Total Synthesis of Natural Lembehyne C and Investigation of Its Cytotoxic Properties

Article abstract of DOI:10.1021/acs.jnatprod.0c00261

The first Z-stereoselective method for the synthesis of the natural marine alkynol lembehyne C, containing a 1Z,5Z,9Z-triene moiety, in 41% yield was developed using the new Ti-catalyzed cross-coupling of oxygenated and aliphatic 1,2-dienes as the key step. It was found for the first time that lembehyne C exhibits moderate cytotoxicity against Jurkat, K562, U937, and HL60 cancer cells and also efficiently induces apoptosis in Jurkat cells, with the cell death mechanism being activated by the mitochondrial pathway. The lembehyne C inhibition of the cell cycle follows the mitotic catastrophe mechanism.

Full text of DOI:10.1021/acs.jnatprod.0c00261

pubs.acs.org/jnp
Article
Total Synthesis of Natural Lembehyne C and Investigation of Its
Cytotoxic Properties
Lilya U. Dzhemileva, Vladimir A. D’yakonov,* Alexey A. Makarov, Elina Kh. Makarova,
Evgeny N. Andreev, and Usein M. Dzhemilev
Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00261
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The first Z-stereoselective method for the synthesis of the natural marine alkynol lembehyne C, containing a
1Z,5Z,9Z-triene moiety, in 41% yield was developed using the new Ti-catalyzed cross-coupling of oxygenated and aliphatic 1,2-
dienes as the key step. It was found for the first time that lembehyne C exhibits moderate cytotoxicity against Jurkat, K562, U937,
and HL60 cancer cells and also efficiently induces apoptosis in Jurkat cells, with the cell death mechanism being activated by the
mitochondrial pathway. The lembehyne C inhibition of the cell cycle follows the mitotic catastrophe mechanism.
urrently, several thousand organic compounds occurring
Cin marine organisms have been isolated and characterized.
Most of these compounds exhibit anticancer, antibacterial,
antiviral, antifungal, antiparasitic, or other types of biological
activity.1 The isolated compounds show pronounced structural
diversity, including steroids, macrolides, lactams, alkaloids,
nucleosides, fatty acids, and various long- and short-chain
Figure 1. Natural acetylenic alcohols lembehynes B and C.
functionally substituted alkenes and alkynes. Most often,
particular biologically active components are present in biomass
in micro- or nanogram amounts; this is a significant obstacle to
their detailed biological testing and fulfillment of their
biomedical potential.
Herein, we developed a total synthesis of lembehyne C
(Figure 1), an alkynol containing three bis-methylene-separated
Z-double bonds, previously isolated in trace amounts from the
Indonesian marine sponge Haliclona sp., with Ti-catalyzed cross-
On the basis of the foregoing, one can state that studying the
cyclomagnesiation of 1,2-dienes as the key step.3 In view of the
natural products isolated from marine species is an important
fact that lembehyne B exhibited anticancer activity against
human leukemia cells by inducing early apoptosis, it seemed
area at the boundary of biology and chemistry. Synthesis and
modification of these compounds and investigations of their
appropriate to produce a sufficient amount of lembehyne C in
effects on biological processes may lead to the development of
order to study its anticancer activity. It is noteworthy that
new biologically active products, which, in turn, would give rise
previously Japanese researchers reported high neuritogenic
to new efficacious drugs.
Previously, it was shown that catalytic cross-cyclomagnesia-
tion of oxygenated and aliphatic 1,2-dienes with Grignard
Received: March 9, 2020
reagents in the presence of Cp2TiCl2 (5−10 mol %) may serve as
the key step for the six-step stereoselective total synthesis of a
natural alkynol, lembehyne B (Figure 1). This, in turn, made it
possible to produce lembehyne B in sufficient amounts to study
its anticancer activity.2
© XXXX American Chemical Society and
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
American Society of Pharmacognosy
J. Nat. Prod. XXXX, XXX, XXX−XXX
A

Journal of Natural Products
pubs.acs.org/jnp
Article
Scheme 1. Retrosynthetic Analysis of Lembehyne C
Scheme 2. Synthesis of (6Z)-Triacosa-1,2,6-triene (10)
Scheme 3. Alternative Approach to the Synthesis of (6Z)-Triacosa-1,2,6-triene (10)
activity of lembehynes and have simultaneously ascertained that
these compounds are efficient low-molecular-weight analogues
of proteins of the neurotrophin family whose function is to
support the viability of neurons.4 Therefore, lembehynes are
considered as potential pharmaceutical drugs for the treatment
of neurodegenerative diseases such as Alzheimer’s and
Parkinson’s diseases and Huntington’s chorea.
the necessity to form three bis-methylene-separated Z-double
bonds. No efficient methods for the formation of the 1Z,5Z,9Z-
triene moiety in lembehyne C were reported, except for the
interesting but laborious approach to unbranched 1,5,9,n-
polyenes developed by A. Stark for the total synthesis of an
acetogenin, chatenaytrienin-4.5 To solve this problem, we
resorted to the new Ti-catalyzed cross-cyclomagnesiation of
functionally substituted and aliphatic 1,2-diynes, previously
developed and which had been used for the total syntheses of
lembehynes A and B, 5Z,9Z-dienoic acids, acetogenins,
macrodiolides, and some insect pest pheromones.6−10
RESULTS AND DISCUSSION
■
Chemistry. Despite the seeming simplicity of the structure of
lembehyne C, its total synthesis is a challenging task because of
B
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Scheme 4. (Z,Z,Z)-Stereoselective Synthesis of Hexaconta-11Z,13Z,17Z-trienal (17)
Scheme 5. Total Synthesis of Natural Lembehyne C (1)
Preliminary retrosynthetic analysis of lembehyne C showed
that the total synthesis can be accomplished by means of Ti-
catalyzed cross-cyclomagnesiation of (6Z)-tricosa-1,2,6-triene
with 11,12-tridecadienoltetrahydropyran ether or 2-dodeca-
10,11-dien-1-yl-1,3- dioxolane (Scheme 1). The subsequent
steps were intended to include generation of the terminal
propargyl moiety to form racemic lembehyne C and oxidation of
the carbinol group followed by stereoselective reduction
(Scheme 1).
It is noteworthy that the synthesis of the key monomer (6Z)-
tricosa-1,2,6-triene is a fairly complicated task; therefore, two
approaches were developed for its synthesis from commercially
available 1-bromohexadecane (2) (Scheme 2) or 4-pentyn-1-ol
(11) (Scheme 3). The former approach comprising eight steps
involves the reaction of 1-bromohexadecane (2) with the
lithium acetylide ethylenediamine complex in DMSO to give 1-
octadecyne (3) in 93% yield.11 Compound 3 is treated with
EtMgBr in THF at reflux for 3 h to give the corresponding
magnesium acetylide 4, which reacts with ethylene oxide and
then undergoes acid hydrolysis to give eicos-3-yn-1-ol (5). Next,
(3Z)-eicos-3-en-1-ol (6) was synthesized via Ni(OAc)2-
catalyzed hydrogenation of alkynol 5; then alcohol 6 was
treated with MeSO2Cl in CH2Cl2 for 4 h to give mesylate 7,
which was converted to 1-bromo-(3Z)-eicos-3-ene (8) in 85%
yield by refluxing with LiBr in acetone for 3 h. The final stage
included preparation of alkyne 9 by the reaction of alkenyl
bromide 8 with lithium acetylide ethylenediamine complex and
the subsequent acetylene−allene rearrangement of alkyne 9
under Mannich reaction conditions to afford the target (6Z)-
tricosa-1,2,6-triene (10).
1-bromohexadecane, this compound was converted to 4-
heneicosyn-1-ol (12), which was subjected to catalytic hydro-
genation with molecular hydrogen in the presence of Ni(OAc)2
to afford 4Z-heneicos-4-en-1-ol (13) in 85% yield over two steps
(Scheme 3). The Dess-Martin periodinane oxidation of alcohol
13 afforded 4Z-heneicos-4-en-1-al (14), which reacted with
dimethyl (1-diazo-2-oxopropyl)phosphonate to afford (5Z)-
docos-5-en-1-yne (9).12 In the last step alkyne 9 underwent
acetylene−allene rearrangement on refluxing with paraformal-
dehyde and dihexylamine in the presence of CuI in dioxane (100
°C, 8 h) to afford allene 10 (Scheme 3).
The next stage of the synthesis involved the intermolecular
cross-cyclomagnesiation of (6Z)-tricosa-1,2,6-triene (10) and
2-dodeca-10,11-dien-1-yl-1,3-dioxolane (15a) or 11,12-trideca-
dienol tetrahydropyran ether (15b) with EtMgBr in the
presence of Mg (powder) and catalytic amounts of Cp2TiCl2
[10:15a (15b):EtMgBr:Mg:[Ti] = 12:10:36:24:0.1, Et2O, 20−
22 °C, 10 h]. The acid hydrolysis of magnesacyclopentanes
16a,b formed in situ afforded aldehyde 17 and tetrahydropyr-
anyl ether 18 containing 1Z,5Z,9Z-diene moieties in 81% and
89% yields, respectively (Scheme 4). The removal of the
tetrahydropyranyl ether protective group and Dess−Martin
oxidation converted ether 18 to aldehyde 17 in ∼64% yield
(Scheme 4), which indicates that the synthesis of aldehyde 17
via dioxolane 15a is preferable.
The reaction of hexaconta-11Z,13Z,17Z-trien-1-al (17) with
lithium (trimethylsilyl)acetylide,13 presynthesized by the
reaction between equimolar amounts of (trimethylsilyl)-
acetylene and n-BuLi in THF, for 3 days at room temperature
gave silane 20 in 91% yield. The removal of the terminal
trimethylsilyl group with trimethylbutylammonium fluoride
(TBAF) in THF for 4 h led to racemic lembehyne C (21) in a
nearly quantitative yield (Scheme 5).
The alternative (five-step) method proposed for the synthesis
of (6Z)-tricosa-1,2,6-triene (10) is based on 4-pentyl-1-ol (11)
as the starting material. By successive treatment with n-BuLi and
C
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
For the synthesis of enantiomerically pure natural lembehyne
C, racemic lembehyne C (21) was oxidized with the Dess−
Martin periodinane (CH2Cl2, rt, 1 h) to give ketone 22 in 87%
yield (Scheme 5). Finally, using (R)-2-methyl-CBS-oxazabor-
olidine,14 stereoselective reduction of ketone 22 afforded natural
lembehyne C (1) in 82% yield with high enantiomeric purity
(96% ee).
(Jurkat, HL-60, U937 and K562). The assays included
determination of IC50, cell viability, effect of the compound on
the cell cycle, and the mechanism of apoptosis induction. This
was done using flow cytofluorimetry and Luminex xMAP
multiplex assays for proteins of the key signaling pathways
responsible for cell growth and proliferation.
Quantitative and qualitative analyses of cell viability, cell cycle,
and apoptosis-inducing activity of lembehyne C were performed
using the Guava Nexin Reagent, Guava Cell Cycle, and Guava
ViaCount kits (Millipore).
The cytotoxic activity of lembehyne C in vitro against Jurkat,
HL-60, U937, and K562 human leukemia cells was studied using
the Guava ViaCount kit (Millipore). Similar to lembehyne B,
lembehyne C showed a pronounced cytotoxicity against all types
of cancer cells. However, lembehyne C had a somewhat higher
cytotoxic action than lembehyne B. The highest IC50 value of
lembehyne C was found for K562 cells (373 nM), whereas the
Table 1. Cytotoxic Activities in Vitro of Natural Lembehyne
C Measured on Tumor Cell Cultures (Jurkat, K562, HL60,
U937) (nM)
compound
Jurkat
K562
U937
HL60
1
161 32
373 57
124 19
93 11
Anticancer Activity of Lembehyne C. Cytotoxicity. The
synthesized lembehyne C (1) was investigated for the first time
for anticancer activity in vitro using human leukemia cell lines
Figure 2. Cytofluorimetric analysis of the apoptosis-inducing activity of lembehyne C in Jurkat cancer cells under the action of the test compounds in
various concentrations. (A) 400 nM; (B) 200 nM; (C) 100 nM; (D) 50 nM; (E) histogram of the phases of apoptosis for Jurkat cells treated with
lembehyne C in various concentrations (for comparison, data for lembehyne B are given). The cells were stained with annexin V/7AAD. The
incubation time was 24 h. The histogram shows the apoptosis rate (%); *P < 0.05 when compared with the control group (n = 5).
D
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Figure 3. Cell cycle phases for Jurkat cells treated with lembehyne C 1 (LC) in various concentrations: (A) 400 nM; (B) 200 nM; (C) 100 nM; (D) 50
nM; (E) diagram of the cell cycle phases; (F) histogram of the cell cycle phases for Jurkat cells treated with lembehyne C in various concentrations
[data for lembehyne B (LB) are given for comparison].2 The incubation time of lembehynes C and B with the cells was 24 h.
values for U937, HL-60, and Jurkat IC50 were 124, 93, and 161
nM, respectively.
The lembehyne C-induced apoptosis in Jurkat, HL-60, U937,
involving annexin V-based detection of phosphatidylserine
externalization on the plasmatic membrane after treatment of
cell cultures with the test compound. The effect of lembehyne C
on the induction of apoptosis is more pronounced in Jurkat cells
and K562 cells was investigated by the versatile method
E
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Figure 4. Detection of accumulation of phosphorylated H2A.X in Jurkat cells treated with lembehynes B and C. Control (intact living cells) (A); cells
treated with etoposide in 0.2 μM concentration (B); cells treated with lembehyne C (1) in 0.4 μM concentration (C); cells treated with lembehyne B in
2 μM concentration (D). The incubation with test compounds lasted for 2 h.
than in other types of cancer cells, which is in line with high
cytotoxicity of the compound against this cell line. As it is known
from literature sources, a specific feature of Jurkat cells is the
presence of a defective TP53 gene, which encodes the tumor
suppressor p53 involved in maintenance of the genome stability.
Thus, Jurkat cells are more stable to some apoptosis-inducing
agents, which initiate apoptosis by the p53-dependent
mechanism.15 As can be seen from Figure 3, treatment of Jurkat
cells with lembehyne C leads to a more pronounced dose-
dependent increase in the number of late apoptotic cells than
observed for lembehyne B. The highest percentage of early
apoptosis (78.6%) is observed when the concentration of
lembehyne B is 200 nM, whereas even at a significantly lower
concentration of lembehyne C (400 nM), a several fold lower
percentage of early apoptosis (15.86%) results, with late
apoptosis predominating (50.24%).
According to cell cycle data determined by Guava Cell Cycle
Reagent, lembehyne C is a potent inducer of hypodiploid cell
population (sub-G1 phase) in all three cell lines after the
appropriate treatment with the test compounds. For compar-
ison, the hypodiploid cell population was somewhat higher for
Jurkat cells at similar concentrations particularly in the case of
lembehyne C: 18.1% (200 nM) (15.1% for 2 μM lembehyne B).
Virtually the same trends were observed for other cancer cell
suspension cultures (HL-60, K562, and U937). Thus,
lembehyne C, like lembehyne B, is an apoptosis inducer in the
given cell lines. Moreover, in the p53-deficient Jurkat cells,
apoptosis develops in response to the treatment with lembehyne
C when incubated for 24 h.
For more in-depth understanding of the mechanisms of action
of lembehynes B and C, the possible DNA damage in Jurkat cells
was studied. The resistance to anticancer agents that induce p53-
dependent apoptosis in Jurkat cells may be caused by not only
the absence of proper p53 protein or the presence of defective
p53 but also by damage of other molecular targets involved in
signal transduction cascade or terminal stages of apoptosis.
Simultaneous detection of phosphorylated and nonphos-
phorylated H2A.X protein in the same cell sample is a sensitive
method for investigating the relationship between DNA
damage, cell cycle checkpoints, and initiation of apoptosis.
Many chemotherapeutic agents, for example, topoisomerase II
inhibitors (etoposide), damage tumor cells by generating DNA
double-strand breaks. It was also demonstrated that the γ-
H2A.X level detected by cytofluorimetry is correlated with DNA
breaks and tumor cell death.16 With increasing DNA damage,
the level of phosphorylated histone H2A.X (γH2AX) increases,
particularly at DNA breaks. The H2A.X protein also plays an
important role in DNA repair processes.17
Stimulation of Jurkat cells with the control compound, that is,
etoposide, a topoisomerase II inhibitor, for 2 h results in a
pronounced increase in the phosphorylation level of H2A.X
(Figure 4, histogram B). Analysis of Jurkat cells treated with
lembehynes B and C demonstrated that these compounds do
not affect H2A.X phosphorylation (Figure 4, histograms C and
D). It is quite obvious that lembehyne C, like lembehyne B, does
not initiate accumulation of DNA breaks followed by activation
of p53-dependent apoptosis.
F
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Figure 5. Detection of changes in the mitochondrial membrane potential (ΔΨ) in Jurkat cells treated with lembehyne C in different concentrations.
(A) Control sample containing living cells; (B) cells treated with staurosporine (200 nM); (C) dead cells (10 μL of H2O2); (D) lembehyne C (1) (200
nM); (E) 1 (400 nM). The cells were stained with MitoSense Red and 7-AAD. The incubation time was 4 h.
Next, the possible participation of lembehyne C in activation
of the mitochondrial apoptosis pathway was analyzed. For this
purpose, the effect of lembehyne C on the mitochondrial
membrane potential (ΔΨ) and formation of reactive oxygen
species (ROS) in mitochondria were investigated. The
mitochondrial regulations of apoptosis and oxidative stress are
closely interrelated, which was repeatedly demonstrated in
numerous studies. Detection of the mitochondrial stress and a
change in the mitochondrial potential (ΔΨ) in combination
with the expression of phosphatidylserine on the cell surface are
considered to be reliable signs of cancer cell apoptosis.
Estimation of the mitochondrial membrane potential (Δψ)
revealed a statistically significant increase in the Jurkat cell
population with a decreased membrane potential in samples
treated with lembehyne C, with the change in the Δψ level being
correlated with the test compound dose (Figure 5, histogram
groups D and E). Lembehyne C decreases the mitochondrial
potential of cancer cells to a minor extent; however, this effect is
dose-dependent and resembles the action of staurosporine, a
known inhibitor of most protein kinases (Figure 5, histogram B).
The percentage of apoptotic cells in lembehyne C-treated (400
nM) samples is 6.19% (3.32% of early apoptosis and 2.87% of
late apoptosis) (Figure 5E). The results attest to activation of the
mitochondrial apoptosis pathway by lembehyne C via
pronounced suppression of the oxidation and phosphorylation
in the mitochondria of the Jurkat cells. It is clear that upon longer
incubation of lembehyne C with the cancer cells, higher early
and late apoptosis values can be obtained, due to a change in the
potential difference of the mitochondrial membrane.
The highest oxidative stress (25.40%) induced in mitochon-
dria by lembehyne C was detected for the cells incubated for 2 h
(Figure 6B). For the 4 h incubation time, lembehyne C proved
to be a much more active inducer of the oxidative stress and
mitochondrial damage than classical apoptosis inducers such as
etoposide (Figure 6D) or camptothecin (Figure 6E) (13.10%
and 1.92%, respectively). Longer incubation (for 24 h) (Figure
6C) leads to a somewhat different situation: most of the cells
successively switch to apoptosis (45.78%) via the stage of
mitochondrial damage (20.58%).
To continue the study of the anticancer activity of lembehyne
C, the protein profiles of the most universal signaling cascades
such as CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3,
and STAT5, responsible for coordinated regulation of the
transcription and translation apparatus of the cancer cell and,
hence, responsible for the growth and protein synthesis function,
which appears especially important for evaluation of tumor
progression, particularly for the p53-deficient cancer cells, were
investigated. The key goal of the investigation of the above-
mentioned signaling cascades was to elucidate the effect of
lembehyne C on the cell cycle and the mechanism of the
lembehyne C anticancer action.
Generally, considering the pairwise comparison of phos-
phorylated and nonphosphorylated fractions of proteins, the
major changes for the ratio between these two fractions occur for
the Akt, p38, CREB, and p70S6K signaling pathways (Figure
7A). Signaling via RTKs (tyrosine kinase receptor) often
enhances metabolism and cell growth. ERK/MAP and Akt are
two key Ser/Thr family kinases activated by the RTK signaling,
which increase the activity of the p70 S6, Msk1, STAT3
G
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
Figure 6. Generation of reactive oxygen species and apoptotic cell death in Jurkat cells treated with lembehyne C in different concentrations and with
different incubation times: (A) control sample; (B) lembehyne C (1) (200 nM), 4 h incubation; (C) (1) (400 nM) 24 h incubation; (D) etoposide
(200 nM), 4 h incubation; (E) camptothecin (200 nM), 4 h incubation. The cells were stained with MitoSox Red and annexin V-CF488A. Q5-1,
apoptotic cells; Q5-2, both apoptotic cells and cells with clear mitochondrial damages; Q5-3, living undamaged cells; Q5-4, cells with damaged
mitochondria.
Figure 7. Histograms of the major signaling pathways in Jurkat tumor cells treated with lembehynes C (LC, 1) and B (LB). Total (A) and
phosphorylated (B) CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3, and STAT5 proteins were determined. Lembehyne C (1) was used in
concentrations of 200 nM (LC_2) and 400 nM (LC_4); lembehyne B was taken in concentrations of 1000 nM (LB_2) and 2000 nM (LB_4). The
images were drawn using xPONENT 4.2 for MAGPIX software.
(Ser727), and CREB kinases and activate other intermediates.
Other signaling pathways induced by stress or death receptor
cause activation of p38, JNK, and NF-κB. As can be seen from
Figure 7(A), lembehyne C in 400 nM concentration causes the
most pronounced decrease for all types of protein kinases in a
cancer cell. Lembehyne B (2 μM) is somewhat inferior to
lembehyne C in activity; however, it also efficiently influences
the contents of phosphorylated forms of various kinases in
comparison with the control.
In conclusion, we developed the first original Z-stereo-
selective total synthesis of a natural alkynol, lembehyne C (1),
with the new Ti-catalyzed cross-cyclomagnesiation of oxy-
genated and aliphatic 1,2-dienes with available Grignard
reagents as the key step. Also, the anticancer properties of
H
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(10.0 g, 34.0 mmol), and CH2Cl2 (52.2 mL) was stirred at 0 °C for 1 h.
The reaction mixture was filtered and the residue washed with CH2Cl2
(40 mL). The combined CH2Cl2 layer was washed with diluted HCl
(30 mL) and water (2 × 25 mL) and dried (Na2SO4), and the solvent
was removed under reduced pressure. To a solution of 7 (11.88 g, 94%)
in dry acetone (104 mL) was added LiBr (6.0 g, 69.2 mmol), and the
mixture refluxed for 3 h. Ice cold water (60 mL) was added, and the oil
that separated was removed. The aqueous layer was extracted with n-
hexane (2 × 50 mL). The n-hexane extract was combined with the oily
liquid, washed with water (20 mL), and dried over anhydrous Na2SO4.
Evaporation of the solvent, followed by column chromatography of the
residue on silica gel, using n-hexane/Et2O (95:5) as eluent, gave 8 (9.8
g, 85%) as a yellow liquid oil: 1H NMR (400 MHz, CDCl3) δ 5.59−5.52
(m, 1H), 5.41−5.35 (m, 1H), 3.38 (t, J = 7.2 Hz, 2H), 2.64 (q, J = 7.1
Hz, 2H), 2.06 (q, J = 7.2 Hz, 2H), 1.38−1.28 (m, 28H), 0.90 (t, J = 6.7
Hz, 3H); 13C NMR (100.62 MHz, CDCl3) δ 133.21, 125.72, 32.52,
31.95, 30.87, 29.72, 29.64, 29.54, 29.39, 29.30, 27.44, 22.71, 14.12;
HRMS (ESI-TOF) calcd for C20H3979Br [M + H]+ 359.2313, found
359.2315. Anal. Calcd for C20H39Br: C, 66.99; H, 10.97. Found: C,
66.93; H, 10.98.
Synthesis of Docos-5-en-1-yne (9). To a suspension of a lithium
acetylide/ethylenediamine complex (3.1 g, 36.9 mmol) in DMSO (13.6
mL) was added 8 (8.0 g, 22.2 mmol) in DMSO (11.2 mL) at 0 °C. The
reaction mixture was stirred for 12 h at 23 °C, saturated NH4Cl (10
mL) was added, and the mixture was extracted with Et2O, washed with
brine, dried over MgSO4, and concentrated. The residue was
chromatographed over silica gel (n-hexane) to afford 9 (4.5 g, 67%)
as a colorless oil: 1H NMR (400 MHz; CDCl3) δ 5.49−5.37 (m, 2H),
2.28 (m, 2H), 2.22 (m, 2H), 2.04 (q, J = 6.8 Hz, 2H), 1.94 (t, J = 2.4 Hz,
1H), 1.39−1.26 (m, 28H), 0.88 (t, J = 6.7 Hz, 3H); 13C NMR (100.62
MHz; CDCl3) δ 131.7, 127.4, 84.2, 68.2, 31.9, 29.7−29.3, 27.3, 26.4,
22.7, 18.9, 14.1; HRMS (ESI-TOF) calcd for C22H40 [M + H]+
305.3208, found 305.3202. Anal. Calcd for C22H40: C, 86.75; H,
13.24. Found: C, 86.77; H, 13.29.
lembehyne C were studied for the first time. The inhibition of
the main kinases of four signaling pathways by lembehyne C has
obviously a suppressing effect on the growth and proliferation of
cancer cells, even in the case of a p-53-deficient cancer cell line.
Lembehyne C, being an efficient inducer of apoptosis activated
via the mitochondrial pathway and an inhibitor of a number of
important signaling pathways of cell growth and proliferation,
may become a drug candidate with a high anticancer potential.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
recorded on a Stuart SMP3 apparatus. IR spectra were recorded on a
Bruker Vertex 70 V spectrometer using KBr discs over the range of
1
400−4000 cm−1. H and 13C NMR spectra were obtained using a
Bruker Ascend 500 spectrometer in CDCl3 operating at 500 MHz for
1H and 125 MHz for 13C and a Bruker AVANCE 400 spectrometer in
1
CDCl3 operating at 400 MHz for H and 100 MHz for 13C. Mass
spectra were obtained with a MALDI TOF/TOF spectrometer in an α-
cyano-4-hydroxycinnamic acid matrix. HR-ESIMS data were measured
on a Bruker maXis instrument using electrospray ionization. In
experiments on selective collisional activation (CAD) the activation
energy was set at maximum abundance of fragment peaks (see figure
legends). A syringe injection was used for solutions in MeCN/H2O,
50/50 vol % (flow rate 3 mL/min). Nitrogen was applied as a dry gas;
the interface temperature was set at 180 °C. Elemental analyses were
measured on a 1106 Carlo Erba apparatus. The purity of the
synthesized compounds was controlled using TLC on Sorbfil plates;
anisic aldehyde in HOAc was used for color development. Column
chromatography was carried out on Acrus silica gel (0.060−0.200 mm).
All solvents were dried (1,4-dioxane, THF, Et2O over Na) and freshly
distilled before use.
Synthesis of Icos-3-yn-1-ol (5). To a solution of octadec-1-yne (20 g,
80 mmol) in THF (50 mL) was slowly added EtMgBr (3.0 M in THF,
32.0 mL, 96.0 mmol). The mixture was heated under reflux for 4 h and
cooled over ice. Ethylene oxide (5.85 g, 130 mmol) was decanted into
ice cold dry THF (30 mL) and slowly added to the cooled Grignard
reagent. A slight exothermic reaction occurred. The mixture was
allowed to warm to room temperature, stirred overnight prior to
cooling over ice, and hydrolyzed by careful addition of HCl (50 mL
5%). The mixture was extracted with EtOAc (2 × 100 mL), dried over
MgSO4, and evaporated to give 5 (16.0 g, 68%) as a colorless oil: 1H
NMR (400 MHz, CDCl3) δ 3.63 (t, J = 6.4 Hz, 2H), 2.64 (s, 1H), 2.40−
2.36 (m, 2H), 2.19−2.07 (m, 2H), 1.47−1.42 (m, 2H), 1.39−1.23 (m,
26H), 0.85 (t, J = 6.7 Hz, 3H); 13C NMR (100.62 MHz, CDCl3) δ
82.32, 76.31, 61.27, 31.92, 29.70, 29.55, 29.37, 29.17, 29.01, 28.91,
23.04, 22.67, 18.70, 14.04; HRMS (ESI-TOF) calcd for C20H38O [M +
H]+ 295.3000, found 295.3006. Anal. Calcd for C22H38O: C, 81.55; H,
13.01. Found: C, 81.65; H, 13.12.
Synthesis of (Z)-Icos-3-en-1-ol (6). To a stirred mixture of
Ni(OAc)2·4H2O (10.7 g, 42.7 mmol) in EtOH (86 mL) was added
NaBH4 (1.2 g, 31 mmol) in EtOH (43 mL) at room temperature under
hydrogen. The mixture was stirred for 60 min, and ethylenediamine
(10.32g, 171.5 mmol) and alkynol 5 (12.6 g, 43 mmol) were added
sequentially. After stirring for 12 h at the same temperature under
hydrogen, the reaction mixture was concentrated and diluted with
EtOAc. The resulting mixture was filtered through a Celite pad, and the
filtrate was concentrated under reduced pressure. The residue was
purified by silica gel chromatography (n-hexane/EtOAc, 5:1) to furnish
6 (12.24 g, 96% yield) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ
5.59−5.53 (m, 1H), 5.40−5.33 (m, 1H), 3.64 (t, J = 6.6 Hz, 2H), 2.33
(q, J = 6.6 Hz, 2H), 2.09−1.96 (m, 2H), 1.27 (m, 28H), 0.89 (t, J = 6.7
Hz, 3H); 13C NMR (100.62 MHz, CDCl3) δ 133.40, 124.94, 62.26,
31.94, 30.77, 29.72, 29.57, 29.39, 29.34, 27.38, 22.70, 14.11; HRMS
(ESI-TOF) calcd for C20H40O [M + H]+ 297.3157, found 297.3152.
Anal. Calcd for C20H40O: C, 80.99; H, 13.60. Found: C, 80.94; H,
13.65.
Synthesis of (Z)-Tricosa-1,2,6-triene (10). Paraformaldehyde (1.0
g), CuI (1.26 g, 6.8 mmol), and dicyclohexylamine (75.7 mL) were
sequentially added to a solution of 9 (5.0 g, 16.4 mmol) in anhydrous
dioxane (75.7 mL). The resulting mixture was heated under reflux for 8
h, the solvent was evaporated in vacuo, 2 M HCl (50 mL) was added,
and the mixture was extracted with Et2O. The ethereal solution was
washed with NaHCO3, water, and brine and dried with anhydrous
MgSO4, and the residue was purified by silica gel column
chromatography (n-hexane/EtOAc, 30:1) to afford 10 (3.96 g, 76%)
1
as a colorless liquid: H NMR (400 MHz, CDCl3) δ 5.50−5.40 (m,
2H), 5.22−5.09 (m, 1H), 4.69 (m, 2H), 2.23−2.16 (m, 2H), 2.10−2.03
(m, 4H), 1.46−1.20 (m, 28H), 0.91 (t, J = 6.7 Hz, 3H); 13C NMR
(100.62 MHz, CDCl3) δ 208.51, 130.77, 128.63, 89.64, 74.80, 31.95,
29.72, 29.63, 29.58, 29.52, 29.38, 29.33, 29.26, 27.29, 26.85, 22.71,
14.12; HRMS (ESI-TOF) calcd forC20H38O [M + H]+ 319.3364,
found 319.3361. Anal. Calcd for C22H38O: C, 86.70; H, 13.29. Found:
C, 86.66; H, 13.28.
Cross-Cyclomagnesiation of (Z)-Tricosa-1,2,6-triene (10) and 2-
(Dodeca-10,11-dien-1-yl)-1,3-dioxolane (15a) or 2-(Dodeca-10,11-
dien-1-yloxy)tetrahydro-2H-pyran (15b) with EtMgBr in the
Presence of Mg Metal and Cp2TiCl2 Catalyst (General Procedure).
Diethyl ether (30 mL), (Z)-tricosa-1,2,6-triene (10) (1.0 g, 4.8 mmol),
2-(dodeca-10,11-dien-1-yl)-1,3-dioxolane (15a) or 2-(dodeca-10,11-
dien-1-yloxy)tetrahydro-2H-pyran (15b) (4.0 mmol), EtMgBr (40.0
mmol) (as a 1.5 M solution in Et2O), Mg powder (0.7 g, 30.0 mmol),
and Cp2TiCl2 (0.1 g, 0.4 mmol) were placed in a glass reactor with
stirring under argon (∼0 °C). The mixture was warmed to room
temperature (20−22 °C) and stirred for 6 h. The reaction mixture was
treated with a 5% solution of NH4Cl in H2O (20 mL) and extracted
with Et2O (2 × 100 mL). The combined organic phases were dried over
MgSO4 and filtered, and the solvents were removed under reduced
pressure. Silica gel column chromatography (n-hexane/EtOAc, 35:1) of
the residue gave compound 17 (1.8 g, 81%) or 18 (2.14 g, 89%) as a
pale yellow oily liquid.
Synthesis of (Z)-1-Bromoicos-3-ene (8). A mixture of CH3SO2Cl
(4.93 g, 43.0 mmol), triethylamine (8.22 mL), (Z)-icos-3-en-1-ol (6)
(11Z,15Z,19Z)-Hexatriaconta-11,15,19-trienal (17): IR νmax 3007,
1
2922, 2852, 1729, 1466, 1091, 910, 722 cm−1; H NMR (400 MHz,
I
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
CDCl3) δ 9.78 (s, 1H), 5.40−5.38 (m, 6H), 2.43 (td, J = 7.4, 1.5 Hz,
2H), 2.10−2.02 (m, 12H), 1.66−1.61 (m, 2H), 1.32−1.28 (m, 40H),
0.90 (t, J = 6.7 Hz, 3H); 13C NMR (100.62 MHz, CDCl3) δ 202.82,
130.41, 130.32, 129.60, 129.13, 129.07, 43.91, 31.93, 29.71, 29.67,
29.58, 29.48, 29.40, 29.37, 29.28, 29.17, 27.45, 27.38, 27.27, 22.69,
22.09, 14.11; HRMS (ESI-TOF) calcd for C36H66O [M + H]+
515.5191, found 515.5194. Anal. Calcd for C36H66O: C, 83.96; H,
12.92. Found: C, 83.93; H, 12.89.
2-[(11Z,15Z,19Z)-Hexatriaconta-11,15,19-trien-1-yloxy]-
tetrahydro-2H-pyran (18): 1H NMR (400 MHz, CDCl3) δ 5.40−5.38
(m, 6H), 4.59 (t, J = 3.2 Hz, 1H), 3.91−3.36 (m, 4H), 2.10−2.03 (m,
12H), 1.86−1.28 (m, 52H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100.62
MHz, CDCl3) δ 130.35, 129.58, 129.05, 98.76, 67.64, 62.20, 31.96,
30.78, 29.74, 29.70, 29.54, 29.41, 29.36, 27.46, 27.39, 27.28, 26.28,
25.54, 22.72, 19.67, 14.12; HRMS (ESI-TOF) calcd for C41H76O2 [M +
H]+ 601.5923, found 601.5925. Anal. Calcd for C41H76O2: C, 81.92; H,
12.75. Found: C, 81.89; H, 12.83.
THP deprotection of ether (18) was carried out with p-TsOH in
CHCl3 /MeOH using a known method13 to afford (11Z,15Z,19Z)-
hexatriaconta-11,15,19-trien-1-ol (19): IR νmax 2925, 2854, 2251, 1718,
1465, 1055, 909, 735 cm−1; 1H NMR (400 MHz, CDCl3) δ 5.40−5.38
(m, 6H), 3.65 (t, J = 6.8 Hz, 2H), 2.75−2.71 (m, 1H), 2.10−2.03 (m,
12H), 1.60−1.28 (m, 44H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR (100.62
MHz, CDCl3) δ 130.41, 130.37, 129.61, 129.08, 63.05, 32.82, 31.94,
29.72, 29.59, 29.45, 29.34, 27.46, 27.38, 27.27, 25.76, 22.70, 14.12;
HRMS (ESI-TOF) calcd for C36H68O [M + H]+ 517.5348, found
517.5345. Anal. Calcd for C36H68O: C, 83.64; H, 13.26. Found: C,
83.67; H, 13.33.
78.13, 77.35, 77.03, 76.71, 45.44, 31.95, 29.72, 29.60, 29.47, 29.39,
29.34, 29.29, 28.90, 27.45, 27.38, 27.27, 23.75, 22.70, 14.11; HRMS
(ESI-TOF) calcd for C38H66O [M + H]+ 539.5191, found 539.5193.
Anal. Calcd for C38H66O: C, 84.67; H, 12.35. Found: C, 84.65; H,
12.31.
The stereoselective reduction of ketone 22 with (R)-2-methyl-CBS-
oxazaborolidine was carried out according to a published procedure.14
Lembehyne C (1): colorless powder; [α]22D +0.5 (c 0.2, CHCl3); IR
νmax 3462, 3301, 2925, 2853, 2252, 2151, 1683, 1464, 1253, 1087, 909,
846, 761, 649 cm−1; yield 82% (96% ee); 1H NMR (400 MHz, CDCl3)
δ 5.41−5.39 (m, 6H), 4.39 (t, J = 5.9 Hz, 1H), 2.47 (d, J = 1.8 Hz, 1H),
2.11−1.94 (m, 12H), 1.83−1.17 (m, 44H), 0.90 (t, J = 6.6 Hz, 3H); 13C
NMR (100.62 MHz, CDCl3) δ 130.42, 130.37, 129.62, 129.12, 129.09,
85.04, 72.80, 62.33, 37.66, 31.94, 29.72, 29.68, 29.59, 29.53, 29.38,
29.35, 29.32, 29.25, 27.46, 27.39, 27.28, 25.02, 22.70, 14.12; HRMS
(ESI-TOF) calcd for C38H64O [M + H]+ 541.5348, found 541.5349.
Anal. Calcd for C38H64O: C, 84.36; H, 12.67. Found: C, 84.33; H,
12.01.
Biology. Cell Culturing. Cells (Jurkat, K562, U937, HL60) were
purchased from HPA Culture Collections (Salisbury, UK) and cultured
according to standard mammalian tissue culture protocols and sterile
technique. All cell lines used in the study were tested and shown to be
free of mycoplasma and other viral contamination. Cells were
maintained in RPMI 1640 (Jurkat, K562, U937, HL60) (Gibco)
supplemented with 4 mM glutamine, 10% FBS (Sigma), and 100 units/
mL penicillin−streptomycin (Sigma). All types of cells were grown in
an atmosphere of 5% CO2 at 37 °C. The cells were subcultured at 2−3-
day intervals. Jurkat, K562, U937, and HL60 cells were subcultured at 2-
day intervals with a seeding density of 1 × 105 cells per 24-well plate in
RPMI with 10% FBS.
The oxidation of alcohol (19) with Dess−Martin periodinane was
carried out according to a known procedure.13
Cytotoxicity Assay. Viability (live/dead) assessment was performed
by staining cells with 7-AAD (7-aminoactinomycin D) (Biolegend).
After treatment cells were harvested, washed one or two times with
phosphate-buffered saline (PBS), and centrifuged at 400g for 5 min.
Cell pellets were resuspended in 200 mL of flow cytometry staining
buffer (PBS without Ca2+ and Mg2+, 2.5% FBS) and stained with 5 μL of
7-AAD staining solution for 15 min at room temperature in the dark.
Samples were acquired on NovoCyte 2000 flow cytometry system
(ACEA) equipped with a 488 nm argon laser. Detection of 7-AAD
emission was collected through a 675/30 nm filter in FL4 channel.
Viability and Apoptosis. Apoptosis was determined by flow
cytometric analysis of annexin V and 7-AAD staining. After treatment
cells during 24 h were harvested, washed one or two times with PBS,
and centrifuged at 400g for 5 min. Cell pellets were resuspended in 200
μL of flow cytometry staining buffer (PBS without Ca2+ and Mg2+, 2.5%
FBS). Then, 200 μL of Guava Nexin reagent (Millipore, Bedford, MA,
USA) was added to 5 × 105 cells in 200 μL, and the cells were incubated
with the reagent for 20 min at room temperature in the dark. At the end
of incubation, the cells were analyzed on a NovoCyte 2000 flow
cytometry system (ACEA).
Cell Cycle Analysis. The cell cycle was analyzed using the method of
propidium iodide staining. After treatment, cells during 24 h were
harvested, washed one or two times with PBS, and centrifuged at 400g
for 5 min. Cell pellets were resuspended in 200 μL of flow cytometry
staining buffer (PBS without Ca2+ and Mg2+, 2.5% FBS). Cells were
plated in 24-well round-bottom plates at a density 10 × 105 cells per
well, centrifuged at 450g for 5 min, and fixed with ice-cold 70% EtOH
for 24 h at 0 °C. Cells were washed with PBS and incubated with 250 μL
of Guava Cell Cycle Reagent (Millipore) for 30 min at room
temperature in the dark. Samples were analyzed on NovoCyte 2000
flow cytometry system (ACEA).
Procedure for Preparation of (15Z,19Z,23Z)-1-(Trimethylsilyl)-
tetraconta-15,19,23-trien-1-yn-3-ol (20). To a solution of trimethyl-
silyl acetylene (0.183 g, 1.87 mmol) in THF (2.13 mL) was added
dropwise a solution of 0.67 mL of n-BuLi (2.5 M in n-hexane) at −78
°C. The temperature was adjusted to −20 °C, and a THF (2.13 mL)
solution of 0.529 g (1.00 mmol) of dienal (17) was added at the same
temperature. The reaction mixture was warmed to room temperature
(20−22 °C) and stirred for 3 days. The mixture was treated with a 5%
solution of NH4Cl in H2O (20 mL) and extracted with Et2O (30 mL).
The combined organic phases were dried over MgSO4 and filtered, and
the solvents were removed under reduced pressure. Silica gel column
chromatography of the residue gave compound 20 (0.55 g, 91%) as a
pale yellow solid.
(15Z,19Z,23Z)-1-(Trimethylsilyl)tetraconta-15,19,23-trien-1-yn-
3-ol (20). IR νmax 3307, 2927, 2855, 2253, 1681, 1466, 1383, 1096, 908,
1
733 cm−1; H NMR (400 MHz, CDCl3) δ 5.40−5.39 (m, 6H), 4.37
(dd, J = 12.1, 6.2 Hz, 1H), 2.11−2.03 (m, 12H), 1.84−1.28 (m, 44H),
0.90 (t, J = 6.7 Hz, 3H), 0.19 (s, 9H); 13C NMR (100.62 MHz, CDCl3)
δ 130.42, 130.38, 129.62, 129.11, 106.97, 89.25, 62.91, 37.72, 31.94,
29.72, 29.68, 29.59, 29.53, 29.38, 29.34, 29.23, 27.46, 27.38, 27.28,
25.12, 22.70, 14.13, −0.11; HRMS (ESI-TOF) calcd for C41H76OSi [M
+ H]+ 613.5743, found 613.5745. Anal. Calcd for C41H76OSi: C, 80.31;
H, 12.50. Found: C, 80.34; H, 12.53.
Procedure for the Preparation of rac-Lembehyne C (21). To a
solution of alkyne (20) (0.55 g, 0.9 mmol) in THF (3.74 mL) was
added TBAF (1 M in THF, 1.2 equv), and the solution stirred for 2 h at
room temperature. The reaction mixture was treated with saturated
aqueous NH4Cl and extracted with Et2O (2 × 10 mL). The combined
organic phases were dried over MgSO4 and filtered, and the solvents
were removed under reduced pressure. Silica gel column chromatog-
raphy of the residue gave compound 21 (0.48 g, 99%) as a colorless
waxy solid.
Histone H2A.X Analysis. Cells for phosphorylation of histone H2A.X
in Jurkat cell analysis were grown in a culture flask for 48 h. The medium
was changed 24 h before drug treatment. Phosphorylation of histone
H2A.X was measured with the FlowCellect DNA damage histone
H2A.X dual detection kit (FCCS025153, Millipore). Briefly, isolated
and treated cells were counted and diluted with assay buffer (5 × 106
cells/2.5 mL). Paraformaldehyde (PFA, Sigma) was added at a final
concentration of 2%, and cells were fixed at 37 °C for 10 min. Cells were
immediately chilled on ice, centrifuged, washed three times with 0.5%
The oxidation of the alcohol (21) with Dess−Martin periodinane
was carried out according to a published procedure.18
(13Z,17Z,21Z)-Octatriaconta-13,17,21-trien-1-yn-3-one (22): IR
νmax 3301, 2925, 2854, 2253, 2095, 1683, 1465, 1370, 1088, 909, 846,
735, 649 cm−1; 1H NMR (400 MHz, CDCl3) δ 5.38 (m, 6H), 3.21 (d, J
= 1.3 Hz, 1H), 2.60 (dd, J = 10.0, 4.7 Hz, 2H), 2.10−2.04 (m, 12H),
1.69 (m, 2H), 1.28 (m, 44H), 0.89 (t, J = 6.9, 3H); 13C NMR (100.62
MHz, CDCl3) δ 187.36, 130.38, 130.29, 129.60, 129.12, 129.06, 81.49,
J
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
bovine serum albumin (BSA; EMD Bioscience, La Jolla, CA, USA)/
PBS, permeabilized with 70% EtOH in distilled water or 90% MeOH in
0.5% BSA/PBS, and kept at 4 °C for EtOH samples and at −20 °C for
MeOH samples until further staining. After that, the cells were fixed,
permeabilized, incubated with anti-phosphohistone H2A.X (Ser139,
Alexa Fluor555, part number CS208203, Millipore) and anti-H2A.X
(PECy5, part number CS209202, Millipore), and then analyzed by flow
cytometry.
Mitochondrial Damage. A cytometric assay, which allowed
multiparametric evaluation of three cell health markerschange in
mitochondrial potential (early apoptosis and cellular stress),
phosphatidylserine expression on the cell surface (late apoptosis),
and membrane permeabilization (cell death)was performed.
Millipore’s FlowCellectMitoDamage kit use of the reagents allows
researchers to obtain information on early and late apoptosis in one
simple assay. Cells were treated with drugs at a concentration of 0.1−
0.4 μM and incubated (37 °C, 5% CO2) for 4 h. After this time, the cells
were dissociated with Accutase solution, stained, and analyzed by flow
cytometry (NovoCyte flow cytometry) according to the manufacturer’s
protocols (FlowCellect MitoDamage kit, Merck, and FlowCellect
oxidative stress characterization kit, Merck).
Multiplex Analysis of Early Apoptosis Markers. The Milliplex MAP
9-Plex multi-pathway kits, total, are used to detect total protein levels or
phosphorylated protein levels for ERK/MAP kinase 1/2, Akt, STAT3,
JNK, p70 S6 kinase, NFκB, STAT5A/B, CREB, and p38 in cell lysates
using the Luminex system. The detection assay is a rapid, convenient
alternative to Western blotting and immunoprecipitation procedures.
Each kit has sufficient reagents for one 96-well plate assay. Multiplexed
immunoassays 9-Plex multi-pathway total magnetic bead kit 96-well
plates from Millipore were used to detect changes in phosphorylated
cAMP response element-binding protein (CREB; pS133), ERK
(pT185/pY187), NF-kB (pS536), JNK (pT183/pY185), p38
(pT180/pY182), p70 S6K (pT412), STAT3 (pS727), STAT5A/B
(pY694/699), and Akt (pS473) (Milliplex 48-680MAG; Merck
Millipore, Germany) in Jurkat cell lysates lysed with RIPA buffer and
protease and phosphatase inhibitors according to the array protocol.
Before lysis in Milliplex MAP lysis buffer, the cells were challenged for 3
min with 100 ng/mL EGF and 10 nM HRG-b1 and prepared for
subsequent analysis.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the Russian Science
Foundation (Grant No. 16-13-10172) and partially for some
biological investigations by the Russian Foundation for Basic
Research (project numbers 18-29-09068, 18-33-20058). The
structural studies of the synthesized compounds were performed
with the use of Collective Usage Centre “Agidel” at the Institute
of Petrochemistry and Catalysis of RAS (grant 2019-05-595-
000-058). The biological studies of lembehyne C were done in
the Laboratory of Molecular Design and Drug Bioscreening at
the Institute of Petrochemistry and Catalysis of RAS, which was
created with the financial support of the Russian Science
Foundation.
REFERENCES
■
(1) (a) Harshad, M. J. Pharm. BioAllied Sci. 2016, 8, 83−91.
(b) Siddiq, A.; Dembitsky, V. Anti-Cancer Agents Med. Chem. 2008, 8,
132−170. (c) Zhou, Z.-F.; Menna, M.; Cai, Y.-S.; Guo, Y.-W. Chem.
Rev. 2015, 115, 1543−1596. (d) Patil, A. D.; Kokke, W. C.; Cochran, S.;
Francis, T. A.; Tomszek, T.; Westley, J. W. J. Nat. Prod. 1992, 55, 1170−
1177. (e) Watanabe, K.; Tsuda, Y.; Yamane, Y.; Takahashi, H.; Iguchi,
K.; Naoki, H.; Fujita, T.; Van Soest, R. W. M. Tetrahedron Lett. 2000,
̂
41, 9271−9276. (f) Listunov, D.; Maraval, V.; Chauvin, R.; Goenisson,
Y. Nat. Prod. Rep. 2015, 32, 49−75.
(2) Dzhemileva, L. U.; D’yakonov, V. A.; Makarov, A. A.; Andreev, E.
N.; Yunusbaeva, M. M.; Dzhemilev, U. M. Org. Biomol. Chem. 2017, 15,
470−476.
(3) D’yakonov, V. A.; Makarov, A. A.; Makarova, E. Kh.; Dzhemilev,
U. M. Tetrahedron 2013, 69, 8516−8526.
(4) (a) Aoki, S.; Matsui, K.; Tanaka, K.; Satari, R.; Kobayashi, M.
Tetrahedron 2000, 56, 9945−9948. (b) Aoki, S.; Matsui, K.; Takata, T.;
Hong, W.; Kobayashi, M. Biochem. Biophys. Res. Commun. 2001, 289,
558−563. (c) Aoki, S.; Matsui, K.; Wei, H.; Murakami, N.; Kobayashi,
M. Tetrahedron 2002, 58, 5417−5422.
(5) Adrian, J.; Stark, C. B. W. J. Org. Chem. 2016, 81, 8175−8186.
(6) D’yakonov, V. A.; Makarov Dzhemileva, L. U.; Makarova, E. Kh.;
Khusnutdinova, E. K.; Dzhemilev, U. M. Chem. Commun. 2013, 49,
8401−8403.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00261.
■
sı
(7) Dzhemilev, U. M.; D’yakonov, V. A.; Dzhemileva, L. U.;
Ishmukhametova, S. R.; Tuktarova, R. A.; Yunusbaeva, M. M.; de
Mejjere, A. J. Nat. Prod. 2016, 79, 2039−2044.
NMR data, NMR spectra, and general procedure (PDF)
(8) D’yakonov, V. A.; Islamov, I. I.; Makarov, A. A.; Dzhemilev, U. M.
Tetrahedron Lett. 2017, 58, 1755−1757.
(9) D’yakonov, V. A.; Makarov, A. A.; Dzhemileva, L. U.; Andreev, E.
N.; Dzhemilev, U. M. Chemistry Select 2017, 2, 1211−1213.
(10) D’yakonov, V. A.; Islamov, I. I.; Dzhemileva, L. U.; Khusainova,
E. M.; Yunusbaeva, M. M.; Dzhemilev, U. M. Tetrahedron 2018, 74,
4606−4612.
(11) Mohammad, I.; Mun, J.; Onorato, A.; Morton, M. D.; Saleh, A. I.;
Smith, M. B. Tetrahedron Lett. 2017, 58, 4162−4165.
(12) Hollowood, C. J.; Yamanoi, S.; Ley, S. V. Org. Biomol. Chem.
2003, 1, 1664−1675.
AUTHOR INFORMATION
Corresponding Author
Vladimir A. D’yakonov − Institute of Petrochemistry and
Catalysis of RAS (IPC RAS), 450075 Ufa, Russian Federation;
orcid.org/0000-0002-7787-5054; Phone: +7(347)
2842750; Email: DyakonovVA@gmail.com
■
Authors
Lilya U. Dzhemileva − Institute of Petrochemistry and Catalysis
of RAS (IPC RAS), 450075 Ufa, Russian Federation
Alexey A. Makarov − Institute of Petrochemistry and Catalysis of
RAS (IPC RAS), 450075 Ufa, Russian Federation
Elina Kh. Makarova − Institute of Petrochemistry and Catalysis of
RAS (IPC RAS), 450075 Ufa, Russian Federation
Evgeny N. Andreev − Institute of Petrochemistry and Catalysis of
RAS (IPC RAS), 450075 Ufa, Russian Federation
Usein M. Dzhemilev − Institute of Petrochemistry and Catalysis
of RAS (IPC RAS), 450075 Ufa, Russian Federation
(13) Brandsma, L. Synthesis of Acetylenes, Allenes and Cumulenes:
Methods and Techniques; Elsevier SPC: Amsterdam, 2004; p 470.
(14) Corey, E. J.; Bakshi, R. K.; Shibata, S. J. Am. Chem. Soc. 1987, 109,
5551−5553.
(15) Cheng, J.; Haas, M. Mol. Cell. Biol. 1990, 10, 5502−5509.
(16) Muslimovic, A.; Ismail, I. H.; Gao, Y.; Hammarsten, O. Nat.
Protoc. 2008, 3, 1187−1193.
(17) Ewald, B.; Sampath, D.; Plunkett, W. Mol. Cancer Ther. 2007, 6,
1239−1248.
(18) Meyer, S. D.; Schreiber, S. L. J. Org. Chem. 1994, 59, 7549−7552.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00261
K
https://dx.doi.org/10.1021/acs.jnatprod.0c00261
J. Nat. Prod. XXXX, XXX, XXX−XXX