Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin

Nov 5, 2015 - Lucie Borkova , Richard Adamek , Petr Kalina , Pavel Drašar , Petr Dzubak , Sona Gurska , Jiri Rehulka , Marian Hajduch , Milan Urban ,...
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Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin Miroslav Soural,†,‡ Jiri Hodon,†,‡ Niall J. Dickinson,† Veronika Sidova,† Sona Gurska,‡ Petr Dzubak,‡ Marian Hajduch,‡ Jan Sarek,‡ and Milan Urban*,†,‡ †

Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, 17. listopadu 1192/12, 771 46 Olomouc, Czech Republic ‡ Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University in Olomouc, Hnevotinska 5, 779 00 Olomouc, Czech Republic S Supporting Information *

ABSTRACT: To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin− streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice.

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INTRODUCTION Triterpenes are natural compoundssecondary metabolites that may be found in almost all living organisms, most often in plants, fungi, and marine animals. Thousands of triterpenes have been isolated from natural sources and many of them are biologically active.1 There are many examples of cytotoxic,2 antimicrobial,3 antifeedant,4 antiulcer,5 antiviral,6 anticariogenic,7 hepatoprotective,8 cardioprotective,9 antiplasmodial,10 and analgesic11 compounds, and if someone was to look for a compound associated with an interesting biological activity, there would almost certainly be triterpenes among them.12 Although the isolated triterpenes are active, their use is complicated by two main drawbacks: 1. Their IC50 is often inferior to that of established therapeutics; 2. They often have inappropriate pharmacological properties: most importantly, their solubility in water based media is low and this causes problems with bioavailability. On the other hand, the toxicity of triterpenes is usually low, which allows higher maximum tolerated dose, and most importantly, they have a variety of mechanisms of action, many of them unusual or yet unknown. Triterpenes with a mechanism of action that is different from that of established therapeutics are of significant interest because they may become alternatives for the treatment of resistant cancers, microbial, parasitic, or viral diseases, and may possibly be used in a combination regime. This is the main reason triterpenes are of interest to many research groups that © 2015 American Chemical Society

are trying to improve the IC50 and pharmacological profile of the mother compounds by chemical modifications13 and by synthesizing prodrugs.14 In our research group, we are trying to develop new triterpenoid anticancer therapeutics. We prepared hundreds of derivatives from natural compounds such as betulinic acid 1, and among them, several had IC50 in low micromolar range.15−18 To improve their solubility, we developed several types of prodrugs or formulations, the most successful of which was the use of a 2-hydroxypropyl-γ-cyclodextrin formulation, which enabled complete dissolution of most triterpenic acids in water.19 At this point, we decided to focus on investigating the mechanism of action of our best compounds. There are several examples in the literature of triterpenes, for which biological activity has been fully explained by understanding the mechanism of action, and in some cases, the specific molecular target was found. The first of such compounds is bevirimat, a derivative of betulinic acid 1 with strong anti-HIV activity.20 It was found that the compound binds noncovalently to the CASP1 cleavage site in immature Gag particles and inhibits maturation of the viral particles.21,22 Unfortunately, mutations develop rapidly and the virus becomes resistant, leading to Received: October 16, 2015 Revised: November 2, 2015 Published: November 5, 2015 2563

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Bioconjugate Chemistry

Figure 1. Triterpenes selected for biotinylation and numbering of the C atoms in the lupane skeleton.

bevirimat being abandoned during the third stage of clinical trials and new derivatives are being sought that would be less susceptible to resistance development.6 Other examples of semisynthetic betulinic acid 1 derivatives with elucidated mechanism of action are topoisomerase inhibitors23 and proteasome inhibitors.24 Betulinic acid 1 also inhibits prostate cancer growth through inhibition of specificity protein transcription factors;25 induces apoptosis through direct interaction with mitochondria in neuroectodermal tumors.26 However, many other mechanisms of action still remain elusive. There are many methods that may help to determine the mechanism of action of an active compound. The ultimate goal is to find a molecular target that the compound interacts with and prove that this interaction is responsible for the biological response. One of the most important methods to find the molecular target is biotin−streptavidin affinity chromatography combined with mass spectroscopy. An improved method uses a combination of quantitative proteomic with stable isotope labeling by amino acids in cell culture (SILAC)27,28 which allows obtaining the knowledge of which protein(s) interact with an active molecule and evaluating how strong the interaction is. This setup also identifies false positives arising from nonspecifically bound proteins. In order to be able to perform such experiments, we have developed a robust technique to synthesize conjugates of active triterpenes with biotin through a linker of our choice. Due to its versatility, we choose solid-phase chemistry to access our desired compounds, despite this methodology having only been reported once in triterpenes,29 though we anticipated complications with reactivity at sterically hindered positions.

Figure 2. Different scenarios of the target scaffold modification.

Previously, we have described a robust method for fast and versatile biotinylation of carboxylic acids using biotin-preloaded resins31 that allows connecting biotin to an active molecule through a PEG linker of chosen length. This work is the first successful and practical application of this methodology. To obtain the preloaded resin, aminomethyl polystyrene resin was equipped with Backbone Amide Linker (4-(4-formyl-3methoxyphenoxy)butanoic acid) which was reductively aminated with 2-(2-aminoethoxy)ethanol. After Fmoc-protection of the amine, the hydroxyl was acylated with D(+)-biotin. Cleavage of Fmoc was followed by amine acylation with [2-[2(Fmoc-amino)ethoxy]ethoxy]acetic acid which yielded the Fmoc-protected, preloaded resin A of high crude purity (above 95%, calculated from LC-UV-MS traces after the cleavage from the polymer support). The loading of the resin A was determined with the use of an external standard (FmocAla-OH) and was 0.26 mmol/g. This value was used to calculate the overall yield of each modification. Finally, the Fmoc-group was cleaved and the preloaded resin A was used to modify the selected derivatives (Scheme 1). Premodification of Triterpenes in Solution-Phase. During the first experiments with betulinic acid 1 it was found that the 28-COOH group is not reactive under proposed conditions due to the inherent steric hindrance of the neopentyl carboxylate which could not react with the amine of our linker. This lack of reactivity can be exploited, because we may biotinylate compounds at position C-3 without the need for protection of 28-COOH. Use of the preloaded resin A required the presence of a suitable functional group on the compounds (preferentially carboxylic group or an active halogen). For this purpose, compounds 1−4 were converted to the corresponding hemisuccinates 6−9.19,32 When it was needed to biotinylate a terpenic acid through its 28-COOH, an extension linker must be used. Therefore, two selected acids (1, 5) were converted to corresponding glyoxalates 10, 11.32,33 Finally, betulinic acid 1 was brominated at C-30 using NBS to give active halogen in compound 14 (Scheme 2).

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RESULTS AND DISCUSSION Molecules Selection. Betulinic acid 1 was used as a standard for our initial experiments because several of its molecular targets are known23−26 and we will be able to use it to optimize and validate the conditions of the pulldown assays. In addition, we chose derivatives from out past research that had low IC50 such as aldehyde 2,15 ketone 3,30 diketone 4,30 and pyrazine 516 (Figure 1) for which the molecular targets are unknown. Synthetic Strategy. When searching for a suitable synthetic strategy, two important criteria were followed: (i) the method should allow modification of the target molecules in different positions of the triterpenoid skeleton; (ii) the method should be applicable for quick and routine use in order to modify a larger number of the active derivatives in a short time frame. Three different conjugation sites of a biotin-PEG label were suggested: attachment via hydroxy group located at the C-3 position, attachment via C-30 position, and attachment via the neopentyl type carboxyl group C-28 (Figure 2). 2564

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Bioconjugate Chemistry Scheme 1. Preparation of the Preloaded Resin A According to a Previously Developed Procedure31a

a

Reagents and conditions: (i) 2-(2-aminoethoxy)ethanol, 10% AcOH/DMF, rt, 16 h, then NaBH(OAc)3, rt, 6 h; (ii) Fmoc-OSu, CH2Cl2, rt, 16 h; (iii) biotin, HOBt, DIC, DIPEA, DMF, rt, 16 h, repeated three times; (iv) 20% piperidine/DMF, rt, 30 min.; (v) [2-[2-(Fmocamino)ethoxy]ethoxy]acetic, HOBt, DIC, CH2Cl2/DMF (1:1), rt, 16 h.

Scheme 2. Solution-Phase Premodification of Selected Compounds for Solid-Phase Biotinylationa

a Reagents and conditions: (i) Succinic anhydride, THF, DMAP, reflux 12 h; (ii) Benzyl bromoacetate, DMF, K2CO3, r.t., 14 h; (iii) cyclohexadiene, Pd/C, THF, 24 h; (iv) Ph3SiCl, imidazole, DMF, 24 h; (v) NBS, CCl4, 2 h; (vi) TBAF, THF, 2 h.

Modification via C-3 Position. Hemisuccinates 6−9 were subjected to the reaction with resin A using HOBt/DIC (Scheme 3). In each case, the acylation provided the corresponding intermediates 15−18. After the cleavage from the polymer support using the standard cleavage procedure for BAL linker (50% TFA in CH2Cl2), the products were purified with semipreparative reverse-phase HPLC and fully characterized. The overall yield calculated from the loading of immobilized 2-(2-aminoethoxy)ethanol was 31% for compound 19, 54% for compound 20, 10% for compound 21, and 30% for compound 22. The limited yields can be explained by unfavorable properties of the target molecules for the reversephase chromatography (they are too lipophilic) and their low reactivity; steric hindrances probably still play significant role. Modification via C-28. Two alternative procedures have been developed. In the first case, the glyoxalic acid derivatives 10, 11 were subjected to acylation of the preloaded resin A, again with the use of HOBt/DIC activation (Scheme 4). It is worth mentioning that in the case of the modification via 28COOH, the standard cleavage conditions led to decomposition

of the molecule due to hydrolysis of the neopentylate ester. However, when the concentration of the cleavage cocktail was decreased to 10% TFA in CH2Cl2, the release of the product from the resin was still quantitative whereas the target molecules were not hydrolyzed. Compound 25 was isolated in an overall yield of 43%; product 26 was obtained in an overall yield of 62%. To avoid the need for solution-phase premodification to obtain glyoxalates 10 and 11, we tried a more direct approach. We converted resin A to its iodoacetyl analog B, which was used for the direct alkylation of the starting neopentyl carboxylates (Scheme 5). The method was successfully tested for both previously used derivatives: betulinic acid 1 and pyrazine derivative 5. Modification via C-30. Finally, a method for the modification via alkyl in position C-30 of the main scaffold was developed. For this purpose, triterpene bromo derivative 14 was used for the alkylation of the resin A (Scheme 6). The target product 28 was obtained in an overall yield of 25%. 2565

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Bioconjugate Chemistry Scheme 3. Modification of Selected Molecules via C-3 Positiona

a

Reagents and conditions: (i) Preloaded resin A, HOBt, DIC, DMF, CH2Cl2, rt, 16 h; (ii) 50% TFA in CH2Cl2, rt, 30 min.

Scheme 4. Modification of Selected Molecules via C-28 Positiona

a

Reagents: (i) Preloaded resin A, HOBt, DIC, DMF, CH2Cl2, rt, 16 h; (ii) 10% TFA in CH2Cl2, rt, 30 min.

Cytotoxicity. Cytotoxic activity of five selected triterpenes and their biotinylated analogs was measured on two cancer cell linesacute lymphocytic leukemia CCRF-CEM and colorectal carcinoma HCT116 (Table 1). These cell lines were chosen since the same cell lines will be used in the target identification procedures. The activity of biotinylated compounds was in the similar range of the activity as their nonbiotinylated parents. Such results indicate that biological activity was not dramatically compromised by biotinylation of the molecule and we can expect identification of molecular targets in the future. In the case of betulinic acid the cytotoxic activity was

low and we can just speculate that modification via the C-28 position is not affecting the activity. Proteomics pull-down affinity experiments will be performed on derivatives biotinylated from different positions which will better characterize the pharmacophore of the molecule. We anticipate isolation of the target protein(s) when the molecule is connected to biotin through a conjugation site far from the pharmacophore, while we expect a negative result when biotinylation is mediated through the pharmacophore and hinders the active function of the molecule(s). In addition to the IC50 of the parent compounds and their biotinylated counterparts, we also 2566

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Bioconjugate Chemistry Scheme 5. Alternative Method for Modification via C-28 with Use of the Preloaded Resin Ba

Reagents and conditions: (i) iodoacetic acid, DIC, CH2Cl2, rt, 2 h; (ii) Betulinic acid 1 or pyrazine derivative 5, DIPEA, DMSO, 60 °C, 16 h; (iii) 10% TFA in CH2Cl2, rt, 30 min.

a

Scheme 6. Modification of Betulinic Acid 1 via C-30 Positiona

a

Reagents and conditions: (i) Preloaded resin A, DIPEA, DMSO, 65 °C, 16 h; (ii) 50% TFA in CH2Cl2, rt, 30 min.

Table 1. Cytotoxic Activity of Biotinylated Triterpenes and Their Parent Compounds IC50 (μmol/La) parental compounds

CCRF-CEM

1

30.0

HCT116 >50

2 3 4 5

1.71 17.6 34.36 0.5

4.14 28.23 >50 11.09

IC50 (μmol/La) biotinylated compounds

position biotinylated C-3 C-28 C-30 C-3 C-3 C-3 C-28 IC50 (μmol/La)

19 25 28 20 21 22 26

CCRF-CEM

HCT116

>50 14.85 >50 3.39 11.03 20.46 5.87

>50 >50 >50 7.77 23.49 49.3 18.01

intermediates

CCRF-CEM

HCT116

6 7 8 9 10 11

29.29 8.96 >50 37.12 43.54 12.25

21.78 13.29 >50 37.49 >50 20.5

a

The lowest concentration that kills 50% of tumor cells. The standard deviation in cytotoxicity assays is typically up to 10% of the average value. Compounds with IC50 50 μM and more are considered inactive.

measured the activities of the intermediateshemisuccinates and glycolates 6−11because their increased hydrophilicity

could have had influence on their activity; however, their IC50 was rather too high. 2567

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CONCLUSIONS We have developed a new method to connect triterpenes to biotin using a solid-phase strategy. The method proved to be universal for connecting a variety of triterpenoid compounds through their 3-OH, 28-COOH, and 30-CH2X groups. This is notable, because for the biotin/streptavidin affinity purification or pulldown assays combined with quantitative proteomics and SILAC, the compound of interest should be biotinylated from two sidesthrough the pharmacophore (negative control) and through the opposite side. Here we describe three options and we should be able to observe which proteins are attracted to each of the three sides of the molecule and possibly conclude which part is responsible for any such activity. In addition, we proved that solid-phase synthesis is a good opportunity to prepare new triterpenoid derivatives in general, and that problems with steric hindrance are avoidable by extending the hindered group farther from the skeleton using the classic solution procedure. This is the second time solid phase was used in terpene chemistry, and here we describe a much more universal procedure. This is the first time that biotinylated lupane derivatives were prepared and they are ready to be used in pulldown assays.

frequencies of 500.16 and 125.77 MHz at ambient temperature (25 °C). 1H and 13C spectra were referenced relative to the signal of TMS. Chemical shifts δ are reported in ppm and coupling constants J in Hz. HRMS analysis was performed using an Orbitrap Elite high-resolution mass spectrometer (Thermo Fischer Scientific, MA, USA) operating at positive full scan mode (120 000 fwmh) in the range of 200−900 m/z. The settings for electrospray ionization were as follows: oven temperature of 300 °C, sheath gas of 8 arb. units, and source voltage of 1.5 kV. The acquired data were internally calibrated with diisooctyl phthalate as a contaminant in methanol (m/z 391.2843). Samples were diluted to a final concentration of 20 μmol/L with 0.1% formic acid in water and methanol (50:50, v/v). The samples were injected by direct infusion into the mass spectrometer. Triterpenoid compounds 1−14 were synthesized from betulin and betulinic acid (purchased from company Betulinines − www.betulinines.com) using previously published15,16,19,30,32,33 procedures. Their structure was confirmed by comparing their melting points and 1H and 13C NMR spectral data to the data in the literature.15,16,19,30,32,33 Preparation of Preloaded Resin A. Preloaded resin A was synthesized according to previously published procedure.31 All properties of the resin were identical to the published data. Preparation of Preloaded Resin B. Iodoacetic acid (186 mg, 1 mmol) was dissolved in CH2Cl2 (2 mL) and DIC (75 μL, 0.5 mmol) was added. The reaction mixture was stirred for 30 min, and then the precipitated N,N-diisopropylurea was filtered off. The filtrate was added to preloaded resin A (200 mg); the resin was shaken 2 h at room temperature and washed with CH2Cl2 (5×). General Procedure for Acylation of Preloaded Resin A with Triterpene Derivatives. Each triterpene 6, 7, 8, 9, 10, or 11 (0.3 mmol) was dissolved in DMF (1.5 mL), HOBt· 2H2O (45 mg, 0.3 mmol), DIC (45 μL, 0.3 mmol), and CH2Cl2 (1.5 mL) was added subsequently. The solution was added to preloaded resin A (250 mg). The resin was shaken overnight, then washed 3× with DMF and 3× with CH2Cl2. General Procedure for Alkylation of Triterpene Derivatives with Preloaded Resin B. Each triterpene 10 or 11 (0.3 mmol) was dissolved in DMF (3 mL) and equivalent of DIPEA (0.3 mmol, 52 μL) was added. The solution was added to preloaded resin B (250 mg). The resin was shaken for 16 h, then washed 3× with DMF and 3× with CH2Cl2. Alkylation of Resin A with Derivative 14. Derivative 14 (0.3 mmol, 160 mg) was dissolved in DMSO (3 mL) and equivalent of DIPEA (0.3 mmol, 52 μL) was added. The solution was added to preloaded resin A (300 mg) and the slurry was shaken at 80 °C for 16 h. The resin was washed 3× with DMF and 3× with CH2Cl2. Cleavage from the Resin (for Stable Intermediates 15−18, 27). Resin 15, 16, 17, 18, or 27 was shaken with 50% TFA in CH2Cl2 (3 mL) for 30 min at room temperature. The cleavage cocktail was isolated and the resin was washed three times with additional 50% TFA in CH2Cl2. Combined washes were evaporated in a stream of nitrogen and the residual material was dissolved in 2 mL of MeCN for the reverse-phase chromatography. Cleavage from the Resin (for Unstable Intermediates 23, 24). Resin 23 or 24 was shaken with 10% TFA in CH2Cl2 (3 mL) for 30 min at room temperature. The cleavage cocktail was isolated and the resin was washed three times with additional 10% TFA in CH2Cl2. Combined washes were

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EXPERIMENTAL SECTION Solvents and chemicals were purchased from Aldrich (Milwaukee, IL, www.sigmaaldrich.com), Acros (Geel, Belgium, www.acros.cz), and Fisher (Pittsburgh, PA, www.fishersci.com). The aminomethylene resin (100−200 mesh, 1% DVB, 0.98 mmol/g), and 4-(4-Formyl-3-methoxyphenoxy)butyric acid were obtained from AAPPTec (Louisville, KY, www.aapptec. com). Solid-phase synthesis was carried out on Domino Blocks in disposable polypropylene reaction vessels (Torviq, Niles, MI, www.torviq.com). Labquake Tube Rotator (Thermolyne, Dubuque, IA, www.barnsteadthermolyne.com) was used for gentle but efficient tumbling of resin slurry. All reactions were carried out at ambient temperature (21 °C) unless stated otherwise. The volume of wash solvent was 10 mL per 1 g of resin. For washing, resin slurry was shaken with the fresh solvent for at least 1 min before changing the solvent. After adding a reagent solution, the resin slurry was manually vigorously shaken to break any potential resin clumps. Resinbound intermediates were dried by a stream of nitrogen for prolonged storage and/or quantitative analysis. For the LC/MS analysis a sample of resin (∼5 mg) was treated by TFA in CH2Cl2, the cleavage cocktail was evaporated by a stream of nitrogen, and cleaved compounds extracted into 1 mL of MeCN. The LC/MS analyses were carried out on UHPLC-MS system consisting of UHPLC chromatograph Acquity with photodiode array detector and single quadrupole mass spectrometer (Waters), using X-Select C18 column at 30 °C and flow rate of 600 μL/min. Mobile phase was (A) 0.01 M ammonium acetate in water and (B) MeCN, linearly programmed from 10% to 80% B over 2.5 min, kept for 1.5 min. The column was re-equilibrated with 10% of solution B for 1 min. The ESI I source operated at discharge current of 5 μA, vaporizer temperature of 350 °C, and capillary temperature of 200 °C. Purification was carried out on C18 reverse-phase column 19 × 100 mm, 5 μm particles; gradient was formed from 10 mM aqueous ammonium acetate and acetonitrile, flow rate 15 mL/min. All 1H and 13C NMR experiments were performed with using Jeol ECX-500SS at magnetic field strengths of 11.749 T corresponding to 1H and 13C resonance 2568

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evaporated in a stream of nitrogen and the residual material was dissolved in 2 mL of MeCN for the reverse-phase chromatography. Purification of Final Products. Each biotinylated derivative was obtained as a solution in 2 mL of MeCN and was further purified on reverse-phase preparative HPLC column using gradient consisting of 10 mM aqueous ammonium acetate and acetonitrile. Pure fractions were evaporated from MeCN under reduced pressure and lyophilized from H2O to obtain the pure compounds as amorphous solids. This procedure gave compound 19 (20.4 mg, 30%), compound 20 (35.5 mg, 54%), compound 21 (7 mg, 10%), compound 22 (20 mg, 30%), compound 25 (30 mg, 43%), compound 26 (40 mg, 62%), and compound 28 (15 mg, 25%). All physical and spectral data are in the Supporting Information file along with 1H, 13C, and HRMS spectra and visual appearance of the compounds. Cell Lines. The CCRF-CEM (CEM) was purchased from the American Tissue Culture Collection (ATCC). Colorectal cancer cell line bearing wild type p53 gene (HCT116) was obtained from Horizon Discovery Ltd. (www.horizondiscovery. com). The cells were maintained in nunc/Corning 80 cm2 plastic tissue culture flasks and cultured in cell culture medium according to ATCC recommendations (DMEM/RPMI 1640 with 5 g/L glucose, 2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 10% fetal calf serum, and NaHCO3). Cytotoxic MTS Assay. MTS assay was performed at Institute of Molecular and Translational Medicine by robotic platform (HighResBiosolutions). Cell suspensions were prepared and diluted according to the particular cell type and the expected target cell density (27 000−33 000 cells/mL based on cell growth characteristics). Cells were added by automatic pipetor (30 μL) into 384 well microtiter plates. All tested compounds were dissolved in 100% DMSO and 4-fold dilutions of the intended test concentration were added in 0.15 μL aliquots at time zero to the microtiter plate wells by the echo-acoustic noncontact dispensor Echo550 (Labcyte). The experiments were performed in technical duplicates and three biological replicates at least. The cells were incubated with the tested compounds for 72 h at 37 °C, in a 5% CO2 atmosphere at 100% humidity. At the end of the incubation period, the cells were assayed by using the MTS test. Aliquots (5 μL) of the MTS stock solution were pipetted into each well and incubated for an additional 1−4 h. After this incubation period, the optical density (OD) was measured at 490 nm with an Envision reader (PerkinElmer). Tumor cell survival (TCS) was calculated by using the following equation: TCS = (ODdrug‑exposed well/mean ODcontrol wells) × 100%. The IC50 value, the drug concentration that is lethal to 50% of the tumor cells, was calculated from the appropriate dose−response curves in Dotmatics software.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +420 585 632 197. Fax: +420 585 632 180. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The scientific part was supported by Czech Science Foundation (15-05620S), except for the synthesis of compounds 21 and 22 that was paid for by the Technology Agency of the Czech Republic (TE01020028). The infrastructural part (Institute of Molecular and Translational Medicine) was supported by the Operational Program Research and Development for Innovations NPUII project LO1304 and NJD was paid by by the Ministry of Education, Youth and Sport of the Czech Republic and by the European Social Fund (CZ.1.07/2.3.00/30.0060).

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REFERENCES

(1) Hill, R. A., and Connolly, J. D. (2015) Triterpenoids. Nat. Prod. Rep. 32, 273−327. (2) Sarek, J., Kvasnica, M., Vlk, M., and Biedermann, D. (2010) Semisynthetic lupane triterpenes with cytotoxic activity. In Pentacyclic Triterpenes as Promising Agents in Cancer (Salvador, J. A. R., Ed.) pp 159−189, Chapter 6, Nova Science Publishers, New York. (3) Zuo, W.-J., Dai, H.-F., Chen, J., Chen, H.-Q., Zhao, Y.-X., Mei, W.-L., and Wang, J.-H. (2011) Triterpenes and Triterpenoid Saponins from the Leaves of Ilex kudincha. Planta Med. 77, 1835−1840. (4) Mallavadhani, U. V., Mahapatra, A., Raja, S. S., and Manjula, S. (2003) Antifeedant Activity of Some pentacyclic Triterpene Acids and Their Fatty Acid Ester Analogues. J. Agric. Food Chem. 51, 1952−1955. (5) Yano, S., Harada, M., Watanabe, K., Nakamaru, K., Hatakeyama, Y., Shibata, S., Takahashi, K., Mori, T., Hirabayashi, K., Takeda, M., et al. (1989) Antiulcer Activity of Glycyrrhetic Acid Derivatives in Experimental Gastric Lesion Models. Chem. Pharm. Bull. 37, 2500− 2504. (6) Dang, Z., Ho, P., Zhu, L., Qian, K., Lee, K.-H., Huang, L., and Chin, C.-H. (2013) New Betulinic Acid Derivatives for BevirimatResistant Human Immunodeficiency Virus Type-1. J. Med. Chem. 56, 2029−2037. (7) Joycharat, N., Thammayong, S., Limsuwan, S., Homlaead, S., Voravuthikunchai, S. P., Yingyongnarongkul, B., Dej-adisai, S., and Subhadhirasakul, S. (2013) Antibacterial substances from Albizia myriophylla wood against cariogenic Streptococcus mutans. Arch. Pharmacal Res. 36, 723−730. (8) Morikawa, T., Ninomiya, K., Imura, K., Yamaguchi, T., Akagi, Y., Yoshikawa, M., Hayakawa, T., and Muraoka, O. (2014) Hepatoprotective medicine from traditional Tibetan medicine Potentilla anserina. Phytochemistry 102, 169−181. (9) Sanchez-Quesada, C., Lopez-Biedma, A., Warleta, F., Campos, M., Beltran, G., and Gaforio, J. J. (2013) Bioactive Properties of the Main Triterpenes Found in Olives, Virgin Olive Oil, and Leaves of Olea europaea. J. Agric. Food Chem. 61, 12173−12182. (10) Chianese, G., Yerbanga, S. R., Lucantoni, L., Habluetzel, A., Basilico, N., Taramelli, D., Fattorusso, E., and Taglialatela-Scafati, O. (2010) Antiplasmodial Triterpenoids from the Fruits of Neem, Azadirachta indica. J. Nat. Prod. 73, 1448−1452. (11) Gaertner, M., Muller, L., Ross, J. F., Santos, A. R. S., Nieto, R., Calixto, J. B., Yunes, R. A., Delle Monache, F., and Cechinel-Filho, V. (1999) Analgesic Triterpenes from Sebastiania schottiana roots. Phytomedicine 6, 41−44. (12) Dzubak, P., Hajduch, M., Vydra, D., Hustova, A., Kvasnica, M., Biedermann, D., Markova, L., Urban, M., and Sarek, J. (2006) Pharmacological activities of natural triterpenoids and their therapeutic implications. Nat. Prod. Rep. 23, 394−411.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00567. Data on synthesis, instruments, calibration, HPLC data, yields, physical and spectral data (mp, HRMS, 1H NMR, 13 C NMR, IR) and pictures of NMR and HRMS spectra of all final products (PDF) 2569

DOI: 10.1021/acs.bioconjchem.5b00567 Bioconjugate Chem. 2015, 26, 2563−2570

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DOI: 10.1021/acs.bioconjchem.5b00567 Bioconjugate Chem. 2015, 26, 2563−2570