A new original approach to the design of anticancer drugs based on energy-rich quadricyclanes

Article abstract of DOI:10.1007/s11172-019-2516-1

Quadricyclane derivatives are shown for the first time to be promising for application as anticancer drugs. The efficiency of such cage compounds is mainly due to thermal action on cancer cells resulting from a C—C bond cleavage in quadricyclane on exposu

Full text of DOI:10.1007/s11172-019-2516-1

Russian Chemical Bulletin, International Edition, Vol. 68, No. 5, pp. 1036—1040, May, 2019
1036
A new original approach to the design of anticancer drugs
based on energy-rich quadricyclanes
U. M. Dzhemilev, A. R. Akhmetov, A. A. Khuzin, V. A. D´yakonov, L. U. Dzhemileva,
M. M. Yunusbaeva, L. M. Khalilov, and A. R. Tuktarov
Institute of Petroleum Chemistry and Catalysis, Russian Academy of Sciences,
141 prosp. Oktyabrya, 450075 Ufa, Republic of Bashkortostan, Russian Federation.
Fax: +7 (347) 284 2750. Е-mail: tuktarovar@gmail.com
Quadricyclane derivatives are shown for the first time to be promising for application as
anticancer drugs. The efficiency of such cage compounds is mainly due to thermal action on
cancer cells resulting from a С—С bond cleavage in quadricyclane on exposure to catalytic
amounts of cisplatin Pt ions.
Key words: norbornadiene, quadricyclane, anticancer activity.
The discovery of valence isomerization of norborna-
diene to quadricyclane1,2 has led to a great number of
studies of their applicability as rocket propellant addi-
tives,3,4 molecular switchers and optical waveguides on
their basis,5—9 photochromic chemosensors,10,11 photo-
switchable organic magnetic materials,12—14 and solar
energy storage materials.3,15,16 This was due to the forma-
tion of a metastable quadricyclane structure containing
highly strained one cyclobutane and two cyclopropane
rings. Despite the so strained structure, the quadricyclane
molecule is stable and, in the absence of a catalyst, the
half-time of reversible transformation to norbornadiene is
14 h at 140 C,1 which is due to orbital forbidding of
[2σ+2σ]-addition. The energy barrier of this thermal
transformation could be lowered using catalysts, such as
transition metal compounds and Lewis acids.
The quadricyclane-to-norbornadiene isomerization
was described first in Ref. 17. To date, a great number of
different Rh, Pt, Pd, and other transition metal complexes
exhibiting high catalytic activity in this reaction have been
described. Their catalytic efficiency depends on the
d-orbital population of the metal atom and the geometric
configuration of the complex.18 For example, among ef-
ficient catalysts18 are metal ions with a d6—d8 electron
shell, including PdII and PtII complexes.
medication cisplatin, at much lower concentration, the
cage molecule will undergo cleavage to release a heat
thereby exercising additional thermal effect on cancer cells.
In order to achieve this aim, namely, the synthesis of
new norbornadiene and quadricyclane derivatives, we
chose 2,3-disubstituted norbornadiene 1 bearing a carboxyl
group as a model compound.
Target quadricyclane adducts 2 and 3 were synthesized
in there steps (Scheme 1): the Diels—Alder reaction to
afford norbornadiene carboxylic acid 1, esterification of
acid 1 with triethylene glycol (TEG) in order to increase
the aqueous solubility, and photoisomerization of the
norbornadiene moiety in esters 4 and 5 into the quadri-
cyclane derivatives 2 and 3.
The structure of the synthesized compounds was well
proved using modern spectral methods of analysis.
The 1H and 13C NMR spectra of compound 4 contain
characteristic signals of the carboxyl group at δC 167.26 and
of sp2-hybridized carbon atoms of the norbornadiene moi-
ety and the phenyl group in a region of δС 128—165, five
signals of the oxyethyl groups at δC 61.73, 69.06, 70.38, 70.54,
70.60, and 72.63, as well as a signal of the bridging carbon
atom (δС 53.00) linked with two geminal protons (δH 2.08
and 2.27). The photoinduced isomerization of the norbor-
nadiene moiety into the quadricyclane one results in up-
field shift of the signals of cage carbon atoms (δC 20—30).
The replacement of the terminal hydroxyl group in
compounds 2 and 4 with the norbornadiene or quadri-
cyclane one results in the appearance of an efficient sym-
metry axis in the molecules of 3 and 5, respectively, which
is manifested in a decrease in the number of signals of the
triethylene glycol spacer to three (δC 63.20, 69.00, 70.39
for the quadricyclane isomer 3 and δС 69.08, 70.58, 70.53
for compound 5).
The fact that strained C—C bonds in the quadricyclane
molecule can be cleaved in the presence of catalytic
amounts of Pd or Pt ions to release a heat of ~100 kJ mol–1
inspired us to synthesize quadricyclane derivatives as
promising anticancer agents. We assumed that, as a result
of active metabolism, cancer cells will accumulate quadri-
cyclane molecules more intensively than healthy cells and,
upon subsequent administration of Pd or Pt ions to the
human body, for example, in the form of a well-known
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1036—1040, May, 2019.
1066-5285/19/6805-1036 © 2019 Springer Science+Business Media, Inc.

Anticancer drugs based on quadricyclanes
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 5, May, 2019
1037
Scheme 1
DCC is dicyclohexylcarbodiimide.
Having synthesized the target quadricyclanes 2 and 3,
we investigated their in vitro anticancer activity vs. the
norbornadiene analogs 4 and 5, respectively, as well as
pure cisplatin against human T-lymphoblastic leukemia
(Jurkat cells) and histiocytic lymphoma (U937) cells.
Taking into account that one mole of compound 3 contains
two-fold more quadricyclane fragments than one mole
of 2, we used a two-fold diluted solution of compound 3
in DMSO.
As the cytotoxicity study showed, compounds 2 and 3
taken alone, as well as cisplatin have no considerable effect
on the viability of Jurkat cells even after 72 h (Table 1).
Having obtained the control parameters of cytotoxicity
of starting compounds 2 and 3 and cisplatin, we studied
whether the exothermic transition of quadricyclanes into
norborna-2,5-dienes in the presence of catalytic amounts
of platinum compounds can be used for thermal action on
cancer cells.
For example, in our first experiments Jurkat cells were
treated simultaneously with solutions of compounds 2 and
3 in combination with cisplatin at different concentrations
and, after incubation for 24 h, were stained with 7-amino-
actinomycin D (7-AAD) using flow cytofluorimetry. We
succeeded in recording the ratio of dead and living cells
(Table 2). The obtained data suggest a synergic effect upon
combined treatment of cancer cells with cisplatin and
quadricyclanes 2 and 3.
Table 2. Joint effect of simultaneously added quadricyclanes 2
and 3 in combination with cisplatin on the viability of Jurkat cells
Table 1. Control parameters of cytotoxicity of compounds 2 and 3,
as well as of cisplatin against Jurkat cells
Com-
pound
Concentration
of compound
2 or 3
Concentration
of cisplatin
Portion of
dead cells
after 24 h (%)
Compound
Concentration
/μmol L–1
Portion of dead cells (%)
24 h 48 h 72 h
11 0.4 12 0.8 12 0.7
12 0.6 14 1.1 15 0.8
0.5 11 0.8 18 1.1
16 0.6 18 0.7 30 0.6
0.7 10 0.9 11 1.0
12 0.6 12 0.8 15 0.5
0.4 0.7 0.6
μmol L–1
2
3
0.30
0.60
0.15
0.30
0.03
0.06
0.03
0.06
0.03
0.06
0.03
0.06
15 0.6
24 0.5
18 0.6
27 0.7
17 0.8
29 0.6
42 0.4
47 0.6
2
0.3
0.6
0.15
0.3
0.03
0.06
.—
3
8
Cisplatin
Control
8
3
5
8

1038
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 5, May, 2019
Dzhemilev et al.
Taking into account the rate of quadricyclane opening
on exposure to platinum and palladium compounds,3,15
one can assume that upon simultaneous mixing of solu-
tions of cisplatin and quadricyclane 2 or 3 the catalytic
transformation of corresponding quadricyclane to norbor-
nadiene occurs outside of a cell, which leads to a negative
result. For this reason, we preliminary incubated cells
with quadricyclanes 2 and 3 at different concentrations for
24 h and then added a solution of cisplatin (0.03 and
0.06 μmol L–1) followed by incubation of the resulting
mixture for additional 24 h. Processing of the cytometric
data (Table 3) showed a statistically significant dose-de-
pendent slight decrease in the number of dead cells in each
group divided in accordance with the concentration of
added cisplatin vs. control, Jurkat cells treated with cis-
platin only (see Table 1).
A more pronounced thermal effect on cancer cells was
observed by the example of quadricyclane 3 containing
simultaneously two quadricyclane moieties. The difference
between the number of living cells after treatment with
cisplatin alone and cisplatin in combination with quadri-
cyclane 3 was from ~35% (the concentrations of Pt and 3
were 0.03 and 0.15 μmol L–1, respectively) to ~50% (the
concentrations of Pt and compound 3 were 0.06 and
0.3 μmol L–1, respectively).
Note that the norbornadiene derivatives 4 and 5 have
no cytotoxic effect on both Jurkat and U937 cells.
We also tested our hypothesis on normal fibroblasts.
Upon separate addition of compound 2 or 3 followed by
addition of cisplatin after incubation for 24 h in the culture
of normal fibroblasts, there were changes similar to those
for the culture of Jurkat cells treated and incubated in
a similar manner. However, statistical processing of data
revealed a statistically significant increase in the number
of viable cells in the fibroblast culture at similar concentra-
tions of compound 2 or 3 and cisplatin compared to Jurkat
cells (the concentration of compound 2 was 0.3 and
0.6 μmol L–1 and the concentration of compound 3 was
0.15 and 0.3 μmol L–1) (p ≤ 0.005).
Thus, we synthesized for the first time quadricyclane
derivatives which, in combination with cisplatin, show
a high cytotoxic effect against human Т-lymphoblastic
leukemia cells (Jurkat cells). The difference between the
numbers of living cells after treatment with cisplatin alone
and cisplatin in combination with quadricyclane was from
35 to 50%.
Experimental
1H and 13C NMR spectra were recorded on a Bruker
Avance-500 spectrometer (500.17 and 125.76 MHz, respectively).
The solvent was CDCl3. Photoisomerization of norbornadi-
ene derivatives to quadricyclane ones was carried out using
a HAMAMATSU LC 8 irradiator at 360 nm. Phenylnorborna-
diene carboxylic acid 1 was synthesized according to the earlier
described procedure19 and its spectral characteristics com-
pletely coincide with the literature data.20 The cytotoxicity of
compounds was studied on a NovoCyteTM 2000 flow cytometry
system (ACEA) (two lasers, red and blue), detection of five
fluorescent parameters.
2-[2-(2-Hydroxyethoxy)ethoxy]ethyl 3-phenylbicyclo
[2.2.1]hepta-2,5-diene-2-carboxylate (4). Compound 1 (1 g,
4.7 mmol) was dissolved in dry CH2Cl2 (10 mL) in a two-necked
glass reactor. Triethylene glycol (3.525 g, 23.5 mmol), 4-dimethyl-
aminopyridine (57.34 mg, 0.47 mmol), and N,N´-dicyclo-
hexylcarbodiimide (968.2 mg, 4.7 mmol) were added with vigor-
ous stirring. The reaction mixture was stirred at ~20 C for ad-
ditional 1 h. All experiments were performed in the stream of dry
argon. After the time expired, the precipitate that formed was
filtered off and washed with CH2Cl2 (310 mL). The combined
filtrate was treated with distilled water (30 mL) to remove the
excess of triethylene glycol and the aqueous layer was separated
and extracted with AcOEt (315 mL). The organic fractions were
combined and concentrated using a vacuum evaporator. Product
4 was purified by column chromatography using petroleum
ether—ethyl acetate (20 : 1) as the eluent. The yield was 1.48 g
(87%). 1H NMR (CDCl3), δ: 7.53 (d, 2 H, CH, J = 7.4 Hz); 7.36
(t, 2 H, CH, J = 7.4 Hz); 7.29—7.33 (m, 1 H, CH); 7.00 (dd, 1 H,
CH, J1 = 5.0 Hz, J2 = 3.1 Hz); 6.93 (dd, 1 H, CH, J1 = 5.0 Hz,
J2 = 3.1 Hz); 4.26 (t, 2 H, CH2, J = 4.1 Hz); 4.08—4.12 (m, 1 H,
CH); 3.72 (t, 2 H, CH2, J = 5.0 Hz); 3.66 (t, 2 H, CH2, J = 5.0 Hz);
3.59—3.62 (m, 2 H, CH); 3.57—3.62 (m, 4 H, 2 CH2); 2.27
(dt, 1 H, CH, J1 = 7.0 Hz, J2 = 1.5 Hz); 2.08 (dt, 1 H, CH,
J1 = 7.0 Hz, J2 = 1.5 Hz). 13C NMR (CDCl3), δ: 167.26, 165.32,
143.75, 140.80, 138.87, 135.71, 128.55, 127.85, 127.69, 72.53,
70.60, 70.54, 70.38, 69.06, 63.27, 61.73, 58.66, 53.00.
Table 3. Joint effect of quadricyclanes 2 and 3 in combination
with cisplatin added to the starting mixture 24 h after addition
of compound 2 or 3 on the viability of Jurkat cells
Com-
pound
Concentration Concentration
Portion
of dead cells
after 48 h (%)
of compound
of cisplatin
2 or 3
Ethane-1,2-diylbis(oxyethane-2,1-diyl) bis(3-phenylbicyclo-
[2.2.1]hepta-2,5-diene-2-carboxylate) (5). Compound 1 (1 g,
4.7 mmol) was dissolved in dry CH2Cl2 (10 mL) in a two-necked
glass reactor. Triethylene glycol (352.5 mg, 2.35 mmol), 4-di-
methylaminopyridine (57.34 mg, 0.47 mmol), and N,N´-di-
cyclohexylcarbodiimide (1.9364 g, 9.4 mmol) were added with
vigorous stirring. The reaction mixture was stirred at ~20 C for
additional 1 h. All experiments were performed in the stream of
dry argon. After the time expired, the precipitate that formed was
filtered off and washed with CH2Cl2 (310 mL). The filtrate was
combined and concentrated using a vacuum evaporator. Product
μmol L–1
2
3
0.3
0.6
0.15
0.3
0.03
0.06
0.03
0.06
0.03
0.06
0.03
0.06
21 0.6
31 0.8
22 0.4
27 0.6
46 1.1
48 1.0
59 0.8
64 1.0

Anticancer drugs based on quadricyclanes
Russ. Chem. Bull., Int. Ed., Vol. 68, No. 5, May, 2019
1039
5 was purified by column chromatography using petroleum
ether—ethyl acetate (20 : 1) as the eluent. The yield was 1.22 g
(90%). 1H NMR (CDCl3), δ: 7.55 (d, 2 H, CH, J = 7.0 Hz); 7.37
(t, 2 H, CH, J = 7.0 Hz); 7.29—7.33 (m, 1 H, CH); 7.00 (dd, 1 H,
CH, J1 = 5.0 Hz, J2 = 3.0 Hz); 6.93 (dd, 1 H, CH, J1 = 5.0 Hz,
J2 = 3.0 Hz); 4.24 (t, 2 H, CH2, J = 5.0 Hz); 4.14 (t, 2 H, CH2,
J = 7.0 Hz); 3.65 (t, 2 H, CH2, J = 5.0 Hz); 2.27 (d, 1 H, CH,
J = 7.0 Hz); 2.08 (d, 1 H, CH, J = 7.0 Hz). 13C NMR (CDCl3),
δ: 167.09, 165.30, 143.74, 140.82, 138.90, 135.71, 128.54, 127.89,
127.69, 70.58, 70.53, 69.08, 63.38, 58.67, 53.03.
Synthesis of 3-phenylquadricyclane-2-carboxylic acid triethyl-
ene glycol esters 2 and 3 (general procedure). In a 10-mL quartz
cuvette, the norbornadiene derivative 4 or 5 (0.3 g, 0.8 or
0.6 mmol, respectively) was dissolved in acetonitrile (7 mL). The
content was UV irradiated (λ = 360 nm) with vigorous stirring
for 15 min for compound 2 and 90 min for compound 3. The
solvent was evaporated under reduced pressure to afford the cor-
responding quadricyclane in quantitative yield.
with intact native membrane. The fluorescence intensity of
7-AAD over the BL-4 channel (PerCP) was estimated on a
NovoCyteTM 2000 flow cytometer (ACEA). The instrument was
adjusted using control samples: living unstained cells (estimation
of autofluorescence), living 7-AAD-stained cells, cells non-ab-
sorbing the dye with a low fluorescence level (gating of living
cells) and cells incubated under the same conditions with addition
of the studied compounds and cisplatin, which results in their
death (gating of dead cells). At least 104 cells were detected in
each sample. Each experiment was performed under the same
conditions in triplicate. If dead cells were present on a bar chart
upon cytometry, the population was divided into two groups:
living cells demonstrated a low fluorescence level, while dead
cells showed a high fluorescence intensity. The statistically
analysis of obtained data was carried out by the t-test using the
Statistica 9.0 program (StatSoft Inc., United States). The data
are presented as mean value standard deviation. The values
were considered to be statistically significant at p ≤ 0.05.
2-[2-(2-Hydroxyethoxy)ethoxy]ethyl 5-phenyltetracyclo-
[3.2.0.02,7.04,6]heptane-1-carboxylate (2). 1H NMR (CDCl3),
δ: 7.28—7.32 (m, 1 H, CH); 7.15—7.20 (m, 1 H, CH); 4.08—4.13
(m, 2 H); 3.57—3.61 (m, 4 H); 3.48—3.52 (m, 4 H); 3.46—3.50
(m, 2 H); 2.62 (dd, 2 H, CH2, J1 = 5.0 Hz, J2 = 2.0 Hz); 2.51
(dd, 1 H, CH, J1 = 5.0 Hz, J2 = 2.0 Hz); 2.40 (d, 1 H, CH, J =
11 Hz); 2.26 (dd, 1 H, CH, J1 = 5.0 Hz, J2 = 2.0 Hz); 2.16 (d, 1
H, CH, J = 11.0 Hz); 1.70—1.76 (m, 1 H, CH). 13C NMR
(CDCl3), δ: 172.00, 137.05, 128.79, 127.64, 126.10, 72.56, 70.43,
70.30, 69.00, 63.10, 61.66, 37.47, 32.85, 31.71, 29.65, 20.89.
Ethane-1,2-diylbis(oxyethane-2,1-diyl) bis(5-phenyltetra-
cyclo[3.2.0.02,7.04,6]heptane-1-carboxylate) (3). 1H NMR
(CDCl3), δ: 7.32—7.27 (m, 1 H, CH); 7.21 (m, 1 H, CH);
4.08—4.16 (m, 2 H, CH); 3.50 (t, 4 H, 2 CH2, J = 5.0 Hz); 3.43
(s, 4 H, 2 CH2); 2.62 (dd, 1 H, CH, J1 = 5.0 Hz, J2 = 2.5 Hz);
2.52 (d, 1 H, CH, J = 5 Hz); 2.42 (d, 1 H, CH, J = 11.6 Hz);
2.27 (dd, 1 H, CH, J1 = 5.0 Hz, J2 = 2.5 Hz); 2.18 (d, 1 H, CH,
J = 11.6 Hz); 1.75 (d, 1 H, CH, J = 5.0 Hz). 13C NMR (CDCl3),
δ: 171.97, 137.07, 128.78, 127.66, 126.10, 70.39, 69.00, 63.19,
37.42, 32.93, 32.28, 31.58, 31.73, 29.54, 20.93.
Cell culturing. Jurkat cell line and normal fibroblast culture
were obtained from European Collection of Cell Cultures (United
Kingdom). Fibroblasts were cultured in DMEM (Gibco) supple-
mented with 10% fetal bovine serum (Sigma), L-glutamine
(2 mmol L–1, Gibco), and antibiotics (50 U mL–1 of penicillin
and 50 μg mL–1 of streptomycin) (Biolot). Jurkat cell line was
cultured in the RPMI 1640 complete medium (Biolot) contain-
ing 10% fetal bovine albumin, L-glutamine (2 mmol L–1), and
antibiotic (50 U mL–1 of penicillin and 50 μg mL–1 of strepto-
mycin). All cells were cultured in vials in a CO2 incubator at 37 C
in the 5% CO2 atmosphere at saturation moisture content. The
cell viability was studied on 5—10-passage cell cultures. To
carry out experiments, Jurkat cells that reached the logarithmic
growth phase were passed into 24-well plates at a volume of
100 thousands cells per well. Fibroblasts were inoculated at
a concentration of 50—100 thousands cells and preincubated in
plates for 24 h to provide cell adhesion and adaptation. After
addition of test substances, cells were incubated for 24—48—72 h
followed by the analysis of data by flow cytometry.
The structural studies of compounds 1—5 were per-
formed at the Center for Shared Use ″Agidel″ at the
Institute of Petroleum Chemistry and Catalysis. The cyto-
toxicity of synthesized compounds was studied at the
Center for Molecular Design and Bioscreening of Candi-
date Substances at the Institute of Petroleum Chemistry
and Catalysis.
This work was financially supported by the Russian
Science Foundation (Project No. 18-33-20027).
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Received December 7, 2018;
in revised form January 21, 2019;
accepted February 14, 2019