Novel triazoles of 3-acetylbetulin and betulone as anticancer agents

The CuAAC reaction of azides and acetylenic triterpenes was used for synthesis of new triazoles of 3-acetylbetulin and betulone. The triazole derivatives were evaluated for their anticancer activity in vitro against amelanotic melanoma C-32, ductal carcinoma T47D and glioblastoma SNB-19 cell lines. 28-[1-(3’-Deoxythymidine-5’-yl)-1H-1,2,3-triazol-4-yl]carbonylbetulone 6e exhibited a significant IC50 value (0.17 µM) against the human glioblastoma SNB-19 cell line, an almost 5-fold higher potency while compared with reference cisplatin.


Introduction
The cycloaddition reaction plays an important role in the synthesis of five-membered heterocyclic structures such as 1,2,3-triazoles. Molecules containing a 1,4-disubstituted 1,2,3-triazole ring are prepared regioselectively from azides and terminal alkynes in the copper-(I)-catalyzed azidealkyne cycloaddition reaction CuAAC (Wei et al. 2012;Marciniec et al. 2017). CuAAC reactions, described by Sharpless and Meldal groups, give high yields under mild conditions and have been used to obtain drugs, photo stabilizers and dyes. Additionally, this reaction occurs in various organic solvents and in aqueous media, in a wide pH area. In contrast to the CuACC reaction, the ruthenium catalyst azide-alkyne cycloaddition is used in the synthesis of the 1,5-disubstituted triazoles (Rostovtsev et al. 2002;Torne et al. 2002;Bonacorso et al. 2013;Bräse et al. 2008;Totobenazara et al. 2015).
Previously, we described a synthetic route and evaluation of cytotoxicity of betulin and betulone derivatives with a propynoyl group at the C-28 position (Boryczka et al. 2013;Bębenek et al. 2016). Expanding our interest to propynoylsubstituted triterpenes, we converted those acetylenic derivatives into the corresponding 1,2,3-triazoles. In this work, we presented application of the CuAAC reaction in the synthesis of new triazoles of pentacyclic triterpenes and their anticancer activity, as well as lipophilicity properties.

General
All organic solvents (from Sigma-Aldrich and P.P.H. STANLAB) were dried and used after purification. Melting points (m.p.) were determined in open capillary tubes on an Electrothermal IA 9300 melting point apparatus and are uncorrected. The 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance III 600 spectrometer in deuterated-d 6 chloroform (CDCl 3 ) or deuterated-d 6 dimethyl sulfoxide (DMSO) solution. The chemical shifts were reported in ppm (δ), and coupling constant (J) values-in hertz (Hz). The spin multiplicity was designated as the singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). High-resolution mass spectra (HR-MS) were recorded on a Bruker Impact II instrument. Infrared spectra (IR) were recorded on a Shimadzu IRAffinity-1 FTIR spectrophotometer (Shimadzu, Japan) using the KBr pellet method. The progress of the reactions was monitored by thin layer chromatography (TLC) using silica gel 60 254 F plates (Merck, Darmstadt, Germany) and detected by spraying with a solution of 5% sulfuric (VI) acid and heating to 120°C . Purity of the obtained compounds was confirmed by column chromatography carried out on silica gel 60, <63 μm (Merck). A mixture of CHCl 3 -EtOH (40:1, 15:1, 5:1 v/v) or CH 2 Cl 2 -EtOH (60:1, 40:1, v/v) was used as the mobile phase.
To an ice-cooled (−10°C) mixture of 3-acetylbetulin 2 (0.48 g, 1 mmol) and propynoic acid (0.12 g, 1.10 mmol) in dichloromethane (5 mL), a freshly prepared solution of dicyclohexylcarbodiimide (0.23 g, 1.12 mmol) and 4dimethylaminopyridine (0.01 g, 0.08 mmol) in dichloromethane (1 mL) was added. The mixture was allowed to react under argon atmosphere at −10°C for 5 h. After warming to room temperature, the mixture was stirred overnight. The reaction was monitored by TLC until completion. The resulting precipitate was filtered and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (CHCl 3 -EtOH 40:1, v/v).
General procedure for the synthesis of triazoles 5a-i and 6a-j Based on the previously reported method, the acetylenic esters 3-4 were converted into triazoles 5a-i and 6a-j by reactions with organic azides in toluene in the presence of copper(I) iodide (Bębenek et al. 2017). The copper(I) iodide (0.1 eqv, 0.004 g, 0.02 mmol) and the organic azide (1.05 eqv, 0.21 mmol) were added to a solution of propynoylated derivatives 3 or 4 (0.20 mmol) in toluene (4 mL). Next, the reaction mixture was stirred for another 72 h under reflux.

WST-1 assay
A WST-1 assay (Roche Diagnostics GmbH, Mannheim, Germany) was used for the evaluate of cytotoxicity against the tested human cancer cell lines. The WST-1 assay was carried out after 72 h incubation of the cells with concentrations ranging from 1 to 100 µg/mL of the tested compounds. The WST-1 tetrazolium salt [sodium 2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium] is reduced by mitochondrial dehydrogenases of viable cells to water-soluble formazan. The amount of formazan produced by viable cells was quantified by measuring the absorbance (λ = 450 nm). The anticancer activity of triterpenes were expressed as an IC 50 in µM (Table 2).

Lipophilicity studies
The theoretical lipophilicity parameters of triazoles 5a-i and 6a-j were calculated using the commercially available ALOGPS 2.1 software program (Tetko et al. 2005) (Table  3).
Subsequently, triterpenes 1-2 were used to prepare the propynoylated derivatives 3-4 according to our published procedures (Boryczka et al. 2013). The triazoles 5a-i and 6a-j were obtained by CuAAC reactions of acetylenic esters with various organic azides in toluene with yields in the range of 45-87%. Synthesis of triazoles 5a-i and 6a-j was depicted in Scheme 1. New compounds were purified by column chromatography on silica gel in CHCl 3 -EtOH or CH 2 Cl 2 -EtOH with various ratios. The chemical characterization of all derivatives was carried out by 1 H-, 13 C-NMR, IR spectroscopies, and HRMS spectra.
In the 1 H NMR spectra of the triazoles 5a-d and 6a-d, singlets of methylene group were observed at δ 5. 48-5.69, which suggests the presence of a bond between C-4 (aryl group) and N-1 of the triazole ring. The signals in the range of δ 7.01-7.72 were assigned to the aromatic protons of the aryl group of derivatives 5a-d and 6a-d. Additionally, for all derivatives 5a-i and 6a-j, signals at δ 7.96-9.08 were Table 2 Anticancer activity (IC 50 ) of acetylenic esters 3-4, triazoles of triterpenes 5a-i and 6a-j and cisplatin as a reference compound against the tested human cancer cell lines observed, corresponding to triazolyl protons in the 1,4disubstituted triazole ring.
Analysis of the 13 C NMR spectra of triazoles 5a-i and 6a-j showed that the signals of acetyl and carbonyl groups are located at 167.5-171.1 p.p.m. and 217.0-218.1 p.p.m., respectively.
The IR spectra of new triazoles 5a-i and 6a-j showed characteristic absorption bands at 1527-1616 cm −1 and 1456-1458 cm −1 , which were attributed to the C=N and the N=N stretching vibrations of the triazole ring, respectively.
The HRMS negative mode was applied to identify all new compounds. In the mass spectra of triterpenes 3, 5a-i,

Biological study
The triazole derivatives of 3-acetylbetulin and betulone were evaluated in vitro for their anticancer activity using a WST-1 assay against three human cancer cell lines: amelanotic melanoma C-32, ductal carcinoma T47D and glioblastoma SNB-19. Cisplatin was used as a positive control. The results of the anticancer activity tests of the studied compounds are reported in Table 2 as IC 50 (µM).
As shown in Table 2, the lowest anticancer activity (IC 50 7.56-83.88 µM) of targeted triazoles was observed in the case of the T47D ductal carcinoma cell line. In the tested group of triazoles, derivative 6i exhibited a highest anticancer activity (IC 50 7.56 µM) against the T47D cells, when compared to the positive control.
For triazoles of 3-acetylbetulin 5a-i, the rank order of the anticancer activity towards the C-32 cell line is as follows: 5g > 5b > 5a > 5d > 5h > 5i > 5f > 5e > 5c. The compound 5g containing a 1-ethylacetyl moiety in triazole ring had the same anticancer activity against the C-32 cell line as the reference cisplatin (IC 50 0.57 µM). Moreover, triazoles 5c, 6a, and 6g had no anticancer activity towards C-32 cell line in the applied concentration range.
According to our studies, compounds 5b, 5g, 6b, and 6e showed a significant activity against human glioblastoma SNB-19 cell line, with IC 50 values from 0.17 to 0.85 µM.
The triazole 6e bearing a 3'-deoxythymidine-5'-yl moiety showed the highest activity in the tested group of compounds against SNB-19 cells, with IC 50 value of 0.17 µM.
Our studies suggest, that the introduction of acetyl or carbonyl group at the C-3 position of triazole derivatives of triterpenes afforded compounds having a higher anticancer activity against amelanotic melanoma C-32 cell line. Additionally, the compounds 5f and 6f containing the 1deoxy-β-D-glucopyranosyl substituted triazole ring had a better activity than their parent 3-hydroxyl substituted analogs against C-32, T47D, and SNB-19 cell lines (Bębenek et al. 2017).  The lipophilicity is one of the important physicochemical parameters in drug development (Andric and Héberger 2015). A lipophilicity study of the tested triazoles was carried out using the ALOGPS 2.1 software program. The predicted log P values were calculated according to the molecular structures of triazoles 5a-i and 6a-j using six computational methods (ALOGPs, AC logP, ALOGP, MLOGP, XLOGP2, and XLOGP3). Considering two triazoles of betulone 6d and 6e, it was observed that their cytotoxic activity increased with the decreasing value of theoretical log P.

Conclusion
In conclusion, on the basis of the CuAAC reaction, a series of new derivatives of 3-acetylbetulin and betulone bearing 1,2,3-triazole moiety has been synthesized. The anticancer activity of the triazoles and cisplatin was tested against the C-32, T47D and SNB-19 cancer cell lines using the WST-1 assay. The triazole 6e with 3'-deoxythymidine-5'-yl substituent proved to be a potent cytotoxic agent with IC 50 value of 0.17 µM in the case of the human glioblastoma SNB-19 cell line. Morever, the triazole 6e can be considered as a promising candidate for anticancer therapy.