg/mL. Ciprofloxacin (50 μg/mL) and fluconazole (25 μg/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.


### 2.3.3. Minimal Inhibitory Concentration

The quantitative assay of the antimicrobial activity of compound2 was determined by microplate assay (in 96-well plate) using the twofold serial dilution technique as described in (CLSI) [30]. Compound2 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 μg/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 × 105 CFU/mL. This suspension was inoculated in each well of a microdilution plate previously prepared with the tested compound to give concentrations from 1000 μg/mL down to 1.95 μg/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.


## 2.3.1. Microorganisms and Media

In this study, we used five reference strains:Escherichia coli ATCC 10536,Pseudomonas aeruginosa ATCC 15442,Staphylococcus aureus ATCC 6538,Candida albicans ATCC 90029, andCandida parapsilosis ATCC 22019. The bacterial strains were grown on Mueller-Hinton agar medium (MHA, Becton Dickinson) at 37°C for 24 h and the yeast on Sabouraud agar medium for 48 h.


## 2.3.2. 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 × 108 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 μL of each compound was loaded into the different wells. Finally, compound2 was dissolved in dimethylsulfoxide (DMSO) to a final concentration of 10 mg/mL. Ciprofloxacin (50 μg/mL) and fluconazole (25 μg/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.


## 2.3.3. Minimal Inhibitory Concentration

The quantitative assay of the antimicrobial activity of compound2 was determined by microplate assay (in 96-well plate) using the twofold serial dilution technique as described in (CLSI) [30]. Compound2 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 μg/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 × 105 CFU/mL. This suspension was inoculated in each well of a microdilution plate previously prepared with the tested compound to give concentrations from 1000 μg/mL down to 1.95 μg/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.


## 2.4. 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% CO2. 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 IC50 values were calculated according to the equation for Boltzmann sigmoidal concentration-response 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 P<0.05.


## 3. Results and Discussion

### 3.1. Chemistry

The title compound2 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 Z geometrical isomers around –C=N– bond and ascis andtrans amide rotomers (conformers) around amide N–H single bond [33–36]. In this study, the 1H NMR (DMSO-d6) spectrum of compound2 showed the presence ofcis andtrans amide rotomers of compound2 in 25% and 75%, respectively (Figure 2). The X-ray analysis showed the Z geometrical isomer of2 around –C=N– forcis andtrans amide rotomers (X-ray section, Figure 3).Figure 1
The synthesis of compound2 which exists ascis andtrans amide rotomers in DMSO-d6 (cis :trans = 25 : 75). The X-ray analysis showed theZ geometrical isomer of2 around –C=N– forcis andtrans amide rotomers.Figure 2
1H NMR (DMSO-d6) of compound2.Figure 3
The asymmetric unit of compound2 showing the atomic numbering and 40% probability displacement ellipsoids,cis :trans = 1 : 1.The1H NMR spectrum of2 showed the signals belonging tocis andtrans amide rotomers (conformers) in 25% and 75%, respectively, (Figure 2). It revealed the aldehydic proton at δ 8.44 and 8.85 forcis andtrans rotomers, respectively, whereas D2O exchangeable signal of hydrazone NH group appeared at δ 12.7 and δ 12.9 fortrans andcis rotomers, respectively (Figure 2).In addition, the IR spectrum of compound2 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 compound2. The mass spectrum of2 revealed a peak corresponding to the molecular ion atm/z = 190.11.


### 3.2. X-Ray Crystallography

The crystal of2 belongs to monoclinic, space groupP21/c, with a=4.5206 (1) Å, b=22.4747 (7) Å, c=17.3637 (5) Å, β=103.752 (1)°, Z=8, V=1713.57 (8) Å3,  Dc=1.467 Mg m−3, μ=0.11 mm−1, F(000)=784, R=0.047, and wR=0.123 for 3798 observed reflections with I>2σ(I) (Table 1).Table 1
Experimental details of X-ray analysis for compound2.
Crystal data
Chemical formula
C9H5N3O2
M
r
187.16
Crystal system, space group
Monoclinic,P21/c
Temperature (K)
293
A, b, c (Å)
4.5206 (1), 22.4747 (7), 17.3637 (5)
B (°)
103.752 (1)
V (Å3)
1713.57 (8)
Z
8
Radiation type
MoKα
μ
(mm−1)
0.11
Crystal size (mm)
0.71 × 0.15 × 0.08
Data collection
Diffractometer
Bruker APEX-II D8 venture
Absorption correction
multiscan, SADABS (Bruker, 2012)
No. of measured, independent, and observed (i) reflections
80166, 5269, 3798
R
int
⁡
0.038
Refinement
R[F2>2σ(F2)], wR(F2), S
0.065, 0.127, 4.80
No. of reflections
5269
No. of parameters
253
No. of restraints
0
H-atom treatment
H-atom parameters constrained
Δ
ρ
max
⁡, Δρmin⁡ (e Å−3)
0.68, −0.26The asymmetric unit of the title compound, C9H7N3O2, contains two moleculescis andtrans conformers of amide function for Z geometrical isomer around the C2=N2 double bond [37] (Figure 3).Although, thetrans conformer of2 is the kinetically favored candidate in free space, a network of intermolecular hydrogen bonds supported the formation of thecis 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 compound2 are illustrated in Table 2.Table 2
Selected bond distances (Å) and bond angles (°) of compound2.
Bond distances
O1A–C1A
1.221 (2)
N2A–C2A
1.281 (2)
O2A–C9A
1.215 (3)
N3A–C9A
1.349 (2)
O1B–C1B
1.215 (2)
N1B–C8B
1.410 (2)
O2B–C9B
1.221 (3)
N1B–C1B
1.357 (2)
N1A–C1A
1.372 (2)
N2B–N3B
1.377 (2)
N1A–C8A
1.406 (2)
N2B–C2B
1.275 (2)
N2A–N3A
1.3712 (19)
N3B–C9B
1.343 (2)
Bond angles
C1A–N1A–C8A
110.35 (13)
N1A–C8A–C7A
128.91 (15)
N3A–N2A–C2A
117.63 (13)
N1A–C8A–C3A
110.21 (13)
N2A–N3A–C9A
114.53 (14)
O2A–C9A–N3A
125.08 (18)
C1B–N1B–C8B
110.41 (13)
O1B–C1B–N1B
127.54 (15)
N3B–N2B–C2B
116.62 (14)
O1B–C1B–C2B
125.83 (15)
N2B–N3B–C9B
117.81 (15)
N1B–C1B–C2B
106.63 (14)
O1A–C1A–C2A
126.91 (15)
N2B–C2B–C1B
128.35 (15)
O1A–C1A–N1A
126.56 (16)
N2B–C2B–C3B
125.65 (15)
N1A–C1A–C2A
106.53 (14)
N1B–C8B–C3B
110.11 (14)
N2A–C2A–C1A
129.10 (14)
N1B–C8B–C7B
128.52 (15)
N2A–C2A–C3A
124.87 (14)
O2B–C9B–N3B
125.67 (19)The torsion angles N2A-N3A-C9A-O2A, N2A-N3A-C9A-H5, H3NA-N3A-C9A-O2A, and H3NA-N3A-C9A-H5 are −175.97°, 4.01°, 3.2°, and 176.82°, respectively. The torsion angles N2B-N3B-C9B-O2B, N2B-N3B-C9B-H10, H3NB-N3B-C9B-O2B, and H3NB-N3B-C9B-H10 are −0.63°, 179.36°, 178.09°, and −1.95°, respectively. The crystal packing is stabilized by intermolecular interactions forming a three-dimensional network (Figure4 and Table 3).Table 3
Hydrogen-bond geometry (Å, °) of compound2.
D–H⋯A
D–H
H⋯A
D
⋯
A
N1AA–H1NA⋯O2BB
0.87 (2)
2.1980
2.981 (2)
N1AA–H1NA⋯N2BB
0.87 (2)
2.6140
3.368 (2)
N1AB–H1NA⋯O2BA
0.891 (19)
2.2010
3.034 (19)
N1AB–H1NA⋯N2BA
0.891 (19)
2.5800
3.392 (2)
N1BA–H1NA⋯O2BD
0.960 (5)
2.3200
3.014 (2)Figure 4
Portion of the crystal packing of compound2 showing hydrogen bonds as dashed lines.


### 3.3. Antimicrobial Activity

The synthesized compound2 is evaluated for its antimicrobial activity against five reference strains:Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,Candida albicans, andCandida parapsilosis using the agar well diffusion method and broth microdilution methods. Compound2 exhibited a moderate activity againstE. coli andP. aeruginosa while its inhibition zone value is close to that of the standard drug ciprofloxacin in case ofS. aureus (Table 4).Table 4
Antibacterial activity of2 and its mean inhibition diameters (mm).
E. coli
P. aeruginosa
S. aureus
C. albicans
C. parapsilosis
Compound2
10
12
18
8
—
Ciprofloxacin
23
24
20
Fluconazole
—
—
—
29
27According to the results obtained by the well diffusion method, the minimal inhibitory concentration (MIC) value of compound2 determined forS. aureus was 15.6 μg/mL compared to ciprofloxacin which is 0.5 μg/mL.


### 3.4. 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 (IC50) 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 compound2 displayed moderate growth inhibitory activity against HepG2 hepatocellular carcinoma cell line. It was found to be that compound2 was less potent than doxorubicin by approximately 3.5-fold with IC50 of 105 μM, while doxorubicin showed IC50 of 29.5 μM. On the other hand, compound2 displayed weak cytotoxic activity against MCF-7 breast cancer cell line with IC50 of 183 μM comparable to doxorubicin which showed IC50 of 3.3 μM.Table 5
In vitro cytotoxic activities of the compound 2 against hepatocellular carcinoma (HepG2) and breast cancer (MCF-7) cell lines.
IC50 (μM)a
HepG2
MCF-7
Compound2
105
183
Doxorubicin
29.5
3.3
aIC50 values are the mean ± S.D. of three separate experiments.


## 3.1. Chemistry

The title compound2 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 Z geometrical isomers around –C=N– bond and ascis andtrans amide rotomers (conformers) around amide N–H single bond [33–36]. In this study, the 1H NMR (DMSO-d6) spectrum of compound2 showed the presence ofcis andtrans amide rotomers of compound2 in 25% and 75%, respectively (Figure 2). The X-ray analysis showed the Z geometrical isomer of2 around –C=N– forcis andtrans amide rotomers (X-ray section, Figure 3).Figure 1
The synthesis of compound2 which exists ascis andtrans amide rotomers in DMSO-d6 (cis :trans = 25 : 75). The X-ray analysis showed theZ geometrical isomer of2 around –C=N– forcis andtrans amide rotomers.Figure 2
1H NMR (DMSO-d6) of compound2.Figure 3
The asymmetric unit of compound2 showing the atomic numbering and 40% probability displacement ellipsoids,cis :trans = 1 : 1.The1H NMR spectrum of2 showed the signals belonging tocis andtrans amide rotomers (conformers) in 25% and 75%, respectively, (Figure 2). It revealed the aldehydic proton at δ 8.44 and 8.85 forcis andtrans rotomers, respectively, whereas D2O exchangeable signal of hydrazone NH group appeared at δ 12.7 and δ 12.9 fortrans andcis rotomers, respectively (Figure 2).In addition, the IR spectrum of compound2 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 compound2. The mass spectrum of2 revealed a peak corresponding to the molecular ion atm/z = 190.11.


## 3.2. X-Ray Crystallography

The crystal of2 belongs to monoclinic, space groupP21/c, with a=4.5206 (1) Å, b=22.4747 (7) Å, c=17.3637 (5) Å, β=103.752 (1)°, Z=8, V=1713.57 (8) Å3,  Dc=1.467 Mg m−3, μ=0.11 mm−1, F(000)=784, R=0.047, and wR=0.123 for 3798 observed reflections with I>2σ(I) (Table 1).Table 1
Experimental details of X-ray analysis for compound2.
Crystal data
Chemical formula
C9H5N3O2
M
r
187.16
Crystal system, space group
Monoclinic,P21/c
Temperature (K)
293
A, b, c (Å)
4.5206 (1), 22.4747 (7), 17.3637 (5)
B (°)
103.752 (1)
V (Å3)
1713.57 (8)
Z
8
Radiation type
MoKα
μ
(mm−1)
0.11
Crystal size (mm)
0.71 × 0.15 × 0.08
Data collection
Diffractometer
Bruker APEX-II D8 venture
Absorption correction
multiscan, SADABS (Bruker, 2012)
No. of measured, independent, and observed (i) reflections
80166, 5269, 3798
R
int
⁡
0.038
Refinement
R[F2>2σ(F2)], wR(F2), S
0.065, 0.127, 4.80
No. of reflections
5269
No. of parameters
253
No. of restraints
0
H-atom treatment
H-atom parameters constrained
Δ
ρ
max
⁡, Δρmin⁡ (e Å−3)
0.68, −0.26The asymmetric unit of the title compound, C9H7N3O2, contains two moleculescis andtrans conformers of amide function for Z geometrical isomer around the C2=N2 double bond [37] (Figure 3).Although, thetrans conformer of2 is the kinetically favored candidate in free space, a network of intermolecular hydrogen bonds supported the formation of thecis 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 compound2 are illustrated in Table 2.Table 2
Selected bond distances (Å) and bond angles (°) of compound2.
Bond distances
O1A–C1A
1.221 (2)
N2A–C2A
1.281 (2)
O2A–C9A
1.215 (3)
N3A–C9A
1.349 (2)
O1B–C1B
1.215 (2)
N1B–C8B
1.410 (2)
O2B–C9B
1.221 (3)
N1B–C1B
1.357 (2)
N1A–C1A
1.372 (2)
N2B–N3B
1.377 (2)
N1A–C8A
1.406 (2)
N2B–C2B
1.275 (2)
N2A–N3A
1.3712 (19)
N3B–C9B
1.343 (2)
Bond angles
C1A–N1A–C8A
110.35 (13)
N1A–C8A–C7A
128.91 (15)
N3A–N2A–C2A
117.63 (13)
N1A–C8A–C3A
110.21 (13)
N2A–N3A–C9A
114.53 (14)
O2A–C9A–N3A
125.08 (18)
C1B–N1B–C8B
110.41 (13)
O1B–C1B–N1B
127.54 (15)
N3B–N2B–C2B
116.62 (14)
O1B–C1B–C2B
125.83 (15)
N2B–N3B–C9B
117.81 (15)
N1B–C1B–C2B
106.63 (14)
O1A–C1A–C2A
126.91 (15)
N2B–C2B–C1B
128.35 (15)
O1A–C1A–N1A
126.56 (16)
N2B–C2B–C3B
125.65 (15)
N1A–C1A–C2A
106.53 (14)
N1B–C8B–C3B
110.11 (14)
N2A–C2A–C1A
129.10 (14)
N1B–C8B–C7B
128.52 (15)
N2A–C2A–C3A
124.87 (14)
O2B–C9B–N3B
125.67 (19)The torsion angles N2A-N3A-C9A-O2A, N2A-N3A-C9A-H5, H3NA-N3A-C9A-O2A, and H3NA-N3A-C9A-H5 are −175.97°, 4.01°, 3.2°, and 176.82°, respectively. The torsion angles N2B-N3B-C9B-O2B, N2B-N3B-C9B-H10, H3NB-N3B-C9B-O2B, and H3NB-N3B-C9B-H10 are −0.63°, 179.36°, 178.09°, and −1.95°, respectively. The crystal packing is stabilized by intermolecular interactions forming a three-dimensional network (Figure4 and Table 3).Table 3
Hydrogen-bond geometry (Å, °) of compound2.
D–H⋯A
D–H
H⋯A
D
⋯
A
N1AA–H1NA⋯O2BB
0.87 (2)
2.1980
2.981 (2)
N1AA–H1NA⋯N2BB
0.87 (2)
2.6140
3.368 (2)
N1AB–H1NA⋯O2BA
0.891 (19)
2.2010
3.034 (19)
N1AB–H1NA⋯N2BA
0.891 (19)
2.5800
3.392 (2)
N1BA–H1NA⋯O2BD
0.960 (5)
2.3200
3.014 (2)Figure 4
Portion of the crystal packing of compound2 showing hydrogen bonds as dashed lines.


## 3.3. Antimicrobial Activity

The synthesized compound2 is evaluated for its antimicrobial activity against five reference strains:Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,Candida albicans, andCandida parapsilosis using the agar well diffusion method and broth microdilution methods. Compound2 exhibited a moderate activity againstE. coli andP. aeruginosa while its inhibition zone value is close to that of the standard drug ciprofloxacin in case ofS. aureus (Table 4).Table 4
Antibacterial activity of2 and its mean inhibition diameters (mm).
E. coli
P. aeruginosa
S. aureus
C. albicans
C. parapsilosis
Compound2
10
12
18
8
—
Ciprofloxacin
23
24
20
Fluconazole
—
—
—
29
27According to the results obtained by the well diffusion method, the minimal inhibitory concentration (MIC) value of compound2 determined forS. aureus was 15.6 μg/mL compared to ciprofloxacin which is 0.5 μg/mL.


## 3.4. 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 (IC50) 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 compound2 displayed moderate growth inhibitory activity against HepG2 hepatocellular carcinoma cell line. It was found to be that compound2 was less potent than doxorubicin by approximately 3.5-fold with IC50 of 105 μM, while doxorubicin showed IC50 of 29.5 μM. On the other hand, compound2 displayed weak cytotoxic activity against MCF-7 breast cancer cell line with IC50 of 183 μM comparable to doxorubicin which showed IC50 of 3.3 μM.Table 5
In vitro cytotoxic activities of the compound 2 against hepatocellular carcinoma (HepG2) and breast cancer (MCF-7) cell lines.
IC50 (μM)a
HepG2
MCF-7
Compound2
105
183
Doxorubicin
29.5
3.3
aIC50 values are the mean ± S.D. of three separate experiments.


## 4. Conclusion

In conclusion, the title compound2 appeared ascis andtrans amide rotomers in 25% and 75%, respectively, in DMSO-d6. X-ray single crystal analysis illustrates the interesting property of compound2 in crystalline state as aZ geometrical isomer around –C=N– bond withcis andtrans amide rotomers due to hydrogen bondings even though in free space thetrans conformer is the kinetically favoured candidate. Compound2 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.



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*Source: 760434-2014-09-03.xml*