Synthesis and biological evaluation of novel N9-heterobivalent β-carbolines as angiogenesis inhibitors

Abstract A series of novel N9-heterobivalent β-carbolines has been synthesized. All the novel compounds were tested for their anticancer activity against six tumour cell lines in vitro. Among these molecules, compounds 5b, and 5w exhibited strong cytotoxic activities with IC50 value of lower than 20 μM. Acute toxicities and antitumor efficacies of the selected compounds in mice were also evaluated, compounds 5b and 5w exhibited that tumour inhibition rate of over 40% in the Sarcoma 180 and Lewis lung cancer animal models. Preliminary structure–activity relationships (SARs) analysis indicated that: (1) C1-methylation and C7-methoxylation were favorable for increased activities; (2) 3-Pyridyl or 2-thienyl group substituent into position-1 of the β-carboline core, and the aryl substituent into another β-carboline ring might be detrimental to cytotoxic effects of this class compounds. Investigation of the preliminary mechanism of action demonstrated that compound 5b had obvious angiogenesis inhibitory effects in the chicken chorioallantoic membrane (CAM) assay.


Introduction
The population growth and aging associated with some risk factors leveraged the incidence of new cases of cancer and related deaths in developed countries and in developing countries 1 . Cancer resistance to therapy is becoming a common phenomenon that threatens the current strategies adopted against this disease. For that reason we need to discover new anticancer agents. One of the successful and effective methods for the discovery of new anticancer drugs from natural products is to synthesize novel compounds through chemical structural modifications on the basis of leading compounds.
Peganum harmala L. have been traditionally used for hundreds of years to treat the alimentary tract cancers and malaria in Northwest China. Harmine, originally isolated from the seeds of Peganum harmala L. in 1847, is the most representative natural occurring b-carboline alkaloid, having a common tricyclic pyrido [3,4-b] indole ring structure. In the past several decades, it has confirmed that harmine was the important active ingredients to treat the alimentary tract cancers 2,3 . Recent reports demonstrated that harmine and its derivatives had remarkable antitumor activities, together with potential neurotoxicity [2][3][4][5] . Moreover, it has been reported that harmine and its derivatives can exert antitumor activities through multiple mechanisms, such as DNA binding [6][7][8] , inhibition topoisomerases I and II 9,10 , CDK (cyclindependent kinase) 11,12 , PLK1 (polo-like kinase) 13 , lipoxygenase 14,15 and IjB kinases 16 .
Our strategy was based on the modification of the prototype B-9-3, following this previous work, we have continued our search for novel antitumor agents endowed with better antitumor activities, and we provide detailed studies of structure-activity relationships (SARs) on the antitumor efficacies in vitro and in vivo of this class of compounds. Here, we designed and synthesized a series of novel N 9 -heterobivalent b-carboline derivatives as potent antitumor agents.
Melting points were determined in capillary tubes on an electrothermal X-5 apparatus and without correction. NMR spectra were recorded at room temperature on a Bruker Avance III HD 400 instrument at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. Chemical shifts (d) are reported in ppm relatively to the residual solvent peak and the multiplicity of each signal is designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Coupling constants (J) are quoted in Hz. High resolution mass spectra (HRMS) were recorded on Bruker ultrafleXtreme MALDI-TOF/TOF-MS and HCCA (alpha-cyano-4hydroxycinnamic acid) is used as matrix. Elemental analyses (C, H, and N) were carried out on an Elementar Vario ELIII CHNS Elemental Analyzer. Column chromatography was performed with silica gel (200-300 mesh) and analytical TLC on silica gel 60-F 254 .
General procedure for the preparation of compounds 4a-h A mixture of 3a (1.68 g, 10 mmol) and anhydrous DMF (50 ml) was stirred at room temperature for 0.5 h, then NaH (0.50 g, 20 mmol) and the 1,4-dibromobutane (20 mmol) were added. The mixture was stirred at room temperature. After that period, the mixture was monitored via TLC and at the end of the reaction the mixture was poured into H 2 O (150 ml) and extracted with ethyl acetate. The organic phase was collected and washed with water and brine, then dried under anhydrous sodium sulfate, filtered, and evaporated. The resulting oil was crystallized from ethyl ether or ethyl ether-petroleum ether to afford the compound 4a. Products 4b-h were prepared according to the same method of 4a.   General procedure for the preparation of compounds 5a-x A solution of compound 3c (2 mmol) in anhydrous DMF (6 ml) was added slowly with stirring to a solution of 4a (3 mmol), NaH (0.25 g, 10 mmol), potassium iodide (1.68 g, 10 mmol) in anhydrous DMF (25 ml). The mixture was stirred at room temperature until the reaction is completed. Then the mixture was poured into icecold water. The reaction mixture was extracted with ethyl acetate (3 Â 30 ml), washed with water and brine, dried over anhydrous Na 2 SO 4 , and filtered. Purification by column chromatography (DCM/MeOH 100:1 as the eluent) furnished the dimeric b-carboline 5a. Compounds 5b-x were synthesized using similar procedure as compound 5a.

Assay of acute toxicities
Specific pathogen-free KM mice (6-8 weeks old) weighing 19-22 g were housed in a mouse room at 24 ± 2 C and 60-70% humidity with 12 h light/dark cycles. The mice were provided rodent laboratory chow pellets and tap water for a week to adapt to the environment of the mouse room. The experimental protocol was approved by the Institutional Animal Ethical Committee, and all of the animals were provided by Laboratory Animal Center of Xinjiang Uygur Autonomous Region. Prior to each experiment, mice were fastened overnight and allowed free access to water. Various doses of the asymmetric dimeric b-carboline derivatives, ranging from 5.0 to 500 mg kg À1 dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) salt solution, were given intraperitoneally to different groups of healthy KM mice, and each group contained 10 mice (5 males and 5 females). After the administration of the compounds, the mice were observed continuously for the first 2 h for any gross behavioral changes and deaths, then intermittently for the next 24 h, and occasionally thereafter for 14 days, and for the onset of any delayed effects. All of the animals were killed on the 14th day after drug administration, and they were checked macroscopically for possible damage to the heart, liver, and kidneys. Mice that experienced immediate death following drug administration were also examined for any possible organ damage. LD 50 values were calculated graphically as described 34 .

In vivo antitumor activity
Sarcoma180 and Lewis lung cancer cell lines were provided by Shanghai Institute of Pharmaceutical Industry. Mice were inoculated with Sarcoma180 and Lewis lung cancer tumour cells. After 7 days, the tumours were removed and the cells were harvested. Mice received subcutaneous injections of viable tumour cells (2 Â 10 6 cells/mouse) in the armpit. Each compound was administered via i.p. injection to different groups of mice (each group contained 10 female mice) 24 h after the inoculation at a dosage about one-fifth of the LD 50 value once a day for seven consecutive days. This dose was the maximum tolerated dose for most of the compounds based on our preliminary studies. CTX at 30 mg kg À1 was used as the positive control and the vehicle as the negative control. The weight of the animals was recorded every three days. All of the animals were killed on the 21st day after tumour inoculation, and the tumours were excised and weighed.
The inhibition rate was calculated as follows: where T is the average tumor weight of the treated group and C is the average tumor weight of the negative control group.

CAM assay in vivo
Anti-angiogenic activity of the selected compounds 5b and 5w were investigated in vivo using a CAM assay. Five-day-old fertilized eggs were obtained from a local hatchery. We injected 5 ml of albumin, and the eggs were incubated horizontally to allow the CAM to detach from the shell to produce a sham chamber. Compounds 5b and 5w were prepared in gelatin sponge discs (5 Â 5 Â 5 mm 3 ) at the concentration of 0.5, 5.0, and 50 mM/disc, respectively. CA4P was used as the positive control drug. Discs containing the vehicle only (DMSO) were used as the negative control. A small window opening was made in the shell, and the discs were directly applied onto the CAM. The square opening was covered with sterilized surgical tape and the embryos were incubated for 48 h at 38.5 C. The CAMs were photographed under a dissecting microscope and blood vessels in each CAM were counted. The results are presented as a mean percentage of inhibition to the control ± SD, (n ¼ 3).

Chemistry
The synthesis of the desired N 9 -heterobivalent b-carbolines (5a-r) were performed in four steps starting from L-tryptophan as outlined in Scheme 1-2. The tetrahydro-b-carbolines 2a-h was prepared by condensation of L-tryptophan with appropriate aldehyde via the Pictet-Spengler condensation, and followed by oxidation and decarboxylation to afford the intermediate 1-substitutedb-carbolines 3a-h 21,29 . Then the N 9 of 3a-d was alkylated by the action of sodium hydride (NaH) in N,N-dimethylformamide (DMF) followed by the addition of the appropriate dibromo alkane to obtain intermediates 4a-f. Finally, 4a-f with appropriate 1-substituted-b-carbolines 3c-h in the presence of sodium hydride in DMF at room temperature to afford the target compounds 5a-r in 47-74% yield.
In Scheme 3, the intermediates 4g-f were synthesized from harmine and 1,4-dibromobutane or 1,5-dibromopentane using the same method of compound 4a, and finally compounds 5s-x were obtained from the similar method of 5a.
The chemical structures of all the novel N 9 -heterobivalent b-carbolines were characterized by 1 H NMR, 13 C NMR, HRMS and the elemental analysis.
In vitro cytotoxicity and structure-activity relationships All the 24 novel synthesized N 9 -heterobivalent b-carbolines (5a-x) were screened for their in vitro cytotoxic activities against six different cancer cell lines, namely BGC-823 (gastric carcinoma), HepG2 (liver carcinoma), MCF-7 (breast carcinoma), HT-29 (colon carcinoma), Eca-109 (esophageal carcinoma) and LLC (Lewis lung carcinoma). Cisplatin and B-9-3 were used as the reference control and the results were expressed as IC 50 values and summarized in Table 1. The IC 50 values were the average of at least three independent experiments.
As illustrated in Table 1, compounds 5b and 5w displayed a broad spectrum of cytotoxic activities with IC 50 value of lower than 20 lM against the tested six tumor cell lines, while compounds 5c, 5s, 5t and 5v only exhibited strong cytotoxic effects with IC 50 value of lower than 20 lM against three or four tumour cell lines. Interestingly, compounds 5d and 5j were selectively active against Eca-109 and LLC cell lines with IC 50 value of lower than 20 lM but fail to show cytotoxic effects in other cell lines. Similarly, compound 5u displayed selective activities against HT-29 and LLC cell lines. Moreover, compound 5f only exhibited strong cytotoxic effects with IC 50 value of lower than 20 lM against LLC cell lines. compounds 5g and 5i showed weak cytotoxic activities with IC 50 values in the range of 18.8-97.6 lM. Unfortunately, compounds 5k-5r were weak or inactive against all tumour cell lines tested. Scheme 2. Synthesis of the N 9 -heterobivalent b-carbolines 5a-r. Scheme 3. Synthesis of the N 9 -heterobivalent b-carbolines 5s-x.
Next, we examined the influence of the spacer length of N 9heterobivalent b-carbolines on cytotoxic activities. According the previous investigation, the synthesized N 9 -heterobivalent b-carbolines were connected at the indolo-N by an alkyl chain, which ranged from 4 to 6 carbon atoms. Comparing the structure of 5b-5d, which bearing different carbon atoms linker and 3-pyridyl group in position-1 of the b-carboline core, compound 5b bearing 4-carbon atoms showed a broad spectrum of cytotoxic activities with IC 50 value of lower than 20 lM against six tumor cell lines, and all the three compounds show selective cytotoxicities with IC 50 value of lower than 20 lM against LLC. Similarly, further introduction of 2-thienyl group in position-1 of the b-carboline ring resulted in dimeric derivatives 5f-5h. Among these derivatives, the IC 50 values of compound 5f against BGC, MCF7, and LLC cells is 38.8, 46.6, and 6.1 lM, respectively, compound 5 g displayed IC 50 values for HepG2 and HT-29 of 18.8 and 32.3 lM, respectively, and compound 5h only exhibited higher cytotoxic effects with IC 50 value of 30.2 lM against Eca-109 than other two compounds. Comparing the structure of 5i with 5j, compound 5i showed weak or moderate cytotoxic activities against all tumor cell lines tested, and compound 5j was selectively active against Eca-109 and LLC cell lines with IC 50 value of lower than 20 lM but fail to show cytotoxic effects in BGC and HepG2 at the concentration of 100 lM. Comparing the structure of 5u with 5v, there is an extra methoxyl group attached to position-7 of the b-carboline, compound 5v demonstrated the higher cytotoxic activities against the tested tumor cell lines than 5u (except for HepG-2 cell line). Similarly, comparing the structure 5w with 5x, compound 5w displayed the higher cytotoxic activities against the tested tumor cell lines than 5x (except for LLC cell line). These results suggested that the length of the spacer had no obvious relationship with the cytotoxic activities against the tumor cell lines.
An overview of the cytotoxic activities data of all new synthesized N 9 -heterobivalent b-carbolines. 11 compounds were found to exhibit selective activity against LLC cell lines with IC 50 value of lower than 20 lM. Moreover, Compound 5b, 5s displayed higher cytotoxic activities against at least four tumour cell lines than the prototype B-9-3, and compound 5c, 5t, 5v, 5w exhibited higher cytotoxic effects against three tumour cell lines than B-9-3.
In summary, a total analysis of the cytotoxic activities of N 9 -heterobivalent b-carbolines in vitro clearly suggest that: (1) C 1 -methylation and C 7 -methoxylation were favorable for increased activities; (2) 3-Pyridyl or 2-thienyl group substituent into position-1 of the b-carboline core, and the aryl (electron withdrawing and donating groups) substituent into position-1' of another b-carboline ring might be detrimental to cytotoxic effects of this class of compounds.

Assessment of acute toxicity
The LD 50 values of the selected N 9 -heterobivalent b-carbolines in mice after intraperitoneal (i.p.) administration are shown in Table 2. All of the tested dimeric b-carbolines resulted in acute toxic manifestation but they did not cause any obvious neurotoxic effects, including tremors, twitch, jumping, and supination. The animals showed a decrease in locomotive activity after the administration of various bivalent b-carbolines. Death occurred mostly in the high dosage group within 4-8 h after injection. All of the surviving animals returned to normal within the next day. Autopsies of the animals that died during the course of experiment and the necropsy findings in the surviving animals at the end of the experimental period (14 days) revealed no obvious changes in any of the organs.
Of all of the investigated asymmetric dimeric b-carbolines, compounds 5b and 5c, which had no substituent in position-7, and bearing different carbon atoms linker and 3-pyridyl group in position-1 of the b-carboline core, demonstrated weaker acute toxicities with the LD 50 values of 150 mg kg À1 and 175 mg kg À1 , respectively. Compounds 5t and 5w, which have a harmine molecular linked with another 1-substituted b-carboline ring, displayed remarkable acute toxicity with the LD 50 values of 35 mg kg À1 and 50 mg kg À1 , respectively. These results suggested that the methoxy substituent in position-7 of b-carboline nucleus played a vital role in determining the remarkable neurotoxic effects. 1 a Data are expressed as mean ± SD. b Acute neurotoxic manifestation were denoted by "þ" and "-". "þ" represents toxic responses including tremble, twitch, jumping and supination, while "-" means no such reaction. c ND ¼ not determined.

Evaluation of antitumor activity of N 9 -heterobivalent b-carbolines in vivo
Based on the in vitro assay results, we further tested the antitumor activity of four N 9 -heterobivalent b-carbolines in vivo against mice bearing Sarcoma 180 and Lewis lung cancer, respectively, and the reference drugs Cyclophosphamide (CTX). Our previous investigation demonstrated that mice bearing Lewis lung cancer were more susceptible to b-carbolines than other animal models; therefore, these animal models were selected and evaluated in the present investigation. 5 The tumor inhibition rates of all of the investigated asymmetric dimeric b-carbolines were summarized in Table 2.
As shown in Table 2, all the tested N 9 -heterobivalent b-carbolines displayed moderate to strong antitumor activities in animal model. Interestingly, compounds 5b and 5c, which having the same substituent and different carbon atoms linker. Compound 5b, which bearing 4-carbon atoms, exhibited the potent antitumor agent with the tumor inhibition rate of 43.6 and 41.9% against Sarcoma 180-bearing mice and Lewis lung cancer-bearing mice, respectively, and compound 5c displayed moderate antitumor activity with the tumor inhibition rate of 33.6% against Sarcoma 180-bearing mice at doses of 35 mg kg À1 . Compound 5t, the harmine and harman linked with five methylene units, showed moderate antitumor activity against mice with Sarcoma 180 with the tumour inhibition rate of 37.2% at doses of 7 mg kg À1 . Particularly, compound 5w was found to be the remarkable antitumor agent with the tumor inhibition rate of 47.1 and 42.3% against mice bearing Sarcoma 180 and Lewis lung cancer, respectively. These results implied that the harmine or harman linked with another of b-carboline nucleus (3-pyridyl or 2-thienyl substituent into position-1) enhanced their antitumor activities. In vivo anti-angiogenic effect of compounds 5b and 5w The most potent compounds, 5b and 5w, were selected to evaluate anti-angiogenic activity by chicken chorioallantoic membrane (CAM) assay. The inhibitory effects of compounds 5b and 5w on the angiogenesis of CAM are shown in Figure 2(A). The anti-angiogenetic activity of compounds 5b and 5w were semi-quantitatively analyzed using Graph Pad Prism 5.0 (shown in Figure 2(B)). The results showed that compound 5b (p < 0.05) could inhibit the angiogenesis of CAM. The anti-angiogenetic activity of compound 5b was comparable to the CA4P in vivo CAM assay at the same dose (50 lM).

Conclusions
In this study, we synthesized 24 new, N 9 -heterodimeric b-carboline derivatives and focused on compounds with 4 À 6 carbon linkers between the indole nitrogen. All of the compounds were screened for their in vitro cytotoxic activity against BGC-823, HepG2, MCF-7, HT-29, Eca-109 and LLC cancer cell lines. The results showed that compounds 5b, and 5w exhibited strong cytotoxic activities with IC 50 value of lower than 20 lM against the six tested tumor cell lines. In addition, four asymmetric dimeric b-carbolines were selected for evaluation in vivo against mice bearing Sarcoma 180 and Lewis lung cancer, compounds 5b and 5w exhibited potent antitumor efficacies with tumour inhibition rate of over 40% in the tested animal models. Moreover, the pharmacological mechanisms showed that compound 5b could retard in the CAM assay, and anti-angiogenetic potency was more potent than the reference drug CA4P. Preliminary structure-activity analysis indicated that: (1) C 1 -methylation and C 7 -methoxylation were favorable for increased activities; (2) 3-Pyridyl or 2-thienyl group substituent into position-1 of the b-carboline core, and the aryl (electron withdrawing and donating groups) substituent into position-1' of another b-carboline ring might be detrimental to cytotoxic effects of this class of compounds. Although most N 9 -heterodimeric b-carbolines presented here showed modest cytotoxic activities, the investigations of these structural modifications and preliminary SARs would be helpful to further design and develop more potent compounds.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the scientific research innovation project in Xinjiang under Grant XJGRI2016052; and the Program for Outstanding Youth Science and Technology Innovation Talents Training in Xinjiang Uygur Region under Grant qn2015jc067.