Synthesis and biological evaluation of new indazole derivatives

New N-methyl and N-ethyl substitutions in the indazole nucleus are reported by reacting 3-(2aminobenzamido)indazole and the appropriate trimethyl/triethyl orthobenzoate. Single crystal Xray analysis confirms the N-ethylation position for the 3-(1-ethyl-1H-indazol-3-yl)-2phenylquinazolin-4(3H)-one derivative 3f. Compounds 11a-d and 3a-d were tested to evaluate their antimicrobial, antiproliferative and COX inhibitory activities, showing scarce or moderately antiproliferative activity and some inhibitory activity against COX-1 and COX-2.


Introduction
The 4(3H)-quinazolinone ring system I represents a very attractive scaffold to obtain new molecules endowed with a wide range of pharmacological properties.
It has been reported that the 4(3H)-quinazolinone moiety is present in a large number of compounds with anticonvulsant and CNS depressant, 1 analgesic and anti-inflammatory, 2 antitumoral, 3 antileukemic, 4 and antifungal 5 activities.As these pharmacological properties mostly depend on the substituents at 2 and 3 positions, we were interested since a long time to study the influence of different substituents on the 4(3H)-quinazolinone ring.Recently, the synthesis and the biological evaluation of some 2-phenyl-3-(substituted-benzothiazol-2-yl)-4(3H)-quinazolinone derivatives, endowed with anti-inflammatory and antimicrobial activities, have been reported. 6In particular, the interesting antiproliferative activity of 3-indazolylsubstituted 4(3H)-quinazolinones bearing hydrogen, methyl or ethyl moieties at C(2) have drawn our attention. 7,8These observations prompted us to study in depth the 3-indazolyl-substituted 4(3H)-quinazolinones through the synthesis of new derivatives bearing a phenyl group at C (2), in order to ascertain the role and the possible advantage of this substitution over the hydrogen, methyl or ethyl-analogs regarding the above mentioned biological activities.
Furthermore, the reaction of compound 1a with trimethyl orthobenzoate 2g gave the Nmethylated indazole nucleus, probably the 3g derivative, as with the ethyl analogue.
A review of the literature showed that orthoesters are potential alkylating agents. 10Our findings represent the first example of methylation/ethylation in the indazole series, evidencing that the methylation/ethylation of indazole nucleus is deeply influenced by the orthoester used in the reaction.In fact, the methylation/ethylation was observed only with the orthobenzoates, while the unethylated 3-indazolyl-4(3H)-quinazolinones were obtained if triethyl orthoformate, triethyl orthoacetate or triethyl orthopropionate were used. 8The possible reason of such result was likely due to the higher boiling points of orthobenzoates with respect to the other orthoesters (Table 1).In order to verify this statement, a mixture of 3-(2-aminobenzamido)indazole 1a in triethyl orthobenzoate 2f was left at 160°C for 5 hours (Scheme 1) giving the 3-(1H-indazol-3-yl)-2phenylquinazolin-4(3H)-one 3a as the main product (20% yield) together with the starting material, instead of the N-ethyl derivative 3f (Scheme 1).

Scheme 4
On the basis of the results of these two synthetic routes we can conclude that the cyclization of 11a-d into 3a-d occurred via thermal rearrangement of the benzoylamidobenzoyl group through the possible intermediate 9a-d (Scheme 5).A similar rearrangement was previously reported in the pyrazole series. 16
The structures of the new compounds 3a-d, 8a, 9a and 11a-d, were characterized by analytical and spectroscopic measurements (see Experimental).

Conclusions
Reactions of 3-(2-aminobenzamido)indazole and the appropriate trimethyl/triethyl orthobenzoate give N-methyl/N-ethylation of the indazole nucleus, representing the first example of Nalkylation in the indazole series.This is probably related to the higher boiling point of the orthobenzoate than the other orthoesters, for which the N-alkylation does not occur.The correct position of the N-alkylation has been clarified by the X-ray analysis of the 3-(1-ethyl-1Hindazol-3-yl)-2-phenylquinazolin-4(3H)-one derivative 3f.The 2-phenyl-3(indazol-3-yl)quinazolin-4(3H)-ones 3a-d and N-(2-(3-amino-1H-indazole-1-carbonyl)phenyl)benzamides 11a-d present scarcely or moderately antiproliferative activity against K-562 (human chronic myelogenus leukemia), HT29 (colon cancer cells) and NCI-H460 (human large cell lung cancer cells).In vitro testing for 3 and 11 indicated that they are endowed with inhibitory activity against COX-1 and COX-2.Further investigation of biology of this kind of products is in progress and the results were published elsewhere.

Experimental Section
General.All melting points were determined on a Buchi 530 capillary melting points apparatus; IR spectra were recorded with a Jasco Spectrum IR-810.System spectrophometer as solid in KBr disc or nujol mull supported on NaCl disks; 1 H and 13 C NMR spectra were obtained in DMSO-d 6 at 300.13 and 75.47 MHz respectively, using a Bruker AC series 300 MHz spectrometer (TMS as internal reference).Mass spectra were recorded on a JEOL jms-0I-SG-2 spectrometer at 75 eV (100µA).Microanalyses (C, H, N), performed in the Redox snc laboratories, were within ±0.4 % of the theoretical values.The microwave reactions were carried out in an Anton Paar Synthos 3000 instrument.

General procedure for 3-(1-alkyl-1H-indazol-3-yl)-2-phenylquinazolin-4(3H)-ones (3f,g)
4 Mmoles of 3-(2-aminobenzamido)indazole 1 were dissolved by heating in 10 ml of orthobenzoate 2f,g and the solution was refluxed for 10 h.Evaporation of the solvent under reduced pressure gave a residue which was recrystallized from ethanol to give pure 3g,f.3f.Yield 55-65 %; mp 208-210°C; MS (m/z) 366 (M + ); IR (KBr) cm -1 1685 (CO); Crystals of 3f were obtained as colorless prisms from a water-methanol 1:1 solution by slow evaporation at room temperature.They were mounted on an Enraf Nonius CAD-4 diffractometer using MoKα (λ MoKα =0.71073Å) radiation at room temperature (293K).The lattice parameters were determined by least-squares refinements of 25 high angle reflections.The structure was solved by direct methods, 19 and the refinement was carried out by full-matrix least-squares with SHELX-97. 20All non-H-atoms were refined anisotropically.A summary of the crystal data, data collection, and structure refinement is presented in Table 3; selected bond lengths and angles are reported in Table 4. Geometrical calculations were carried out with the program PARST. 21