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

Scientific Reports volume 6, Article number: 36238 (2016) | Download Citation

Abstract

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.

Introduction

Natural products have been a rich source of compounds for drug discovery and the majority of anticancer drugs currently used in clinical are derived from natural product scaffolds. β-Carbolines alkaloids, a family of natural products possessing common parent nucleus of a planar tricyclic pyrido[3,4-b]indole ring system, are abundant in the plant kingdom or other organisms1. Some β-carboline derivatives, both natural and synthetic compounds such as harmine, harmane, norharman, and β-carboline-benzimidazole conjugates (Fig. 1), have been reported to display antitumor activities through multiple mechanisms such as intercalating into DNA or inhibiting Topoisomerase I and II, cyclin-dependent kinases (CDK), PLK, or MAO2,3,4,5,6. According to the SARs summarized from the reported β-carboline derivatives, the modification on positions-1 or -3 of the parent nucleus would make great influence on their antitumor activities6,7,8,9.

Figure 1: The design of novel β-carbolines/salicylic acid hybrids.
Figure 1

Many natural and synthetic anticancer agents with the ability to interact with DNA, including β-carbolines, often have relatively low therapeutic index, most likely due to the unspecific manner of these agents in causing DNA damage both in neoplastic and in highly proliferative normal tissues10. Hybrid molecules, which combine structural motifs from two or more biologically active agents, often have improved potency and/or reduced toxicity, and have received increased attention recently. A variety of hybrids have been reported to date that displayed improved anti-tumor activities than the individual agents2,9,11,12,13.

Recently, we have shown that hybrids of β-carboline and hydroxamic acid showed synergistic effects and displayed increased anticancer activity9. In this manuscript, we report the design and synthesis of hybrid compounds featuring structurally moieties of β-carboline and acetylsalicylic acid (aspirin). Aspirin, a well known nonsteroidal anti-inflammatory agent, has been revealed to exhibit high potency in the treatment of cancer14,15,16. Epidemiological studies suggested that the regular intake of aspirin was associated with a reduction in the incidence of malignancies, including colorectal, gastrointestinal, and lung cancer17,18. Recent studies demonstrated that aspirin and its metabolite salicylic acid (SA), and their derivatives could induce apoptosis in several colorectal carcinoma cell lines19,20,21,22.

A series of novel β-carbolines/salicylic acid hybrids 7a-l and 8a-l have been designed by introducing the salicylic acid fragment into the β-carboline molecule at the 3-position with diamines linkers of different length. We hypothesized that the novel types of hybrids could selectively inhibit tumor cell proliferation and efficaciously induce tumor cell apoptosis with a synergy for the treatment of cancer. In addition, given the importance of the 1-position of the β-carboline for its antitumor activity, hybrids bearing different substituents at this position were also investigated. Therefore, twenty β-carbolines/salicylic acid hybrids were designed and synthesized, and their in vitro antitumor activities were evaluated. Herein, the synthesis and biological evaluation of these compounds were reported.

Results and Discussion

Chemistry

The synthetic route to compounds 7a-l, 8a-l is depicted in Fig. 2. The substituted β-carbolines 4a-c were prepared in a three-step sequence. First, commercially available L-tryptophan 1 was converted to 1-substituted-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid 2a-c in a Pictet-Spengler reaction using differently substituted aldehydes (formaldehyde, acetaldehyde or 4-methoxyphenaldehyde). Second, methyl esterification of compounds 2a-c using methanol and SOCl2 afforded compound 3a-c. Intermediates 3a-c were oxidized by KMnO4 in DMF to afford compounds 4a-c, which were then reacted with different diamines in EtOH to provide amides 5a-l. Reaction of acetylsalicylic acid 6 with 5a-l in the presence of ethyl chloroformate and N-methylmorpholine yielded compounds 7a-l. Finally, hydrolysis of 7a-l afforded compounds 8a-l. The acetamide 10a and benzamide 10b were prepared by the treatment of 5h with acetyl chloride 9a or benzoyl chloride 9b, respectively, in the presence of triethylamine. All target compounds were purified by column chromatography, and their structures were confirmed by IR, 1H NMR, MS, HRMS, and elemental analyses.

Figure 2: Reagents and conditions.
Figure 2

(a) H+ or OH, RCHO, reflux, 2–4 h, 81–86%; (b) SOCl2, MeOH, 0 °C, 1 h, and then reflux 6 h, 90–95%; (c) KMnO4, DMF, reflux, 6 h, 60–70%; (d) different diamines, EtOH, reflux, 3–5 h, 81–89%; (e) i) N-methylmorpholine, ethyl chloroformate, THF, 0 °C, 1 h; ii) Et3N, THF, 0 °C, 1–3 h, 55–68%; (f) NaOH, MeOH, r.t., 2 h, 90–96%; (g) triethylamine, CH2Cl2, r.t., 2 h, 75–79%.

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 IC50 values (the effective concentration that inhibiting 50% of tumor cell proliferation) are listed in Table 1.

Table 1: The IC50 values of synthetic compounds 7a-l, 8a-l, and 10a-b against five human cancer cell lines.

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 μM, 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 IC50 values of 6.97–8.25 μM, which was 4~6 fold lower than that of 5-FU (IC50 = 19.6–35.2 μM). Importantly, when the salicylamide was replaced with either an acetamide (10a) or benzamide (10b), the antitumor activities were significantly weaker (>50 μM), 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 μM. However, 8h displayed apparent anti-proliferation activity on SMMC-7721 cells with IC50 of 6.97 μM, which is 9 fold lower than against liver normal LO2 cells (IC50 = 55.2 μM), confirming that 8h can selectively suppress the proliferation of tumor cells in vitro.

Figure 3
Figure 3

(A) Inhibitory effects of 8h on the proliferation of SMMC-7721 and LO2 cells. Cells were incubated with the indicated concentrations of 8h for 48 h. Cell proliferation was assessed using the MTT assay. Data are means ± SD of the inhibition (%) from three independent experiments. (B) Inhibitory effects of 5h, 8h, and SA or the vehicle control against SMMC-7721 and LO2 cells. SMMC-7721 and LO2 cells were incubated with the indicated compounds at 25 μM for 48 h, and cell proliferation was assessed by the MTT assay. Data are means ± SD of the inhibition (%) from three independent experiments. *P < 0.01 versus control of SMMC-7721, #P < 0.01 inhibitory effects of 8h in SMMC-7721 versus LO2 cells.

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 μM 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 (IC50 = 27.2 ± 3.4 μM) was also more potent in inhibiting SMMC-7721 cell growth than the two “capped” analogs 10a (IC50 = 58.6 ± 6.7 μM) or 10b (IC50 > 100 μM), in which the primary amino group was acylated to afford either an acetamide or a benzamide. However, the inhibitory effect of 5h appears to be non-specific for tumor cells, as a similar effect was seen in the non-tumor LO2 cells. Such an increase on both potency and cytotoxicity with the introduction of an alkyl amino group is consistent with previous reports23,24, 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 μM), or 14 μM 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 μM) induced 46% SMMC-7721 cell apoptosis, while the higher dose (14 μM) of 8h induced 63% SMMC-7721 cell apoptosis. Both are significantly greater than those induced by 5-FU at 14 μM (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.

Figure 4: Compound 8h induced SMMC-7721 cell apoptosis in vitro.
Figure 4

SMMC-7721 cells were incubated with the indicated concentrations of 8h and 14 μM 5-FU for 48 h, and the cells were stained with FITC-Annexin V and PI, followed by flow cytometry analysis. (A) Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means ± SD of the percentages of apoptotic cells from three independent experiments. *P < 0.01 vs control.

The mitochondrial pathway plays an important role in cell apoptosis25. 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 μM), or 14 μM 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 μM. 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 apoptosis26. 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.

Figure 5: Effect of 8h on the expression of apoptosis-related proteins in SMMC-7721 cells.
Figure 5

(A) The expression of Bax, Bcl2, caspase 3 and β-actin was examined by western blot analysis. SMMC-7721 cells were incubated with, or without, 8h at the indicated concentrations for 48 h and the levels of protein expression were detected using specific antibodies, and β-actin was used as the control. Data shown are representative images of each protein for three separate experiments. (B) Quantitative analysis: the relative levels of each protein compared to control β-actin were determined by densimetric scanning. Data are expressed as means ± SD from three separate experiments. *P < 0.01 vs control.

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 IC50 values of 6.97–8.25 μM which greater than that of 5-FU (IC50 = 19.6–35.2 μM), 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). 1H 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 1H 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 literatures2,7,9. 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 μm); 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.

N-(3-Aminopropyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5a)

A mixture of 4a (2.3g, 10 mmol) and 1,3-propanediamine 8.3 mL (100 mmol) in 15 mL methanol was refluxed for 10 h. After cooled, the solvent was evaporated. The residue was purified by silica chromatography (CH2Cl2-CH3OH = 6:1, v/v as the eluent) to afford a yellowish solid 2.3 g, yield: 85%. MS (ESI) m/z = 269 [M+H]+.

N-(3-Aminopropyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide(5b)

Refer to the synthesis of 5a, the title product was obtained from 4b and 1,3-propanediamine to afford a pale yellow solid 2.3 g, yield: 83%. MS (ESI) m/z = 283 [M+H]+.

N-(3-Aminopropyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5c)

Refer to the synthesis of 5a, the title product was obtained from 4c and 1,3-propanediamine to afford a pale yellow solid 3.1 g, yield: 82%. MS (ESI) m/z = 375 [M+H]+.

N-(4-Aminobutyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5d)

Refer to the synthesis of 5a, the title product was obtained from 4a and 1,4-diaminobutane to afford the pale yellow solid 2.4 g, yield: 87%. MS (ESI) m/z = 283 [M+H]+.

N-(4-Aminobutyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide(5e)

Refer to the synthesis of 5a, the title product was obtained from 4b and 1,4-diaminobutane to afford the pale yellow solid 2.5 g, yield: 84%. MS (ESI) m/z = 297 [M+H]+.

N-(4-Aminobutyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5f)

Refer to the synthesis of 5a, the title product was obtained from 4c and 1,4-diaminobutane to afford the pale yellow solid 3.1 g, yield: 81%. MS (ESI) m/z = 389 [M+H]+.

N-(5-Aminopentyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5g)

Refer to the synthesis of 5a, the title product was obtained from 4a and 1,5-pentanediamine to afford the pale yellow solid 2.6 g, yield: 87%. MS (ESI) m/z = 297 [M+H]+.

N-(5-Aminopentyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide(5h)

Refer to the synthesis of 5a, the title product was obtained from 4b and 1,5-pentanediamine to afford the pale yellow solid 2.6 g, yield: 84%. 1H NMR (DMSO-d6, 300 MHz): δ 8.66 (m, 1H, Ar-H), 8.59 (m, 1H, NH), 8.33 (d, 1H, J = 6.0 Hz, Ar-H), 7.55–7.63 (m, 2H, Ar-H), 7.28 (m, 1H, Ar-H), 3.36 (m, 2H, NCH2), 2.84 (s, 3H, ArCH3), 2.57 (m, 2H, NCH2), 1.42–1.60 (m, 4H, 2 × NCH2CH2), 1.15–1.22 (m, 2H, NHCH2CH2CH2); MS (ESI) m/z = 311 [M+H]+; HRMS (ESI): m/z calcd for C18H23N4O: 311.1872; found: 311.1887 [M+H]+.

N-(5-aminopentyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide(5i)

Refer to the synthesis of 5a, the title product was obtained from 4c and 1,5-pentanediamine to afford the pale yellow solid 3.3 g, yield: 82%. MS (ESI) m/z = 403 [M+H]+.

N-(6-Aminohexyl)-9H-pyrido[3,4-b]indole-3-carboxamide (5j)

Refer to the synthesis of 5a, the title product was obtained from 4a and 1,6-hexamethylene diamine to afford the pale yellow solid 2.7 g, yield: 87%. MS (ESI) m/z = 311 [M+H]+.

N-(6-Aminohexyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (5k)

Refer to the synthesis of 5a, the title product was obtained from 4b and 1,6-hexamethylene diamine to afford the pale yellow solid 2.8 g, yield: 85.3%. MS (ESI) m/z = 325 [M+H]+.

N-(6-Aminohexyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (5l)

Refer to the synthesis of 5a, the title product was obtained from 4c and 1,6-hexamethylene diamine to afford the pale yellow solid 3.5 g, yield: 85%. MS (ESI) m/z = 417 [M+H]+.

N-(3-(2-Methylbenzamido)propyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7a)

To a solution of acetylsalicylic acid 6 (1.8 g, 10 mmol) in 20 mL anhydrous THF/DMF (v:v = 3:1) was added N-methyl morpholine (1.5 g, 15 mmol) under ice bath. After slowly added ethyl carbonochloridate (1.3 g, 12 mmol), the mixture was stirred at room temperature for 0.5 h, and then added dropwise to 10 mL anhydrous THF solution of 5a (2.7 g, 10 mmol) and triethylamine (1.5 g, 15 mmol) at 0 °C. The mixture was continued reacted for 2 h and added 100 mL water, then extracted with ethyl acetate (3 × 50 mL). The combined organic layer was washed with brine, dried with Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 2:3, v/v) to afford a pale yellow solid 3.4 g, yield: 77%. Analytical data for 7a: IR (KBr, cm-1): 3451, 2963, 1657, 1574, 1485, 1428, 1301, 1265, 1028. 1H NMR (CDCl3, 300 MHz): δ 11.21 (s, 1H, NH), 8.89 (s, 1H, Ar-H), 8.71 (s, 1H, Ar-H), 8.11–8.19 (m, 3H, CONH, Ar-H), 7.58–7.76 (m, 4H, Ar-H), 7.26–7.48 (m, 3H, Ar-H), 3.63 (m, 2H, NCH2). 3.52 (m, 2H, NCH2), 2.38 (s, 3H, CH3CO), 1.92 (m, 2H, CH2); MS (ESI) m/z = 446 [M+H]+; Anal. Calcd. for C25H25N4O4: C, 67.40; H, 5.66; N, 12.58; Found: C, 67.23; H, 5.77; N, 12.47.

1-Methyl-N-(3-(2-methylbenzamido)propyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7b)

Refer to the synthesis of 7a, the title product was obtained from 5b and acetylsalicylic acid to afford a pale yellow solid 3.3 g, yield: 73%. Analytical data for 7b: IR (KBr, cm−1): 3391, 2943, 1618, 1503, 1480, 1398, 1310, 1236, 1028. 1H NMR (CDCl3, 300 MHz): δ 9.36 (s, 1H, NH), 8.87 (s, 1H, CONH), 8.73 (s, 1H, Ar-H), 8.47 (s, 1H, CONH), 8.15 (m, 1H, Ar-H), 8.06 (m, 1H, Ar-H), 7.58–7.63 (m, 2H, Ar-H), 7.42–7.45 (m, 2H, Ar-H), 7.30 (m, 1H, Ar-H), 7.06 (m, 1H, Ar-H), 3.70 (m, 2H, CH2N), 3.56 (m, 2H, NHCH2), 2.83 (s, 3H, ArCH3), 2.35 (s, 3H, CH3CO), 1.92 (m, 2H, NHCH2CH2); MS (ESI) m/z = 460 [M+H]+; Anal. Calcd. for C26H27N4O4: C, 67.96; H, 5.92; N, 12.19; Found: C, 67.88; H, 6.08; N, 12.01.

1-(4-Methoxyphenyl)-N-(3-(2-methylbenzamido)propyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7c)

Refer to the synthesis of 7a, the title product was obtained from 5c and acetylsalicylic acid to afford a pale yellow solid 3.8 g, yield: 71%. IR (KBr, cm−1): 3450, 2904, 1626, 1549, 1485, 1406, 1313, 1220, 1020. Analytical data for 7c: 1H NMR (DMSO-d6, 300 MHz): δ−11.05 (s, 1H, NH), 8.75 (s, 1H, Ar-H), 8.35–8.41 (m, 2H, Ar-H, CONH), 8.19 (d, 2H, J = 7.5 Hz, Ar-H), 7.84 (m, 1H, Ar-H), 7.57–7.65 (m, 3H, Ar-H), 7.41–7.46 (m, 3H, Ar-H), 7.06 (d, 2H, J = 7.5 Hz, Ar-H), 3.89 (s, 3H, OCH3), 3.68 (m, 2H, CH2N), 3.55 (m, 2H, NCH2), 2.32 (s, 3H, CH3CO), 1.89 (m, 2H, NCH2CH2). MS (ESI)−m/z = 552 [M+H]+; Anal. Calcd. for C32H31N4O5: C, 69.68; H, 5.66; N, 10.16; Found: C, 69.59; H, 5.77; N, 10.01.

N-(4-(2-Methylbenzamido)butyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7d)

Refer to the synthesis of 7a, the title product was obtained from 5d and acetylsalicylic acid to afford a pale yellow solid 3.5 g, yield: 77%. Analytical data for 7d: IR (KBr, cm−1): 3451, 2883, 1676, 1514, 1456, 1400, 1343, 1260, 1010. 1H NMR (CDCl3, 300 MHz): δ 10.87 (br, 1H, NH), 8.88 (s, 1H, Ar-H), 8.74 (s, 1H, Ar-H), 8.28 (s, 1H, CONH), 8.16 (m, 1H, Ar-H), 7.69–7.71 (m, 1H, Ar-H), 7.49–7.57 (m, 3H, Ar-H), 7.30–7.38 (m, 2H, Ar-H), 7.09 (m, 1H, Ar-H), 3.57 (m, 2H, NCH2), 3.28 (m, 2H, CH2N), 2.29 (s, 3H, CH3CO), 1.58–1.67 (m, 4H, 2 × NCH2CH2); MS (ESI) m/z = 460 [M+H]+; Anal. Calcd. for C26H27N4O4: C, 67.96; H, 5.92; N, 12.19; Found: C, 67.82; H, 6.06; N, 12.11.

1-Methyl-N-(4-(2-methylbenzamido)butyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7e)

Refer to the synthesis of 7a, the title product was obtained from 5e and acetylsalicylic acid to afford a pale yellow solid 3.7 g, yield: 77%. Analytical data for 7e: IR (KBr, cm−1): 3478, 2952, 1677, 1542, 1494, 1446, 1311, 1265, 1028. 1H NMR (CDCl3, 300 MHz): δ 10.65 (s, 1H, NH), 8.75 (s, 1H, Ar-H), 8.70 (s, 1H, CONH), 8.30 (s, 1H, CONH), 8.14 (d, 1H, J = 6.0 Hz, Ar-H), 7.73 (d, 1H, J = 6.0 Hz, Ar-H), 7.53 (m, 2H, Ar-H), 7.42 (m, 1H, Ar-H), 7.29 (m, 2H, Ar-H), 7.06 (m, 1H, Ar-H), 3.48–3.59 (m, 4H, 2 × NCH2), 2.80 (s, 3H, ArCH3), 2.31 (s, 3H, CH3CO), 1.71–1.85 (m, 4H, 2 × CH2); MS (ESI) m/z = 474 [M+H]+; Anal. Calcd. for C27H29N4O4: C, 68.48; H, 6.17; N, 11.83; Found: C, 68.37; H, 6.23; N, 11.99.

1-(4-Methoxyphenyl)-N-(4-(2-methylbenzamido)butyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7f)

Refer to the synthesis of 7a, the title product was obtained from 5f and acetylsalicylic acid to afford a pale yellow solid 4.1 g, yield: 72%. Analytical data for 7f: IR (KBr, cm−1): 3451, 2963, 1599, 1504, 1435, 1428, 1333, 1265, 1011. 1H NMR (CDCl3, 300 MHz): δ 8.76 (s, 1H, Ar-H), 8.33 (s, 1H, CONH), 8.16 (m, 1H, Ar-H), 7.89–7.92 (d, 2H, J = 9.0 Hz, Ar-H), 7.47–7.69 (m, 4H, Ar-H), 7.30–7.35 (m, 2H, Ar-H), 7.12 (d, 2H, J = 9.0 Hz, Ar-H), 7.00 (m, 1H, Ar-H), 3.88 (s, 3H, OCH3), 3.57–3.59 (m, 2H, NCH2), 3.44–3.46 (m, 2H, NCH2), 2.30 (s, 3H, COCH3), 1.69–1.77 (m, 4H, 2 × NHCH2CH2); MS (ESI) m/z = 566 [M+H]+; Anal. Calcd. for C33H33N4O5: C, 70.07; H, 5.88; N, 9.91; Found: C, 69.92; H, 5.97; N, 9.82.

N-(5-(2-Methylbenzamido)pentyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7g)

Refer to the synthesis of 7a, the title product was obtained from 5g and acetylsalicylic acid to afford a pale yellow solid 3.5 g, yield: 73%. Analytical data for 7g: IR (KBr, cm−1): 3467, 2966, 1625, 1545, 1480, 1421, 1321, 1235, 1018. 1H NMR (CDCl3, 300 MHz): δ 12.45 (br, 1H, NH), 8.93 (s, 1H, Ar-H), 8.76 (s, 1H, Ar-H), 8.26 (s, 1H, CONH), 8.11 (m, 1H, Ar-H), 7.48–7.63 (m, 3H, Ar-H), 7.25–7.38 (m, 3H, Ar-H), 7.06 (m, 1H, Ar-H), 3.61 (m, 2H, NCH2). 3.50 (m, 2H, NCH2), 2.33 (s, 3H, CH3CO), 1.73–1.82 (m, 4H, 2 ×NCH2CH2), 1.30 (m, 2H, NCH2CH2CH2); MS (ESI) m/z = 474 [M+H]+; Anal. Calcd. for C27H29N4O4: C, 68.48; H, 6.17; N, 11.83; Found: C, 68.32; H, 6.32; N, 11.69.

1-Methyl-N-(5-(2-methylbenzamido)pentyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7h)

Refer to the synthesis of 7a, the title product was obtained from 5h and acetylsalicylic acid to afford a pale yellow solid 3.6 g, yield: 75%. Analytical data for 7h: IR (KBr, cm−1): 3441, 2963, 1651, 1574, 1485, 1427, 1299, 1261, 1011. 1H NMR (CDCl3, 300 MHz): δ 10.42 (s, 1H, NH), 8.76 (s, 1H, Ar-H), 8.12–8.21 (m, 2H, Ar-H, CONH), 7.87 (m, 1H, Ar-H), 7.48–7.65 (m, 3H, Ar-H), 7.19–7.32 (m, 3H, Ar-H), 3.56 (m, 2H, NCH2), 3.38 (m, 2H, CH2N), 2.79 (s, 3H, ArCH3), 2.30 (s, 3H, COCH3), 1.53–1.69 (m, 4H, 2 × NCH2CH2), 1.27 (m, 2H, NHCH2CH2CH2); MS (ESI) m/z = 488 [M+H]+; Anal. Calcd. for C28H31N4O4: C, 68.97; H, 6.41; N, 11.49; Found: C, 68.82; H, 6.53; N, 11.63.

1-(4-Methoxyphenyl)-N-(5-(2-methylbenzamido)pentyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7i)

Refer to the synthesis of 7a, the title product was obtained from 5i and acetylsalicylic acid to afford a pale yellow solid 4.2 g, yield: 73%. Analytical data for 7i: IR (KBr, cm−1): 3400, 2961, 1677, 1574, 1484, 1428, 1321, 1264, 1020. Analytical data for 7i: 1H NMR (CDCl3, 300 MHz): δ 9.76 (s, 1H, NH), 8.71–8.75 (m, 2H, Ar-H, CONH), 8.15 (d, 1H, J = 7.5 Hz, Ar-H), 8.06 (m, 1H, Ar-H), 7.85 (d, 2H, J = 7.5 Hz, Ar-H), 7.56–7.63 (m, 3H, Ar-H), 7.41–7.47 (m, 3H, Ar-H), 7.05 (d, 2H, J = 7.5 Hz, Ar-H), 3.85 (s, 3H, OCH3), 3.59 (m, 2H, NCH2). 3.44 (m, 2H, NCH2), 2.31 (s, 3H, CH3CO), 1.70–1.85 (m, 4H, 2 ×NCH2CH2), 1.28 (m, 2H, NCH2CH2CH2). MS (ESI) m/z = 580 [M+H]+; Anal. Calcd. for C34H35N4O5: C, 70.45; H, 6.09; N, 9.67; Found: C, 70.32; H, 5.92; N, 9.52.

N-(6-(2-Methylbenzamido)hexyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7j)

Refer to the synthesis of 7a, the title product was obtained from 5j and acetylsalicylic acid to afford a pale yellow solid 3.8 g, yield: 77%. Analytical data for 7j: IR (KBr, cm−1): 3466, 2960, 1650, 1574, 1483, 1436, 1300, 1267, 1028. Analytical data for 7j: 1H NMR (CDCl3, 300 MHz): δ 11.08 (s, 1H, NH), 8.89 (s, 1H, Ar-H), 8.70 (s, 1H, Ar-H), 8.38 (m, 1H, CONH), 8.12–8.17 (m, 2H, CONH, Ar-H), 8.03 (m, 1H, Ar-H), 7.77 (m, 1H, Ar-H), 7.53–7.60 (m, 2H, Ar-H), 7.36–7.47 (m, 3H, Ar-H), 3.55 (m, 2H, NCH2), 3.41 (m, 2H, CH2N), 2.28 (m, 3H, CH3CO), 1.53–1.68 (m, 2H, NCH2CH2), 1.23–1.40 (m, 4H, 2 × NHCH2CH2CH2); MS (ESI) m/z = 488 [M+H]+; Anal. Calcd. for C28H31N4O4: C, 68.97; H, 6.41; N, 11.49; Found: C, 68.82; H, 6.53; N, 11.36.

1-Methyl-N-(6-(2-methylbenzamido)hexyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7k)

Refer to the synthesis of 7a, the title product was obtained from 5k (3.2 g, 10 mmol) and acetylsalicylic acid (1.8 g, 10 mmol) to afford a pale yellow solid 3.6 g, yield: 73%. Analytical data for 7k: IR (KBr, cm−1): 3451, 2925, 1669, 1576, 1475, 1427, 1316, 1265, 1028. 1H NMR (CDCl3, 300 MHz): δ 8.78 (s, 1H, Ar-H), 8.67 (s, 1H, CONH), 8.10 (m, 1H, Ar-H), 7.90 (m, 1H, Ar-H), 7.53–7.66 (m, 3H, Ar-H), 7.29–7.35 (m, 2H, Ar-H), 7.06 (m, 1H, Ar-H), 3.53–3.59 (m, 2H, NCH2), 3.36–3.40 (m, 2H, CH2N), 2.81 (s, 3H, ArCH3), 2.30 (s, 3H, COCH3), 1.59–1.70 (m, 4H, 2 × NCH2CH2), 1.25–1.46 (m, 4H, 2 × NHCH2CH2CH2); MS (ESI) m/z = 502 [M+H]+; Anal. Calcd. for C29H33N4O4: C, 69.44; H, 6.63; N, 11.17; Found: C, 69.26; H, 6.75; N, 11.32.

1-(4-Methoxyphenyl)-N-(6-(2-methylbenzamido)hexyl)-9H-pyrido[3,4-b]indole-3-carboxamide (7l)

Refer to the synthesis of 7a, the title product was obtained from 5l and acetylsalicylic acid to afford a pale yellow solid 4.2 g, yield: 70%. Analytical data for 7l: IR (KBr, cm−1): 3451, 2965, 1651, 1514, 1463, 1403, 1289, 1229, 1021. 1H NMR (CDCl3, 300 MHz): δ 8.67–8.73 (m, 2H, CONH, Ar-H), 8.12 (m, 1H, Ar-H), 8.06 (m, 1H, Ar-H), 7.90 (d, 2H, J = 9.0 Hz, Ar-H), 7.61–7.73 (m, 3H, Ar-H), 7.43–7.56 (m, 2H, Ar-H), 7.29 (m, 1H, Ar-H), 7.07 (d, 2H, J = 9.0 Hz, Ar-H), 3.89 (s, 3H, OCH3), 3.60 (m, 2H, NCH2), 3.42 (m, 2H, CH2N), 2.30 (s, 3H, COCH3), 1.59–1.70 (m, 4H, 2 × NCH2CH2), 1.23–1.45 (m, 4H, 2 × NCH2CH2CH2); MS (ESI) m/z = 594 [M+H]+; Anal. Calcd. for C35H37N4O5: C, 70.81; H, 6.28; N, 9.44; Found: C, 70.69; H, 6.49; N, 9.33.

N-(3-(2-Hydroxybenzamido)propyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8a)

To a solution of 7a (2.2 g, 5.0 mmol) in 20 mL methanol was added NaOH (0.4 g, 10.0 mmol) in 5 mL water and the mixture was stirred at 60 °C for 2 h, then added 10 mL water, neutralized with 2 M HCl and extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1 to 1:3, v/v) to to give a pale yellow solid 1.7 g, yield: 90%. Purity: 97.2% (by HPLC). Analytical data for 8a: 1H NMR (CDCl3, 300 MHz): δ 11.39 (s, 1H, NH), 8.78 (s, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 8.49 (s, 1H, CONH), 8.10 (m, 2H, Ar-H), 7.90 (m, 1H, Ar-H), 7.44–7.62 (m, 3H, Ar-H), 7.30 (s, 1H, Ar-H), 7.05 (m, 1H, Ar-H), 3.53–3.72 (m, 4H, 2 × NCH2), 1.90–1.95 (m, 2H, NCH2CH2); MS (ESI) m/z = 389 [M+H]+; HRMS (ESI): m/z calcd for C22H21N4O3: 389.1614; found: 389.1630 [M+H]+.

N-(3-(2-Hydroxybenzamido)propyl)−1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (8b)

Refer to the synthesis of 8a, the title product was obtained from 7b to afford a pale yellow solid 1.8 g, yield: 88%. Purity: 98.2% (by HPLC). Analytical data for 8b: 1H NMR (CDCl3, 300 MHz): δ 8.87 (s, 1H, Ar-H), 8.73 (s, 1H, Ar-H), 8.48 (s, 1H, NH), 8.14 (d, 1H, J = 6.0 Hz, Ar-H), 8.06 (m, 1H, Ar-H), 7.56 (m, 2H, Ar-H), 7.34 (m, 2H, Ar-H), 7.05 (m, 1H, Ar-H), 3.71 (m, 2H, CH2N), 3.56 (m, 2H, NCH2), 2.83 (s, 3H, ArCH3), 1.91 (m, 2H, NCH2CH2). MS (ESI) m/z = 403 [M+H]+; HRMS (ESI): m/z calcd for C23H23N4O3: 403.1770; found: 403.1787 [M+H]+.

N-(3-(2-Hydroxybenzamido)propyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8c)

Refer to the synthesis of 8a, the title product was obtained from 7c to afford a pale yellow solid 2.2 g, yield: 88%. Purity: 98.1% (by HPLC). Analytical data for 8c: 1H NMR (CDCl3, 300 MHz): δ 12.45 (s, 1H, NH), 8.77 (s, 1H, Ar-H), 8.68 (s, 1H, NH), 8.60 (s, 1H, CONH), 8.22–8.30 (m, 3H, Ar-H), 8.11 (m, 1H, Ar-H), 7.90 (m, 1H, Ar-H), 7.48–7.56 (m, 3H, Ar-H), 7.33–7.39 (m, 3H, Ar-H), 7.13 (m, 1H, Ar-H), 6.99 (m, 1H, Ar-H), 3.84 (s, 3H, OCH3), 3.63 (m, 2H, CH2N), 3.51 (m, 2H, NCH2), 2.01 (m, 2H, NCH2CH2). MS (ESI) m/z = 495 [M+H]+; HRMS (ESI): m/z calcd for C29H27N4O4: 495.2032; found: 495.2046 [M+H]+.

N-(4-(2-Hydroxybenzamido)butyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8d)

Refer to the synthesis of 8a, the title product was obtained from 7d (2 g, 10 mmol) to afford a pale yellow solid 2.0 g, yield: 88%. Purity: 97.3% (by HPLC). Analytical data for 8d: 1H NMR (CDCl3, 500 MHz): δ 11.57 (s, 1H, NH), 9.00 (s, 1H, CONH), 8.88 (s, 1H, Ar-H), 8.77 (s, 1H, Ar-H), 8.30 (m, 1H, Ar-H), 7.18 (d, 1H, J = 5.0 Hz, Ar-H), 7.54–7.61 (m, 3H, Ar-H), 7.33 (m, 1H, Ar-H), 7.17 (s, 1H, Ar-H), 7.03 (m, 1H, Ar-H), 3.57–3.61 (m, 4H, 2 × NCH2), 1.45–1.68 (m, 4H, 2 × NCH2CH2); MS (ESI) m/z = 403 [M+H]+; HRMS (ESI): m/z calcd for C23H23N4O3: 403.1770; found: 403.1791 [M+H]+.

N-(4-(2-Hydroxybenzamido)butyl)−1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (8e)

Refer to the synthesis of 8a, the title product was obtained from 7e (2.4 g, 10 mmol) to afford a pale yellow solid 1.8 g, yield: 87%. Purity: 96.5% (by HPLC). Analytical data for 8e: 1H NMR (CDCl3, 300 MHz): δ 11.92 (s, 1H, NH), 8.66 (s, 1H, CONH), 8.58 (m, 1H, Ar-H), 8.33 (m, 1H, Ar-H), 7.58–7.65 (m, 4H, Ar-H), 7.28 (m, 1H, Ar-H), 6.83 (m, 1H, Ar-H), 3.06–3.18 (m, 4H, 2 × NCH2), 2.85 (s, 3H, ArCH3), 1.43–1.59 (m, 4H, 2 × NCH2CH2); MS (ESI) m/z = 417 [M+H]+; HRMS (ESI): m/z calcd for C24H25N4O3: 417.1927; found: 417.1918 [M+H]+.

N-(4-(2-Hydroxybenzamido)butyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8f)

Refer to the synthesis of 8a, the title product was obtained from 7f (2.8 g, 10 mmol) to afford a pale yellow solid 2.3 g, yield: 90%. Purity: 95.4% (by HPLC). Analytical data for 8f: 1H NMR (CDCl3, 300 MHz): δ 12.22 (s, 1H, NH), 8.67 (s, 1H, Ar-H), 8.51 (s, 1H, CONH), 8.17 (m, 2H, Ar-H), 8.02 (s, 1H, Ar-H), 7.59 (m, 2H, Ar-H), 7.33–7.46 (m, 4H, Ar-H), 7.13 (m, 2H, Ar-H), 6.91 (m, 1H, Ar-H), 3.86 (s, 3H, OCH3), 3.24–3.39 (m, 4H, 2 × NCH2), 1.61 (m, 4H, 2 × NCH2CH2). MS (ESI) m/z = 509 [M+H]+; HRMS (ESI): m/z calcd for C30H29N4O4: 509.2189; found: 509.2208 [M+H]+.

N-(5-(2-Hydroxybenzamido)pentyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8g)

Refer to the synthesis of 8a, the title product was obtained from 7g (2.4 g, 10 mmol) to afford a pale yellow solid 1.9 g, yield: 91%. Purity: 95.4% (by HPLC). Analytical data for 8g: 1H NMR (CDCl3, 500 MHz): δ 12.01 (s, 1H, NH), 8.86 (s, 1H, Ar-H), 8.66–8.72 (m, 2H, NH, Ar-H), 8.37 (s, 1H, Ar-H), 7.18 (d, 1H, J = 6.0 Hz, Ar-H), 7.55–7.68 (m, 4H, Ar-H), 7.16–7.22 (m, 3H, NH, Ar-H), 3.40–3.72 (m, 4H, 2 × NCH2), 1.45–1.65 (m, 4H, 2 × NCH2CH2), 1.21–1.27 (m, 2H, NCH2CH2CH2); MS (ESI) m/z = 417 [M+H]+ ; HRMS (ESI): m/z calcd for C24H25N4O3: 417.1927; found: 417.1944 [M+H]+.

N-(5-(2-Hydroxybenzamido)pentyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (8h)

Refer to the synthesis of 8a, the title product was obtained from 7h (2.4 g, 10 mmol) to afford a pale yellow solid 2.0 g, yield: 92%. Purity: 96.0% (by HPLC). Analytical data for 8h: 1H NMR (CDCl3, 300 MHz): δ 8.78 (s, 1H, NH), 8.39 (s, 1H, NH), 8.17 (m, 1H, Ar-H), 8.02 (m, 1H, Ar-H), 7.58–7.60 (m, 2H, Ar-H), 7.36 (m, 1H, Ar-H), 7.19 (m, 2H, Ar-H), 7.05 (m, 1H, Ar-H), 3.58–3.72 (m, 4H, 2 × NCH2), 2.87 (s, 3H, ArCH3), 1.53 (m, 4H, 2 × NCH2CH2), 1.18 (m, 2H, NHCH2CH2CH2); MS (ESI) m/z = 431 [M+H]+; HRMS (ESI): m/z calcd for C25H27N4O3: 431.2083; found: 431.2102 [M+H]+.

N-(5-(2-Hydroxybenzamido)pentyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8i)

Refer to the synthesis of 8a, the title product was obtained from 7i (2.9 g, 10 mmol) to afford a pale yellow solid 2.3 g, yield: 89%. Purity: 98.4% (by HPLC). Analytical data for 8i: 1H NMR (CDCl3, 300 MHz): δ 12.60 (s, 1H, NH), 8.76 (m, 1H, Ar-H), 8.62 (m, 1H, Ar-H), 8.13–8.24 (m, 2H, CONH, Ar-H), 8.02 (s, 1H, Ar-H), 7.79 (m, 1H, Ar-H), 7.43–7.58 (m, 5H, Ar-H), 7.09 (m, 2H, Ar-H), 6.93 (m, 1H, Ar-H), 3.91 (s, 3H, OCH3), 3.55–3.65 (m, 4H, 2 × NCH2), 1.78–1.84 (m, 4H, 2 × NCH2CH2), 1.24 (m, 2H, NHCH2CH2CH2); MS (ESI) m/z = 523 [M+H]+; HRMS (ESI): m/z calcd for C31H31N4O4: 523.2345; found: 523.2328 [M+H]+.

N-(6-(2-Hydroxybenzamido)hexyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8j)

Refer to the synthesis of 8a, the title product was obtained from 7j (2.4 g, 10 mmol) to afford a pale yellow solid 1.8 g, yield: 87%. Purity: 95.4% (by HPLC). Analytical data for 8j: 1H NMR (CDCl3, 300 MHz): δ 11.35 (s, 1H, NH), 8.86 (s, 1H, Ar-H), 8.77 (s, 1H, Ar-H), 8.57 (m, 2H, CONH, Ar-H), 8.15 (m, 1H, Ar-H), 7.78 (m, 1H, Ar-H), 7.45–7.67 (m, 3H, Ar-H), 7.13 (m, 1H, Ar-H), 6.91 (m, 1H, Ar-H), 3.52–3.71 (m, 4H, 2 × NCH2), 1.60–1.77 (m, 4H, 2 × NCH2CH2), 1.19–1.32 (m, 4H, 2 × NHCH2CH2CH2); MS (ESI) m/z = 431 [M+H]+; HRMS (ESI): m/z calcd for C25H27N4O3: 431.2083; found: 431.2098 [M+H]+.

N-(6-(2-Hydroxybenzamido)hexyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (8k)

Refer to the synthesis of 8a, the title product was obtained from 7k (2.5 g, 10 mmol) to afford a pale yellow solid 1.9 g, yield: 87%. Purity: 98.5% (by HPLC). Analytical data for 8k: 1H NMR (CDCl3, 500 MHz): δ 11.87 (s, 1H, NH), 8.66 (m, 1H, Ar-H), 8.56 (m, 1H, Ar-H), 8.35 (d, 1H, J = 10.0 Hz, Ar-H), 7.62–7.76 (m, 4H, Ar-H), 7.31 (m, 1H, Ar-H), 6.98 (m, 2H, CONH, Ar-H), 3.36 (m, 2H, NCH2), 2.85 (s, 3H, ArCH3), 3.02 (m, 2H, NCH2), 1.53–1.66 (m, 4H, 2 × NCH2CH2), 1.23–1.41 (m, 4H, 2 × NHCH2CH2CH2); MS (ESI) m/z = 445 [M+H]+; HRMS (ESI): m/z calcd for C26H29N4O3: 445.2240; found: 445.2223 [M+H]+.

N-(6-(2-Hydroxybenzamido)hexyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (8l)

Refer to the synthesis of 8a, the title product was obtained from 7l (3.0 g, 10 mmol) to afford a pale yellow solid 2.4 g, yield: 88%. Purity: 96.2% (by HPLC). Analytical data for 8l: 1H NMR (CDCl3, 300 MHz): δ 11.80 (s, 1H, NH), 10.43 (s, 1H, NH), 8.77 (m, 3H, Ar-H, NH), 8.40 (d, 1H, J = 6.0 Hz, Ar-H), 8.17 (d, 2H, J = 6.3 Hz, Ar-H), 7.57–7.70 (m, 3H, Ar-H), 7.19–7.32 (m, 4H, Ar-H), 6.85 (m, 1H, Ar-H), 3.89 (s, 3H, OCH3), 3.35–3.63 (m, 4H, 2 × NCH2), 1.61–1.83 (m, 4H, 2 × NCH2CH2), 1.22–1.38 (m, 4H, 2 × NHCH2CH2CH2); MS (ESI) m/z = 537 [M+H]+; HRMS (ESI): m/z calcd for C32H33N4O4: 537.2502; found: 537.2488 [M+H]+.

N-(5-acetamidopentyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (10a)

To a solution of 5h (0.31 g, 1.0 mmol) and triethylamine (0.15 g, 1.5 mmol) in 3 mL anhydrous DMF was slowly added acetyl chloride (86 mg, 1.1 mmol) in 1 mL anhydrous CH2Cl2 under ice bath. After addition, the mixture was stirred at room temperature for 2 h, and then poured into 30 mL water, then extracted with ethyl acetate (3 × 25 mL). The combined organic layer was washed with brine, dried with Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1 to 1:3, v/v) to afford a pale yellow solid, yield :79%. Purity: 95.6% (by HPLC). Analytical data for 10a: IR (KBr, cm−1): 3432, 2941, 1628, 1476, 1420, 1250, 1018. 1H NMR (CDCl3, 300 MHz): δ 11.91 (s, 1H, NH), 8.63–8.66 (m, 2H, Ar-H, NH), 8.35 (s, 1H, Ar-H), 7.63 (d, 1H, J = 6.0 Hz, Ar-H), 7.59 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 5.76 (s, 1H, NH), 3.66 (t, 2H, J = 6.0 Hz, CONHCH2), 3.40 (m, 2H, CH2N), 2.84 (s, 3H, Ar-CH3), 2.33 (s, 3H, COCH3), 1.69–1.82 (m, 4H, 2 × NCH2CH2), 1.23–1.30 (m, 2H, NCH2CH2CH2); MS (ESI) m/z = 353 [M+H]+.

N-(5-benzamidopentyl)-1-methyl-9H-pyrido[3,4-b]indole-3-carboxamide (10b)

Refer to the synthesis of 10a, the title product was obtained from 5h (0.31 g, 1.0 mmol) and benzoyl chloride (0.15 g, 1.1 mmol) to afford a pale yellow solid, yield: 75%. Purity: 98.0% (by HPLC). Analytical data for 10b: IR (KBr, cm−1): 3466, 2958, 1620, 1468, 1418, 1244, 1011. 1H NMR (CDCl3, 300 MHz): δ 11.91 (s, 1H, NH), 8.67 (s, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 8.49 (m, 1H, NHCO), 8.34 (m, 1H, Ar-H), 7.84 (d, 1H, J = 6.0 Hz, Ar-H), 7.64 (d, 1H, J = 6.0 Hz, Ar-H), 7.51–7.58 (m, 2H, Ar-H), 7.44 (m, 2H, Ar-H), 7.28 (m, 1H, Ar-H), 3.40 (m, 2H, NCH2), 3.31 (m, 2H, NCH2), 2.84 (s, 3H, CH3), 1.65–1.78 (m, 4H, 2 × NCH2CH2), 1.22–1.28 (m, 2H, NCH2CH2CH2); MS (ESI) m/z = 415 [M+H]+.

Biological evaluation

MTT assay

Hepatocellular carcinoma cells (SMMC-7721 and Hep G2), Human colon cancer cell lines (HCT116), human bladder carcinoma cells (EJ) or human lung cancer cells (H460) at 104 cells per well were cultured in 10% FBS DMEM in 96-well flat-bottom microplates overnight. The cells were incubated in triplicate with, or without, different concentrations of each test compound for 48 h. During the last 4 h incubation, 30 μL of tetrazolium dye (MTT) solution (5 mg mL−1) was added to each well. The resulting MTT-formazan crystals were dissolved in 150 μL DMSO, and absorbance was measured spectrophotometrically at 570 nm using an ELISA plate reader. The inhibition induced by each test compound at the indicated concentrations was expressed as a percentage. The concentration required for 50% inhibition (IC50) was calculated using the software (GraphPadPrism Version 4.03).

Flow cytometry assay of cell apoptosis

SMMC-7721 cells were cultured overnight and incubated in triplicate with compound 8h (7.0 and 14 μM), 5-FU (14 μM), or vehicle for 48 h. The cells were harvested, and stained with FITC-Annexin V and PI (BioVision) at room temperature for 15 min. The percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter) analysis.

Mitochondrial membrane potential assay

Mitochondrial membrane potential was assessed using a Beyotime (China) kit with JC-1. Briefly, the SMMC-7721 cells in the logarithmic growth phase were treated with 8h (7.0 or 14 μM), 5-FU (14 μM), or vehicle control for 24 h. The cells were harvested, and then labeled with JC-1 in accordance with the manufacturer’s instructions. The samples labeled with JC-1 were analyzed via flow cytometry. For the detection of JC-1, excitation was set at 530 nm, and emissions collected at 585 nm.

Western blot assay

The mechanisms of the cell apoptosis were determined by western blot assay. SMMC-7721 cells at 1.5 × 105/mL were treated with 3.5, 7.0, or 14 μM 8h or vehicle control for 8 h. After harvested and lyzed, the cell lysates (50 μg/lane) were separated by SDS-PAGE (12% gel) and transferred onto nitrocellulose membranes. After blocked with 5% fat-free milk, the target proteins were probed with anti-Bcl-2, anti-Bax, anti-caspase-3, anti-PARP, and anti-β-actin antibodies (Cell Signaling, Boston), respectively. The bound antibodies were detected by HRP-conjugated second antibodies and visualized using the enhanced chemiluminescent reagent.

Additional Information

How to cite this article: Xu, Q.-B. et al. Design, synthesis and biological evaluation of hybrids of β-carboline and salicylic acid as potential anticancer and apoptosis inducing agents. Sci. Rep. 6, 36238; doi: 10.1038/srep36238 (2016).

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References

  1. 1.

    , , & β-Carboline alkaloids: biochemical and pharmacological functions. Curr. Med. Chem. 14, 479−500 (2007).

  2. 2.

    et al. Design and synthesis of C3-pyrazole/chalcone-linked beta-carboline hybrids: antitopoisomerase-I, DNA-interactive, and apoptosis-inducing anticancer agents. Chem. Med. Chem. 9(9), 2084–2098 (2014).

  3. 3.

    et al. Ruthenium-Arene-beta-Carboline Complexes as Potent Inhibitors of Cyclin-Dependent Kinase 1: Synthesis, Characterization and Anticancer Mechanism Studies. Chemistry 19, 12152–12160 (2013).

  4. 4.

    et al. A series of beta-carboline derivatives inhibit the kinase activity of PLKs. PLoS One 7, e46546 (2012).

  5. 5.

    , , , & beta-Carboline alkaloids in Peganum harmala and inhibition of human monoamine oxidase (MAO). Food Chem. Toxicol. 48, 839–845 (2010).

  6. 6.

    , , & Alkaloid-metal based anticancer agents. Curr. Top. Med. Chem. 13(17), 2104–2115 (2013).

  7. 7.

    et al. Synthesis of beta-carboline-benzimidazole conjugates using lanthanum nitrate as a catalyst and their biological evaluation. Org. Biomol. Chem. 12, 2370–2387 (2014).

  8. 8.

    et al. Novel N-(3-carboxyl-9-benzyl- beta-carboline-1-yl)ethylamino acids: Synthesis, anti-tumor evaluation, intercalating determination, 3D QSAR analysis and docking investigation. Eur. J. Med. Chem. 44, 4153–4161 (2009).

  9. 9.

    et al. β-Carboline/Hydroxamic Acid Hybrids Targeting Both Histone Deacetylase and DNA Display High Anticancer Activity via Regulation of the P53 Signaling Pathway. J. Med. Chem. 58(23), 9214−9227 (2015).

  10. 10.

    & DNA minor groove binding ligands: a new class of anticancer agents. Expert Opin. Investig Drugs. 6(7), 875–884 (1997).

  11. 11.

    et al. Synthesis of CDDO-amino acid-nitric oxide donor trihybrids as potential antitumor agents against both drug-sensitive and drug-resistant colon cancer. J. Med. Chem. 58(5), 2452–2464 (2015).

  12. 12.

    et al. Synthesis and biological evaluation of hybrids from farnesylthiosalicylic acid and hydroxylcinnamic acid with dual inhibitory activities of Ras-related signaling and phosphorylated NF-κB. Org. Biomol. Chem. 12(25), 4517–4530 (2014).

  13. 13.

    , , & 1,3,5-Triazine inhibitors of histone deacetylases: synthesis and biological activity. Med. Chem. Res. 23(12), 5188–5196 (2014).

  14. 14.

    et al. Aspirin and non-steroidal anti-inflammatory drugs for cancer prevention: an international consensus statement. Lancet Oncol. 10, 501–507 (2009).

  15. 15.

    , , , & Mechanisms underlying aspirin-mediated growth inhibition and apoptosis induction of cyclooxygenase-2 negative colon cancer cell line SW480. World J. Gastroenterol. 14, 4227–4233 (2008).

  16. 16.

    et al. Aspirin and cancer: has aspirin been overlooked as an adjuvant therapy? Br. J. Cancer 105, 1107–1113 (2011).

  17. 17.

    , , , & Aspirin and cancer risk: a quantitative review to 2011. Ann. Oncol. 23, 1403–1415 (2012).

  18. 18.

    & Effects of regular aspirin on long-term cancer incidence and metastasis: a systematic comparison of evidence from observational studies versus randomised trials. Lancet Oncol. 13, 518–527 (2012).

  19. 19.

    et al. The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells. Eur. J. Clin. Invest. 32, 838–846 (2002).

  20. 20.

    , , & Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures. Planta 228, 89–97 (2008).

  21. 21.

    et al. Novel Resveratrol-Based Aspirin Prodrugs: Synthesis, Metabolism, and Anticancer Activity. J. Med. Chem. 58(16), 6494–6506 (2015).

  22. 22.

    et al. Synthesis and biological evaluation of novel farnesylthiosalicylic acid/salicylic acid hybrids as potential anti-tumor agents. Chin. Chem. Lett. 25(12), 1545–1549 (2014).

  23. 23.

    et al. Design, synthesis, and biological evaluation of indenoisoquinoline topoisomerase I inhibitors featuring polyamine side chains on the lactam nitrogen. J. Med. Chem. 46(26), 5712–5724 (2003).

  24. 24.

    et al. Synthesis of new indeno[1,2-c]isoquinolines: cytotoxic non-camptothecin topoisomerase I inhibitors. J. Med. Chem. 43(20), 3688–3698 (2000).

  25. 25.

    , , & Doxycycline in mitochondrial mediated pathway of apoptosis: a systematic review. Anticancer Agents Med. Chem. 10(7), 556–563 (2010).

  26. 26.

    et al. Withaferin A induces apoptosis by activating p38 mitogen-activated protein kinase signaling cascade in leukemic cells of lymphoid and myeloid origin through mitochondrial death cascade. Apoptosis 13, 1450–1464 (2008).

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Acknowledgements

We gratefully acknowledge the financial support by the Natural Science Foundation of China (Grant Nos 81302628 and 81473089), the Project of “Jiangsu Six Peaks of Talent” (2014-SWYY-044), China Pharmaceutical University for the Open Project Program of State Key Laboratory of Natural Medicines (SKLNMKF201611), China and Jiangsu Province Postdoctoral Science Foundation (2016M590488 and 1601136B), Jiangsu Government Scholarship for Overseas Studies (JS-2014-212), Applied Research Projects of Nantong City (MS12015060), and also thank a project funded by the Priority Academic Programs Development of Jiangsu Higher Education Institutions (PAPD).

Author information

Author notes

    • Qi-Bing Xu
    •  & Xiang-Fan Chen

    These authors contributed equally to this work.

Affiliations

  1. School of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, China

    • Qi-Bing Xu
    • , Xiang-Fan Chen
    • , Jiao Feng
    • , Ji Liu
    • , Feng-Tao Liu
    • , Bi-Xi Niu
    • , Jin-Yang Cai
    • , Chao Huang
    • , Yanan Zhang
    •  & Yong Ling
  2. State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P.R. China

    • Qi-Bing Xu
    •  & Yong Ling
  3. Tumor chemotherapy Department, Affiliated Hospital of Nantong University, China

    • Jie-Fei Miao

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Contributions

Q.X., J.F., Y.-A.Z. and Y.L. conceived and designed the project. Q.X., X.-F.C., J.L., F.-T.L., L.-N.M., J.-Y.C. and C.H. conducted the experiments. J.F., B.-X.L., Y.-A.Z. and Y.L. analyzed the data and prepared the manuscript. All authors read and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Yanan Zhang or Yong Ling.

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https://doi.org/10.1038/srep36238

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