Triphenylphosphonium Cations of the Diterpenoid Isosteviol: Synthesis and Antimitotic Activity in a Sea Urchin Embryo Model

Article abstract of DOI:10.1021/acs.jnatprod.5b00124

A series of novel triphenylphosphonium (TPP) cations of the diterpenoid isosteviol (1, 16-oxo-ent-beyeran-19-oic acid) have been synthesized and evaluated in an in vivo phenotypic sea urchin embryo assay for antimitotic activity. The TPP moiety was applied as a carrier to provide selective accumulation of a connected compound into mitochondria. When applied to fertilized eggs, the targeted isosteviol TPP conjugates induced mitotic arrest with the formation of aberrant multipolar mitotic spindles, whereas both isosteviol and the methyltriphenylphosphonium cation were inactive. The structure-activity relationship study revealed the essential role of the TPP group for the realization of the isosteviol effect, while the chemical structure and the length of the linker only slightly influenced the antimitotic potency. The results obtained using the sea urchin embryo model suggested that TPP conjugates of isosteviol induced mitotic spindle defects and mitotic arrest presumably by affecting mitochondrial DNA. Since targeting mitochondria is considered as an encouraging strategy for cancer therapy, TPP-isosteviol conjugates may represent promising candidates for further design as anticancer agents.

Full text of DOI:10.1021/acs.jnatprod.5b00124

Article
pubs.acs.org/jnp
Triphenylphosphonium Cations of the Diterpenoid Isosteviol:
Synthesis and Antimitotic Activity in a Sea Urchin Embryo Model
Irina Yu. Strobykina,† Mayya G. Belenok,† Marina N. Semenova,‡,§ Victor V. Semenov,*,⊥
Vasiliy M. Babaev,† Ildar Kh. Rizvanov,† Vladimir F. Mironov,† and Vladimir E. Kataev†
†A. E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center of Russian Academy of Sciences, Arbuzov Street,
8, 420088, Kazan, Russian Federation
‡N. K. Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, Vavilov Street, 26, 119334, Moscow, Russian
Federation
§Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus
⊥N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect, 47, 119991, Moscow, Russian
Federation
S
* Supporting Information
ABSTRACT: A series of novel triphenylphosphonium (TPP)
cations of the diterpenoid isosteviol (1, 16-oxo-ent-beyeran-19-
oic acid) have been synthesized and evaluated in an in vivo
phenotypic sea urchin embryo assay for antimitotic activity.
The TPP moiety was applied as a carrier to provide selective
accumulation of a connected compound into mitochondria.
When applied to fertilized eggs, the targeted isosteviol TPP
conjugates induced mitotic arrest with the formation of
aberrant multipolar mitotic spindles, whereas both isosteviol
and the methyltriphenylphosphonium cation were inactive.
The structure−activity relationship study revealed the essential role of the TPP group for the realization of the isosteviol effect,
while the chemical structure and the length of the linker only slightly influenced the antimitotic potency. The results obtained
using the sea urchin embryo model suggested that TPP conjugates of isosteviol induced mitotic spindle defects and mitotic arrest
presumably by affecting mitochondrial DNA. Since targeting mitochondria is considered as an encouraging strategy for cancer
therapy, TPP-isosteviol conjugates may represent promising candidates for further design as anticancer agents.
umerous natural terpenoids, such as sesquiterpenoids,1
acid hydrolysis of the Stevia rebaudiana (Bertoni) Bertoni
(Asteraceae) glycoside stevioside,16 a component of various
nonsugar low-calorie sweeteners manufactured on a large scale
in a number of countries. Isosteviol markedly inhibited in vivo
chemically induced mouse skin carcinogenesis, suggesting its
potential chemopreventive properties.17 However, in vitro
biological evaluation of isosteviol in human cancer cell lines
failed to reveal cytotoxic effects,18−23 except for very weak
cytotoxicity against HL60 human leukemia cells (IC50 = 75
μM),24 MOLT-4 T-lymphoblastic leukemia cells (LD50 = 84
μM),25 BALL-1 B-lymphoblastic leukemia cells (LD50 = 87
μM),25 and NUGC-3 human gastric cells (LD50 = 167 μM).25
The cytotoxicity of 1 was considered to be associated with the
inhibition of mammalian DNA metabolic enzymes topoisomer-
ase II and DNA polymerases α, β, and λ.25 In spite of the
negligible antiproliferative potency of 1, chemical modifications
of the isosteviol ring D,18−22,26,27 the synthesis of 16,19-
dihydroxy-ent-beyerane and cinnamic acid esters,24 and a
coupling of two isosteviol molecules by a diamide linker23
2−4
pimarane,2,5 and ent-
Nditerpenoids of the abietane,
kaurane6 series, tetracyclic triterpenoids of the lanostane7 and
cucurbitane8 series, and pentacyclic triterpenoids of the
lupane,9 oleanane,7,10,11 ursane,7 and friedelane1 series, affect
the in vitro growth and viability of human tumor cells,
suggesting their potential as anticancer agents. Typically, these
molecules are extracted from the respective natural sources in
small quantities, thereby restricting their further investigation
for the treatment of cancer. The diterpenoid abietic acid7,12 and
triterpenoid acids, including betulinic,9 oleanolic,7 glycyrrhe-
tinic,7,10,11 and ursolic7 acids, are readily extracted from higher
plants in high yields and represent favorable exceptions. These
natural products display cytotoxicity against panels of human
cancer cell lines.7,13 In in vivo preclinical trials, betulinic acid
showed a pronounced antitumor effect together with the
absence of systemic toxicity.14 Notably, betulinic acid induces
apoptosis via a direct effect on mitochondria, leading to
cytochrome c release into the cytosol.14
Isosteviol (1, 16-oxo-ent-beyeran-19-oic acid15) is a tetracy-
clic natural product hydrolytic derivative related structurally to
the diterpenoid acids (Scheme 1). It can be easily obtained by
Received: February 6, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00124
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a
Scheme 1.
a
Reagents and conditions: (a) MeCN, K2CO3, reflux, 10−12 h. (b) LiBr, acetone, reflux, 14−16 h. (c) PPh3, toluene, reflux, 37−75 h. (d) NEt3,
CH3CN, reflux, 17−23 h.
furnished a series of derivatives that demonstrated cytotoxic
activity against a panel of human cancer cell lines with IC50
values in the range 1−7 μM.18−24,26,27 The mechanisms of
action and cellular targets of isosteviol derivatives remain
unclear. Only for C-19 O-acylated isosteviol analogues has an
apoptosis-inducing effect in HL60 leukemia cells been
reported.24
It is worth noting that among natural products only 20,29-
dihydrobetulinic acid and 3-epi-20,29-dihydrobetulinic acid
have been conjugated with the TPP cation through a
trimethylene linker, yielding derivatives with a markedly
increased cytotoxicity against human cancer cells.13c
A
lipophilic polymethylene linker is the only one reported to
connect the TPP cation to a small molecule,13c,30−40 whereas
for a water-soluble isosteviol triethylammonium salt, a hydro-
philic poly(ethylene glycol) (PEG) linker binds together the
diterpenoid skeleton and the N+Et3 moiety.41 It was found that
this amphiphilic isosteviol derivative readily integrated with a
lipid bilayer of dipalmitoylphosphatidylcholine liposomes and
formed micelle-like aggregates in water, mediating DNA
transfer into bacterial cells.41 Considering such evidence, it
was supposed that partially water-soluble amphiphilic TPP
conjugates of isosteviol containing a long hydrophilic PEG
linker might also form micelles, facilitating their penetration
into the mitochondrial matrix.
This hypothesis was also based on the observation that the
incorporation of the diterpenoid paclitaxel in liposomes
modified with stearyl triphenyl phosphonium enhanced its
accumulation in mitochondria, triggering apoptosis in the
paclitaxel-resistant OVCAR-3 ovarian cancer cell line.42 Hence,
it was aimed to synthesize a series of novel biologically active
isosteviol derivatives, with the isosteviol scaffold conjugated
with a TPP cation by polymethylene or PEG linkers.
All synthesized isosteviol derivatives were evaluated for
antimitotic activity using the sea urchin embryo model. Due to
the abundance and accessibility of these gravid test animals,
their straightforward artificial spawning, fertilization, and
rearing, the relatively large size of the eggs and embryos, and
their rapid synchronous development, optical transparency, and
high penetrability to different chemicals, this simple organism
model has been applied extensively in a wide range of biological
and ecological tests of natural products and synthetic
Generally, DNA-damaging agents initiate the mitochondria-
related intrinsic apoptotic pathway, which involves an increase
of mitochondrial outer membrane permeability and the release
of cytochrome c into the cytosol, thereby triggering down-
stream apoptotic events including caspase activation.28 There-
fore, the effective intracellular trafficking and targeted delivery
to mitochondria may improve the cytotoxicity of a given
molecule. It was found that tetraphenylphosphonium and
methyltriphenylphosphonium bromides easily penetrate into
mitochondria due to a highly negative membrane potential and
a pH gradient across the mitochondrial inner membrane.29
Moreover, a markedly higher mitochondrial transmembrane
potential in malignant cells in comparison to that in normal
epithelial cells provides the selective accumulation of the
phosphonium cations inside cancer cell mitochondria.29 Hence,
the bulky lipophilic triphenylphosphonium (TPP) cations are
assumed to be promising carriers for the predominant drug
accumulation in mitochondria.29 Notably, a conjugation of
several well-known antioxidants and anticancer agents with
TPP has resulted in both the targeting of mitochondria in
cancer cells29−40 and the increase of cytotoxicity.13c,38−40 The
application of mitochondrion-targeting TPP-conjugated meso-
tetraphenylporphyrin derivatives34 essentially improved capa-
bilities of photodynamic therapy in oncology.35 In addition,
64Cu-labeled triphenylphosphonium cations containing
1,4,7,10-tetraazacyclododecane moieties were used successfully
as novel radiotracers for imaging tumors by positron-emission
tomography.36,37
B
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a
Scheme 2.
a
Reagents and conditions: (a) Br(CH2)nBr, MeCN, K2CO3, reflux, 3−13 h. (b) PPh3, toluene, reflux, 37−75 h.
Table 1. Effects of Methyltriphenylphosphonium Bromide (MTPPB), Isosteviol (1), and Its TPP (14−16, 25−27) and
Ammonium (17, 18) Conjugates on Sea Urchin Embryos
a
EC, μM
b
compound
linker length (number of atoms)
cleavage alteration
full mitotic arrest
embryo spinning
posthatching lethality
isosteviol
MTPPB
14
>4
>4
1
>4
>4
2
>4
>4
>4
>4
2
8
11
14
8
>10
>10
>10
>100
15
1
2
2
16
1
4
4
17
>100
>100
>100
c
40
18
11
>100
>100
>100
>100
c
100
25
3
6
8
2
4
4
>4
>4
4
26
2
1
27
2
4
>4
0.5
40
etoposide
0.5
>68
>68
c
4
nocodazole
paclitaxel
0.005
0.5
0.01
5
0.1
>0.5
d
0.02
>10
2
a
The sea urchin embryo assay was conducted as described previously.44 Fertilized eggs and hatched blastulae were exposed to 2-fold decreasing
b
concentrations of compounds. Duplicate measurements showed no differences in effective threshold concentration (EC) values. Death of embryos
exposed to compounds at the hatched blastula stage. Inhibition of skeletal spiculae growth. Developmental arrest at the late gastrula stage.
c
d
compounds, especially in the study of antiproliferative/
antimitotic activity. The well-known development of the sea
urchin embryos facilitates the interpretation of the aberrant
morphogenetic events caused by a given test molecule.
Moreover, the easily observed in vivo phenotypic abnormalities
of eggs and embryos exposed to a test compound can provide
information about its mode of action and a putative cellular
target.43 The in vivo phenotypic sea urchin embryo assay used
in the present study provided information on antiproliferative,
antimitotic, cytotoxic, and microtubule-destabilizing activities of
the molecules tested along with their solubility and
permeability potential.44 The assay yields highly reproducible
results that correlate well with values obtained by conventional
cell-based and in vitro tubulin polymerization assays.45 Using
the above animal model, the preliminary structure−activity
relationship of the resulting compounds was used to reveal the
contributions of the diterpenoid ent-beyerane moiety, the linker
structure, and the TPP group to the resultant antimitotic
effects.
RESULTS AND DISCUSSION
■
The targeted amphiphilic TPP conjugates of isosteviol (14−
16) with PEG as a linker were obtained by three steps (Scheme
1). Reaction of isosteviol (1) with a double excess of the
ditosylates of various polyethylene glycols (2−4), which were
obtained by interaction of appropriate polyethylene glycols with
tosyl chloride,46 gave a mixture of tosylates 5−7 and diketones
8−10. Tosylates 5−7 were further converted into the
corresponding bromides 11−13 in boiling acetone in the
presence of LiBr. In the final step, bromides 11−13 were
refluxed with triphenylphosphine in toluene to obtain the TPP
cations 14−16. The isosteviol derivatives 17 and 18 with a
triethylammonium group instead of a triphosphonium moi-
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etythe analogues of TPP conjugates 14 and 15were
synthesized according to a reported procedure.41
Conjugates of isosteviol (25−27) with the TPP group
attached to the diterpenoid ent-beyerane skeleton by hydro-
phobic polymethylene linkers were synthesized via a reported
procedure (Scheme 2).47 Isosteviol (1) was refluxed in CH3CN
with a 2-fold excess of dibromoalkanes in the presence of
K2CO3 to afford products of monoalkylation (19−21) and
dialkylation (22−24). Further interaction of compounds 19−
21 with Ph3P in toluene under reflux yielded the TPP
conjugates 25−27.
Since numerous structurally diverse antimitotic agents have
been found to target microtubules,48 the possible involvement
of microtubules in the antiproliferative effects of isosteviol
derivatives was investigated. Microtubules are cytoskeletal
organelles involved in cell shape maintenance, intracellular
transport, cell adhesion and migration, and ciliary and flagellar
motility. An essential role for microtubules in cell division is the
formation of the mitotic spindle, responsible for the correct
chromosomal orientation and separation into daughter cells.49
Antiproliferative effects of isosteviol (1), methyltriphenyl-
phosphonium bromide (MTPPB), TPP-isosteviol conjugates
14−18 and 25−27, and dialkylation products 23 and 24 were
studied using the sea urchin embryo model. The assay includes
(i) a fertilized egg test for antimitotic activity and (ii) a means
of assessing embryo viability, morphogenesis, and swimming
pattern after exposure to a compound just after hatching, ∼9 h
postfertilization. Specific embryo motility changes, namely, the
lack of forward movement, settlement to the bottom of the
culture vessel, and rapid spinning around the animal−vegetal
axis, indicate the microtubule-destabilizing activity caused by a
molecule.44 The test results are shown in Table 1. An inhibitor
of tubulin polymerization, nocodazole, a microtubule stabilizer,
paclitaxel, and a topoisomerase II inhibitor, etoposide, served as
reference compounds.
In addition, throughout the study, mitotic spindle patterns in
fertilized eggs arrested by the test molecules were analyzed.
Importantly, sea urchin embryos offer the experimental
possibility for direct in vivo observation of the mitotic apparatus
using conventional light microscopy (Figure 1). Mitotic
phenotypes of fertilized eggs exposed to a compound can be
easily monitored, yielding the opportunity to suggest a
mechanism of action. During the first division of intact eggs,
normal bipolar mitotic spindles can be seen as two large light
spots (Figure 1A). Tubulin-targeting agents interfere with
microtubule assembly, resulting in diverse mitotic spindle
defects and cleavage arrest. Microtubule stabilizers cause the
formation of multipolar mitotic asters,44,50 as shown for
paclitaxel-treated eggs (Figure 1C). In contrast, inhibitors of
tubulin polymerization, e.g., nocodazole, completely block
spindle assembly, producing eggs devoid of mitotic apparatus,
with small clear spots corresponding to nuclei43 in the center of
arrested eggs (Figure 1D).
Figure 1. Mitotic spindle defects in the arrested sea urchin eggs
exposed to isosteviol derivative 26 and microtubule-targeted agents.
(A) Intact eggs with normal bipolar mitotic apparatus at the first
cleavage anaphase, 1 h postfertilization. (B−G) At 2.5 h postfertiliza-
tion. (B) Control eight-cell embryos. (C) Multipolar mitotic spindles
caused by the microtubule stabilizer paclitaxel (5 μM). (D) Eggs
devoid of mitotic apparatus in the presence of the microtubule
destabilizer nocodazole (50 nM). (E) Formation of aberrant mitotic
spindles initiated by 26 (2 μM). Samples were incubated at 21 °C. The
average egg/embryo diameter was 115 μm.
Specifically, the replacement of TPP in 14 and 15 with
triethylamine afforded the inactive conjugates 17 and 18, which
caused only weak effects, namely, retardation of skeletal
spiculae growth at the pluteus stage. Although compounds
with PEG and polymethylene linkers displayed closely
comparable effects, the PEG-containing conjugates 14−16
were slightly more active when applied to fertilized eggs.
Alternatively, molecules with polymethylene fragments (25−
27) affected posthatching development in a more potent
manner than 14−16, especially compound 27, with the longest
octamethylene linker (Table 1). The antimitotic activity of all
TPP conjugates was independent of linker length.
It is worth noting that compounds 14−16 and 25−27 failed
to block mitotic spindle assembly or to induce embryo spinning
typical of microtubule destabilizers. In contrast to isosteviol
derivatives, nocodazole was unable to kill embryos at
posthatching stages even at a concentration 1000 times higher
than that sufficient for cleavage alterations. Multipolar mitotic
spindles were observed in eggs treated by microtubule-
stabilizing agents, but not in eggs exposed to TPP-isosteviol
conjugates. Therefore, the differences in both developmental
As shown in Table 1, both isosteviol (1) and methyl-
triphenylphosphonium bromide were inactive up to a 4 μM
concentration, whereas the TPP conjugates of isosteviol (14−
16 and 25−27) caused cleavage alteration, cleavage arrest, and
posthatching embryo death in the low micromolar concen-
tration range. Interestingly, compounds 14−16 and 25−27
generated a distinct arrested egg phenotype with a mitotic
apparatus as a centrally located large light spot (Figure 1E).
Preliminary structure−activity relationship studies showed
that the TPP moiety is essential for antimitotic activity.
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alterations and mitotic spindle patterns caused by TPP
conjugates in comparison with the tubulin-targeting com-
pounds paclitaxel and nocodazole suggested that the antimitotic
effect of the synthesized isosteviol derivatives is not mediated
by the direct interaction with tubulin/microtubules.
Considering the ability of isosteviol (1) to inhibit the DNA-
related enzymes topoisomerase II and DNA polymerase α,25
the effects of TPP-isosteviol conjugates and DNA targeting
molecules were compared. In the assay system used, the
topoisomerase II inhibitor etoposide displayed cleavage
alterations but failed to induce mitotic arrest. When applied
to hatched blastulae, etoposide caused embryo death at a
concentration significantly higher than that for cleavage
alteration (Table 1). According to literature data, a specific
inhibitor of DNA polymerase α, the tetracyclic diterpene
aphidicolin,51 and an inhibitor of DNA replication, tribromoin-
dole carbaldehyde, initiated the formation of arrested eggs with
homogeneous cytoplasm, lacking both mitotic spindle and
condensed chromatin.52,53 In contrast to these DNA-targeting
agents, compounds 14−16 and 25−27 blocked cell division,
inducing the formation of aberrant mitotic spindles (Figure
1E). These data suggest that the antimitotic effects of TPP-
isosteviol conjugates are unlikely to be related to the inhibition
of either topoisomerase II or DNA polymerase α.
The conjugation of anticancer agents with TPP leads to their
selective accumulation in cancer cell mitochondria, thereby
consistently enhancing cytotoxicity. Specifically, this was
reported for TPP derivatives of the DNA-alkylating agent
chlorambucil,39 the proapoptotic natural product curcumin,40
and an inhibitor of DNA topoisomerases I and II,
dihydrobetulinic acid.13c,54 The present study demonstrated
that isosteviol (1) did not affect sea urchin embryo develop-
ment at any stage up to a 4 μM concentration, while TPP-
isosteviol conjugates 14−16 and 25−27 each exhibited strong
antimitotic activity. The data suggested the predominant
contribution of the diterpenoid ent-beyerane moiety to the
antimitotic properties of TPP-isosteviol conjugates, since the
TPP cation itself was inactive, as determined from MTPPB
tests (Table 1). In addition, the accumulation in mitochondria
ensured by a TPP transporter could play a key role in the
response of the sea urchin embryos to isosteviol derivatives.
It was reported recently that mitochondria are implicated in
the regulation of centrosome duplication and mitotic spindle
assembly.55 Specifically, the depletion of mitochondrial DNA
induces the formation of aberrant multipolar spindles followed
by mitotic arrest in the G2/M phase. Considering these
literature data, it was decided to analyze the mitotic spindle
morphology in sea urchin eggs arrested by isosteviol conjugates.
At first glance, the test compounds initiated the formation of
monopolar mitotic spindles (Figure 1E). However, a detailed
microscopic study using the phase-contrast imaging technique
revealed irregular multipolar asters in a certain number of eggs
(Figure 2), suggesting the possible interaction of isosteviol
derivatives with mitochondrial DNA.
Figure 2. Aberrant mitotic spindles (asterisks) in the eggs arrested by
26 (2 μM). Phase-contrast imaging at higher magnification.
on antimitotic activity. The mechanism of action and cellular
target(s) of isosteviol derivatives require further clarification.
Nevertheless, the results obtained in the assay system used
suggest that TPP conjugates of isosteviol might affect
mitochondrial DNA, thereby inducing mitotic spindle defects
and mitotic arrest. As targeting mitochondria is an encouraging
strategy for cancer therapy,56 TPP-isosteviol conjugates
represent a promising starting point for their further design
as possible anticancer agents.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Compound melting points
were determined on a Boetius compact heating table. Optical rotations
were determined on a PerkinElmer 341 polarimeter (concentration c is
1
given as g/mL). H and 31P NMR spectra were collected on Bruker
Avance-400 and Bruker Avance-500 NMR spectrometers. Mass spectra
were obtained on an UltraFlex III TOF/TOF time-of-flight mass
spectrometer (Bruker Daltonik, Bremen, Germany). Positively charged
ions were registered. High-resolution 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. The dried-droplet spotting
technique was applied in preparing a matrix and analyte solutions.
After application of 0.5 μL of each solution to the target plate MTP
AnchorChip (Bruker Daltonik GmbH, Bremen, Germany), the solvent
was allowed to evaporate under ambient conditions, and the plate was
inserted into the mass spectrometer for analysis. The data were
processed using the FlexAnalysis 3.0 software (Bruker Daltonik,
Therefore, a series of TPP conjugates of the diterpenoid
isosteviol (1) were synthesized, where TPP was introduced as a
carrier for the preferential delivery of the diterpenoid ent-
beyerane moiety into mitochondria. The targeted molecules
exhibited pronounced antimitotic effects in the sea urchin
embryo model. A preliminary structure−activity relationship
study revealed the essential role of the TPP group for the
realization of the isosteviol effect, whereas the chemical
structure and the length of the linker had only limited impact
E
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Bremen, Germany). Elemental analysis was accomplished with the
automated EuroVector EA3000 CHNS-O elemental analyzer (Euro-
Vector, 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 5% H2SO4, followed by heating to 120 °C. The
targeted compounds were isolated using dry-column flash chromatog-
raphy on KSKG silica gel (<0.063 mm, Crom-Lab Ltd., Lyubertsy,
Russian Federation).
2 × H-20), 0.97 (6H, s, 2 × H-17), 1.19 (6H, s, 2 × H-18), 0.87−1.93
(36H, m, ent-beyerane skeleton), 2.18 (2H, d, J = 13.3 Hz, 2 × H-3),
2.62 (2H, dd, J = 3.7, 18.6 Hz, 2 × Hα-15), 3.59−3.62 (4H, br s,
OCH2CH2O), 3.65−3.71 (4H, m, 2 × C(O)OCH2CH2), 4.13−4.23
(4H, m, 2 × C(O)OCH2); MALDIMS m/z 773.66 [M + Na]+, 789.62
[M + K]+; anal. C 73.58; H 9.42%, calcd for C46H70O8, C 73.56; H
9.39%.
3′,6′,9′-Trioxaundecane-1′,11′-diylbis(16-oxo-ent-beyeran-19-
oate) (9): amorphous powder; 0.57 g, 23% yield; mp 85−88 °C;
1
[α]20 −78.3 (c 0.69; CH2Cl2); H NMR (CDCl3, 400 MHz) δ 0.71
All solvents were dried according to standard protocols. Isosteviol
(1) was prepared from the sweetener Sweta (Stevian Biotechnology
Corp., Labu, Negeri Sembilan, Malaysia) as described previously:57 mp
D
(6H, s, 2 × H-20), 0.97 (6H, s, 2 × H-17), 1.20 (6H, s, 2 × H-18),
0.85−1.92 (36H, m, ent-beyerane skeleton), 2.19 (2H, d, J = 13.5 Hz,
2 × H-3), 2.62 (2H, dd, J = 3.7, 18.6 Hz, 2 × Hα-15), 3.59−3.65 (8H,
m, 2 × OCH2CH2O), 3.66−3.70 (4H, m, 2 × C(O)OCH2CH2),
4.14−4.23 (4H, m, 2 × C(O)OCH2); MALDIMS m/z 817.52 [M +
Na]+; anal. C 72.58; H 9.40%; calcd for C48H74O9, C 72.51; H 9.38%.
3′,6′,9′,12′-Tetraoxatetradecane-1′,14′-diylbis(16-oxo-ent-beyer-
an-19-oate) (10): viscous oil; 0.05 g, 19% yield; [α]20D −75.5 (c 0.64;
CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 0.70 (6H, s, 2 × H-20), 0.95
(6H, s, 2 × H-17), 1.18 (6H, s, 2 × H-18), 0.86−1.90 (36H, m, ent-
beyerane skeleton), 2.17 (2H, d, J = 13.3 Hz, 2 × H-3), 2.60 (2H, dd, J
= 18.6, 3.7 Hz, 2 × Hα-15), 3.60−3.62 (8H, m, 2 × OCH2CH2O),
3.62−3.64 (4H, m, OCH2CH2O), 3.64−3.69 (4H, m, 2 × C(O)-
OCH2CH2), 4.12−4.21 (4H, m, 2 × C(O)OCH2); MALDIMS m/z
861.50 [M + Na]+; anal. C 71.60; H 9.40%; calcd for C50H78O10, C
71.56; H, 9.37%.
234 °C (lit.57 mp 234−235 °C); [α]20 −72.3 (c 1.02, EtOH) (lit.58
D
[α]20 −72, c 0.12, MeOH). Triethylene glycol, tetraethylene glycol,
D
and dibromoalkanes were purchased from Vekton Closed Corporation
(Krasnodar, Russian Federation), ABCR GmbH & Co. (Karlsruhe,
Germany), and Alfa Aesar (Heysham, United Kingdom), respectively.
Ditosylates of polyethylene glycols 2−4 were synthesized according to
a literature procedure.46 Ditosylate of triethylene glycol 2: mp 80 °C
(lit.46 mp 80−81 °C); ditosylate of tetraethylene glycol 3: oil;
MALDIMS m/z 502.1 (calcd for C22H30O9S2, 502.133); ditosylate of
pentaethylene glycol 4: oil, MALDIMS m/z 546.2 (calcd for
C24H34O10S2, 546.159). MTPPB was synthesized as previously
described:59 mp 238−240 °C (lit.59 mp 230−233 °C).
General Procedure for the Synthesis of Tosylates 5−7 and
Diketones 8−10. Poly(ethylene glycol) ditosylate (2 mmol) was
added to a stirred warm solution (50 °C) of isosteviol (1, 1 mmol) in
dry CH3CN. The reaction mixture was cooled to room temperature,
and K2CO3 (2 mmol) was added. The resulting mixture was heated at
70 °C for 10−12 h, the precipitate was filtered off, and CH3CN was
removed under reduced pressure. The crude oily syrup obtained was
purified by dry-column flash chromatography (step gradient of 6:1 to
1:2.5 petroleum ether/ethyl acetate) to afford the targeted tosylates
(5−7) and diketones (8−10).
General Procedure for the Synthesis of Bromides 11−13.
Lithium bromide (1.1 mmol) was added to a stirred solution of each
tosylate (1 mmol) in acetone, and the mixture was refluxed for 14−16
h. The resulting precipitate was filtered off, and acetone was removed
under reduced pressure. The separated oily syrup was purified by dry-
column flash chromatography on silica gel (petroleum ether/ethyl
acetate, 5:1−1:1.5) to afford pure bromides 11−13.
8′-Bromo-3′,6′-dioxaoctan-1′-yl 16-oxo-ent-beyeran-19-oate
(11): viscous oil; 3.7 g, 88% yield; [α]20 −60.2 (c 1.42, MeOH);
8′-p-Toluenesulfonyloxy-3′,6′-dioxaoctan-1′-yl 16-oxo-ent-be-
D
1H NMR (CDCl3, 400 MHz) δ 0.8−2.0 (18H, m, ent-beyerane
skeleton), 0.71 (3H, s, H-20), 0.97 (3H, s, H-17), 1.19 (3H, s, H-18),
2.18 (1H, d, J = 13.5 Hz, H-3), 2.62 (1H, dd, J = 3.7, 18.6 Hz, Hα-15),
3.46 (2H, t, J = 6.3 Hz, CH2Br), 3.61−3.67 (4H, m, OCH2CH2O),
3.67−3.71 (2H, m, CH2O), 3.80 (2H, br t, J = 6.2 Hz, CH2O), 4.16−
4.21 (2H, m, C(O)OCH2); MALDIMS m/z 535.2 [M + Na]+; anal. C
60.27; H 8.09; Br 15.45%, calcd for C26H41BrO5, C 60.81; H 8.05; Br
15.56%.
yeran-19-oate (5): viscous oil; 5.0 g, 53% yield; [α]20 −52.3 (c
D
1
0.55, MeOH); H NMR (CDCl3, 400 MHz) δ 0.62−2.14 (18H, m,
ent-beyerane skeleton), 0.70 (3H, s, H-20), 0.96 (3H, s, H-17), 1.18
(3H, s, H-18), 2.17 (1H, d, J = 13.2 Hz, H-3), 2.44 (3H, s, CH3Car),
2.60 (1H, dd, J = 3.4, 18.6 Hz, Hα-15), 3.56 (4H, br s, OCH2CH2O),
3.64 (2H, t, J = 4.8 Hz, CH2O,), 3.68 (2H, t, J = 4.8 Hz, CH2O),
4.08−4.24 (4H, m, C(O)OCH2 and CH2OTs), 7.33 (2H, d, J = 8.0
Hz, H−Car), 7.78 (2H, d, J = 7.9 Hz, H-Car); MALDIMS, m/z 627.4
[M + Na]+, 643.4 [M + K]+; anal. C 65.56; H 8.24; S 5.16%, calcd for
C33H48O8S, C 65.54; H 8.0; S 5.30%.
11′-Bromo-3′,6′,9′-trioxaundecan-1′-yl 16-oxo-ent-beyeran-19-
oate (12): viscous oil; 0.99 g, 78% yield; [α]20 −59.3 (c 1.02,
D
1
MeOH); H NMR (CDCl3, 400 MHz) δ 0.81−1.89 (18H, m, ent-
11′-p-Toluenesulfonyloxy-3′,6′,9′-trioxaundecan-1′-yl 16-oxo-
ent-beyeran-19-oate (6): viscous oil; 1.75 g, 43% yield; [α]20
−52.0 (c 0.58, MeOH); H NMR (CDCl3, 400 MHz) δ 0.82−1.90
beyerane skeleton), 0.69 (3H, s, H-20), 0.94 (3H, s, H-17), 1.17 (3H,
s, H-18), 2.16 (1H, d, J = 13.5 Hz, H-3), 2.59 (1H, dd, J = 3.7, 18.6
Hz, Hα-15), 3.43 (2H, t, J = 6.3 Hz, CH2Br), 3.58−3.69 (10H, m,
CH2O), 3.77 (2H, t, J = 6.3 Hz, CH2O), 4.13−4.17 (2H, m,
C(O)OCH2); MALDIMS, m/z 579.2 [M + Na]+, 597.2 [M + K]+;
anal. C 60.37; H 8.19; Br 14.45%, calcd for C28H45BrO6, C 60.32; H
8.14; Br 14.33%.
D
1
(18H, m, ent-beyerane skeleton), 0.71 (3H, s, H-20), 0.97 (3H, s, H-
17), 1.19 (3H, s, H-18), 2.18 (1H, d, J = 13.2 Hz, H-3), 2.44 (3H, s,
CH3Car), 2.61 (1H, dd, J = 3.8, 18.6 Hz, Hα-15), 3.58 (4H, s,
OCH2CH2O), 3.60 (4H, s, OCH2CH2O), 3.65−3.70 (4H, m, 2 ×
CH2O), 4.13−4.19 (4H, m, C(O)OCH2 and CH2OTs), 7.33 (2H, d, J
= 8.5 Hz, H-Car), 7.79 (2H, d, J = 8.4 Hz, H-Car); MALDIMS m/z
671.3 [M + Na]+, 687.3 [M + K]+; anal. C 64.87; H 8.05; S 5.01%,
calcd for C35H52O9S, C 64.79; H 8.08; S 4.94%.
14′-Bromo-3′,6′,9′,12-tetraoxatetradecan-1′-yl 16-oxo-ent-be-
yeran-19-oate (13): 0.10 g, 67% yield; viscous oil; [α]20 −39.0 (c
D
1
2.27, CH2Cl2); H NMR (CDCl3, 500 MHz) δ 0.82−1.90 (18H, m,
ent-beyerane skeleton), 0.71 (3H, s, H-20), 0.96 (3H, s, H-17), 1.19
(3H, s, H-18), 2.18 (1H, d, J = 13.3 Hz, H-3), 2.62 (1H, dd, J = 3.7,
18.6 Hz, Hα-15), 3.46 (2H, t, J = 6.3 Hz, CH2Br), 3.60−3.71 (14H, m,
CH2O), 3.80 (2H, t, J = 6.3 Hz, CH2O), 4.14−4.21 (2H, m,
C(O)OCH2); MALDIMS, m/z 623.2 [M + Na]+, 639.2 [M + K]+;
anal. C 60.01; H 8.19; Br 13.34%, calcd for C30H49BrO7, C 59.89; H
8.21; Br 13.28%.
14′-p-Toluenesulfonyloxy-3′,6′,9′,12′-tetraoxatetradecan-1′-yl
16-oxo-ent-beyeran-19-oate (7): viscous oil; 0.18 g, 41% yield; [α]20
−51.5 (c 1.85, CH2Cl2); H NMR (CDCl3, 500 MHz) δ 0.82−1.90
D
1
(18H, m, ent-beyerane skeleton), 0.71 (3H, s, H-20), 0.96 (3H, s, H-
17), 1.19 (3H, s, H-18), 2.18 (1H, d, J = 13.3 Hz, H-3), 2.44 (3H, s,
CH3Car), 2.61 (1H, dd, J = 3.7, 18.6 Hz, Hα-15), 3.57 (4H, s,
OCH2CH2O), 3.59−3.65 (8H, m, 2 × OCH2CH2O), 3.65−3.70 (4H,
m, 2 × CH2O), 4.13−4.19 (4H, m, C(O)OCH2 and CH2OTs), 7.33
(2H, d, J = 7.9 Hz, H-Car), 7.79 (2H, d, J = 8.2 Hz, H-Car); MALDIMS
m/z 715.3 [M + Na]+; anal. C 64.25; H 8.10; S 4.56%, calcd for
C37H56O10S, C 64.14; H 8.15; S 4.63%.
General Procedure for the Synthesis of TPP Conjugates of
Isosteviol (14−16, 25−27). Triphenylphosphine (1.5−3 mmol) was
added to the solution of bromide (11−13 or 19−21) (1 mmol) in dry
toluene, and the mixture was stirred under reflux for 37−75 h. Toluene
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
3′,6′-Dioxaoctane-1′,8′-diylbis(16-oxo-ent-beyeran-19-oate) (8):
amorphous powder; 1.63 g, 28% yield; mp 127−129 °C; [α]20D −76.0
(c 0.19; MeOH+CH2Cl2); 1H NMR (CDCl3, 400 MHz) δ 0.71 (6H, s,
F
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Article
resulting precipitate was washed with diethyl ether (3 mL) and dried
in vacuo to give the pure TPP conjugate.
literature procedure.60 Triethylamine (1.5 mL, 10 mmol) was added to
a stirred solution of bromide 12 (0.56 g, 1 mmol) in CH3CN (12 mL).
The reaction mixture was refluxed for 23 h, CH3CN was removed
under reduced pressure, and the resulting oily syrup was purified by
column chromatography on silica gel (0.060−0.200 mm, 60 Å (Acros
Organics, NJ, USA), step gradient of 30:1 to 10:1 CH2Cl2/methanol),
to isolate ammonium compound 18 in 27% yield (0.18 g, 0.27 mmol)
as a viscous oil: [α]20D −49.0 (c 1.31, MeOH); 1H NMR (CDCl3, 400
MHz) δ 0.83−1.90 (18H, m, ent-beyerane skeleton), 0.70 (3H, s, H-
20), 0.95 (3H, s, H-17), 1.18 (3H, s, H-18), 1.39 (9H, t, J = 7.2 Hz, 3
× NCH2CH3), 2.16 (1H, d, J = 13.7 Hz, H-3), 2.59 (1H, dd, J = 3.7,
18.5 Hz, Hα-15), 3.52−3.62 (12H, m, 3 × NCH2CH3, CH2CH2N and
OCH2CH2O), 3.63−3.68 (4H, m, OCH2CH2O), 3.78−3.80 (2H, m,
OCH2CH2N), 3.94−3.98 (2H, m, C(O)OCH2CH2O), 4.12−4.20
(2H, m, C(O)OCH2); HRMS m/z 578.4488 (calcd for C34H60NO6,
578.4415).
General Procedure for the Synthesis of Bromides 19−21.
K2CO3 (2 mmol) was added to a solution of α,ω-dibromoalkane (2−4
mmol) in dry CH3CN; then a warm solution (30 °C) of isosteviol (1)
(1 mmol) in dry CH3CN was added dropwise, and the reaction
mixture was stirred under reflux for 3−13 h. A solid residue was
filtered off, CH3CN was removed under reduced pressure, and the
resulting oily syrup was purified by dry-column flash chromatography
on silica gel (step gradient of 50:1 to 4:1 petroleum ether/ethyl
acetate) to afford the target bromides 19−21. Dialkylation byproducts
23−24 were detected in the reaction mixtures without further
isolation.
19-(9′-Triphenylphosphonium-1′,4′,7′-trioxanonyl)-16,19-dioxo-
ent-beyerane bromide (14): amorphous powder; 0.01 g, 1% yield; mp
60−66 °C; [α]20 −44.0 (c 0.47, MeOH); 31P NMR (CDCl3) δ
D
1
−26.05; H NMR (CDCl3, 400 MHz) δ 0.68−1.90 (18H, m, ent-
beyerane skeleton), 0.67 (3H, s, H-20), 0.96 (3H, s, H-17), 1.18 (3H,
s, H-18), 2.16 (1H, d, J = 13.5 Hz, H-3), 2.56 (1H, dd, J = 3.5, 18.4
Hz, Hα-15), 3.25−3.30 (2H, m, OCH2), 3.31−3.35 (2H, m, OCH2),
3.37−3.44 (2H, m, OCH2CH2P), 3.89−4.00 (2H, m, CH2P), 4.01−
4.06 (2H, m, C(O)OCH2CH2O), 4.18−4.25 (2H, m, C(O)OCH2),
7.61−7.68 (6H, m, H-Car), 7.72−7.78 (3H, m, H-Car), 7.80−7.88 (6H,
m, H-Car); HRMS m/z 695.3870 (calcd for C44H56O5P, 695.3860).
19-(12′-Triphenylphosphonium-1′,4′,7′,10′-tetraoxadodecyl)-
16,19-dioxo-ent-beyerane bromide (15): amorphous powder; 0.04 g,
2% yield; mp 65−70 °C; [α]20 −29.5 (c 0.41, MeOH); 31P NMR
D
1
(CDCl3) δ −26.29; H NMR (CDCl3, 500 MHz) δ 0.69 (3H, s, H-
20), 0.96(3H, s, H-17), 1.18 (3H, s, H-18), 0.84−1.92 (18H, m, ent-
beyerane skeleton), 2.16 (1H, d, J = 13.4 Hz, H-3), 2.60 (1H, dd, J =
3.6, 18.4 Hz, Hα-15), 3.27−3.35 (4H, m, 2 × OCH2), 3.35−3.40 (2H,
m, OCH2), 3.48−3.51 (2H, m, OCH2), 3.60−3.65 (2H, m, OCH2),
3.89−3.99 (2H, m, CH2P), 4.12−4.16 (2H, m, C(O)OCH2CH2O),
4.18−4.25 (2H, m, C(O)OCH2), 7.61−7.67 (6H, m, H-Car), 7.72−
7.78 (3H, m, H-Car), 7.82−7.88 (6H, m, H-Car); HRMS m/z 739.4124
(calcd for C46H60O6P, 739.4122).
19-(15′-Triphenylphosphonium-1′,4′,7′,10′,13′-pentaoxapenta-
decacyl)-16,19-dioxo-ent-beyerane bromide (16): viscous oil; 0.01 g,
7% yield; [α]20D −27.3 (c 0.37, MeOH); 31P NMR (CDCl3) δ −25.68;
1H NMR (CDCl3, 500 MHz) δ 0.70 (3H, s, H-20), 0.97 (3H, s, H-
17), 1.19 (3H, s, H-18), 0.80−1.90 (18H, m, ent-beyerane skeleton),
2.17 (1H, d, J = 13.5 Hz, H-3), 2.60 (1H, dd, J = 13.8, 18.6 Hz, Hα-
15), 3.26−3.35 (4H, m, 2 × OCH2), 3.37−3.40 (2H, m, OCH2),
3.50−3.54 (2H, m, OCH2), 3.55−3.62 (4H, m, OCH2CH2), 3.63−
3.67 (2H, m, OCH2), 3.90−3.99 (2H, m, CH2P), 4.13−4.19 (2H, m,
C(O)OCH2CH2O), 4.21−4.28 (2H, m, C(O)OCH2), 7.62−7.68 (6H,
m, H-Car), 7.72−7.78 (3H, m, H-Car), 7.83−7.90 (6H, m, H-Car);
HRMS m/z 783.4413 (calcd for C48H64O7P, 783.4384).
19-(3′-Triphenylphosphoniumpropyloxy)-16,19-dioxo-ent-beyer-
ane bromide (25): powder; 0.28 g, 18% yield; mp 114−117 °C;
[α]20D −41.5 (c 1.05, CHCl3); 31P NMR (CDCl3) δ −24.69; 1H NMR
(CDCl3, 400 MHz) δ 0.44 (3H, s, H-20), 0.97 (3H, s, H-17), 1.11
(3H, s, H-18), 0.82−2.01 (20H, m, ent-beyerane skeleton and CH2
fragment of linker), 2.11 (1H, d, J = 13.5 Hz, H-3), 2.42 (1H, dd, J =
3.8, 18.6 Hz, Hα-15), 3.90−4.04 (1H, m, CH2P), 4.12−4.25 (1H, m,
CH2P), 4.40−4.51 (2H, m, C(O)OCH2), 7.67−7.73 (6H, m, H-Car),
7.77−7.83 (3H, m, H-Car), 7.83−7.91 (6H, m, H-Car); HRMS m/z
621.3511 (calcd for C41H50O3P, 621.3492).
3′-Bromo-n-propyl 16-oxo-ent-beyeran-19-oate (19): powder;
1
2.04 g, 46% yield; mp 97−99 °C; [α]20 −61.7 (c 1.10, CHCl3); H
D
NMR (CDCl3, 400 MHz) δ 0.70 (3H, s, H-20), 0.96 (3H, s, H-17),
1.18 (3H, s, H-18), 0.82−1.90 (18H, m, ent-beyerane skeleton), 2.13−
2.19 (3H, m, H-3 and CH2 fragment of linker), 2.61 (1H, dd, J = 3.9,
18.6 Hz, Hα-15), 3.46 (2H, t, J = 6.5 Hz, CH2Br), 4.08−4.14 (1H, m,
C(O)OCH), 4.18−4.23 (1H, m, C(O)OCH); anal. C 62.68; H 8.32;
Br 18.02%; calcd for C23H35BrO3, C 62.87; H 8.03; Br 18.18%.
6′-Bromo-n-hexyl 16-oxo-ent-beyeran-19-oate (20): viscous oil;
1
1.91 g, 40% yield; [α]20 −46.6 (c 1.52, CHCl3); H NMR (CDCl3,
D
400 MHz) δ 0.70 (3H, s, H-20), 0.96 (3H, s, H-17), 1.18 (3H, s, H-
18), 0.80−1.90 (22H, m, ent-beyerane skeleton and (CH2)4 fragment
of linker), 2.17 (1H, d, J = 13.5 Hz, H-3), 2.62 (1H, dd, J = 3.7, 18.6
Hz, Hα-15), 2.40 (2H, t, J = 6.8 Hz, CH2Br), 3.95−4.07 (2H, m,
C(O)OCH2); anal. C 64.88; H 8.62; Br 16.42%, calcd for C26H41BrO3,
C 64.85; H 8.58; Br 16.59%.
8′-Bromo-n-octyl 16-oxo-ent-beyeran-19-oate (21): viscous oil;
1
2.62 g, 51% yield; [α]20 −48.2 (c 1.67, CHCl3); H NMR (CDCl3,
D
400 MHz) δ 0.71 (3H, s, H-20), 0.98 (3H, s, H-17), 1.19 (3H, s, H-
18), 0.80−1.93 (24H, m, ent-beyerane skeleton and (CH2)6 fragment
of linker), 2.19 (1H, d, J = 13.1 Hz, H-3), 2.63 (1H, dd, J = 3.7, 18.5
Hz, Hα-15), 3.41 (2H, t, J = 6.8 Hz, CH2Br), 3.96−4.06 (2H, m,
C(O)OCH2); anal. C 66.08; H 8.82; Br 15.58%, calcd for C28H45BrO3,
C 66.00; H 8.90; Br 15.68%.
19-(3′-Triphenylphosphoniumpropyloxy)-16,19-dioxo-ent-beyer-
ane bromide (26): powder; 0.3 g, 20% yield; mp 72−74 °C; [α]20
D
−32.7 (c 1.09, CHCl3); 31P NMR (CDCl3) δ −24.51; 1H NMR
(CDCl3, 400 MHz) δ 0.64 (3H, s, H-20), 0.97 (3H, s, H-17), 1.15
(3H, s, H-18), 0.81−1.91 (22H, m, ent-beyerane skeleton and (CH2)4
fragment of linker), 2.15 (1H, d, J = 13.3 Hz, H-3), 2.54 (1H, dd, J =
3.6, 18.5 Hz, Hα-15), 3.85−4.03 (4H, m, CH2P and C(O)OCH2),
7.65−7.73 (6H, m, H-Car), 7.74−7.81 (3H, m, H-Car), 7.83−7.92 (6H,
m, H-Car); HRMS m/z 663.3511 (calcd for C44H56O3P, 663.3492).
19-(8′-Triphenylphosphoniumoctyloxy)-16,19-dioxo-ent-beyer-
Sea Urchin Embryo Assay.44 Adult sea urchins, Paracentrotus
lividus L. (Echinidae), were collected from the Mediterranean Sea on
the Cyprus coast in March−April and November 2014 and kept in an
aerated seawater tank. Gametes were obtained by intracoelomic
injection of 0.5 M KCl. Eggs were washed with filtered seawater and
fertilized by adding drops of diluted sperm. Embryos were cultured at
room temperature under gentle agitation with a motor-driven plastic
paddle (60 rpm) in filtered seawater. The embryos were observed with
a Biolam light microscope (LOMO, St. Petersburg, Russian
Federation). For treatment with the test compounds, 5 mL aliquots
of embryo suspension were transferred to six-well plates and incubated
as a monolayer at a concentration up to 2000 embryos/mL. Stock
solutions of MTPPB, isosteviol (1), and compounds 23−27 were
prepared in DMSO at 10 mM concentration followed by a 10-fold
dilution with 96% EtOH. This procedure enhanced the solubility of
the test compounds in the salt-containing medium (seawater), as
evidenced by microscopic examination of the samples. The maximal
tolerated concentrations of DMSO and EtOH in the in vivo assay were
determined to be 0.05% and 1%, respectively. Higher concentrations
ane bromide (27): powder; 0.56 g, 37% yield; mp 83−84 °C; [α]20
D
−37.2 (c 1.03, CHCl3); 31P NMR (CDCl3) δ −24.63; 1H NMR
(CDCl3, 500 MHz) δ 0.68 (3H, s, H-20), 0.96 (3H, s, H-17), 1.16
(3H, s, H-18), 0.82−1.91 (24H, m, ent-beyerane skeleton and (CH2)6
fragment of linker), 2.16 (1H, d, J = 13.7 Hz, H-3), 2.59 (1H, dd, J =
3.8, 18.5 Hz, Hα-15), 3.82−3.90 (2H, m, CH2P), 3.91−4.01 (2H, m,
C(O)OCH2), 7.67−7.72 (6H, m, H-Car), 7.76−7.81 (3H, m, H-Car),
7.83−7.90 (6H, m, H-Car); HRMS m/z 691.4275 (calcd for
C46H60O3P, 691.4271).
19-(9′-Triethylammonium-1′,4′,7′-trioxanonyl)-16,19-dioxo-ent-
beyerane bromide (17): This was synthesized as previously
described:60 mp 90−93 °C (lit.60 mp 90−93 °C).
19-(12′-Triethylammonium-1′,4′,7′,10′-tetraoxadodecyl)-16,19-
dioxo-ent-beyerane bromide (18): This was prepared according to a
G
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of either DMSO (≥0.1%) or EtOH (>1%) caused nonspecific
alterations and retardation of the sea urchin embryo development
independent of the treatment stage. Stock solutions of compounds
14−16 (5 mM) and 17 and 18 (both 10 mM) were prepared in
distilled water. Etoposide (LANS, Veropharm, Moscow, Russian
Federation, 20 mg/mL in 96% EtOH), nocodazole, and paclitaxel
(both Sigma-Aldrich Co., St. Louis, MO, USA) served as reference
compounds. Stock solutions of nocodazole and paclitaxel, both at 5
mM, were prepared in DMSO and 96% ethanol, respectively. The
antimitotic activity was assessed by exposing fertilized eggs (8−15 min
postfertilization, 45−55 min before the first mitotic cycle completion)
to 2-fold decreasing concentrations of the compound. Cleavage
alteration and arrest were clearly detected at 2.5−5.5 h after
fertilization. The effects were estimated quantitatively as an effective
threshold concentration, resulting in cleavage alteration and embryo
death before hatching or full mitotic arrest. At these concentrations, all
tested compounds caused 100% cleavage alteration and embryo death
before hatching, whereas at 2-fold lower concentrations the
compounds failed to produce any effect. For microtubule-destabilizing
activity and developmental defects, the compounds were tested on
free-swimming blastulae just after hatching (8−9 h postfertilization),
which originated from the same embryo culture. Embryos were
observed until the four-arm pluteus stage (34−36 h postfertilization).
The specific changes in swimming pattern, namely, both spinning on
the bottom of the well and lack of forward movement, were
interpreted to be the result of the microtubule-destabilizing activity
of a molecule. Microphotographs were obtained using an AmScope
binocular microscope with an MU500 digital camera (United Scopes
LLC, Irvine, CA, USA). Video illustrations are available at http://
www.chemblock.com. The sea urchin embryo assay data are available
free of charge via the Internet at http://www.zelinsky.ru.
(5) Ge, Y.-Z.; Zhang, H.; Liu, H.-C.; Dong, L.; Ding, J.; Yue, J.-M.
Phytochemistry 2014, 100, 120−125.
(6) Aoyagi, Y.; Ozawa, K.; Kobayashi, T.; Hasuda, T.; Gui, M.-Y.; Jin,
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of compounds 5−16, 18−21, and 25−27; 31
P
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NMR spectra of compounds 14−16 and 25−27. The
Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00124.
(17) Takasaki, M.; Konoshima, T.; Kozuka, M.; Tokuda, H.;
Takayasu, J.; Nishino, H.; Miyakoshi, M.; Mizutani, K.; Lee, K.-H.
Bioorg. Med. Chem. 2009, 17, 600−605.
AUTHOR INFORMATION
Corresponding Author
*Tel: +7 916 620 9584. Fax: +7 499 137 2966. E-mail: vs@
zelinsky.ru.
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by Russian Scientific Foundation
(grant no. 14-50-00014).
■
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