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RESEARCH ARTICLE View Journal | View Issue

Mitochondria-targeted betulinic and ursolic acid

derivatives: synthesis and anticancer activity†
Open Access Article. Published on 13 September 2017. Downloaded on 08/06/2018 10:16:21.

Cite this: Med. Chem. Commun.,

2017, 8, 1934
Darya A. Nedopekina, a Rinat R. Gubaidullin,a Victor N. Odinokov, a

Polina V. Maximchik,b Boris Zhivotovsky, bc Yuriy P. Bel'skii,d

Veniamin A. Khazanov,d Arina V. Manuylova,d
Vladimir Gogvadze*bc and Anna Yu. Spivak *a

A series of new betulinic and ursolic acid conjugates with a lipophilic triphenylphosphonium cation, meant to
enhance the bioavailability and mitochondriotropic action of natural triterpenes, have been synthesized. The
in vitro experiments on three human cancer cell lines (MCF-7, HCT-116 and TET21N) revealed that all the
obtained triphenylphosphonium triterpene acid derivatives not only showed higher cytotoxicity as compared to
betulinic acid but were also markedly superior in triggering mitochondria-dependent apoptosis, as assessed
Received 15th May 2017, using a range of apoptosis markers such as cytochrome c release, stimulation of caspase-3 activity, and cleav-
Accepted 17th August 2017
age of polyIJADP-ribose) polymerase, which is one of the targets of caspase 3. The IC50 was much lower for all
triphenylphosphonium derivatives when compared to betulinic acid. Out of the tested group of conjugates, the
DOI: 10.1039/c7md00248c
most potent toxicity was exhibited by the betulinic acid conjugate 9 (for 9, the IC50 values against MCF-7 and
rsc.li/medchemcomm TET21N cells were 0.70 μM and 0.74 μM; for betulinic acid (BA), IC50 > 25 μM against MCF-7 cells).

1. Introduction ing cytochrome c) from the intermembrane space of mito-

chondria are regarded as key events in apoptosis induction.
Apoptosis is an evolutionarily conserved and genetically regu- Once in the cytosol, cytochrome c interacts with its adaptor
lated process of critical importance for embryonic develop- molecule, Apaf-1, resulting in the recruitment, processing
ment and maintenance of tissue homeostasis in the adult or- and activation of pro-caspase-9. Active caspase-9, in turn,
ganism.1 Apoptosis and cancer are antagonistic processes in cleaves and activates pro-caspase-3 and -7; these effector
cell physiology. Stimulation of apoptosis is responsible for caspases are responsible for the cleavage of cellular proteins
the elimination of potentially harmful or premalignant cells, leading to biochemical and morphological characteristic fea-
whereas suppression of apoptotic pathways can lead to tures of apoptosis. Other proteins released from the inter-
uncontrolled cell proliferation and formation of malignancies. membrane space of mitochondria are the Apoptosis Inducing
Thus, searching for compounds that can specifically stimulate Factor (AIF), Smac/Diablo and Omi. AIF and the serine prote-
tumor cell death is very important for developing antitumor ase Omi catalyze caspase-independent downstream events in
strategies.2,3 the apoptotic process. Therefore, permeabilization of the
Although it appears that distinct pathways leading to cell outer mitochondrial membrane (OMM) is considered a cru-
death are triggered by different signals, they often merge at a cial event during the early phase of the apoptotic process. It
common “regulator” of this multistep process – mitochon- should be mentioned that caspase-8 cleaves not only caspase-
dria. Specifically, permeabilization of the outer mitochondrial 3 but also Bid, a cytosolic protein and a proapoptotic mem-
membrane (OMM) and the release of certain proteins (includ- ber of Bcl-2 family proteins.4 Truncated Bid (tBid)
oligomerizes another proapoptotic protein, Bax, which causes
a
permeabilization of the OMM and cytochrome c release. The
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences, 141
prosp. Oktyabrya, Ufa 450075, Russian Federation. E-mail: [email protected]
pore-forming capacity of Bax and subsequent release of cyto-
b
Faculty of Fundamental Medicine, MV Lomonosov Moscow State University, chrome c can be prevented by overexpression of Bcl-2. The
11999 Moscow, Russia. E-mail: [email protected] overexpression of Bcl-2 and Bcl-XL counteracts OMM perme-
c
Division of Toxicology, Institute of Environmental Medicine, Karolinska abilization and contributes to drug resistance in high-risk NB
Institutet, Box 210, 17177 Stockholm, Sweden
d
cells.5 Another mode of OMM permeabilization is stimulation
Innovative Pharmacology Research (IPHAR), 79/4 Elizarova, Tomsk 634021,
Russian Federation
of the so-called mitochondrial permeability transition, a
† Electronic supplementary information (ESI) available. See DOI: 10.1039/ Ca2+-dependent process which results in mitochondrial swell-
c7md00248c ing and OMM rupture. Targeting of mitochondria and OMM

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permeabilization via induction of mitochondrial permeability triterpenoid skeleton through the hydrophobic n-butyl or hy-
transition (MPT) might be powerful tool against protective drophilic triethylene glycol spacer. In an attempt to enhance
effects of Bcl-2 and Bcl-XL.5–7 the antitumor activity of triphenylphosphonium salts, we
Pentacyclic lupane and ursane type triterpenoids (betulin, linked the 3β-OH group in compounds 6, 10, 14, and 16 with
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betulinic and ursolic acids) are among the most abundant a molecule of dichloroacetate, a known pyruvate dehydroge-
terpenoids found in the plant kingdom. These compounds nase kinase (PDK) inhibitor.30 Inhibition of PDK stimulates
are of interest for pharmacological research, as they exhibit a the activity of pyruvate dehydrogenase (PDH) facilitating oxi-
broad spectrum of biological properties.8–13 Among these dation of pyruvate in mitochondria. Dichloroacetate was
properties of triterpenoids, of special interest is their antican- shown to reduce high mitochondrial membrane potential in
cer activity and the ability to trigger the mitochondrial apo- tumor cells and to increase mitochondrial ROS production in
Open Access Article. Published on 13 September 2017. Downloaded on 08/06/2018 10:16:21.

ptosis pathway in various types of human cancer cells. Thus, malignant but not in normal cells.31 Previously,32 it has been
betulinic acid is capable of inducing apoptosis in tumor cells shown that dichloroacetate appended to the C-3 hydroxy
of certain types such as melanoma, lung cancer, ovarian can- group of betulinic acid provided a synergistic cytotoxic effect
cer and neuroectodermal tumors.14–16 Betulinic acid stimu- against a broad spectrum of cancer cells.
lates apoptosis with the participation of reactive oxygen spe- The betulinic and ursolic acid triphenylphosphonium salts
cies (ROS). ROS contribute to permeabilization of the outer 9 and 15 containing a triethylene glycol bridge were
mitochondrial membrane (OMM), releasing apoptogenic mi- obtained by alkylation of the 28-carboxyl group of the
tochondrial proteins (cytochrome c, Smac, apoptosis induc- triterpenoids with triethylene glycol dibromide in DMF in the
ing factor (AIF)) from the intermembrane space, and subse- presence of K2CO3 at 50 °C for 3 h. The dibromides were syn-
quent activation of the caspase cascade.14–18 thesized from triethylene glycol ditosylate33 by treatment with
In vitro, ursolic acid was shown to inhibit the proliferation LiBr in boiling acetone for 14 hours. In the synthesis of
of various cancer cell types by suppressing the STAT3 activa- triterpene acid conjugates 6 and 14, the C(28)-carboxyl group
tion pathway.19,20 It can also induce apoptosis, autophagy, was alkylated with a twofold excess of 1,4-dibromobutane in
and cell cycle arrest through various pathways, such as inhi- DMF and acetonitrile in the presence of K2CO3 at 50 °C. The
bition of DNA replication, stimulation of reactive oxygen spe- bromides thus obtained were refluxed with a fivefold molar
cies (ROS) production, and affecting the balance between excess of triphenylphosphine in acetonitrile. The phospho-
pro- and antiapoptotic proteins.21–23 nium salts with 3-dichloroacetates 6, 10, 14, and 16 were pre-
The special attractiveness of these natural compounds is pared by esterification of the C(3)-hydroxyl in the triterpenoid
due to the absence of their cytotoxic action towards normal skeleton with dichloroacetic acid using the carbodiimide
human cells (fibroblasts or proliferating normal lymphocytes) method (Schemes 1 and 2).
and negligible systemic toxicity in in vivo experiments, using
xenograft models.24–27 However, the relatively low anticancer
potential, high hydrophobicity, and poor blood serum solubi- 2.2. Biological evaluation
lization of triterpene acids markedly hamper their advance- 2.2.1. Cytotoxicity of BA- and UA-derivatives. The cytotoxic
ment as anticancer drug candidates. activity was studied for all of the compounds against two hu-
Previously, we synthesized conjugates of lupane man cancer cell lines: MCF-7 (breast adenocarcinoma) and
triterpenoids with a triphenylphosphonium (TPP+) group, TET21N (neuroblastoma), as well as normal mouse
which showed cytotoxicity in vitro at low micromolar concen- splenocytes, using the MTT test. The results are shown in
trations and significantly exceeded the antitumor activity of Fig. 1 and in Table 1.
betulinic acid.28,29 In this paper, we discuss the results of in- High cytotoxic activity against each of the cell lines was
vestigations into probable mechanisms underlying the cyto- found for the betulinic acid conjugate 9 (IC50 values of 0.70–
toxic effect of triphenylphosphonium cations of triterpenoids. 0.74 μM). A considerable activity (<1 μM) was also inherent
We have synthesized a new series of triphenylphosphonium in the triphenylphosphonium salts of lupane triterpenoids
salts of betulinic and ursolic acids, studied their cytotoxic ac- with an acetate or a dichloroacetate function at the
tivity in two human cancer cell lines and their ability to in- triterpenoid's C-3 atom: 6 and 7 against MCF-7 (IC50 values
duce programmed cancer cell death, using markers of apo- of 0.80 μM and 0.85 μM) and 7, 10 and 15 against TET21N
ptosis such as activation of caspase-3, PARP-1 cleavage, (IC50 values of 0.98 μM, 0.95 μM, and 0.81 μM). Contrary to
permeabilization of the outer mitochondrial membrane, re- our expectations, the conjugation of the lupane and ursane
lease of cytochrome c and inhibition of the mitochondrial re- triphenylphosphonium salts with dichloroacetate did not
spiratory chain. bring about a synergistic cytotoxicity. The cytotoxic activity of
conjugate 7 against TET21N cells was comparable with that
2. Results and discussion of conjugate 10, while the activity of 9 was even somewhat
higher than that of 10 (Table 1). We did not find any notice-
2.1. Chemistry able difference between the anticancer activities of n-butyl-
The triphenylphosphonium group was linked to a molecule and triethylene glycol-bridged conjugates. The triterpenoid
of betulinic or ursolic acid at the C-28 position of the skeleton structure had a pronounced effect on the cytotoxicity

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Scheme 1 Synthesis of betulinic acid derivatives. Reagent and conditions: a 1,4-dibromobutane, K2CO3, MeCN, DMF, 50 °C, 3h; b PPh3, CH3CN,
reflux, Ar, 20–35 h; c Cl2COOH, DMAP, DCC, CH2Cl2, 2–7 h, rt; d AcCI, Py, DMAP, THF, 20 °C; e tri(ethylene glycol) dibromide, K2CO3, DMF, 50 °C,
3 h; f tri(ethylene glycol) ditosylate, K2CO3, MeCN, 70 °C, 7 h.

of phosphonium salts. The antitumor activity of phospho- the IC50 values were 4.91 μM and 15.89 μM for BA-DCA and
nium lupane triterpenoid derivatives was markedly higher >10 μM and >20 μM for UA-DCA, respectively.
than the activities of the corresponding ursane derivatives,
14, 15 and 16. Betulinic acid and C(3)-dichloroacetyl betulinic
and ursolic acid derivatives, BA-DCA and UA-DCA, exhibited
markedly lower cytotoxicity than any of the triphenylphosph-
onium salts. At the concentrations we tested, these com-
pounds were inactive: in the case of TET21N and MCF-7 cells,

Scheme 2 Synthesis of ursolic acid derivatives. Reagents and Fig. 1 Dose-dependent cytotoxic action of triphenylphosphonium
conditions: a 1,4-dibromobutane, K2CO3, MeCN, DMF, 50 °C, 3 h; b salts of betulinic and ursolic acids on TET21N (neuroblastoma) (A) and
PPh3, CH3CN, reflux, Ar, 20–35 h; c Cl2COOH, DMAP, DCC, CH2Cl2, 2– MCF-7 (breast adenocarcinoma) (B) cells. The results are expressed as
7 h, rt; d tri(ethylene glycol) dibromide, K2CO3, DMF, 50 °C, 3 h. the percentage of viable cells.

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Table 1 Cytotoxic activity IC50* of the triphenylphosphonium betulinic

and ursolic acid derivatives against the cancer cells TET21N and MCF-7

Test compound TET21N MCF-7

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6 1.26 ± 0.18 0.80 ± 0.08

7 0.98 ± 0.11 0.85 ± 0.09
8 1.28 ± 0.18 1.51 ± 0.13
9 0.74 ± 0.14 0.70 ± 0.11
10 0.95 ± 0.15 2.31 ± 0.09
14 4.4 ± 0.34 2.76 ± 0.21
15 0.81 ± 0.08 1.59 ± 0.11
16 1.18 ± 0.16 3.90 ± 0.06
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BA-DCA 4.9 ± 0.2 15.89 ± 0.19

UA-DCA >10 >20
BA — >25

Note: *IC50 (μM) is the half maximal inhibitory concentration for

viable cells. Each IC50 (mean ± SE) has been derived from at least
three experiments in duplicate.

However, the cytotoxic activity of the newly obtained

triphenylphosphonium salts was comparable to their cyto-
Fig. 2 Assessment of oxygen consumption by HCT116 (human colon
toxic activity against normal mouse splenocytes. Thus the
carcinoma) cancer cells after incubation with betulinic acid and its
IC50 values were 1.95 μM, 0.66 μM, 1.75 μM and 1.13 μM for derivatives, 7 (upper panel) and 9 (lower panel), for 24 hours.
compounds 6, 10, 14 and 16, respectively.
Because the antitumor activities of betulinic acid deriva-
tives 6–10 were higher than the activities of the correspond-
ing ursane conjugates 14–16, we have further focused on the of many key cellular proteins during apoptosis. Since
ability of BA and its analogues to stimulate cancer cell death. caspase-3 cleaves PARP, it is possible to assess the intensity
2.2.2. Analysis of mitochondrial respiration. Next, consid- of apoptosis by means of the quantity of cleaved 85 kDa
ering that the targets of BA are mitochondria, we analyzed fragments by using western blotting with anti-PARP anti-
their functional activity, in particular oxygen consumption, bodies.34 All cell lines revealed different resistances to BA
upon treatment of cells with BA and its analogs. Evaluation (Fig. 3A). The most sensitive were neuroblastoma Tet21N
was performed using Seahorse Analyzer (Fig. 2). Oligomycin, cells. For apoptosis stimulation in other cell lines, signifi-
an inhibitor of mitochondrial ATP synthetase, a cantly higher concentrations of BA were needed. Based on
protonophore CCCP, which dissipates the mitochondrial these results, working concentrations of BA derivatives were
membrane potential allowing analysis of spare respiratory ca- chosen at which BA had a minimal effect on apoptosis
pacity, and inhibitors of mitochondrial respiratory chain, ro- induction.
tenone (complex I) and antimycin (complex III), were added As has been mentioned in the introduction, the caspase
consequently after assessment of basal respiration. cascade is triggered by the release of cytochrome c. In order
As shown in Fig. 2, the derivatives of BA, 7 (upper panel) to prove that administration of BA and its analogs results in
and 9 (lower panel), suppressed respiration in a OMM permeabilization, assessment of cytochrome c release
concentration-dependent manner. At the same time, the was performed (Fig. 3B). The content of cytochrome c in cyto-
same concentrations of BA were ineffective. These experi- sol and mitochondria was assessed by western blotting after
ments clearly demonstrate mitochondrial involvement in trig- fractionation of HCT116 cells (see 4.2. Biology; 4.2.5.). As can
gering apoptosis by BA triphenylphosphonium derivatives. be seen from the results, at 3.5 μM concentration BA failed to
2.2.3. Analysis of antitumor activity of BA derivatives. trigger the release of cytochrome c, whereas the
Since various cell lines can respond to treatment differently, triphenylphosphonium derivatives of BA were significantly
first we analyzed how the cell lines used in our experiments more effective. These experiments clearly demonstrate mito-
respond to BA. This is important for choosing working con- chondrial involvement in triggering apoptosis by BA
centrations allowing demonstration of the advantage of BA derivatives.
derivatives. Apoptosis was assessed by cleavage of polyIJADP- Next, we analyzed the ability of BA and its derivatives to
ribose)polymerase (PARP) – a nuclear enzyme involved in stimulate caspase-3 activity in HCT116 and TET21 cells.
DNA repair and a well-known caspase-3 target. Caspases are DEVD-AMC was used as a fluorogenic substrate for caspase-3.
a family of proteins, which are important for maintaining In agreement with previous data, the results revealed that the
homeostasis through regulating cell death and inflamma- triphenylphosphonium salt of betulinic acid 9 considerably
tion. Among them, caspase-3 is one of the crucial mediators surpassed betulinic acid in the caspase-3 activation (Fig. 3C).
of programmed cell death, catalyzing the specific cleavage In addition, the advantage of the derivatives of BA was

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Fig. 4 Apoptosis stimulated by betulinic acid (BA) and its derivatives in

HCT116 carcinoma cells, shown as the percentage of cells with a
reduced amount of DNA (SubG1 population, left part of each
quadrant).

Finally, the antitumor activity of BA and its derivatives

was analyzed by the assessment of cells with decreased
DNA content. During apoptosis, DNA is cleaved by
caspase-activated DNases (CAD), with formation of small
fragments. The fragmented 182 bp DNA multimers leak
out of the apoptotic cell upon fixation. This results in
the appearance of a subpopulation of cells with a re-
duced DNA content, which can be detected upon staining
with propidium iodide. An assessment of the SubG1 pop-
ulation in HCT116 cells is demonstrated in Fig. 4. BA
did not stimulate cell death (second quadrant in the sec-
ond row), whereas its several triphenylphosphonium deriv-
Fig. 3 Stimulation of apoptosis (A) by BA in various tumor cell lines: atives, such as 6–9, triggered apoptosis. Thus, the advan-
HCT WT (human colon carcinoma cell line and RKO (human rectal tage of triphenylphosphonium derivatives of BA in cell
carcinoma cell line, TET21N and SK-N-BE (2) (neuroblastoma cell
death stimulation was confirmed by a number of
lines); (B) release of cytochrome c from the mitochondria to the cyto-
sol under the action of betulinic acid (BA) and its derivatives with a
parameters.
concentration of 3.5 μM; (C) activation of caspase-3 in HCT and
TET21N cells by betulinic acid (BA) and its derivatives, 9 and BA-DCA.
The mean values of three experiments ± standard error are given. The
statistical significance of the differences was estimated using the Stu-
3. Conclusions
dent's t-test, *<0.05, **<0.01; (D) cleavage of PARP by betulinic and
ursolic acid derivatives. The assay was based on determining the con-
A range of anticancer agents named mitocans (an abbrevia-
tent of the PARP cleavage product (cl-PARP). Glyceraldehyde 3-phos- tion formed from MITOchondria and CANcer) were shown to
phate dehydrogenase (GAPDH) was used as the blot loading control. cause cell death via targeting mitochondria.35 Thus, one of
the mitocans, D-α-tocopherol succinate, a redox-silent ana-
logue of vitamin E (α-TOS), induces multiple changes in tu-
demonstrated by their ability to cleave PARP (Fig. 3D). The re- mor cells leading to cell death.36 This compound was shown
sults clearly show that the antitumor activity of the original to selectively kill malignant cells at concentrations which are
BA is markedly lower than that of some of its derivatives. non-toxic to normal cells. This faculty of α-TOS was explained

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by its ability to produce ROS (more toxic to malignant cells and on a Bruker Avance-400 spectrometer (1H, 400.13 MHz;
13
due to lower antioxidant activity) via interaction with the re- C, 100.62 MHz; 31P, 161.98 MHz) using Me4Si as the inter-
spiratory chain of mitochondria. Non-malignant cells are nal standard and CDCl3 as the solvent. Mass spectra were
protected from α-TOS due to higher levels of esterases which recorded on a Bruker-Autoflex III instrument in the MALDI-
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cleave α-TOS with formation of succinate and an antioxidant TOF regime with positive ionization recording using 2,5-
– α-tocopherol. In the case of BA, the mechanisms might be dihydroxybenzoic and α-cyano-4-hydroxycinnamic acids as ma-
different, although mitochondrial involvement in cell death trices. Optical rotation was measured using a Perkin-Elmer-
execution has been demonstrated by several groups.14–17 141 polarimeter. Elemental analysis was performed using a
Recently, conjugation with a lipophilic mitochondriotropic Carlo Erba 1106 analyzer. Sorbfil plates (Sorbpolimer, Kras-
triphenylphosphonium cation (TPP+) has been actively used nodar, Russia) were used for TLC, visualized with anis-
Open Access Article. Published on 13 September 2017. Downloaded on 08/06/2018 10:16:21.

to enhance the effectiveness and selectivity of mitochondria- aldehyde/sulfuric acid. Silica gel L (50–160 lm, KSKG) was
targeted anti-cancer agents.37,38 Making a compound posi- used for column chromatography.
tively charged by coupling with TPP+ bearing three positive 4.1.1. General procedure for the synthesis of bromides 2, 4
charges enhances mitochondrial targeting and allows de- and 12. Lithium bromide (2.00 g, 23.1 mmol) was added to a
creasing doses of the drug. The triphenylphosphonium moi- stirred solution of triethylene glycol ditosylate (4.00 g, 8.70
ety was used to enhance the bioavailability and cytotoxic ac- mmol) in acetone (40 mL), and the mixture was refluxed for
tivity against cancer cells of such compounds as α-tocopheryl 14–16 h. The resulting precipitate was filtered off, and ace-
succinate,39 curcuminoids,40 alkyl gallates41 and the tone was removed under reduced pressure. The separated oily
diterpenoid isosteviol.42 Selective accumulation of lipophilic syrup was purified by column flash chromatography on silica
cationic compounds in mitochondria is associated with the gel [petroleum ether/ethyl acetate, 30 : 1] to afford dibromide.
relatively high transmembrane potential of these organelles Triethylene glycol dibromide (3 mmol) was added to a stirred
(Δψ 150–180 mV) in comparison with other organelles and suspension of BA or UA (1 mmol) in DMF (5 mL) and K2CO3
cells. In addition, the transmembrane potential of mitochon- (1 mmol), and the mixture was kept at 50 °C for 3 h [TLC
dria of solid tumor cells is higher than the transmembrane monitoring hexane/EtOAc 1 : 1]. The mixture was then poured
potential of normal cells, which can contribute to the selec- into cold H2O (10× volume) and extracted with CHCl3 (2 × 15
tive cytotoxicity of antitumour substances. mL each). The combined CHCl3 layers were washed with H2O
Indeed, under the conditions of our experiments, conjuga- (100 mL) and concentrated. The crude residue was co-
tion of native triterpenoids, betulinic and ursolic acids with a evaporated with hexane (100 mL) to remove residual DMF.
TPP+ group led to the significant enhancement of cytotoxicity The residue was chromatographed on silica gel [hexane/
to MCF-7 and TET-21N cancer cells, when compared to BA. EtOAc, 30 : 1], to obtain a pure compound.
The mitochondriotropic analogues of BA were markedly supe- 8′-Bromo-3′,6′-dioxaoctan-1′-yl-3β-acetoxylup-20IJ29)-en-28-
rior to the native triterpenoid in triggering mitochondria- oate (2). Amorphous powder, 49% yield; [α]21 D +12.42° (c 1.21,
dependent apoptosis, as assessed by a range of apoptosis CHCl3); IR (CHCl3) νmax: 1729 (CO) cm−1; 1H NMR (400
markers, such as cytochrome c release, stimulation of MHz, CDCl3) δ 0.84, 0.85, 0.86, 0.92, 0.97 (3H each, all s, H-
caspase-3 activity and cleavage of polyIJADP-ribose) polymer- 23–H-27), 0.78–2.28 (m, 24H, CH, CH2 in pentacyclic
ase. Thus, structural modifications of BA by conjugation with skeleton), 1.69 (s, 3H, H-30), 2.05 (s, 3H, CH3CO–), 2.96–3.05
TPP+ markedly enhance its anticancer properties. (m, 1H, H-19), 3.47 (t, 2H, J = 6 Hz, H-8′), 3.65–3.73 (m, 6H,
H-2′, H-4′, H-5′), 3.82 (t, 2H, J = 6 Hz, H-7′), 4.24–4.27 (m, 2H,
H-1′), 4.45–4.49 (m, 1H, H-3), 4.61 (s, 1H, H-29), 4.74 (s, 1H,
4. Experimental section H-29); 13C NMR (100 MHz, CDCl3) δ 14.66 (C-27), 16.01 (C-
26), 16.20 (C-25), 16.49 (C-24), 18.18 (C-6), 19.34 (C-30), 20.91
4.1. Chemistry (C-11), 21.33 (CH3CO–), 23.70 (C-12), 25.48 (C-2), 27.94 (C-23),
All reagents and solvents were of the purest grade available 29.62 (C-15), 30.24 (C-8′), 30.56 (C-16), 32.09 (C-21), 34.27 (C-
and generally were used without further treatment. The 7), 37.01 (C-22), 37.10 (C-1), 37.80 (C-4), 38.22 (C-13), 38.40
starting compounds betulinic, ursolic acids and reagents: (C-10), 40.72 (C-8), 42.40 (C-14), 46.98 (C-18), 49.40 (C-19),
LiBr, triethylene glycol, p-toluenesulfonyl chloride, PPh3, 1,4- 50.45 (C-9), 55.42 (C-5), 56.54 (C-17), 62.78 (C-1′), 69.38,
dibromobutane, dimethylaminopyridine (DMAP), 70.50, 70.56, 71.27 (C-2′, C-4′, C-5′, C-7′), 80.91 (C-3), 109.65
dichloroacetic acid, N,N′-dicyclohexylcarbodiimide (DCC) and (C-29), 150.55 (C-20), 170.99 (CH3 C _ O–), 176.00 (C-28); anal.
acetonitrile were purchased from Sigma-Aldrich, Europe. Tri- calcd. for C38H61BrO6: C, 65.78; H, 8.86. Found: C, 65.74; H,
ethylene glycol ditosylate was prepared by a reported 8.84.
method.33 3-Acetylbetulinic and 3-acetylursolic acids were syn- 8′-Bromo-3′,6′-dioxaoctan-1′-yl-3β-hydroxylup-20IJ29)-en-28-
thesized from betulinic and ursolic acids according to the oate (4). White crystals, 61% yield; mp 88–90 °C (EtOH); [α]21 D
typical procedures. IR spectra were recorded on a Specord IR- +5° (c 1.02, CHCl3); IR (CHCl3) νmax: 3447 (–OH), 1723 (CO)
75 spectrometer for neat samples or for solutions in CHCl3. cm−1; 1H NMR (400 MHz, CDCl3) δ 0.82, 0.83, 0.93, 0.93, 0.97
1
H, 13C, and 31P NMR spectra were recorded on a Bruker (3H each, all s, H-23–H-27), 0.77–2.29 (m, 24H, CH, CH2 in
Avance-500 spectrometer (1H, 500.13 MHz; 13C, 125.78 MHz) pentacyclic skeleton), 1.69 (s, 3H, H-30), 2.96–3.56 (m, 1H, H-

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19), 3.14–3.22 (m, 1H, H-3), 3.47–3.48 (m, 2H, H-8′), 3.67–3.72 CH3), 23.69 (C-12), 25.47 (C-2), 27.94 (C-23), 29.60 (C-15),
(m, 6H, H-2′, H-4′, H-5′), 3.81 (t, 2H, J = 6.4 Hz, H-7′), 4.25– 30.56 (C-16), 32.07 (C-21), 34.26 (C-7), 36.98 (C-22), 37.09 (C-
4.27 (m, 2H, H-1′), 4.61 (s, 1H, H-29), 4.74 (s, 1H, H-29); 13C 1), 37.79 (C-4), 38.21 (C-13), 38.39 (C-10), 40.71 (C-8), 42.39
NMR (100 MHz, CDCl3) δ 14.91 (C-27), 15.38 (C-26), 16.02 (C- (C-14), 46.98 (C-18), 49.38 (C-19), 50.44 (C-9), 55.42 (C-5),
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25), 16.14 (C-24), 18.30 (C-6), 19.37 (C-30), 20.90 (C-11), 25.53 56.53 (C-17), 62.72 (C-1′), 68.76, (C-8′), 69.18, 69.32, 70.45,
(C-12), 27.41 (C-2), 27.99 (C-23), 29.64 (C-21), 30.24 (C-8′), 70.78 (C-2′, C-4′, C-5′, C-7′), 80.91 (C-3), 109.64 (C-29), 127.97,
30.61 (C-15), 32.11 (C-16), 34.35 (C-7), 37.02 (C-22), 37.19 (C- 129.82, 133.15, 145.20, (OTs-Ph), 150.42 (C-20), 170.99
1), 38.25 (C-4), 38.72 (C-13), 38.86 (C-10), 40.71 (C-8), 42.41 (CH3C _ O–), 176.02 (C-28); anal. calcd. for C45H68O8S: C, 68.84;
(C-14), 46.96 (C-18), 49.41 (C-19), 50.55 (C-9), 55.35 (C-5), H, 8.73. Found: C, 68.86; H, 8.71.
56.55 (C-17), 62.79 (C-1′), 69.38, 70.50, 70.59, 71.27 (C-2′, C-4′, 4.1.2. General procedure for the synthesis of bromides 1
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C-5′, C-7′), 78.94 (C-3), 109.59 (C-29), 150.58 (C-20), 176.01 (C- and 11. 1,4-Dibromobutane (4 mmol) was added to a stirred
28); anal. calcd. for C36H59BrO5: C, 66.34; H, 9.12. Found: C, suspension of BA or UA (1 mmol) in DMF (6 mL), acetonitrile
66.32; H, 9.13. (2 mL), and K2CO3 (1 mmol) and the mixture was kept at 50
8′-Bromo-3′,6′-dioxaoctan-1′-yl-3β-hydroxyurs-12-en-28-oate °C for 3 h [TLC monitoring hexane/EtOAc 1 : 1]. The mixture
(12). White crystals; 52% yield; mp 52–54 °C (EtOH); [α]21 D was then poured into cold H2O (10× volume) and extracted
+49.92° (c 0.66, CHCl3); IR (CHCl3) νmax: 3448 (–OH), 1722 with CHCl3 (2 × 15 mL each). The combined CHCl3 layers
(CO) cm−1; 1H NMR (400 MHz, CDCl3) δ 0.75, 0.77, 0.91, were washed with H2O (100 mL) and concentrated. The crude
0.98, 1.07 (3H each, all s, H-23–H-27), 0.84 (d, 3H, J = 6 Hz, residue was co-evaporated with H2O (100 mL) to remove re-
H-30), 0.93 (d, 3H, J = 6 Hz, H-29), 0.70–2.02 (m, 22H, CH, sidual DMF. The residue was chromatographed on silica gel
CH2 in pentacyclic skeleton), 2.23 (d, 1H, J = 11.6 Hz, H-18), [hexane/EtOAc, 30 : 1 → 1 : 1] to obtain a pure compound.
3.18–3.22 (m, 1H, H-3), 3.46 (t, 2H, J = 6.4 Hz, H-8′), 3.65– 4-Bromobutyl-3β-hydroxylup-20IJ29)-en-28-oate (1). White
3.67 (m, 6H, H-2′, H-4′, H-5′), 3.81 (t, 2H, J = 6.4 Hz, H-7′), crystals; 75% yield; mp 78–80 °C (EtOH); [α]21 D +27° (c 0.95,
4.15 (t, 2H, J = 4.8 Hz, H-1′), 5.24 (br s, 1H, H-12); 13C NMR CHCl3); IR (CHCl3) νmax: 3448 (–OH), 1726 (CO) cm−1; 1H
(100 MHz, CDCl3) δ 15.48 (C-25), 15.66 (C-24), 17.02 (C-26), NMR (400 MHz, CDCl3) δ 0.77, 0.83, 0.90, 0.92, 0.97 (3H
17.09 (C-29), 18.31 (C-6), 21.19 (C-30), 23.31 (C-11), 23.54 (C- each, all s, H-23–H-27), 0.68–2.26 (m, 24H, CH, CH2 in
27), 24.17 (C-16), 27.21 (C-2), 27.98 (C-15), 28.15 (C-23), 30.24 pentacyclic skeleton, 4H, H-2′, H-3′), 1.70 (s, 3H, H-30), 2.99–
(C-8′), 30.66 (C-21), 33.04 (C-7), 36.61 (C-22), 36.95 (C-10), 3.04 (m, 1H, H-19), 3.17–3.21 (m, 1H, H-3), 3.46 (t, 2H, J = 7
38.63 (C-1), 38.74 (C-20), 38.83 (C-19), 39.04 (C-4), 39.54 (C-8), Hz, H-4′), 4.08–4.18 (m, 2H, H-1′), 4.61 (s, 1H, H-29), 4.75 (s,
42.03 (C-14), 47.53 (C-9), 48.05 (C-17), 52.82 (C-18), 55.21 (C- 1H, H-29); 13C NMR (100 MHz, CDCl3) δ 14.71 (C-27), 15.37
5), 63.22 (C-1′), 69.23, 70.52, 70.57, 71.26 (C-2′, H-4′, C-5′, H- (C-26), 16.04 (C-25), 16.14 (C-24), 18.30 (C-6), 19.38 (C-30),
7′), 78.94 (C-3), 125.59 (C-12), 138.07 (C-13), 177.42 (C-28); 20.91 (C-11), 25.54 (C-12), 27.42 (C-2), 27.50 (C-3′), 27.99 (C-
anal. calcd. for C36H59BrO5: C, 66.34; H, 9.12. Found: C, 23), 29.50 (C-2′), 29.67 (C-21), 30.64 (C-15), 32.18 (C-16), 33.02
66.38; H, 9.11. (C-4′), 34.35 (C-7), 37.05 (C-22), 37.20 (C-1), 38.32 (C-4), 38.73
8′-Tosyl-3′,6′-dioxaoctan-1′-yl-3β-acetoxylup-20IJ29)-en-28- (C-13), 38.86 (C-10), 40.72 (C-8), 42.42 (C-14), 46.01 (C-18),
oate (3). Prepared by a reported method.42 Triethylene glycol 49.42 (C-19), 50.56 (C-9), 55.36 (C-5), 56.57 (C-17), 62.84 (C-
ditosylate (0.73 g, 1.6 mmol) was added to a stirred warm 1′), 78.96 (C-3), 109.64 (C-29), 150.51 (C-20), 176.09 (C-28);
solution (50 °C) of betulinic acid (0.40 g, 0.8 mmol) in dry anal. calcd. for C34H55O3Br: C, 69.02; H, 9.37. Found: C,
acetonitrile (12 mL). The reaction mixture was cooled to 69.12; H, 9.31.
room temperature, and then K2CO3 (0.062, 2 mmol) was 4-Bromobutyl-3β-hydroxyurs-12-en-28-oate (11). White
added. The resulting mixture was heated at 70 °C for 7 h crystals; 69% yield; mp 66–68 °C (EtOH); [α]20 D +53.44° (c 0.36,
[TLC monitoring hexane/EtOAc 2 : 1], the precipitate was CHCl3); IR (CHCl3) νmax: 3448 (–OH), 1719 (C=O) cm−1; 1H
filtered off and acetonitrile was removed under reduced NMR (400 MHz, CDCl3) δ 0.76, 0.79, 0.93, 1.00, 1.09 (3H
pressure. The residue was chromatographed on silica gel each, all s, H-23–H-27), 0.88, 0.96 (3H each, both d, J = 6 Hz,
[hexane/EtOAc, 10 : 1 → 5 : 1], to obtain a pure compound. H-29, H-30); 0.64–2.05 (m, 22H, CH, CH2 in pentacyclic
White crystals; 60% yield; mp 38–40 °C (EtOH); [α]17.5 D +96° (c skeleton, 4H, H-2′, H-3′); 2.24 (d, 1H, J = 11 Hz, H-18), 3.23
0.49, CHCl3); IR (CHCl3) νmax: 1727 (CO) cm−1; 1H NMR (dd, 1H, J = 10.8, 4.8 Hz, H-3), 3.43 (t, 2H, J = 7 Hz, H-4′),
(400 MHz, CDCl3) δ 0.84, 0.85, 0.86, 0.91, 0.96 (3H each, all s, 4.03 (m, 2H, H-1′), 5.26 (br s, 1H, H-12); 13C NMR (100 MHz,
3H, H-23–H-27), 0.78–2.26 (m, 24H, CH, CH2 in pentacyclic CDCl3) δ 15.50 (C-25), 15.63 (C-24), 17.02 (C-26), 17.16 (C-29),
skeleton), 1.69 (s, 3H, H-30), 2.05 (s, 3H, CH3CO–), 2.46 (s, 18.32 (C-6), 21.18 (C-30), 23.30 (C-11), 23.56 (C-27), 24.23 (C-
3H, OTs-CH3), 2.99–3.03 (m, 1H, H-19), 3.59 (s, 4H, H-4′, H- 16), 27.23 (C-2), 27.33 (C-3′), 27.97 (C-15), 28.15 (C-23), 29.52
5′), 3.66, 3.70 (2H each, both t, J = 4.8 Hz, H-2′, H-7′), 4.16, (C-2′), 30.67 (C-21), 33.05 (C-7), 33.10 (C-4′), 36.77 (C-22),
4.22 (2H each, both t, J = 4.8 Hz, H-1′, H-8′), 4.15–4.24 (m, 36.98 (C-10), 38.62 (C-1), 38.75 (C-20), 38.88 (C-19), 39.09 (C-
1H, H-3), 4.60 (s, 1H, H-29), 4.72 (s, 1H, H-29) 7.34, 7.80 (2H 4), 39.56 (C-8), 42.07 (C-14), 48.13 (C-9), 48.13 (C-17), 52.88
each, both d, J = 8 Hz, Ph); 13C NMR (100 MHz, CDCl3) δ (C-18), 55.22 (C-5), 63.17 (C-1′), 79.03 (C-3), 125.62 (C-12),
14.98 (C-27), 16.11 (C-26), 16.19 (C-25), 16.49 (C-24), 18.17 (C- 138.23 (C-13), 177.44 (C-28). Anal. calcd. for C34H55O3Br: C,
6), 19.33 (C-30), 20.90 (C-11), 21.32 (CH3CO–), 21.65 (OTs- 69.02; H, 9.37. Found: C, 69.11; H, 9.34.

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4.1.3. General procedure for the synthesis of 61 °C (EtOH); [α]17.5D +7.5° (c 0.39, CHCl3); IR (CHCl3) νmax:
triphenylphosphonium salts 5, 7, 8, 9, 13 and 15. A mixture 1727 (CO) cm−1; 31P NMR (161 MHz, CDCl3) δ 25.57; 1H
of triterpenoids 1–4, 11 or 12 (0.40 mmol), acetonitrile (13 NMR (500 MHz, CDCl3) δ 0.80, 0.82, 0.83, 0.89, 0.95 (3H
mL) and Ph3P (2 mmol) was refluxed for 20–35 h to prepare each, all s, H-23–H-27), 0.77–2.25 (m, 24H, CH, CH2 in
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the triphenylphosphonium salts [TLC monitoring hexane/ pentacyclic skeleton), 1.68 (s, 3H, H-30), 2.03 (s, 3H,
EtOAc 10 : 1]. The solution was cooled and the solvent was CH3CO–), 2.30 (s, 3H, OTs-CH3), 2.96–2.99 (m, 1H, H-19),
evaporated under vacuum. The solid product obtained was 3.26–3.38 (m, 6H, H-4′, H-5′, H-7′), 3.84–4.07 (m, 6H, H-1′, H-
washed with hot hexane (2 × 10 mL), dissolved in EtOAc (2–4 2′, H-8′), 4.44–4.48 (m, 1H, H-3), 4.60 (s, 1H, H-29), 4.71 (s,
mL), and diluted with hexane (12 ml). The precipitate was fil- 1H, H-29), 7.05 (d, 2H, J = 2 Hz, Ph), 7.62–7.80 (m, 15H, Ph
tered to obtain the title product. and 2H, OTs-Ph); 13C NMR (126 MHz, CDCl3) δ 14.65 (C-27),
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4-(Triphenylphosphonio)butyl-3β-hydroxylup-20IJ29)-en-28- 16.02 (C-26), 16.21 (C-25), 16.49 (C-24), 18.17 (C-6), 19.28 (C-
oate bromide (5). White crystals; 79% yield; mp 150–152 °C 30), 20.90 (C-11), 21.29 (CH3CO–), 21.32 (OTs-CH3), 23.67 (C-
(EtOH); [α]21D −0.38° (c 1.85, CHCl3); IR (CHCl3) νmax: 3446 12), 24.43 (C-8′, J = 54 Hz), 25.45 (C-2), 27.93 (C-23), 29.60 (C-
(–OH), 1718 (C=O); 31P NMR (161 MHz, CDCl3) δ 24.49; 1H 15), 30.53 (C-16), 32.04 (C-21), 34.27 (C-7), 36.99 (C-22), 37.09
NMR (400 MHz, CDCl3) δ 0.73, 0.77, 0.79, 0.90, 0.94 (3H (C-1), 37.78 (C-4), 38.20 (C-13), 38.38 (C-10), 40.70 (C-8), 42.39
each, all s, H-23–H-27), 0.63–2.12 (m, 24H, CH, CH2 in (C-14), 46.99 (C-18), 49.34 (C-19), 50.42 (C-9), 55.40 (C-5),
pentacyclic skeleton, 4H, H-2′, H-3′), 1.63 (s, 3H, H-30), 56.51 (C-17), 62.53 (C-1′), 63.06 (C-7′, J = 7.4 Hz), 68.97, 69.84,
2.87–2.89 (m, 1H, H-19), 3.15–3.18 (m, 1H, H-3), 3.96–4.13 70.08 (C-2′, C-4′, C-5′), 80.88 (C-3), 109.75 (C-29), 119.11 (Ph, J
(m, 4H, H-4′, H-1′), 4.56 (s, 1H, H-29), 4.66 (s, 1H, H-29), = 86 Hz), 130.00 (Ph, J = 12.7 Hz), 134.00 (Ph, J = 10.2 Hz),
7.69–7.86 (m, 15H, Ph); 13C NMR (100 MHz, CDCl3) δ 14.65 134.53 (Ph, J = 2.6 Hz), 126.11, 128.32, 138.54, 144.31, (OTs-
(C-27), 15.41 (C-26), 15.96 (C-25), 16.13 (C-24), 18.27 (C-6), Ph), 150.41 (C-20), 171.01 (CH3CO–), 175.89 (C-28); MS: m/z
19.27 (C-30), 19.42 (d, J = 3 Hz, C-3′), 20.81 (C-11), 22.30 (d, [M − TsO]−, found 875.32. [C63H83O8PS]+ requires 1030.55.
J = 50 Hz, 4′), 25.41 (C-12), 27.37 (C-2), 27.99 (C-23), 29.28 9′-(Triphen ylph ospho nio-3′,6′-dioxaoctan-1′-yl)-3β-
(d, J = 17 Hz, 2′), 29.57 (C-21), 30.52 (C-15), 32.00 (C-16), hydroxylup-20IJ29)-en-28-oate bromide (9). White crystals; 69%
34.29 (C-7), 36.91 (C-22), 37.14 (C-1), 38.13 (C-4), 38.68 (C- yield; mp 95–97 °C (EtOH); [α]18 D +3.32° (c 0.62, CHCl3); IR
13), 38.83 (C-10), 40.61 (C-8), 42.31 (C-14), 46.91 (C-18), (CHCl3) νmax: 3370 (−OH), 1722 (CO) cm−1; 31P NMR (161
49.30 (C-19), 50.46 (C-9), 55.28 (C-5), 56.42 (C-17), 62.75 (C- MHz, CDCl3) δ 25.45; 1H NMR (500 MHz, CDCl3) δ 0.72, 0.77,
1′), 78.81 (C-3), 109.70 (C-29), 117.85 (d, J = 85 Hz, Ph), 0.86, 0.93, 0.94 (3H each, all s, H-23–H-27), 0.64–2.19 (m,
130.50 (d, J = 13 Hz, Ph), 133.70 (d, J = 10 Hz, Ph), 135.03 24H, CH, CH2 in pentacyclic skeleton), 1.65 (s, 3H, H-30),
(br s, Ph), 150.37 (C-20), 175.91 (C-28); MS: m/z [M−Br]−, 2.93–2.95 (m, 1H, H-19), 3.15–3.17 (m, 1H, H-3), 3.15–3.39
found 773.49. [C52H70BrO3P]+ requires 852.42. (m, 6H, H-4′, H-5′, H-7′), 3.88–3.93 (m, 2H, H-8′), 4.06–4.14
8′-(Triphenylphosphonio-3′,6′-dioxaoctan-1′-yl)-3β-acetoxylup- (m, 4H, H-1′, H-2′), 4.57 (s, 1H, H-29), 4.69 (s, 1H, H-29),
20IJ29)-en-28-oate bromide (7). White crystals; 70% yield; mp 7.64–7.83 (m, 15H, Ph); 13C NMR (125 MHz, CDCl3) δ 14.68
108–110 °C (EtOH); [α]18 D +9.6° (c 0.50, CHCl3); IR (CHCl3) (C-27), 15.42 (C-26), 16.01 (C-25), 16.14 (C-24), 18.27 (C-6),
νmax: 1727 (CO) cm−1; 31P NMR (161 MHz, CDCl3) δ 25.50; 19.28 (C-30), 20.87 (C-11), 25.41 (C-8′, J = 52 Hz), 25.46 (C-12),
1
H NMR (500 MHz, CDCl3) δ 0.81, 0.82, 0.83, 0.88, 0.94 (3H 27.34 (C-2), 27.99 (C-23), 29.59 (C-21), 30.52 (C-15), 32.04 (C-
each, all s, H-23–H-27), 0.76–2.25 (m, 24H, CH, CH2 in 16), 34.32 (C-7), 36.97 (C-22), 37.15 (C-1), 38.21 (C-4), 38.68
pentacyclic skeleton), 1.67 (s, 3H, H-30), 2.02 (s, 3H, (C-13), 38.82 (C-10), 40.67 (C-8), 42.38 (C-14), 46.96 (C-18),
CH3CO–), 3.28–3.29 (m, 1H, H-19), 3.33–3.40 (m, 6H, H-4′, H- 49.33 (C-19), 50.47 (C-9), 55.29 (C-5), 56.51 (C-17), 62.51 (C-
5′, H-7′), 3.89–3.96 (m, 2H, H-8′), 4.07–4.19 (m, 4H, H-1′, H- 1′), 64.06 (C-7′, J = 7 Hz), 69.01, 69.84, 70.24 (C-2′, C-4′, C-5′),
2′), 4.43–4.47 (m, 1H, H-3), 4.59 (s, 1H, H-29), 4.70 (s, 1H, H- 78.80 (C-3), 109.70 (C-29), 118.82 (Ph, J = 87 Hz), 130.07 (Ph, J
29), 7.64–7.83 (m, 15H, Ph); 13C NMR (126 MHz, CDCl3) δ = 12.7 Hz), 134.03 (Ph, J = 10.2 Hz), 134.69 (Ph, J = 2.6 Hz),
14.64 (C-27), 16.02 (C-26), 16.20 (C-25), 16.48 (C-24), 18.16 (C- 150.42 (C-20), 175.88 (C-28); MS: m/z [M−Br]−, found 833.32.
6), 19.26 (C-30), 20.89 (C-11), 21.31 (CH3CO–), 23.66 (C-12), [C54H74BrO5P]+ requires 912.45.
25.42 (C-8′, J = 52 Hz), 25.43 (C-2), 27.92 (C-23), 29.59 (C-15), 4-(Triphenylphosphonio)butyl-3β-hydroxyurs-12-en-28-oate
30.51 (C-16), 32.03 (C-21), 34.26 (C-7), 36.98 (C-22), 37.08 (C- bromide (13). White crystals; 85% yield; mp 166–168 °C (EtOH);
1), 37.77 (C-4), 38.19 (C-13), 38.36 (C-10), 40.70 (C-8), 42.38 D +3.02° (c 0.50, CHCl3); IR (CHCl3) νmax: 3367 (–OH), 1716
[α]20
(C-14), 46.98 (C-18), 49.33 (C-19), 50.41 (C-9), 55.39 (C-5), (CO); 31P NMR (161 MHz, CDCl3) δ 24.48; 1H NMR (400 MHz,
56.51 (C-17), 62.50 (C-1′), 64.06 (C-7′, J = 7 Hz), 69.01, 69.85, CDCl3) δ 0.64, 0.77, 0.87, 0.98, 1.03 (3H each, all s, H-23–H-27),
70.24 (C-2′, C-4′, C-5′), 80.88 (C-3), 109.75 (C-29), 118.91 (Ph, J 0.82, 0.93 (3H each, both d, J = 6 Hz, H-29, H-30), 0.67–2.10 (m,
= 86 Hz), 130.07 (Ph, J = 12.7 Hz), 134.04 (Ph, J = 10.2 Hz), 22H, CH, CH2 in pentacyclic skeleton, 4H, H-2′, H-3′), 2.08 (d,
134.65 (Ph, J = 2.6 Hz), 150.40 (C-20), 171.01 (CH3C _ O–), 1H, J = 10.4 Hz, H-18), 3.20 (dd, 1H, J = 10.4, 4.4 Hz, H-3), 3.94–
175.88 (C-28); MS: m/z [M−Br]−, found 875.33. [C56H76BrO6P]+ 4.08 (m, 4H, H-4′, H-1′), 5.07 (br s, 1H, H-12), 7.68–7.80 (m, 15H,
requires 954.46. Ph); 13C NMR (100 MHz, CDCl3) δ 15.47 (C-25), 15.67 (C-24),
8′-(Triphenylphosphonio-3′,6′-dioxaoctan-1′-yl)-3β-acetoxylup- 17.01 (C-26), 17.02 (C-29), 18.29 (C-6), 19.43 (d, J = 3 Hz, C-3′),
20IJ29)-en-28-oate tosyl (8). White crystals; 91% yield; mp 59– 21.16 (C-30), 22.30 (d, J = 50 Hz, 4′), 23.27 (C-11), 23.52 (C-27),

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24.10 (C-16), 27.18 (C-2), 27.93 (C-15), 28.15 (C-23), 29.37 (d, J = 9), 55.33 (C-5), 56.39 (C-17), 62.72 (C-1′), 64.81 (C _ HCl2CO–),
17 Hz, 2′), 30.59 (C-21), 32.95 (C-7), 36.66 (C-22), 36.91 (C-10), 84.93 (C-3), 109.77 (C-29), 118.15 (d, J = 85 Hz, Ph), 130.53 (d,
38.60 (C-1), 38.72 (C-20), 38.84 (C-19), 38.95 (C-4), 39.44 (C-8), J = 12.4 Hz, Ph), 133.66 (d, J = 10 Hz, Ph), 135.07 (br s, Ph),
41.96 (C-14), 47.45 (C-9), 48.00 (C-17), 52.82 (C-18), 55.15 (C-5), 150.31 (C-20), 164.29 (CHCl2C _ O–), 175.88 (C-28); MS: m/z [M
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62.85 (C-1′), 78.91 (C-3), 118.14 (d, J = 86 Hz, Ph), 125.46 (C-12), −Br]−, found 883.51. [C54H70BrCl2O4P]+ requires 962.36.
130.51 (d, J = 13 Hz, Ph), 133.64 (d, J = 9 Hz, Ph), 135.03 (br s, 8′-(Triphen ylph ospho nio-3′,6′-dioxaoctan-1′-yl)-3β-
Ph), 138.09 (C-13), 177.44 (C-28); MS: m/z [M−Br]−, found 773.49. dichloroacetoxylup-20IJ29)-en-28-oate bromide (10). White
[C52H70BrO3P]+ requires 852.42. crystals; 82% yield; mp 98–100 °C (EtOH); [α]17.5 D +8.4° (c
8′-(Triphe nylphosphonio-3′,6′-dioxaoctan-1′-yl)-3β- 0.49, CHCl3); IR (CHCl3) νmax: 1722 (CO) cm−1; 31P NMR
hydroxyurs-12-en-28-oate bromide (15). White crystals; 69% (161 MHz, CDCl3) δ 25.49; 1H NMR (500 MHz, CDCl3) δ 0.83,
Open Access Article. Published on 13 September 2017. Downloaded on 08/06/2018 10:16:21.

yield; mp 81–83 °C (EtOH); [α]18 D +28.61° (c 0.57, CHCl3); IR 0.86, 0.87, 0.88, 0.94 (3H each, all s, H-23–H-27), 0.77–2.21
(CHCl3) νmax: 3151 (−OH), 1722 (CO) cm−1; 31P NMR (161 (m, 24H, CH, CH2 in pentacyclic skeleton), 1.66 (s, 3H, H-30),
MHz, CDCl3) δ 25.46; 1H NMR (500 MHz, CDCl3) δ 0.71, 0.75, 2.93–2.97 (m, 1H, H-19), 3.27–3.39 (m, 6H, H-4′, H-5′, H-7′),
0.88, 0.96, 1.06 (3H each, all s, H-23–H-27), 0, 83 (d, 3H, J = 6 3.90–4.15 (m, 6H, H-2′, H-8′, H-1′), 4.54–4.57 (m, 2H, H-3, H-
Hz, H-30), 0.93 (d, 3H, J = 6 Hz, H-29), 0.69–2.01 (m, 22H, 29), 4.69 (s, 1H, H-29), 5.91 (s, 1H, CH _ Cl2CO–), 7.64–7.80 (m,
CH, CH2 in pentacyclic skeleton), 2.19 (d, 1H, J = 11.5 Hz, H- 15H, Ph); 13C NMR (125 MHz, CDCl3) δ 14.64 (C-27), 15.99
18), 3.19–3.21 (m, 1H, H-3), 3.27–3.36 (m, 6H, H-4′, H-5′, H- (C-26), 16.16 (C-25), 16.33 (C-24), 18.05 (C-6), 19.26 (C-30),
7′), 3.90–3.98 (m, 4H, H-2′, H-8′), 4.13–4.15 (m, 2H, H-1′), 5.24 20.91 (C-11), 23.19 (C-12), 25.25 (C-8′, J = 52 Hz), 27.38 (C-2),
(br s, 1H, H-12), 7.64–7.84 (m, 15H, Ph); 13C NMR (125 MHz, 27.80 (C-23), 29.56 (C-21), 30.49 (C-15), 32.00 (C-16), 34.19 (C-
CDCl3) δ 15.48 (C-25), 15.69 (C-24), 17.02 (C-26), 17.12 (C-29), 7), 36.96 (C-22), 37.06 (C-1), 38.15 (C-4), 38.21 (C-13), 38.21
18.29 (C-6), 21.15 (C-30), 23.29 (C-11), 23.50 (C-27), 24.18 (C- (C-10), 40.68 (C-8), 42.39 (C-14), 46.96 (C-18), 49.30 (C-19),
16), 25.42 (C-8′, J = 52 Hz), 27.18 (C-2), 27.96 (C-15), 28.15 (C- 50.37 (C-9), 55.33 (C-5), 56.48 (C-17), 62.51 (C-1′), 64.03 (C-7′,
23), 30.61 (C-21), 33.03 (C-7), 36.66 (C-22), 36.93 (C-10), 38.59 J = 7 Hz), 64.81 (CHCl2CO–), 69.01, 69.84, 70.23 (C-2′, C-4′, C-
(C-1), 38.72 (C-20), 38.85 (C-19), 39.01 (C-4), 39.52 (C-8), 42.03 5′), 84.94 (C-3), 109.76 (C-29), 118.82 (Ph, J = 87 Hz), 130.00
(C-14), 47.46 (C-9), 48.07 (C-17), 52.86 (C-18), 55.15 (C-5), (Ph, J = 12.7 Hz), 134.03 (Ph, J = 10.2 Hz), 134.67 (Ph, J = 2.6
62.92 (C-1′), 64.06 (C-7′, J = 7 Hz), 68.86, 69.87, 70.27 (C-2′, C- Hz), 164.28 (CHCl2CO–), 150.37 (C-20), 175.86 (C-28); MS: m/z
4′, C-5′), 78.86 (C-3), 118.81 (Ph, J = 87 Hz), 125.57 (C-12), [M−Br]−, found 943.59. [C56H74BrCl2O6P]+ requires 1022.38.
130.08 (Ph, J = 13 Hz), 134.07 (Ph, J = 10 Hz), 134.66 (Ph, J = 4′-(Triphenylphosphonio)butyl-3β-dichloroacetoxyurs-12-en-
3 Hz), 138.06 (C-13), 177.38 (C-28); MS: m/z [M−Br]−, found 28-oate bromide (14). White crystals; 71% yield; mp 138–140
833.32 [C54H74BrO5P]+ requires 912.45. °C (EtOH); [α]17.5
D +24.7° (c 0.63, CHCl3); IR (CHCl3) νmax:
4.1.4. General procedure for the synthesis of 1721 (CO) cm−1; 31P NMR (161 MHz, CDCl3) δ 24.52; 1H
triphenylphosphonium salts 6, 10, 14, 16 and derivatives BA- NMR (400 MHz, CDCl3) δ 0.65, 0.80, 0.90, 0.91, 1.03 (3H
DCA and UA-DCA. Dichloroacetic acid (1.20 mmol) was added each, all s, HIJ23)–HIJ27)), 0.82, 0.92 (3H each, both d, J = 6
to a stirred suspension of derivatives 5, 9, 13, 15, BA or UA (1 Hz, H-29, H-30), 0.67–2.10 (m, 22H, CH, CH2 in pentacyclic
mmol) in CH2Cl2 (20 mL), DMAP (0.13 mmol), and DCC (1.20 skeleton, 4H, H-2′, H-3′), 2.08 (d, 1H, J = 10.4 Hz, H-18), 3.96–
mmol); the mixture was kept at room temperature for 2–7 h 4.07 (m, 4H, H-4′, H-1′), 4.58–4.61 (m, 1H, H-3), 5.09 (br s,
[TLC monitoring CHCl3/MeOH, 5 : 1], the precipitate was fil- 1H, H-12), 5.94 (s, 1H, CH _ Cl2CO–), 7.69–7.87 (m, 15H, Ph);
tered off and CH2Cl2 was removed under reduced pressure. 13
C NMR (100 MHz, CDCl3) δ 15.49 (C-25), 16.60 (C-24), 17.00
The residue was chromatographed on silica gel [CHCl3/ (C-26), 17.04 (C-29), 18.07 (C-6), 19.36 (d, J = 3 Hz, C-3′),
MeOH, 50 : 1 → 5 : 1], to obtain a pure compound. 21.16 (C-30), 22.36 (d, J = 50 Hz, 4′), 23.06 (C-2), 23.26 (C-11),
4′-(Triphenylphosphonio)butyl-3β-dichloroacetoxylup-20IJ29)- 23.50 (C-27), 24.09 (C-16), 27.95 (C-15), 27.96 (C-23), 29.17 (d,
en-28-oate bromide (6). White crystals; 70% yield; mp 123–125 J = 17 Hz, 2′), 30.57 (C-21), 32.82 (C-7), 36.64 (C-22), 36.80 (C-
°C (EtOH); [α]17.5
D +4.3° (c 0.53, CHCl3); IR (CHCl3) νmax: 1722 10), 38.10 (C-1), 38.11 (C-20), 38.84 (C-19), 38.94 (C-4), 39.45
(CO) cm−1; 31P NMR (161 MHz, CDCl3) δ 24.56; 1H NMR (C-8), 41.96 (C-14), 47.38 (C-9), 47.97 (C-17), 52.81 (C-18),
(500 MHz, CDCl3) δ 0.80, 0.81, 0.86, 0.87, 0.90 (3H each, all s, 55.21 (C-5), 62.80 (C-1′), 64.80 (C _ HCl2CO–), 84.91 (C-3),
H-23–H-27), 0.75–2.11 (m, 24H, CH, CH2 in pentacyclic 118.65 (d, J = 85.5 Hz, Ph), 125.26 (C-12), 130.12 (d, J = 12.4
skeleton, 4H, H-2′, H-3′), 1.63 (s, 3H, H-30), 2.86–2.88 (m, 1H, Hz, Ph), 133.10 (d, J = 9.9 Hz, Ph), 134.98 (br s, Ph), 138.15
H-19), 3.90–4.13 (m, 4H, H-4′, H-1′), 4.54–4.55 (m, 2H, H-29, (C-13), 164.32 (CHCl2C _ O–), 177.40 (C-28); MS: m/z [M−Br]−,
H-3), 4.66 (s, 1H, H-29), 5.92 (s, 1H, CH _ Cl2CO–), 7.68–7.85 found 883.54. [C54H70BrCl2O4P]+ requires 962.36.
(m, 15H, Ph); 13C NMR (100 MHz, CDCl3) δ 14.61 (C-27), 8′-(Triphen ylph ospho nio-3′,6′-dioxaoctan-1′-yl)-3β-
15.95 (C-26), 16.14 (C-25), 16.34 (C-24), 18.03 (C-6), 19.23 (C- dichloroacetoxyurs-12-en-28-oate bromide (16). White crystals;
30), 19.50 (d, J = 3 Hz, C-3′), 20.85 (C-11), 22.30 (d, J = 50 Hz, 87% yield; mp 96–98 °C (EtOH); [α]17.5D +24.3° (c 0.61, CHCl3);
4′), 23.19 (C-12), 25.32 (C-2), 27.81 (C-23), 29.34 (d, J = 17 Hz, IR (CHCl3) νmax: 1722 (CO) cm−1; 31P NMR (161 MHz,
2′), 29.54 (C-21), 30.49 (C-15), 31.97 (C-16), 34.15 (C-7), 36.90 CDCl3) δ 25.52; 1H NMR (500 MHz, CDCl3) δ 0.72, 0.75, 0.88,
(C-22), 37.04 (C-1), 38.05 (C-4), 38.20 (C-13), 38.21 (C-10), 0.96, 1.06 (3H each, all s, H-23–H-27), 0.83 (d, 3H, J = 6 Hz,
40.62 (C-8), 42.32 (C-14), 46.90 (C-18), 49.26 (C-19), 50.36 (C- H-30), 0.93 (d, 3H, J = 6 Hz, H-29), 0.70–2.13 (m, 22H, CH,

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CH2 in pentacyclic skeleton), 2.19 (d, 1H, J = 11.5 Hz, H-18), roblastoma cells (SK-N-BEIJ2)) were purchased from the
3.27–3.37 (m, 6H, H-4′, H-5′, H-7′), 3.90–3.98 (m, 4H, H-2′, H- European Collection of Authenticated Cell Cultures (ECACC),
8′), 4.14–4.15 (m, 2H, H-1′), 4.58–4.59 (m, 1H, H-3), 5.21 (br s, human colon carcinoma (HCT116) cells were kindly provided
1H, H-12), 5.93 (s, 1H, CH _ Cl2CO–), 7.64–7.84 (m, 15H, Ph); by Professor Bert Vogelstein (Johns Hopkins Medical School),
This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.

13
C NMR (125 MHz, CDCl3) δ 15.51 (C-25), 16.60 (C-24), 17.03 human colon carcinoma (RKO) and human colon neuroblas-
(C-26), 17.09 (C-29), 18.08 (C-6), 21.14 (C-30), 23.05 (C-2), toma (Tet21N) cells were purchased from American Type Cul-
23.29 (C-11), 23.47 (C-27), 24.15 (C-16), 25.42 (C-6′, J = 52 Hz), ture Collection (ATCC). Cells used in these experiments were
27.95 (C-15), 27.94 (C-23), 30.59 (C-21), 32.90 (C-7), 36.64 (C- cultured in RPMI 1640 complete medium (Tet21N and SK-N-
22), 36.81 (C-10), 38.11 (C-1), 38.10 (C-20), 38.84 (C-19), 39.00 BE(2) cells) or DMEM medium (HCT116 and RKO cells)
(C-4), 39.52 (C-8), 42.04 (C-14), 47.38 (C-9), 48.06 (C-17), 52.84 supplemented with 10% (w/v) heat-inactivated fetal calf se-
Open Access Article. Published on 13 September 2017. Downloaded on 08/06/2018 10:16:21.

(C-18), 55.21 (C-5), 62.92 (C-1′), 64.06 (C-5′, J = 7 Hz), 64.80 rum and penicillin/streptomycin (100 U ml−1). For Tet21N
(C
_ HCl2CO–), 68.85, 69.86, 70.26 (C-2′–C-4′), 84.89 (C-3), cells, 100 μg ml−1 hygromycin and 200 μg ml−1 geneticin were
118.70 (d, J = 69 Hz, Ph), 125.33 (C-12), 129.10 (d, J = 10 Hz, also added to the medium. Cells were grown in a humidified
Ph), 133.08 (d, J = 8 Hz, Ph), 134.67 (br s, Ph), 138.14 (C-13), air/CO2 (5%) atmosphere at 37 °C and maintained in a loga-
164.28 (CHCl2C _ O–), 177.35 (C-28); MS: m/z [M−Br]−, found rithmic growth phase for all experiments. Suspensions of
943.59. [C56H74BrCl2O6P]+ requires 1022.38. splenocyte cell lines were prepared by treating BALB/c mouse
3β-Dichloroacetoxyurs-12-en-28-oic acid (UA-DCA). White spleens in a glass homogenizer; the suspensions were fil-
crystals; 71% yield; mp 258–260 °C (EtOH); [α]17.5 D +75.5° (c tered, washed with cold phosphate buffer saline (Mediatech),
0.48, CHCl3); IR (CHCl3) νmax: 1748 (CO) cm−1; 1H NMR and resuspended in the culture medium, and the cell viabil-
(500 MHz, CDCl3) δ 0.79, 0.92, 0.95, 0.99, 1.09 (3H each, all s, ity was estimated using the 0.1% trypan blue test. Suspen-
H-23–H-27), 0.88 (d, 3H, J = 6 Hz, H-30), 0.97 (d, 3H, J = 6.5 sions containing at least 95% viable cells were used in experi-
Hz; H-29), 1.27–2.03 (m, 22H, CH, CH2 in pentacyclic ments. The cells were cultured in the above-described
skeleton), 2.20 (d, 1H, J = 11 Hz, H-18), 4.62–4.65 (m, 1H, H- medium.
3), 5.25 (t, 1H, J = 3.5 Hz, H-12), 5.96 (s, 1H, CH_ Cl2CO–); 13C 4.2.2. Gel electrophoresis and western blot analysis. Cell
NMR (125 MHz, CDCl3) δ 15.51 (C-25), 16.56 (C-24), 17.01 (C- were harvested, washed in phosphate-buffered saline (PBS)
26), 17.09 (C-29), 18.05 (C-6), 21.19 (C-30), 23.10 (C-2), 23.27 and lysed for 10 min at room temperature in lysis buffer
(C-11), 23.62 (C-27), 23.99 (C-16), 27.95 (C-15), 27.96 (C-23), supplemented with complete protease inhibitors (Roche Di-
30.58 (C-21), 32.77 (C-7), 36.71 (C-22), 36.90 (C-10), 38.10 (C- agnostics). Cell extracts were centrifuged at 13 000 rpm for 20
1), 38.15 (C-20), 38.82 (C-19), 39.00 (C-4), 39.48 (C-8), 41.88 min at 4 °C to separate the insoluble material, followed by
(C-14), 47.43 (C-9), 47.97 (C-17), 52.45 (C-18), 55.25 (C-5), the determination of protein concentration using the BSA as-
64.82 (C _ HCl2CO–), 84.94 (C-3), 125.58 (C-12), 137.99 (C-13), say (Pierce). Equal amounts of protein from each sample
164.32 (CHCl2C _ O–), 184.31 (C-28); anal. calcd. for were mixed with Laemmle's loading buffer, boiled for 5 min
C32H48Cl2O4: C, 67.71; H, 8.52. Found: C, 67.69; H, 8.49. and subjected to SDS-PAGE. Membranes were blocked for 1 h
3β-Dichloroacetoxylup-20IJ29)-en-28-oic acid (BA-DCA). White with 5% non-fat milk in PBS at room temperature and subse-
crystals; 65% yield; mp 267–269 °C (EtOH), lit.32 mp 269 °C; quently probed with the primary antibody of interest. Blots
[α]17.5
D +17.2° (c 0.58, CHCl3); IR (CHCl3) νmax: 1742 (CO) were revealed either using the ECL Western Blotting Sub-
cm−1, lit. 1745 cm−1; 1H NMR (500 MHz, CDCl3) δ 0.89, 0.91, strate (Promega, USA) or SuperSignal West Dura Extended
0.92, 0.96, 1.00 (3H each, all s, H-23–H-27), 0.83–2.32 (m, Duration Substrate (Thermo Scientific, USA).
24H, CH, CH2 in pentacyclic skeleton), 1.72 (s, 3H, H-30), 4.2.3. Assessment of oxygen consumption using a
3.00–3.06 (m, 1H, H-19), 4.60–4.64 (m, 2H, H-29, H-3), 4.76 Seahorse XF-96 Analyzer. Approximately 15 000 cells per well
(s, 1H, H-29), 5.96 (s, 1H, CH _ Cl2CO–); 13C NMR (125 MHz, were grown in 96-well plates (Seahorse Bioscience, Billerica,
CDCl3) δ 14.68 (C-27), 16.03 (C-26), 16.15 (C-25), 16.33 (C-24), MA), allowed to adhere overnight, and treated with BA or its
18.06 (C-6), 19.36 (C-30), 20.88 (C-11), 23.24 (C-2), 25.40 (C- derivatives for 24 h. The cells were then washed with assay
12), 27.85 (C-23), 29.69 (C-21), 30.57 (C-15), 32.15 (C-16), medium (for Mitotest: unbuffered DMEM supplemented with
34.19 (C-7), 37.06 (C-22), 37.12 (C-1), 38.26 (C-4), 38.27 (C-13), 5 mM glucose, 2 mM glutamine, pH 7.4) before incubation
38.42 (C-10), 40.70 (C-8), 42.45 (C-14), 46.96 (C-18), 49.25 (C- with assay medium (0.175 mL) for 1 h at 37 °C in a CO2-free
19), 50.36 (C-9), 55.37 (C-5), 56.44 (C-17), 64.83 (C _ HCl2CO–), incubator. The following states of respiration were evaluated:
84.97 (C-3), 109.79 (C-29), 150.34 (C-20), 164.33 (CHCl2C _ O–), basal respiration, proton leak after oligomycin injection,
182.76 (C-28); anal. calcd. for C32H48Cl2O4: C, 67.71; H, 8.52. spare respiratory capacity after injection of the uncoupler
Found: C, 67.73; H, 8.54. CCCP (carbonyl cyanide 3-chlorophenylhydrazone), and non-
mitochondrial respiration after injection of rotenone and
antimycin A. The data were normalized to the total protein in
4.2. Biology (materials and methods) each well.
4.2.1. Cells. The work was conducted exclusively on hu- 4.2.4. Flow cytometric analysis of sub-G1 population. For
man cancer cell lines. Human Caucasian breast adenocarci- the analysis of the SubG1 subpopulation, cells were
noma cells (MCF7) and human Caucasian bone marrow neu- harvested, fixed in 70% ethanol overnight and stained with

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