Design, synthesis and biological evaluation of hybrids of β-carboline and salicylic acid as potential anticancer and apoptosis inducing agents

A novel series of hybrids (7a-l, 8a-l) from β-carboline and salicylic acid (SA) were designed and synthesized, and their in vitro biological activities were evaluated. Most of the hybrids displayed potent antiproliferative activity against five cancer cell lines in vitro, showing potencies superior to 5-FU and harmine. In particular, compound 8h selectively inhibited proliferation of liver cancer SMMC-7721 cells but not normal liver LO2 cells, and displayed greater inhibitory selectivity than intermediate 5h and SA. 8h also induced cancer cell apoptosis in an Annexin V-FITC/propidium iodide flow cytometry assay, and triggered the mitochondrial/caspase apoptosis by decreasing mitochondrial membrane potential which was associated with up-regulation of Bax, down-regulation of Bcl-2 and activation levels of the caspase cascade in a concentration-dependent manner. Our findings suggest that the β-carboline/SA hybrids may hold greater promise as therapeutic agents for the intervention of human cancers.

Biological evaluation and structure-activity relationships (SARs) analysis. The in vitro antitumor activities of the synthesized compounds 7a-l and 8a-l against five human cancer cells were evaluated in MTT assays using 5-fluorouracil (5-FU) and harmine as positive controls. These cells are human hepatocellular carcinoma cells (SMMC-7721 and Hep G2), human colon cancer cell lines (HCT116), human bladder carcinoma cells (EJ), and human lung cancer cells (H460). Their IC 50 values (the effective concentration that inhibiting 50% of tumor cell proliferation) are listed in Table 1.
Most of the synthesized compounds exhibited higher inhibitory potency than 5-FU and harmine in all five cell lines. For instance, 7g-h, 7k, 8d-e, 8g-h, and 8k displayed anti-proliferation activities in the low micromolar range of 6.97-19.4 μ M, which were significantly greater than the parent compound harmine. In particular, 8h showed the greatest inhibition activities for liver and colon cancer cells growth with IC 50 values of 6.97-8.25 μ M, which was 4~6 fold lower than that of 5-FU (IC 50 = 19.6-35.2 μ M). Importantly, when the salicylamide was replaced with either an acetamide (10a) or benzamide (10b), the antitumor activities were significantly weaker (> 50 μ M), indicating the contribution of the salicylamide structural moiety in 8h to its antitumor activity and the improved anti-proliferation potency.
Analysis of SAR reveals that the length of hybrids linkers influences the antitumor activities. Consistent with previous hybrid compounds, the potency first increases then decreases with the elongation of the linker. For example, hybrids linked with butanediamine (n = 3) and amyl diamine (n = 4) exhibited greater antitumor activities than that with propane diamine (n = 2) or hexanediamine (n = 5). Compounds with a methyl group at the 1-position of the β -carboline showed greater potency than the other two classes containing either hydrogen or p-methoxyphenyl (e.g. 8h vs. 8g vs. 8i; 7h vs. 7g vs. 7i). The potency order is methyl > hydrogen > p-methoxyphenyl for substitutes at the 1-position of the β -carboline. These results suggest that an appropriate substitution is preferred at the 1-position for antitumor activities of these hybrids. Interestingly, compounds 8a-l in general displayed greater antitumor activity than their acetylated compounds 7a-l, suggesting the phenolic hydroxyl group is preferred for antitumor activities.
Given the strong growth inhibitory activity of 8h in vitro, it was profiled for antitumor selectivity by examining its inhibitory effects on the growth of SMMC-7721 cells and LO2 cells (human hepatocellular normal cells). The dose-response curve is shown in Fig. 3A. As can be seen, no apparent growth inhibitory activity was observed on the non-cancer LO2 cells at concentrations up to 25 μ M. However, 8h displayed apparent anti-proliferation activity on SMMC-7721 cells with IC 50 of 6.97 μ M, which is 9 fold lower than against liver normal LO2 cells (IC 50 = 55.2 μ M), confirming that 8h can selectively suppress the proliferation of tumor cells in vitro.
The inhibitory activity of 8h, SA, and 5h, an analog of 8h without the SA structural moiety, against SMMC-7721 cells and LO2 cells were evaluated at a concentration of 25 μ M for 48 h. Figure 3B shows that treatment with SA alone induced little proliferation of both SMMC-7721 and LO2 cells, similar to those of the cells treated with vehicle. Treatment with 5h caused much greater inhibitory effects (~42%) against SMMC-7721 cells than SA. Compound 5h (IC 50 = 27.2 ± 3.4 μ M) was also more potent in inhibiting SMMC-7721 cell growth than the two "capped" analogs 10a (IC 50 = 58.6 ± 6.7 μ M) or 10b (IC 50 > 100 μ M), in which the primary amino group potency and cytotoxicity with the introduction of an alkyl amino group is consistent with previous reports 23,24 , and is likely the result of non-specific interactions between the primary amino group in 5h (mostly protonated at pH 7.4) and residues around the carboline binding site in both cell types. At 25 uM, hybrid 8h displayed significant inhibitory effects on tumor cell proliferation (~90%). This is markedly greater than SA (~15%) and carboline analog 5h (~42%), and also greater than the combination of SA and 5h (~55%, Fig. 3B). Given that the inhibitory activity of 8h is significantly higher than SA, it is likely that the β -carboline moiety may provide greater contribution to the overall antitumor activity. These results suggest that the increased anti-tumor activity of 8h most likely result from the presence of both the β -carboline and SA moieties in 8h, suggesting the existence of the synergetic effects by these two agents.
Importantly, 8h showed little inhibitory effects on the LO2 cells, in contrast to 5h which had high toxicities to these normal cells. These results indicate that the SA structural moiety in 8h contributes to the improved anti-proliferation potency and reduced toxicity (8h vs 5h), which therefore represents a more potent and safer antitumor agent than β -carboline alone, which may warrant further investigation and development.
To determine whether apoptosis is involved in the anti-proliferative effects of 8h, apoptosis assay was performed using SMMC-7721 cells. The cells were incubated with either vehicle alone, different concentrations of 8h (7.0 or 14 μ M), or 14 μ M 5-FU for 48 h, and then stained with FITC-Annexin V and propidium iodide (PI). The percentages of apoptotic SMMC-7721 cells were determined by flow cytometry analysis. The results are shown in Fig. 4A. In the vehicle group, the occurrence of SMMC-7721 cell apoptosis was minimal. However, in the 8h-treated SMMC-7721 cells, the population of the apoptosis cells clearly increased. Low concentration of 8h (7.0 μ M) induced 46% SMMC-7721 cell apoptosis, while the higher dose (14 μ M) of 8h induced 63% SMMC-7721 cell apoptosis. Both are significantly greater than those induced by 5-FU at 14 μ M (only 22%). These results revealed that the antitumor activity of 8h is associated with the apoptosis effect which appeared to be a concentration-dependent manner.
The mitochondrial pathway plays an important role in cell apoptosis 25 . To determine whether mitochondria is involved in the apoptosis-inducing effect of 8h, changes in mitochondrial membrane potential were investigated with a fluorescent probe JC-1. JC-1 is a lipophilic cationic dye which facilitates the penetration of mitochondrial membrane due to the decrease of membrane potential, indicated by a fluorescence shift from red to green. The cells were treated with vehicle alone, with different concentrations of 8h (7.0 or 14 μ M), or 14 μ M 5-FU for 48 h, and then stained with JC-1. As shown in Fig. 4B, in the control group, the percentage of cells emitting green fluorescence was only 4.1%, whereas cells treated with 8h exhibited stronger green fluorescence. The percentage of cells emitting green fluorescence reached 57% at the higher dose of 14 μ M. The observations indicate that 8h plays a role in the dissipation of mitochondrial membrane potential and the apoptosis effect of 8h is associated with mitochondrial depolarization in cells.
Next, to investigate the preliminarily molecular mechanisms underlying the cell apoptosis profiles in 8h-treated cells, we conducted western blot analysis to examine the expression of apoptosis proteins in SMMC-7721 cells. It is well known that Bcl-2 and Bax are anti-apoptotic or pro-apoptotic regulator proteins, respectively, and caspase-3 is the execution factor of apoptosis. In addition, PARP is one of several known cellular substrates for caspase-3 and the cleavage of PARP by caspase-3 is considered to be a hallmark of apoptosis 26 . As shown in Fig. 5, the protein levels of Bax proteins significantly increased in 8h-treated cells, whereas the expression of Bcl-2 was dramatically reduced in a concentration-dependent manner. Moreover, compound 8h resulted in more significant cleavage of both PARP and caspase-3 than the control group. These results indicate that 8h could significantly induce cell apoptosis through the regulation of apoptosis proteins.

Conclusions
A novel series of hybrids (7a-l, 8a−l) from β -carboline and SA were synthesized, and their in vitro biological activities were evaluated. It was found that most of the hybrids showed potent anti-proliferation activities against five human cancer cells in vitro. The most potent compound 8h exhibited the highest inhibition activities against liver and colon cancer cells with IC 50 values of 6.97-8.25 μ M which greater than that of 5-FU (IC 50 = 19.6-35.2 μ M), and selectively inhibited cancer cells but not non-tumor liver cell proliferation in vitro. Furthermore, 8h dose-dependently induced cancer cell apoptosis which is associated with mitochondrial depolarization in tumor cells by up-regulating Bax, down-regulating Bcl-2, in addition to activating levels of the caspase cascade in a concentration-dependent way. Therefore, our novel findings provide a proof of principle in the design of new β -carboline/SA hybrids for the intervention of human cancers.

Methods
Chemistry. Infrared (IR) spectra (KBr) were recorded on a Nicolet Impact 410 instrument (KBr pellet). 1 H NMR spectra were recorded with a Bruker Avance 300 MHz spectrometer at 300 K, using TMS as an internal standard. MS spectra were recorded on a Mariner Mass Spectrum (ESI). High resolution mass spectra were recorded using an Agilent Technologies LC/MSD TOF. Element analysis was performed on an Eager 300 instrument. All compounds were routinely checked by TLC and 1 H NMR. TLCs and preparative thin-layer chromatography were performed on silica gel GF/UV 254, and the chromatograms were conducted on silica gel (200-300 mesh, Merck) and visualized under UV light at 254 and 365 nm. All solvents were reagent grade and, when necessary, were purified and dried by standards methods. L-tryptophan 1, acetylsalicylic acid 6 and different substituted aldehydes were commercially available. Compounds 2a-c, 3a-c, and 4a-c were synthesized according literatures 2,7,9 . Solutions after reactions and extractions were concentrated using a rotary evaporator operating at a reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulfate. High-performance liquid chromatography (HPLC) analysis conditions: column: Shimadzu C18 (150 mm × 4.6 mm × 5 μ m); mobile phase: methanol: water = 75: 25; wavelength: 254 nm; flow rate: 1 mL/min. All target compounds were of > 95% purity as determined by HPLC.