Synthesis of new Schiff bases bearing 1,2,4-triazole, thiazolidine and chloroazetidine moieties and their pharmacological evaluation

Abstract New compounds based on oxindole moiety were synthesized via the reaction of 5-substitued isatins 1a–e with different nucleophiles such as benzidine, 3,3′-dimethoxybenzidine 2a,b and 2,6-diaminopyridine 3 to afford three different classes of bis-Schiff bases 4a–e, 5a–e and 6a–e, respectively. The structures of the new compounds were elucidated on the basis of their FTIR, 1H NMR, 13C NMR, GC/MS spectral data and elemental analysis. The in vitro antimicrobial activity of the new compounds was evaluated using a broth dilution technique in terms of minimal inhibitory concentration (MIC) against four bacterial and two fungal pathogens and anticancer activities against HELA cervix. The revealed data showed that compound 9d has excellent activity against Gram + ve and Gram –ve bacteria, and compounds 11b presented promising anticancer activity against HELA cervix.


Introduction
The oxo-derivatives of indole particularly isatins and related compounds are important class of compounds due to their biological effects, including antifungal, antiviral, anticancer and antiproliferative activities 1,2 . These compounds are of great interest in oncology, microbiology and immunology 2 . Hence a significant rising research interest in the design of different oxindoles and related compounds as drugs is currently observed in the field of medicinal chemistry 3 . Schiff-bases and spiro-thiadiazoline derivatives of isatins have shown remarkable biological activities [4][5][6] . Also oxindoles were reported earlier due to their marked cytotoxicity 7-10. The compounds carrying azomethine functional group -C¼Nwhich are known as Schiff bases have gained importance in medicinal and pharmaceutical fields due to the most versatile organic synthetic intermediates and also showing a broad range of biological activities, such as antituberculosis 11,12 , anticancer 13 , analgesic and anti-inflammatory 14 , anticonvulsant 15,16 , antibacterial and antifungal activities 17,18 .
Schiff bases are good intermediates for the synthesis of many heterocyclic ring systems like thiazolidinones 19 and azetidinones 20 etc.
Schiff bases are used as substrates in the preparation of a number of industrial and biologically active compounds via ring closure, cycloaddition and replacement reactions 21 . Moreover, Schiff bases derived from various heterocycles have been reported to possess cytotoxic 22 , anticonvulsant 23 , antiproliferative 24 , antimicrobial 25 and anticancer activities 26 .
Schiff bases are reported to possess antimicrobial activities. Heterocycles bearing nitrogen, sulfur and thiazole moieties constitute the core structure of a number of biologically interesting compounds 27 . Schiff base complexes derived from heterocyclic compounds have found increased interest in the context of bioinorganic chemistry [28][29][30][31] .
The chemistry of 1,2,4-triazole and its fused heterocyclic derivatives has received considerable attention owing to their synthetic and effective biological importance. 1,2,4-triazole moieties have been incorporated into a variety of therapeutically interesting drug candidates including antiviral (ribavarin), anti-migraine (rizatriptan), antifungal (fluconazole) and antianxiety compounds (alprazolam).The pharmacological importance of heterocycles derived from 1,2,4-triazole paved the way towards active research in a triazole chemistry 32 .
In view of these reports and in continuation of our research on synthesis of biologically active molecules [33][34][35][36][37][38][39][40][41] , we hereby report the synthesis of some new Schiff bases bearing triazole, thiazolidine and chloroazetidine moieties and evaluation of their biological activities as antimicrobial and anticancer agents.

General remarks
All melting points are uncorrected and were determined on a Gallenkamp Instrument (London, UK). IR and NMR recorded on Perkin-Elmer-1430 infrared spectrophotometer (Waltham, MA) using the potassium bromide wafer or the Nujol mull technique for metal complexes and 1 H NMR, 13 C NMR spectra were measured in DMSO-d 6 measured on a Varian Genini-300, 500 MHz spectrophotometer (Varian, Palo Alto, CA) and chemical shifts d are in ppm. The mass spectra were measured on a HP GC MS-QPL000EX (Shimadzu, Tokyo, Japan) mass spectrophotometer at 70 ev. Microanalyses were carried out using a Perkin Elmer 2400 CHN elemental analyzer (Waltham, MA). The metal percentage was estimated using inductively coupled argon plasma (ICP) technique on a 6500 Duo apparatus, Thermo Scientific (Mahwah, NJ).
A 1000 mg/L multi-element and certified standard solution (Merck, Darmstadt, Germany) was used as the stock solution for instrument standardization. A microwave Digestion Lab Station closed system, Ethos Pro; Milestone, Italy was used to digest the organic matter in aqua regia. UV-Vis spectra were measured on UV-1600 spectrophotometer. The solid reflectance spectra were measured on a Shimadzu 3101 pc spectrophotometer. Magnetic susceptibilities of the metal complexes were measured at room temperature using a magnetic susceptibility Sherwood Scientific apparatus (Cambridge, UK). The molar conductance values of the metal chelates were calculated using a conductivity meter ORION model 150 with a 0.6 cell constant.
In this study, Explorer Automated Microwave Synthesis Work station (CEM) was used for the synthesis of the compounds.
Four bacterial strains and two fungal strains from the Basic Science Department, Faculty of Applied Medical Science, October 6th University were employed for minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) microbial counts.
The primary evaluation of the in vitro cytotoxicity of the compounds under investigation against human tumor cells was tested at the National Cancer Institute (NCI), Cairo University, Egypt.
Using microwave irradiation A mixture of pyridine-2,6-diamine 3 (0.004 mol, 0.436 g) and 5-substituted isatins 1a-e (0.008 mol) in the minimum quantity of ethanol (5 ml, required to form a slurry) was irradiated with microwave radiation under controlled condition. On cooling, pure crystals were separated (TLC).
[Method B] A mixture of Schiff bases 4b, 5a (0.001 mol) and mercaptoacetic acid (0.002 mol) was taken in DMF in a round-bottom flask fitted with a Dean Stark apparatus. The mixture was refluxed for 6 h with removal of water azeotropically. A sticky solid was formed on evaporating of solvent and was treated with a solution of sodium bicarbonate to remove excess of acid. The solid formed was filtered, washed with water, dried and recrystallized from ethanol to give 7a,b.

Pharmacological evaluation
In vitro antimicrobial measurement The compounds were tested for their in vitro antimicrobial activity by the broth-dilution technique in terms of minimum inhibitory concentrations (MIC). Experimentally for potent Drugs 42 . The antimicrobial activities of the compounds in this study were evaluated against six pathogenic microbial species: Gram þ ve bacteria Staphylococcus aureus and Staphylococcus epidermidis, Gram -ve bacteria Escherichia coli and Klebsiella pneumonia and fungi Aspergillus fumigatu, and Candida albicans. Reference drugs used were sulfamethoxazole as an antibacterial standard and fluconazole as an antifungal standard.

In vitro cytotoxicity evaluation
The primary evaluation of in vitro cytotoxicity of the selected new compounds against human tumor cells was carried out at the NCI (Cairo University, Cairo, Egypt) using the method of Skehan and Storeng 43 . The cytotoxicity evaluation also involved the use of vinblastine sulfate or doxorubicin 44 as antitumor drug reference standards. The procedure used was as follows: at 37 C and in atmosphere of 5% CO 2 . 4. After 48 h, the cells were fixed, washed and stained with sulfo-Rhodamine-B. 5. Excess stain was washed away with acetic acid and attached stain was recovered with Tris-EDTA buffer. 6. Color intensity was measured with an ELISA reader. 7. The relationship between the surviving fraction and drug concentration is plotted to give the survival curve of cancer breast cell line.
The results of the in vitro cytotoxicity activity on human tumor cell line HELA (cervix) were determined according to the dose values of the drug exposure required to reduce survival in the cell lines to 50%.
This study was extended to prepare new heterocycles such as bis spirothiazolidin-4-one derivatives 7a,b, bis spiroazetidinone derivatives 8a,b and bis spiro1,2,4-triazole derivatives 9a-e, respectively by the reaction of bis-Schiff bases 4b, 5a-d with mercaptoacetic acid in the presence of anhydrous ZnCl 2 , chloroacetyl chloride in presence of triethylamine at temperature 80 C and thiosemicarbazide as illustrated in Scheme 2. The structure of these compounds was confirmed by FTIR, 1 H NMR 13 C NMR, and MS spectra and elemental analysis. The 1 H NMR spectrum of bis Scheme 1. Synthesis of the target compounds 4a-e, 5a-e and 6a-e. The reaction of bis-Schiff bases 6a,b with formaldehyde in the presence of a secondary amines such as piperidine and morpholine yielded the N-Mannich bases 10a-d, respectively, as illustrated in Scheme 4. The structure of these compounds was established from FTIR, 1 H NMR, 13  The complexes 11a-f were prepared by condensation reaction of 2,6-diamine pyridine with 5-substituted isatins 1b,c in the presence of MCl 2 .nH 2 O salts (where M ¼ Cu, Co or Ni).The structure of metal complexes 11a-f was confirmed by elemental analysis and spectral studies. The elemental analysis showed a ratio of 2:2:1 [isatins:DAP:MCl 2 ] as shown in Scheme 4. The theoretical values were in a good agreement with the found values. The presence of chlorine confirmed from element analysis and the low molar conductance values (15-82.30 S cm 2 mol À1 ) for the complexes 11a-f supports the non-electrolytic nature of the metal complexes.
IR spectral studies and mode of coordination of complexes 11a-f In the spectrum of 2,6-diaminopyridine a pair of medium intensity bands present at 3375-3400 cm À1 corresponding to (NH 2 ) but these are absent in the infrared spectra of all the complexes. Further, no strong absorption band was observed at 1750-1700 cm À1 indicating the absence of (C ¼ O) group of 5-substituted isatins. This indicates that the condensation of carbonyl groups of 5-substituted isatins and amino groups of pyridine -2, 6-diamino might have taken place. These results provide strong evidence for the formation of macrocyclic frame. A strong absorption band in the region 1620-1612 cm À1 may be attributed to the (C¼N) group. The lower values of (C¼N) may be explained on the basis of drift of lone pair density of azomethine nitrogen towards the metal atom. The presence of band in the region 3213-3136 cm À1 in the isatin complexes may be assigned due to (N-H) stretching. New bands in the 555-505 cm À1 regions are assigned to stretching frequencies of (M-N) bonds. The unchanged pyridine ring vibrations in the complexes indicate noncoordination of the pyridine nitrogen atom. Moreover, the coordination through pyridine nitrogen is also ruled out on the basis that it will result in the formation of four membered heterocyclic rings, which are sterically unstable. Thus, in the presence of metal salts, a quadridentate macrocycle is formed which coordinates through suitably placed azomethine nitrogen while pyridine nitrogens do not take part in the coordination.

Electronic spectra and magnetic moment studies 11a-f
The magnetic susceptibility measurement for the solid Cu (II) complexes (1.8-2.1 B.M) is indicative of octahedral environment. The diffuse reflectance spectrum of the copper complexes 11a,b showed two band groups at 393-314 and 242-234 nm, these bands can be attributed to p-p Ã and n-p Ã transition states within the hydrazone ligand. Bands at 767-472 nm can be attributed to d-d transition states and ligand to metal charge transfer.
The magnetic susceptibility measurement for the solid Co (II) complexes (3.50-3.90 B.M) is indicative of three unpaired electrons per Co (II) ion suggesting consistency with their octahedral environment. The diffuse reflectance spectrum of the cobalt complexes 11c,d showed two band groups at 396-313 and 254-243 nm, these bands can be attributed to p-p Ã and n-p Ã transition states within the hydrazone ligand. Bands at 769-400 nm can be attributed to d-d transition states and ligand to metal charge transfer.
The magnetic susceptibility measurement for the solid Ni (II) complexe (2.60-2.74 B.M) is indicative of octahedral environment. The diffuse reflectance spectrum of the nickel complexes 11e,f showed two band groups at 411-320 and 266-212 nm, these bands can be attributed to p-p Ã and n-p Ã transition states within the hydrazone ligand. Bands at 768-432 nm can be attributed to d-d transition states and ligand to metal charge transfer.
In vitro cytotoxicity evaluation of mononuclear Cu(II), Co(II) and Ni(II) macrocyclic complexes 11a-f The activities of three different macrocyclic metal complexes 11a-f were performed against the cervix human cancer cell line (HELA). Unexpected low values of activity were obtained with the three series of macrocyclic complexes although the copper complex 11b gave remarkable activity comparable to the reference drug, also the activity obtained with the copper complex 11a may be considered as moderate one Figure 1.

Conclusion
In this study, we report a convenient route for the synthesis of some novel heterocycles incorporating oxindole moiety in order to investigate their antimicrobial, antifungal activity. The in vitro evaluation of their antimicrobial against several pathogenic bacterial and fungal strains revealed that compound 9d showed the highest activity against Gram þ ve and Gram -ve bacteria. The activities of three different macrocyclic metal complexes 11a-f were performed against the cervix human cancer cell line (HELA) .Unexpected low values of activity were obtained with the three series of macrocyclic complexes although the copper complex 11b gave remarkable activity comparable to the reference drug, also the activity obtained with the copper complex 11a may be considered as moderate one.