Synthesis and Antiproliferative Activity of Chlorinated Maprotiline Analogues

A novel chlorinated tetracyclic compound 13 of the class ethanoanthracene as analogue of maprotiline was prepared via multistep syntheses. The tetracyclic key intermediate 5 with its 2.2.2 system was built nicely and economically in one-step via a Diels-Alder reaction between acrolein and 1,8-dichloroanthracene, followed by Wittig C-2 homologation. The synthesized chlorinated maprotiline analogues 6 , 7 and 13 as well intermediates 4 and 5 exerted antiproliferative activity in cancer cell lines A549 and HePG2 at low micromolar concentrations. In addition, the intermediates 4 and 5 were also had high activity against HCT cell line. Interestingly, the intermediate 4 was the most active against all cell lines studied. caspase BL lines as well some of multi-drug resistant many of these active than maprotiline that all tested analogues well intermediates inhibit growths the cell lines A549 analogues cell and 4.44 µg/ml analogues on HCT cell intermediates compounds 4 and 5 formyl compounds 6, 7 and against A549 cell lines, IC50 compound 4 and and 3.71 against A549 and µg/ml

to distinguish between the Z 8 and E 9 isomers. The ratio of the isomers, as deduced from integration of the vinylic proton signals, is approximately 1:2. The 1H-NMR spectrum of the Z 8 showed double doublet signal at δ 5.47 ppm with coupling constants J = 11.3, 9.5 Hz integrated for the proton assigned for olefinic proton (-CH=CH-) and a doublet signal at δ 5.61 ppm with coupling constant J = 11.7 Hz integrated for the proton assigned for olefinic proton (-CH=CH-) attached to ester group (-COO-CH 2 -CH 3 ). Whereas these signals of the E 9 appeared at δ 6.36 ppm as double doublet with coupling constants J = 15.4, 9.5 Hz and at δ 5.75 ppm as doublet with coupling constant J = 15.4 Hz. The α,β unsaturated esters 8 and 9, products of the Wittig reaction, were then subjected to hydrogenation to reduce the double bond by stirring for 24 hours at room temperature in ethanol in presence of Pd/C under H 2 (balloon). After filtration of the reaction mixture through a pad of celite and solvent was removed in vacuo, the saturated ester Ethyl 3-(1,8-dichloro-9,10-dihydro-9,10-ethanoanthracen-11-yl)propanoate 10 was obtained in an excellent yield of 92 %. Reduction of ester 10 with reducing agent diisobutylaluminium hydride (DIBAL) at room temperature gave the alcohol 3-(1,8-Dichloro-9,10-dihydro-9,10-ethanoanthracen-11-yl)propan-1-ol (11) in 53 % yield, which was completely oxidized using Pyridinium Chlorochromate (PCC) at room temperature in dichloromethane to give the desired aldehyde 3-(1,8-Dichloro-9,10dihydro-9,10-ethanoanthracen-11-yl)propanal (12). An attempt to transform the ester 10 to the aldehyde 12 in one step according to the known literature procedure (22) using DIBAL at -78 0 C resulted in a mixture of aldehyde 12 and its corresponding alcohol 11. Direct reductive amination of the aldehyde 12 by the same procedure applied to synthesize the chlorinated maprotiline analogues 6 and 7 led to the desired chlorinated maprotiline analogue 13. The overall yield of the synthesis of the target (76) using DIBAL at -78 0 C and at room temperature was 19 % and 15.3 % respectively.

In vitro anticancer activity evaluation
The cancer cell lines were incubated with serial dilution of each compound (from 7 313 pg ML -1 to 5 mg ML -1 ) in a 96-well plate for 4 days, and then tested for growth inhibition by MTT-Test. Maprotiline 1 had been used as a positive control. Previous studies reported that maprotiline showed a potential antiproliferative activity against BL lymphoma cell line DG-75 (6,7). A number of 9,10-dihydro-9,10ethanoanthracenes were exhibited potent antiproliferative activity through inducing apoptosis and caspase activation in BL cell lines as well some of these compounds displayed activity in multi-drug resistant (MDR) cells. Furthermore many of these compounds were more active than maprotiline (8). Our results showed that all tested maprotiline analogues as well intermediates were able to inhibit the growths of the cancer cell lines A549 and HePG2 at low micromolar concentrations. In addition, the intermediates compounds 4 and 5 were also able to inhibit the growth of a third cell line (HCT). The IC 50 values of all compounds, and that from maprotiline, are given in Table 1. The chlorinated maprotiline analogues 6, 7 and 13 were found to exhibit a potent antiproliferative effect on A549 cell line with IC50 values 25.5, 18.9 and 7.8 µg/ml respectively as well as against HePG2 cell line with IC50 values 12.66, 13.8 and 4.44 µg/ml while these maprotiline analogues had no effect on HCT cell line. The results showed that intermediates compounds 4 and 5 with formyl group were more potent than compounds 6, 7 and 13 against A549 and HepG2 cell lines, the IC50 of compound 4 and 5 were 1.1 and 3.71 against A549 and 0.12 and 0.65 µg/ml against HepG2 respectively. that's mean the sensitivities of the treated cancer cells to 4 and 5 were 6 times higher than maprotiline in case of the breast cancer cell line A459, and even 40 times higher in case of hepatocyte carcinoma cell line HepG2. Furthermore, The IC50 of compounds 4 and 5 against HCT were 0.4 and 0.7 µg/ml respectively. Importantly the intermediates compound 4 with formyl group above on chlorine atom was the most potent against all three tested cancer cell lines. This result shows a direct or an indirect role of the formyl group and its position in the biological activity of the two compounds 4 and 5.
Further investigations for these compounds are suggested.  The compounds 3-7 were synthesized according to (14,20) and characterization is also recorded in supplementary data.

Conclusion
In conclusion, a simple, economical and flexible synthesis route of tetracyclic chlorinated maprotiline analogue was described. The key cyclisation step was achieved through Diels-Alder reaction at room temperature followed by Wittig C-2 homologation. This analogue and intermediates were found to exert potent Maprotiline and chlorinated tetracyclic analogues.

Supplementary Files
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Graphical Abstract.pdf Supplemantray Data.pdf