3 ′-[ 4-( { [ 3 β , 28-Bis ( acetyloxy ) lup-20 ( 29 )-en-30-yl ] oxy } carbonyl )-1 H-1 , 2 , 3-triazol-1-yl ]-3 ′-deoxythymidine

The reaction of the azidothymidine (AZT) with the 30-propynoylated derivative of 3,28-O, O′-diacetylbetulin gave a 1,4-disubstituted 1,2,3-triazole. The chemical structure of new derivative was characterized by 1H NMR, 13C NMR and HR-MS. The triterpene-AZT conjugate was tested against a human cancer cell lines such as glioblastoma (SNB-19), amelanotic melanoma (C-32), ovarian adenocarcinoma (SKOV-3) and breast cancer (T47D, and MCF-7). 3′-[4-({[3β,28-Bis(acetyloxy)lup20(29)-en-30-yl]oxy}carbonyl)-1H-1,2,3-triazol-1-yl]-3′-deoxythymidine shown significant activity against MCF-7 cells, with an IC50 value of 4.37 μM.


Introduction
Natural pentacyclic lupane-type triterpenes are often the major constituents of medicinal plants. These compounds are characterized by a wide range of pharmacological activity and diversity in chemical structures [1]. The lupane family includes triterpenes that exhibit anticancer [2,3], antiviral [4,5], antibacterial [6,7], anti-inflammatory [8,9] and hepatoprotective properties [10]. Scientific research aimed at improving the activity of triterpenes is based on the modification of the triterpene scaffold or functionalization of the hydroxyl or carboxyl groups present in them. Improving bioactivity can also be achieved by introduction a triazole ring into the triterpene moiety. 1,2,3-Triazoles are prepared from the corresponding azides by copper catalyzed 1,3-dipolar cycloaddition (CuAAC) with alkyne derivatives of triterpenes [1,11,12].
One of the possibilities for the synthesis of new biologically active compounds is the combination of two bioactive moieties into hybrid molecule. It is assumed, that the obtained hybrid molecule should have more enhanced biological activity. Due to the anticancer effects of pentacyclic triterpenes on various types of cancer cells, the triterpene scaffold is an attractive moiety for the synthesis of such system. Another a bioactive substance that can be used in the preparation of hybrid compounds is azidothymidine (AZT, Figure 1). This nucleoside reverse transcriptase inhibitor applied in anti-HIV therapy also has an anticancer effect [13].
This study describes the synthesis and anticancer activity of the novel 30-substituted 3,28-O,O -diacetylbetulin derivative linked to AZT via a 1,2,3-triazole ring. This study describes the synthesis and anticancer activity of the novel 30-substituted 3,28-O,O′-diacetylbetulin derivative linked to AZT via a 1,2,3-triazole ring.

Results and Discussion
The 3,28-O,O′-diacetyl-30-propynoylbetulin 1 was obtained by the Steglich reaction as previously described in the literature [14]. Propynyl-functionalized derivative 1 was used as starting compound in the 1,3-dipolar cycloaddition (CuAAC-Cooper Catalyzed Azide-Alkyne Cycloaddition). The CuAAC reaction of alkyne derivative 1 with azidothymidine in the presence of copper(I) iodide in refluxing toluene led to the formation of triterpene-AZT conjugate at a 54% yield. The synthesis of 1,2,3-triazole derivative 2 is outlined in Scheme 1. The chemical structure of compound 2 was confirmed by NMR, IR and HR-MS analysis. The new 1,2,3-triazole derivative 2 and cisplatin (reference drug) were evaluated in vitro for cytotoxic activity against the following human cancer cell lines such as glioblastoma (SNB-19), amelanotic melanoma (C-32), ovarian adenocarcinoma (SKOV-3) and breast cancer (T47D, and MCF-7). The obtained cytotoxicity results expressed as IC50 values (half-maximum inhibitory concentrations) are presented in Table 1.

Results and Discussion
The 3,28-O,O -diacetyl-30-propynoylbetulin 1 was obtained by the Steglich reaction as previously described in the literature [14]. Propynyl-functionalized derivative 1 was used as starting compound in the 1,3-dipolar cycloaddition (CuAAC-Cooper Catalyzed Azide-Alkyne Cycloaddition). The CuAAC reaction of alkyne derivative 1 with azidothymidine in the presence of copper(I) iodide in refluxing toluene led to the formation of triterpene-AZT conjugate at a 54% yield. The synthesis of 1,2,3-triazole derivative 2 is outlined in Scheme 1. This study describes the synthesis and anticancer activity of the novel 30-substituted 3,28-O,O′-diacetylbetulin derivative linked to AZT via a 1,2,3-triazole ring.

Experimental Procedures
Melting point was detected on an Electrothermal IA 9300 apparatus (Bibby Scientific Limited, Stone, Southhampton, UK), and was uncorrected. 1 H and 13 C NMR spectra were recorded in DMSO-d6 on a Bruker Avance III 600 spectrometer (Bruker, Billerica, MA, USA) at 600 MHz and 150 MHz, respectively. High resolution mass data was performed on a Bruker Impact II instrument (Bruker) using an APCI method (negative mode). Infrared spectrum (KBr, pellet) was measured on an IRAffinity-1 Shimadzu spectrometer (Shimadzu Corporation, Kyoto, Japan). The progress of a reaction and the purity of the compound were monitored by TLC method on silica gel 60 254F plates (Merck, Darmstadt, Germany).

WST-1 Assay
The WST-1 test (Roche Molecular Biochemicals, Mannheim, Germany) was applied to evaluate the cytotoxic activity. This colorimetric test determines the ability of viable cells to cleavage of tetrazolium salt of WST-1 to dark red formazan. The cells with compound 2 (concentrations ranging from 1 to 100 µg/mL) and WST-1 were incubated for 72 h. The amount of formazan was quantified by measuring the absorbance at λ = 450 nm with a UVM340 microplate reader (Biogenet, Józefów, Poland).