Natural trienoic acids as anticancer agents: First stereoselective synthesis, cell cycle analysis, induction of apoptosis, cell signaling and mitochondrial targeting studies

Article abstract of DOI:10.3390/cancers13081808

The first Z-stereoselective method was developed for the synthesis of unsaturated acids containing a 1Z,5Z,9Z-triene moiety in 61–64% yields using the new Ti-catalyzed cross-coupling of oxygen-containing and aliphatic 1,2-dienes as the key synthetic step. It was shown for the first time that trienoic acids with non-methylene-interrupted Z-double bonds show moderate cytotoxic activities against tumor cell lines (Jurkat, K562, U937, HL60, HeLa), human embryonic kidney cells (Hek293), normal fibroblasts and human topoisomerase I (hTop1) inhibitory activity in vitro. The synthesized acids efficiently initiate apoptosis of Jurkat tumor cells, with the cell death mechanism being activated by the mitochondrial pathway. A probable mechanism of topoisomerase I inhibition was also hypothesized on the basis of in silico studies resorting to docking. The activation and inhibition of the most versatile intracellular signaling pathways (CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5 tyrosine kinases) responsible for cell proliferation and for initiation of apoptosis were studied by multiplex assay technology (Luminex xMAP).

Full text of DOI:10.3390/cancers13081808

cancers
Article
Natural Trienoic Acids as Anticancer Agents: First
Stereoselective Synthesis, Cell Cycle Analysis, Induction of
Apoptosis, Cell Signaling and Mitochondrial Targeting Studies
Vladimir A. D’yakonov * , Alexey A. Makarov, Lilya U. Dzhemileva * , Ilfir R. Ramazanov
Elina Kh. Makarova and Usein M. Dzhemilev
,
Institute of Petrochemistry and Catalysis, Russian Academy of Sciences pr. Oktyabrya 141, 450075 Ufa, Russia;
makarovalexink@yandex.ru (A.A.M.); ilfir.ramazanov@gmail.com (I.R.R.); makarovaelina87@yandex.ru (E.K.M.);
dzhemilev@anrb.ru (U.M.D.)
* Correspondence: DyakonovVA@rambler.ru (V.A.D.); Dzhemilev@mail.ru (L.U.D.);
Tel.: +7-347-284-2750 (V.A.D.)
Simple Summary: Currently, the whole world is acutely concerned with the selection of effective
treatment regimens for oncological diseases. This problem is becoming more and more catastrophic
every year due to the phenomenon of multidrug resistance, the consequence of which is the loss of
the effectiveness of drugs against tumor cells. One of the solutions to the problem described above is
the synthesis of new low molecular weight compounds that can effectively affect molecular cellular
targets, for example, enzymes of the cell cycle, and, as a consequence, interrupt DNA synthesis,
contributing to tumor death. Within the framework of this article, we carried out the Z-stereoselective
synthesis of natural unsaturated acids containing a 1Z,5Z,9Z-triene moiety, for which it was shown
that they are effective inhibitors of human topoisomerase I, and also affect mitochondria. At the
same time, using multiplex analysis, the activation of signaling pathways was studied and a probable
mechanism of the antitumor action of the synthesized trienoic acids was proposed.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: D’yakonov, V.A.; Makarov,
A.A.; Dzhemileva, L.U.; Ramazanov,
I.R.; Makarova, E.K..; Dzhemilev,
U.M. Natural Trienoic Acids as
Anticancer Agents: First
Stereoselective Synthesis, Cell Cycle
Analysis, Induction of Apoptosis, Cell
Signaling and Mitochondrial
Abstract: The first Z-stereoselective method was developed for the synthesis of unsaturated acids
containing a 1Z,5Z,9Z-triene moiety in 61–64% yields using the new Ti-catalyzed cross-coupling
of oxygen-containing and aliphatic 1,2-dienes as the key synthetic step. It was shown for the first
time that trienoic acids with non-methylene-interrupted Z-double bonds show moderate cytotoxic
activities against tumor cell lines (Jurkat, K562, U937, HL60, HeLa), human embryonic kidney cells
(Hek293), normal fibroblasts and human topoisomerase I (hTop1) inhibitory activity in vitro. The
synthesized acids efficiently initiate apoptosis of Jurkat tumor cells, with the cell death mechanism
being activated by the mitochondrial pathway. A probable mechanism of topoisomerase I inhibition
was also hypothesized on the basis of in silico studies resorting to docking. The activation and
inhibition of the most versatile intracellular signaling pathways (CREB, JNK, NFkB, p38, ERK1/2,
Akt, p70S6K, STAT3 and STAT5 tyrosine kinases) responsible for cell proliferation and for initiation
of apoptosis were studied by multiplex assay technology (Luminex xMAP).
Targeting Studies. Cancers 2021, 13,
1808. https://doi.org/10.3390/
cancers13081808
Academic Editor:
Noriyoshi Sawabata
Received: 2 February 2021
Accepted: 6 April 2021
Published: 10 April 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: Z,Z,Z-trienoic acids; cross-cyclomagnesiation; topoisomerase inhibitors; anticancer activ-
ity; apoptosis; cell signaling
Copyright:
© 2021 by the authors.
1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
According to reports and predictions of the World Health Organization, cancer dis-
eases permanently rank first among human fatal diseases and this sad statistic does not tend
to improve in the near future (Causes of Death. Available online: https://ourworldindata.
org/causes-of-death, accessed on 8 April 2021). The mortality rate caused by malignant
neoplasms is also very high; now, the worldwide average is 150 per 100,000 population. By
as soon as 2030, the number of people diagnosed with cancer may increase 1.5-fold. About
Cancers 2021, 13, 1808. https://doi.org/10.3390/cancers13081808
https://www.mdpi.com/journal/cancers

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10,000 scientific publications dealing with cancer diagnosis, treatment and prevention
appear every month, which confirms high importance and relevance of this problem.
In this regard, a necessary condition for the search for new highly effective low-toxic
drugs is the development of compounds with a clearly defined effect on the molecular
targets of carcinogenesis. In the context of the above, human topoisomerases I (hTopI) and
II (hTop2α), which are one of the key enzymes of DNA synthesis, are promising molecular
targets for new generation anticancer drugs.
Currently, quite a few compounds of various classes able to inhibit topoisomerases
are known. Some of these compounds are isolated from natural objects, e.g., camptothecin,
podophyllotoxin, anthracyclines, polyenoic acids and many other.
Among numerous classes of compounds, polyunsaturated fatty acids attract special
attention of researchers, as they are significant for the vital activity of all living organisms,
as they are precursors of potent eicosanoid bioregulators such as prostaglandins and
leukotrienes [
1
–
4], are involved in lipid metabolism [
5], exhibit antihistaminic [6] and
anti-inflammatory effects [
7], possess antitumor properties [8–10] and stimulate immune
response [11]. They are widely used for prevention and treatment of cardiovascular
diseases [11–13] and are promising for the treatment of brain diseases [14].
Among the whole diversity of polyunsaturated acids, of most interest, in our opin-
ion, are non-methylene-interrupted fatty acids (up to C34), which have unique structure
and are found in mollusks [15,16], sea sponges [17,18] and gymnosperm seeds [19,20].
The most recent studies demonstrated the efficiency of these compounds as antimicro-
bial [21], antibacterial, antimalarial, antifungal, anti-inflammatory and trypanocidal and
anti-leishmaniasis agents [7,22]. In addition, several research groups performed intense
studies of the biomedical potential of 5Z,9Z-dienoic acids acting as topoisomerase in-
hibitors. Topoisomerases are a class of enzymes called isomerases that have a marked effect
on DNA topology. Topoisomerases are able to relax supercoiled DNA molecules, thereby
affecting transcription, replication and recombination and are considered by modern phar-
macology as molecular targets for antitumor agents [23–27]. At the same time, the high
complexity of the isolation of natural 5Z,9Z-dienoic acids, as well as their extremely low
concentration in natural objects, greatly complicate and often make it almost impossible
to establish a relationship between their biological activity and structure. Earlier, in our
laboratory, we developed an original stereoselective method for the synthesis of fatty acids
with a given position of the 1Z,5Z-diene group relative to the carboxyl group. The key
step in this synthetic pathway of aliphatic and oxygen-containing 1,2-dienes was a new
catalytic reaction of intermolecular cross-cyclomagnesiation [28] (Figure 1).
Figure 1. Stereoselective synthesis of dienoic acids.
In continuation of these studies, we assumed that this method of synthesis of unsatu-
rated acids could be extended to unique trienoic acids containing a 1Z,5Z,9Z-triene moiety
in the molecule, particularly, natural acids that were found in phospholipids of the sea
anemone Stoichactis helianthus [29] (Figure 2).

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Figure 2. Natural 1Z,5Z,9Z-trienoic acids from the sea anemone S. helianthus.
The minor content of these acids in natural products and the difficulty of isolation are
the key factors that hamper investigations of their biomedical potential.
Therefore, development of stereoselective methods for the preparative synthesis of
natural trienoic acids and studies of the biological activities of the products were among
the primary goals.
2. Results
2.1. Chemistry
Our study started with the retrosynthetic analysis of the structure of trienoic acids with
non-methylene-interrupted Z-double bonds, which demonstrated that the total synthesis
can be implemented through Ti-catalyzed cross-cyclomagnesiation of (6Z)-alka-1,2,6-trienes
with tetrahydropyran ethers of allene alcohols (Scheme 1). The final step of the synthesis of
the desired acids is the Jones oxidation of the trienol tetrahydropyran ether that is formed
upon acid hydrolysis of the reaction mixture (Scheme 1).
Scheme 1. Retrosynthetic analysis of the synthesis of natural trienoic acids.
It is noteworthy that the synthesis of key monomers, (6Z)-alka-1,2,6-trienes, was
a separate task. Therefore, we developed an approach to these compounds based on
commercially available 1-bromoalkanes (1-bromodecane, 1-bromododecane) and 1-alkynes
(1-hexyne, 1-octyne, 1-decyne) (Scheme 2). This eight-step method includes the reaction
of 1-bromoalkanes 1d
sulfoxide (DMSO), which gives 1-alkynes 2d
of the resulting compounds 2d or commercially available 1-alkynes 2a
in THF and refluxing for 3 h affords magnesium acetylides 3a , which are then allowed
to react with ethylene oxide. The subsequent acid hydrolysis leads to alk-3-yn-1-ols 4a
. Then, (3Z)-alk-3-en-1-ols 5a are prepared by Ni(OAc)-catalyzed hydrogenation of
alkynols 4a–e; the resulting alcohols 5a are treated with methanesulfonyl chloride in
CH2Cl2 to afford mesylates 6a–e within 4 h. Refluxing of the latter with lithium bromide in
acetone for 3 h results in the formation of 1-bromo-(3Z)-alk-3-enes 7a in ~85% yields. At
the final stage, alkynes 8a are synthesized by the reaction of alkenyl bromides 7a with
lithium acetylide ethylenediamine complex, while the acetylene-allene rearrangement of
alkynes 8a under the Mannich reaction conditions yields the target (6Z)-alka-1,2,6-trienes
9a–e (Scheme 2).
,e
with the lithium acetylide ethylenediamine complex in dimethyl
in >90% yields. The subsequent treatment
with EtMgBr
,e
,e
–c
–e
–
e
–e
–
e
–e
–e
–e
–
e

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Scheme 2. Synthesis of (6Z)-alka-1,2,6-trienes (9a–e).
According to the synthetic route to 1Z,5Z,9Z-trienoic acids (5Z,9Z,13Z-octadecatrienoic
13a, 5Z,9Z,13Z-eicosatrienoic 13b, 5Z,9Z,13Z-docosatrienoic 13c, 9Z,13Z,17Z-triacontatrienoic
13d, 11Z,15Z,19Z-dotriacontatrienoic 13e, 11Z,15Z,19Z-triacontatrienoic 13f and 13Z,17Z,21Z-
dotriacontatrienoic 13g acids) (Scheme 3), developed on the basis of retrosynthetic analy-
sis, we carried out intermolecular cross-cyclomagnesiation of (6Z)-alka-1,2,6-trienes 9a–e
((6Z)-undeca-1,2,6-triene 9a, (6Z)-trideca-1,2,6-triene 9b, (6Z)-pentadeca-1,2,6-triene 9c, (6Z)-
heptadeca-1,2,6-triene 9d, (6Z)-nonadeca-1,2,6-triene 9e) with allene alcohol tetrahydropyran
ethers 10a-d on treatment with EtMgBr in the presence of Mg (powder) and catalytic amounts
of Cp2TiCl2 (9a
acid hydrolysis of magnesacyclopentanes 11a
12a with 1Z,5Z,9Z-triene moieties in 81–89% yields (Scheme 3). Finally, the Jones oxidation
of ethers 12a–g furnished the target acids 13a–g in 61–64% yields (Scheme 3).
–e:10a–d
:EtMgBr:Mg:[Ti] = 12:10:36:24:0.1, Et2O, 20–22 ◦C, 10 h). The in situ
–g thus formed gave tetrahydropyran ethers
–g
Scheme 3. (Z,Z,Z)-Stereoselective synthesis of trienoic acids 13a–g.
An unusual fact is that a mixture of alkyl-substituted allene with the tetrahydropyran
ether of allene alcohol selectively gives the cross-coupling product under the reaction
conditions. Meanwhile, these allene derivatives taken in a pure state without the partner
are smoothly converted to homo-coupling products. This intriguing feature of this system,
which allowed us to successfully prepare dienoic and trienoic acids, can be explained in
the following way:

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Titanocene generated upon the reduction of titanocene dichloride is a coordinatively
unsaturated low-valence titanium complex. In our opinion, because of the coordina-
tive unsaturation, titanocene forms a stronger complex with the bidentately coordinated
tetrahydropyran ether of allene alcohol than with alkyl-substituted allene. The complex
thus formed is already largely coordinatively saturated because of the strong binding of
titanium to the allene double bond. Therefore, this complex would react at a higher rate
with more nucleophilic alkyl-substituted allene. Meanwhile, the presence of oxygen in the
tetrahydropyran ether of allene alcohol decreases the double bond nucleophilicity and,
hence, reactivity towards the addition of the second allene molecule to titanocene. Thus,
the formation of the cross-coupling product is quite expectable, if one takes into account
the nature and the energy of the frontier orbitals of the intermediate titanocene (Scheme 4).
Scheme 4. Putative scheme of the predominant formation of cross-cyclomagnesiation products from
aliphatic and oxygen-containing allenes.
Thus, the key point is the original (Z,Z,Z)-stereoselective total synthesis of trienoic
acids, which contain the 1Z,5Z,9Z-triene system, developed by us, using a new Ti-catalyzed
cross-cyclomagnetation 1,2-dienes with readily available Grignard reagents. This method
is an important step for the development of new highly effective anticancer drugs.
2.2. Biological Evaluation
2.2.1. Cytotioxic Activity In Vitro
Trienoic acids 13a–g, which we synthesized, were evaluated for antitumor activity
in vitro using the Jurkat, K562, U937, HL60, HeLa and HEK293 cell lines and normal
fibroblasts (Table 1). The assays included determination of IC50 by flow cytofluorimetry.

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Table 1. Cytotoxic activity in vitro of trienoic acids 13a–g (48 h).
Jurkat,
IC50, µM
K562,
IC50, µM
U937,
IC50, µM
HL60,
IC50, µM
HEK293,
IC50, µM
HeLa,
IC50, µM
Fibroblasts,
IC50, µM
Acids
13a
13b
13c
13d
13e
13f
13g
0.178 ± 0.022 0.211 ± 0.021 0.165 ± 0.019 0.159 ± 0.014 0.617 ± 0.054 0.574 ± 0.052 1.119 ± 0.104
0.147 ± 0.015 0.172 ± 0.016 0.139 ± 0.013 0.127 ± 0.011 0.523 ± 0.049 0.489 ± 0.041 0.937 ± 0.086
0.105 ± 0.009 0.136 ± 0.012 0.098 ± 0.010 0.086 ± 0.008 0.451 ± 0.039 0.311 ± 0.027 0.754 ± 0.069
0.448 ± 0.043 0.479 ± 0.046 0.456 ± 0.041 0.409 ± 0.037 1.297 ± 0.114 1.029 ± 0.091 1.738 ± 0.154
0.421 ± 0.038 0.467 ± 0.043 0.394 ± 0.037 0.387 ± 0.039 1.082 ± 0.093 0.983 ± 0.086 1.597 ± 0.139
0.454 ± 0.046 0.471 ± 0.051 0.461 ± 0.043 0.404 ± 0.040 1.211 ± 0.106 1.087 ± 0.094 1.914 ± 0.176
0.122 ± 0.011 0.154 ± 0.014 0.117 ± 0.010 0.104 ± 0.009 0.487 ± 0.046 0.354 ± 0.032 0.739 ± 0.068
Results are expressed as mean ± SE (n = 3). Data are presented as mean plus standard error p > 0.01.
The assays showed that trienoic acids 13a–g exhibit a cytotoxic effect against the
chosen cell lines, which is comparable with or somewhat higher than the effect of the
previously synthesized 5Z,9Z-dienoic acids. Acid 13c had the highest cytotoxicity against
all of the cell lines chosen for the assays. The selectivity index (SI) of the therapeutic
and cytotoxic action for the Jurkat, K562, U937 and HL60 cell lines was 4–9 times higher
than that for normal fibroblasts and conditionally normal human embryonic kidney cells
Hek293 (Table 1).
2.2.2. Apoptosis and Cell Cycle Research
In accordance with the results of the cytotoxicity of the studied compounds, we more
deeply assessed the ability of compound 13c to induce apoptosis in Jurkat cell culture and
to influence the phases of the cell cycle (Figure 3).
Figure 3. Cont.

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Figure 3. Analysis of the induction of apoptosis in Jurkat cells after exposure to compound 13c
in various concentrations. (
13c in 0.2 M concentration; (
0.05 M concentration; ( ) compound 13c in 0.025
A
) control sample untreated with the test compound; (
) compound 13c in 0.1 M concentration; ( ) compound 13c in
M concentration; ( ) histogram of apoptosis
B) compound
µ
C
µ
D
µ
E
µ
F
phases in Jurkat cells treated with compound 13c. The cells treated with compound 13c in various
concentrations were stained with Annexin V/7-AAD. The incubation time of compound 13c with the
cells was 24 h.
The effect of compound 13c resulted in apoptosis of Jurkat cells and this effect was com-
parable to that of camptothecin and etoposide, the popular cytostatic agents in medicine.
The highest rate of apoptosis in Jurkat cells (71.41%) was the exposure to test compound
13c at a concentration of 0.2 µM and a concentration of 0.025 µM caused the lowest per-
centage of Jurkat cells in the late apoptosis stage (6.56%) (Figure 3). High activity of
compound 13c towards live cells may be attributable to the presence of cis-double bonds in
the molecule because, as already known, these compounds show high inhibitory activity
against topoisomerase I.
Jurkat cells in the control sample show the balance of all phases of the cell cycle
with a predominance of cells in the G0–G1 phase (Figure 4). The analysis of the cell cycle
with the addition of compound 13c to the Jurkat cell culture demonstrates a significant
predominance of the population of hypodiploid cells (sub-G0-G1), a dramatic increase
in the S-phase and a pronounced decrease in the percentage of G2 + M cells, which
corresponds to the standard picture of the effect of classical cytostatics-camptothecin and
etoposide. In summary, it can be argued that the compound 13c has antitumor activity
against cell T cell leukemia caused by apoptosis-inducing activity.

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Figure 4. Cytofluorometric analysis of cell cycle phases (
A
–
E) in Jurkat tumor cells treated with com-
pound 13c. The cells treated with various concentrations of 13c and stained with PI. The incubation
time of compound 13c with the cells was 24 h. ( ) Control sample untreated with the test compound;
) Compound 13c in 0.1 M concentration; ( ) Com-
) Compound 13c in 0.025 M concentration; ( ) Histogram
SE
A
(B
) Compound 13c in 0.2
µ
M concentration; (
C
µ
D
pound 13c in 0.05
µ
M concentration; (
E
µ
F
of cell cycle phases in Jurkat cells treated with compound 13c. Results are expressed as mean
(n = 3).
±
2.2.3. Topoisomerase I Inhibition Assay and Molecular Docking Studies
The ability of trienoic acids 13a
–g (Table 2) to interact with topoisomerase I in vitro
with the formation of relaxation of the supercoiled plasmid pHOT1 was studied (Figure 5).

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Table 2. Inhibitory activity of trienoic acids 13a–g in vitro.
Acids
13a
13b
13c
13d
13e
13f
13g
0.4
hTopI inhibition, µM
0.2
0.2
0.1
0.8
0.6
0.8
Figure 5. Electrophoretic separation in 2% agarose gel of the relaxation products of 250 ng of plasmid
DNA (pHOT1) in vitro under the action of 4 U of topoisomerase I (TG-1018-2 Topoisomerase I Drug
Screening Kit, Topogen, Buena Vista, CO, USA) in the presence of natural 5Z,9Z,13Z-docosatrienoic
acid 13c (the test compounds were added before the addition of topoisomerase I). (Lane 1) Relax
DNA; (Lane 2) Supercoiled DNA; (Lane 3) 13c, 0.1
µM; (Lane 4) 13c, 0.05 µM; (Lane 5) 13c, 0.02 µM;
(Lane 6) No compound; (Lane 7) Camptotecin, 0.5 µM.
The data of this study, showing the dependence of the amount of relaxed DNA on the
structure of the obtained trienoic acids, will serve as further chemical modifications that
will significantly improve the chemotherapeutic properties of these fatty acids (Figure 5).
The addition of trienoic acids to the relaxation reaction of supercoiled DNA at a con-
centration of IC50 makes it possible to detect a gradual decrease in the formed topoisomers
and, as a consequence, to observe an increase in the open circular form of the plasmid,
which directly indicates a decrease in the enzyme activity. When no test compound was
added, no such effect was observed (Figure 5, line 6). All seven unsaturated acids we
prepared behaved almost identically in the given concentration range; they started to in-
hibit topoisomerase I when present in concentrations below 0.1–0.8
activity against human topoisomerase I was found for 5Z,9Z,13Z-docosatrienoic acid 13c
in concentrations above 0.1 M. Thus, we found that even micromolar concentrations of
µM. A high inhibitory
µ
compounds 13a–g are able to suppress the catalytic activity of topoisomerase I.
Molecular dynamics studies of the mechanism of topoisomerase I inhibition by eicos-
apentaenoic acid (cEPA) showed that the K443, K587 and N722 residues are the preferable
binding sites (Figure 6) [30]. Presumably, the interaction of the carboxylate group of unsat-
urated carboxylic acids with the above-mentioned sites of Topo I gives a stable complex,
which prevents the catalytic center at Y723 from performing a nucleophilic attack on the
DNA phosphate. Similar results were obtained by molecular docking for the mechanism
of topoisomerase I inhibition by unsaturated cholesterol-containing acids and synthetic
analogues of natural 5Z,9Z-dienoic acids [27
,31]. It can be assumed that synthetic Z,Z,Z-
trienoic acids 13a should inhibit human Topo I by similar mechanisms. A truncated
–
g
hTopI form (70 kDa) complexed with the 22-pair duplex oligonucleotide (PDBID 1A36),
from which the duplex oligonucleotide fragment and water molecules were removed,
served as the initial model for the molecular docking (Figure 6). The interaction of synthetic
Z,Z,Z-trienoic acids 13a
(Glide XP). We found that the preferable binding sites for the carboxylate group in the com-
pounds 13a are the K443, K587 and N722 residues. The docking score calculated by the
OPLS3 (Glide XP) method increases in the following series of compounds: 13b 7.5) < 13c
–g with the 1A36 fragment were analyzed using the OPLS3 method
–g
(
−
(−6.8) < 13e (−5.9) < 13g (−5.1) < 13a (−4.6) < 13d (−4.4) < 13f (−2.9), which is generally
correlated with the experimentally observed activities of the synthetic Z,Z,Z-trienoic acids.

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Figure 6. Ligand interaction diagram for acids 13b (A), 13c (B) and 13f (C).
2.2.4. Studying the Effect of Trienoic Acids on Mitochondria
Mitochondria are extremely important as the main organelles that ensure the energy
balance of the cell. Moreover, they regulate a number of apoptotic and proapoptotic
proteins, are the main source of ATP and interact with reactive oxygen species (ROS).
Today mitochondria are key organelles involved in the pathogenesis of many forms of
cancer, inflammatory and neurodegenerative processes [32].
The mitochondrial regulation of apoptosis and permeability of mitochondrial mem-
brane upon variation of the potential difference between the inner and outer membrane
parts are closely interrelated, which has been demonstrated by a large number of research
groups all over the world. According to Mitchell’s theory, the proton electrochemical
potential ∆Ψ, arising during the transfer of electrons through the inner membrane, deter-
mines the interaction of oxidation and phosphorylation processes, the conjugation of which
serves as a source of energy for the formation of adenosine triphosphate [33]. Various
changes in ∆Ψ detected by the MitoSense Red dye, in combination with the externalization
of phosphatidylserine on the cell membrane surface, is a reliable sign of apoptosis due to
mitochondrial damage and the formation of ROS ions. The expression of phosphotidylser-
ine on the membrane is assessed by the binding of annexin V and the permeability of the
plasma membrane to the 7AAD dye is a clear indicator of apoptosis in the cell. Multivariate
analysis carried out using three dyes—annexin V Alexa fluor 488, 7AAD and MitoSense
Red—makes it possible to clearly establish the event of apoptosis depending on damage to
the mitochondrial membrane and dysfunction of mitochondria.
The change of potential (∆Ψ) in Jurkat cells after treatment with compound 13c was
assessed using the fluorescent cationic dye MitoSense Red, which selectively accumulates
only in intact mitochondria of living cells and does not penetrate into dead objects.
As a result of the experiment, it was found that in the samples treated with compound
13c, the number of cells with damaged membrane potential (∆Ψ) increased dramatically
(84.18%), while when treated with staurosporin, this number of cells was 58.15% (Figure 7,
histograms B and C). Out of all trienoic acids, compound 13c induced the most pronounced
decrease in the mitochondrial potential; moreover, this effect from compound 13c was
dose-dependent and significantly exceeded the values of cells treated with staurosporin, a
well-known inhibitor of protein kinases (Figure 7, histogram B). The percentage of apop-
totic cells in the samples treated with compound 13c (0.079 µM) was 48.24% (40.00% early
apoptosis and 8.24% late apoptosis, respectively) (Figure 7, histogram C). The results indi-
cate that compound 13c activates the mitochondrial pathway of apoptosis via disjunction
of oxidation and phosphorylation pathways in the mitochondria of Jurkat cells.

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Figure 7. Cont.

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Figure 7. Changes in the mitochondrial potential (∆Ψ) and related early and late apoptosis in Jurkat
cells induced by compound 13c. (A) Control sample containing live cells; (B) Staurosporine (1 µM);
(C
)
13c (0.08 M); ( ) Histogram showing the percentage of damaged mitochondria and phases of
µ
D
apoptosis. The cells were stained by MitoSense Red, Anexin V, CF488A and 7-AAD. The incubation
time was 4 h. Results are expressed as mean ± SE (n = 3).
2.2.5. Cytochrome C Release from Mitochondria
An important role of mitochondria in apoptosis is associated with the release of
the transmembrane protein cytochrome C, which is an activator of procaspase-9 [34].
Cytochrome c is a soluble heme protein that carries electrons in oxidative phosphorylation
during respiration and transfers electrons from the cytochrome bc1 complex to cytochrome
oxidase on the surface of the inner mitochondrial membrane [35]. Dissipative changes
in the transmembrane potential of mitochondria (∆Ψm) indicate the appearance of signs
of apoptosis in the cell [36]. Flow cytometry using detection of mitochondrial potential
dissipation and cytochrome release into the cytoplasm is the method of choice for this
analysis and proves the mitochondrial pathway of apoptosis in cells [37].
A quantitative assessment of the release of cytochrome c from the mitochondria
of cells with apoptosis was used to detect the mitochondria-dependent pathway of cell
death using flow cytometry. We used labeled antibodies against cytochrome c FITC,
control of the isotype anti-IgG1-FITC, as well as optimized fixation, permeabilization and
blocking buffer, allowing the detection of cytochrome c by flow cytometry. In the work,
buffers were used to achieve selective mitochondrial permeability, while simultaneously
leaving the mitochondrial membrane intact. Viable or living cells show higher levels of
cytochrome c fluorescence, while apoptotic cells that have released their cytochrome c from
the mitochondria into the cytoplasm will show a reduced staining intensity when probed
with an anti-cytochrome c antibody FITC.
The detection of cytochrome c-negative cells in samples incubated with the test com-
pound directly attests to the loss of cytochrome c with mitochondria and, as a consequence,
to the intrinsic pathway of apoptosis. The histograms depicted in Figure 8 show the
downward changes for the cytochrome c fluorescence for the cells incubated with test
compound 13c in a concentration of 0.08 µM. The assay was carried out using the FlowCel-
lect Cytochrome c Kit for flow cytofluorimetry. The histograms show live cells (negative
control) stained with cytochrome c-FITC and demonstrating a high level of fluorescence
(Figure 8, histograms 1). These cells had intact mitochondria. In the cells that show reduced
fluorescence, the membrane has been damaged by the test compound, resulting in the
loss of cytochrome c. Comparison of the effects of staurosporine and the test compound
13c shows that in 2 h, compound 13c induces the loss of 94.93% of cytochrome c from
all cells, while in the case of staurosporine, only 80% of cytochrome c is lost (Figure 8,
histograms 2, 3).

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Figure 8. Loss of cytochrome c from the cells after incubation with trienoic acid 13c. (
1) untreated Jurkat cells; (2) cells
treated with staurosporine (0.1 µM); (3) cells treated with compound 13c. The incubation time was 2 h.
2.2.6. Major Kinases and Their Phosphorylation Status of Nine Signaling Pathways for Cell
Growth and Proliferation
Nine major kinases of signaling pathways responsible for cell growth and differen-
tiation were studied in Jurkat tumor cells incubated with novel trienoic acid 13c. The
study was carried out using multiplex analysis using Luminex xMAP technology. This
technology makes it possible to simultaneously detect in the same experiment a number
of protein analytes per unit of time, which significantly reduces the probability of errors
associated with sample preparation. This method is based on the use of special polystyrene
spheres coated with antibodies with special fluorophores, followed by laser detection and
digital processing. In our experiment, the following kinases were analyzed: CREB, JNK,
NFkB, p38, ERK1/2, Akt, p70S6K, STAT3 and STAT5, more precisely, their phosphorylated
and non-phosphorylated forms in cells. Often, in many tumors, it is the activation of these
kinases, all at once or only some, that gives the cell immortality, completely excluding the
Hayflick limit.
We performed a pairwise comparison of all nine kinases CREB, JNK, NFkB, p38,
ERK1/2, Akt, p70S6K, STAT3 and STAT5 in an inactive and phosphorylated state in three
samples of Jurkat cell lysate (samples containing 0.04 µM and 0.08 µM test compound and
control). All of these samples have been tested using Luminex Assay (MILLIPLEX® MAP
9-Plex Multi-Pathway 9-plex Magnetic Bead Kit) (Figure 9).
Pairwise comparison of the active and inactive forms of the protein shows that the
most significant changes are detected for two signaling pathways, namely Akt and p38
(Figure 9). Kinases ERK/MAP and Akt are two key families of serine-threonine kinases
Ser that are activated by RTK signals and, in turn, activate the kinases p70S6, Msk1, STAT3
(Ser727) and CREB. In addition, the simultaneous activation of FAS receptors or other
stressful stimuli generated in response to intracellular signaling can cause the activation of
p38, JNK and NF-kB. Figure 9 shows that compound 13c significantly reduces all types of
kinase proteins at a concentration of 0.08 µM compared to the control sample. Obviously, a
slight increase in the phosphorylated form of p38 is a response to stress exposure of the test
compound. Akt kinase is a key enzyme of the PI3K/Akt signaling pathway involved in cell
growth and differentiation [38]. In recent years, this kinase has been attributed an important
role as a pro-oncogene and a participant in oncogenic cell transformation [39]. In Figure 9,
it is plainly seen that the phosphorylated Akt kinase fraction considerably decreases, with
this decrease being dose-dependent, in comparison with the concentration of this kinase in
the control sample. Akt is an important component of phosphatidylinositol-3-kinase (PI3K)
signaling pathway; it has numerous substrates and promotes signal transduction along this
pathway. The PI3K and Akt kinases are important targets for the therapeutic action [40].

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Figure 9. Effect of compound 13c on intracellular signaling (CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3) through
the activation of tyrosine kinases of the main pathways of cell proliferation and differentiation. Phosphorylation levels of key
members of the 9 most important signaling pathways were determined using the MILLIPLEX® MAP 9-Plex Multi-Pathway
9-plex Magnetic Bead Kit (Luminex) in protein harvested from control cells and cells treated with the substance after 24 h of
incubation (Jurkat cells were used). Phosphorylation levels in control HeLa cells were taken as 100%. Phosphorylation
levels were adjusted for protein load by the levels of total protein (non-phosphorylated) determined using the 9-Plex
Multi-Pathway Total Magnetic Bead Kit (Luminex) in the same sample. Data are presented as mean plus standard error
(* p > 0.05, ** p > 0.01, *** p > 0.001).
P38 is a MAPK kinase, that is, a serine threonine protein kinase activated by numerous
external stimuli towards signal transduction from the membrane surface to the nucleus.
The MAPK signaling pathway kinases are the central components of the Ras/ERK/MAPK
cascade responsible for differentiation and cell growth as well. The Ras and Raf proteins are
important prognostic markers of tumor diseases and promising targets for the therapy [41].
3. Materials and Methods
3.1. Chemistry
IR spectra were recorded on Bruker VERTEX 70V using KBr discs over the range
1
of 400–4000 cm−1. H and 13C NMR spectra were obtained using a Bruker Ascend 500
1
spectrometer in CDCl3 operating at 500 MHz for H and 125 MHz for 13C and Bruker
AVANCE 400 spectrometer in CDCl3 operating at 400 MHz for 1H and 100 MHz for 13C.
High resolution mass spectra (HRMS) were measured on a Bruker maXis instrument
using electrospray ionization (ESI). In experiments on selective collisional activation (CAD)
activation energy was set at maximum abundance of fragment peaks (see figures legend). 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; 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 AcOH 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.

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3.2. Cell Culturing
Cell culturing were carried out following the known procedure [42]. Cells (Jurkat,
K562, U937, HL60, HeLa, HEK293 and normal fibroblasts) were purchased from HPA
Culture Collections (Salisbury, UK) and cultured according to standard mammalian tissue
culture protocols and sterile technique.
3.3. DNA Topoisomerase I Assay
Inhibition of DNA topoisomerase I tests were carried out following the known proce-
dure [25]. Human topoisomerase I inhibition studies were carried out using commercially
available Topoisomerase I Drug Screening Kit (TG-1018-2, Topogen, CO, USA).
3.4. Cytotoxicity Assay
Cytotoxicity tests were carried out following the known procedure [42]. Viability
(live/dead) assessment was performed by staining cells with 7-AAD (7-Aminoactinomycin
D) (Biolegend). After treatment cells were harvested, washed 1–2 times with phosphate-
buffered saline (PBS) and centrifuged at 400 g 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 NovoCyteTM 2000 FlowCytometry System (ACEA) equipped
with 488 nm argon laser. Detection of 7-AAD emission was collected through a 675/30 nm
filter in FL4 channel.
3.5. Viability and Apoptosis
Induction of apoptosis tests were carried out following the known procedure [42]. Apop-
tosis was determined by flow cytometric analysis of Annexin V and 7-aminoactinomycin
D staining. After treatment cells during 24 h were harvested, washed 1–2 times with
phosphate-buffered saline (PBS) and centrifuged at 400 g for 5 min. Cell pellets were
resuspended in 200
2.5% FBS). Then, 200
added to 5
105 cells in 200
at room temperature in the dark. At the end of incubation, the cells were analyzed on
NovoCyteTM2000 FlowCytometry System (ACEA).
µ
L of flow cytometry staining buffer (PBS without Ca2+and Mg2+,
L of Guava Nexin reagent (Millipore, Bedford, MA, USA) was
L and the cells were incubated with the reagent for 20 min
µ
×
µ
3.6. Cell Cycle Analysis
Cell cycle analysis was carried out following the known procedure [42]. Guava Cell
Cycle Reagent (Millipore) was used. Samples were analyzed on NovoCyteTM 2000Flow-
Cytometry System (ACEA).
3.7. Mitochondrial Damage
Mitochondrial damage tests were carried out following the known procedure [42]. Cy-
tometric assay (Millipore’s FlowCellect™MitoDamage Kit) 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 used.
3.8. Histone H2A.X Analysis
Histone H2A.X analysis was carried out following the known procedure [42]. Cell
preparation for phosphorylation of Histone H2A.X in Jurkat cells analysis were grown in
culture flask at 48 h. The medium was changed at 24 h before drug treatment. Phospho-
rylation of histone H2A.X was measured with the FlowCellect™ DNA Damage Histone
H2A.X Dual Detection Kit (FCCS025153, Millipore, MA, USA).

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3.9. Cytochrome C Release Analysis
The commercially available Millipore FlowCellect
™
Cytochrome c kit measures the
loss of mitochondrial cytochrome c in cells in which apoptosis has been induced. The kit
includes a directly labeled anti-cytochrome cFITC antibody, an anti-IgG1-FITC isotype
control, as well as buffers for fixation and permeabilization of cells, allowing the detection
of cytochrome by flow cytometry. Viable or living cells show higher levels of fluorescence
of cytochrome c and the mitochondria themselves, while apoptotic cells that have released
their cytochrome c from the mitochondria into the cytoplasm will show a reduced intensity
of staining when probed with an anti-cytochrome c FITC antibody. The incubation time of
the cells with the test substances was 4 h.
3.10. Multiplex Analysis of Early Apoptosis Markers
Multiplex analysis was carried out following the known procedure [42]. The MILLIPLEX®
MAP 9-Plex Multi-Pathway Kits, total, are used to detect total protein levels or phospho-
rylated 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-PathwayTotal Magnetic Bead Kit 96-well Platefrom 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.
3.11. Chemical Experimental Data
3.11.1. Procedure for Preparation of Alka-3-in-1-ols (4a–e)
To a solution of alk-1-yne (80 mmol) in THF (50 mL) was slowly added ethyl magne-
sium bromide (3.0 M in THF, 32.0 mL, 96.0 mmol). The mixture was heated under reflux for
a further 4 h then 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 exotherm
was observed on this addition. The mixture was allowed to warm to room temperature
then stirred overnight prior to cooling over ice then hydrolyzed by careful addition of HCl
(50 mL 5%). The mixture was extracted with EtOAc (2
evaporated to give 4a–e as a colorless oil.
×
100 mL), dried over MgSO4 and
Oct-3-yn-1-ol (4a)
1
88% yield. H NMR (CDCl3, 400 MHz)
δ 3.67 (2H, t, J = 6.4 Hz), 2.44–2.42 (2H, m),
2.18–2.11 (2H, m), 1.49–1.39 (4H, m), 0.91 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
82.65 (C-4), 76.26 (C-3), 61.37 (C-1), 31.06 (C-6), 23.15 (C-2), 21.94 (C-7), 18.40 (C-5), 13.57
δ
(C-8); anal. calcd for C8H14O: C, 76.13; H, 11.18; found: C, 76.09; H, 11.11; HRMS (ESI-TOF):
calcd for C8H14O [M + H]+127.2041; found 127.2038.
Dec-3-yn-1-ol (4b)
1
85% yield. H NMR (CDCl3, 400 MHz)
δ 3.66 (2H, t, J = 6.4 Hz), 2.44–2.40 (2H, m),
2.18–2.13 (2H, m), 1.52–1.24 (8H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
82.65 (C-4), 76.27 (C-3), 61.35 (C-1), 31.33 (C-8), 28.94 (C-7), 28.54 (C-6), 23.13 (C-2), 22.52
δ
(C-9), 18.71 (C-5), 13.99 (C-10); anal. calcd for C10H18O: C, 77.86; H, 11.76; found: C, 77.69;
H, 11.63; HRMS (ESI-TOF): calcd for C10H18O [M + H]+155.2572; found 155.2568.

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Dodec-3-yn-1-ol (4c)
1
79% yield. H NMR (CDCl3, 400 MHz)
δ
3.59 (2H, t, J = 6.4 Hz), 2.36–2.32 (2H, m),
2.10–2.06 (2H, m), 1.46–1.22 (12H, m), 0.82 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ
82.14 (C-4), 76.34 (C-3), 61.23 (C-1), 31.79 (C-10), 29.15 (C-7), 29.07 (C-9), 28.96 (C-8), 28.84
(C-6), 22.98 (C-2), 22.58 (C-11), 18.65 (C-5), 13.95 (C-12); anal. calcd for C12H22O: C, 79.06;
H, 12.16; found: C, 78.91; H, 12.02; HRMS (ESI-TOF): calcd for C12H22O [M + H]+183.3104;
found 183.3100.
Tetradec-3-yn-1-ol (4d)
1
83% yield. H NMR (CDCl3, 400 MHz)
δ 3.68 (2H, t, J = 6.4 Hz), 2.46–2.41 (2H, m),
2.21–2.14 (2H, m), 1.53–1.27 (16H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
82.72 (C-4), 76.25 (C-3), 61.37 (C-1), 31.89 (C-12), 29.57 (C-8), 29.52 (C-10), 29.30 (C-9),
δ
29.14 (C-11), 29.00 (C-8), 28.89 (C-6), 23.17 (C-2), 22.66 (C-13), 18.72 (C-5), 14.06 (C-14); anal.
calcd for C14H26O: C, 79.93; H, 12.45; found: C, 79.85; H, 12.33; HRMS (ESI-TOF): calcd for
C14H26O [M + H]+211.3635, found 211.3630.
Hexadec-3-yn-1-ol (4e)
1
89% yield. H NMR (CDCl3, 400 MHz)
δ 3.68 (2H, t, J = 6.4Hz), 2.45–2.40 (2H, m),
2.18–2.13 (2H, m), 1.52–1.27 (20H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
82.71 (C-4), 76.26 (C-3), 61.36 (C-1), 31.92 (C-14), 29.67 (C-9), 29.64 (C-7, C-11), 29.54 (C-12),
δ
29.35 (C-13), 29.16 (C-10), 29.00 (C-8), 28.91 (C-6), 23.16 (C-2), 22.68 (C-15), 18.73 (C-5),
14.09 (C-16); anal. calcd for C16H30O: C, 80.60; H, 12.68; found: C, 80.52; H, 12.60; HRMS
(ESI-TOF): calcd for C16H30O [M + H]+239.4167, found 239.4161.
3.11.2. Synthesis of (Z)-alk-3-en-1-ols (5)
To a stirred mixture of Ni(OAc)2
added NaBH4 (1.2 g, 31 mmol) in ethanol (43 mL) at room temperature under hydrogen.
The mixture was stirred for 60 min and ethylenediamine (10.32 g, 171.5 mmol) and alkynol
×
4H2O (10.7 g, 42.7 mmol) in ethanol (86 mL) was
4a–e (43 mmol) were added sequentially. After stirring for 12 h at the same temperature
under hydrogen, the reaction mixture was concentrated and diluted with ethyl acetate. 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/ethyl
acetate 5:1) to furnish 5a–e as a colorless oil.
(Z)-Oct-3-en-1-ol (5a)
1
96% yield. H NMR (CDCl3, 400 MHz)
δ
5.57–5.26 (2H, m), 3.68 (2H, dt, J = 6.6 Hz),
2.37–2.29 (2H, m), 2.08–2.02 (2H, m), 1.35–1.20 (4H, m), 0.89 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.23 (C-4), 124.97 (C-3), 62.22 (C-1), 31.84 (C-6), 30.72 (C-2), 27.03
δ
(C-5), 22.31 (C-7), 13.90 (C-8); anal. calcd for C8H16O: C, 74.94; H, 12.57; found: C, 74.87; H,
12.49; HRMS (ESI-TOF): calcd for C8H16O [M + H]+129.2199, found 129.2192.
(Z)-Dec-3-en-1-ol (5b)
1
91% yield. H NMR (CDCl3, 400 MHz)
δ 5.62–5.32 (2H, m), 3.67 (2H, t, J = 6.6 Hz),
2.38–2.33 (2H, m), 2.11–2.06 (2H, m), 1.37–1.28 (8H, m), 0.90 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.63 (C-4), 124.91 (C-3), 62.37 (C-1), 31.89 (C-8), 30.81 (C-2), 29.71
δ
(C-7), 29.32 (C-6), 27.39 (C-5), 22.68 (C-9), 14.11 (C-10); anal. calcd for C10H20O: C, 76.86; H,
12.90; found: C, 76.80; H, 12.82; HRMS (ESI-TOF): calcd for C10H20O [M + H]+157.2714,
found 157.2710.
(Z)-Dodec-3-en-1-ol (5c)
1
94% yield. H NMR (CDCl3, 400 MHz)
δ 5.62–5.32 (2H, m), 3.66 (2H, t, J = 6.6 Hz),
2.38–2.33 (2H, m), 2.11–2.06 (2H, m), 1.43–1.26 (12H, m), 0.90 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.35 (C-4), 124.96 (C-3), 62.25 (C-1), 31.91 (C-10), 30.76 (C-2),
29.71 (C-6), 29.64 (C-7), 29.55 (C-8), 29.35 (C-9), 27.37 (C-5), 22.68 (C-11), 14.09 (C-12); anal.
δ

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calcd for C12H24O: C, 78.19; H, 13.12; found: C, 78.10; H, 13.02; HRMS (ESI-TOF): calcd for
C12H24O [M + H]+185.3263, found 185.3258.
(Z)-Tetradec-3-en-1-ol (5d)
1
98% yield. H NMR (CDCl3, 400 MHz)
δ
5.58–5.33 (2H, m), 3.62 (2H, t, J = 6.6 Hz,),
2.35–2.30 (2H, m, J = 6.6 Hz), 2.10–2.00 (2H, m), 1.37–1.23 (16H, m), 0.88 (3H, t, J = 6.7 Hz);
13C NMR (CDCl3, 100.62 MHz) δ 133.35 (C-4), 124.96 (C-3), 62.25 (C-1), 31.91 (C-12), 30.76
(C-2), 29.71 (C-8), 29.64 (C-6), 29.55 (C-7), 29.35 (C-9), 29.32 (C-10, C-11), 27.37 (C-5), 22.68
(C-13), 14.09 (C-14); anal. calcd for C14H28O: C, 79.17; H, 13.28; found: C, 79.09; H, 13.19;
HRMS (ESI-TOF): calcd for C14H28O [M + H]+213.3794, found 213.3790.
(Z)-Hexadec-3-en-1-ol (5e)
1
98% yield. H NMR (CDCl3, 400 MHz)
δ 5.59–5.33 (2H, m), 3.63 (2H, t, J = 6.6 Hz),
2.35–2.30 (2H, m), 2.09–2.04 (2H, m), 1.37–1.27 (20H, m), 0.88 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.38 (C-4), 124.98 (C-3), 62.27 (C-1), 31.93 (C-14), 30.81 (C-2), 29.72
δ
(C-8), 29.69 (C-6, C-7), 29.65 (C-10, C-11), 29.56 (C-12), 29.36 (C-9), 29.33 (C-13), 27.38 (C-5),
22.69 (C-15), 14.10 (C-16); anal. calcd for C16H32O: C, 79.92; H, 13.41; found: C, 79.86; H,
13.32; HRMS (ESI-TOF): calcd for C16H32O [M + H]+241.4326, found 241.4320.
3.11.3. Synthesis of (Z)-1-Bromoalk-3-enes (7)
A mixture of methane sulphonyl chloride (4.93 g, 43.0 mmol), triethylamine (8.22 mL),
(Z)-alk-3-en-1-ol (5a
reaction mixture was filtered and the residue washed with CH2Cl2 (40 mL). The combined
CH2Cl2 layer was washed with dil. HCl (30 mL), water (2 25 mL), dried (Na2SO4) and the
–e
) (34.0 mmol) and CH2Cl2 (52.2 mL) was stirred at 0 ◦C for 1 h. The
×
solvent removed under reduced pressure. The solution of the 6a–e 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 out 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 sodium sulphate. Evaporation of the solvent,
followed by column chromatography of the residue on silica gel, using n-hexane-diethyl
ether (95:5) as eluent, gave 7a–e as a yellow liquid oil.
(Z)-1-Bromoct-3-ene (7a)
1
91% yield. H NMR (CDCl3, 400 MHz)
δ 5.57–5.32 (2H, m), 3.36 (2H, t, J = 7.2 Hz),
2.65–2.59 (2H, m), 2.18–2.03 (2H, m), 1.38–1.24 (4H, m), 0.90 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.11 (C-4), 125.74 (C-3), 32.52 (C-1), 31.69 (C-2), 30.84 (C-6), 27.12
δ
(C-5), 22.31 (C-7), 13.94 (C-8); anal. calcd for C8H15Br: C, 50.27; H, 7.99; found: C, 50.17; H,
7.88; HRMS (ESI-TOF): calcd for C8H15Br [M + H]+192.1087, found 192.1076.
(Z)-1-Bromdec-3-ene (7b)
1
90% yield. H NMR (CDCl3, 400 MHz)
δ 5.59–5.34 (2H, m), 3.38 (2H, t, J = 7.2 Hz),
2.64–2.61 (4H, m), 1.39–1.28 (8H, m), 0.90 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
133.21 (C-4), 125.73 (C-3), 32.54 (C-1), 31.73 (C-8), 30.86 (C-2), 29.49 (C-7), 28.95 (C-6),
δ
27.44 (C-5), 22.63 (C-9), 14.07 (C-10); anal. calcd for C10H19Br: C, 54.80; H, 8.73; found: C,
54.73; H, 8.66; HRMS (ESI-TOF): calcd for C10H19Br [M + H]+220.1698, found 220.1690.
(Z)-1-Bromdodec-3-ene (7c)
1
89% yield. H NMR (CDCl3, 400 MHz)
δ 5.59–5.34 (2H, m), 3.38 (2H, t, J = 7.2 Hz),
2.66–2.61 (2H, m), 2.11–2.03 (2H, m), 1.38–1.26 (12H, m), 0.91 (3H, t, J= 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.22 (C-4), 125.72 (C-3), 32.55 (C-1), 31.88 (C-10), 30.86 (C-2), 29.53
δ
(C-7), 29.48 (C-8), 29.28 (C-6, C-9), 27.43 (C-5), 22.67 (C-11), 14.09 (C-12); anal. calcd for
C12H23Br: C, 58.30; H, 9.38. Found: C, 58.22; H, 9.31; HRMS (ESI-TOF): calcd for C12H23Br
[M + H]+248.2229, found 248.2220.

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(Z)-1-Bromtetradec-3-ene (7d)
1
93% yield. H NMR (CDCl3, 400 MHz)
δ
5.59–5.35 (2H, m), 3.38 (2H, t, J = 7.2 Hz),
2.67–2.61 (2H, m), 2.09–2.01 (2H, m), 1.45–1.29 (16H, m), 0.91 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) δ 133.18 (C-4), 125.73 (C-3), 32.44 (C-1), 31.93 (C-12), 30.88 (C-2), 29.64
(C-6, C-7), 29.54 (C-8, C-9), 29.36 (C-10), 29.30 (C-11), 27.44 (C-5), 22.70 (C-13), 14.10 (C-14);
anal. calcd for C14H27Br: C, 61.08; H, 9.88; found: C, 60.00; H, 9.79; HRMS (ESI-TOF): calcd
for C14H27Br [M + H]+276.2761, found 276.2758.
(Z)-1-Bromhexadec-3-ene (7e)
1
95% yield. H NMR (CDCl3, 400 MHz)
δ 5.59–5.35 (2H, m), 3.37 (2H, t, J = 7.2 Hz),
2.66–2.61 (2H, m), 2.08–2.03 (2H, m), 1.40–1.29 (20H, m), 0.90 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 133.17 (C-4), 125.73 (C-3), 32.42 (C-1), 31.97 (C-14), 30.89 (C-2), 29.73
δ
(C-6, C-7),29.67(C-10, C-11), 29.56 (C-8, C-12), 29.41 (C-9), 29.32 (C-13), 27.45 (C-5), 22.73
(C-15), 14.13 (C-16); anal. calcd for C16H31Br: C, 63.35; H, 10.30; found: C, 63.28; H, 10.22;
HRMS (ESI-TOF): calcd for C16H31Br [M + H]+304.3292, found 304.3287.
3.11.4. Synthesis of Alk-5-en-1-ynes (8a–e)
To a suspension of a lithium acetylide-ethylenediamine complex (3.1 g, 36.9 mmol) in
DMSO (13.6 mL) was added 7a ◦
–e
(22.2 mmol) in DMSO (11.2 mL) at 0 ◦C. The reaction
mixture was stirred for 12 h 23 C. After the reaction had been completed, sat. NH4Cl
(10 mL) was added to the mixture. The mixture was extracted with ether, washed with
brine, dried over MgSO4 and concentrated. The residue was chromatographed over silica
gel (hexane) to afford 8a–e as a colorless oil.
(Z)-Dec-5-en-1-yne (8a)
1
67% yield. H NMR (CDCl3, 400 MHz)
δ
5.50–5.40 (2H, m), 2.31–2.17 (6H, m), 2.07–2.03
131.59
(1H, m), 1.40–1.20 (4H, m), 0.91 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ
(C-6), 129.11 (C-5), 84.18 (C-2), 68.25 (C-1), 31.75 (C-8), 27.41 (C-7), 26.99 (C-4), 22.27 (C-9),
18.84 (C-3), 13.89 (C-10); anal. calcd for C10H16: C, 88.16; H, 11.83; found: C, 88.10; H, 11.78;
HRMS (ESI-TOF): calcd for C10H16 [M + H]+137.2419, found 137.2412.
(Z)-Dodec-5-en-1-yne (8b)
1
68% yield. H NMR (CDCl3, 400 MHz)
δ
5.52–5.09 (2H, m), 2.32–2.18 (6H, m), 2.11–2.04
133.09
(1H, m), 1.45–1.33 (10H, m), 0.91 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ
(C-6), 129.10 (C-5), 84.27 (C-2), 68.25 (C-1), 31.73 (C-10), 29.59 (C-9), 28.92 (C-8), 27.75 (C-7),
27.32 (C-4), 22.63 (C-11), 18.86 (C-3), 14.08 (C-12); anal. calcd for C12H22: C, 86.66; H, 13.33;
found: C, 86.59; H, 13.25; HRMS (ESI-TOF): calcd for C12H22 [M + H]+167.3110, found
167.3107.
(Z)-Tetradec-5-en-1-yne (8c)
1
72% yield. H NMR (CDCl3, 400 MHz)
δ
5.52–5.09 (2H, m), 2.32–2.18 (6H, m), 2.10–
2.04 (1H, m), 1.43–1.29 (12H, m), 0.91 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ
133.04 (C-6), 129.12 (C-5), 84.19 (C-2), 68.25 (C-1), 31.90 (C-12), 29.64 (C-9, C-10),29.49 (C-8),
29.27 (C-11), 27.75 (C-7), 27.32 (C-4), 22.63 (C-3), 18.86 (C-13), 14.09 (C-14); anal. calcd for
C14H24: C, 87.42; H, 12.57; found: C, 87.38; H, 12.49; HRMS (ESI-TOF): calcd for C14H24
[M + H]+193.3483, found 193.3479.
(Z)-Hexadec-5-en-1-yne (8d)
1
69% yield. H NMR (CDCl3, 400 MHz)
δ
5.51–5.09 (2H, m), 2.33–2.19 (6H, m), 2.09–2.05
132.34
(1H, m), 1.47–1.30 (16H, m), 0.92 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ
(C-6), 129.14 (C-5), 84.08 (C-2), 68.22 (C-1), 31.94 (C-14), 29.70 (C-10), 29.65 (C-9, C-11),
29.54 (C-8), 29.38 (C-12), 29.28 (C-13), 27.74 (C-7), 27.32 (C-4), 22.71 (C-3), 18.86 (C-15),
14.08 (C-16); anal. calcd for C16H28: C, 87.19; H, 12.80; found: C, 87.11; H, 12.73; HRMS
(ESI-TOF): calcd for C16H28 [M + H]+221.4014, found 221.4009.

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(Z)-Octadec-5-en-1-yne (8e)
1
70% yield. H NMR (CDCl3, 400 MHz)
δ
5.51–5.09 (2H, m), 2.32–2.20 (6H, m), 2.10–2.06
(1H, m), 1.48–1.32 (20H, m), 0.92 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ 132.31
(C-6), 129.21 (C-5), 83.92 (C-2), 68.21 (C-1), 32.01 (C-16), 29.75 (C-10, C-12, C-9), 29.71 (C-13),
29.60 (C-14), 29.46 (C-8), 29.38 (C-11), 29.32 (C-15), 27.76 (C-7), 27.35 (C-4), 22.75 (C-17),
18.87 (C-3), 14.09 (C-18); anal. calcd for C18H32: C, 87.01; H, 12.98; found: C, 86.97; H, 12.88;
HRMS (ESI-TOF): calcd for C18H32 [M + H]+249.4546, found 249.4540.
3.11.5. Synthesis of (Z)-alk-1,2,6-trienes (9a–e)
Paraformaldehyde (1.0 g), copper iodide (1.26 g, 6.8 mmol) and dicyclohexylamine
(75.7 mL) were sequentially added to a solution of 8a–e (16.4 mmol) in anhydrous dioxane
(75.7 mL). The resulting mixture was heated under reflux for 8 h. The solvent was evap-
orated in vacuo. The addition of 2 M HCl (50 mL) and extraction with diethyl ether was
followed by an extraction of the organic layer with NaHCO3, water and brine and a drying
with anhydrous MgSO4 and the residue was purified by silica gel column chromatography
(hexane/ethyl acetate = 30/1) to afford 9a–e as colorless liquid.
(Z)-Undeca-1,2,6-triene (9a)
1
76% yield. H NMR (CDCl3, 400 MHz)
δ
5.44–5.32 (2H, m), 5.15–5.12 (1H, m), 4.71–
4.67 (2H, m), 2.20–2.04 (6H, m), 1.38–1.33 (4H, m), 0.92 (3H, t, J = 6.7 Hz); 13C NMR
(CDCl3, 100.62 MHz) 208.50 (C-2), 130.71 (C-7), 128.65 (C-6), 89.65 (C-3), 74.82 (C-1),
δ
31.90 (C-9), 28.41 (C-5), 26.99 (C-8), 26.84 (C-4), 22.34 (C-10), 13.99 (C-11); anal. calcd for
C11H18: C, 87.92; H, 12.07; found: C, 87.87; H, 11.98; HRMS (ESI-TOF): calcd for C11H18
[M + H]+151.2685, found 151.2680.
(Z)-Trideca-1,2,6-triene (9b)
1
72% yield. H NMR (CDCl3, 400 MHz)
δ
5.49–5.21 (m, 2H), 5.15–5.08 (1H, m), 4.71–4.67
(2H, m), 2.23–2.05 (6H, m), 1.45–1.29 (8H, m), 0.89 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3,
100.62 MHz) 205.56 (C-2), 133.08 (C-7), 129.10 (C-6), 89.64 (C-3), 74.79 (C-1), 31.72 (C-11),
δ
29.67 (C-5), 29.58 (C-9), 28.91 (C-10), 27.74 (C-8), 27.29 (C-4), 22.61 (C-12), 14.07 (C-13); anal.
calcd for C13H22: C, 87.56; H, 12.43; found: C, 87.48; H, 12.37; HRMS (ESI-TOF): calcd for
C13H22 [M + H]+179.3217, found 179.3211.
(Z)-Pentadeca-1,2,6-triene (9c)
1
74% yield. H NMR (CDCl3, 400 MHz)
δ
5.51–5.18 (2H, m), 5.17–5.08 (1H, m), 4.71–4.68
(2H, m), 2.24–2.03 (6H, m), 1.45–1.29 (12H, m), 0.91 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3,
100.62 MHz) 204.19 (C-2), 133.09 (C-7), 129.10 (C-6), 89.64 (C-3), 74.79 (C-1), 31.89 (C-13),
δ
29.62 (C-5), 29.47 (C-9), 29.32 (C-10), 29.28 (C-11), 29.25 (C-12), 27.74 (C-8), 27.29 (C-4), 22.67
(C-14), 14.09 (C-15); anal. calcd for C15H26: C, 87.30; H, 12.69; found: C, 87.24; H, 12.58;
HRMS (ESI-TOF): calcd for C15H26 [M + H]+207.3748, found 207.3742.
(Z)-Heptadeca-1,2,6-triene (9d)
1
77% yield. H NMR (CDCl3, 400 MHz)
δ
5.50–5.38 (2H, m), 5.22–5.09 (1H, m), 4.71–4.68
(2H, m), 2.24–2.04 (6H, m), 1.46–1.30 (16H, m), 0.92 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3,
100.62 MHz) 208.54 (C-2), 133.01 (C-7), 129.12 (C-6), 89.62 (C-3), 74.75 (C-1), 31.93 (C-15),
δ
29.72 (C-5), 29.63 (C-11), 29.58 (C-9), 29.52 (C-12), 29.35 (C-13), 29.33 (C-14), 29.26 (C-10),
27.74 (C-8), 27.29 (C-4), 22.69 (C-16), 14.08 (C-17); anal. calcd for C17H30: C, 87.10; H, 12.89.
Found: C, 87.03; H, 12.80; HRMS (ESI-TOF): calcd for C17H30 [M + H]+235.4280, found
235.4273.
(Z)-Nonadeca-1,2,6-triene (9e)
1
75% yield. H NMR (CDCl3, 400 MHz)
δ
5.53–5.42 (2H, m), 5.22–5.10 (1H, m), 4.72–4.69
(2H, m), 2.26–2.07 (6H, m), 1.49–1.33 (20H, m), 0.95 (3H, t, J = 6.7 Hz); 13C NMR (CDCl3,
100.62 MHz) 208.59 (C-2), 132.86 (C-7), 129.21 (C-6), 89.59 (C-3), 74.71 (C-1), 32.02 (C-17),
δ

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29.78 (C-5, C-11), 29.75 (C-13, C-9), 29.71 (C-14, C15), 29.60 (C-12), 29.46 (C-16), 29.33 (C-10),
27.78 (C-8), 27.33 (C-4), 22.76 (C-18), 14.10 (C-19); anal. calcd for C19H34: C, 86.94; H, 13.05;
found: C, 86.87; H, 12.97; HRMS (ESI-TOF): calcd for C19H34 [M + H]+263.4812, found
263.4807.
3.11.6. Cross-Cyclomagnesiation of (Z)-alk-1,2,6-trienes (9a–e) and Allene Alcohol
Tetrahydropyran Ethers (10a–d) with EtMgBr in the Presence of Mg Metal and Cp2TiCl2
Catalyst (General Procedure)
Diethyl ether (30 mL), (Z)-alk-1,2,6-trienes (9a
–e) (4.8 mmol), allene alcohol tetrahy-
dropyran ethers (10a ) (4.0 mmol), EtMgBr (40.0 mmol) (as 1.5 M solution in Et2O), Mg
–d
powder (0.7 g, 30.0 mmol) an◦d Cp2TiCl2 (0.1 g, 0.4 mmol) were placed in a glass reactor
with stirring under argon (~0 C). The reaction 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, filtered and the solvents were removed under reduced
pressure. Silica gel column chromatography (n-hexane/EtOAc (35/1)) of the residue gave
compound 12a–g as a pale yellow oily liquid.
2-((5Z,9Z,13Z)-Octadeca-5,9,13-trien-1-yloxy)tetrahydro-2H-pyran (12a)
1
75% yield. H NMR (CDCl3, 400 MHz)
δ 5.43–5.37 (6H, m), 4.58 (1H, t, J = 3.2 Hz),
3.87–3.37 (4H, m), 2.09–2.02 (12H, m), 1.63–1.26 (14H, m), 0.90 (3H, t, J = 7.2 Hz,); 13C NMR
(CDCl3, 100.62 MHz) 130.29 (C-9), 129.95 (C-10), 129.61 (C-5), 129.52 (C-14), 129.39 (C-6),
δ
129.06 (C-13), 98.74 (C-19), 67.42 (C-1), 62.17 (C-23), 31.91 (C-16), 30.74 (C-2), 29.37 (C-20),
27.45 (C-4), 27.40 (C-12), 27.36 (C-7), 27.04 (C-15), 26.94 (C-8, C-11), 26.38 (C-3), 25.51 (C-22),
22.32 (C-17), 19.60 (C-21), 13.96 (C-18); anal. calcd for C23H40O2: C, 78.57; H, 12.32; found:
C, 78.50; H, 12.26; HRMS (ESI-TOF): calcd for C23H40O2 [M + H]+352.5942, found 352.5936.
2-((5Z,9Z,13Z)-Icosa-5,9,13-trien-1-yloxy)tetrahydro-2H-pyran (12b)
1
80% yield. H NMR (CDCl3, 400 MHz)
δ 5.41–5.32 (6H, m), 4.60 (1H, t, J = 3.2 Hz),
3.91–3.38 (4H, m), 2.11–1.97 (12H, m), 1.65–1.28 (18H, m), 0.90 (3H, t, J = 7.2 Hz). 13C
NMR (CDCl3, 100.62 MHz) 130.43 (C-14), 130.00 (C-5), 129.67 (C-10), 129.57 (C-9), 129.44
δ
(C-6), 129.09 (C-13), 98.82 (C-21), 67.49 (C-1), 62.29 (C-25), 31.79 (C-18), 30.78 (C-2), 29.72
(C-22), 29.40 (C-16), 29.00 (C-17), 27.47 (C-4), 27.42 (C-15), 27.38 (C-12), 27.28 (C-7), 27.08
(C-8, C-11), 26.39 (C-3), 25.52 (C-24), 22.66 (C-19), 19.66 (C-23), 14.11 (C-20); anal. calcd
for C25H44O2: C, 79.72; H, 11.77; found: C, 79.67; H, 11.69; HRMS (ESI-TOF): calcd for
C25H44O2 [M + H]+377.6236, found 377.6230.
2-((5Z,9Z,13Z)-Docosa-5,9,13-trien-1-yloxy)tetrahydro-2H-pyran (12c)
1
78% yield. H NMR (CDCl3, 400 MHz)
δ 5.43–5.36 (6H, m), 4.59 (1H, t, J = 3.2 Hz),
3.89–3.36 (4H, m), 2.13–1.96 (12H, m), 1.64–1.27 (22H, m), 0.89 (3H, t, J = 7.2 Hz); 13C NMR
(CDCl3, 100.62 MHz) 130.36 (C-14), 129.95 (C-5), 129.62 (C-10), 129.52 (C-9),129.40 (C-6),
δ
129.04 (C-13), 98.74 (C-23), 67.43 (C-1), 62.17 (C-27), 31.89 (C-10), 30.75 (C-20), 29.73 (C-2),
29.51 (C-24), 29.38 (C-16), 29.31 (C-17, C-18), 29.23 (C-19), 27.45 (C-4), 27.40 (C-15), 27.36
(C-12), 27.25 (C-7), 27.05 (C-8, C-11), 26.38 (C-3), 25.52 (C-26), 22.66 (C-21), 19.61 (C-25),
14.06 (C-22); anal. calcd for C27H48O2: C, 80.13; H, 11.95; found: C, 80.09; H, 11.87; HRMS
(ESI-TOF): calcd for C27H48O2 [M + H]+405.6767, found 405.6760.7293.
2-((9Z,13Z,17Z)-Triaconta-9,13,17-trien-1-yloxy)tetrahydro-2H-pyran (12d)
1
83% yield. H NMR (CDCl3, 400 MHz)
δ 5.42–5.36 (6H, m), 4.59 (1H, t, J = 3.2 Hz),
3.91–3.33 (4H, m), 2.12–1.98 (12H, m), 1.63–1.28 (38H, m), 0.89 (3H, t, J = 7.2 Hz); 13C NMR
(CDCl3, 100.62 MHz) 130.39 (C-9), 130.33 (C-18), 129.60 (C-14, C-13), 129.10 (C-10), 129.07
δ
(C-17), 98.80 (C-31), 67.65 (C-1), 62.26 (C-35), 31.95 (C-28), 30.79 (C-32), 29.77 (C-2), 29.71
(C-4, C-22), 29.68 (C-7, C-20, C-24), 29.59 (C-25, C-26), 29.52 (C-6), 29.49 (C-23), 29.38 (C-27),
29.34 (C-21), 29.28 (C-5), 27.46 (C-8, C-19), 27.39 (C-11, C-16), 27.27 (C-12, C-15), 26.26 (C-3),
25.54 (C-34), 22.70 (C-29), 19.66 (C-33), 14.12 (C-30); anal. calcd for C35H64O2: C, 81.32; H,

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12.48; found: C, 81.27; H, 12.41; HRMS (ESI-TOF): calcd for C35H64O2 [M + H]+517.8894,
found 517.8891.
2-((11Z,15Z,19Z)-Triaconta-11,15,19-trien-1-yloxy)tetrahydro-2H-pyran (12e)
1
81% yield. H NMR (CDCl3, 400 MHz)
δ 5.43–5.36 (6H, m), 4.59 (1H, t, J = 3.2 Hz),
3.91–3.37 (4H, m,), 2.10–1.98 (12H, m), 1.64–1.28 (38H, m), 0.90 (3H, t, J = 7.2 Hz); 13C NMR
(CDCl3, 100.62 MHz) 130.39 (C-11, C-20), 129.61 (C-15, C-16), 129.08 (C-12, C-19), 98.81
δ
(C-31), 67.68 (C-1), 62.27 (C-35), 31.94 (C-28), 30.80 (C-2), 29.78 (C-32), 29.77 (C-4), 29.72 (C-7,
C-24), 29.68 (C-9, C-22), 29.66 (C-25), 29.62 (C-5), 29.59 (C-6), 29.57 (C-26), 29.52 (C-27), 29.37
(C-8), 29.34 (C-23), 27.47 (C-10, C-21), 27.39 (C-13, C-18), 27.28 (C-14, C-17), 26.27 (C-3),
25.54 (C-34), 22.70 (C-29), 19.69 (C-33), 14.12 (C-30); anal. calcd for C35H64O2: C, 81.32; H,
12.48; found: C, 81.29; H, 12.40; HRMS (ESI-TOF): calcd for C35H64O2 [M + H]+517.8894,
found 517.8890.
2-((11Z,15Z,19Z)-Dotriaconta-11,15,19-trien-1-yloxy)tetrahydro-2H-pyran (12f)
1
77% yield. H NMR (CDCl3, 400 MHz)
δ 5.42–5.37 (6H, m), 4.59 (1H, t, J = 3.2 Hz),
3.91–3.36 (4H, m), 2.10–1.98 (12H, m), 1.64–1.28 (42H, m), 0.90 (3H, t, J = 7.2 Hz); 13C NMR
(CDCl3, 100.62 MHz) 130.37 (C-11, C-20), 129.59 (C-15, C-16), 129.07 (C-12, C-19), 98.78
δ
(C-33), 67.66 (C-1), 62.23 (C-37), 31.95 (C-30), 30.79 (C-2), 29.77 (C-34), 29.72 (C-4), 29.69 (C-7,
C-24), 29.66 (C-9, C-22, C-26), 29.62 (C-27, C-5), 29.58 (C-28, C-6), 29.52 (C-25), 29.39 (C-29),
29.35 (C-8, C-23), 27.47 (C-10, C-21), 27.39 (C-13, C-18), 27.28 (C-14, C-17), 26.28 (C-3), 25.55
(C-36), 22.71 (C-31), 19.68 (C-35), 14.12 (C-32); anal. calcd for C37H68O2: C, 81.55; H, 12.57;
found: C, 81.49; H, 12.50; HRMS (ESI-TOF): calcd for C37H68O2 [M + H]+545.9425, found
545.9420.
2-((13Z,17Z,21Z)-Dotriaconta-13,17,21-trien-1-yloxy)tetrahydro-2H-pyran (12g)
1
79% yield. H NMR (CDCl3, 400 MHz)
δ 5.42–5.37 (6H, m), 4.59 (1H, t, J = 3.2 Hz),
3.91–3.36 (4H, m), 2.10–1.97 (12H, m), 1.62–1.28 (42H, m), 0.90 (3H, t, J = 7.2 Hz). 13C NMR
(CDCl3, 100.62 MHz) 130.35 (C-13, C-22), 129.58 (C-17, C-18), 129.07 (C-14, C-21), 98.75
δ
(C-33), 67.64 (C-1), 62.18 (C-37), 31.94 (C-30), 30.79 (C-2, C-34), 29.78 (C-4), 29.68 (C-9, C-26),
29.64 (C-5, C-6), 29.60 (C-7, C-11, C-24), 29.53 (C-8, C-27), 29.38 (C-28, C29), 29.35 (C-10,
C-25), 27.47 (C-12, C-23), 27.39 (C-15, C-20), 27.28 (C-16, C-19), 26.28 (C-3), 25.55 (C-36),
22.71 (C-31), 19.66 (C-35), 14.11 (C-32); anal. calcd for C37H68O2: C, 81.55; H, 12.57; found:
C, 81.48; H, 12.48; HRMS (ESI-TOF): calcd for C37H68O2 [M + H]+545.9425, found 545.9419.
3.11.7. General Procedures for the Preparation of 1Z,5Z,9Z-Dienoic Acids 13a–g
To a solution of tetrahydropyran ester (0.5 mmol) in acetone (12 mL) and CH2Cl2
(2 mL) at room temperature (20–22 ◦C) Jones reagent (0.5 mL) was added dropwise. The
◦
reaction mixture was stirred at room temperature (20–22 C) for 1 h, then treated with water
(2.5 mL), excess solvent was evaporated under vacuum (acetone, CH2Cl2), the aqueous
layer was extracted with diethyl ether, dried over MgSO4. The reaction product was
isolated by column chromatography (silica gel, Acrus (0.060–0.200 mm), eluent petroleum
ether/ethyl acetate) [43,44].
(5Z,9Z,13Z)-Octadeca-5,9,13-trienoic acid (13a)
77% yield. IR (KBr)
ν
3524, 2900, 2866, 1740, 1722, 1665, 1463, 1384, 1365, 1250,
max
1241, 1034, 737 cm−1; H NMR (CDCl3, 400 MHz)
δ 5.43–5.38 (6H, m), 2.42–2.35 (2H,
1
m), 2.13–2.05 (12H, m), 1.76–1.69(2H, m), 1.36–1.28 (4H, m), 0.92 (3H, t, J = 6.6 Hz); 13C
NMR (CDCl3, 100.62 MHz) 179.63 (C-1), 130.56 (C-6), 130.39 (C-9), 129.80 (C-10), 129.40
δ
(C-14), 129.08 (C-5),128.65 (C-13), 33.36 (C-2), 31.93 (C-16), 27.46 (C-7, C-12), 27.36 (C-15),
27.32 (C-4), 26.97 (C-11), 26.49 (C-8), 24.58 (C-3), 22.35 (C-17), 13.99 (C-18); anal. calcd
for C18H30O2: C, 77.64; H, 10.86; found: C, 77.58; H, 10.80; HRMS (ESI-TOF): calcd for
C18H30O2 [M + H]+279.4375, found 279.4370.

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(5Z,9Z,13Z)-Icosa-5,9,13-trienoic acid (13b)
79% yield. IR (KBr) 3378, 3045, 2930, 2838, 1780, 1740, 1651, 1465, 1367, 1242,
1130, 748 cm−1; 1H NMR (CDCl3, 400 MHz)
(12H, m), 1.76–1.62 (8H, m), 1.42–1.28 (2H, m),0.91 (3H, t, J = 6.6 Hz); 13C NMR (CDCl3,
100.62 MHz) 179.88 (C-1), 130.57 (C-6), 130.45 (C-9), 129.81 (C-10), 129.40 (C-14), 129.07
ν
max
δ
5.95.34 (6H, m), 2.47–2.35 (2H, m), 2.15–1.97
δ
(C-5), 128.65 (C-13), 33.40 (C-2), 31.79 (C-18), 29.71 (C-16), 29.00 (C-17), 27.46 (C-7), 27.36
(C-12), 27.32 (C-15), 27.28 (C-4), 26.49 (C-8, C-11), 24.58 (C-3), 22.66 (C-19), 14.10 (C-20);
anal. calcd for C20H34O2: C, 78.37; H, 11.18; found: C, 78.30; H, 11.11; HRMS (ESI-TOF):
calcd for C20H34O2 [M + H]+307.4907, found 307.4900.
(5Z,9Z,13Z)-Docosa-5,9,13-trienoic acid (13c)
81% yield. IR (KBr)
1168, 1030, 752 cm−1; 1H NMR (CDCl3, 400 MHz)
2.15–2.02 (12H, m), 1.76–1.62 (12H, m), 1.40–1.28 (2H, m), 0.90 (3H, t, J = 6.6 Hz); 13C NMR
(CDCl3, 100.62 MHz) 179.82 (C-1), 130.56 (C-6), 130.45 (C-9), 129.81 (C-10), 129.39 (C-14),
ν
3520, 2918, 2865, 1748, 1735, 1670, 1463, 1390, 1358, 1253,
max
δ
5.46–5.35 (6H, m), 2.42–2.35 (2H, m),
δ
129.07 (C-5), 128.65 (C-13), 33.39 (C-2), 31.91 (C-20), 29.75 (C-16), 29.53 (C-17, C-18), 29.33
(C-19), 29.06 (C-16), 27.46 (C-7), 27.36 (C-12), 27.32 (C-15), 27.28 (C-4), 26.49 (C-8, C-11),
24.58 (C-3), 22.69 (C-21), 14.11 (C-22); anal. calcd for C22H38O2: C, 78.98; H, 11.44; found:
C, 78.91; H, 11.37; HRMS (ESI-TOF): calcd for C22H38O2 [M + H]+335.5438, found 335.5433.
(9Z,13Z,17Z)-Triaconta-9,13,17-trienoic acid (13d)
76% yield. IR (KBr)
ν
3520, 2920, 2865, 1730, 1670, 1463, 1378, 1330, 1248, 1158,
max
1025, 736 cm−1; H NMR (CDCl3, 400 MHz)
δ 5.42–5.36 (6H, m), 2.37 (2H, t), 2.12–2.02
1
(12H, m), 1.67–1.62 (28H, m), 1.42–1.28 (2H, m), 0.91 (3H, t, J = 6.6 Hz); 13C NMR (CDCl3,
100.62 MHz) 179.95 (C-1), 130.43 (C-10), 130.25 (C-14), 129.65 (C-13), 129.60 (C-18), 129.22
δ
(C-9), 129.09 (C-17), 34.02 (C-2), 31.95 (C-28), 29.77 (C-22), 29.71 (C-7), 29.68 (C-20, C-24,
C-25), 29.59 (C-23, C-26), 29.38 (C-27), 29.35 (C-21), 29.17 (C-5), 29.09 (C-4), 29.05 (C-6),
27.48 (C-8, C-19), 27.45 (C-11), 27.39 (C-16), 27.29 (C-12), 27.23 (C-15), 24.67 (C-3), 22.71
(C-29), 14.13 (C-30); anal. calcd for C30H54O2: C, 80.65; H, 12.18; found: C, 80.60; H, 12.11;
HRMS (ESI-TOF): calcd for C30H54O2 [M + H]+447.7565, found 447.7560.
(11Z,15Z,19Z)-Triaconta-11,15,19-trienoic acid (13e)
81% yield. IR (KBr)
735 cm−1; 1H NMR (CDCl3, 400 MHz)
1.70–1.59 (28H, m), 1.38–1.28 (2H, m), 0.90 (3H, t, J = 6.6 Hz); 13C NMR (CDCl3, 100.62 MHz)
180.06 (C-1), 130.42 (C-11), 130.35 (C-20), 129.62 (C-15, C-16), 129.13 (C-12), 129.08 (C-19),
ν
3360, 3035, 1854, 1718, 1640, 1564, 1478, 1360, 1265, 1018,
max
δ
5.43–5.36 (6H, m), 2.36 (2H, t), 2.11–1.98 (12H, m),
δ
34.07 (C-2), 31.93 (C-28), 29.76 (C-7), 29.74 (C-24), 29.66 (C-9, C-22), 29.58 (C-25), 29.50 (C-6),
29.42 (C-26), 29.37 (C-27), 29.34 (C-8), 29.29 (C-23), 29.25 (C-5), 29.08 (C-4), 27.47 (C-10,
C-21), 27.43 (C-13), 27.39 (C-18), 27.27 (C-14, C-17), 24.69 (C-3), 22.70 (C-29), 14.11 (C-30);
anal. calcd for C30H54O2: C, 80.65; H, 12.18; found: C, 80.59; H, 12.10; HRMS (ESI-TOF):
calcd for C30H54O2 [M + H]+447.7565, found 447.7561.
(11Z,15Z,19Z)-Dotriaconta-11,15,19-trienoic acid (13f)
−1
77% yield. IR (KBr)
ν
3360, 3030, 1850, 1718, 1642, 1470, 1365, 1263, 1020, 742 cm
;
max
1H NMR (CDCl3, 400 MHz)
δ
5.44–5.38 (6H, m), 2.37 (2H, t), 2.14–2.02 (12H, m), 1.69–1.61
180.22
(32H, m), 1.39–1.28 (2H, m), 0.91 (3H, t, J = 6.6 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ
(C-1), 130.41 (C-11), 130.35 (C-20), 129.62 (C-15, C-16), 129.13 (C-12), 129.08 (C-19), 34.09
(C-2), 31.95 (C-30), 29.77 (C-7), 29.72 (C-24), 29.68 (C-9, C22), 29.60 (C-26, C-27), 29.50 (C-28),
29.43 (C-6), 29.38 (C-25), 29.35 (C-29), 29.30 (C-8), 29.26 (C-23), 29.08 (C-4), 27.47 (C-10,
C-21), 27.40 (C-13, C-18), 27.28 (C-14, C-17), 24.68 (C-3), 22.71 (C-31), 14.12 (C-32); anal.
calcd for C32H58O2: C, 80.94; H, 12.31; found: C, 80.87; H, 12.28; HRMS (ESI-TOF): calcd
for C32H58O2 [M + H]+475.8096, found 475.8091.

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(13Z,17Z,21Z)-Dotriaconta-13,17,21-trienoic acid (13g)
82% yield. IR (KBr) 3360, 3030, 1852, 1720, 1646, 1474, 1360, 1265, 1020, 741 cm
−1
ν
;
max
1H NMR (CDCl3, 400 MHz) δ 5.47–5.38 (6H, m), 2.36 (2H, t), 2.12–2.02 (12H, m), 1.70–1.62
(32H, m), 1.46–1.30 (2H, m), 0.91 (3H, t, J = 6.6 Hz); 13C NMR (CDCl3, 100.62 MHz)
δ 180.19
(C-1), 130.39 (C-13, C-22), 129.62 (C-17, C-18), 129.09 (C-14, C-21), 34.11 (C-2), 31.94 (C-30),
29.77 (C-9, C-26), 29.68 (C-11, C-24), 29.62 (C-8, C-27), 29.61 (C-6), 29.46 (C-7), 29.38 (C-28,
C-29), 29.35 (C-10, C-25), 29.27 (C-5), 29.09 (C-4), 27.47 (C-12, C-23), 27.40 (C-15), 27.39
(C-20), 27.29 (C-16, C-19), 24.70 (C-3), 22.71 (C-31), 14.11 (C-32); anal. calcd for C32H58O2:
C, 80.94; H, 12.31; found: C, 80.89; H, 12.25; HRMS (ESI-TOF): calcd for C32H58O2 [M +
H]+475.8096, found 475.8090.
4. Conclusions
Compound 13c exhibits a pronounced antitumor effect due to the multi-target effect
on the main intracellular targets. The levels of all main kinases responsible for cell growth
and proliferation markedly decrease under the action of the synthesized trienoic acid
13c; simultaneously, acid 13c actively inhibits topoisomerase I and induces apoptosis of
tumor cells according to the intrinsic mitochondrial pathway. Using molecular docking,
we found that the preferable binding sites for the carboxylate group in the acids 13a–g
are the K443, K587 and N722 residues. Among the activated signaling pathways, the
phosphorylation levels of ERK1/2, Akt, JNK and p38 were characterized by different basic
kinetics in response to the action of the test compound, but differed in lower values than in
the control sample. Interestingly, STAT-3 and STAT-5 phosphorylation, characterized by a
progressive increase over time in untreated cultures, was inhibited in samples containing
the test compound. In general, these results are consistent with the results of our earlier
studies of higher fatty acids with diene and triene groups, showing the ability to suppress
these signaling pathways in various tumor cultures. The synthesized trienoic acids can
claim the role of multitarget compounds that are able to simultaneously penetrate through
various biological membranes, including mitochondrial, uncouple oxidation and phospho-
rylation, thereby reducing mitochondrial potential, as well as inhibiting topoisomerase I
and affecting the main signaling pathways of cell proliferation.
Author Contributions: Conceptualization, V.A.D., L.U.D. and U.M.D.; methodology, A.A.M.; vali-
dation, A.A.M., V.A.D. and L.U.D.; formal analysis, E.K.M.; docking studies, I.R.R.; investigation,
L.U.D.; writing—original draft preparation, V.A.D. and L.U.D.; writing–review and editing, V.A.D.
and L.U.D.; visualization, V.A.D.; supervision, V.A.D. and L.U.D.; project administration, V.A.D.;
funding acquisition, L.U.D. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by Russian Science Foundation, grant number 18-73-10030. 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. The anticancer activity
studies of the synthesized compounds were performed in Laboratory of Molecular Design and
Biological Screening of Candidate Substances for the Pharmaceutical Industry at the Institute of
Petrochemistry and Catalysis of RAS.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in this article.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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