3-(1,2,3-Triazol-4-yl)-β-Carbolines and 3-(1H-Tetrazol-5-yl)-β-Carbolines: Synthesis and Evaluation as Anticancer Agents

Herein, the synthesis and anticancer activity evaluation of a series of novel β-carbolines is reported. The reactivity of nitrosoalkenes towards indole was explored for the synthesis of novel tryptophan analogs where the carboxylic acid was replaced by a triazole moiety. This tryptamine was used in the synthesis of 3-(1,2,3-triazol-4-yl)-β-carbolines via Pictet–Spengler condensation followed by an oxidative step. A library of compounds, including the novel 3-(1,2,3-triazol-4-yl)-β-carbolines as well as methyl β-carboline-3-carboxylate and 3-tetrazolyl-β-carboline derivatives, was evaluated for their antiproliferative activity against colorectal cancer cell lines. The 3-(1H-tetrazol-5-yl)-β-carbolines stood out as the most active compounds, with values of half-maximal inhibitory concentration (IC50) ranging from 3.3 µM to 9.6 µM against colorectal adenocarcinoma HCT116 and HT29 cell lines. The results also revealed a mechanism of action independent of the p53 pathway. Further studies with the 3-tetrazolyl-β-carboline derivative, which showed high selectivity for cancer cells, revealed IC50 values below 8 μM against pancreatic adenocarcinoma PANC-1, melanoma A375, hepatocarcinoma HEPG2, and breast adenocarcinoma MCF-7 cell lines. Collectively, this work discloses the 3-tetrazolyl-β-carboline derivative as a promising anticancer agent worthy of being further explored in future works.


Introduction
Cancer is one of the leading causes of death worldwide. In 2020, the number of new cases of cancer was estimated to be around 19.3 million, with approximately 10 million deaths globally being attributed to cancer [1]. Moreover, colorectal cancer (CRC) is the third most common cancer type, and one of the deadliest (in 2020, CRC accounted for over 2 million new cancer cases and 1 million deaths worldwide). Therefore, the continuous effort towards the discovery of novel and effective anticancer agents against CRC is of great importance [1].
In recent years, several synthetic methodologies have been applied to the development of novel β-carboline derivatives. However, the most commonly explored synthetic route involves the Pictet-Spengler reaction between an aldehyde and a tryptamine derivative, followed by oxidation of the initially formed tetrahydro-β-carboline [7,19,20]. In our group, the reactivity of nitrosoalkenes towards heterocycles has been extensively studied, and it was thought that it could be explored for the synthesis of functionalized tryptophan analogs [21][22][23]. The nitrosoalkenes are generated in situ through the treatment of αbromooximes with sodium carbonate, followed by the hetero-Diels-Alder reaction with indoles, subsequent 1,2-oxazine ring-opening, and concomitant rearomatization to afford 3-alkylated indoles incorporating an oxime moiety [24,25]. This open-chain oxime is then reduced to afford the desired tryptamine analog. In fact, the synthesis of a tryptophan analog, where the carboxylic acid was replaced by the bioisosteric tetrazole group, was recently reported by our group, and was used as a building block for the synthesis of novel 3-tetrazolyl-β-carbolines (Scheme 1a) [8]. Using a similar synthetic strategy, 6-substitutedβ-carboline-3-carboxylates were also synthesized via the Pictet-Spengler approach, using tryptophan ethyl esters functionalized in the indole moiety (Scheme 1a) [8].

Chemistry
In order to develop the proposed 3-triazolyl-β-carboline derivatives, firstly, the αbromooxime 5 was prepared following previously reported procedures [21]. Treatment of oxime 5 with sodium carbonate led to the in situ formation of the nitrosoalkene 6, which reacted with indole (7) to give 3-alkylated indole 9 via hetero-Diels-Alder reaction, followed by a 1,2-oxazine ring-opening reaction. Oxime 9 reacted further with another molecule of nitrosoalkene 6 to afford functionalized indole 10, which was isolated in 57% yield (overall yield from 5). The reduction of the alkylated oxime 10 was achieved using an excess of metallic zinc/aqueous formic acid in tetrahydrofuran at room temperature for 24 h, affording the novel tryptamine 11 an 81% yield (Scheme 2). the latter β-carbolines, as well as of a range of methyl β-carboline-3-carboxylates, as a cancer agents against human colon cancer cells is also reported.

Chemistry
In order to develop the proposed 3-triazolyl-β-carboline derivatives, firstly, the bromooxime 5 was prepared following previously reported procedures [21]. Treatmen oxime 5 with sodium carbonate led to the in situ formation of the nitrosoalkene 6, wh reacted with indole (7) to give 3-alkylated indole 9 via hetero-Diels-Alder reaction, lowed by a 1,2-oxazine ring-opening reaction. Oxime 9 reacted further with another m ecule of nitrosoalkene 6 to afford functionalized indole 10, which was isolated in 57% y (overall yield from 5). The reduction of the alkylated oxime 10 was achieved using excess of metallic zinc/aqueous formic acid in tetrahydrofuran at room temperature fo h, affording the novel tryptamine 11 an 81% yield (Scheme 2). Scheme 2. Synthesis of a tryptamine bearing a 1,2,3-triazole group.

Anticancer Activity
The synthesized 3-triazolyl-β-carbolines 13a-f and 3-tetrazolyl-β-carbolines 1a-c [8] were evaluated for their anticancer activity in several colon cancer cells. In order to better evaluate and establish structure-activity relationships, methyl β-carboline-3-carboxylates 14a-e were also synthesized from L-tryptophan methyl ester following a previously reported procedure, and their evaluation as colorectal anticancer agents was carried out (Figure 3) [50]. Impairment of the tumor suppressor p53 protein pathway is a critical event in cancer. Therefore, re-establishing p53 activity has become one of the most appealing therapeutic anticancer strategies [51][52][53]. Moreover, some β-carboline derivatives are known for their capability to activate the p53 activity [54,55]. In this context, we set out to determine whether the anticancer activity of the synthesized β-carbolines could involve selective activation of the p53 pathway. For that, the antiproliferative activity of β-carbolines 1a-c, 13a-f, and 14a-e was evaluated against the human colorectal carcinoma HCT116 cell line expressing wild type (wt) p53, as well as the respective p53 knockout HCT116 derivative. The comparison of the activity of the compounds was made by analyzing the corresponding IC50 values calculated from the dose-response curves (Table 1).  Impairment of the tumor suppressor p53 protein pathway is a critical event in cancer. Therefore, re-establishing p53 activity has become one of the most appealing therapeutic anticancer strategies [51][52][53]. Moreover, some β-carboline derivatives are known for their capability to activate the p53 activity [54,55]. In this context, we set out to determine whether the anticancer activity of the synthesized β-carbolines could involve selective activation of the p53 pathway. For that, the antiproliferative activity of β-carbolines 1a-c, 13a-f, and 14a-e was evaluated against the human colorectal carcinoma HCT116 cell line expressing wild type (wt) p53, as well as the respective p53 knockout HCT116 derivative. The comparison of the activity of the compounds was made by analyzing the corresponding IC 50 values calculated from the dose-response curves (Table 1). In this initial screening, it was possible to observe that, despite our initial assumptions, the 3-triazolyl-β-carboline derivatives 13a-f were not active against the studied cancer cell lines, with IC 50 values ranging from 32 µM to values higher than 50 µM. Furthermore, among the methyl β-carboline-3-carboxylates, β-carboline 14c was the one that presented the most promising results, with IC 50 values of 15 µM and 18 µM against p53 +/+ and The 3-(1H-tetrazol-5-yl)-β-carbolines 1a-c proved to be the most active compounds, with values of IC 50 ranging from 3.3 µM to 4.6 µM against both cancer cell lines studied. It should be highlighted that the replacement of the methyl ester group of β-carbolines 14a and 14b by the bioisosteric benzyl-tetrazole group, leading to 3-(1H-tetrazol-5-yl)β-carbolines 1a and 1b, respectively, resulted in a huge increase in anticancer activity. However, the results allowed us to conclude that none of the tested 3-(1H-tetrazol-5-yl)-βcarbolines selectively activate the p53 pathway.
The study was extended to evaluation of the anticancer activity of the 3-triazolyl-and 3-tetrazolyl-β-carbolines (13a-f and 1a-c) against other human colorectal adenocarcinoma cell lines (SW837 and HT29), as well as the cytotoxicity against a normal human colon cell line (CCD-18Co), to determine the selectivity of these derivatives to cancer cells. The results of this study are summarized in Table 2. Table 2. Antiproliferative activity of β-carbolines 13a-f and 1a-c against colorectal adenocarcinoma and normal colon cell lines. From this study, we could confirm that the introduction of a triazole moiety directly attached to the β-carboline core has a negative impact on anticancer activity. In fact, it was observed that the 3-triazolyl-β-carboline derivatives 13a-f were not active against the SW837 and HT29 cancer cell lines, nor did they show cytotoxicity against normal colon cell lines.
Interestingly, a different activity profile was observed with the 3-tetrazolyl-β-carboline derivatives 1a-c. These β-carbolines proved to be active against the HT29 cells, showing IC 50 values ranging from 5.9 to 9.6 µM. However, moderate activity was observed against the SW837 cells (IC 50 from 29 µM to 44 µM).
The study of the cytotoxicity against a normal colon cell line (CCD-18Co) revealed that 3-tetrazolyl-β-carboline 1a has the highest selectivity for cancer cells, showing an IC 50 value of 26 µM in these cells. In fact, this selectivity of compound 1a to cancer cells was further evidenced in the normal human foreskin fibroblasts (HFF-1) cell line, in which compound 1a presented an IC 50 value of 29 µM (Table 3). Moreover, the results showed that the antiproliferative activity of compound 1a was associated with apoptotic cell death (Figure 4).

General Information
NMR spectra were recorded on a Bruker Avance III instrument, operating at 400 MHz ( 1 H) or 100 MHz ( 13 C). Chemical shifts are expressed in ppm relative to tetramethylsilane (TMS), and coupling constants (J) are in Hz. Infrared spectra (IR) were recorded using a Fourier Transform spectrometer coupled with a diamond Attenuated Total Reflectance (ATR) sampling accessory. High-resolution mass spectra (HRMS) were obtained on a TOF VG Autospect M spectrometer with electrospray ionization (ESI). Melting points were recorded in open glass capillaries. Thin Layer Chromatography (TLC) was performed using precoated silica gel plates. Flash chromatography was performed using silica gel 60 as a stationary phase. 3-Tetrazolyl-β-carbolines 1 [8], 3-triazolyl-α-bromooxime 10 [8], and β-carboline-3-carboxylates 14 [50] were prepared as described in the literature.
The study of the cytotoxicity against a normal colon cell line (CCD-18Co) revealed that 3-tetrazolyl-β-carboline 1a has the highest selectivity for cancer cells, showing an IC50 value of 26 µM in these cells. In fact, this selectivity of compound 1a to cancer cells was further evidenced in the normal human foreskin fibroblasts (HFF-1) cell line, in which compound 1a presented an IC50 value of 29 µM (Table 3). Moreover, the results showed that the antiproliferative activity of compound 1a was associated with apoptotic cell death ( Figure 4). The exact mechanism of action for these molecules was not studied; however, the selectivity of these compounds against cancer cells may be explained by the ability of βcarboline derivatives to bind to the DNA by intercalation. In fact, the DNA intercalation of β-carboline derivatives is associated with structural damage to the DNA of cancer cells, as well as with an inhibition of the DNA repair mechanism, inducing apoptosis [18,56,57].
Taking this into account, the anticancer activity of compound 1a was further analyzed by testing its antiproliferative activity against distinct cell lines from distinct tumor tissues. As observed in Table 3, compound 1a showed IC50 values below 8 µM against pancreatic carcinoma PANC-1, melanoma A375, hepatocarcinoma HEPG2, and breast adenocarcinoma MCF-7 cell lines. These results indicate that compound 1a may be highly effective against distinct cancer types. The exact mechanism of action for these molecules was not studied; however, the selectivity of these compounds against cancer cells may be explained by the ability of β-carboline derivatives to bind to the DNA by intercalation. In fact, the DNA intercalation of β-carboline derivatives is associated with structural damage to the DNA of cancer cells, as well as with an inhibition of the DNA repair mechanism, inducing apoptosis [18,56,57].
Taking this into account, the anticancer activity of compound 1a was further analyzed by testing its antiproliferative activity against distinct cell lines from distinct tumor tissues. As observed in Table 3, compound 1a showed IC 50 values below 8 µM against pancreatic carcinoma PANC-1, melanoma A375, hepatocarcinoma HEPG2, and breast adenocarcinoma MCF-7 cell lines. These results indicate that compound 1a may be highly effective against distinct cancer types.

Experimental Section
3.1. Chemistry 3.1.1. General Information NMR spectra were recorded on a Bruker Avance III instrument, operating at 400 MHz ( 1 H) or 100 MHz ( 13 C). Chemical shifts are expressed in ppm relative to tetramethylsilane (TMS), and coupling constants (J) are in Hz. Infrared spectra (IR) were recorded using a Fourier Transform spectrometer coupled with a diamond Attenuated Total Reflectance (ATR) sampling accessory. High-resolution mass spectra (HRMS) were obtained on a TOF VG Autospect M spectrometer with electrospray ionization (ESI). Melting points were recorded in open glass capillaries. Thin Layer Chromatography (TLC) was performed using precoated silica gel plates. Flash chromatography was performed using silica gel 60 as a stationary phase. 3-Tetrazolyl-β-carbolines 1 [8], 3-triazolyl-α-bromooxime 10 [8], and β-carboline-3-carboxylates 14 [50] were prepared as described in the literature. (11) Oxime 10 (1.25 g, 2.13 mmol) was dissolved in the smallest amount of THF, 70% aqueous formic acid (40 mL) was added, and the solution was cooled to 0 • C. Then, zinc powder (4.18 g, 63.9 mmol) was added portion-wise over 30 min. The reaction mixture was stirred for 24 h at room temperature. After this time, the mixture was filtered on a Celite pad and the celite was washed with ethyl acetate (3 × 15 mL). The filtrate was neutralized with a concentrated ammonia solution to pH 8, and then extracted with ethyl acetate (3 × 40 mL). The combined organic phases were washed with water, then dried over anhydrous Na 2 SO 4 , and the solvent evaporated off.   Figure S1 and S2.

General Procedure for the Synthesis of Tetrahydro-β-carbolines 12
Trifluoroacetic acid (2 equiv.) was added to a solution of amine 11 (1 equiv.) and the appropriate aldehyde (1 equiv.) to dry dichloromethane (8 mL/mmol). The reaction mixture was stirred at room temperature, monitored by TLC, until all of the amine 11 was consumed. Upon completion, the reaction mixture was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (7 mL) and neutralized with an aqueous solution of Na 2 CO 3 10%. The resulting solution was extracted with ethyl acetate (3 × 25 mL), dried over anhydrous Na 2 SO 4 , and the solvent evaporated off. The products were purified by recrystallization in MeOH and obtained as a mixture of cis/trans isomers, unless otherwise stated. Sulfur powder (3 equiv.) was added to a solution of the appropriate tetrahydro-βcarboline 12 (1 equiv.) in dimethylformamide (20 mL/mmol). The reaction mixture was stirred under reflux for 24 h. After this time, the solvent was evaporated off. The product was precipitated by addition of diethyl ether, then filtered and dried under vacuum to obtain the products in a pure form, unless otherwise stated.
Human cell lines were seeded in 96-well plates at a density of 5.0 × 10 3 (HCT116 p53 −/− , HCT116 wt, HT29, SW837, CCD-18Co, HFF-1, and MCF-7 cell lines) or 4.5 × 10 3 (A375, HEPG2 and PANC-1 cell lines) cells per well, for 24 h. Cells were treated with the appropriate compound, with serial dilutions ranging from 0.2 to 50 µM, for 48 h. Following the incubation, the effects on cell proliferation were evaluated through the SRB assay as stated by Ramos et al. [58]. The IC 50 values were determined using the software GraphPad Prism version 9.0.

Annexin-V Assay
The analysis of apoptotic cell death was performed essentially as described by Ramos et al. [58]. Briefly, 1.5 × 10 5 HCT116 cells/well were seeded in 6-well plates, allowed to adhere overnight, and then treated with 4.5 and 9.0 µM of compound 1a. After 48 h of treatment, cells were stained using the Annexin V-FITC Apoptosis Detection Kit I from BD Biosciences (Enzifarma, Porto, Portugal), according to the manufacturer's instructions. The AccuriTM C6 flow cytometer and the BD Accuri C6 software (BD Biosciences) were used.

Conclusions
The synthesis of β-carboline derivatives and their activity against several human colorectal adenocarcinoma cell lines has been disclosed. A synthetic route to novel 3-(1,2,3-triazol-4-yl)-β-carboline derivatives was established via the Pictet-Spengler approach using a tryptophan analog, where the carboxylic acid was replaced by a triazole moiety. In order to better evaluate and establish structure-activity relationships, methyl β-carboline-3-carboxylates and 3-tetrazolyl-β-carbolines were also synthesized from the corresponding tryptamine analogs.
The antiproliferative activity of the synthesized β-carbolines against colorectal cancer cells revealed that the 3-(1H-tetrazol-5-yl)-β-carbolines, particularly compound 1a, were the most active molecules, and that they act through a p53-independent apoptotic pathway. Moreover, compound 1a demonstrated a high selectivity to cancer cells, which may be attributed to the DNA intercalating ability of β-carbolines, with subsequent inhibition of DNA repair mechanisms, in cancer cell lines.
The disclosed results also indicate that the presence of a triazole moiety at β-carboline's C-3 carbon severely hinders the overall biological activity of these heterocycles, while a tetrazole moiety at the same position greatly increases the cytotoxicity against human colon cancer cell lines when compared with the methyl ester β-carboline derivatives.
Further studies on the 3-tetrazolyl-β-carboline derivative, with the highest selectivity for cancer cells, unveiled an interesting anticancer profile by targeting cancer cells from distinct tissues.
This study, therefore, unveils a promising anticancer agent worthy of being explored in future works.