Carcinogenic effects of N-nitroso-3-(substituted phenylimino)-indolin-2-one derivatives.

AIM: A novel series of N-nitroso-3-(substituted phenylimino)-indolin-2-one 3a-h was synthesized and tested for carcinogenic effects.
MATERIALS AND METHODS: The synthesized pyrazole derivatives' chemical structures were proved by means of their infra red (IR), proton nuclear magnetic resonance ((1)H-NMR), and mass,and confirmed by elemental analyses. The carcinogenic activity was assessed by 3-(4,5dimethyl thiazole-2yl)-2,5-diphenyltetrazoliumbromide (MTT) cell-viability assay.
RESULTS: The results show that most of the synthesized compounds exhibit significant carcinogenic activities. Among the synthesized compounds, N-nitroso-3-(2,4-dinitrophenylimino)-indolin-2-one 3h exhibited the most potent carcinogenic activity.
CONCLUSION: The structure-activity relationship (SAR) studies show that the nature as well as the position of the amine are important for deciding the activity profile of the indolin-2-one derivatives, which reiterates the need for further experimental investigations.

Nitroso (NO) compounds are divided into the nitrosamines, derived from dialkyl, alkaryl, diaryl, or cyclic secondary amines; and the nitrosamides, derived from N-alkylureas, N-alkylcarbamates, and simple N-alkylamides. Most tested nitrosamines and nitrosamides have proved to be strong carcinogens.[1] N-nitrosoamines are an important class of environmental carcinogens, and may play a role in carcinogenesis in the nasal cavity, larynx, trachea, intestine, Harderian gland and lips. Heterocyclic N-nitrosamines are a well-characterized class of carcinogens with varying organ specificities.[2-12] They require metabolic activation for their biological activity.[13-19] Isatin and its derivatives show diverse and marked biological activities such as anticancer, anticholinesterase, anti[convulsant, anti-inflammatory, antihypertensive, hypoxia, antimicrobial, antineoplastic, antiulcer, and antiviral activities, as well as some central nervous system activities.[20] However, nitrosoisatin with substituted aromatic primary amine results in the formation of the corresponding biologically inactive compounds.

In view of these important applications and as a continuation of our earlier work we report, in the present paper, the results of nitrosoisatin with aromatic primary amine on their carcinogenic effects.

Materials and Methods

Chemistry

Melting points were measured in open capillaries on Thomas-Hoover melting point apparatus and are uncorrected. Infrared spectra were recorded on the ABB-Bomem Fourier transform infrared (FT-IR) spectrometer MB 104. 1H-NMR and 13C-NMR spectra were recorded on the Bruker spectrometer. Mass spectra were recorded on JEOL GCmate™. Elemental analysis was performed on the Perkin-Elemer 2400 CHN analyser, and the values were within the acceptable limits (±0.4%) of the calculated values. The purity of the synthesized compounds was checked by thin layer chromatography (TLC), using E-Merk TLC aluminum sheets coating with silica gel 60 F254 (0.2 mm)with chloroform/methanol (9:1) as eluent and visualized in an iodine chamber. All the chemicals used were of analytical grade.

Synthesis of N-nitrosoisatin 1

To a solution of 0.1 mol of isatin or 5-bromoisatin in chloroform (200 ml) were added concentrated hydrochloric acid (30 ml) and water (30 ml) and, while stirring this mixture, solid NaNO2 (1.65 g, 0.024 mol) was added in portions over 30 min. The stirring was continued for 4 h. The organic layer was washed with water and saturated aqueous NaHCO3 and dried over MgSO4. After removal of the chloroform, the residue was recrystallized from ethanol.[21]

General procedure for synthesis of the final compounds 3a-h

Equimolar quantities (0.01mol) of N-nitrosoisatin and the aromatic primary amine 2 were dissolved in 10 ml of warm ethanol and refluxed for 3 h and then checked for completion of reaction by TLC. After allowing to stand for approximately 24 h at room temperature, the products were separated by filtration, vacuum dried, and recrystallized from warm ethanol.

N-nitroso-3-(4-chlorophenylimino)-indolin-2-one 3a

Yield 96.4%; melting point (MP) 254–256°C; dmf/dmso; IR (KBR, cm−1): 3178 (Ar-CH), 1592 (C=C), 1331 (C-N), 1461 (N-NO), 1738 (C=O), 1611 (C=N), 751 (C-Cl); 1H-NMRδ (ppm): 6.58-7.74 (m, 8H,Ar-H); 13C-NMR δ (ppm):119.2, 122.7, 123.4, 123.7, 124.5, 129.6, 130.2, 131.0, 132.3, 133.8, 138.7, 151.3, 161.5, 163.3; MASS m/z: 285(M+) (5%), 286 (M+1) (5%); Analytical Calculated for C14H8ClN3O2 : C, 58.86; H, 2.82; Cl, 12.41; N, 14.71; O, 11.20. Found: C, 58.82; H, 2.77; Cl, 12.38; N, 14.68; O, 11.17.

N-nitroso-3-(4-bromophenylimino)-indolin-2-one 3b

Yield 92.6%; MP 220–222°C; dmf/dmso; IR (KBR, per centimeter): 3268 (Ar-CH), 1592 (C=C), 1334 (C-N), 1460 (N-NO), 1739 (C=O), 1610 (C=N), 582 (C-Br); 1H-NMR δ (ppm): 6.42-7.71 (m,8H, Ar-H); 13C-NMR δ (ppm):119.8, 121.4, 121.6, 123.7, 124.1, 124.5,129.6, 131.2, 133.0, 133.3, 138.8, 152.8, 161.7, 163.9; MASS m/z: 330 (M+) (1.1%),332 (M+1) (5%); Anal. Calcd. for C14H8BrN3O2: C, 50.93; H, 2.44; Br, 24.20; N, 12.73; O, 9.69. Found: C, 50.90; H, 2.41; Br, 24.18; N, 12.72; O, 9.68.

N-nitroso-3-(4-fluorophenylimino)-indolin-2-one 3c

Yield 89.2%; MP 156-158°C; dmf/dmso; IR (KBR, cm−1): 2987 (Ar-CH), 1497 (C=C), 1385 (C-N), 1463 (N-NO), 1728 (C=O), 1615 (C=N), 1288(C-F); 1H-NMR δ (ppm): 6.41-7.61 (m, 8H, Ar-H); 13C-NMR δ (ppm):116.4, 116.5, 118.7, 121.6, 123.7, 123.9, 124.5, 129.6, 131.2, 138.8, 149.0, 161.3, 161.9, 163.9; MASS m/z: 270 (M+) (3%); Anal. Calcd.for C14H8FN3O2: C, 62.46; H, 3.00; F, 7.06; N, 15.61; O, 11.89. Found: C, 62.42; H, 2.98; F, 7.02; N, 15.58; O, 11.88.

N-nitroso-3-(3-chloro-4-fluorophenylimino)-indolin-2-one 3d

Yield 66.6%; MP 210–212°C; dmf/dmso; IR (KBR, cm -1): 3196 (Ar-CH), 1590 (C=C), 1334 (C-N), 1463 (N-NO), 1726 (C=O), 1611 (C=N), 758 (C-Cl), 1204 (C-F); 1H-NMR δ (ppm): 6.76–8.21 (m, 7H, Ar-H); 13C-NMR δ (ppm):117.8, 118.4, 121.4, 122.2, 122.3, 124.1, 124.5, 129.6, 131.2, 138.5, 150.8, 161.2, 161.7, 163.4; MASS m/z: 303 (M+) (10%),304 (M+1) (7%); Anal. Calcd. for C14H7ClFN3O2: C, 55.37; H, 2.32; Cl, 11.67; F, 6.26; N, 13.84; O, 10.54. Found: C, 55.34; H, 2.30; Br, 11.66; N, 13.84; O, 10.53.

N-nitroso-3-(4-methylphenylimino)-indolin-2-one 3e

Yield 37.7%; MP 216–218°C; dmf/dmso; IR (KBR, cm−1): 3257 (Ar-CH), 1592 (C=C), 1333 (C-N), 1463 (N-NO), 1742 (C=O), 1611 (C=N), 2910 (C-CH3); 1H-NMR δ (ppm): 6.58-8.04 (m, 8H, Ar-H), 2.2 (s.3H, CH3); 13C-NMR δ (ppm):24.3, 117.6, 121.9, 122.2, 122.3, 124.1, 129.6, 130.2, 130.4, 131.3, 136.8, 138.6, 150.4, 161.0, 163.4; MASS m/z: 265 (M +) (15%); Anal. Calcd.for C15H11N3O2: C, 67.92; H, 4.18; N, 15.84; O, 12.06. Found: C, 67.90; H, 4.17; N, 15.82; O, 12.04.

N-nitroso-3-(4-methoxyphenylimino)-indolin-2-one 3f

Yield 61.3%; MP 225-227°C; dmf/dmso; IR (KBR, cm−1): 3118 (Ar-CH), 1499 (C=C), 1332 (C-N), 1460 (N-NO), 1738 (C=O), 1611 (C=N), 2833 (CO-CH3); 1H-NMR δ (ppm): 6.78-8.21 (m, 8H, Ar-H), 3.3 (s, 3H, OCH3); 13C-NMR δ (ppm): 55.8, 115.4, 115.6, 117.6, 121.7, 123.1, 123.3, 124.6, 129.4, 131.2, 138.8, 145.6, 159.3, 161.8, 163.6; MASS m/z: 281(M+) (3%); Anal. Calcd. for C15H11N3O3: C, 64.05; H, 3.94; N, 14.94; O, 17.07. Found: C, 64.03; H, 3.93; N, 14.92; O, 17.05.

N-nitroso-3-(4-nitrophenylimino)-indolin-2-one 3g

Yield 95.4%; MP 88-90°C; dmf/dmso; IR (KBR, cm−1): 3370 (Ar-CH), 1584 (C=C), 1301 (C-N), 1471 (N-NO), 1734 (C=O), 1621 (C=N), 1367 (C-NO2); 1H-NMR δ (ppm): 6.92–8.21 (m, 8H, Ar-H); 13C-NMR δ (ppm): 117.8, 121.4, 122.6, 122.7, 123.1, 123.3, 124.5, 129.6, 131.2, 138.8, 146.8, 158.8, 161.7, 163.9; MASS m/z: 296 (M+) (7%); Anal. Calcd. for C14H8N4O4: C, 56.76; H, 2.72; N, 18.91; O, 21.60. Found: C, 56.74; H, 2.71; N, 18.90; O, 21.58.

N-nitroso-3-(2, 4-dinitrophenylimino)-indolin-2-one 3h

Yield 76.4%; MP120-122°C; dmf/dmso; IR (KBR, cm−1): 3335 (Ar-CH), 1584 (C=C), 1301 (C-N), 1464 (N-NO), 1732 (C=O), 1624 (C=N), 1388 (C-NO2); 1H-NMR δ (ppm): 7.03-8.28 (m, 7H, Ar-H); 13C-NMR δ (ppm): 117.8, 120.4, 121.6, 124.1, 124.5, 128.3, 129.6, 131.2, 138.0, 142.3, 147.8, 154.2, 161.0, 163.3; MASS m/z: 341 (M+) (2%); Anal. Calcd. for C14H8N5O6: C, 49.28; H, 2.07; N, 20.52; O, 28.13. Found: C, 49.26; H, 2.07; N, 20.50; O, 28.11.

Pharmacology

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay measures the metabolic activity of the viable cells.[22] The assay can be performed entirely in a microtiter plate (MTP). It is suitable for measuring cell proliferation, cell viability, or cytotoxicity. The reaction between MTT and mitochondrial dehydrogenase produces water-insoluble formazan salt. This method involves culturing the cells in a 96-well MTP and then incubating the culture with MTT solution for approximately 2 h. During the incubation period, viable cells convert MTT to a water-insoluble formazan dye. The formazan dye in the MTP is solubilized and quantified with an ELISA plate reader. The absorbance directly correlates with the cell number. This is applicable for adherent cells cultured in MTP.

MTT assay

The MTT cell viability assay was performed as previously described.[22] In brief, MTT was supplied as a stock solution (5 mg/ml PBS; pH 7.2) and sterile-filtered. First, 25 μl of MTT solution was added to each well and then, after 4 h at 37°C, 100 μl of solubilizing buffer (10% Sodium dodecyl sulphate (SDS) dissolved in 0.01 N HCl) was added to each well. After overnight incubation, the absorbance was determined by an ELISA plate reader (FLASHScan® S12, Analytik Jena, Germany) at 570 nm as a read-out for cell viability. The viable cells produced dark blue formazan products, whereas no such staining was observed in dead cells. Cell viability of treated samples (SampleV) was calculated in reference to the untreated control cell line, which was defined as 100% viability (maximal viability; MaxV). The H2O2–treated cell control was defined as minimal viability reference (minimal viability; MinV). Thus, the degree of inhibition of drug-treated cells can be expressed as a percentage of the untreated cell control, using the formula:

Viability (%) = [100 × (SampleV-MinV)/(MaxV-MinV)]

Results and Discussion

N-Nitrosoisatins were prepared from isatin, sodium nitrite, and hydrochloric acid by nitrosation and subjected to Schiff reaction with aromatic amines. The series of novel isatin derivatives 3a–h are presented in Scheme, Table 1. The compound of this study were characterized by IR, 1H-NMR, 13C-NMR, and mass spectral data and confirmed by elemental analysis. The biological evaluation leads to better understanding of the importance of the interaction of chemical moieties with structural features of the synthesized compounds. Substitution of aryl amine in the 3rd position of the nitrosoisatin ring [Figure 1] significantly affects the anticancer activity. N-Nitrosamines have been drawing considerable interest in recent years due to their strong carcinogenic and mutagenic properties. In the present investigation we have substituted aryl amine at the 3rd position, which enhances the carcinogenic activity. The carcinogenic effect of compounds 3a-h was assessed by comparing them with comparing them with control. The results are summarised in Table 2. The percentage viability of cells was compared with control (100% viable cell). The results show that at 200 μm dose, compounds 3h, 3a, 3c, 3e, and 3g exhibit high carcinogenic activity, compounds 3b and 3f exhibit moderate activity, and compound 3d relatively less activity. At a dose of 100 μm, compound 3h exhibits high activity,compounds 3e and 3g exhibit moderate activity, and compound 3b exhibits less activity. At 50 μm dose, compound 3h exhibits high activity, while compounds 3e, 3g, 3b, and 3f relatively less activity. At low dose, compounds 3a, 3c, and 3h exhibit high activity, compound 3b exhibits moderate activity, and compound 3g exhibits less activity. Among these compound N-nitroso-3-(2, 4-dinitrophenylimino)-indolin-2-one 3h [Figure 2] exhibits high activity. All the Schiff bases were to be devoid of anticancer activity at the experimental dose levels due to the effect of the aryl amine substituents in the 3rd position of N-nitrosoisatin.

Table 1

Carcinogenic studies of synthesized compounds

Figure 1

The active position of nitrosoisatin ring for carcinogenic activity

Table 2

Carcinogenic studies of synthesised compounds

Figure 2

The active substituent's of aryl ring for carcinogenic activity

Novel indolin-2-one derivatives

In conclusion, the present study highlights the importance of the structural features in the carcinogenic activity of N-nitrosoisatins. Further investigations involving quantitative structural activity relationship (QSAR) and pharmacokinetic parameters are required to identify the exact chemical entities responsible for the potent anticancer activity, which may form a novel lead to anticancer therapy in this millennium.