Anti-Inflammatory Activity : Assessment of Benzo [ 6 , 7 ] Cyclohepta [ 1 , 2-b ] Pyridine Derivatives

A variety of benzo [6,7] cyclohepta [1,2-b] pyridine derivatives have been evaluated for their in vitro anti-proliferative activity against four cancer cell lines such as HeLa (cervical), MIAPACA (pancreatic), MDA-MB-231 (breast) and IMR32 (neuroblastoma). Among tested compounds, a exhibited potent antiproliferative activity against IMR32 with GI50 value less than 0.01 μM and compounds d, l and n exhibited promising anti-proliferative activity against IMR32 with GI50 values 0.1, 0.21 and 0.21 μM, respectively. This is the first report on in vitro antiproliferative evaluation of benzo [6,7] cyclohepta [1,2-b] pyridine derivatives in addition to anti-inflammatory activity.


Effects of the Compounds on the Viability of Human Cancer Cells
All the synthesized compounds of benzo [6,7] cyclohepta [1,2-b] pyridine derivatives (a-n) were screened for their in vitro antiproliferative activity against four cancer cell lines such as HeLa(cervical), MIAPACA (pancreatic), MDA-MB-231 (breast) and IMR32 (neuroblastoma) summarized in Table 1. The compounds were picked for an advanced assay against these four human cancer cell lines at five concentrations (0.01, 0.1, 1, 10, 100 μM). GI 50 (growth inhibitory activity) was calculated and these values corresponded to the concentration of the compound causing 50% decrease in the net cell growth as compared with the standard drugs, doxorubicin and paclitaxel. Results were calculated for each of these parameters if the level of activity was reached; however, if the effect was not achieved, the value was expressed as greater or less than the maximum or minimum concentration tested.
Based on Table 1, the synthesized compounds a-n showed potent to significant cancer cell growth inhibition with GI 50 values ranging from <0.01 to >100 μM. Among tested compounds a exhibited potent antiproliferative activity against IMR32 with GI 50 value less than 0.01 μM, which was more active than standards Doxorubicin and Paclitaxel. Compound d showed anti-proliferative activity against IMR32 with GI 50 value 0.1 μM, which was close to the standard Paclitaxel. Five compounds l, n, m, k and b exhibited promising anti-proliferative activity against IMR32 with GI 50 values 0.21, 0.21, 0.69, 0.98 and 1.1μM.
The Structure-Activity Relationship (SAR) revealed that. The presences of ester group/cyclic group on pyridine ring in combination with methyl group on C-2 position appear to be crucial for observed activity. Further studies are underway to optimize the lead molecule ( Figure 1).

Figure 1: Structures of anti-proliferative compounds. RB Proliferative Assay Method
The cell lines, HeLa, MIAPACA, MDA MB 231 and IMR 32 (cervical, pancreatic, breast and neuroblastoma) which were used in this study were procured from American Type Culture Collection (ATCC), USA. The synthesized test compounds were evaluated for their in vitro anti-proliferative activity in these four different human cancer cell lines. A protocol of 48 h continuous drug exposure was used and an SRB cell proliferation assay was used to estimate cell viability or growth. All the cell lines were grown in Dulbecco's modified Eagle's medium (containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C). Cells were trypsinized when subconfluent from T25 flasks/60 mm dishes and seeded in 96-well plates in 100 μL aliquots at plating densities depending on the doubling time of individual cell lines. The microtiter plates were incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to addition of experimental drugs and were incubated for 48 h with different doses (0.01, 0.1, 1, 10, 100 μM) of prepared derivatives. After 48 h incubation at 37 °C, the cell monolayers were fixed by the addition of 10% (wt/vol) cold trichloroacetic acid and incubated at 4 °C for 1 h and were then stained with 0.057% SRB dissolved in 1% acetic acid for 30 min at room temperature. Unbound SRB was washed with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader (Enspire, Perkin Elmer, USA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz [(Ti-Tz)/Tz] x 100 for concentrations for which Ti>Tz The dose response parameter, growth inhibition of 50% (GI 50 ) was calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drugconcentration resulting in a 50% reduction in the net protein increase(as measured by SRB staining) in control cells during the drug incubation. Values were calculated for this parameter if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested.

Conclusion
Taken together, we have synthesized a series of benzo [6,7]cyclohepta [1,2-b] pyridine derivatives (a-n) in good yields and performed there in vitro anti-proliferative activity against four different human cancer cell lines, HeLa, MIAPACA, MDA-MB-231 and IMR32. Compound a exhibited potent anti-proliferative activity against IMR32 with GI 50 value less than 0.01 μM, which was more active than standards Doxorubicin and Paclitaxel in addition to antiinflammatory activity.