Synthesis of new pyrido[4,3-g and 3,4-g ]quinoline-5,10-dione and dihydrothieno[2,3-g and 3,2-g ]quinoline-4,9-dione derivatives and preliminary evaluation of cytotoxic activity

Several pyrido[4,3-g and 3,4-g ]quinoline-5,10-dione and dihydrothieno[2,3-g and 3,2-g ]quinoline-4,9-dione derivatives were synthesized and evaluated for their potential cytotoxic properties. A number of these compounds exhibited significant in vitro antiproliferative activity at submicromolar concentration in a preliminary evaluation for their cytotoxic activity using the MT-4 cell line. These structures represent potential scaffolds in discovery of new agents with antitumoral activity and the synthetic strategy developed could be used to prepare libraries of new derivatives by combinatorial chemistry.


Introduction
The quinone nucleus is an important structural moiety in a number of complex chemotherapeutic agents target the DNA, playing an important role in determining their biological activities.Doxorubicin and mitoxantrone are representative of this class and are widely used in the treatment of several leukaemia and lymphomas as well as in combination chemotherapy of solid tumors. 1 The importance of this class of antitumour agents has stimulated a number of studies, aimed to developing new agents that retain the core quinonic moiety yet exhibit different spectra of potency, together with reduced overall toxicity. 24][5] These bioisosteres retain the planarity, spatial, and electronic characteristics required for molecular recognition at the cellular level and would clearly differ from their carbocyclic counterparts in their interaction with specific targets as well as in their reduction potential.
On the other hand, the dihydrothieno[2,3-g and 3,2-g]quinoline-4,9-dione derivatives (DTQQs, 4 and 5) were obtained as 2.2:1 regioisomeric mixture with yields varying from 15-40%, and separated chromatographically.The regiochemistry of compounds 4 and 5 have been assigned by reference to theoretical considerations and by analogy with the work of different authors. 8Thus, the formation of major compound 4 may be attributed to the electronwithdrawing effect of the quinoline nitrogen atom making the C-8 carbonyl group more electron deficient, with preferential attack of the sulphur at the C-6 position.A series of acyl DTQQs derivatives containing different aromatic moiety, were prepared by coupling of 4 and 5 with both 2-iodo (6 and 7) and 3-fluorobenzoyl chloride (8 and 9) using TEA as base, and Boc-L-Phe amino acid using HBTU/HOBt as coupling agents.After Boc-deprotection, the compounds 10 and 11 were obtained as HCl salts.Subsequently, reaction of these compounds with phenylisothiocyanate gave the N-carbamoyl derivatives 12 and 13, respectively.The structures of all compounds were confirmed from their analytical and spectroscopic data.
In this preliminary study, the compounds were tested in vitro for the grow inhibition of MT-4 cell lines, as monitored by the MTT method, 9,10 and the results are reported in Tables 1 and 2.
For comparative purposes, we evaluated the cytotoxic activities of compounds relative to doxorubicine.
As shown in Table 1, the regioisomer mixtures 2,3-b and 2,3-e and the compounds 2c, 3c, and 3d exhibited similar activity against of MT-4 cell line at µM range.Only the compounds supporting a chloride atom at position 4 of phenyl ring showed certain specificity in the interaction with the target.In fact, the compound 3d showed to be 18 times more active compared to the regioisomers 2d.However, more encouraging results were obtained by the DTQQ derivatives.In fact, the regioisomers 4 and 5 showed similar activity at submicromolar concentration (Table 2) and were 4 time more potent that the carbocyclic analogue, (DTNQ, CC 50 = 1.2 µM). 7he incorporation in DTQQs of 2-iodo or 3-fluorobenzoyl residues (6-9) conserved practically the activity in the same range, whereas the incorporation of Phe residues (10, 11)  significantly reduced the cytotoxicity by 10 and 100 fold, respectively.This activity was recovered with derivatives supporting of phenylthio carbamoyl moiety (12, 13).Table 2. Cytotoxic Activity of 3-Amino-3-ethoxycarbonyl-2,3-dihydrothieno[2,3-g] quinoline-4,9-dione (4, 6, 8, 10, 12) and 3-Amino-3-ethoxycarbonyl-2,3-dihydrothieno [3,2- Although the compounds here reported are less active compared to doxorubicine, the dihydrothieno[2,3-g and 3,2-g]quinoline-4,9-dione (DTQQ) derivatives represent a potential starting point in discovering of new antitumoral agents.In particular, the synthetic method developed to produce these compounds could be used to perform libraries by combinatorial approach to optimize the activity of the DTQQ derivatives.In fact, the results obtained indicate that it is possible to design analogues that could be more effective on tumoral cells by introducing of appropriate structural modifications on dihydrothiophene ring.
In conclusion, we report the synthesis and in vitro biological evaluation of new quinone derivatives as potential cytotoxic agents.The first results confirm the validity of our synthetic method providing practical access to quinone-based derivatives of intense current interest in antitumoral therapy.Further experiments aimed at defining the target and the mechanisms of the inhibitory effect showed by these molecules are in progress and the results will be reported in a forthcoming paper.Reagents, starting material and solvents were purchased from commercial suppliers and used as received.Analytical TLC was performed on a 0.25 mm layer of silica gel 60 F 254 Merck and silica gel 60 (300-400 mesh), Merck, was used for flash chromatography.Melting points were taken on a Kofler apparatus and are uncorrected. 1H NMR and 13 C NMR spectra were recorded with a Bruker-500 spectrometer, operating at 500 and 125 MHz respectively.Chemical shifts are reported in δ values (ppm) relative to internal Me 4 Si and J values are reported in Herz (Hz).Mass spectra were obtained using a FAB-MS spectrometer.IR spectra were measured on a Nicolet Avatar 360 FT-IR; values are expressed in wavenumbers (cm -1 ).

Biological Activity Assays
Assays of antiviral and cytotoxic activities were carried out following established procedures. 10,11 he compounds were dissolved in DMSO at an initial concentration of 200 µM and then were serially diluted in culture medium.Tumour cell growth at each drug concentration was expressed as percentage of untreated controls, and the concentration resulting in 50% (CC 50 ) growth inhibition was determined by linear regression analysis.

Figure 1 .
Figure 1.Structures of compounds I and II.

Table 1 .
Cytotoxic a Compound concentration (µM) required to reduced the viability of mock-infected cells by 50%, as determined by MTT method.