Synthesis and Cytotoxic Potential of 3-oxo-19 β -Triﬂuoroacetoxy-18 α H -oleane-28-oic Acid

: Triﬂuoroacetic acid-promoted Wagner-Meerwein rearrangement of betulonic acid carboxamide led to the formation of the expected 19 β ,28-lactam along with a new germanicane-type 3-oxo-19 β -triﬂuoroacetoxy-18 α H -oleane-28-oic acid. The structure of this triterpenoid was conﬁrmed by 2D NMR analyses. A primary evaluation of biological potency revealed an anticancer activity with GI 50 < 5 µ M against leukemia, colon cancer, breast cancer, and prostate cancer cell lines, while the parent compounds were not active.

On the other hand, treatment of betulonic acid carboxamide with trifluoroacetic acid (TFA) led to E-ring lactam and its dimethylsuccinoyl ester demonstrated antiretroviral activity [12]. Interested by this type of rearrangement, we report herein our investigations, as well as the discovery of another product in this reaction.

Results and Discussion
In the presence of Lewis or mineral acids, the rearrangement of betulin ring E takes place, leading to allobetulin, as described for the first time by Schulze and Pieroh in 1922 and which is now known as Wagner-Meerwein rearrangement [13]. Various acidic conditions have been investigated for this rearrangement, such as hydrobromic acid in chloroform, sulfuric acid in acetic acid, concentrated hydrochloric acid in ethanol, different "solid acids", etc., and up to 2010 they are summarized in a review [3]. In a recent article, ditions have been investigated for this rearrangement, such as hydrobromic acid in chloroform, sulfuric acid in acetic acid, concentrated hydrochloric acid in ethanol, different "solid acids", etc., and up to 2010 they are summarized in a review [3]. In a recent article, Wagner-Meerwein rearrangement was also conducted using HCl, montmorillonite K10, and BF3·Et2O to the synthesis of cyclopentyl units by contraction of the A-ring [14]. In this work, we tried to obtain the E-ring lactam derivative based on betulonic acid carboxamide 1 under similar rearrangement.
Reaction of betulonic acid carboxamide 1 with TFA in refluxing chloroform led to a mixture of 3-oxo-18α-oleane-19β,28-lactam 2 and a new compound 3 with yields of 65% and 32% (Scheme 1), respectively. The change of amount of TFA, temperature, or reaction time did not have any influence on the ratio of compounds 2 and 3. The structure of compound 2 was similar to those described in [12]; the structure of compound 3 was confirmed by HSQC and HMBC spectra (Figure 1), (for NMR spectra of 3, please see Supplementary Materials). In the 1 H NMR spectrum, the signal of H-19 connected with the trifluoroacetate group was observed at δH 4.50 ppm. In the 13 C NMR spectrum, the signal of C19 at δC 96.11 ppm and two quartet signals splited by the three 19   ditions have been investigated for this rearrangement, such as hydrobromic acid in chloroform, sulfuric acid in acetic acid, concentrated hydrochloric acid in ethanol, different "solid acids", etc., and up to 2010 they are summarized in a review [3]. In a recent article, Wagner-Meerwein rearrangement was also conducted using HCl, montmorillonite K10, and BF3·Et2O to the synthesis of cyclopentyl units by contraction of the A-ring [14]. In this work, we tried to obtain the E-ring lactam derivative based on betulonic acid carboxamide 1 under similar rearrangement. Reaction of betulonic acid carboxamide 1 with TFA in refluxing chloroform led to a mixture of 3-oxo-18α-oleane-19β,28-lactam 2 and a new compound 3 with yields of 65% and 32% (Scheme 1), respectively. The change of amount of TFA, temperature, or reaction time did not have any influence on the ratio of compounds 2 and 3. The structure of compound 2 was similar to those described in [12]; the structure of compound 3 was confirmed by HSQC and HMBC spectra (Figure 1), (for NMR spectra of 3, please see Supplementary Materials). In the 1 H NMR spectrum, the signal of H-19 connected with the trifluoroacetate group was observed at δH 4.50 ppm. In the 13 C NMR spectrum, the signal of C19 at δC 96.11 ppm and two quartet signals splited by the three 19   Therefore, according to NMR data, compound 3 is 3-oxo-19β-trifluoroacetoxy-oleane-28-oic acid. The formation of compound 2 could be explained by a Wagner-Meerwein Therefore, according to NMR data, compound 3 is 3-oxo-19β-trifluoroacetoxyoleane-28-oic acid. The formation of compound 2 could be explained by a Wagner-Meerwein rearrangement of amide 1 under acidic conditions. The formation of compound 3 could be explained by the further rearrangement, including ring opening of the lactam cycle under trifluoroacetic acid condition with the following hydrolysis of amide intermediate A to 19-trifluoroacetoxy-olean-28-oic acid (Figure 2). It is known that lupane-type triterpenoids betulin and betulinic acid possess a broad range of biological effects, particularly anticancer activities [15]. Betulinic acid induces the internal apoptosis pathway in cancer cells while sparing normal cells, and now is under clinical evaluation in Phase I/II clinical trials (NCT00346502) as 20% betulinic acid ointment (BA ointment) for treatment of dysplastic nevi that have potential to transform into melanoma [16,17]. Its oxidized product betulonic acid, having a carbonyl moiety at C-3 position, and which was used as a starting material for the synthesis of compound 1 in this work, possesses a better anticancer profile than betulinic acid [18]. Moreover, small structural changes of lupane type derivatives, including betulonic acid, lead to significant differences in their anticancer properties [19].
Compounds 1-3 were selected by the National Cancer Institute (NCI) Developmental Therapeutics Program (www.dtp.nci.nih.gov, accessed on 1 November 2018) for in vitro cell line screening to investigate their anticancer activity. As betulonic acid is a wellknown compound, it was not selected by NCI. Anticancer assays were performed according to the US NCI protocol, which was described elsewhere [20,21]. The first step, during which compounds were tested at 10 μM, revealed that compounds showed cytotoxicity against cancer cells. The results of NCI screening for carboxamide of betulonic acid 1 (first assay) were presented recently [22] and revealed no cytotoxicity. The highest activity of compound 2 was against non-small cell lung cancer cell lines (HOP-92), with over 32% of cell growth, while compound 3 demonstrated cytotoxicity with lethality against leukemia cell lines (HL-60(TB)) ( Table 1). Compound 3 being promising, it was further investigated in a five-dose testing mode and exhibited moderate activity with GI50 ranges from 3.16 to 26.40 μM against all cell lines of NCI-60 (Table 2) Figure 2. Plausible way of rearrangement of compound 1 to compounds 2 and 3.
It is known that lupane-type triterpenoids betulin and betulinic acid possess a broad range of biological effects, particularly anticancer activities [15]. Betulinic acid induces the internal apoptosis pathway in cancer cells while sparing normal cells, and now is under clinical evaluation in Phase I/II clinical trials (NCT00346502) as 20% betulinic acid ointment (BA ointment) for treatment of dysplastic nevi that have potential to transform into melanoma [16,17]. Its oxidized product betulonic acid, having a carbonyl moiety at C-3 position, and which was used as a starting material for the synthesis of compound 1 in this work, possesses a better anticancer profile than betulinic acid [18]. Moreover, small structural changes of lupane type derivatives, including betulonic acid, lead to significant differences in their anticancer properties [19].
Compounds 1-3 were selected by the National Cancer Institute (NCI) Developmental Therapeutics Program (www.dtp.nci.nih.gov, accessed on 1 November 2018) for in vitro cell line screening to investigate their anticancer activity. As betulonic acid is a well-known compound, it was not selected by NCI. Anticancer assays were performed according to the US NCI protocol, which was described elsewhere [20,21]. The first step, during which compounds were tested at 10 µM, revealed that compounds showed cytotoxicity against cancer cells. The results of NCI screening for carboxamide of betulonic acid 1 (first assay) were presented recently [22] and revealed no cytotoxicity. The highest activity of compound 2 was against non-small cell lung cancer cell lines (HOP-92), with over 32% of cell growth, while compound 3 demonstrated cytotoxicity with lethality against leukemia cell lines (HL-60(TB)) ( Table 1). Compound 3 being promising, it was further investigated in a five-dose testing mode and exhibited moderate activity with GI 50 ranges from 3.16 to 26.40 µM against all cell lines of NCI-60 (Table 2)  The compounds 2 and 3 also were evaluated at the University of Queensland (Australia) using five bacterial strains, including Gram-negative Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii and Pseudomonas aeruginosa; and Gram-positive methicillinresistant Staphylococcus aureus (MRSA). The antifungal activity was determined against Candida albicans and Cryptococcus neoformans. The primary screening of the antimicrobial activity of compounds 2 and 3 was carried out in a single concentration of 32 mg/mL in tests of the inhibition of cell reproduction. Samples with inhibition value above 80% were classed as active. Samples with inhibition values between 50 and 80% were classed as partial actives. The techniques for testing the antimicrobial and antifungal activities of compounds are given in the website http://www.co-add.org [23], accessed on 1 November 2018. It was found that none of the tested compounds inhibited growth of the pathogenic microorganisms in the studied concentration (Table 3).

Materials and Methods
The spectra were recorded at the Center for the Collective Use "Chemistry" of the UIC UFRC RAS and RCCU "Agidel" of the UFRC RAS. 1 H, 13 C, and 19 F NMR spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bruker, Billerica, MA, USA, 500, 125.5 and 470 MHz) with a Z-axis gradient unit, operated with a 5-mm broad-band multinuclear (PABBO) probe in CDCl3, using tetramethylsilane as the internal standard. A complete and unambiguous assignment of 1 H and 13 C nuclear magnetic resonance (NMR) signals is reported on the basis of two-dimensional NMR techniques ( 1 H-1 H COSY, 1 H-1 H NOESY, 1 H-13 C HSQC, 1 H-13 C HMBC). Melting points were detected on a micro table "Rapido PHMK05" (Nagema, Dresden, Germany). Optical rotations were measured on a polarimeter "Perkin-Elmer 241 MC" (PerkinElmer, Waltham, MA, USA) in a tube length of 1 dm. Elemental analysis was performed on a Euro EA-3000 CHNS analyzer (Euro vector, Milan, Italy); the main standard is acetanilide. Thin-layer chromatography analyses were performed on Sorbic plates (Copolymer, Krasnodar, Russian Federation), using the solvent system chloroform-ethyl acetate, 40:1. Substances were detected by a 10% solution of a sulfuric acid solution with subsequent heating at 100-120 • C for 2-3 min. Compound 1 was obtained according to the method described previously [5].