Synthesis and Cytotoxic Distinction of Benzo[h]naphtho[1,2-b] [1,6] Naphthyridine and its Isomeric Benzo[b]naphtho[1,2-h][1,6] Naphthyridines

Benzo[h]naphtho[1,2-b][1,6]naphthyridine and its isomeric benzo[b]naphtho[1,2-h][1,6]naphthyridine with aliphatic, aromatic and hetero substitution were synthesized and screened for its antiproliferative activity against four human cancer cell lines. Among these, HeLa cells are more susceptible to compounds 3a, 3b, 9a and 9b with IC50 values of 3.62, 1.05, 6.21 and 1.41 μM respectively. Interestingly chloro substituted compound 9b showed IC50 values of 5.93, 7.01, and 6.81 μM against MCF7, K562 and Hep-G2 cancer cells, which is more active than the standard adriamycin. Furthermore chloro substituted compound 3b displayed good activity against MCF7 (IC50 6.63 μM) and K562 (IC50 7.23 μM) cancer cell lines. This study also revealed that, benzo[h]naphtho[1,2-b][1,6] naphthyridine series were more active than its isomeric benzo[b]naphtho[1,2-h][1,6] naphthyridines.


Introduction
The need of exploring novel synthetic strategies to make new heterocycles is still expanding owing to meet the challenges in identifying new lead compounds for various therapeutic areas. It is also quite evident from literature that closely related isomers/heterocycles behave quite differently to the biological target [1]. For instance, it was demonstrated that 6-isomers of 5, 8-O dimethyl acylshikonin derivatives exhibit higher inhibitory effects on DNA topoisomerase-I and also had upper hand in vitro IC 50 values against L1210 cell than its corresponding 2-isomer ( Figure 1). This triggers further interest to study one of the potent isomers which lead to potential candidate both in vitro and in vivo studies using KM mice model [2,3]. When screened a small collection of tricyclic 4-(phenylamino)furo [2,3-b] quinolone and its positional isomer, 2-(furan-2-yl)-4-(phenylamino) quinolone against 60 NCI cancer cells ( Figure 1). Tzeng et al. found that one of the former isomer turns out to be more cytotoxic whereas its corresponding isomer is inactive [4]. A comparison of the biological activity of isomers with varying alkyl substitutions on the heterocyclic nitrogen of benzhydro[f]quinoline derivatives was made. The trans-isomer was effective rather than the cis-isomer in relaxing methacholine contracted guinea-pig trachea through a β-adrenergic mechanism since propranol blocked this response [5].
These interesting facts coupled with our current interest in unraveling the interesting anticancer properties of various nitrogen heterocycles prompted us to explore the biological activities of two positional isomers in greater detail. A general strategy to obtain both benzo napthonaphthyridines and its isomer was developed and their in vitro cytotoxicity we studied systematically and the results are presented in this manuscript.

Experimental Protocols General
Melting points (m.p.) were determined on Mettler FP 51 apparatus (Mettler Instruments) and are uncorrected. They are expressed in degree centigrade (°C). A Nicolet Avatar Model FT-IR spectrophotometer was used to record the IR spectrum (4000-400 cm -1 ). 1 H NMR and 13 C NMR spectra were recorded on Bruker AV 400 [400 MHz ( 1 H) and 100 MHz ( 13 C)] and AV 500 [500 MHz ( 1 H) and 125 MHz ( 13 C)] spectrometer using tetramethylsilane (TMS) as an internal reference. The chemical shifts are expressed in parts per million (ppm). Mass spectra (MS) were recorded on Auto Spec EI+ Shimadzu QP 2010 PLUS GC-MS mass spectrometer. Microanalyses were performed on a Vario EL III model CHNS analyzer (Vario, Germany). The solvent and reagents used for the preparations were of reagent grade and were purified by standard methods; petroleum ether used was of boiling range 60-80 °C. Anhydrous sodium sulphate was used to dry the solution of organic extracts. Thin layer chromatography (TLC) was performed using glass plates coated with silica gel-G containing 13% calcium sulphate as binder. Ethyl acetate and petroleum ether were used as developing solvents. A chamber containing iodine vapour was used to locate the spots. Separation and purification of the crude products was carried out using chromatographic columns packed with activated silica gel (60-120 mesh). In the case of mixture of solvents used for elution, the ratio of the mixture is given in brackets.

In vitro cytotoxicity
Experimental procedure for SRB assay: The cell lines (K562, MCF7, Hep-G2, and HeLa) were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. For present screening experiment, cells were inoculated into 96 well microtiter plates in 90 µL at plating densities as shown in the study details above, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37°C, 5% CO 2 , 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, one plate of each cell line was fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs were solubilized in appropriate solvent at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to 10 times the desired final maximum test concentration with complete medium containing test compound at a concentration of 10 -3 . Additional three, 10-fold serial dilutions were made to provide a total of four drug concentrations plus control. Aliquots of 10 µl of these different drug dilutions were added to the appropriate micro-titer wells already containing 90 µL of medium, resulting in the required final drug concentrations.

Endpoint measurement:
After compound addition, plates were incubated at standard conditions for 48 hours and assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded; the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) 28,29 solution (50 µl) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing five times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on an Elisa plate reader at a wavelength of 540 nm with 690 nm reference wavelength. The results were expressed as the concentration at which there was 50% inhibition (IC 50 ).
Our aim is to introduce hetero ring in benzonaphthonaphthyridine analogues. In order to achieve this the potential intermediate (3a) was reacted with pyridine-3-carboxylic acid in the presence of PPA (Polyphosphoric acid) at 110°C for an hour. IR spectrum of compound 4a showed three sharp bands at 1634 cm -1 , 1598 cm -1 , 1547 cm -1 confirms the presence of three C=N groups. In its 1 H NMR spectrum two singlets at δ 2.36 and 2.92 accounts for C 4 and C 8 -CH 3 respectively. All other aromatic protons resonated in the region at δ 7.15-9.68. Its 13 C NMR spectrum clearly showed the presence of 27 carbons. All the spectral and analytical details attest the structure of the compound as 2,6-dimethyl-7-(pyridin-3'-yl)benzo[h]naphtho[1,2-b] [1,6]naphthyridine (7a). The same reaction was carried out with other hetero substituted carboxylic acids like furan-2-carboxylic acid and thiophen-2-carboxylic acid, the reaction conditions (including time and temperature) is represented in Table 1. The structures of all the compounds (7a,b-9a,b) were established by elemental and spectral analysis (Refer experimental section).
We envisaged the synthesis of benzo[b]naphtho [1,2-h] [1,6] naphthyridine isomer, the second isomer by treating 4-Chloro-2methylbenzo[h]quinoline [28] (10) with p-toluidine (11a) under neat condition at 190°C (Scheme 2). As expected compound 12a was obtained as a brown solid in 73% yield. In IR spectrum the absorption bands at 3371 cm -1 and 1628 cm -1 confirms the presence of NH and C=N functional groups. Its 1 H NMR spectrum showed the presence of methyl groups at δ 2.37 and 2.70 for C 4 ' and C 2 -CH 3 . The peculiar C 3 -H appeared as a singlet at δ 6.63. All the 10 aromatic protons appeared at δ 7.62-9.27 while two broad singlets each for one proton integration observed at δ 10.35 and δ 13.56 were assigned for C 4 -NH amino form and N 1 -H imino form respectively. The ratio of amino and imino form was found to be 1:1. Its 13 C NMR spectrum confirmed the presence of 21 carbons.
The IR Spectrum of 13a showed the absorption bands at 1624 cm -1 and 1561 cm -1 which were due to two C=N functional groups. The 1 H NMR spectrum of 13a exhibited two singlets each at δ 2.38 and 3.15 for C 6 -CH 3 and C 11 -CH 3 respectively. All the aromatic protons resonated at δ 7.35-8.25 except for two proton doublets which were very much deshielded at δ 9.39 (J=8.00 Hz) and δ 9.45 (J=9.00 Hz). With the help of 2D NMR studies (H,H-COSY, C,H-COSY, HSQC and HMBC) the deshielded proton at δ 9.39 was assigned for C 4 -H while the proton at δ 9.45 for C 13 -H. Its 13 C NMR spectrum showed the appearance of 28 carbon signals and the mass spectrum identified the molecular ion peak at m/z 384. From its elemental analysis the molecular formula was deduced as C 28 H 20 N 2 . All the above spectral and analytical details attest the structure of the compound as 6,9-dimethyl-7-phenylbenzo[b] naphtho [1,2-h] [1,6]naphthyridine (13a). The generality of the reaction was tested with 4-chloroaniline (12b) in order to get the corresponding benzonaphthonaphthyridines (13b). The similar set of reaction was also extended to other carboxylic acids i.e., acetic acid and 1-naphthoic acid to get 6,7 -dimethylbenzo[b]naphtho [1,2- [1,6] naphthyridine 15 respectively (Scheme 3). In all cases the C 4 -H and C 13 -H were deshielded. The reason for the two protons to get deshielded very much could be due to the interaction of these protons with the nitrogen atom at 5 th and 12 th position.
Further substrate scope of the reaction was examined using pyridine-3-carboxylic acid, furan-2-carboxylic acid and thiophen-2-carboxylic acid the reaction conditions (including time and temperature) are represented in Table 2. The structures of all compounds were confirmed by elemental and spectral analysis (Refer experimental section).
The precursors for the cyclization 3a (quinoline moiety and its 4 th position was substituted by naphthyl amine) and 12a (benzoquinoline core moiety and its 4 th position was substituted with aniline derivatives) showed good activity against all the four cell lines with IC 50 range of 3.62-17.80 µM and 28.77-49.20 µM (Entry 1 in Tables 3 and 4) respectively. Very interestingly, incorporation of chlorine in 3a i.e., compound 3b (Entry 2 in Table 3) showed very good activity with IC 50 values of 1.05, 11.65 µM against HeLa and Hep-G2 cell lines which are 10 and 2 fold more active than adriyamycin (11.52 and 21.73 µM) whereas for K562 and MCF7 cell lines its IC 50 values are 7.23 and 6.63 µM respectively, which is comparable with positive control ADR (8.71 and 9.93 µM). Similarly, 12b (chlorine derivative) is more active than its methyl derivative compound 12a, (Entries 1 and 2 in Table 4). The overall comparision of intermediates depicts that 3b is more potential than 12b which is pictorially represented in Figure 2.
Substitution of methyl, phenyl substituents at 7 th position (Entries 3-6 in Tables 3 and 4) did not give beneficial results in both the isomers (4a,b-5a,b and 13a,b-14a,b), whereas increasing the hydrophobicity from phenyl to naphthyl, increases the activity marginally in both isomers (Entries 7, 8 in Tables 3 and 4).
As evident from the Tables 3 and 4 (Entries 9-14), a clear trend was found in cytotoxicity when the substitution at 7 th position containing pyridine moiety (7a,b and 16a,b) was replaced by furan ring (8a,b and 17a,b) which in turn is replaced by thiophene moiety (9a,b and 18a,b). Compound 7a derived from pyridine carboxylic acid showed moderate anticancer activity (IC 50 value range 18.06-49.01 µM) and the activity further increases marginally by a chlorine substitution at 2 nd position (7b Interestingly when pyridine carboxylic acid is replaced by furan and thiophene carboxylic acids the activity shoots up steeply. For compounds 8a,b similar pattern was observed that chloro substituted compound 8b was more active than methyl substituted compound 8a as mentioned earlier. Compound 8b displayed stronger cytotoxicity against MCF7 and Hep-G2 cell lines with IC 50 values 9.03 and 10.76 µM. It showed almost equipotent activity with the control against MCF7 and tenfold more active against Hep-G2 cancer cell. For K562 and HeLa cancer lines 8b showed moderate activity. Its isomeric compound 17b showed moderate activity against all the four cell lines. To our delight, thiophene substituted isomers, (9a,b and 18a,b) showed the best anti-proliferative activity among the compounds screened in this study. Among them 9b is the most outstanding compound which showed highest range of activity in this series with IC 50 values 7.01, 5.93, 6.86 and 1.41 µM against K562, MCF7, Hep-G2 and HeLa cell lines which were 15 fold active than standard against Hep-G2 and 10 fold more active against HeLa cancer cell lines. Its isomeric compound 18b also showed excellent activity against Hep-G2 cancer cell line with an IC 50 value 17.85 which was four fold active when compared to standard and showed significant activity against MCF7 and HeLa cell lines with IC 50 13.68 and 14.91 and least activity against K562 cell line. This observation strongly support that the presence of thiophene moiety [31,32] enhance the cytotoxic activity. By comparing the two isomers, various substituted benzo[h]naphtho [1,2-b] [1,6] naphthyridine series (4-9) were more active than its isomeric various substituted benzo [b] naphtho [1,2-h] [1,6] naphthyridines (13)(14)(15)(16)(17)(18) and is pictorially depicted in Figure 3.

Conclusion
In conclusion, We have synthesized 7-substituted benzo[h] naphtho [1,2- bearing chloro group and the compound 14 holds chloro group and thiophene moiety in naphthyridine nucleus turns out to be the best candidate in the series screened. All the above results indicates that these new compounds represent useful templates for development of new anticancer agents.