Cytotoxic evaluation of substituted azetopyrroloazepinones

.


Introduction
Cancer is one of the main causes of death in the world despite considerable progress in the understanding of its biology and pharmacology.The traditional therapeutic strategies for the treatment of this disease are surgery, radiotherapy, immunotherapy and chemotherapy.For the time being, 50% of the patients diagnosed with cancer are cured either through one of these methods or by a combination of them.For some types of disseminated cancers, chemotherapy is the only effective therapy because it distributes anticancer drugs through the circulatory system. 1 We are currently engaged in a program aimed at synthesizing novel heterocyclic compounds that inhibit the growth of cancer cells. 2 One of these compounds is azetopyrroloazepinone 1a, which showed in vitro cytotoxic activity against PC-3 (prostate) and U251 (CNS) cancer cells lines. 3he mechanism of action of antiproliferative activity of compound 1a is still unknown, therefore, further exploration of the azetopyrroloazepinone pharmacophore is advisable.The aim of the present study was both to explore the role of the substituents attached to the 3-phenyl ring of compounds 1a-g on the inhibition of cancer cell growth, and to identify an optimal candidate among currently available compounds.It was also intended to ascertain potential directions for synthetic lead-optimization studies. 4ompounds 1b-e were prepared in order to study the tendency of halogens to present activity.In addition, compounds 1f (R= CH 3 ) and 1g (R= OCH 3 ) were included in the study to investigate the electron donating effects of -CH 3 and -OCH 3 groups.

Antiproliferative activity
Azeto-pyrroloazepinones 1a-g were evaluated in vitro for their ability to inhibit the growth of PC-3 prostate, U251 central nervous system, K652 leukemia, HCT-15 colon and MFC7 breast cancer cells.The percentage of inhibition of the growth of the five tumoral cell lines after treatment with each compound at a concentration of 100 µM is given in Table 1 and the IC 50 values (µM) on Table 2 Compounds 1a-g were examined to analyze the importance of the relative position of the substituents attached to the 3-phenyl ring of these compounds and their cytotoxic activity (Table 2).The inhibition resulted by 1a lead to the conclusion that electronegativity and/or size of halogens could be implicated in the cytotoxic activity.To test this idea, compounds 1b and 1c were synthesized.The series of 4-halogen derivatives showed that activity followed the order 1c(Cl)>1b(Br)>1a(I).To complete the series of halogen compounds, 1d(3-Br) and 1e(3-Cl) were obtained and they showed a greater activity than the 4-halogens derivatives.
These results indicated an apparent relationship between both the electronegativity and the size of the halogen versus the cytotoxic activity of the 4-halogen derivatives.However, this statement is not clear in the case of 3-halogen derivatives.On other hand, compound 1f (4-CH 3 ) was the most selective displaying a relatively good inhibition in the PC-3 cell line.In contrast, compound 1g (4-OCH 3 ) was the less selective.An analysis of the cytotoxic activity by cell line type indicated that all compounds 1a-g are active on the PC-3 cell line, derivative 1d being four times more active than the leading compound 1a.In contrast, on U-251 cell line, all compounds 1b-g tested showed a complete lack of activity.Compound 1g (4-OCH 3 ) is the only one that induces cytotoxic activity on the HCT-15 cell line.In the case of K-562 and MFC-7 cell lines the compounds 1a-g did not show any tendency.

Conclusions
The data presented here are inconclusive in regard to the relationship between electronegativity and size of the halogens substituents on the 3-phenyl group of azeto-pyrroloazepinones 1a-g and the inhibition of the growth in tumor cell lines.However, the results show a clear link between the presence of halogens on position 3-or 4-and the cytotoxicity of these compounds.Likewise, this study reveals the interesting finding that the compounds that presented cytotoxic activity were primarily those containing halogens mainly on position 3-or a CH 3 -/ -OCH 3 group on position.4-.

Experimental Section
General Procedures.All reactions were performed in oven-dried glassware under a positive pressure of nitrogen.

Cytotoxic activity
Tumoral cell lines were supplied by the National Cancer Institute.The cytotoxicity assays were carried out at 5000 to 7500 cells/ml.as reported by Skehan et al and Monks et al using the sulforhodamine B (SRB) protein assay to estimate cell growth. 8,9 ompounds were dissolved in DMSO which has not effect on the inhibition has shown by the control.The % inhibition of the growth described for all compounds were obtained from three different experiments.The percentage growth was evaluated spectrophotometrically in a Bio kinetics reader spectrophotometer.Daunomicyne was used as reference.This compound under the described conditions gave 100% of inhibition.Each experiment was made two times by triplicate.

Table 1 . % Inhibition of growth of compounds 1a-g to the five cancer cell lines
.

Table 2 .
The IC 50 values (µM) of compounds 1a-g to the five cancer cell lines