Design, Synthesis, and Cancer Cell Growth Inhibitory Activity of

Article pubs.acs.org/jnp

Design, Synthesis, and Cancer Cell Growth Inhibitory Activity of Triphenylphosphonium Derivatives of the Triterpenoid Betulin Olga V. Tsepaeva,† Andrey V. Nemtarev,*,†,‡ Timur I. Abdullin,‡ Leysan R. Grigor’eva,‡ Elena V. Kuznetsova,‡ Rezeda A. Akhmadishina,‡ Liliya E. Ziganshina,‡ Hanh H. Cong,‡ and Vladimir F. Mironov†,‡

J. Nat. Prod. 2017.80:2232-2239. Downloaded from pubs.acs.org by KAOHSIUNG MEDICAL UNIV on 09/29/18. For personal use only.

†

A. E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences, Arbuzov Street 8, 420088, Kazan, Russian Federation ‡ Kazan (Volga Region) Federal University, Kremlevskaya Street 18, 420008, Kazan, Russian Federation S Supporting Information *

ABSTRACT: A series of new triphenylphosphonium (TPP) derivatives of the triterpenoid betulin (1, 3-lup-20(29)-ene-3β,28diol) have been synthesized and evaluated for cytotoxic effects against human breast cancer (MCF-7), prostate adenocarcinoma (PC-3), vinblastine-resistant human breast cancer (MCF-7/Vinb), and human skin fibroblast (HSF) cells. The TPP moiety was applied as a carrier group through the acyl linker at the 28- or 3- and 28-positions of betulin to promote cellular and mitochondrial accumulation of the resultant compounds. A structure−activity relationship study has revealed the essential role of the TPP group in the biological properties of the betulin derivatives produced. The present results showed that a conjugate of betulin with TPP (3) enhanced antiproliferative activity toward vinblastine-resistant MCF-7 cells, with an IC50 value as low as 0.045 μM.

T

diseases.7−10 Most of the antitumor agents that are being developed or used in clinical trials show low efficacies and safety profiles due to limited bioavailability, as a result of low water solubility, extensive metabolism, poor cell permeability, or rapid excretion.11 Prodrug-based synthetic approaches have been used to improve the pharmacokinetic properties of drug compounds.12 They are based on the covalent attachment of different substituents to a molecule of interest to increase its solubility in water or lipid membranes, blood serum stability, and selectivity of action. In spite of diverse biological activities, the triterpenoids suffer from a general low bioavailability, which restricts their possible therapeutic use. Efforts are being made to increase the bioavailability of triterpenoid-based compounds by developing their prodrugs with improved solubility and selectivity.13 Lipophilic cations are promising molecules to improve pharmacokinetic properties of

riterpenoids are an important class of naturally occurring compounds, with over 200 different skeletons known covering aldehydes, ketones, alcohols, ethers, lactones, and other functionalities.1 They are found in fungi, ferns, seaweeds, angiosperms, and animals. Several recent publications have reported the ability of certain triterpenoids, such as ursolic, oleanolic, and betulinic acids and their derivatives, to inhibit the growth of tumor cells in vitro and in vivo.2,3 The potential antitumor activity of triterpenoids results not only from their direct cytotoxic effects on tumor cells but also from inhibiting tumor angiogenesis and dissemination. There is evidence that triterpenoids and their derivatives cause the death of tumor cells mainly due to activation of apoptosis via a mitochondrial pathway.4−6 Mitochondria are key cellular organelles that support ATP biosynthesis, Ca2+ homeostasis, active oxygen species (AOS) production, cell signaling, and apoptosis activation. Mitochondrial AOS cause oxidative damage of cell components and are involved in various pathologies, including diabetes, cardiovascular disorders, infarction, stroke, and neurodegenerative © 2017 American Chemical Society and American Society of Pharmacognosy

Received: February 7, 2017 Published: August 7, 2017 2232

DOI: 10.1021/acs.jnatprod.7b00105 J. Nat. Prod. 2017, 80, 2232−2239

Journal of Natural Products

Article

Scheme 1. General Synthesis of Compounds 2−7

active betulin derivatives, in which the betulin scaffold is conjugated with the TPP group through an acyl linker at the 28- or 3- and 28-positions.

triterpenoids due to high lipid solubility and the ability to readily pass cellular membranes and accumulate in mitochondria.14,15 The intracellular transport rate of the phosphonium cation is 107−108 times higher compared with hydrophilic cations such as Na+.16 However, with increasing hydrophobicity of the alkyl group, a more powerful driving force of an entropic nature enhances the degree of binding to the membrane.17 The triphenylphosphonium (TPP) group was found at the same place on the hydrophobic side of the membrane lipid/water interface, with the hydrophobic tail penetrating into the hydrophobic core of the membrane.18 The TPP cation is one of the most well characterized lipophilic cations for mitochondrial delivery of compounds with antioxidant,19−21 spin trapping,22,23 and other properties.24,25 An important approach to the development of selective anticancer prodrugs is the synthesis of drug conjugates, which target biochemical and physicochemical features of cancer cells. Many studies have shown that cancerous cells possess increased mitochondrial and plasma membrane potential compared with “normal” cells.26−28 The lipophilic cations administered in vivo accumulate in cancer cells in a more effective way, causing their selective death. The TPP moiety was also shown to promote mitochondrial accumulation and exhibit preferable toxicity toward cancer cells compared to control cells.27 An increase in the relative concentration of the conjugates of toxic components to TPP occurs within cancer cells to selectively promote their death.29,30 The ability of bioactive molecules to accumulate into mitochondria is of particular interest in both research and therapeutic applications.31,32 Conjugation of nonionic bioactive compounds with lipophilic cations, including TPP, could lead to their enhanced selective transport into the mitochondrion.14 This approach was proposed by Chen et al., who showed that modification of the anticancer drug cisplatin with a rhodamine lipophilic cation promoted transportation of the anticancer drug into cancer cells.26 Several ammonium and triphenylphosphonium derivatives of triterpenoids with a lupane structure have been obtained, which exhibit considerably more potent cytotoxic effects on cancer cells of different origin when compared with betulinic acid.33−39 The aim of the present study was to synthesize new biologically

■

RESULTS AND DISCUSSION Chemistry. Betulin [3-lup-20(29)-ene-3β,28-diol], with demonstrated cytotoxic and antiproliferative effects on tumor cells,40 was used as a scaffold compound to synthesize new antitumor substances. Mono- and diether derivatives of betulin and betulinic acid have been shown to possess anti-HIV, immunomodulating, hepatoprotective, anti-inflammatory, and antitumor activities.41−53 Recently, 28-O-chloroacetylbetulin was synthesized with an increased cytotoxic effect on MCF-7 cancer cells in comparison with betulinic acid.54 C-3,C-28-Functionalized derivatives of betulin and lupane were obtained by the interaction of betulin with ωbromoalkanecarboxylic acids in dichloromethane in the presence of dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC), using a Steglich esterification.55 This esterification method was found to have high selectivity in the production of polysubstituted compounds with an equimolar ratio of betulin and bromoalkanecarboxylic acids. For esterification, regiochemistry derivatives of the primary hydroxy group at C-28 are commonly produced, with the secondary hydroxy group at C-3 not affected. Comparison of the 1H NMR spectra of compounds 2−4 and betulin showed a considerable downfield shift of the resonance of the C-28 protons, which form an AX spin system as a result of the introduction of an acyl substituent into the molecule. Moreover, the signal position of the H-3 proton in the 1H NMR spectra of compounds 2−4 closely resembled that in the betulin 1H NMR spectrum. This gave good evidence of specific introduction of the acyl function at position 28. The products were purified using silica gel column chromatography. The reaction of betulin with two equivalents of a bromoalkanecarboxylic acid generates C-3, C-28 betulin diesters in over 90% yield. In the 1H NMR spectra of compounds 5−7 in the downfield region there were signals of the H-3 (δH 4.5 ppm) and H-28 (δH 3.9, 4.4 ppm) protons, indicating the formation of ester bonds. Hence, the phosphonium and biphosphonium derivatives of betulin were obtained by nucleophilic substitution of 2233



Article

Scheme 2. General Synthesis of Compounds 8−13

Table 1. Inhibitory Concentrations (IC50, μM) of Test Compounds for Different Human Cells in Vitro (MTT Assay) IC50, μM compound 8 9 10 11 12 13 doxorubicin vinblastine

PC-3 6.8 0.16 0.12 6.9 0.54 0.47

± ± ± ± ± ±

1.89 0.02 0.01 0.76 0.11 0.05

MCF-7 >10 0.43 0.70 8.7 0.35 0.48 0.15 0.5

± ± ± ± ± ± ±

MCF-7/Vinb

0.13 0.24 1.46 0.05 0.07 0.02 0.02

>10 0.045 0.75 4.3 0.88 0.84 0.3 9.2

± ± ± ± ± ± ±

0.01 0.12 0.66 0.22 0.17 0.05 1.1

HSF 9.9 2.1 1.5 5.6 0.86 0.76

± ± ± ± ± ±

0.93 0.57 0.16 0.46 0.158 0.23

respectively. Half-maximal inhibitory concentrations (IC50) of betulin derivatives determined using an MTT assay are summarized in Table 1. Figure S1 (Supporting Information) shows representative concentration−viability curves of compound 9. The conjugation of betulin with ω-bromoalkanoic acid and TPP at positions 28 or 3 and 28 led to a decrease in IC50 values of the synthesized compounds, indicating an increase in their cytotoxicity. It can be assumed that increased cytotoxicity of the betulin derivatives arises from bioactivity of the introduced TPP moiety and/or increased intracellular uptake of the conjugates. The results showed that the conjugates of betulin with TPP (8−13) exhibited considerably more potent cytotoxic effects, with an IC50 value as low as 0.045 μM compared with other acylated derivatives of betulin (2−7 with the lowest IC50 value of 22 μM). The TPP group may impart enhanced intramitochondrial penetration to these test compounds in accordance with literature data.15 Cytotoxic activity of the synthesized compounds increased in the order 8 ∼ 11 < 10 ∼ 13 ∼ 12 < 9 for the betulin−TPP conjugates (Table 1). The following main structure−activity relationship inferences could be revealed from these data. In particular, the betulin derivatives modififed with a TPP group at the 28- or 28- and 3-positions showed noticeably higher cytotoxic effects compared with the starting triterpenoid and the acylated derivatives. The length of the alkyl linker in the ester group (n = 3, 4) affected the activity, where the methylene linker (8, 11) exhibited relatively lower effects than that of the

halogenated betulin with a 4-fold molar excess of triphenylphosphine in CH3CN upon heating for 4−5 h (as controlled by a TLC method), resulting in 83−95% yields, as detailed in Scheme 1 and Scheme 2. The 31P{1H} NMR spectra of biphosphonium salts 12 and 13 contained only one signal for the phosphorus atoms of the phosphonium moieties. There were two singlets belonging to the phosphorus atoms of phosphonium moieties at the 3- and 28-positions of the betulin scaffold in the 31P{1H} NMR spectrum of biphosphonium salt 11, because of the influence of the chiral betulin scaffold. For the interpretation of the 13C{1H} and 13C NMR spectra, the spectroscopic data for bromoalkanecarboxylic acids and betulin56 and the multiplicity of the signals were considered. The 13C{1H} NMR spectra of the compounds showed signals from the aromatic carbons in the field of δC 135−118 ppm. A characteristic doublet of ipsocarbons appeared in the relatively high-field region of the spectrum (δC 118 ppm). It should be noted that these ipsocarbons of the initial triphenylphosphine and its oxidation product, the triphenylphosphine oxide, resonate in a more downfield region (δC 137.8 and 135.6 ppm with coupling constants 1JPC 12.5 and 104.4 Hz, respectively).57 Cytotoxic Activity of Betulin Derivatives. A comparative study of cytotoxicity of betulin derivatives was carried out on different cell lines and cultures. PC-3 (prostate adenocarcinoma), MCF-7 (human breast cancer), and vinblastine-resistant MCF-7 (MCF-7/Vinb) cells, as well as human skin fibroblasts (HSFs), were used as model cancer cells and “normal” cells, 2234



Article

Figure 1. Distribution of TMRE fluorescence in PC-3 cells treated with betulin derivatives: (A) compound 1 (betulin), (B) compound 7, (C) compound 10, (D) compound 12, (E) compound 13. Cells were cultured with compounds at the IC50 concentration for 48 h and stained with 100 nM TMRE.

Effect of Betulin Derivatives on Mitochondrial Potential and Cell Cycle in Cancer Cells. In order to assess the potential interaction of betulin derivatives with cell mitochondria, a tetramethylrhodamine ethyl ester (TMRE) fluorescent probe was used, which penetrates cellular membranes and accumulates in the mitochondria in proportion to their transmembrane potential. This potential reflects the functional activity of the mitochondria and decreases upon apoptosis induction.58 Figure 1 shows flow cytometry data of TMRE fluorescence in PC-3 cells treated with randomly selected compounds (1, 7, 10, 12, 13). Under experimental conditions (48 h culturing at IC50 concentration), betulin did not alter mitochondrial potential in the cells, in contrast to its derivatives containing a TPP moiety and oligomethylene fragments, which induced decreases in the potential. These decreases observed could be explained by direct interaction of the compounds studied with mitochondria as well as apoptosis induction. The mean channel fluorescence of TMRE in treated cells decreased as follows: control (untreated cells) (210.8) > compound 1 (205.9) > compound 7 (187.9) > compound 10 (186.6) > compound 13 (112.2) > compound 12 (90.1). These results indicate that compound 10, with a single phosphonium group, and the acylated product 7 induce relatively weak decreases in the mitochondrial potential by ca. 10% compared with untreated cells, whereas compounds bearing two phosphonium groups (13 and 12) reduced these factors by about 2-fold (Figure 1). Taking into account a similar cytotoxicity profile of betulin−TPP conjugates (Table 1), these results indicate that the compounds with two phosphonium groups affect mitochondrial function in a more effective way, presumably due to their increased tropism to mitochondria. According to cell cycle analysis, the following effects on the cell phases in growing populations of PC-3 cells were observed

propylene (9, 12) and butylene (10, 13) linkers, which provide enhanced cytotoxicity with similar IC50 values. Hence, extended alkyl linkers in betulin−TPP conjugates ensure enhanced ability of the compounds to affect the viability of mammalian cells. The second phosphonium group at position 28 similarly affects the cytotoxicity of the corresponding compounds with only a single phosphonium group (8−10) for the cancer cells, irrespective of the length of the alkyl linker (Table 1). However, the compounds 12 and 13 decrease viability of HSFs to a higher extent compared with 9 and 10, respectively. Similar cytotoxic effects of betulin derivatives modified with single (28position) and double (28-, 3-positions) TPP groups could be explained by the crucial role of the 28-position in generating bioactive conjugates with TPP and/or the similar cellular uptake of these compounds. We believe that enhancement promotion in the transportation of the betulin derivatives into the cytosol and presumably to mitochondria by the TPP group contributes to enhanced cellular effect of the compounds synthesized. The betulin−TPP conjugates, as well as the acylated and diacylated compounds, generally exhibited comparable IC50 values for the cancer cells and HSFs. Certain cancer versus normal selectivity was observed only for the conjugates of betulin (4, 9, 10). In particular, there was almost a 10-fold difference in cytotoxicity for 9 toward HSFs (IC50 2.1 μM) and PC-3 cells (IC50 0.2 μM) as well as between MCF-7 (IC50 0.43 μM) and MCF-7/Vinb (IC50 0.045 μM) (Table 1). Relatively low cytotoxicity toward HSFs compared with cancer cells was also detected for 4 (IC50 < 100 μM) and 10 (IC50 1.5 μM). The differences observed could be explained by increased accumulation of the above compounds and/or preferable interaction with intracellular targets in cancer cells compared with HSFs. 2235



Article

C(O)CH2Br), 3.98 (1H, d, J = 10.8 Hz, HA-28), 4.31 (1H, d, J = 11.0 Hz, HX-28), 4.61 (1H, s, HA-29), 4.71 (1H, s, HB-29); anal. C 68.0, H 9.3, Br 14.1%, calcd for C32H51BrO3%, C 68.19, H 9.12, Br 14.18%. 3β-Hydroxylup-20(29)-en-28-yl 4-bromobutanoate (3): amorphous powder; 0.95 g, 95% yield; mp 85−87 °C; IR (KBr) νmax 3419, 3071, 2942, 2869, 1733, 1641, 1455, 1389, 1249, 1200, 1130,1033, 983 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.77, 0.83, 0.98, 0.99, 1.04 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.69 (3H, s, CH3-30), 0.90−2.23 (28H, m, betulin scaffold and (CH2) fragment of linker), 2.45 (1H, m, J = 10.7, 5.6 Hz, H-19), 2.54 (2H, t, J = 7.2 Hz, C(O)CH2), 3.20 (1H, m, J = 11.0, 5.0 Hz, H-3), 3.48 (2H, t, J = 6.5 Hz, CH2Br), 3.88 (1H, d, J = 11.0 Hz, HA-28), 4.31 (1H, d, J = 11.0 Hz, HX-28), 4.60 (1H, s, HA-29), 4.70 (1H, s, HB-29); 13C NMR (CDCl3, 100 MHz) δ 14.8 (CH3, C-27), 15.4 (CH3, C-24), 16.0 (CH3, C-26), 16.1 (CH3, C-25), 18.3 (CH2, C-6), 19.2 (CH3, C-30), 20.8 (CH2, C-11), 25.2 (CH2, C-12), 27.1 (CH2, C-15), 27.4 (CH2, C2), 27.8 (CH2CH2Br), 28.0 (CH3, C-23), 29.6 (CH2, C-16), 29.8 (CH2, C-21), 32.65 (OC(O)CH2), 32.7 (CH2Br), 34.2 (CH2, C-22), 34.6 (CH2, C-7), 37.2 (C, C-10), 37.6 (CH, C-13), 38.7 (CH2, C-1), 38.9 (C, C-4), 40.9 (C, C-8), 42.7 (C, C-14), 46.4 (C, C-17), 47.8 (CH, C-18), 48.8 (CH, C-19), 50.4 (CH, C-9), 55.3 (CH, C-5), 63.0 (CH2, C-28), 79.0 (CH, C-3), 109.9 (CH2, C-29), 150.1 (C, C-20), 172.9 (C, CO); anal. C 68.6, H 8.9, Br 13.3%, calcd for C34H55BrO3, C 69.0, H 9.37, Br 13.5%. 3β-Hydroxylup-20(29)-en-28-yl 5-bromopentanoate (4): amorphous powder; 1.0 g, 85% yield; mp 112−115 °C; IR (KBr) νmax 3438, 2940, 2869, 1733, 1628, 1454, 1389, 1250, 1193, 1044, 983 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.78, 0.84, 0.98, 0.99, 1.05 (15H, s, CH323, CH3-24, CH3-25, CH3-26, CH3-27), 1.70 (3H, s, CH3-30), 0.91− 2.01 (30H, m, betulin scaffold and (CH2)2 fragment of linker), 2.39 (2H, t, J = 7.3 Hz, C(O)CH2), 2.45 (1H, m, J = 11.0, 5.7 Hz, H-19), 3.19 (1H, m, J = 11.1, 5.0 Hz, H-3), 3.43 (2H, t, J = 6.5 Hz, CH2Br), 3.87 (1H, d, J = 11.0 Hz, HA-28), 4.27 (1H, d, J = 11.0 Hz, HX-28), 4.60 (1H, s HA-29), 4.70 (1H, s, HB-29); 13C NMR (CDCl3, 100 MHz) δ 14.8 (CH3, C-27), 15.4 (CH3, C-24), 16.1 (CH3, C-26), 16.1 (CH3, C-25), 18.3 (CH2, C-6), 19.2 (CH3, C-30), 20.8 (CH2, C-11), 23.6 (CH2, OC(O)CH2CH2), 25.2 (CH2, C-12), 27.1 (CH2, C-15), 27.4 (CH2, C-2), 28.0 (CH3, C-23), 29.6 (CH2, C-16), 29.9 (CH2, C21), 32.1 (CH2CH2Br), 32.9 (OC(O)CH2), 33.5 (CH2Br), 34.2 (CH2, C-22), 34.6 (CH2, C-7), 37.2 (C, C-10), 37.6 (CH, C-13), 38.7 (CH2, C-1), 38.9 (C, C-4), 40.9 (C, C-8), 42.7 (C, C-14), 46.4 (C, C-17), 47.7 (CH, C-18), 48.8 (CH, C-19), 50.4 (CH, C-9), 55.3 (CH, C-5), 62.8 (CH2, C-28), 79.0 (CH, C-3), 109.9 (CH2, C-29), 150.1 (C, C20), 173.5 (C, CO); anal. C 68.8, H 8.9, Br 13.1%, calcd for C35H57BrO3, C 69.4, H 9.48, Br 13.19%. General Procedure for the Synthesis of Diesters (5−7). To a mixture of betulin (1, 0.88 g, 2 mmol) and ω-bromoalkanoic acids (4.20 mmol) in dry dichlomethane (15 mL) was added a solution of DCC (0.46 g, 4.2 mmol) and DMAP (0.04 g, 0.32 mmol) in dry dichloromethane (10 mL). Stirring at room temperature was continued for 1−2 h. After the reaction was complete, the reaction mixture was filtered and solvent was removed under reduced pressure. The crude product was purified by column chromatography (chloroform−ethanol, 100:1) to give the pure corresponding diesters (5−7). Lup-20(29)-ene-3β,28-diyl bis(2-bromoethanoate) (5): amorphous powder; 1.16 g, 85% yield; mp 165 °C; IR (KBr) νmax 2944, 2871, 1733, 1640, 1452, 1424, 1389, 1375, 1349, 1317, 1277, 1219, 1107, 980, 884 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.88, 0.89, 0.90, 1.00, 1.06 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.70 (3H, s, CH3-30), 2.45 (1H, m, J = 10.8, 5.6 Hz, H-19), 3.81 (1H, d, J = 12.0 Hz, C(O)CH2(A)Br) 3.86 (1H, d, J = 12.0 Hz C(O)CH2(B)Br), 3.87 (2H, s, C(O)CH2Br), 3.96 (1H, d, J = 11.3 Hz, HA-28), 4.40 (1H, d, J = 11.0 Hz, HX-28), 4.55 (1H, m, J = 9.2, 7.0 Hz, H-3), 4.62 (1H, HA-29), 4.71 (1H, HB-29); anal. C 61.0, H 7.8, Br 23.0%, calcd for C34H52Br2O4, C 59.65, H 7.66, Br 23.34%. Lup-20(29)-ene-3β,28-diyl bis(4-bromobutanoate) (6): amorphous powder; 0.88 g, 60% yield; mp 58−60 °C; IR (KBr) νmax 2946, 2871, 1731, 1641, 1455, 1391, 1376, 1307, 1281, 1251, 1202, 1176, 1129, 1008, 980 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.86,

(%): resting phase G0/G1 (59), synthetic phase (10), and growth phase (28). Cell treatment with compounds 1, 7, 10, 12, and 13 was not accompanied by a significant alteration in phase ratios, indicating a lack of ability of these compounds to induce cell cycle blocking or increase the proliferative potential of PC-3 cells in culture. The present results indicate that the modification of betulin with a TPP or moieties can be used to generate betulin derivatives with enhanced cytotoxic effects toward cancer cells including drug-resistant ones.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Boetius compact heating table. Optical rotations were determined on a PerkinElmer 341 polarimeter (concentration c is given as g/mL). The IR spectra were recorded using a Bruker Tenzor 27 spectrometer. The 1H, 13C, and 31P NMR spectra were recorded using a Bruker Avance-400 NMR spectrometer. Solutions of analyte samples in CHCl3 at a concentration of 1 mg/mL were used. Highresolution MALDI mass spectra were acquired in the reflectron mode with Triton X-100 as a reference. Solutions of analyte samples in CHCl3 at a concentration of 1 mg/mL were used. A mixture of Triton X-100 solution and analyte sample (1:1 v/v) was used for internal calibration. 2,5-Dihydroxybenzoic acid (5 mg/mL in methanol) was used as a matrix. Elemental analysis was accomplished with the automated EuroVector EA3000 CHNS-O elemental analyzer (EuroVector, Milano, Italy). The progress of reactions and the purity of products were monitored by TLC on Sorbfil plates (IMID Ltd., Krasnodar, Russian Federation). The TLC plates were visualized by treatment with phosphotungstic acid (solution in ethanol), followed by heating to 120 °C. The targeted compounds were isolated using column chromatography on silica gel (60A, 60−200 μm, Acros, Belgium). All solvents were dried according to standard protocols. MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)], propidium iodide, Triton X100, and tetramethylrhodamine ethyl ester were purchased from Sigma-Aldrich. Vinblastine was obtained from Verofarm-OJSC Moscow. RNase was purchased from Fermentas. Cell culture reagents were obtained from PAA Laboratories. Plant Material. The bark of Betula pendula Roth (Betulaceae) was collected in Kazan, Republic of Tatarstan, Russian Federation, in July− August 2015 and was identified by Assist. Prof. V. N. Poluyanova, Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Russian Federation. A voucher specimen (Bp-0916-1) was deposited in the Herbarium of the Botanical Garden of Kazan (Volga Region) Federal University. Extraction and Isolation. Air-dried and powdered bark of B. pendula (50 g) was extracted with CHCl3 in a Soxhlet extractor for 4 h under reflux. The extract was concentrated under vacuum to yield a residue, which was recrystallized from 2-propanol to provide purified betulin as a white powder (10 g): mp 256−258 °C; [α]20D +10.4 (c 0.65, CHCl3); its spectroscopic data were consistent with the published literature data.59 General Procedure for the Synthesis of Monoesters (2−4). To a mixture of betulin (1, 0.88 g, 2 mmol) and ω-bromoalkanoic acid (2.10 mmol) in dry dichlomethane (12 mL) was added a solution of DCC (0.46 g, 2.2 mmol) and DMAP (0.02 g, 0.16 mmol) in dry dichloromethane (5 mL). Stirring at room temperature was continued for 1−2 h. After the reaction was complete, the reaction mixture was filtered and solvent was removed under reduced pressure. The crude product was purified by column chromatography (chloroform− ethanol, 100:1) to give the pure corresponding monoesters (2−4). 3β-Hydroxylup-20(29)-en-28-yl 2-bromoethanoate (2): amorphous powder; 1.0 g, 90% yield; mp 115 °C; IR (KBr) νmax 3477, 2943, 2870, 1736, 1642, 1455, 1388, 1286, 1175, 1107, 1044, 1014, 995, 983, 885 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.78, 0.84, 0.98, 1.00, 1.05 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26,CH3-27), 0.80− 2.02 (26H, m, betulin scaffold), 1.70 (3H, s, CH3-30), 2.45 (1H, m, J = 10.8, 5.6 Hz, H-19), 3.2 (1H, m, J = 11.0, 5.0 Hz, H-3), 3.87 (2H, s, 2236



Article

C(O)CH2), 3.19 (1H, dd, J = 11.0, 5.0 Hz, H-3), 3.77 (1H, d, J = 11.0 Hz, HA-28), 3.88 (2H, m, CH2P), 4.18 (1H, d, J = 11.0 Hz, HB-28), 4.59 (1H, HA-29), 4.67 (1H, HB-29), 7.65−7.87 (15H, m, H−Car); 13 C NMR (CDCl3, 100 MHz) δ 14.7, 15.3, 16.0, 18.2, 19.1, 20.7, 22.0, 22.6, 24.7, 25.1, 25.4, 27.0, 27.3, 27.9, 29.5, 29.7, 33.2, 33.4, 34.2, 34.5, 37.6, 38.7, 38.8, 40.8, 42.6, 46.3, 47.7, 48.7, 50.3, 55.2, 62.6, 78.9, 109.9, 118.3 (CH, d, J = 85.8 Hz, Cipso), 130.0 (CH, d, J = 13.2 Hz, Cmeta), 133.0 (CH, d, J = 11.0 Hz, Cortho), 135.0 (CH, d, J = 2.9 Hz, Cpara), 150.0, 173.5; 31P NMR (CDCl3, 162 MHz) δ 24.3; MALDIMS m/z 788.03 [M − Br]+; anal. C 73.2, H 8.3, Br 9.25%, calcd for C53H72BrO3P, C 73.3, H 8.36, Br 9.20%. {2,2′-[Lup-20(29)-ene-3β,28-diyldiox]-2,2′-dioxoethyl}hexaphenyldiphosphonium dibromide (11): amorphous powder; 1.1 g, 90% yield; mp 185−190 °C; IR (KBr) νmax 2941, 2871, 1727, 1639, 1587, 1438, 1391, 1318, 1268, 1192, 1110, 997, 978 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.63, 0.66, 0.70, 0.82, 0.85 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.58 (3H, s, CH3-30), 0.79−1.72 (26H, m, betulin scaffold), 2.23 (1H, m, H-19), 3.72 (1H, d, J = 10.9 Hz, HA-28), 4.1 (1H, d, J = 11.1 Hz, HB-28), 4.32 (1H, m, H-3), 4.51 (1H, s, HA-29), 4.58 (1H, s, HB-29), 5.26−5.6 (4H, m, CH2P), 7.64− 7.67 (12H, m, H−Car), 7.74−7.78 (6H, m, H−Car), 7.84−7.90 (12H, m, H−Car); 13C NMR (CDCl3, 100 MHz) δ 14.6, 15.9, 16.0, 16.4, 18.0, 19.0, 20.6, 22.5, 23.2, 26.8, 27.8, 29.3, 33.3, 33.4, 34.0, 34.1, 36.8, 37.5, 37.7, 38.2, 40.7, 42.5, 46.0, 47.4, 48.6, 50.0, 55.2, 65.3, 84.8, 110.0, 117.9 (CH, d, J = 88.8 Hz, Cipso), 118.0 (CH, d, J = 88.8 Hz, Cipso), 130.2 (CH, d, J = 13.2 Hz, Cmeta), 133.9 (CH, d, J = 10.6 Hz, Cortho), 134.0 (CH, d, J = 10.6 Hz, Cortho), 135.2 (CH, d, J = 2.9 Hz, Cpara), 149.6, 164.2, 164.79; 31P NMR (CDCl3, 162 MHz) δ 20.7, 21.0; MALDIMS m/z 1145.3 [M − Br]+, 1065.4 [M − 2Br]+; anal. C 68.6, H 6.8, Br 12.8%, calcd for C71H86Br2O4P2, C 69.6, H 7.08, Br 13.04%. {4,4′-[Lup-20(29)-ene-3β,28-diyldiox])-4,4′-dioxobutyl}hexaphenyldiphosphonium dibromide (12): amorphous powder; 1.06 g, 83% yield; mp 175−180 °C; IR (KBr) νmax 3383, 2944, 2870, 1723, 1640, 1587, 1439, 1390, 1321, 1232, 1184, 1112, 996, 980, 883 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.80, 0.81, 0.84, 0.96, 1.02 (15H, s, H CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.68 (3H, s, CH3-30), 0.88−2.01 (30H, m, betulin scaffold and CH2 fragment of linker), 2.41 (1H, m, H-19), 2.87−2.95 (4H, m, C(O)CH2), 3.83 (1H, d, J = 11.1 Hz, HA-28), 4.01−4.12 (4H, m, CH2P), 4.23 (1H, d, J = 10.6 Hz, HB-28), 4.42−4.46 (1H, m, H-3), 4.59 (1H, s, HA-29), 4.68 (1H, s, HB-29), 7.69−7.74 (12H, m, H−Car), 7.79−7.82 (6H, m, H− Car), 7.89−7.94 (12H, m, H−Car); 13C NMR (CDCl3, 100 MHz) δ 14.0, 14.7, 16.0, 16.1, 16.5, 18.1, 19.1, 22.1, 22.6, 23.8, 25.1, 27.0, 28.1, 29.6, 31.5, 33.3, 33.6, 34.1, 34.5, 34.6, 37.0, 37.6, 37.8, 38.4, 40.9, 42.7, 46.3, 47.7, 48.8, 50.3, 55.5, 66.5, 81.6, 109.8, 118.3 (CH, d, J = 86.2 Hz, Cipso), 130.4 (CH, d, J = 12.4 Hz, Cmeta), 133.9 (CH, d, J = 10.0 Hz, Cortho), 135.0, 150.1, 173.0, 173.8; 31P NMR (CDCl3, 162 MHz) δ 24.5; MALDIMS m/z 1201.0 [M − Br]+; anal. C 69.8, H 7.1, Br 12.2%, calcd for C75H94Br2O4P2, C 70.3, H 7.39, Br 12.47%. {5,5′-[Lup-20(29)-ene-3β,28-diyldioxy]-5,5′-dioxopentyl}hexaphenyldiphosphonium dibromide (13): amorphous powder; 1.04 g, 80% yield; mp 125 °C; IR (KBr) νmax 3370, 2942, 2869, 1723, 1640, 1588, 1438, 1390, 1184, 1113, 1043, 997, 881 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.79, 0.80, 0.83, 0.95, 1.02 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.67 (3H, s, CH3-30), 0.87−1.97 (34H, m, betulin scaffold and (CH2)3 fragment of linker), 2.38−2.41 (1H, m, H-19), 2.85−2.94 (4H, m, C(O)CH2), 3.82 (1H, d, J = 10.4 Hz, HA-28), 4.0−4.11 (4H, m, CH2P), 4.22 (d, J = 11.4 Hz, HB-28), 4.41−4.44 (1H, m, H-3), 4.57 (1H, s, HA-29), 4.67 (1H, s, HB-29), 7.68−7.73 (12H, m, H−Car), 7.78−7.80 (6H, m, H−Car), 7.89−7.92 (12H, m, H−Car); 13C NMR (CDCl3, 100 MHz) δ 14.7, 15.2, 16.0, 16.1, 16.5, 18.1, 19.0, 20.8, 22.0, 22.7, 23.6, 25.1, 27.0, 28.0, 29.5, 29.7, 32.0, 32.9, 33.0, 33.4, 34.1, 34.5, 37.0, 37.5, 37.7, 38.3, 40.8, 42.6, 46.3, 47.7, 48.7, 50.2, 55.3, 65.8, 80.8, 109.8, 118.3 (CH, d, J = 85.8 Hz, Cipso), 130.5 (CH, d, J = 12.4 Hz, Cmeta), 133.7 (CH, d, J = 10.2 Hz, Cortho), 135.0, 150.0, 172.8, 173.5; 31P NMR (CDCl3, 162 MHz) δ 23.8; MALDIMS m/z 1228.6 [M − Br]+, 1148.7 [M − 2Br]+; anal. C 70.8, H 7.3, Br 12.2%, calcd for C77H98Br2O4P2, C 70.6, H 7.54, Br 12.21%.

0.87, 0.99, 1.06 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.70 (3H, s, CH3-30), 2.45 (1H, m, H-19) 2.51 (2H, td, J = 6.7, 1.6 Hz, OC(O)CH2), 2.54 (2H, t, J = 7.2 Hz, OC(O)CH2), 3.48 (2H, t, J = 6.5 Hz, CH2Br), 3.49 (2H, t, J = 6.4 Hz, CH2Br), 3.89 (1H, d, J = 10.8 Hz, HA-28), 4.31 (1H, d, J = 10.8 Hz, HX-28), 4.50 (1H, dd, J = 11.0, 6.4 Hz, H-3), 4.61 (1H, s, HA-29), 4.71 (1H, d, 1.8 Hz, HB-29); anal. C 60.58, H 8.49, Br 21.0%, calcd for C38H60Br2O4, C 61.6, H 8.1, Br 21.6%. Lup-20(29)-ene-3β,28-diyl bis(5-bromopentanoate) (7): amorphous powder; 1.0 g, 70% yield; mp 49−50 °C; IR (KBr) νmax 2940, 2869, 1733, 1628,1454, 1389, 1250, 1193, 1044, 983 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.87, 0.88, 1.00, 1.06 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.70 (3H, s, CH3-30), 2.35 (2H, t, J = 7.16 Hz, OC(O)CH2), 2.38 (2H, t, J = 7.4 Hz, OC(O)CH2), 2.45 (1H, m, J = 11.1, 6.1 Hz, H-19), 3.43 (2H, t, J = 6.7, CH2Br), 3.43 (2H, t, J = 6.6, CH2Br), 3.87 (1H, d, J = 11.1 Hz, HA-28), 4.30 (1H, dd, J = 11.3, 1.3 Hz, HX-28), 4.52 (1H, ddd, J = 9.6, 6.3, 1.4 Hz, H-3), 4.61 (1H, m, HA-29), 4.70 (1H, d, 2.0 Hz, HB-29); anal. C 61.58, H 7.9, Br 20.7%, calcd for C40H64Br2O4, C 62.5, H 8.4, Br 20.8%. General Procedure for the Synthesis of TPP Conjugates of Betulin (8−13). Triphenylphosphine (4−6 mmol) was added to the solution of bromide derivatives (2−4 or 5−7) (1 mmol) in dry acetonitrile under argon, and the mixture was stirred under reflux for 6−8 h. Acetonitrile was removed under reduced pressure, and the precipitate was washed with hot petroleum ether (3 × 5 mL) and dissolved in ethyl acetate. Petroleum ether (5 mL) was added to the reaction mixture. The resulting precipitate was washed with diethyl ether (3 mL) and dried in vacuo to give the pure TPP conjugate. {2-[3β-Hydroxylup-20(29)-en-28-yl-oxy]-2-oxoethyl}triphylphosphonium bromide (8): amorphous powder; 0.78 g, 95% yield; mp 165−170 °C; IR (KBr) νmax 3383, 2941, 2870, 1731, 1641, 1588, 1484, 1439, 1388, 1298, 1249, 1192, 1111, 1046, 997, 920, 883 cm−1; 1 H NMR (CDCl3, 400 MHz) δ 0.73, 0.77, 0.88, 0.89, 0.94 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.60 (3H, s, CH3-30), 0.65−1.74 (26H, m, betulin scaffold), 2.24 (1H, m, H-19), 3.15 (1H, dd, J = 11.1, 4.7 Hz, H-3), 3.75 (1H, d, J = 11.3 Hz, HA-28), 4.13 (1H, d, J = 10.6 Hz, HX-28), 4.5 (1H, s, HA-29), 4.60 (1H, s, HB-29), 5.45− 5.63 (2H, m, CH2P), 7.63−7.68 (6H, m, H−Car), 7.75−7.83 (3H, m, H−Car), 7.87−7.92 (6H, m, H−Car); 13C NMR (CDCl3, 100 MHz) δ 14.6, 15.3, 15.9, 16.0, 18.2, 19.0, 20.6, 25.0, 26.9, 27.3, 27.9, 29.3, 30.8, 33.4, 34.2, 37.0, 37.5, 38.6, 38.8, 40.7, 42.5, 46.0, 47.4, 48.7, 50.2, 55.2, 65.4, 78.8, 110.0, 118.0 (CH, d, J = 88.8 Hz, Cipso), 130.3 (CH, d, J = 13.2 Hz, Cmeta), 134.0 (CH, d, J = 11.0 Hz, Cortho), 135.2 (CH, d, J = 2.9 Hz, Cpara), 149.6, 164.8; 31P NMR (CDCl3, 162 MHz) δ 21.2; MALDIMS m/z 746.03 [M − Br]+; anal. C 72.6, H 8.1, Br 9.6%, calcd for C50H66BrO3P, C 72.71, H 8.05, Br 9.67%. {4-[3β-Hydroxylup-20(29)-en-28-yl-oxy]-4-oxobutyl}triphylphosphonium bromide (9): amorphous powder; 0.81 g, 95% yield; mp 165 °C; IR (KBr) νmax 3397, 2939, 2869, 1727, 1644, 1559, 1439, 1389, 1183, 1113, 1046, 996 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.77, 0.83, 0.98, 0.98, 1.03 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.68 (3H, s, CH3-30), 0.81−1.98 (28H, m, betulin scaffold and (CH2)2 fragment of linker), 2.41 (1H, m), 2.93 (2H, m, C(O)CH2), 3.19 (1H, dd, J = 11, 5.0 Hz, H-3), 3.87 (1H, d, J = 11 Hz, HA-28), 4.11 (2H, m, CH2P), 4.21 (1H, d, J = 11 Hz, HX-28), 4.59 (1H, HA-29), 4.68 (1H, HB-29), 7.68−7.94 (15H, m, H−Car); 13C NMR (CDCl3, 100 MHz) δ 14.7, 15.3, 16.0, 18.1, 19.0, 20.8, 25.2, 27.0, 28.0, 29.6, 34.2, 34.5, 37.1, 37.6, 38.8, 40.9, 42.7, 46.3, 47.7, 48.8, 50.3, 55.3, 63.1, 78.9, 109.7, 118.3 (CH, d, J = 88.8 Hz, Cipso), 130.0 (CH, d, J = 13.2 Hz, Cmeta), 133.0 (CH, d, J = 11.0 Hz, Cortho), 135 (CH, d, J = 2.9 Hz, Cpara), 150.1, 173.8; 31P NMR (CDCl3, 162 MHz) δ 24.4; MALDIMS m/z 774.08 [M − Br]+; anal. C 73.0, H 8.20, Br 9.3% calcd for C52H70BrO3P, C 73.13, H 8.26, Br 9.35%. {5-[3β-Hydroxylup-20(29)-en-28-yloxy]-5-oxopentyl}triphylphosphonium bromide (10): amorphous powder; 0.82 g, 95% yield; mp 130 °C; IR (KBr) νmax 3447, 3329, 2942, 2866, 1733, 1627, 1575, 1455, 1389, 1247, 1192, 1044, 1015, 983 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.76, 0.82, 0.96, 0.97, 1.0 (15H, s, CH3-23, CH3-24, CH3-25, CH3-26, CH3-27), 1.67 (3H, s, CH3-30), 0.89−2.06 (30H, m, betulin scaffold and (CH2)3 fragment of linker), 2.43 (2H, t, J = 7.2 Hz, 2237



Article

Cell Isolation and Culturing. The MCF-7 breast cancer and PC-3 prostate adenocarcinoma human cell lines were obtained from ATCC. MCF-7 and PC-3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. The cells were grown aseptically under standard conditions (37 °C, 5% CO2, humidified air atmosphere). The grown cells were washed with Hank’s balanced salt solution (HBSS) and collected by treating with trypsin−EDTA dissociation solution. Vinblastine-resistant MCF-7 cells (MCF-7/Vinb) were obtained by continuous passaging of MCF-7 cells at gradually increased concentrations of vinblastine. MCF-7/Vinb cells were maintained by culturing them in the presence of 1 nM vinblastine. Postsurgical full-thickness human skin fragments were obtained from the Republican Clinical Hospital (Kazan, Russia). Human skin fibroblasts were used under a protocol approved by the Institutional Ethical Review Board of the Kazan Federal University (Protocol #1, Apr 30, 2015). HSFs were isolated from skin explants according to the procedure60 with some modifications. The explants (about 0.2−0.6 cm2 in area) were collected and kept in HBSS containing antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin) prior to treatment. All manipulations with the samples collected were further performed aseptically in a laminar hood using sterile materials and solutions. The samples were washed with HBSS, placed onto a 10 cm Petri dish precovered with DMEM, and carefully minced with fine scissors into small pieces (∼1 mm3). These skin pieces were evenly spread onto the bottom of a 75 cm culture flask and gently covered with α-MEM containing 20% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. The skin samples were continuously cultured to allow fibroblasts to migrate from the tissues and proliferate until they reached an almost confluent monolayer. HSFs were collected by treating with trypsin−EDTA dissociation solution and used for cytotoxicity study. HSFs were cultured under standard conditions in fully supplemented α-MEM with 10% FBS. Cytotoxicity Study. Cytotoxicity of the compounds was evaluated using an MTT assay performed as follows. Cells were collected from the culture probed flask by treating with trypsin−EDTA solution, then seeded in a 96-well plate at a density of 1000 cells per well in DMEM and cultured overnight. Aqueous compound solutions with a starting concentration of 10 mg/mL were prepared and sterilized using a syringe filter. The culture medium in plate was replaced with a fresh one, and the tested compounds were added to cells. Sterile water was added as a control. Cells were cultured in the presence of compounds in standard conditions for 72 h; then the medium was replaced by the fresh one containing MTT reagent at a concentration of 0.5 mg/mL. Cells were additionally cultured for 3 h to allow them to reduce MTT into a water-insoluble product (formazan) followed by discarding the medium and formazan dissolution with 100 μL of DMSO per well. The absorbance of formazan solution in each well is proportional to the number of viable cells and was measured using an Infinite M200 PRO microplate analyzer (Tecan) at a wavelength of 555 nm. Cell viability was calculated as a percentage of control cells grown without compounds (100% viability). Mitochondrial Potential and Cell Cycle Analysis. Changes in transmembrane potential of mitochondria and cell cycle were assessed by a flow cytometry technique using TMRE and propidium iodide fluorescent probes, respectively. Briefly, cells were seeded in six-well plates at the density of 100 000 cells per well and the next day were treated with tested compounds at concentrations equivalent to IC50 for 48 h. Treated cells were collected by trypsinization and washed with phosphate-buffered saline (PBS) by means of centrifugation. The resulting cell suspension (1 × 106 cells/mL) was supplemented with 100 nM TMRE and incubated for 30 min in the dark, prior to analysis. TMRE fluorescence intensity in treated cells was detected on a Guava EasyCyte flow cytometer (Merck Millipore) in the yellow channel as a measure of mitochondrial potential. For cell cycle analysis, cells were seeded and cultured with test compounds as described above. Cells were collected, washed in PBS, and fixed in 70% ethanol in an ice bath for 2 h. Fixed cells were treated

with 0.1 mg/mL RNase solution in the presence of 0.5% Triton X100, then washed and stained with 10 μg/mL propidium iodide. Propidium iodide fluorescence in fixed cells was quantified on a Guava EasyCyte flow cytometer using the red channel.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00105. 1 H NMR spectra of compounds 2−13; 13C{1H} NMR spectra of compounds 2−4, 8−13; 31P NMR spectra of compounds 8−13; IR spectra of compounds 2−13; inhibitory concentrations of compounds 2−7 for PC-3, MCF-7, MCF-7/Vinb, and HSF cells in vitro (MTT assay) (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +7 (843) 272 73 84. Fax: +7 (843) 273-18-72. E-mail: a. [email protected]. ORCID

Andrey V. Nemtarev: 0000-0001-8478-2705 Liliya E. Ziganshina: 0000-0003-1999-0705 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Ministry of Education and Science of the Russian Federation and was funded by a subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities and also is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University.

■

REFERENCES

(1) Connolly, J. D.; Hill, R. A. Nat. Prod. Rep. 2007, 24, 465−486. (2) Hata, K.; Hori, K.; Takahashi, S. J. Nat. Prod. 2002, 65, 645−648. (3) Laszczyk, M. N. Planta Med. 2009, 75, 1549−1560. (4) Mullauer, F. B.; Kessler, J. H.; Medema, J. P. Apoptosis 2009, 14, 191−202. (5) Chen, J. Y.; Zhang, L.; Zhang, H.; Su, L.; Qin, L. P. Phytother. Res. 2014, 28, 1468−1478. (6) Liu, J.; Wu, N.; Ma, L. N.; Zhong, J. T.; Liu, G.; Zheng, L. H.; Lin, X. K. Asian Pac. J. Cancer Prev. 2014, 15, 4519−4525. (7) Balaban, R. S.; Nemoto, S.; Finkel, T. Cell 2005, 120, 483−495. (8) Murphy, M. P. Biochem. J. 2009, 417, 1−13. (9) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 7915−7922. (10) Beckman, K. B.; Ames, B. N. Physiol. Rev. 1998, 78, 547−581. (11) Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen, T.; Savolainen, J. Nat. Rev. Drug Discovery 2008, 7, 255−270. (12) Huttunen, K. M.; Raunio, H.; Rautio, J. Pharmacol. Rev. 2011, 63, 750−771. (13) Dai, L.; Li, D.; Cheng, J.; Liu, J.; Deng, L. H.; Wang, L. Y.; Lei, J. D.; He, J. Polym. Chem. 2014, 5, 5775−5783. (14) Murphy, M. P. Trends Biotechnol. 1997, 15, 326−330. (15) Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cochemé, H. M.; Filipovska, A.; Da Ros, T.; Hurd, T. R.; Smith, R. A.; Murphy, M. P. Biochemistry 2005, 70, 222−230. (16) Honig, B. H.; Hubbell, W. L.; Flewelling, R. F. Annu. Rev. Biophys. Biophys. Chem. 1986, 15, 163−193. (17) Ono, A.; Miyauchi, S.; Demura, M.; Asakura, T.; Kamo, N. Biochemistry 1994, 33, 4312−4318. 2238



Article

(18) Asin-Cayuela, J.; Manas, A. R.; James, A. M.; Smith, R. A.; Murphy, M. P. FEBS Lett. 2004, 571, 9−16. (19) Smith, R. A.; Porteous, C. M.; Coulter, C. V.; Murphy, M. P. Eur. J. Biochem. 1999, 263, 709−716. (20) Kelso, G. F.; Porteous, C. M.; Coulter, C. V.; Hughes, G.; Porteous, W. K.; Ledgerwood, E. C. J. Biol. Chem. 2001, 276, 4588− 4596. (21) Filipovska, A.; Kelso, G. F.; Brown, S. E.; Beer, S. M.; Smith, R. A.; Murphy, M. P. J. Biol. Chem. 2005, 280, 24113−24126. (22) Murphy, M. P.; Echtay, K. S.; Blaikie, F. H.; Asin-Cayuela, J.; Cochemé, H. M.; Green, K. J. Biol. Chem. 2003, 278, 48534−48545. (23) Quin, C.; Trnka, J.; Hay, A.; Murphy, M. P.; Hartley, R. C. Tetrahedron 2009, 65, 8154−8160. (24) Robinson, K. M.; Janes, M. S.; Pehar, M.; Monette, J. S.; Ross, M. F.; Hagen, T. M. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15038− 15043. (25) Cochemé, H. M.; Quin, C.; McQuaker, S. J.; Cabreiro, F.; Logan, A.; Prime, T. A. Cell Metab. 2011, 13, 340−350. (26) Chen, L. B. Annu. Rev. Cell Biol. 1988, 4, 155−181. (27) Rideout, D. C.; Calogeropoulou, T.; Jaworski, J. S.; Dagnino, R. J.; McCarthy, M. R. Anti-Cancer Drug Des. 1989, 4, 265−280. (28) Modica-Napolitano, J. S.; Singh, K. K. Expert Rev. Mol. Med. 2002, 4, 1−19. (29) Manetta, A.; Gamboa, G.; Nasseri, A.; Podnos, Y. D.; Emma, D.; Dorion, G.; Rawlings, L.; Carpenter, P. M.; Bustamante, A.; Patel, J.; Rideout, D. Gynecol. Oncol. 1996, 60, 203−212. (30) Rideout, D.; Bustamante, A.; Patel, J. Int. J. Cancer 1994, 57, 247−253. (31) Weissig, V.; Torchilin, V. P. Adv. Drug Delivery Rev. 2001, 49, 127−149. (32) Murphy, M. P.; Smith, R. A. J. Adv. Drug Delivery Rev. 2000, 41, 235−250. (33) Serafim, T. L.; Carvalho, F. S.; Bernardo, T. C.; Pereira, G. C.; Perkins, E.; Holy, J.; Krasutsky, D. A.; Kolomitsyna, O. N.; Krasutsky, P. A.; Oliveira, P. J. Bioorg. Med. Chem. 2014, 22, 6270−6287. (34) Bernardo, T. C.; Cunha-Oliveira, T.; Serafim, T. L.; Holy, J.; Krasutsky, D.; Kolomitsyna, O.; Krasutsky, P.; Moreno, A. M.; Oliveira, P. J. Bioorg. Med. Chem. 2013, 21, 7239−7249. (35) Drag-Zalesinska, M.; Kulbacka, J.; Saczko, J.; Wysocka, T.; Zabel, M.; Surowiak, P.; Drag, M. Bioorg. Med. Chem. Lett. 2009, 19, 4814−4817. (36) Holy, J.; Kolomitsyna, O.; Krasutsky, D.; Oliveira, P. J.; Perkins, E.; Krasutsky, P. A. Bioorg. Med. Chem. 2010, 18, 6080−6088. (37) Biedermann, D.; Eignerova, B.; Hajduch, M.; Sarek, J. Synthesis 2010, 22, 3839−3848. (38) Suresh, C.; Zhao, H.; Gumbs, A.; Chetty, S. C.; Bose, H. S. Bioorg. Med. Chem. Lett. 2012, 22, 1734−1738. (39) Spivak, A. Yu.; Nedopekina, D. A.; Shakurova, E. R.; Khalitova, R. R.; Gubaidullin, R. R.; Odinokov, V. N.; Dzhemilev, U. M.; Bel’skii, Yu. I.; Bel’skaya, I. V.; Stankevich, S. A.; Korotkaya, E. V.; Khazanov, V. A. Russ. Chem. Bull. 2013, 62, 188−198. (40) Król, S. K.; Kiełbus, M.; Rivero-Müller, A.; Stepulak, A. BioMed Res. Int. 2015, 2015, 1−11. (41) Flekhter, O. B.; Medvedeva, N. I.; Tret’yakova, E. V.; Galin, F. Z.; Tolstikov, G. A. Chem. Nat. Compd. 2006, 42, 706−709. (42) Petrenko, N. I.; Elantseva, N. V.; Petukhova, V. Z.; Shakirov, M. M.; Shul’ts, E. E.; Tolstikov, G. A. Chem. Nat. Compd. 2002, 38, 331− 339. (43) Kvasnica, M.; Sarek, J.; Klinotova, E.; Dzubak, P.; Hajduch, M. Bioorg. Med. Chem. 2005, 13, 3447−3454. (44) Fotie, J.; Bohle, D. S.; Leimanis, M. L.; Georges, E.; Rukunga, G.; Nkengfack, A. E. J. Nat. Prod. 2006, 69, 62−67. (45) Drag-Zalesinska, M.; Kulbacka, J.; Saczko, J.; Wysocka, T.; Zabel, M.; Surowiak, P.; Drag, M. Bioorg. Med. Chem. Lett. 2009, 19, 4814−4817. (46) Santos, R. C.; Salvador, J. A. R.; Marin, S.; Cascante, M. Bioorg. Med. Chem. 2009, 17, 6241−6250. (47) Ahmad, F. B. H.; Moghaddam, M. G.; Basri, M.; Rahman, M. B. A. Biosci., Biotechnol., Biochem. 2010, 74, 1025−1029.

(48) Kashiwada, Y.; Sekiya, M.; Ikeshiro, Y.; Fujioka, T.; Kilgore, N.; Wild, C.; Allaway, G.; Lee, K.-H. Bioorg. Med. Chem. Lett. 2004, 14, 5851−5853. (49) Amjad, M.; Carlson, R.; Krasutsky, P.; Karim, M. J. Microbiol. Biotechnol. 2004, 14, 1086−1088. (50) Flekhter, O. B.; Karachurina, L. T.; Poroikov, V. V.; Nigmatullina, L. R.; Baltina, L. A.; Zrudii, F. S.; Davydova, V. A.; Spirikhin, L. V.; Baikova, I. P.; Galin, F. Z.; Tolstikov, G. A. Russ. J. Bioorg. Chem. 2000, 26, 192−200. (51) Alakurtti, S.; Mäkelä, T.; Koskimies, S.; Yli-Kauhaluoma, J. Eur. J. Pharm. Sci. 2006, 29 (1), 1−13. (52) Kommera, H.; Kaluderovic, G.; Dittrich, S.; Kalbitz, J.; Dräger, B.; Mueller, T.; Paschke, R. Bioorg. Med. Chem. Lett. 2010, 20, 3409− 3412. (53) Zhang, D.-M.; Xu, H.-G.; Wang, L.; Li, Y.-J.; Sun, P.-H.; Wu, X.M.; Wang, G.-J.; Chen, W.-M.; Ye, W.-C. Med. Res. Rev. 2015, 35, 1127−1155. (54) Kommera, H.; Kaluđerović, G. N.; Kalbitz, J.; Paschke, R. Invest. New Drugs 2011, 29, 266−272. (55) Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 522−524. (56) Patra, A.; Chaudhuri, S. K.; Panda, S. K. J. Nat. Prod. 1988, 51, 217−220. (57) Schraml, J.; Č apka, M.; Blechta, V. Magn. Reson. Chem. 1992, 30, 544−547. (58) Johnson, I.; Spence, M. T. Z., Eds. The Molecular Probes Handbook. A Guide to Fluorescent Probes and Labeling Technologies, 11th ed.; Life Technologies Corporation: Carlsbad, CA, 2010. (59) Green, B.; Bentley, M. D.; Chung, B. Y.; Lynch, N. G.; Jensen, B. L. J. Chem. Educ. 2007, 84, 1985−1987. (60) Rittie, L.; Fisher, G. J. Fibrosis Res.: Methods Protoc. 2005, 117, 83−98.

2239


Design, Synthesis, and Cancer Cell Growth Inhibitory Activity of

Recommend Documents