New 1Z,5Z-diene macrodiolides: Catalytic synthesis, anticancer activity, induction of mitochondrial apoptosis, and effect on the cell cycle

Article abstract of DOI:10.1016/j.bioorg.2020.103832

An original scheme was developed for the synthesis of previously undescribed unsaturated macrodiolides containing a 1Z,5Z-diene moiety in 44–80% yields and with high stereoselectivity (>95%) based on the intermolecular esterification of α,ω-diols with α,ω-alka-nZ,(n + 4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-dienedicarboxylic acid, 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid, 1,18-octadeca-7Z,11Z-dienedicarboxylic acid) catalyzed by hafnium triflate [Hf(OTf)₄]. The unsaturated dicarboxylic acids were prepared via homo-cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the presence of Mg metal and the Cp₂TiCl₂ catalyst (10 mol.%) and the subsequent Jones oxidation of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The thus prepared macrodiolides exhibit high cytotoxic activity in vitro against Jurkat, K562, U937, Hek293 and HeLa cancer cell lines. It was found that induction of the programmed cell death in Jurkat cells by macrodiolides corresponds to the mitochondrial apoptosis pathway. Also, it was shown that the prepared macrodiolides efficiently suppress phosphorylation of Akt and p38 kinases and CREB transcription factor in cancer cells.

Full text of DOI:10.1016/j.bioorg.2020.103832

Journal Pre-proofs
New 1Z,5Z-Diene Macrodiolides: Catalytic Synthesis, Anticancer Activity,
Induction of Mitochondrial Apoptosis, and Effect on the Cell Cycle
Lilya U. Dzhemileva, Vladimir A. D'yakonov, Ilgiz I. Islamov, Milyausha M.
Yunusbaeva, Usein M. Dzhemilev
PII:
S0045-2068(19)32201-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103832
YBIOO 103832
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
23 December 2019
5 April 2020
6 April 2020
Please cite this article as: L.U. Dzhemileva, V.A. D'yakonov, I.I. Islamov, M.M. Yunusbaeva, U.M. Dzhemilev,
New 1Z,5Z-Diene Macrodiolides: Catalytic Synthesis, Anticancer Activity, Induction of Mitochondrial
Apoptosis, and Effect on the Cell Cycle, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.103832
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New 1Z,5Z-Diene Macrodiolides: Catalytic Synthesis, Anticancer
Activity, Induction of Mitochondrial Apoptosis, and Effect on the
Cell Cycle
Lilya U. Dzhemileva,* Vladimir A. D’yakonov,* Ilgiz I. Islamov, Milyausha M. Yunusbaeva
and Usein M. Dzhemilev
Institute of Petrochemistry and Catalysis of RAS (IPC RAS), Prospect Oktyabrya, 141, 450075
Ufa, Russian Federation
ABSTRACT: An original scheme was developed for the synthesis of previously undescribed
unsaturated macrodiolides containing a 1Z,5Z-diene moiety in 44-80% yields and with high
stereoselectivity (>95%) based on the intermolecular esterification of α,ω-diols with α,ω-alka-
nZ,(n+4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-dienedicarboxylic acid, 1,14-tetradeca-
5Z,9Z-dienedicarboxylic acid, 1,18-octadeca-7Z,11Z-dienedicarboxylic acid) catalyzed by
hafnium triflate [Hf(OTf)4]. The unsaturated dicarboxylic acids were prepared via homo-
cyclomagnesiation of tetrahydropyran ethers of O-containing 1,2-dienes with EtMgBr in the
presence of Mg metal and the Cp2TiCl2 catalyst (10 mol. %) and the subsequent Jones oxidation
of pyran ethers formed after the acid hydrolysis of magnesacyclopentanes. The thus prepared
macrodiolides exhibit high cytotoxic activity in vitro against Jurkat, K562, U937, Hek293 and
HeLa cancer cell lines. It was found that induction of the programmed cell death in Jurkat cells
by macrodiolides corresponds to the mitochondrial apoptosis pathway. Also, it was shown that
the prepared macrodiolides efficiently suppress phosphorylation of Аkt and p38 kinases and
CREB transcription factor in cancer cells.
Keywords: Macrodiolides, Anticancer Activity, Mitochondrial Apoptosis, Cell Cycle,
Cytotoxicity.
INTRODUCTION
Macrodiolides are attracting ever increasing attention of researchers because of their wide
occurrence in nature, high structural diversity, and the impressive range of biological activities
they exhibit. For example, macrodiolides such as pyrenophorol (1), vermiculine (2), colletodiol
(3), grahamimycin A1 (4), verbalactone (5), and conglobatin (6) isolated from various fungi, sea
sponges, and algae possess antibacterial,1-3 antifungal,4,5 antitumor,6 and antiparasitic7,8
activities.
In most cases, these macrodiolides contain one or several double bonds and various alkyl,
hydroxy, or keto groups or, more rarely, heterocyclic subsituents.
1

O
O
O
O
HO
O
O
O
O
O
O
O
O
O
OH
O
O
O
O
O
O
O
OH
O
O
HO
4
1
2
3
O
OH
O
O
O
O
O
N
N
O
O
O
HO
O
5
6
Figure 1. Structures of some natural macrodiolides
In addition, oxygen-containing macrocyclic compounds, being highly lipophilic, are able
to deeply penetrate and to be accumulated in various organs and tissues; this can be used to
generate high concentrations of active substances there. Owing to these properties, macrolides
are also employed as targeted drug carriers for anticancer agents, histone deacetylase inhibitors,9-
11
and hybrid molecules with multitarget properties.12-14
Recently,15,16 we developed an efficient synthetic route to previously undescribed
macrodiolides via intermolecular cyclization of aliphatic α,ω-dicarboxylic acids with α,ω-alka-
nZ,(n+4)Z-dienediols catalyzed by hafnium triflate [Hf(OTf)4]. We found that the unsaturated
macrodiolides that we prepared exhibit high cytotoxic activity in vitro against Jurkat, K562,
U937, Hek293, and HeLa cell lines, in particular, in comparison with the saturated analogues.
O
O
(CH2)m
O
O
(CH2)m
OH
HO
Hf(OTf)4
57-78%
O
O
+
( )n
( )n
HO
OH
n
n
Figure 2. Previously developed synthesis of macrodiolides
In view of the foregoing and to pursue our research towards development of
stereoselective syntheses of biologically active compounds containing 1Z,5Z-diene moieties in
the molecules,17-26 here we report development of a synthesis of macrodiolides by catalytic
intermolecular esterification of α,ω-diols with α,ω-alka-nZ,(n+4)Z-dienedicarboxylic acids and
study of the obtained compounds for cytotoxic activity, induction of apoptosis, and effect on the
cell cycle in order to elucidate the relationship between the macrodiolide structure and anticancer
activity.
RESULTS AND DISCUSSION
2

Chemistry
The planned strategy towards the synthesis of unsaturated macrocyclic compounds was
started with the preparation of α,ω-alka-nZ,(n+4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-
dienedicarboxylic acid (4a), 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid (4b), and 1,18-
octadeca-7Z,11Z-dienedicarboxylic acid (4c)). The intermolecular homo-cyclomagnesiation of
the tetrahydropyran ethers of allenic alcohols 1a-c (hexa-4,5-dienol (1a), hepta-5,6-dienol (1b),
nona-7,8-dienol (1c)) with EtMgBr in the presence of magnesium metal and the Cp2TiCl2
о
catalyst (1 : EtMgBr : Mg : [Ti] = 10 : 20 : 24 : 0.5; Et2O, 6 h, 20−22 С) affords
magnesacyclopentanes 2, which undergo acid hydrolysis to give bis-tetrahydropyran ethers 3a-c
in ~85-92% yields and with high stereoselectivity (>95%) (Scheme 1). Finally, the Jones
oxidation of tetrahydropyran ethers 3a-c gave the target α,ω-alka-nZ,(n+4)Z-dienedicarboxylic
acids 4a-c in ~50% yields (Scheme 1).
Scheme 1. Synthesis of α,ω-alka-nZ,(n+4)Z-diendicarboxylic acids 4a-c using Ti-
catalyzed intermolecular cyclomagnesiation of 1,2-dienes.
Cp2TiCl2 (10 mol%)
H3O+
EtMgBr, Mg
.
THPO
OTHP
THPO
n
n
n
~85%
Mg
1a-c
2a-c
O
O
Jones ox.
~50%
THPO
OTHP
HO
OH
n
n
n-1
0-1
3a-c
4a-c
a
b
c
n = 2( ), 3( ), 5( )
The next stage of our research towards the synthesis of target macrodiolides was the Hf-
catalyzed intermolecular esterification of the synthesized dicarboxylic acids 4a-c with aliphatic
and aromatic α,ω-diols according to the protocol reported by Collins and co-workers.27
The reactions of α,ω-alka-nZ,(n+4)Z-dienedicarboxylic acids (1,12-dodeca-4Z,8Z-
dienedicarboxylic acid (4a), 1,14-tetradeca-5Z,9Z-dienedicarboxylic acid (4b), 1,18-octadeca-
7Z,11Z-dienedicarboxylic acid (4c)) with 1,6-hexanediol 5e (4:5 = 1:1, 16 h, toluene) catalyzed
by Hf(OTf)4 (10 mol. %) at reflux resulted in the formation of macrodiolides 6e-8e in 55-76%
yields (Scheme 2).
Scheme 2. Hf-Catalyzed intermolecular esterification of α,ω-alka-nZ,(n+4)Z-
dienedicarboxylic acids 4a-c with aliphatic α,ω-diols 5a-h.
(CH2)m
Hf(OTf)4 (10 mol%)
toluene, 110 oC, 16 h
(CH2)m
2
OH
HO
5a-h
O
1O
O
O
+
O
O
44-80%
( )n
( )n
HO
OH
n-1
n-1
6a-h
7a-h
8a-h
(n = 2)
(n = 3)
(n = 5)
4a-c
3

n
m
Macrodiolide
Yield (%)
2 (4a)
2 (4a)
2 (4a)
2 (4a)
2 (4a)
2 (4a)
2 (4a)
2 (4a)
3 (4b)
3 (4b)
3 (4b)
3 (4b)
3 (4b)
3 (4b)
3 (4b)
3 (4b)
5 (4c)
5 (4c)
5 (4c)
5 (4c)
5 (4c)
5 (4c)
5 (4c)
5 (4c)
0 (5a)
1 (5b)
2 (5c)
3 (5d)
4 (5e)
5 (5f)
6 (5g)
8 (5h)
0 (5a)
1 (5b)
2 (5c)
3 (5d)
4 (5e)
5 (5f)
6 (5g)
8 (5h)
0 (5a)
1 (5b)
2 (5c)
3 (5d)
4 (5e)
5 (5f)
6 (5g)
8(5h)
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
-
-
-
44
55
64
65
-
-
-
65
71
76
78
80
79
-
-
65
69
72
75
79
79
8g
8h
In order to elucidate the influence of the structure of the initial saturated aliphatic α,ω-
diols (that is, the length of the hydrocarbon chain connecting the α and ω hydroxy groups) in the
reactions with α,ω-dienedicarboxylic acids on the yields of the target macrodiolides, we studied
the cyclocondensation of acids 4a-c with α,ω-diols (С2-С10) 5a-h under conditions developed
previously (Scheme 2).
It was found that ethylene glycol 5a and 1,3-propanediol 5b do not react with α,ω-
dicarboxylic acids 4a-c. Similar results were obtained for the reactions of 1,12-dodeca-4Z,8Z-
dienedicarboxylic acid 4a with diols 5с and 5h. Cyclocondensation of chosen α,ω-diols 5a-h
with acids 4a-c proceeds to give the target macrocycles starting with 1,4-butanediol, with the
yields of macrodiolide increasing with increasing size of α,ω-diols to reach 80%.
We could not accomplish cyclocondensation of α,ω-alka-nZ,(n+4)Z-dienedicarboxylic
acids 4a-c with dihydroxybenzenes, naphthalenediols, or decahydro-2,7-naphthalenediol.
Meanwhile, p-phenylenediethanol 5i undergoes cyclocondensation with acids 4a-c rather easily
to give macrodiolides 6i-8i in 55-64% yields (Scheme 3).
Scheme 3. Hf-Catalyzed intermolecular esterification of α,ω-alka-nZ,(n+4)Z-
dienedicarboxylic acids 4a-c with p-phenylenediethanol 5i.
4

Hf(OTf)4 (10 mol%)
toluene, 110 oC, 16 h
HO
OH
5i
+
O
O
O
O
O
O
55-64%
( )n
( )n
HO
OH
n-1
n-1
4a-c
6i
7i
8i
(n = 2)
(n = 3)
(n = 5)
n
Macrodiolide
Yield (%)
2 (4a)
3 (4b)
5 (4c)
6i
7i
8i
55
63
64
Biological studies
Previously, we showed15 that macrodiolides containing 1Z,5Z-diene moieties in the
molecules exhibit high cytotoxicity against various cancer cell lines. Therefore, in this work, we
set ourselves the task not only to study the effect of the macrodiolide structure on the in vitro
cytotoxicity against cancer cells (Jurkat, K562, HeLa, and U937), conditionally normal cells
(HEK293), and normal cells (fibroblasts), but also to evaluate the influence of this class of
compounds on the cell cycle and ability to induce apoptosis and to elucidate the putative
mechanism of interaction of new synthesized macrodiolides with DNA and mitochondria.
The in vitro cytotoxicity of macrodiolides (6-8).
First, we estimated the in vitro cytotoxicity of all macrodiolides 6-8 against Jurkat, K562,
HEK293, HeLa, and U937 cells and fibroblasts by determination of IC50 values using flow
cytofluorimetry with the Guava ViaCount kit (Millipore).
The in vitro cytotoxic activity of compounds 6-8 against Jurkat, K562, U937, and HeLa
cancer cells, HEK293 human embryonic kidney cells, and fibroblasts is shown in Table 1.
Table 1. Cytotoxic activities in vitro of synthesized macrodiolides 6-8 measured on
tumor cell cultures (Jurkat, K562, Hek293, HeLa, U937) (µM).
Comp.
Jurkat
K562
U937
HeLa
Hek293
Fibrobl.
(IC50, µM) (IC50, µM) (IC50, µM) (IC50, µM) (IC50, µM) (IC50, µM)
0.08±0.011 0.22±0.021 0.32±0.012 0.28±0.015 0.48±0.026 0.78±0.034
0.03±0.012 0.17±0.017 0.15±0.011 0.74±0.065 0.58±0.026 0.98±0.018
0.14±0.016 0.18±0.011 0.11±0.016 0.23±0.017 0.39±0.015 0.84±0.025
0.28±0.021 0.64±0.014 0.19±0.014 0.72±0.010 0.40±0.022 0.76±0.027
0.26±0.022 0.26±0.012 0.17±0.021 0.29±0.013 0.52±0.020 1.02±0.031
0.21±0.012 0.29±0.017 0.07±0.013 0.30±0.014 0.44±0.015 0.84±0.021
0.14±0.017 0.10±0.019 0.06±0.011 0.24±0.018 0.37±0.016 0.96±0.019
0.18±0.012 0.53±0.019 0.20±0.010 0.58±0.019 0.62±0.013 1.12±0.043
6e
6f
6g
7c
7d
7e
7f
7g
5

7h
8c
8d
8e
8f
8g
8h
6i
0.44±0.014 0.58±0.011 0.52±0.014 0.61±0.036 0.79±0.017 1.29±0.026
0.31±0.017 0.36±0.014 0.16±0.018 0.38±0.018 0.47±0.011 0.86±0.021
0.48±0.013 0.52±0.013 0.31±0.012 0.72±0.018 0.86±0.012 1.42±0.033
0.50±0.014 0.61±0.017 0.34±0.017 0.61±0.027 0.76±0.013 0.99±0.024
0.48±0.011 0.38±0.015 0.29±0.014 0.48±0.019 0.69±0.014 0.87±0.027
0.10±0.019 0.11±0.014 0.03±0.014 0.21±0.013 0.51±0.019 1.23±0.039
0.22±0.018 0.29±0.018 0.44±0.017 0.34±0.034 0.46±0.011 0.96±0.019
0.14±0.012 0.34±0.018 0.28±0.016 0.39±0.013 0.93±0.019 1.57±0.027
0.05±0.015 0.14±0.011 0.06±0.015 0.28±0.029 0.56±0.016 1.36±0.026
0.04±0.032 0.12±0.016 0.10±0.022 0.17±0.021 0.31±0.033 1.24±0.024
7i
8i
The highest activities against Т-cell leukemia (Jurkat), chronic myeloid leukemia (K562),
histiocytic lymphoma (U937), and cervical cancer (HeLa) cells were found for compounds 7f,
8g, 7i, and 8i (IC50 = 0.03-0.29 µM). In addition, it was found that macrocycles 6e and 6f exhibit
selective cytotoxicity against Jurkat cells (IC50 = 0.03-0.08 µM), while macrocycle 7e selectively
affects U937 cells (IC50 = 0.07 µM). For the indicated cell lines, the therapeutic selectivity index
of cytotoxic action (SI) was in the range from 2 to 18 in comparison with normal fibroblasts
(Table 1). Therefore, for further studies of the induction of apoptosis, influence on the cell cycle,
and possible mechanism of cytotoxic action, we chose macrodiolide 8g containing the 1Z,5Z-
diene moiety and macrocycle 8i containing an aromatic ring apart from the diene moiety. In
addition, for comparison of the effects made by these unsaturated compounds on the cancer cell
lines, we included in the study the previously synthesized saturated analogue, 1,9-
dioxacyclohenicosane-2,8-dione 9.15
Induction of apoptosis and cell cycle analysis
The apoptosis-inducing activity of new macrodiolides was studied using Jurkat T-
lymphoblasts. In these tumor cells, the TP53 gene, which encodes the tumor suppressor р53,
involved in maintenance of the genome stability, is mutated. Back in 1990, Cheng and Haas
discovered a mutation in exon 6 of TP53 (rs397516435, p.R196X) that resulted in the formation
of a stop codon and a shortened not-functional p53 isoform in Jurkat cells.28 Later it was shown
that this mutation is associated with the Li-Fraumeni syndrome, which is an autosomal dominant
hereditary disease, characterized by appearance of various types of malignant tumors in young
people.29 Although the loss of р53 protective effects is usually taken as a mechanism of
oncogenesis and p53 protein is a classical tumor suppressor, in some cases, as was shown in
2014,30 it is the defective short p53 isoform that can become oncogenic. Also it was shown that
the shortened protein product, Ψp53, resulting from mutation in TP53 exon 6 can induce
expression of epithelial–mesenchymal transition markers, which in turn increases the mobility
and invasive ability of cancer cells, that is, directly affects the ability to metastasis. Moreover,
these mutant Ψp53 isoforms are located in mitochondria in which they activate the pro-
oncogenic cellular program by regulating the permeability of the mitochondrial membrane pores,
like Cyclophilin D, and thus block apoptotic processes.31 Thus, expression of the mutant Ψp53
isoform in Jurkat cell line may increase the stability of this cell line against classical inducers of
apoptosis initiated by the p53-dependent pathway.32
6

Numerous published data indicate that among macrocyclic compounds, one can find
molecules that can easily penetrate the mitochondrial membrane, uncouple oxidation and
phosphorylation, cause mitochondrial stress, and initiate apoptosis by exactly the mitochondrial
pathway. Macrocyclic polyethers with different substituents at the methylene groups (crowns)
are representatives of such ionophores or channel formers.
Considering the obtained results on cytotoxicity, we carried out a more detailed study of
the apoptosis-inducing activity and influence on the cell cycle in Jurkat cells for compounds 8i,
8g, and 9 (Fig. 1).
We showed that all of the above-listed macrodiolides exhibit pronounced apoptosis-
inducing activity towards Jurkat cells, their effect exceeding the effects of classical apoptosis-
inducing agents such as camptothecin and etoposide. Moreover, these compounds initiate the
mitotic catastrophe described in 2004 by Castedo and co-workers,33 who studied the cell cycle in
Jurkat cells. The highest percentage of late apoptosis (58.51%) and the lowest percentage of
living cells (5.43%) were observed upon the addition of compound 8i (0.1 µМ concentration) to
Jurkat cells; the lowest percentage of necrotic cells was found for the sample treated with 0.1 µМ
of compound 9 (0.97%) (Fig. 1А). Apparently, this high activity of compound 8i towards living
cells is attributable to the presence of the phenyl group in the molecule and, as a consequence,
higher cytotoxicity compared to other test compounds.
А
7

В
Figure 1. Histograms of cytofluorimetric assay of the apoptosis-inducing activity (А) and cell
cycle phases (B) in Jurkat tumor cells under the action of compounds 8i, 8g, and 9. The cells
were treated with different concentrations of compound 8i, 8g, and 9 and stained with annexin
V/7AAD. The incubation time of compounds 8i, 8g, and 9 with the cells was 24 hours. (A) The
histogram shows the apoptosis rate (%); *P<0.05 when compared with the control group (n=10).
(B) Flow cytometric histograms are representative of 3 separate experiments. Proliferation index:
PI = [S+(G2+M)]/[(G0/G1)+S+(G2+M)]×100%. Data are presented with the means ±SD and
mean values of three independent experiments. *p<0.05 and **p<0.01 in comparison with the
control group.
For the control sample, cell cycle characteristics of Jurkat cells included considerable
predominance of cells in the G0-G1 phase and a balance between the synthesis (S-phase) and
apoptosis (sub-G0-G1 phase) processes.
Within 24 hours after exposure to compounds 8i, 8g, and 9, apoptosis processes
predominated (increased sub-G0-G1 phase) with retained or even somewhat enhanced ability of
cells to DNA synthesis (S-phase) (Fig. 1В).
The highest value for the sub-G0-G1 phase was found for cell samples treated with
compound 8g in 0.15 µM concentration (19.68%) and compound 9 in 0.1 µM concentration
(13.52%) (Fig. 1В). Virtually for all cells, the fraction of cells in the G0-G1 phase was found to
decrease, while the proliferation block increased and proliferation index decreased due to the
decrease in the number of cells in the G2+M phase. All the foregoing may be indicative of a
cytotoxic activity of compounds 8i, 8g, and 9 against chronic myeloid and Т-cell leukemia cells
caused by the ability of these compounds to induce apoptosis.
Histones
8

For more detailed investigation of the mechanisms of action of the test macrodiolides, we
investigated the possible DNA damage in Jurkat tumor cells. Determination of the levels of both
total and phosphorylated H2A.Х protein in the same cell makes it possible to confirm the target
specificity of the phosphorylation event for this class of histones. The use of simultaneous
determination of phosphorylated and non-phosphorylated H2A.Х protein is a sensitive and
valuable tool for studying the factors that induce DNA damage and/or affect the DNA repair,
which can shed light on the relationship between the DNA damage, cell cycle checkpoints, and
initiation of apoptosis. Many chemotherapeutic agents such as etoposide damage tumor cells by
generating DNA double-strand breaks. Furthermore, it was also shown that the γ-H2A.X level
detected by flow cytofluorimetry is correlated with the number of DNA breaks and tumor cell
death.34 The H2A.X protein is a representative of H2A histone family. Serine phosphorylation in
position 139 of the H2A.X protein is a reliable indicator of DNA damage.35 As the content of
damaged DNA increases, the phosphorylation level of H2A.X (also known as γH2AX) increases,
particularly at the DNA damage sites. Hence, accumulation of phosphorylated H2A.X is often
used as an indication of the level of DNA damage inside the cell. H2A.X is also significant for
DNA repair processes.36
Stimulation of Jurkat cells with etoposide for 2 hours results in a pronounced growth of
the phosphorylated fraction of the H2A.X histone (Fig. 2). Analysis of Jurkat cells treated with
compounds 8i, 8g, and 9 demonstrated that these compounds affect the H2A.X phosphorylation
only slightly and virtually do not increase the cell population with the phosphorylated protein
(Fig. 2). Thus, it can be stated that compounds 8g, 8i, and 9 do not initiate accumulation of DNA
breaks followed by activation of р53-dependent apoptosis.
В
С
A
E
D
9

Figure 2. Detection of accumulation of phosphorylated H2A.X in Jurkat cells treated with
compounds 8i, 8g, and 9. The incubation with test compounds lasted for 2 hours. Control (non-
stained living cells) (A); cells treated with etoposide in 2 µM concentration (B); cells treated
with compound 8g in 0.2 µM concentration (C); cells treated with compound 8i in 0.2 µM
concentration (D); cells treated with compound 9 in 0.4 µM concentration (Е);
Q4-2, cells containing phosphorylated H2A.X histones (the DNA damage marker is staining
with phosphate-specific Anti-Phospho-Histone H2A.x antibodies (Ser139)–PerCP); Q4-3, cells
with no stain (intact living cells); Q4-4, cells containing non-phosphorylated H2A.X histones
(Anti-Histone H2A.X-FITC).
Mitochondrial damage
Mitochondria are known to play a critical role in the cell energy balance, regulation of
apoptotic proteins and to be the main source of reactive oxygen species, which are now
considered to be the damaging agent of many systemic inflammatory, tumor, and
neurodegenerative diseases.37 Superoxide is byproduct by disfunction produced upon oxidative
phosphorylation on the mitochondrial inner membrane during molecular oxygen reduction in the
respiratory chain.38 Superoxide can initiate a reaction cascade giving rise to a number of reactive
oxygen species (ROS) including peroxynitrite, hydrogen peroxide, and hydroxyl radical. The
vicious circle of oxidative stress and cell structure damage may lead to either cell death upon
apoptosis or energy deficiency of the cells and cell aging.39 The mitochondrial regulation of
apoptosis and oxidative stress are closely interrelated, which was demonstrated in numerous
studies. By detection of mitochondrial stress with MitoSox Red and annexin V, it is possible to
measure simultaneously two important characteristics of the state of cells: (1) formation of
mitochondrial superoxide, which is detected by the MitoSox Red dye penetrating the membrane
(2) and phosphatidylserine expression on the apoptotic cell surface, which is estimated by
binding annexin V to externalized phosphatidylserine; these two characteristics are measured in
the same cell sample at the same time. The multiparameter estimation of the cell viability
characteristics reveals the correlation and relationship between the oxidative stress and
apoptosis.
The highest mitochondrial oxidative stress (98.95%) was detected in the cells incubated
with compound 8g (Fig. 3B). Compounds 8i and 9 proved to be less active than 8g (22.17% and
25.9%, respectively) (Figs 3C and 3D).
10

B
A
C
D
Figure 3. Generation of reactive oxygen species and apoptotic cell death in Jurkat cells treated
with compounds 8i, 8g, and 9. (A, control sample; В, 8g (0.2 µM); С, 9 (0.4 µM), D, 8i (0.2
µM). The cell incubation time with test compounds was 2 hours.
We also studied the effect of the synthesized macrocyclic compounds on the change in
the mitochondrial membrane potential (ΔΨ) in Jurkat cells. According to the Mitchell and
Skulachev chemiosmotic theory, coupling of oxidation with phosphorylation is related to the
electrochemical proton potential ΔΨ arising on the mitochondrial inner membrane during the
electron transport. The proton gradient creating the chemical and electrical potential difference
serves as the energy source for ATP formation. A change in the mitochondrial potential in
combination with phosphatidylserine expression on the cell surface is considered to be reliable
indication of apoptosis initiated by mitochondrial damage and oxidative stress, which is
evaluated from annexin V binding, plasmatic membrane permeability, and the presence of dead
cells.
The change in the mitochondrial membrane potential (ΔΨ) in Jurkat cells on treatment
with compounds 8i, 8g, and 9 was measured using the MitoSense Red fluorescent cationic dye,
which is accumulated in mitochondria and responds to changes in the membrane potential.
Living, normally functioning cells with unchanged mitochondrial membrane potential
demonstrate high fluorescence of MitoSense Red, whereas cells with the violated mitochondrial
11

membrane potential show lower MitoSense Red fluorescence or no flurorescence at all. Annexin
V is calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine,
a membrane component usually located on the inner surface of living cell membrane. The
control cells do not show green fluorescence, whereas apoptotic cells exhibit positive green
fluorescence due to phosphatidylserine externalization. The DNA intercalator 7-
aminoactinomycin (7-AAD), which does not penetrate the cell, detects changes in the membrane
permeability for late apoptotic and dead cells. Thus, the multiparameter detection of three dyes,
MitoSense Red, Annexin V-CF488A, and 7-AAD, was used to analyze the key parameters of the
change in the mitochondrial membrane potential and the induction of apoptosis in Jurkat tumor
cells in response to the action of the macrodiolides (Fig. 4).
1
2
3
A
В
С
12

D
E
F
Figure 4. Detection of changes in the mitochondrial membrane potential (ΔΨ) and the
coupled early and late apoptosis in Jurkat cells treated with compounds 8i, 8g, and 9. A, control
sample containing living cells; B, staurosporine (0.2 µM); С, 8g (0.1 µM); D, 8g (0.2 µM); E, 8i
(0.2 µM); F, 9 (0.4 µM). The cells were stained with MitoSense Red, Annexin V– CF488A, and
7-AAD. The incubation time was 4 hours.
Evaluation of the membrane potential (Δψ) for mitochondria showed a reliable increase
in the content of Jurkat cells with decreased Δψ for samples treated with compounds 8g
(28.81%), 8i (14.22%), and 9 (15.35%). (Fig. 4, histogram groups D, E, and F). The most
pronounced decrease in the mitochondrial potential was induced by 8g; furthermore, this effect
was dose-dependent and was comparable with the action of staurosporine, a known inhibitor of
most protein kinases in the cell (Fig. 4, histogram groups B, C, and D). The percentage of
apoptotic cells in samples treated with 8g (2µL) was 92.85% (23.48% of early apoptosis and
69.37% of late apoptosis) (Fig. 4D). These results attest to activation of the mitochondrial
apoptosis pathway by compound 8g via pronounced suppression of the oxidation and
phosphorylation in the mitochondria of the Jurkat cells.
13

А
B
Figure 5. Histograms and heat maps of key signaling pathways in Jurkat tumor cells
treated with macrodiolides 8i (M40), 8g (M36), and 9 (M34). Phosphorylated (А) and total (B)
proteins CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3, and STAT5 were analyzed.
The images were drawn using the xPONENT® 4.2 for MAGPIX® software.
Activation and inhibition of the most universal cell signaling pathways responsible for
cell proliferation and apoptosis initiation were studied for Jurkat cancer cells after treatment with
the specified macrodiolides using multiplex assay by the Luminex xMAP technology, which
allows simultaneous detection of several protein analytes in the same sample. The Luminex
xMAP multiplex assay is based on flow fluorimetry of special polystyrene microspheres labeled
by specific fluorophores and subsequent laser detection and digital processing of the signal. We
studied two types of protein analytes that form phosphorylated and non-phosphorylated protein
fractions of key signaling pathways: CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3,
STAT5.
Expression of CREB, JNK, NFkB, p38, ERK1/2, Akt, p70S6K, STAT3, STAT5, and
phosphorylated ERK/MAP kinases (Thr185/Tyr187), Akt (Ser473), STAT3 (Ser727), JNK
(Thr183/Tyr185), p70 S6 kinase (Thr412), NFkB (Ser536), STAT5A/B (Tyr694/699), CREB
(Ser133), and p38 (Thr180/Tyr182) in six Jurkat cancer cell lysate samples was studied by the
Luminex technology (MILLIPLEX® MAP 9-Plex Multi-Pathway 9-plex Magnetic Bead Kit).
14

This granule-based assay uses fluorescence beads with color encoding precoated by specific
antibodies targeting 9 main kinases in the signaling pathways.
Generally, on the basis of pairwise comparison of phosphorylated and non-
phosphorylated protein fractions, the major changes of the ratio between these two fractions
occur for Аkt, p38, and CREB (Fig. 5А). 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 p70 S6, Msk1, and STAT3
(Ser727) kinases and CREB transcription factor and enhance also the activation of other
intermediates. Other signaling pathways induced by stress or death receptor (e.g., TNFа) activate
p38, JNK, and NF-кB. As can be seen from 5А, compound 8g in 0.2 µM concentration causes a
most pronounced decrease for all types of protein kinases in a cancer cell. Macrocycle 8i (0.2
µM) and 9 (0.4 µM) are somewhat inferior to 8g in activity; however, they also efficiently affect
the amount of phosphorylated forms of various kinases compared with the control. Compound
8g most strongly suppresses Аkt, p38, and CREB. Аkt (RAC-alpha serine/threonine-protein
kinase, Protein kinase B alpha) is a cellular enzyme, one of the three representatives of protein
kinase B family. Аkt is a key enzyme of the PI3K/Akt signaling pathway and is involved in the
regulation of cell proliferation, growth, and survival.40 Functioning of this enzyme attracts
considerable research attention, since it acts as an oncogen for many malignant diseases.41
Compound 9 (Fig. 5) is more selective than 8g or 8i in the action on the protein profile of the
considered signaling pathways; it suppresses ERK1/2, NFkB, and JNK kinases, while p38
phosphorylation and CREB transcription factor are affected to a somewhat lesser extent. This
may be attributable to its molecular structure containing a 1Z,5Z-diene moiety and an aromatic
ring, which differs from the structures of 8g and 8i.
CONCLUSIONS
Thus, we developed an original stereoselective synthesis of previously undescribed
macrodiolides containing a 1Z,5Z-diene moiety in the molecule in high yields and with high
stereoselectivity (>98%). The developed method includes synthesis of α,ω-alka-nZ,(n+4)Z-
dienedicarboxylic acids based on the use of original Ti-catalyzed intermolecular homo-
cyclomagnesiation of O-containing 1,2-dienes with Grignard reagents followed by their
intermolecular esterification with aliphatic and aromatic α,ω-diols catalyzed by hafnium triflate
Hf(OTf)4. The biological activity of the new macrodiolides was studied. The prepared
macrodiolides were found to exhibit high cytotoxic activity in vitro against Jurkat, K562, U937,
Hek293, and HeLa cell lines and fibroblasts. Moreover, in relation to compounds 8i, 8g, and 9,
which exhibited the highest cytotoxic effect, it was found that these macrodiolides acting as
inducers of programmed cell death initiate the mitochondrial apoptosis pathway in Jurkat cells.
The most pronounced decrease in the mitochondrial potential is attained for macrocycle 8g, this
effect being dose-dependent and comparable with the effect of staurosporine, a known inhibitor
of most protein kinases in the cell. By means of multiplex assay, macrodiolides 8i, 8g, and 9
were shown to efficiently suppress phosphorylation of the Аkt and p38 kinases and the CREB
transcription factor in р53-deficient Jurkat cells. Thus, inhibition of the key kinases of three
signaling pathways initiated by compounds 8g, 8i, and 9 evidently depresses the growth and
proliferation of tumor cells; compound 9 suppresses more selectively the phosphorylation of
15

ERK1/2, NFkB, and JNK signaling and to a lesser extent affects p38 and CREB. The data
obtained suggest that, most likely, the studied macrocyclic compounds affected the expression of
proteins of the signaling pathways and suppressing it, since we see that both the quantitative
content of the total pool of protein kinases and the amount of the phosphorylated proteins
fraction are reduced in the treated cells. We believe that further detailed investigation of
participation of compounds 8g, 8i, and 9 in the change of activity of intracellular signaling would
shed light on the change in the proliferative activity of p53-deficient tumor cells under the action
of these macrocycles.
EXPERIMENTAL SECTION
1
13
General: H, C NMR spectra were obtained using a Bruker AVANCE 400 in CDCl3
1
13
operating at 400.13 MHz for H, 100.62 MHz for C and Bruker Ascend -500 (500 MHz (1H),
125 MHz (13C). Elemental analyses were measured on a 1106 Carlo Erba apparatus. Mass
spectra were obtained on MALDI TOF/TOF spectrometer in a sinapic acid matrix. All solvents
were dried (diethyl ether, toluene over Na, dioxane over NaOH, methanol over Mg, chloroform
over P2O5) and freshly distilled before use. All reactions were carried out under a dry argon
atmosphere. Commercially available alkynols, Hf(OTf)4 (Aldrich), Cp2TiCl2 (Aldrich) were
used. α,ω-Alca-nZ,(n+4)Z-diendicarboxylic acid synthesized according to procedures described
in literature.26
Synthetic procedures for compounds 4a-c
To a solution of THP ethers 3a-c1 (0.5 mmol) in acetone (12 mL) and CH2Cl2 (2 mL) at
rt, Jones reagent (1.0 mL) was added dropwise. The reaction mixture was stirred at rt for 1 h,
then quenched with water (5 mL), concentrated under reduced pressure to remove the excess of
acetone and CH2Cl2, and the aqueous layer extracted with diethyl ether (3 × 10 mL). The organic
layer was dried over MgSO4 and concentrated in vacuo. The residue was purified by column
chromatography using hexane/ethyl acetate = 3/1 as the elution solvent to afford dienedioic acids
4a-c.
(4Z,8Z)-dodeca-4,8-dienedioic acid (4a). White waxy solid; yield 50 %. Rf = 0.33,
1
hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.48 – 5.35 (m, 4H), 2.45 – 2.34 (m, 8H),
2.15 – 2.05 (m, 4H).13C NMR (101 MHz, CDCl3): δ = 179.6, 130.9, 127.6, 34.2, 27.1, 22.6. MS
(MALDI-TOF): calcd. for С12H18O4 + Na+ [M + Na]+ 249.110; found 249.121. Anal. Calcd for
C12H18O4: C, 63.70; H, 8.02. Found: C, 63.67; H, 8.06.
(5Z,9Z)-tetradeca-5,9-dienedioic acid (4b). White waxy solid; yield 52 %. Rf = 0.36,
1
hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.45 – 5.35 (m, 4H), 2.38 (t, J = 7.3 Hz,
13
4H), 2.14 – 2.08 (m, 8H), 1.76 – 1.68 (m, 4H). C NMR (101 MHz, CDCl3): δ = 180.3, 130.4,
128.8, 33.4, 27.3, 26.4, 24.5. MS (MALDI-TOF): calcd. for С14H22O4 + Na+ [M + Na]+ 277.142;
found 277.134. Anal. Calcd for C14H22O4: C, 66.12; H, 8.72. Found: C, 66.21; H, 8.64.
(7Z,11Z)-octadeca-7,11-dienedioic acid (4c). White waxy solid; yield 56 %. Rf = 0.37,
1
hexane/EtOAc 5:1. H NMR (500 MHz, CDCl3): δ = 5.38 – 5.35 (m, 4H), 2.35 (t, J = 7.3 Hz,
4H), 2.10 – 1.99 (m, 8H), 1.68 – 1.62 (m, 4H), 1.39 – 1.34 (m, 8H). 13C NMR (101 MHz,
CDCl3): δ = 180.3, 129.9, 129.4, 34.1, 29.3, 28.7, 27.4, 27.0, 24.6. MS (MALDI-TOF): calcd.
for С18H30O4 + Na+ [M + Na]+ 333.204; found 333.192. Anal. Calcd for C18H30O4: C, 69.64; H,
9.74. Found: C, 69.47; H, 9.86.
16

General procedure synthesis of macrodiolides. Dienedicarboxylic acid (0.2 mmol, 1.0
equiv.) and diol (0.2 mmol, 1.0 equiv.) were dissolved in toluene (40 mL, 5 mM). Then Hf(OTf)4
(0.015 g, 0.02 mmol, 0.1equiv.) was added to the solution and the reaction mixture heated to 110
oC for 16-18 h. After cooling to room temperature, silica gel (~ 0.7 g) was added and the slurry
was concentrated under reduced pressure and purified by column chromatography (elution with
petroleum ether/EtOAc (15/1)) to afford the desired product as a colorless oil.
1,7-dioxacyclononadeca-11Z,15Z-diene-8,19-dione (6d). Colorless oil; yield 44%. Rf =
0.52, hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 5.62 – 5.31 (m, 4H), 4.16 – 4.09 (m,
13
4H), 2.46 – 2.26 (m, 8H), 2.17 – 1.95 (m, 4H), 1.71 – 1.61 (m, 4H), 1.54 – 1.48 (m, 2H). C
NMR (101 MHz, CDCl3): δ = 173.1, 130.5, 128.1, 64.0, 35.06, 28.2, 27.3, 23.3, 23.0. MS
(MALDI-TOF): calcd. for С17H26O4 + Na+ [M + Na]+ 317.173; found 317.192. Anal. Calcd for
C17H26O4: C, 69.36; H, 8.90. Found: C, 69.27; H, 8.96.
1,8-dioxacycloicosa-12Z,16Z-diene-9,20-dione (6e). Colorless oil; yield 55%. Rf = 0.54,
hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 5.50 – 5.31 (m, 4H), 4.13 (t, J = 6.1 Hz,
4H), 2.42 – 2.31 (m, 8H), 2.13 – 2.06 (m, 4H), 1.69 – 1.64 (m, 4H), 1.47 – 1.41 (m, 4H) ppm.
13C NMR (101 MHz, CDCl3): δ = 173.2, 130.7, 127.9, 64.0, 34.8, 28.2, 27.3, 25.3, 23.2 ppm.
MS (MALDI-TOF): calcd. for С18H28O4 + Na+ [M + Na]+ 331.189; found 331.202. Anal. Calcd
for C18H28O4: C, 70.10; H, 9.15. Found: C, 70.03; H, 9.23.
1,9-dioxacyclohenicosa-13Z,17Z-diene-10,21-dione (6f). Colorless oil; yield 64%. Rf =
0.54, hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 5.52 – 5.33 (m, 4H), 4.12 (t, J = 6.1
Hz, 4H), 2.46 – 2.30 (m, 8H), 2.16 – 2.02 (m, 4H), 1.66 – 1.61 (m, 4H), 1.42 – 1.35 (m, 6H)
13
ppm. C NMR (101 MHz, CDCl3): δ = 173.2, 130.6, 128.0, 64.2, 34.9, 28.3, 27.2, 25.8, 23.2
ppm. MS (MALDI-TOF): calcd. for С19H30O4 + Na+ [M + Na]+ 345.204; found 345.291. Anal.
Calcd for C19H30O4: C, 70.77; H, 9.38. Found: C, 70.69; H, 9.27.
1,10-dioxacyclodocosa-14Z,18Z -diene-11,22-dione (6g). Colorless oil; yield 65%. Rf =
0.54, hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 5.91 – 5.12 (m, 4H), 4.12 (t, J = 5.9
Hz, 4H), 2.42 – 2.32 (m, 8H), 2.20 – 1.90 (m, 4H), 1.68 – 1.60 (m, 4H), 1.44 – 1.32 (m, 8H).
13
ppm. C NMR (126 MHz, CDCl3): δ = 23.7, 24.9, 27.2, 28.4, 28.5, 34.5, 63.5, 128.8, 130.1,
173.8 ppm. MS (MALDI-TOF): calcd. for С20H32O4 + Na+ [M + Na]+ 359.220; found 359.234.
Anal. Calcd for C20H32O4: C, 71.39; H, 9.59. Found: C, 71.28; H, 9.52.
1,6-Dioxacycloicosa-11Z,15Z-diene-7,20-dione (7c). Colorless oil; yield 65%. Rf =
0.52, hexane/EtOAc 5:1.1H NMR (400 MHz, CDCl3): δ = 5.53 – 5.29 (m, 4H), 4.20 – 4.09 (m,
4H), 2.34 (t, J = 5.9 Hz, 4H), 2.17 – 1.91 (m, 8H), 1.74 – 1.60 (m, 8H) ppm. 13C NMR
(101MHz, CDCl3): δ = 172.1, 130.3, 128.9, 63.8, 33.4, 29.7, 27.4, 25.5, 24.7 ppm. MS (MALDI-
TOF): calcd. for С18H28O4 + Na+ [M + Na]+ 331.188; found 331.201. Anal. Calcd for С18H28O4:
С, 70.10; Н, 9.15. Found: C, 70.18; H, 9.21.
1,7-Dioxacyclohenicosa-12Z,16Z-diene-8,21-dione (7d). Colorless oil; yield 71%. Rf =
1
0.52, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ= 5.49 – 5.30 (m, 4H), 4.12 (t, J = 6.1
Hz, 4H), 2.41 – 2.25 (m, 4H), 2.14 – 1.96 (m, 8H), 1.79 – 1.62 (m, 8H), 1.55 – 1.47 (m, 2H)
ppm. 13C NMR (100.62 MHz, CDCl3): δ= 173.8, 130.3, 129.0, 64.0, 33.6, 28.2, 27.4, 26.3, 24.7,
23.1 ppm. MS (MALDI-TOF): calcd. for С19H30O4 + Na+ [M + Na]+ 345.204; found 345.301.
Anal. Calcd for С19H30O4: С, 70.77; Н, 9.38. Found: C, 70.71; H, 9.31.
17

1,8-Dioxacyclodocosa-13Z,17Z-diene-9,22-dione (7e). Colorless oil; yield 76%. Rf =
1
0.52, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.51 – 5.33 (m, 4H), 4.13 (t, J = 6.0
Hz, 4H), 2.34 (t, J = 6.8 Hz, 4H), 2.15 – 1.96 (m, 8H), 1.76 – 1.68 (m, 4H), 1.61 – 1.51 (m, 4H),
1.47 – 1.41 (m, 4H) ppm. 13C NMR (100.62 MHz, CDCl3): δ = 173.7, 130.2, 129.0, 64.1, 33.7,
28.3, 27.3, 26.4, 25.6, 24.9 ppm. MS (MALDI-TOF): calcd. for С20H32O4 + Na+ [M + Na]+
359.219; found 359.321. Anal. Calcd for С20H32O4: С, 71.39; Н, 9.59. Found: C, 71.46; H, 9.64.
1,9-Dioxacyclotricosa-14Z,18Z-diene-10,23-dione (7f). Colorless oil; yield 78%. Rf =
1
0.51, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.50 – 5.32 (m, 4H), 4.11 (t, J = 5.9
Hz, 4H), 2.39 – 2.29 (m, 4H), 2.16 – 1.96 (m, 8H), 1.75 – 1.60 (m, 8H), 1.45 – 1.35 (m, 6H)
13
ppm. C NMR (101 MHz, CDCl3): δ = 173.8, 130.2, 129.0, 64.2, 33.7, 28.5, 28.3, 27.3, 26.4,
25.9, 24.8 ppm. MS (MALDI-TOF): calcd. for С21H34O4 + Na+ [M + Na]+ 373.235; found
373.331. Anal. Calcd for С21H34O4: С, 71.96; Н, 9.78. Found: C, 71.84; H, 9.71.
1,10-Dioxacyclotetracosa-15Z,19Z-diene-11,24-dione (7g). Colorless oil; yield 80%. Rf
1
= 0.52, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.45 – 5.28 (m, 4H), 4.15 – 4.10
(m, 4H), 2.33 (t, J = 7.1 Hz, 4H), 2.12 – 1.97 (m, 8H), 1.75 – 1.60 (m, 8H), 1.45 – 1.28 (m, 8H)
13
ppm. C NMR (100.62 MHz, CDCl3): δ = 173.8, 130.2, 129.0, 64.3, 33.7, 28.64, 28.4, 27.3,
26.4, 25.7, 24.8 ppm. MS (MALDI-TOF): calcd. for С22H36O4 + Na+ [M + Na]+ 387.251; found
387.313. Anal. Calcd for С22H36O4: С, 72.49; Н, 9.95. Found: C, 72.37; H, 9.86.
1,12-dioxacyclohexacosa-17Z,21Z-diene-13,26-dione (7h). Colorless oil; yield 79%. Rf
= 0.51, hexane/EtOAc 5:1. 1H NMR (500 MHz, CDCl3): δ = 5.53 – 5.24 (m, 4H), 4.08 (t, J = 6.4
Hz, 4H), 2.37 – 2.28 (m, 4H), 2.15 – 1.96 (m, 8H), 1.76 – 1.65 (m, 4H), 1.68 – 1.56 (m, 4H),
13
1.42 – 1.29 (m, 12H) ppm. C NMR (125 MHz, CDCl3): δ = 173.8, 130.2, 129.0, 64.4, 33.7,
29.4, 29.2, 28.6, 27.3, 26.6, 25.9, 24.9 ppm. MS (MALDI-TOF): calcd. for С24H40O4 + Na+ [M +
Na]+ 415.282; found 415.346. Anal. Calcd for С24H40O4: С, 73.43; Н, 10.27. Found: C, 73.29; H,
10.18.
1,6-Dioxacyclotetracosa-13Z,17Z-diene-7,24-dione (8c). Colorless oil; yield 65%. Rf =
1
0.52, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.47 – 5.32 (m, 4H), 4.14 (s, 4H),
2.32 (t, J = 6.8 Hz, 4H), 2.11 – 1.99 (m, 8H), 1.76 – 1.62 (m, 8H), 1.37 - 1.32 (m, 8H) ppm. 13C
NMR (101 MHz, CDCl3): δ = 173.7, 129.9, 129.5, 63.8, 34.4, 29.7, 29.2, 28.5, 27.6, 26.8, 25.5,
24.9 ppm. MS (MALDI-TOF): calcd. for С22H36O4 + Na+ [M + Na]+ 387.251; found 387.323.
Anal. Calcd for C22H36O4: C, 72.49; H, 9.95. Found: C, 72.37; H, 10.02.
1,7-Dioxacyclopentacosa-14Z,18Z-diene-8,25-dione (8d). Colorless oil; yield 69%. Rf =
1
0.55, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.48 – 5.33 (m, 4H), 4.12 (t, J = 6.1
Hz, 4H), 2.32 (t, J = 7.3 Hz, 4H), 2.13 – 1.98 (m, 8H), 1.72 – 1.61 (m, 8H), 1.53 – 1.30 (m, 10H)
13
ppm. C NMR (101 MHz, CDCl3): δ = 173.8, 129.9, 129.5, 63.9, 34.4, 29.1, 28.6, 28.1, 27.5,
26.9, 24.9, 22.7 ppm. MS (MALDI-TOF): calcd. for С23H38O4 + Na+ [M + Na]+ 401.266; found
401.343. Anal. Calcd for C23H38O4: C, 72.98; H, 10.12. Found: C, 73.06; H, 10.17.
1,8-Dioxacyclohexacosa-15Z,19Z-diene-9,26-dione (8e). Colorless oil; yield 72%. Rf =
1
0.56, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.45 – 5.34 (m, 4H), 4.11 (t, J = 6.4
Hz, 4H), 2.32 (t, J = 7.1 Hz, 4H), 2.13 – 1.97 (m, 8H), 1.70 – 1.61 (m, 8H), 1.49 – 1.32 (m, 12H)
13
ppm. C NMR (101 MHz, CDCl3): δ = 173.9, 129.9, 129.4, 64.1, 34.4, 29.1, 28.4, 28.4, 27.5,
26.8, 25.6, 24.9 ppm. MS (MALDI-TOF): calcd. for С24H40O4 + Na+ [M + Na]+ 415.282; found
415.323. Anal. Calcd for C24H40O4: C, 73.43; H, 10.27. Found: C, 73.29; H, 10.37.
18

1,9-dioxacycloheptacosa-16Z,20Z-diene-10,27-dione (8f). Colorless oil; yield 75%. Rf
= 0.56, hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 5.48 – 5.32 (m, 4H), 4.10 (t, J = 5.9
Hz, 4H), 2.32 (t, J = 7.3 Hz, 4H), 2.15 – 1.97 (m, 8H), 1.71 – 1.62 (m, 10H), 1.46 – 1.21 (m,
13
12H) ppm. C NMR (101 MHz, CDCl3): δ = 173.9, 129.9, 129.5, 64.2, 34.5, 29.2, 28.8, 28.5,
28.4, 27.5, 26.9, 25.8, 24.9 ppm. MS (MALDI-TOF): calcd. for С25H42O4 + Na+ [M + Na]+
429.298; found 429.334. Anal. Calcd for C25H42O4: C, 73.85; H, 10.41. Found: C, 73.74; H,
10.48.
1,10-Dioxacyclooctacosa-17Z,21Z-diene-11,28-dione (8g). Colorless oil; yield 79%. Rf
1
= 0.55, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 5.47 – 5.32 (m, 4H), 4.10 (t, J =
6.1 Hz, 4H), 2.32 (t, J = 7.1 Hz, 4H), 2.13 – 1.91 (m, 8H), 1.68 – 1.57 (m, 10H), 1.35 – 1.28 (m,
14H) ppm.13C NMR (101 MHz, CDCl3): δ = 173.9, 129.9, 129.4, 64.2, 34.5, 29.1, 28.8, 28.5,
28.5, 27.5, 26.9, 25.8, 24.9 ppm. MS (MALDI-TOF): calcd. for С26H44O4 + Na+ [M + Na]+
443.313; found 443.328. Anal. Calcd for C26H44O4: C, 74.24; H, 10.54. Found: C, 74.09; H,
10.43.
1,12-dioxacyclotriaconta-19Z,23Z-diene-13,30-dione (8h). Colorless oil; yield 79%. Rf
= 0.51, hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 5.43 – 5.29 (m, 4H), 4.10 (t, J = 5.9
Hz, 4H), 2.32 (t, J = 7.1 Hz, 4H), 2.14 – 1.96 (m, 8H), 1.69 – 1.59 (m, 10H), 1.42 – 1.29 (m,
13
18H) ppm. C NMR (100 MHz, CDCl3): δ = 173.9, 129.9, 129.4, 64.3, 34.5, 29.2, 29.0, 28.9,
28.6, 27.4, 26.9, 25.9, 25.0 ppm. MS (MALDI-TOF): calcd. for С28H48O4 + Na+ [M + Na]+
471.345; found 471.388. Anal. Calcd for С28H48O4: С, 74.95; Н, 10.78. Found: C, 74.72; H,
10.61.
4,17-dioxabicyclo[18.2.2]tetracosa-1(22),8,12,20,23-pentaene-5,16-dione
(6i).
Colorless oil; yield 55%. Rf = 0.42, hexane/EtOAc 5:1. 1H NMR (500 MHz, CDCl3): δ = 7.17 (s,
4H), 5.41 – 5.29 (m, 4H), 4.42 – 4.34 (m, 4H), 2.97 – 2.92 (m, 4H), 2.33 – 2.23 (m, 8H), 2.07 –
2.01 (m, 4H) ppm.13C NMR (125 MHz, CDCl3): δ = 173.1, 136.6, 130.5, 128.9, 128.0, 64.5,
34.8, 34.5, 27.0, 22.9 ppm. MS (MALDI-TOF): calcd. for С22H28O4 + Na+ [M + Na]+ 379.189;
found 379.205. Anal. Calcd for C22H28O4: C, 74.13; H, 7.92. Found: C, 74.21; H, 7.81.
4,19-dioxabicyclo[20.2.2]hexacosa-1(24),9,13,22,25-pentaene-5,18-dione
(7i).
1
Colorless oil; yield 63%. Rf = 0.44, hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 7.16
(s, 4H), 5.48 – 5.13 (m, 4H), 4.35 (d, J = 4.9 Hz, 4H), 2.99 – 2.90 (m, 4H), 2.32 – 2.21 (m, 4H),
13
2.13 – 1.87 (m, 8H), 1.69 – 1.54 (m, 4H) ppm. C NMR (101 MHz, CDCl3) δ = 173.6, 136.6,
130.2, 129.4, 128.9, 64.3, 34.5, 33.6, 27.3, 26.4, 24.7 ppm. MS (MALDI-TOF): calcd. for
С24H32O4 + Na+ [M + Na]+ 407.220; found 407.197. Anal. Calcd for C24H32O4: C, 74.97; H, 8.39.
Found: C, 75.12; H, 8.27.
4,23-dioxabicyclo[24.2.2]triaconta-1(28),11,15,26,29-pentaene-5,22-dione
(8i).
Colorless oil; yield 64%. Rf = 0.46, hexane/EtOAc 5:1. 1H NMR (400 MHz, CDCl3): δ = 7.16 (s,
4H), 5.45 – 5.30 (m, 4H), 4.33 (t, J = 6.1 Hz, 4H), 2.93 (t, J = 6.1 Hz, 4H), 2.26 (t, J = 7.1 Hz,
13
4H), 2.12 – 1.93 (m, 8H), 1.61 – 1.51 (m, 4H), 1.35 – 1.24 (m, 8H) ppm. C NMR (101 MHz,
CDCl3): δ = 173.7, 136.3, 129.9, 129.4, 128.9, 64.4, 34.7, 34.4, 29.1, 28.4, 27.5, 26.9, 24.9 ppm.
MS (MALDI-TOF): calcd. for С28H40O4 + Na+ [M + Na]+ 463.282; found 463.344. Anal. Calcd
for C28H40O4: C, 76.33; H, 9.15. Found: C, 76.21; H, 9.09.
1,9-Dioxacyclohenicosane-2,8-dione (9). Colorless oil; yield 59%. Rf = 0.51,
1
hexane/EtOAc 5:1. H NMR (400 MHz, CDCl3): δ = 1.12– 1.44 (m, 18H), 1.52– 1.84 (m, 8H),
19

2.33 (t, J = 7.3 Hz, 4H), 4.13 (t, J = 5.9 Hz, 4H) ppm.13C NMR (101 MHz, CDCl3): δ = 24.8,
25.9, 28.1, 28.2, 28.3, 28.4, 28.7, 34.4, 64.3, 173.6 ppm. MS (MALDI-TOF): calcd. for С19H34O4
+ Na+ [M + Na]+ 349.235; found 349.298. Anal. Calcd for C19H34O4: C, 69.90; H, 10.50. Found:
C, 69.87; H, 10.46.
Cell culturing
Cells (Jurkat, K562, U937, 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. All cell lines used in the study were tested and shown to
be free of mycoplasma and other viral contamination. HeLa and HEK293 cell lines were cultured
as monolayers and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL)
supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin solution at 37 °C in
a humidified incubator under a 5% CO2 atmosphere. Cells were maintained in RPMI 1640
(Jurkat, K562, U937) (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 days intervals. Adherent cells (HeLa, HEK293
and normal fibroblasts) were suspended using trypsin/EDTA and counted after they have
reached 80% confluency. Cells were then seeded in 24 well plates at 5×104 cells per well and
incubated overnight. Jurkat, K562, U937 cells were subcultured at 2 day intervals with a seeding
density of 1×105 cells per 24 well plates in RPMI with 10% FBS.
Cytotoxicity assay
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 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 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.
Viability and apoptosis
Apoptosis 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 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 NovoCyteTM2000 FlowCytometry System (ACEA).
Cell cycle analysis
Cell cycle was analyzed using the method of propidium iodide staining. After treatment cells
during 24 h were harvested, washed 1–2 times with phosphate-buffered saline (PBS) and
centrifuged at 400g for 5 min. Cell pellets were resuspended in 200 μL of flow cytometry
20

staining buffer (PBS without Ca2+ and Mg2+, 2.5% FBS). Then, 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% ethanol for 24 h at 0 °C. Cells were then 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 NovoCyteTM 2000FlowCytometry System (ACEA).
Histone H2A.X analysis
Cell preparation for phosphorylation of Histone H2A.X in Jurkat cells analysis were grown in
culture flask at 48 hours. The medium was changed at 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, MA, USA). 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 then immediately chilled on ice, centrifuged, washed three times with 0.5% bovine serum
albumin (BSA; EMD Bioscience, La Jolla, CA)-PBS, permeabilized with 70% ethanol in
distilled water or 90% methanol in 0.5% BSA-PBS, and kept at 4°C for ethanol samples and at –
20°C for methanol samples until further staining. After that, the cells were fixed, permeabilized,
and incubated with antiphosphohistone H2A.X (Ser139, Alexa Fluor®555, part number
CS208203, Millipore, Billerica, MA, USA) and anti-H2A.X (PECy5, part number CS209202,
Millipore, Billerica, MA, USA), and then analyzed by were analyzed by flow cytometry.
Mitochondrial damage
In our work we performed 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). Millipore’s FlowCellect™MitoDamage Kit use of the reagents
allows researchers to obtain information on early and late apoptosis in one simple assay. Cells
were treated with testing compounds 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-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
21

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.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available on website at DOI:
AUTHOR INFORMATION
Corresponding Authors
*E-mail: DyakonovVA@gmail.com (Chemistry, VAD), Dzhemilev@mail.ru (Biology, LUD)
Notes: The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Russian Science Foundation (grant No. 17-73-
10136). 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 and
biological screening of candidate substances for the pharmaceutical industry at the Institute of
Petrochemistry and Catalysis of RAS.
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Graphical Abstract
26

Declaration of interests
☐The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
27

Highlights
−
−
−
−
Catalytic synthesis of new 1Z,5Z-diene macrodiolides have been developed.
New molecules are efficient apoptosis inducers in cancer cells.
Macrodiolides suppress phosphorylation of Аkt and p38 kinases and CREB transcription factor.
The structures of all novel compounds were confirmed by NMR measurements.
28