Usnic Acid Conjugates with Monoterpenoids as Potent Tyrosyl-DNA Phosphodiesterase 1 Inhibitors

Article abstract of DOI:10.1021/acs.jnatprod.9b01089

Hybrid molecules created from different pharmacophores of natural and synthetic equivalents are successfully used in pharmaceutical practice. One promising target for anticancer therapy is tyrosyl-DNA phosphodiesterase 1 (Tdp1) because it can repair DNA lesions caused by DNA-topoisomerase 1 (Top1) inhibitors, resulting in drug resistance. In this study, new hybrid compounds were synthesized by combining the pharmacophoric moiety of a set of natural compounds with inhibitory properties against Tdp1, particularly, phenolic usnic acid and a set of different monoterpenoid fragments. These fragments were connected through a hydrazinothiazole linker. The inhibitory properties of the new compounds mainly depended on the structure of the terpenoid moieties. The two most potent compounds, 9a and 9b, were synthesized from citral and citronellal, which contain acyclic fragments with IC50 values in the range of 10-16 nM. Some synthesized derivatives showed low cytotoxicity against HeLa cells and increased the effect of the Top1 inhibitor topotecan in vitro by three to seven times. These derivatives may be considered as potential agents for the development of anticancer therapies when combined with Top1 inhibitors.

Full text of DOI:10.1021/acs.jnatprod.9b01089

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Article
Usnic Acid Conjugates with Monoterpenoids as Potent Tyrosyl-DNA
Phosphodiesterase 1 Inhibitors
Olga Luzina, Alexander Filimonov, Alexandra Zakharenko, Arina Chepanova, Olga Zakharova,
Ekaterina Ilina, Nadezhda Dyrkheeva, Galina Likhatskaya, Nariman Salakhutdinov, and Olga Lavrik*
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ABSTRACT: Hybrid molecules created from different pharma-
cophores of natural and synthetic equivalents are successfully used
in pharmaceutical practice. One promising target for anticancer
therapy is tyrosyl-DNA phosphodiesterase 1 (Tdp1) because it can
repair DNA lesions caused by DNA-topoisomerase 1 (Top1)
inhibitors, resulting in drug resistance. In this study, new hybrid
compounds were synthesized by combining the pharmacophoric
moiety of a set of natural compounds with inhibitory properties
against Tdp1, particularly, phenolic usnic acid and a set of different
monoterpenoid fragments. These fragments were connected
through a hydrazinothiazole linker. The inhibitory properties of
the new compounds mainly depended on the structure of the
terpenoid moieties. The two most potent compounds, 9a and 9b,
were synthesized from citral and citronellal, which contain acyclic fragments with IC50 values in the range of 10−16 nM. Some
synthesized derivatives showed low cytotoxicity against HeLa cells and increased the effect of the Top1 inhibitor topotecan in vitro
by three to seven times. These derivatives may be considered as potential agents for the development of anticancer therapies when
combined with Top1 inhibitors.
he creation of hybrid molecules from natural compounds is
a modern and a highly relevant trend in medicinal
chemical transformation, and its availability in high optical
purity.8,9 The primary obstacle to its widespread use in
pharmacology is its high toxicity. Therefore, the successes
achieved in the field of research and utilization of the biological
properties of this compound during the past decades have been
focused on the synthesis of its derivatives and the identification
of structure−activity relationships.10,11
T
chemistry. This concept presents new perspectives regarding
the use of natural compounds in pharmacology and can
potentially improve the safety and efficacy of drugs.1 A rational
combination of ≥ two components of natural origin can lead to
hybrid molecules that synergistically increase the efficacy of each
other and consequently reduce the required dosage of the drug.2
One approach regarding the concept of molecular hybridization
is based on combining two different pharmacophores, with
similar or different biological functions, directly or through a
linker fragment.3 This approach can resolve several common
issues associated with the bioavailability of a drug, its transport
through the membranes of cellular organelles, and protection
against enzymatic degradation; consequently, it can improve its
pharmacokinetic and pharmacodynamic profiles.4 Additionally,
the expected positive effects of hybrid molecules can include
minimization of the risk of drug interaction and the develop-
ment of resistance to therapy.5,6 Several examples of hybrid
molecules that exist in nature and form through various
metabolic pathways (e.g., Taxol and cannabinoids) and are
created from natural synthetic equivalents,7 successfully used in
pharmaceutical practice are present.
Recently, the use of UA (1)12 and its derivatives 2,12−14
3,12,15,16 and 4a (Figure 1),17 in the form of a combination
therapy with cytotoxic drugs, has been widely studied using in
vitro and in vivo experiments. This approach has been shown to
result in pronounced effects that are associated with negating the
tolerance of drug-resistant cancer cells18 and the inhibition of
tumor metastasis.13,15 The mechanisms of action of these
compounds have not been fully elucidated but include
increasing the expression of E-cadherin18 and inhibiting the
growth of endovascular vessels,19 as well as inhibiting DNA
repair enzymes.14,15,17
Received: November 3, 2019
For several decades, usnic acid (UA) has attracted
considerable research interest, due to the broad scope of its
native biological activity, the susceptibility of its structure to
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. Natural and semisynthetic Tdp1 inhibitors.
The use of DNA repair enzyme inhibitors as chemotherapy
components is a promising and an intensively developing area of
modern oncopharmacology.20 Using such compounds is
particularly relevant for the treatment of drug-resistant tumors.
One promising target for the treatment of oncological diseases
involves the DNA repair enzyme tyrosyl-DNA phosphodiester-
ase 1 (Tdp1), which plays a key role in repairing damage to DNA
caused by exposure to the camptothecin drugs, irinotecan and
topotecan, which are widely used in clinical practice and are also
involved in the repair of damage caused by drugs such as
Temozolomide, bleomycin, and etoposide.21 UA derivatives
have proven to be promising inhibitors of repair enzymes. In
particular, there are reports of their moderate inhibitory activity
against PARP122 and significantly more pronounced activity
(IC50 10−6−10−8 M) against Tdp1,14−17,23,24 whereas native UA
(1) does not demonstrate such an effect. Recent studies have
revealed certain structural patterns of potential inhibitors based
on UA, leading to increased activity. In particular, it was found
that thiazole derivatives 3 of UA, with arylhydrazone
substituents attached to the thiazole ring, exhibit Tdp1
inhibitory activity in the nanomolar range.15,16 In vitro studies
with lung carcinoma cell line A-54916 and in vivo studies in mice
with Lewis lung carcinoma15 have confirmed the synergistic
antitumor effect of thiazole derivatives of UA when combined
with topotecan. Studies on the purified enzyme have also
demonstrated that the structure of the aryl or heteroaryl
substituent significantly affected the inhibitory potential of
compounds.15,16
(6), an amino linker,26 amide,27 or ester bond,28 or an aromatic/
heteroaromatic moiety.29 These compounds showed inhibitory
activity in the 0.6−21 μM range and demonstrated a synergistic
effect in vitro in human cell lines when used in combination with
a Top1 inhibitor, camptothecin (coumarins)25 or topotecan
(adamantanes).26,27 The advantages of terpenoids as natural
compounds include their structural affinity to many biological
receptors developed during the course of evolution and their
own wide range of biological activities, which have often inspired
the development of synthetic drugs based on them.30 The
introduction of terpenoid fragmentswhich often have a bulky
chiral 3D structureonto the Tdp1 inhibitor molecule may also
contribute to more selective binding of the ligand to the enzyme.
It has been demonstrated that the use of the available and cheap
monoterpenoids as starting molecules for synthesizing new and
effective agents to treat a number of diseases is a rather
promising and topical direction of medicinal chemistry. It
should also be mentioned that the use of monoterpenoids to
design drugs often reduces toxicity of the resulting compound,
which may indicate that there is complementarity between
natural molecules and living organisms that have coexisted for
thousands of years.
The spectrum of UA derivatives described in the literature is
fairly broad; however, UA derivatives that combine the structure
of phenols and terpenoids are represented only by alkylidenfur-
anone derivatives 4b.24 These compounds display Tdp1
inhibitory properties that are not inferior to the analogues
featuring a benzylidenefuranone fragment 4a.17
Herein, we synthesized new hybrid derivatives of terpeno-
phenol, i.e., UA and monoterpenoids that are joined into a single
molecule by a hydrazinothiazole linker. The synthesized
compounds include substances containing both cyclic (mono-
cyclic and bicyclic) and acyclic terpene fragments. A compound
with a non-terpene but saturated linear substituent was
synthesized to enable a broader investigation of the structure−
This study aimed at the synthesis and study of the Tdp1
inhibitory activity of the hydrazinothiazole derivatives of UA
containing terpenoid substituents of natural origin. The choice
of monoterpenoids as the second component of the inhibitor
molecule was based on the literature data related to the activity
of Tdp1 inhibitors combining monoterpenoid moieties with
substituted 7-hydroxycoumarin (5),25 adamantane fragments
B
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activity relationship. Studies of Tdp1 inhibitory activity and
cytotoxicity, concerning transplantable cell lines, were con-
ducted for the series of new UA derivatives.
conducted by the treatment of bromo-UA 8 with thiosemi-
carbazones 7a−h (Scheme 1).32 The reaction was conducted in
MeOH at reflux for 1 h, and the final products, isolated by
column chromatography, with yields, are reported in Table 1.
Column chromatography was used to purify the final products.
Compound 9f was obtained according to the procedure
provided above.32 The structures of all new compounds were
confirmed by 1H and 13C NMR and HRMS data. The
regioselectivity of bromination and further cyclization to
thiazole derivatives were proven early for analogues of
compounds 9a−h, which were characterized on the basis of X-
ray structural analysis (RSA).33
RESULTS AND DISCUSSION
■
Chemistry. Terpenoid thiosemicarbazones 7a−h were
synthesized from commercially available terpenoid aldehydes
and ketones (Figure 2; Scheme 1), according to published
Biology. A fluorophore/quencher-coupled DNA biosensor
for real-time measurement of Tdp1 cleavage activity was used.
The substrate was a 16-mer single-strand oligonucleotide
containing both a 5′-FAM fluorophore donor and a quenching
3′-BHQ1 moiety. The IC50 values (half-maximal inhibition
concentrations) for compounds 9a−h are collated in Table 1.
High Tdp1 inhibitory activity (in the nanomolar concen-
tration range) was established for terpenic derivatives of UA
with a hydrazinothiazole linker 9. Apparent relationships were
observed between the effective inhibitory concentrations of the
compounds and the structure of the terpene fragment;
conjugates of (+)-UA with acyclic fragments 9a and 9b were
among the compounds with high inhibitory ability. The
compound with monocyclic substituent 9e was marginally less
active. Derivatives with bicyclic substituents 9f−h had three to
five times higher IC50 values. Remarkably, both the replacement
of the isoprene fragment with a saturated linear fragment (9d)
and the introduction of a polar hydroxy group (9c) led to a
significant decrease in activity; the inhibitory properties of the
conjugates decreased by an order of magnitude in both cases.
Cytotoxicity. Because Tdp1 inhibitors are supposed to be
used in therapeutic cocktails, they should possess the lowest
possible toxicity and should not enhance the existing side effects
of therapy. Intrinsic cytotoxicity was studied for substances that
showed anti-Tdp1 activity in concentrations of <80 nM. The
Figure 2. Structures of monoterpenoids used for synthesis.
methods or under conditions similar to those described. In all
cases, except for verbenone thiosemicarbazone, only one set of
signals, corresponding to one isomer of thiosemicarbazone, was
observed in the NMR spectra. In the case of verbenone (7g),
two sets of peaksfor the E and Z isomerswere observed in a
3:2 ratio. Citronellal and citronellal hydrates were used as
racemates, and the resulting derivatives, 9b and 9c, were present
as a mixture of two diastereomers; no doubling of signals in the
NMR spectra was observed. The compounds were further tested
as a mixture of diastereomers.
UA bromoketone 8 was obtained from R-(+)-UA 1 by the
action of bromine in dioxane, in the presence of hydrogen
bromide, according to the method described by Luzina et al.31
The synthesis of target hydrazinothiazole derivatives 9a−h was
Scheme 1. Synthesis of Hydrazonothiazole Derivatives 9a−h
C
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a
Table 1. Influence of UA Derivatives on Tdp1 Activity
a
*Topotecan (Tpc) in the presence of 5 μM Tdp1 inhibitors. **nd = not determined.
cytotoxicity of compounds was investigated using HeLa
(cervical cancer) cells (Figure 3).
UA conjugates with terpenoids had a moderate cytotoxic
effect in concentrations from 20 μM, which exceeded inhibitory
concentrations by 2 to 3 orders of magnitude. Compound 9f,
synthesized from myrtenal, was the least toxic for both cell types
(CC50 > 100 μM), and for the remaining compounds, the values
of CC50 were in the 20−70 μM range. Notably, compounds
based on myrtenal were reported as the most promising in the
series of terpenoid derivatives with coumarins and adamantanes,
D
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Figure 3. (A) Dose-dependent impact of UA derivatives on the viability of HeLa cells. (B) Dose-dependent action of topotecan in combination with
UA derivatives on HeLa cells’ viability.
previously synthesized as Tdp1 inhibitors, which may indicate
the important role of this structural unit for the development of
potential Tdp1 inhibitors.25,26
Thus, the binding of the hydroxy group of the aliphatic
fragment of inhibitor molecule 9c to the active site of the enzyme
led to the loss of contact of the dibenzofuran part of the inhibitor
molecule with Tdp1 catalytic residues, which in turn gave rise to
a decrease in total binding energy. Inhibitors 9b and 9c had the
same binding site to the catalytic domain (Figure 5). The
binding energies of compounds 9b and 9c with the enzyme
correlated with the Tdp1-inhibiting activity of compound 9b at a
lower concentration, compared with compound 9c (IC50 values
of 16.4 and 139 nM).
In conclusion, new compounds in the form of conjugates of
UA and monoterpenoids were developed that can serve as
effective inhibitors of Tdp1, in the concentration range of 10−
139 nM. These compounds serve as one of the most effective
inhibitors of Tdp1 published to date. Summarizing the data on
the inhibitory effect and toxicity, compound 9b can be
considered as the most promising inhibitor of Tdp1 because it
was weakly toxic to the studied cells, demonstrated an inhibitory
effect on Tdp1 at a concentration of 16 nM, and enhanced the
cytotoxicity of topotecan 7-fold.
Topoisomerase 1 (Top1) poisons (topotecan and irinotecan)
are used as anticancer drugs for the treatment of a wide range of
oncological diseases.34,35 Because Tdp1 is involved in the
removal of DNA damage caused by Top1 poisons, it is believed
to be responsible for the drug resistance of some cancers.36
Thus, a combination of Top1 poisons and Tdp1 inhibitors may
be able to improve the effectiveness of chemotherapy.
Next, the effect of Tdp1 inhibitors on the cytotoxic effect of
topotecan, a Top1 poison used in clinical practice, was
investigated. Nontoxic concentrations of UA derivatives (5
μM) and different concentrations of topotecan were used to
estimate CC50 values for HeLa cells. Tdp1 inhibitors in nontoxic
concentrations increased the cytotoxic effect of topotecan from
1.7 to 7 times (Figure 3B and Table 1). The best results were
obtained for compound 9b, which reduced the CC50 of
topotecan 7-fold.
Thus, terpenophenol UA derivatives represent a promising
class of compounds for development as sensitizers of Top1
poisons to achieve enhanced therapeutic effects.
EXPERIMENTAL SECTION
■
1
Chemistry. The H and 13C NMR spectra for solutions of the
Molecular Modeling. Molecules 9b and 9c were docked
into the binding site of Tdp1 (PDB ID: 6N19, resolution 1.5 Å)
to clarify the 10-fold difference in the values of their effective
inhibitory concentrations. The structures of the complexes of
the catalytic domain of Tdp1 with compounds 9b and 9c with
the lowest energy were used to analyze contacts in the complex.
An analysis of the contacts of compound 9b with the active
center showed that the O5 oxygen atom of this compound
formed a hydrogen bond with the catalytic residue His 263,
along with Lys 265 and Asn 283, whereas the sulfur atom formed
hydrogen bonds with Pro 461 and Thr 466 (Figure 4A). The
energy of the hydrogen bonds of compound 9b with catalytic
domain Tdp1 was −10.0 kcal/mol.
The O6 of the hydroxy group of the aliphatic fragment 9c
formed hydrogen bonds with the catalytic residues His 263 and
Lys 265, and O5 formed a hydrogen bond with Lys 519,
alongside Tdp1 (Figure 4B). The Tyr 204 residue of the Tdp1
formed an arene−H bond with compound 9c. The energy of
hydrogen bonds of compound 9c alongside the catalytic domain
of Tdp1 was −4.2 kcal/mol.
compounds in CDCl3 were recorded using a Bruker AV-400
spectrometer (400.13 and 100.61 MHz, respectively). The residual
signals of the solvent were used as references (δH 2.48 and δC 39.52 for
DMSO-d6 and δH 7.27 and δC 77.1 for CDCl3). The mass spectra (70
eV) were recorded on a DFS Thermo Scientific high-resolution mass
spectrometer. Electron impact ionization was used in the measurement
of mass spectra. Merck silica gel (63−200 μm) was used for column
chromatography. Thin-layer chromatography was performed on Silufol
plates (UV-254).
(+)-UA 1 ([α]D +478 (c 0.1, CHCl3)) was isolated from a mixture of
lichens of the genus Usnea, using the procedure employed in
Salakhutdinov et al.37
The atom numbers in the compound were provided for the
assignment of signals in the NMR spectra and were different from
numbering in the nomenclature name. The target compounds (9a−h)
had a purity of at least 97% (high-performance liquid chromatography).
General Procedure for the Synthesis of Compounds 7a−h.
Aldehyde (1 mmol) and thiosemicarbazide (1 mmol) were added to a
flask. The mixture was dissolved in 10 mL of EtOH (95%). The mixture
was boiled, and after 60 min, it was cooled, and water was added. The
precipitate was filtered off, washed with water, and then air-dried. The
thiosemicarbazones 7a−h obtained were isolated in 81−98% yields.
E
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Figure 4. 2D diagrams of the contacts of compounds 9b (A) and 9c (B) with catalytic domain Tdp1, obtained using the “ligand interaction” module of
the MOE 2019 software package. Pin designations are indicated in the figure.
F
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8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-pentaen-3-one (9b).
1
Yellow, amorphous powder, yield 44%; mp 120−124 °C; H NMR
(CDCl3, 400 MHz) δ 0.94 (3H, d, J = 6.3 Hz), 1.10−1.28 (3H,
m),1.34−1.42 (1H, m), 1.59 (3H, s), 1.67 (3H, s), 1.73 (3H, s), 1.90−
2.05 (2H, m), 2.13−2.22 (4H, m), 2.63 (3H, s), 5.08 (1H, t, J = 7.1 Hz),
5.90 (1H, s), 7.09 (1H, s), 7.21 (1H, t, J = 5.7 Hz), 8.64 and 8.80 (1H,
bs), 10.24 (1H, s), 13.29 (1H, bs), 18.77 (1H, s); 13C NMR (CDCl3,
100 MHz) δ 8.2, 17.6, 19.5, 25.3, 25.6, 27.8, 30.9, 32.1, 36.6, 39.0, 59.4,
97.3, 97.4, 103.2, 104.2, 105.1, 108.9, 124.1, 131.5, 145.6, 146.1, 151.2,
151.5, 156.5, 166.6, 180.7, 191.6, 198.1, 201.3; HRMS m/z 551.2082
(calcd for C29H33O6N3S, 551.2085).
(2R)-4-Acetyl-5,11,13-trihydroxy-10-{2-[(E)-2-(7-hydroxy-3,7-
dimethyloctylidene)hydrazin-1-yl]-1,3-thiazol-4-yl}-2,12-dimethyl-
8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-pentaen-3-one (9c).
1
Yellow, amorphous powder, yield 63%; mp 135−139 °C; H NMR
Figure 5. Superposition of compounds 9b and 9c in the active center of
the catalytic domain of Tdp1. The structures of the compounds are
shown in stick form in white (9b) and yellow (9c). The molecular
surface of Tdp1 at the binding site is shown as hydrophobic (green),
polar (pink), and exposed (red).
(CDCl3, 400 Hz) δ 0.94 (3H, d, J = 6.7 Hz), 1.18−1.22 (7H, m), 1.34−
1.46 (7H, m), 1.72 (3H, s), 2.13 (3H, s), 2.16−2.19 (1H, m), 2.64 (3H,
s), 5.89 (1H, s), 7.08 (1H, s), 7.17 (1H, t, J = 5.6 Hz), 8.73 (1H, bs),
10.24 (1H, s), 12.37 (1H, bs), 18.77 (1H, s); 13C NMR (CDCl3, 100
MHz) δ 8.3, 19.6, 21.5, 27.8, 29.2, 29.2, 31.3, 32.1, 36.9, 39.1, 43.9, 59.5,
70.9, 97.3, 97.5, 103.2, 104.2, 105.1, 108.9, 143.5, 146.3, 151.2, 151.5,
156.6, 166.6, 180.7, 191.6, 198.1, 201.3; HRMS m/z 569.2187 (calcd
for C29H35O7N3S, 569.2190).
Compounds 7a and 7b were assigned as described in Tarasconi et al.38
Compound 7d was assigned as described in Wiles et al.39 Compound 7f
was assigned as described by Oliveira et al.40 Compound 7h was
assigned as described by Lazkowski et al.41
(R,E)-2-{[4-(Prop-1-en-2-yl)cyclohex-1-enyl]methylene}-
hydrazinecarbothioamide (7e). White crystals, yield 81%; mp 138−
140 °C; 1H NMR (CDCl3, 400 MHz) δ 1.46 (1H, m), 1.72 (3H, s),
1.85−2.46 (6H, m), 4.69 (2H, d, J = 18.8 Hz), 6.17 (1H, s), 6.51 (1H,
s), 7.05 (1H, s), 7.55 (1H, s), 10.02 (1H, s); 13C NMR (CDCl3, 100
MHz) δ 20.5, 23.3, 26.5, 31.4, 40.6, 109.0, 134.0, 138.4, 147.2, 148.6,
177.8.
{[(1S,2E,5S)-4,6,6-Trimethylbicyclo[3.1.1]hept-3-en-2-ylidene]-
amino}thiourea and {[(1S,2Z,5S)-4,6,6-trimethylbicyclo[3.1.1]hept-
3-en-2-ylidene]amino}thiourea (3:2) (7g). White, amorphous pow-
der, yield 98%; 1H NMR (DMSO-d6, 400 MHz) δ 0.81 (3H, s), 1.42
(3H, s), 1.49 (1H, m), 1.87 (3H, s), 2.23 (1H, m), 2.65 (2H, m), 3.41
(1H, m), 6.68 (1H, s), 7.50 (1H, s), 7.93 (1H, s), 10.36 (1H, s); 13C
NMR (DMSO-d6, 100 MHz) δ 21.7, 23.6, 25.9, 37.3, 47.9, 48.6, 50.7,
110.7, 153.9, 160.3, 177.9; 1H NMR (DMSO-d6, 400 MHz) δ 0.81 (3H,
s), 1.39 (3H, s), 1.58 (1H, m), 1.90 (3H, s), 2.29 (1H, m), 2.62 (2H,
m), 3.53 (1H, m), 5.77 (1H, s), 7.50 (1H, s), 7.93 (1H, s), 10.27 (1H,
s); 13C NMR (DMSO-d6, 100 MHz) δ 21.9, 22.9, 25.5, 36.5, 43.1, 47.7,
48.4, 118.5, 155.7, 156.7, 178.1.
General Procedure for the Synthesis of Compounds 9a−h.
(+)-UA 1 (1 mM) was treated with the bromine−dioxane complex
(2 mmol Br2, 0.10 mL, dissolved in dioxane, 14 mL) and several drops
of HBr and left for 7 d at room temperature. The reaction mixture was
concentrated in a rotary evaporator and chromatographed over silica
gel with CH2Cl2. The yield of compound 8 was 67%.
(2R)-4-Acetyl-5,11,13-trihydroxy-2,12-dimethyl-10-{2-[(E)-2-
octylidenehydrazin-1-yl]-1,3-thiazol-4-yl}-8-oxatricyclo[7.4.0.02,7]-
trideca-1(9),4,6,10,12-pentaen-3-one (9d). Yellow, amorphous pow-
der, yield 82%; mp 100−105 °C; 1H NMR (CDCl3, 400 MHz) δ 0.86−
0.89 (3H, m), 1.20−1.35 (8H, m), 1.54 (2H, bt), 1.73 (3H, s), 2.13
(3H, s), 2.31 (2H, bt), 2.64 (3H, s), 5.91 (1H, s), 7.09 (1H, s), 7.18
(1H, bt), 8.53 (1H, bs), 10.23 (1H, s), 18.77 (1H, s); 13C NMR
(CDCl3, 100 MHz) δ 8.2, 13.9, 22.4, 26.3, 27.7, 28.8, 28.9, 31.5, 31.9,
32.0, 59.3, 97.2, 97.4, 103.1, 104.9, 105.0, 108.8, 143.3, 147.1, 151.1,
151.4, 156.5, 166.5, 180.7, 191.5, 198.0, 201.3; HRMS m/z 525.1927
(calcd for C27H31O6N3S, 525.1928).
(2R)-4-Acetyl-5,11,13-trihydroxy-2,12-dimethyl-10-{2-[(E)-2-
{[(4R)-4-(prop-1-en-2-yl)cyclohex-1-en-1-yl]methylidene}hydrazin-
1-yl]-1,3-thiazol-4-yl}-8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-
pentaen-3-one (9e). Yellow, amorphous powder, yield 73%; mp 143−
145 °C; 1H NMR (CDCl3, 400 MHz) δ 1.50 (1H, bs), 1.75 (3H, s),
1.76 (3H, s), 1.92 (1H, bs), 2.12−2.26 (5H, m), 2.28−2.36 (1H, m),
2.52−2.62 (1H, m), 2.67 (3H, s), 4.76 (2H, d, J = 18.5 Hz), 5.93 (1H,
s), 6.01 (1H, s), 7.11 (1H, s), 7.34 (1H, s), 8.72 (1H, s), 10.28 (1H, s),
12.58 (1H, s), 18.81 (1H, s); 13C NMR (CDCl3, 100 MHz) δ 8.2, 20.6,
23.4, 26.6, 27.7, 31.2, 32.1, 40.7, 59.4, 97.2, 97.5, 103.2, 104.3, 105.1,
108.8, 108.9, 134.3, 135.2, 143.4, 146.1, 148.8, 151.1, 151.4, 156.4,
166.5, 180.6, 191.5, 198.0, 201.2; HRMS m/z 547.1767 (calcd for
C29H29O6N3S, 547.1772).
(2R)-4-Acetyl-10-{2-[(E)-2-{[(1R,5S)-6,6-dimethylbicyclo[3.1.1]-
hept-2-en-3-yl]methylidene}hydrazin-1-yl]-1,3-thiazol-4-yl}-
5,11,13-trihydroxy-2,12-dimethyl-8-oxatricyclo[7.4.0.02,7]trideca-1-
(9),4,6,10,12-pentaen-3-one (9f). Yellow, amorphous powder, yield
66%; mp 155−160 °C; 1H NMR (CDCl3, 400 MHz) δ 0.78 (3H, s),
1.35 (3H, s), 1.75 (3H, s), 2.17 (5H, bs), 2.38−2.60 (3H, m), 2.67 (3H,
s), 2.94 (1H, t, J = 5.6 Hz,), 5.88 (1H, m), 5.91 (1H, s), 7.07 (1H, s),
7.43 (1H, s), 9.05 (1H, bs), 10.27 (1H, s), 18.77 (1H, s); 13C NMR
(CDCl3, 100 MHz) δ 8.2, 20.7, 25.9, 27.7, 30.9, 32.0, 32.2, 37.5, 40.0,
40.6, 59.3, 97.2, 97.5, 103.2, 104.3, 105.1, 108.9, 131.7, 143.2, 144.7,
144.7, 151.2, 151.5, 156.4, 166.5, 180.6, 191.5, 198.1, 201.2; HRMS m/
z 547.1728 (calcd for C29H29O6N3S, 547.1772).
(2R)-4-Acetyl-5,11,13-trihydroxy-2,12-dimethyl-10-(2-{2-[(2Z)-
4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-ylidene]hydrazin-1-yl}-1,3-
thiazol-4-yl)-8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-pen-
taen-3-one and (2R)-4-acetyl-5,11,13-trihydroxy-2,12-dimethyl-10-
(2-{2-[(2E)-4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-ylidene]-
hydrazin-1-yl}-1,3-thiazol-4-yl)-8- oxatricyclo[7.4.0.02,7]trideca-1-
(9),4,6,10,12-pentaen-3-one (3:2) (9g). Yellow, amorphous powder,
yield 62%; 1H NMR (CDCl3, 400 MHz) δ 0.90 (3H, s), 1.44 (3H, s),
1.46−1.54 (1H, m), 1.72 (3H,s), 1.92 (3H, s), 2.12 (3H, s), 2.39 (1H,
bt), 2.55−2.73 (5H, m), 2.79 (2H, m), 5.89 (1H, s), 6.09 (1H, s), 7.09
(1H, s), 8.58 (1H, bs), 10.22 (1H, s), 12.37 (1H, bs), 18.79 (1H, s); 13C
NMR (CDCl3, 100 MHz) δ 8.2, 21.2, 23.9, 25.9, 27.7, 32.0, 37.6, 49.5,
A 1 mmol amount of compound 8 was added to 1 mmol of the
thiosemicarbazone of aldehyde 7a−h, and the mixture was boiled in
10 mL of MeOH for 1 h. After 60 min the mixture was cooled, and
water was added to the mixture. The precipitate that formed was filtered
off, washed with water, and air-dried.
The NMR assignment is in the Supporting Information (Tables S1−
S3).
(2R)-4-Acetyl-10-{2-[(E)-2-[(2E)-3,7-dimethylocta-2,6-dien-1-
ylidene]hydrazin-1-yl]-1,3-thiazol-4-yl}-5,11,13-trihydroxy-2,12-di-
methyl-8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-entaen-3-one
1
(9a). Yellow, amorphous powder, yield 64%; mp 145−150 °C; H
NMR (CDCl3, 400 MHz) δ 1.59 (3H, s), 1.66 (3H, s), 1.71 (3H, s),
1.84 (3H, s), 2.14 (7H, s), 2.63 (3H, s), 5.09 (1H, bs), 5.89 (1H, s),
6.00 (1H, d, J = 9.1 Hz), 7.08, (1H, s), 7.70 (1H, d, J = 9.1 Hz), 9.09
(1H, bs), 10.25 (1H, bs), 18.78 (1H, s); 13C NMR (CDCl3, 100 MHz)
δ 8.0, 16.7, 17.3, 25.2, 25.7, 27.4, 31.7, 39.7, 59.1, 97.0, 97.3, 102.9,
104.0, 104.8, 108.5, 120.4, 122.6, 131.8, 142.2, 143.1, 147.6, 150.9,
151.2, 156.1, 165.9, 180.4, 191.3, 197.8, 201.0; HRMS m/z 549.1925
(calcd for C29H31O6N3S, 549.1928).
(2R)-4-Acetyl-10-{2-[(E)-2-(3,7-dimethyloct-6-en-1-ylidene)-
hydrazin-1-yl]-1,3-thiazol-4-yl}-5,11,13-trihydroxy-2,12-dimethyl-
G
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50.9, 57.4, 59.4, 97.2, 97.6, 103.0, 104.2, 105.1, 108.1, 108.9, 143.5,
143.6, 151.1, 151.5, 154.1, 156.3, 156.7, 166.5, 167.4, 180.8, 191.6,
198.1, 201.2. 1H NMR (CDCl3, 400 MHz) δ 0.91 (3H, s), 1.45 (3H, s),
1.48 (1H, m), 1.72 (3H,s), 1.92 (3H, s), 2.12 (3H, s), 2.32 (1H, bt),
2.55−2.73 (5H, m), 5.89 (1H, s), 5.93 (1H, s), 7.08 (1H, s), 8.45 (1H,
bs), 10.22 (1H, s), 12.37 (1H, bs), 18.79 (1H, s); 13C NMR (CDCl3,
100 MHz) δ 8.2, 22.1, 23.4, 26.5, 27.8, 32.0, 36.7, 40.7, 49.6, 57.5, 59.5,
97.2, 97.6, 103.0, 104.2, 105.1, 108.9, 118.6, 143.5, 143.6, 151.1, 151.5,
155.0, 156.3, 161.9, 166.5, 167.6, 180.8, 191.6, 198.1, 201.2; HRMS m/
z 547.1777 (calcd for C29H29O6N3S, 547.1772).
for Tpc in the absence of Tdp1 inhibitor to CC50 for Tpc in the
presence of 5 μM Tdp1 inhibitor.
Molecular Modeling. Models of the 3D structure of compounds
9b and 9c were obtained using the crystalline structure of UA45,46
(CCDC ID: USNICA01) and using the molecular editor of the
Molecular Operating Environment (MOE)47 software, version
2019.01. Compound structures were optimized using an Amber10:-
EHT (extended Hueckel theory) force field. The Tdp1 catalytic
domain structure obtained at a resolution of 1.5 Å (PDB ID: 6N19)48,49
was used for molecular docking. Docking was conducted using the
Dock module of the MOE 2019.01 software. The structures of 30
complexes were calculated with the score parameter London dG; then,
for five complexes, the structures of the complexes were refined with the
score parameter GBVI/WSA dG. The analysis of the complexes was
performed using the Ligand interactions module of the MOE 2019.01
software. The results were obtained using Institute of Automation and
Control Processes of Far East Branch of Russian Academy of Sciences
Shared Resource Center’s “Far Eastern Computing Resource” equip-
ment (https://cc.dvo.ru).
(2R)-4-Acetyl-5,11,13-trihydroxy-2,12-dimethyl-10-(2-{2-[(2Z)-
1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene]hydrazin-1-yl}-1,3-
thiazol-4-yl)-8-oxatricyclo[7.4.0.02,7]trideca-1(9),4,6,10,12-pen-
taen-3-one (9h). Yellow, amorphous powder, yield 91%; mp 176−178
1
°C; H NMR (CDCl3, 400 MHz) δ 0.77 (3H, s), 0.94 (3H, s), 1.05
(3H, s), 1.17−1.29 (1H, m), 1.39−1.49 (1H, m), 1.71 (3H, s), 1.74−
1.80 (1H, m), 1.82−1.92 (2H, m), 2.00−2.08 (1H, m), 2.13 (3H, s),
2.36−2.46 (1H, m), 2.63 (3H, s), 5.88 (1H, s), 7.06 (1H,s), 7.95 (1H,
bs),.10.21 (1H, s), 12.52 (1H, bs), 18.77 (1H, s); 13C NMR (CDCl3,
100 MHz) δ 8.2, 10.9, 18.5, 19.4, 27.1, 27.8, 32.1, 32.4, 33.3, 44.0, 48.0,
52.6, 59.4, 97.2, 97.5, 103.0, 104.2, 105.1, 108.9, 143.4, 151.1, 151.5,
156.7, 165.5, 167.6, 180.8, 191.6, 198.1, 201.3; HRMS m/z 549.1917
(calcd for C29H31O6N3S, 549.1928).
Real-Time Detection of Tdp1 Activity. A fluorophore quencher-
coupled DNA biosensor for real-time measurement of Tdp1 cleavage
activity was recently designed in our laboratory.42 The biosensor is a 16-
mer single-stranded oligonucleotide containing both a 5′-FAM
fluorophore donor and a quenching 3′-BHQ1 moiety.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01089.
■
sı
1H and 13C NMR spectra of compounds 7e,g; 1H and 13C
NMR and mass spectra of compounds 9a−h (PDF)
Recombinant protein Tdp1 was expressed in Escherichia coli (pET
16B plasmid containing Tdp1 cDNA was provided by Dr. K. W.
Caldecott, University of Sussex, UK) and isolated as described.43,44 The
reaction mixture in a final volume of 200 μL contained a Tdp1 reaction
buffer (50 mM Tris-HCl, 50 mM NaCl, and 7 mM β-mercaptoethanol),
a 50 nM oligonucleotide substrate, and varied concentrations of
potential inhibitor. Purified Tdp1 was added in a final concentration of
1.5 nM.
The reaction mixtures were incubated at a constant temperature of
26 °C in a POLARstar OPTIMA fluorimeter (BMG LABTECH,
GmbH, Germany), to measure fluorescence intensity each minute
(Ex485/Em520 nm). Tdp1 inhibition was calculated by comparing the
rate of increase in the fluorescence of a biosensor in the presence of a
compound similar to that of DMSO (1.5%) control wells. The
measurements were conducted in at least two independent experi-
ments. IC50 values were determined using an 11-point concentration
response curve. The data were imported into the MARS Data Analysis
2.0 software package (BMG LABTECH), and the slope during the
linear phase (using data from 0 to 7 min) was calculated.
Cytotoxicity Assay. Analysis of the intrinsic toxicity of the
compounds was conducted against the human cell line HeLa (cervical
cancer), using the EZ4U cell proliferation and cytotoxicity assay
(Biomedica, Austria), as per the manufacturer’s protocols. Briefly, cells
were grown in a Dulbecco’s modified Eagle medium, using an
antibiotic−antimycotic mixture (Gibco, Thermo Fisher Scientific,
USA), and in the presence of 10% fetal bovine serum (Biolot) in a 5%
CO2 atmosphere. After formation of a 20−30% monolayer, tested
compounds were added to the medium (the volume of added reagents
was 1/100 of the total volume of the culture medium, and the amount of
DMSO was 1% of the final volume), and the cell culture was monitored
for 3 days. Control cells were grown in the presence of 1% DMSO.
Further, an EZ4U (substrate) was added into each well, and cells were
incubated at 37 °C for 2 h; absorbance was read at 450 or 492 nm, with
620 nm used as a reference, using a POLARstar OPTIMA fluorimeter.
The compound concentration that caused 50% cell growth inhibition
(CC50) was determined using the OriginPro 8.6.0 software package.
The measurements were conducted in three parallel experiments.
To study the influence of inhibitors on the cytotoxic effect of
topotecan (“ACTAVIS GROUP PTC ehf.”), the CC50 for Tpc was
determined in the presence and absence of Tdp1 inhibitors.
Enhancement of cytotoxity of Tpc was calculated as the ratio of CC50
AUTHOR INFORMATION
Corresponding Author
■
Olga Lavrik − Novosibirsk Institute of Chemical Biology and
Fundamental Medicine, Siberian Branch of the Russian Academy
of Sciences, Novosibirsk 630090, Russian Federation; Novosibirsk
State University, Novosibirsk 630090, Russian Federation;
orcid.org/0000-0001-5980-8889; Phone: +7(383)
3635196; Email: lavrik@niboch.nsc.ru
Authors
Olga Luzina − N. N. Vorozhtsov Novosibirsk Institute of Organic
Chemistry, Siberian Branch of the Russian Academy of Sciences,
Novosibirsk 630090, Russian Federation
Alexander Filimonov − N. N. Vorozhtsov Novosibirsk Institute of
Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, Novosibirsk 630090, Russian Federation; Novosibirsk
State University, Novosibirsk 630090, Russian Federation
Alexandra Zakharenko − Novosibirsk Institute of Chemical
Biology and Fundamental Medicine, Siberian Branch of the
Russian Academy of Sciences, Novosibirsk 630090, Russian
Federation
Arina Chepanova − Novosibirsk Institute of Chemical Biology
and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, Novosibirsk 630090, Russian Federation
Olga Zakharova − Novosibirsk Institute of Chemical Biology and
Fundamental Medicine, Siberian Branch of the Russian Academy
of Sciences, Novosibirsk 630090, Russian Federation
Ekaterina Ilina − Novosibirsk Institute of Chemical Biology and
Fundamental Medicine, Siberian Branch of the Russian Academy
of Sciences, Novosibirsk 630090, Russian Federation
Nadezhda Dyrkheeva − Novosibirsk Institute of Chemical
Biology and Fundamental Medicine, Siberian Branch of the
Russian Academy of Sciences, Novosibirsk 630090, Russian
Federation
Galina Likhatskaya − Pacific Institute of Bioorganic Chemistry,
Far Eastern Branch of Russian Academy of Sciences, Vladivostok
690022, Russian Federation
H
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Novosibirsk State University, Novosibirsk 630090, Russian
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Notes
̌
E.; Zakharenko, A.; Kuprushkin, M.; Nilov, D.; Gushchina, I.; Svedas,
The authors declare no competing financial interest.
V.; Salakhutdinov, N.; Lavrik, O. Planta Med. 2019, 85, 103−111.
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ACKNOWLEDGMENTS
■
This study was funded by Russian State funded budget project
(AAAA-a17-117020210022-4) and by Russian Foundation for
Basic Research grant No. 18-44-540023. Analytical and
spectrometric studies were conducted at the Chemical Service
Center for collective use of the Siberian Branch of the Russian
Academy of Sciences.
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