Synthesis and Cytotoxicity Testing of New Amido-Substituted Triazolopyrrolo[2,1-c][1,4]benzodiazepine (PBDT) Derivatives

A series of amido-substituted triazolopyrrolo[2,1-c][1,4]benzodiazepine (PBDT) derivatives was synthesized from isatoic anhydride, and their cytotoxicity against the MRC-5 and Mahlavu cell lines was evaluated. The results suggest that compound PBDT-7i with the meta-trifluoromethylbenzoyl substituent can selectively inhibit the growth of Mahlavu cells and has low toxicity towards MRC-5 cells.


Introduction
Cancer is a leading cause of death globally, accounting for 7.6 million deaths in 2008. Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and occurs at a high rate (3-5% annually) in patients with chronic viral hepatitis and cirrhosis. HCC is also a major cause of morbidity and mortality in patients with advanced liver disease [1,2]. Therefore, there is a desire to design and develop more potent therapeutic agents for cancer. We are interested in investigating N-bridged heterocycles composed of two bioactive heterocyclic moieties, such as benzodiazepines and triazoles, in a single molecular scaffold because such bridged molecules are endowed with a variety of biological activities and have a wide range of therapeutic properties.
Owing to the biological significance of these two classes of compounds, we planned to synthesize a combined molecular framework. We report herein the synthesis of tetracyclic triazolopyrrolo[2,1-c] [1,4]benzodiazepines (PBDTs) and their amido-substituted derivatives. The cytotoxicity of the newly synthesized derivatives was investigated using the Mahlavu (human hepatocellular carcinoma) and MRC-5 (normal human fibroblast) cell lines.

Cytotoxicity
The cytotoxic activities of the newly synthesized amido-substituted triazolopyrrolo [ Our initial results showed that percentages of viable Mahlavu and MRC-5 cells exposed to the compounds PBDT-7a,b and PBDT-7j,k were above 75%, although the concentration was increased to 100 µM. This revealed that the aliphatic and aromatic amides seem no significant difference observed in cytotoxicity between Mahlavu and MRC-5 cell lines. Intriguingly, cells exposed to compounds with various substituents [-F (PBDT-7c-e), -OMe (PBDT-7f), -CN (PBDT-7g), -Cl (PBDT-7h) and -CF 3 (PBDT-7i)] on the phenyl ring of the PBDT exhibited different viabilities for both cell lines. Compounds with the fluoro substituent at various positions (compounds PBDT-7c-e) exhibited different levels of toxicity against Mahlavu and MRC-5 cells. The viability of Mahlavu cells exposed to the compound with a fluoro substituent at the meta-position (compound PBDT-7d) was approximately 61% at 100 µM, whereas the ortho-and para-fluoro-substituted compounds PBDT-7c and e had little effect at the same concentration.
Based on the preliminary results, we selected to further investigate the meta-position of the phenyl group (compounds PBDT-7f-i). The cell viabilities were reduced in the presence of the chloro-substituted PBDT-7h in a dose-dependent manner, and the survival rate of Mahlavu cells was below 50% at 50 µM. PBDT-7h was a potent inhibitor of Mahlavu cells, but was also highly toxic for normal cells. To increase the specificity, we evaluated other three meta-substituted PBDTs, including compounds with the -OMe (PBDT-7f), -CN (PBDT-7g) and -CF 3 (PBDT-7i) substituents. It should be noted that the cell viabilities of Mahlavu cells were decreased to 37%, 36%, and 2% at 100 µM, respectively, and the IC 50 values of PBDT-7g and PBDT-7i were 50 µM for Mahlavu cells. These compounds were slightly toxic to MRC-5 cells, with cell viabilities above 70% at 50 µM. These revealed compound PBDT-7i with the meta-trifluoromethylbenzoyl substituent can selectively inhibit the growth of Mahlavu cells and is low-toxic to MRC-5 cells. This novel compound certainly deserves further careful investigation to determine its mechanism of action.

Conclusions
We have synthesized a series of novel amido-substituted triazolopyrrolo [2,1-c] [1,4]benzodiazepines from isatoic anhydride in high total yields. The biological evaluation of these compounds based on the growth of Mahlavu and MRC-5 cells revealed that compound PBDT-7i selectively inhibited the proliferation of Mahlavu cells. More interestingly, compound PBDT-7i might inhibit not only Mahlavu cell growth, but also shows low toxicity towards MRC-5 cells.