Synthesis, Molecular Docking and Biological Evaluation of Some New Benzotriazines

Methyl 2-(4-oxobenzotriazin-3(4H)-yl) alkanoates4a-c proved to be important intermediates for the preparation of some biologically interesting compounds containing the benzotriazinone ring system. Esters 4a-c were prepared by direct diazotization of methyl anthranilate followed by addition of amino acid esters hydrochloride in a one-pot strategy. An equivocal synthesis of methyl 2-(4oxobenzotriazin-3(4H)-yl) acetate 4a was achieved by alkylation of benzotriazin-4(3H)one with methyl chloroacetate on the basis of chemoselective reaction of heterocyclic amide with electrophiles. A series of N-alkyl-2-(4-oxobenzotriazin-3(4H)-yl) alkanamides7-8(a-h) and methyl 2-(2-(4oxobenzotriazin-3(4H)-yl)alkanamido)alkanoates (dipeptides) 9-10(a-d) were prepared via azide coupling from 4a-b. Esters 4a-b were converted into the corresponding hydrazides followed by condensation with aldehydes; 4-methoxybenzaldehyde, 4-dimethylamino benzaldehyde and arabinose to afford the corresponding hydrazone derivatives11-13. All the synthesized compounds were subjected to the molecular docking using MOE 2008-10 software as agonist for; E. coli Fab-H receptor and Vitamin D receptor for antibacterial and anticancer evaluation, respectively. The most pronounced strong binding affinity towards the target E. coli Fab-H receptor were compounds 7a, 11a, 11b,10a,10cand 12b. On the other hand, the most pronounced strong binding affinity towards the target Vitamin D receptor were compounds 3, 9c, 11a and 10d. The in vitro antibacterial activity of highest binding affinity docked compounds were tested against E. coli, Staphylococcus aureus and Salmonella spp. All the tested compounds gave effective positive results against E. coli with inhibitory zone of about 1.1 cm, while were inactive against Staphylococcus aureus and Salmonella spp. The in vitro cytotoxic activity of the highest binding affinity docked compounds were tested against human liver carcinoma cell line (HepG2) cancer cell lines. Many compounds showed potent cytotoxic activity with low IC50 values, especially for 3(6.525μM) and 11a (10.97 μM), while for standard drug doxorubicin (5.8 μM).


1-Introduction
One of the most corner stone principles in our research group is based on searching for new anticancer drugs 1,2 where the search for new anticancer drugs is never ending task with the aim to obtain products with lower toxicity and more selectivity towards tumor cells. Benzotriazine and its derivatives possess a diverse range of biological activities of pharmacological activities including antimicrobial, 3 antiinflammatory 4 , anti-depressant 5 , anti-ulcer 6 , antidiarrhoeal 7 , anaesthetics 8 , anticancer 9 . Some current commercial benzotriazinone anticancer drugs such as α-hydroxylatedbenzotriazinone 10  Beside the established applications of benzotriazin-4(3H)-one in dying, 15 imaging and recording material. 16 One of the most important benzotriazin-4(3H)-one derivative is 3-Hydroxy-1,2,3benzotriazin-4(3H)-one where it is a versatile reagent employed for peptide synthesis. 17 In the present work, we aimed to synthesize a series of compounds containing benzotriazinone moiety on the basis of structure modification of methyl 2-(4-oxobenzotriazin-3(4H)-yl) alkanoates 4a-c for biological evaluation as antimicrobial and anticancer agents.

2-Discussion.
Early attempts for preparation of 4a-c as the target molecules for structure modification of benzotriazinone was achieved from isatoic anhydride 1. Isatoic anhydride1 was reacted with ammonia in the presence of ammonium carbonate to give anthranilamide 2, which subsequently diazotized using sodium nitrite and HCl solution at 0 o C to give benzotriazinone 3. The benzotriazinone 3 was reacted with methyl chloroacetate in DMF in the presence of potassium carbonate at 100 o C to give the chemoselective N-alkylated ester; methyl 2-(4-oxobenzotriazin-3(4H)-yl) acetate 4a as the only product on the basis of chemoselective reactivity of heterocyclic amides towards electrophiles, Scheme1 18,19 . The multi-step reactions mentioned gave 4a in an overall high yield from available isatoic anhydride but the alkyl halides needed to prepare other methyl 2-(4-oxobenzotriazin-3(4H)-yl) alkanoates 4a-c were not available. Methyl2-(4-oxobenzotriazin-3(4H)-yl)alkanoates 4a-c are excellent precursors for structure modification of benzotriazinone ring system via azide coupling method by an attachment of either amines or amino acid through a peptide bond. Thus, the ester 4a-b reacted with hydrazine hydrate in ethanol under reflux condition for 6h afforded the corresponding hydrazides 5a,b, Scheme2.
Hydrazides 5a,b were reacted with NaNO 2 and HCl in water at 0 o C for 1 h. to afford the corresponding azides 6a,b and were extracted with ethyl acetate. The insitu generated azide 6a,b solution was successively added to primary amines; isopropyl, n-butyl, tert-butyl, n-decyl, cyclohexyl and benzyl amines and secondary amines; piperidine and morpholine to give N-alkyl-2-(4-
Next, a number of hydrazones 11-12(a,b) was prepared by condensation of hydrazides 5a,b with aldehydes; 4-methoxybenzaldehyde and 4-dimethylaminobenzaldehyde in ethanol under reflux condition for 6h, and gave 11-12(a,b) in excellent yields, Scheme 5.  Finally, the 2-(4-oxobenzotriazin-3(4H)-yl) acetic acid hydrazide 5a was condensed with arabinose in ethanol under reflux condition for 6h, and gave 13, Scheme 6. The results were evaluated based on binding affinity calculation together with cluster size determination and visually through possible interaction with key residues at the active site.

In vitro anti-bacterial activity:
The

In vitro anti-cancer activity:
Potential cytotoxicity of the newly synthesized compounds was tested against human liver carcinoma cell line (HepG2) using the method of Hansen etal. 21,22 The in vitro anticancer screening was done by planting tissue unit in vacsera, Cairo A. R. Egypt.
This work was performed by a modification (Hansen et al., 1989) 21 of the tetrazolium salt (MTT) method Mosmann, 1983. 22 Preferably, cells should be plated in triplicate wells. Relative cell proliferation/viability was measured when eg-treated cells are compared with untreated cells.
The in vitro cytotoxic activity of the highest binding affinity docked compounds were tested against human liver carcinoma cell line (HepG2) cancer cell lines. Many compounds showed potent cytotoxic activity with low IC 50 values, especially for 5 (6.525µM) and 13a (10.97µM), while for standard drug doxorubicin (5.8 µM).

4-Acknowledgements
We would like to thank the Science & Technology Development Fund in Egypt STDF Project ID: 22909 for funding this research proposal.