New inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 7-hydroxycoumarin and monoterpenoid moieties

Article abstract of DOI:10.1016/j.bmc.2016.09.016

A number of derivatives of 7-hydroxycoumarins containing aromatic or monoterpene substituents at hydroxy-group were synthesized based on a hit compound from a virtual screen. The ability of these compounds to inhibit tyrosyl-DNA phosphodiesterase I (Tdp 1), important target for anti-cancer therapy, was studied for the first time. It was found that the 7-hydroxycoumarin derivatives with monoterpene pinene moiety are effective inhibitors of Tdp 1 with the most active derivative (+)-25c with IC₅₀value of 0.675?μM. This compound has low cytotoxicity (CC₅₀?>100?μM) when tested against human cancer cells which is crucial for presupposed application in combination with clinically established anticancer drugs. The ability of the new compounds to enhance the cytotoxicity of camptothecin, an established topoisomerase 1 poison, was demonstrated.

Full text of DOI:10.1016/j.bmc.2016.09.016

Accepted Manuscript
New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 7-Hy-
droxycoumarin and Monoterpenoid Moieties
Tatyana Khomenko, Alexandra Zakharenko, Tatyana Odarchenko, Homayon
John Arabshahi, Victoriya Sannikova, Olga Zakharova, Dina Korchagina,
Jóhannes Reynisson, Konstantin Volcho, Nariman Salakhutdinov, Olga Lavrik
PII:
S0968-0896(16)30703-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.016
BMC 13269
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 June 2016
31 July 2016
8 September 2016
Please cite this article as: Khomenko, T., Zakharenko, A., Odarchenko, T., Arabshahi, H.J., Sannikova, V.,
Zakharova, O., Korchagina, D., Reynisson, J., Volcho, K., Salakhutdinov, N., Lavrik, O., New Inhibitors of Tyrosyl-
DNA Phosphodiesterase I (Tdp 1) Combining 7-Hydroxycoumarin and Monoterpenoid Moieties, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.016
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Graphical Abstract
New Inhibitors of Tyrosyl-DNA Phosphodiesterase I
(Tdp 1) Combining 7-Hydroxycoumarin and
Monoterpenoid Moieties
Leave this area blank for abstract info.
Tatyana Khomenkoa,d, Alexandra Zakharenkob, Tatyana Odarchenko,a,d, Homayon John Arabshahic, Victoriya
Sannikovad, Olga Zakharovab, Dina Korchaginaa, Jóhannes Reynissonc, Konstantin Volchoa,d, Nariman
Salakhutdinova,d, Olga Lavrikb,d
aN. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, 9, Akademika Lavrentieva Ave., Novosibirsk, 630090, Russian Federation
bNovosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian
Academy of Sciences, 8, Akademika Lavrentieva Ave., Novosibirsk, 630090, Russian Federation
cSchool of Chemical Sciences, University of Auckland, New Zealand
dNovosibirsk State University, Pirogova 2, Novosibirsk, 630090, Russian Federation
O
O
O
Compound (+)-25c. IC50 against Tdp1 0.675±0.007µM. (+)-25c enhances the cytotoxic effect of
camptothecin in 8 times

Bioorganic & Medicinal Chemistry
New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 7-
Hydroxycoumarin and Monoterpenoid Moieties
Tatyana Khomenkoa,d, Alexandra Zakharenkob, Tatyana Odarchenkoa,d, Homayon John Arabshahic,
Victoriya Sannikovad, Olga Zakharovab, Dina Korchaginaa, Jóhannes Reynissonc, Konstantin Volchoa,d,
Nariman Salakhutdinova,d, Olga Lavrikb,d
aN. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, 9, Akademika Lavrentieva Ave., Novosibirsk,
630090, Russian Federation
bNovosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8, Akademika Lavrentieva Ave.,
Novosibirsk, 630090, Russian Federation
cSchool of Chemical Sciences, University of Auckland, New Zealand
dNovosibirsk State University, Pirogova 2, Novosibirsk, 630090, Russian Federation
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A number of derivatives of 7-hydroxycoumarins containing aromatic or monoterpene
substituents at hydroxy-group were synthesized based on a hit compound from a virtual screen.
The ability of these compounds to inhibit tyrosyl-DNA phosphodiesterase I (Tdp 1), important
target for anti-cancer therapy, was studied for the first time. It was found that the 7-
hydroxycoumarin derivatives with monoterpene pinene moiety are effective inhibitors of Tdp 1
with the most active derivative (+)-25c with IC50 value of 0.675 μM. This compound has low
cytotoxicity (CC50 > 100 µM) when tested against human cancer cells which is crucial for
presupposed application in combination with clinically established anticancer drugs. The ability
of the new compounds to enhance the cytotoxicity of camptothecin, an established
topoisomerase 1 poison, was demonstrated.
Available online
Keywords:
Tyrosyl-DNA Phosphodiesterase I inhibitor
coumarin
monoterpene
molecular modeling
2016 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors that stabilize the Topo1-DNA covalent complex and
catalytically inhibit Tdp1, for example compound 3.7,8,9,10
Human tyrosyl-DNA phosphodiesterase (Tdp1) is a member
of the phospholipase D superfamily.1 Tdp1 hydrolyses the
phosphodiester bond between a catalytic tyrosine Tyr723
(human) of topoisomerase 1 (Topo1) and DNA 3′-phosphate.2
Tdp1 plays a key role in the removal of DNA damage, resulting
from Topo1 inhibition with camptothecin and its derivatives
irinotecan and topotecan. Furthermore, Tdp1 is also involved in
the removal of DNA damage caused by other anticancer drugs
commonly used in clinical practice, e.g., temozolomide,
bleomycin, etoposide, etc.3 The mechanisms of action are
different for these drugs, as are the various repair ensembles of
proteins that remove DNA damage. It is believed that Tdp1 is
responsible for the drug resistance of some cancers,4 making it a
promising target to enhance anticancer treatment in conjunction
with DNA damaging therapies.
Recently we have found that benzopentathiepine derivatives are
effective inhibitors of Tdp1,13 with compound 5 (Fig. 1) being the
most active (IC50 = 0.2 μM). Compound 5 causes apoptotic cell
death in MCF-7 and Hep G2 tumor cells.13 Specific inhibition of
Tdp1 is not expected be cytotoxic in itself because Tdp1-/- mice
are indistinguishable from wild-type mice, physically,
histologically, behaviorally, and electrophysiologically.14
Compared to wild-type mice, Tdp1-/- mice are hypersensitive to
camptothecin and bleomycin.14 The absence of increased
incidence of cancer or other health problems in Tdp1-/- mice
suggests that compound 5 toxicity is associated with other
cellular targets.
The combination of effective Tdp1 inhibition with general
cytotoxic potential opens the possibility for an antitumor
monotherapy based on derivative 5.
The literature describes relatively few Tdp1 inhibitors,5-12 with
potency varying from submicromolar to millimolar values.
Structures of some of these Tdp1 inhibitors (compounds 1-4) are
presented in Fig.1. Some were shown to be dual Tdp1/Topo1
———
At the same time, it is highly desirable to develop Tdp1
inhibitors with little or no inherent cytotoxicity for application in
combination with clinically established anticancer drugs such as
camptothecin.
 Corresponding author. Tel.: +7-383-363-5196; fax: +7-383-363-5153; e-mail: lavrik@niboch.nsc.ru

The aim of this work is to identify and develop a new class of
potent inhibitors against Tdp1 capable of enhancing the effect of
camptothecin on the tumor cells.
as containing other substituents at a hydroxyl group as shown in
Scheme 1.
Scheme 1. General scheme of preparation of proposed inhibitors.
Figure 1. Structures of reported Tdp1 inhibitors.
Preliminary molecular modeling against the Tdp1 binding
pocket indicated that the replacement of the aromatic substituent
by bulky aliphatic substituents, for example with
bicyclo[3.1.1]heptane framework, could improve the efficacy of
the inhibitors.
2. Results and Discussion
2.1. Virtual Screen
One of the problems encountered during the search for new
biologically active compounds by screening commercially
available libraries of small molecules is their lack of chemical
diversity, combined with a low proportion of biologically
relevant compounds.15 To sidestep this problem, we used the
library of natural products and their derivatives (42,000
compounds, InterBioScreen). Interestingly, many antitumor
agents used for chemotherapy are natural products, or their
analogues.16-18 Furthermore, many researchers are interested in
2.2. Chemistry
Synthesis of the unsubstituted at hydroxy-group coumarins
was carried out by the reaction of resorcinol 7 with esters of
corresponding β-keto acids 8-10 (Scheme 2), the yields of
products 11-13 amounted to 63%-95%.
Bromides required for reaction with the phenolic group of
coumarins were synthesized by reduction of the corresponding
aldehydes to alcohols which were then reacted with PBr3
(Scheme 3). As the starting compound for the preparation of bulk
aliphatic substituents we chose monoterpenoid myrtenal 18
having a bicyclo[3.1.1]heptane framework, which is accessible as
both enantiomers. (-)-Enantiomer (-)-19 was synthesized from
commercially available (-)-myrtenal (-)-18, and for its (+)-
enantiomer (+)-19 we first obtained (+)-myrtenal (+)-18 from
(+)-α-pinene 20 (Scheme 3).
developing new cytotoxic agents from this class of compounds.19-
22
The molecules of the library were screened to the binding
pocket of the crystal structure of Tdp1 (PDB ID: 1MU7,
resolution 2.0 Å).23 From the InterBioScreen natural product
library 10179 compounds were used. The GoldScore (GS),23
ChemScore (CS),25,26 ChemPLP27 and ASP28 scoring functions
were used to assess the binding of the ligands. The virtual screen
was done in two phases using first relatively low screening
efficiency weeding out compounds that were unlikely to fit the
binding pocket followed by a more robust search for the
remaining ligands. First, all ligands were screened and those with
no or weak predicted hydrogen bonding (<1) were eliminated as
well as those with low predicted binding energies (GS<43,
CS<28, ChemPLP<78 and ASP<38). This left 470 candidates,
which were screened again with high search efficiency and again
were eliminated based on their binding energies (GS<54, CS<32,
ChemPLP<85 and ASP<43) and poor hydrogen bonding (<1)
resulting in 111 candidates. They were inspected visually for
consensus of the best predicted configuration of the ligands
between the four scoring functions, that the ligands had plausible
configurations, i.e., not strained, for lipophilic moieties pointing
into the water environment and, finally for undesirable moieties
linked to cell toxicity and chemical reactivity.29 The screening
approach used has been previously successfully applied to find
active ligands against the Phosphoinositide specific-
phospholipase C-γ2 enzyme and Autophagy.30,31 A detailed
description of the virtual screen is given in the Methodology
section. Sixteen compounds were selected for experimental
testing and compound 6, which is 3-methoxybenzyl derivative of
7-hydroxycoumarin annelated with the cyclohexane ring
(Scheme 1), was identified as a promising inhibitor with an IC50
value of 5 μM using our oligonucleotide-based fluorescence
assay.13 Based on this result, we decided to synthesize structural
analogs of compound 6 modified at the coumarin skeleton as well
Compound 22 which is an analogue of (-)-myrtenal (-)-18
with additional methylene group was synthesized based on nopol
21.
Scheme 2. Synthesis of substituted 7-hydroxycoumarins.
Target compounds 6, 24b-e, 25a-e and 26a-d were prepared
by reaction of coumarins 11-13 with the synthesized bromides
16, 17, (-)- and (+)-19, and 22 as well as with benzyl bromide 23
according to the procedure shown in Scheme 1. The products

were purified by recrystallization or column chromatography.
The structures of the compounds obtained are given in Table 1.
compare, we chose cell line RPMI 8226 (human multiple
myeloma) with Tdp1 expression level 10 times lower.
Table 1.
Inhibitory activities of compounds 6, 24b-e, 25a-e and 26a-d against Tdp1.
Compound
R
IC50, µM
4.93 ± 0.49
6
5.62 ± 0.15
1.23 ± 0.34
24b
(-)-24c
1.20 ± 0.53
> 10
(+)-24c
24d
Scheme 3. Synthesis of bromides.
We did not succeed in the reaction of bromide 22 with
methylcoumarin 13 due to the formation of complex reaction
mixture with high level of resinification.
1.45 ± 0.15
24e
2.3. Biology
Tdp1 activity
> 10
25a
25b
9.17 ± 0.69
Previously, we designed real-time oligonucleotide biosensor
based on the capability of Tdp1 to remove fluorophore quenchers
from the 3’-end of DNA.32 Hexadecameric oligonucleotide were
chosen as Tdp1 substrate, which had 5(6)-carboxyfluorescein
(FAM) at the 5’ end and fluorophore quencher BHQ1 (Black
Hole Quencher-1) at the 3’-end.13 This approach was used to
measure Tdp1 activity and to determine inhibitory properties of
the synthesized compounds.
1.37 ± 0.29
(-)-25c
(+)-25c
0.675±0.007
The results of the Tdp1 assay for the coumarin derivatives are
shown in Table 1. The removal of the methoxy group, 24b
compared to 6, did not result in a significant change in activity.
However, adding two additional methoxy groups to the phenyl
group led to loss of activity (24d). Replacement of aromatic
substituents by monoterpene residue (compounds (-)- and (+)-
24c) quadrupled the inhibitor potency, both enantiomers were
equally active. The introduction of an additional methylene link
in the aliphatic chain did not affect the inhibition (see (-)-24c and
24e).
> 10
25d
1.09 ± 0.24
>10
25e
26a
We observed decrease in efficacy of compounds 25a and 25b,
containing aromatic substituents, when replacing the cyclohexane
ring annelated with the coumarin scaffold with a cyclopentane
moiety (Table 1). However, the efficiency of compounds (-)-25c
and 25e is similar as for the type 24 analogues (Table 1).
Enantiomer (+)-25c was twice as active as its (-)-25c counterpart.
5.28 ± 0.25
1.56 ± 0.50
4.31 ± 0.46
26b
(-)-26c
(+)-26c
Compound 26b was as active as its analogue 24b, while 26a
and 26d were inactive. The inhibitory effectiveness of
compounds (-)- and (+)-26c is close to the efficiency of its 24 and
25, counterparts. Thus the most active compounds from these
series is (+)-25c with the impressive IC50 of 0.675 µM.
> 10
26d
Cell growth and viability
To investigate the cytotoxic effect of Tdp1 inhibitors we chose
the cell line MCF- 7 (human breast adenocarcinoma) with higher
than the average level of Tdp1 gene expression according to
BioGPS data base http://biogps.org/#goto=welcome. To
The cells MCF-7 and RPMI 8226 were exposed to the
inhibitors in concentrations up to 100 µM. None of the four

tested compounds ((+)-24c, (+)-25c, (+)-26c and (-)-26c) had
effect on RPMI 8226 cells viability (data not shown) Part of them
do not show any antiproliferative effect for concentration up to
100 µM and are therefore not cytotoxic for MCF-7 cells (Fig. 2,
(+)-24c, black, (-)-24c, red, (-)-25c, pink).
concentrations of coumarins (5 µM) and different concentrations
of CPT to estimate CC50 values for MCF-7 and RPMI 8226 cells.
MTT tests showed marked reduction of CC50 values for MCF-7
cells in the presence of the coumarine derivatives as shown in
Fig. 3 and Table 2.
Compounds (+)-26c and (-)-26c have some influence on
MCF-7 cells (Fig. 2, dark blue and purple), but no concentration
dependence was observed. Derivatives 24e and 25e (Fig. 2, blue
and green) demonstrated weak concentration-dependence on
MCF-7 cells as evaluated by the MTT cytotoxicity assay, but it
was not possible to determine the 50% cytotoxic concentration
(CC50 value) in the concentration range used.
Table 2.
CC50 values for MCF-7 cells in the presence of coumarine derivatives in
combination with CPT.
CC50 for CPT,
R
Compound
nM
Synergistic activity with camptothecin against tumor cells
CPT
1430 ± 430
Camptothecin (CPT) and its derivatives are important Topo1
inhibitors, recognized for their anticancer activities. For instance,
irinotecan is key chemotherapeutic drug for metastatic colorectal
cancer and other types of tumors.33 Topotecan is used to treat
cancer of the ovaries when other treatments have failed and may
also be used in certain types of lung cancer (small cell lung
cancer). Since Tdp1 is involved in the removal of DNA damage
caused by CPT, it is believed that Tdp1 is responsible for drug
resistance of some cancers. Thus, a combination of these
anticancer drugs and Tdp1 inhibitors could significantly improve
the effectiveness of chemotherapy.
410 ± 165
(+)-24c
2300 ± 840
330 ± 20
24e
(-)-25c
180 ± 70
340 ± 80
(+)-25c
(-)-26c
110
100
90
80
70
60
(+)-24c
320 ± 95
(+)-26c
50
40
30
20
10
0
(-)-24c
24e
(-)-25c
25e
(+)-26c
(-)-26c
It is clear that most of the coumarins enhance the cytotoxicity
of camptothecin on MCF-7 cells. Only compound 24e did not
enhance the CPT activity. The best results were again obtained
for compound (+)-25c, which was able to reduce the CC50 of
camptothecin eight fold.
1
10
100
concentration of compounds, M
Figure 2. Dose-dependent action of the coumarins on MCF-7 cells viability
measured with the MTT assay.
In contrast to MCF-7 cells, RPMI 8226 cells did not show
increased sensitivity to camptothecin in the presence of inhibitor
(+)-25c (Fig. 4). Most likely, such differences can be explained
by the different levels of Tdp1 gene expression in these cell lines.
It is indirect proof that the obtained growth inhibition occurs due
to specific targeting of Tdp1.
120
100
80
Thus, coumarins are a promising class of compounds to
develop as sensitizers of Topo1 inhibitors for an enhanced
therapeutic effect.
cpt
(+)-24c
24e
(-)-25c
(+)-25c
(+)-26c
(-)-26c
60
40
20
0
2.4. Molecular modelling
All of the derivatives were docked against the binding pocket
of the Tdp1 crystal structure (PDB ID: 1MU7, resolution 2.0
Å),23 and the results of the scoring functions used are given in
Table S1 in the SI. The docked configuration of the most active
derivative (+)-25c is shown in Fig. 5. As can be seen, three
hydrogen bonding interactions are predicted with Asparagine
(Asn) 283, Histidines (His) 493 and 263, respectively.
10
100
1000
concentration of camptothecin, nM
Figure 3. Dose-dependent action of CPT in combination with the coumarins
on MCF-7 cell viability.
To check this hypothesis, we evaluated the cellular effect of
CPT in the presence of the Tdp1 inhibitors. We used nontoxic

3. Experimental section
3.1. General chemical methods
110
100
90
80
70
60
50
40
30
20
10
0
Reagents and solvents were purchased from commercial
suppliers (Sigma-Aldrich, Acros) and used as received. GC-MS:
Agilent 7890A gas chromatograph equipped with a quadrupole
mass spectrometer Agilent 5975C as a detector; quartz column
HP-5MS (copolymer 5%–diphenyl–95%–dimethylsiloxane) of
length 30 m, internal diameter 0.25 mm and stationary phase film
thickness 0.25 µm. Optical rotation: polAAr 3005 spectrometer;
CPT
CTP + (+)-25c
1
CHCl3 soln. H and 13C NMR: Bruker DRX-500 apparatus at
500.13 MHz (1H) and 125.76 MHz (13C), J in Hz; structure
determinations by analyzing the 1H NMR spectra, including 1H –
1H double resonance spectra and 1H – 1H 2D homonuclear
correlation, J-modulated 13C NMR spectra (JMOD), and 13C – 1H
2D heteronuclear correlation with one-bond and long-range spin-
spin coupling constants (C – H COSY, 1J(C,H) = 160 Hz,
COLOC, 2,3J(C,H) = 10 Hz). HR-MS: DFS Thermo Scientific
spectrometer in a full scan mode (15-500 m/z, 70 eV electron
impact ionization, direct sample administration).
10
100
1000
concentration of camptothecin, nM
Figure 4. Dose-dependent action of CPT in combination with the
coumarin (+)-25c on RPMI 8226 cell viability.
Previous molecular modelling studies strongly indicate that
hydrogen bonding to Histidine 263 is required for effective
inhibition.5,12 Also, the hydrophobic pocket in the binding site is
occupied by the terpene moiety. It can therefore be stated that a
plausible binding mode is predicted.
Spectral and analytical investigations were carried out at
Collective Chemical Service center of Siberian Branch of
Russian Academy of Sciences. All product yields are given for
pure compounds purified by recrystallization or isolated by
column chromatography. Column chromatography (CC): silica
gel (SiO2; 60-200 μ; Macherey-Nagel); hexane, solution
containing from 25 to 100% chloroform in hexane, ethanol. The
purity of the target compounds was determined by GC-MS
methods. All of the target compounds reported in this paper have
a purity of no less than 95%.
Considering the difference of the 24, 25 and 26 series it is
clear from the modelling that the aliphatic ring systems and the
methyl group are accommodated by a lipophilic cleft. Regarding,
the analogues containing the 3,4,5-trimethoxy substituted phenyl
ring (24d, 25d and 26d) a good fit was not predicted to the
binding site, the phenyl moiety was not inserted into the
lipophilic pocket, which can explain their inactivity.
Synthesis of (1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-ene-
2-carbaldehyde (+)-18.
The mainstream molecular descriptors are given in Table S2
in the Supplementary Information. The coumarins are relatively
small all with MW < 400 g mol-1. The log P values range from
3.5 to 5.5 with only three derivatives exceeding 5. None of them
have hydrogen bond donors but have three to six hydrogen bond
acceptors. In general, the coumarins are within drug-like
chemical space.
(+)-Myrtenal (+)-18 was synthesized according to the
procedure33 by oxidation of (+)-α-pinene (20) (
(neat)) using t-BuOOH/SeO2 system with 57% yield.
30.8 =50.4
[]589
Synthesis of compounds 11-13.
2.5. Conclusions
Syntheses were carried out from resorcinol 7 (45 mmol) and
appropriate β-keto esters (8 -10) in accordance with article.35
Conc. H2SO4 (5 mL, 94 mmol) was added dropwise to cooled (0-
5 °C) solution of resorcinol 7 (45 mmol) and appropriate β-keto
esters (8-10) (45 mmol) in dry ethanol (15 mL) with vigorously
stirring. The mixture was stirred until to be congealed, left
overnight at r.t., and poured into ice water (150 mL). The
resulting solid was filtered off and crystallized from ethanol-
water (75%). The yields of 11, 12 and 13 were 70%, 64% and
95% respectively.
It was found that the 7-hydroxycoumarin derivative (+)-25c
with monoterpene pinene moiety is an effective inhibitor of
tyrosyl-DNA phosphodiesterase I (Tdp 1) with IC50 value 675
nM. Furthermore, it has low cytotoxicity (CC50 > 100 µM) when
tested against human cancer cells. Finally, the ability of the
coumarin derivatives to substantially enhance the cytotoxicity of
camptothecin was demonstrated, with (+)-25c by an order of a
magnitude.
Synthesis of compounds 16, 17, (+)-19, (-)-19, 22.
Bromides 16, 17, (+)- and (-)-19, 22 were synthesized from
the corresponding monoterpene or methoxyphenyl aldehydes (14,
15, (+)- and (-)-18) via reduction to alcohols with NaBH4,34
followed by the reaction with PBr3.36
NaBH4 (10.3 mmol) was added to cooled (0-5°C ) solution of
10.3 mmol of appropriate aldehyde in methanol (20 ml) and the
reaction mixture was stirred for 3 h at r.t. Then 5% aqueous HCl
was added to reach pH 4-5. The solvent was distilled off and the
product was extracted by ether, dried with Na2SO4. The solvent
was evaporated; resulting alcohols were used in the synthesis
without purification (yields of alcohols– 34-73%).
Figure 4. The docked configuration of (+)-25c to the binding site of Tdp1
using the ASP scoring function. (A) The protein surface is rendered. The
terpene group is occupying a lipophilic pocket and the coumarin is in a cleft.
Red depicts a positive partial charge on the surface, blue depicts negative
partial charge and grey shows neutral/lipophilic areas. (B) Hydrogen bonds
are depicted as green lines between the ligand and the amino acids His263,
Asn282 and His493.

PBr3 (2.5 mmol) was added to cooled (0-5°C) solution of 7.5
mmol of appropriate alcohol in dry ether (10 ml) and the reaction
mixture was stirred for 2 h at r.t. Saturated aqueous NaHCO3 was
added and the product was extracted with ether. The extracts
were washed with brine, dried with Na2SO4 and evaporated.
Compounds 16, 17, (+)- and (-)-19 (the yields 65%, 61%, 55%
and 60%, respectively) were sufficiently pure and used for the
next step without purification. The compound 22 was purified by
column chromatography on SiO2 (yield 24%).
J(18,17a)=J(18,17s)=2.8, J(18,16)=1.3, H-C(18)); 2.20 (ddd, 1H,
J(20,18)=J(20,21s)=5.6, J(20,16)=1.4, H-C(20)); 2.24 (dm, 1H,
2J=18.0, H-C(17a)); 2.32 (dm, 1H, 2J=18.0, H-C(17s)); 2.39(ddd,
2
1H, J=8.7, J(21s,18)=J(21s,20)=5.6, H-C(21s)); 2.53 (tm, 2H,
J(10,11)=6.3, 2H-C(10)); 2.72 (tm, 2H, J(13,12)=6.3, 2H-C(13));
2
4.41 (dm, 1H, J=12.4, other J≤2.0, H-C(14)); 4.43 (dm, 1H,
2J=12.4, other J≤2.0, H-C(14’)); 5.59-5.62 (m, 1H, H-C(16)),
6.79 (d, 1H, J(9,7)=2.4, H-C(9)); 6.81 (dd, 1H, J(7,6)=8.7,
J(7,9)=2.4, H-C(7)); 7.40 (d, 1H, J(6,7)=8.7, H-C(6)). 13C-NMR
(CDCl3, δC): 153.30 (s, C(1)); 162.12 (s, C(2)); 120.29 (s, C(3));
147.17 (s, C(4)); 113.54 (s, C(5)); 123.79 (d, C(6)); 112.62 (d,
C(7)); 160.69 (s, C(8)); 101.49 (d, C(9)); 23.72 (t, C(10)); 21.60
(t, C(11)); 21.29 (t, C(12)); 25.10 (t, C(13)); 70.93 (t, C(14));
143.12 (s, C(15)); 121.12 (d, C(16)); 31.16 (t, C(17)); 40.69 (d,
C(18)); 37.98 (s, C(19)); 43.06 (d, C(20)); 31.38 (t, C(21)); 26.01
(q, C(22)), 20.96 (q, C(23)). HRMS 350.1875 ([M]+, m/z calcd
for C23H26O3 350.1877).
Synthesis of compounds 6, 24b-e, 25a-e and 26a-d.
Compounds 6, 24b-e, 25a-e and 26a-d were synthesized
according to the procedure.37 To 0.5 mmol of corresponding
compound 11, 12 or 13 in dry ethanol (5 mL) 0.75 mmol of
K2CO3, and 0.75 mmol of bromide (16, 17, (-)-19, (+)-19, 22)
were added at r.t. under stirring. The reaction mixture was stirred
at r.t. for 15 minutes, and then heated at 60 °C for 5 hours. The
hot solution was filtered; the filtrate was kept at -18 °C for 48
hours. The products were isolated in the individual form a) by
recrystallization from ethanol; or b) by column chromatography
on silica gel, eluent - solution containing from 25 to 100%
chloroform in hexane.
3-(((1S,5R)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
yl)methoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one
((+)-24c). Yield 38%, the method a. M.p. 105°C.
31.7
589
1
[] =+32.6 (c=0.65. EtOH). H and 13C NMR spectra of
((+)-24c) correspond to the spectra of ((-)-24c). HRMS 350.1872
([M]+, m/z calcd for C23H26O3 350.1876).
3-((3,4,5-Trimethoxybenzyl)oxy)-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-6-one (24d). Yield 44%, method b. M.p. 182
°C. H-NMR (CDCl3, δH): 1.74-1.86 (m, 4H, 2H-C(11), 2H-
3-((3-Methoxybenzyl)oxy)-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-6-one (6). Yield 56%, method a. M.p. 90 °C.
1H-NMR (CDCl3, δH):1.74-1.86 (m, 4H, 2H-C(11), 2H-C(12));
2.53 (tm, 2H, J(10,11)=6.2, 2H-C(10)); 2.72 (tm, 2H,
J(13,12)=6.2, 2H-C(13)); 3.80 (s, 3H-C(21)); 5.07 (s, 2H, 2H-
C(14)); 6.84 (d, 1H, J(9,7)=2.5, H-C(9)); 6.85 (ddd, 1H,
J(18,19)=8.3, J(18,16)=2.6, J(18,20)=0.9, H-C(18)); 6.88 (dd,
1H, J(7,6)=8.8, J(7,9)=2.5, H-C(7)); 6.95 (dd, 1H, J(16,18)=2.6,
J(16,20)=1.6, H-C(16)); 6.98 (ddd, 1H, J(20,19)=7.5,
J(20,16)=1.6, J(20,18)=0.9, H-C(20)); 7.28 (dd, 1H,
J(19,18)=8.3, J(19,20)=7.5, H-C(19)); 7.43 (d, 1H, J(6,7)=8.8, H-
C(6)). 13C-NMR (CDCl3, δC): 153.33 (s, C(1)); 161.97 (s, C(2));
120.58 (s, C(3)); 147.07 (s, C(4)); 113.89 (s, C(5)); 124.01 (d,
C(6)); 112.48 (d, C(7)); 160.31 (d, C(8)); 101.65 (d, C(9)); 23.73
(t, C(10)); 21.57 (t, C(11)); 21.28 (t, C(12)); 25.10 (t, C(13));
70.13 (t, C(14)); 137.53 (s, C(15)); 112.83 (d, C(16)); 159.81 (s,
C(17)); 113.59 (d, C(18)); 129.66 (d, C(19)); 119.49 (d, C(20));
55.14 (q, C(21)). HRMS: 336.1363 ([M]+, m/z calcd for C21H20O4
336.1356).
1
C(12)); 2.54 (tm, 2H, J(10,11)=6.2, 2H-C(10)); 2.73 (tm, 2H,
J(13,12)=6.2, 2H-C(13)); 3.83 (s, 3H-C(22)); 3.85 (s, 6H-C(23), -
C(21)); 5.01 (s, 2H, 2H-C(14)); 6.63 (s, 2H, H-C(16), H-C(20));
6.85 (d, 1H, J(9,7)=2.5, H-C(9)); 6.89 (dd, 1H, J(7,6)=8.8,
J(7,9)=2.5, H-C(7)); 7.44 (d, 1H, J(6,7)=8.8, H-C(6)). 13C-NMR
(CDCl3, δC): 153.32 (s, C(1)); 162.00 (s, C(2)); 120.64 (s, C(3));
147.12 (s, C(4));113.95 (s, C(5)); 124.05 (d, C(6)); 112.50 (d,
C(7)); 160.27 (s, C(8)); 101.54 (d, C(9)); 23.72 (t, C(10)); 21.55
(t, C(11)); 21.26 (t, C(12)); 25.11 (t, C(13)); 70.53 (t, C(14));
131.44 (s, C(15)); 104.52 (d, C(16), C(20)); 153.42 (s, C(17),
C(19)); 137.84 (s, C(18)); 56.05 (q, C(21), C(23)); 60.72 (q,
C(22)). HRMS 396.1563 ([M]+ m/z calcd for C23H26O3
396.1567).
3-(2-((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
yl)ethoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (24e).
Yield 32%, method a. M.p. 120 °C. []5287.91 =-12.7 (c=0.55,
1
3-(Benzyloxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-
one (24b). Yield 84%, method a. M.p. 140 °C. 1H-NMR (CDCl3,
δH):1.74-1.85 (m, 4H, 2H-C(11), 2H-C(12)); 2.53 (tm, 2H,
J(10,11)=6.2, 2H-C(10)); 2.71 (tm, 2H, J(13,12)=6.2, 2H-C(13));
5.09 (s, 2H, 2H-C(14)); 6.84 (d, 1H, J(9,7) = 2.5, H-C(9)); 6.88
(dd, 1H, J(7,6)= 8.8, J(7,9) =2.5, H-C(7)); 7.32 (tt, 1H,
J(18,17(19))=7.1, J(18,16(20))=1.5, H-C(18)); 7.37 (tm, 2H,
EtOH). H-NMR (CDCl3, δH): 0.80 (s, 3H-C(24)); 1.15 (d, 1H,
2J=8.5, H-C(22a)); 1.25 (s, 3H-C(23)); 1.73-1.86 (m, 4H, 2H-
C(11), 2H-C(12)); 2.05-2.10 (m, 2H, H-C(19), H-C(21)); 2.19
2
2
(dm, 1H, J=17.7, H-C(18)); 2.26 (dm, 1H, J=17.7, H-C(18’));
2.36 (ddd, 1H, 2J=8.5, J(22s,19)=J(22s,21)=5.6, H-C(22s)); 2.39-
2.50 (m, 2H-C(15)); 2.50-2.55 (m, 2H-C(10)); 2.69-2.75 (m, 2H-
C(13)); 3.95-4.00 (m, 2H-C(14)); 5.32-5.35 (m, 1H, H-C(17));
6.75 (d, 1H, J(9,7)=2.5, H-C(9)); 6.79 (dd, 1H, J(7,6)=8.8,
J(7,9)=2.5, H-C(7)); 7.41 (d, 1H, J(6,7)=8.8, H-C(6)). 13C-NMR
(CDCl3, δC): 153.36 (s, C(1)); 162.12 (s, C(2)); 120.24 (s, C(3));
147.24 (s, C(4)); 113.49 (c, C(5)); 123.93 (d, C(6)); 112.32 (d,
C(7)); 160.64 (s, C(8)); 100.94 (d, C(9)); 23.69 (t, C(10)); 21.27
(t, C(11)); 21.58 (t, C(12)); 25.10 (t, C(13); 66.68 (t, C(14));
36.11 (t, C(15)); 143.95 (s, C(16)); 118.89 (d, C(17)); 31.24 (t,
C(18)); 40.56 (d, C(19)); 37.96 (s, C(20)); 45.69 (d, C(21));
31.52 (t, C(22)); 26.15 (q, C(23)); 21.08 (q, C(24)). HRMS:
363.1947 ([M]]+, m/z calcd for C24H27O3 363.1955).
J=7.1,
H-C(17),
H-C(19));
7.41
(br.
d,
2H,
J(16,17)=J(20,19)=7.1, H-C(16), H-C(20)). 13C-NMR (CDCl3,
δC): 153.31 (s, C(1)); 161.95 (s, C(2)); 120.54 (s, C(3)); 147.05
(s, C(4)); 113.85 (s, C(5)); 124.00 (d, C(6)); 112.46 (d, C(7));
160.33 (s, C(8)); 101.60 (d, C(9)); 23.71 (t, C(10)); 21.56 (t,
C(11)); 21.26 (t, C(12)); 25.08 (t, C(13)); 70.24 (t, C(14));
135.93 (s, C(15)); 127.36 (d, C(16), C(20)); 128.57 (d, C(17),
C(19)); 128.13 (d, C(18)). HRMS: 306.1249 ([M]+, m/z calcd for
C20H18O3 306.1251).
3-(((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
yl)methoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one ((-)-
7-((3-Methoxybenzyl)oxy)-2,3-dihydrocyclopenta[c]chromen-
1
24c). Yield 38%, method a. M.p. 110 °C, []3580.98 =-25 (c=0.75,
4(1H)-one (25a). Yield 40%, method a. M.p. 122 °C. H-NMR
(CDCl3, δH): 2.14-2.22 (m, 2H, 2H-C(11)); 2.84-2.89 (m, 2H,
2H-C(10)); 2.99-3.04 (m, 2H, 2H-C(12)); 3.80 (s, 3H-C(20));
5.08 (s, 2H, 2H-C(13)); 6.86 (ddd, 1H, J(17,18)=8.3,
J(17,15)=2.6, J(17,19)=1.0, H-C(17)); 6.89 (dd, 1H, J(7,6)=9.2,
1
EtOH). H-NMR (CDCl3, δH): 0.80 (s, 3H-C(23)); 1.16 (d, 1H,
2J=8.7, H-C(21a)); 1.27 (d, 3H-C(22)); 1.74-1.85 (m, 4H, 2H-
C(11), 2H-C(12)); 2.09 (ddddd, 1H, J(18,20)=J(18,21s)=5.6,

J(7,9)=2.5, H-C(7)); 6.90 (d, 1H, J(9,7)=2.5, H-C(9)); 6.95 (dd,
1H, J(15,17)=2.6, J(15,19)=1.6, H-C(15)); 6.98 (ddd, 1H,
J(19,18)=7.6, J(19,15)=1.6, J(19,17)=1.0, H-C(19)); 7.29 (dd,
1H, J(18,17)=8.3, J(18,19)=7.6, H-C(18)); 7.32 (d, 1H,
J(6,7)=9.2, H-C(6)).13C-NMR (CDCl3, δC): 155.60 (s, C(1));
160.32 (s, C(2)); 124.57 (s, C(3)); 156.10 (s, C(4)); 112.55 (s,
C(5)); 125.49 (d, C(6)); 112.70 (d, C(7)); 160.89 (s, C(8));
101.81 (d, C(9)); 30.26 (t, C(10)); 22.45 (t, C(11)); 31.93 (t,
C(12)); 70.19 (t, C(13)); 137.45 (s, C(14)); 112.85 (d, C(15));
159.83 (s, C(16)); 113.62 (d, C(17)); 129.68 (d, C(18)); 119.50
(d, C(19)); 55.15 (q, C(20)). HRMS 322.1203 ([M]+, m/z calcd
for C20H18O4 322.1200).
7-(Benzyloxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one
(25b). Yield 35%, method a. M.p. 223 °C. 1H-NMR (CDCl3, δH):
2.13-2.21 (m, 2H, 2H-C(11)); 2.85-2.90 (m, 2H, 2H-C(10));
2.99-3.04 (m, 2H, 2H-C(12)); 5.11 (s, 2H, 2H-C(13)); 6.90 (dd,
1H, J(7,6) = 8.4, J(7,9) = 2.5, H-C(7)); 6.92 (d, 1H, J(9,7)= 2.5,
H-C(9)); 7.28-7.35 (m, 2H, H-C(6), H-C(17)); 7.38 (br.t, 2H,
160.87 (s, C(8)); 101.73 (d, C(9)); 30.28 (t, C(10)); 22.47 (t,
C(11)); 31.96 (t, C(12)); 70.62 (t, C(13)); 131.37 (s, C(14));
104.56 (d, C(15), C(19)); 153.46 (c, C(16), C(18)); 137.91 (s,
C(17)); 56.08 (q, C(20), C(22)); 60.75 (q, C(21)). HRMS:
382.1410 ([M]+ , m/z calcd for C22H22O6 382.1411).
7-(2-((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
yl)ethoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one (25e).
28.3
Yield 29%, method b. M.p. 149 °C. [] =-9.7 (c=0.31,
589
1
MeOH). H-NMR (CDCl3, δH): 0.81 (s, 3H, H-C(23)); 1.16 (d,
1H, J=8.5, H-C(21a)); 1.26 (s, 3H, H-C(22)); 2.06-2.10 (m, 2H,
2
H-C(18), H-C(20)); 2.13-2.23 (m, 3H, 2H-C(11), H-C(17)); 2.27
(dm, 1H, 2J=17.6, H-C(17’)); 2.36 (ddd, 1H, 2J=8.5,
J(21s,18)=J(21s,20)=5.6, H-C(21s)); 2.40-2.51 (m, 2H, H-
C(14)); 2.84-2.90 (m, 2H, H-C(10)); 2.99-3.04 (m, 2H, H-C(12));
4.00 (td, 2H, J(13,14)=7.0, J(13,9)=0.7; H-C(13)); 5.32-5.36 (m,
1H, H-C(16)); 6.80 (dd, 1H, J(7,6)=8.5, J(7,9)=2.4, H-C(7));
6.82 (br.d, 1H, J(9,7)=2.4, H-C(9)); 7.30 (d, 1H, J(6,7)=8.5, H-
C(6)). 13C-NMR (CDCl3, δC): 155.72 (s, C(1)); 160.44 (s, C(2));
124.29 (s, C(3)); 156.23 (s, C(4)); 112.21 (s, C(5)); 125.40 (d,
C(6)); 112.56 (d, C(7)); 161.29 (s, C(8)); 101.20 (d, C(9)); 30.25
(t, C(10)); 22.49 (t, C(11)); 31.94 (t, C(12)); 66.80 (t, C(13);
36.14 (t, C(14)); 143.99 (s, C(15)); 118.93 (d, C(16)); 31.29 (t,
C(17)); 40.66 (d, C(18)); 37.99 (s, C(19)); 45.81 (d, C(20));
31.55 (t, C(21)); 26.19 (q, C(22)); 21.09 (q, C(23)). HRMS:
349.1797 ([M-H]+, m/z calcd for C23H25O3 349.1798).
J=7.5,
H-C(16),
H-C(18));
7.41
(br.d,
2H,
J(15,16)=J(19,18)=7.5, H-C(15), H-C(19)). 13C-NMR (CDCl3,
δC): 155.64 (s, C(1)); 160.36 (s, C(2)); 124.59 (s, C(3)); 156.13
(s, C(4)); 112.55 (s, C(5)); 125.50 (d, C(6)); 112.75 (d, C(7));
160.96 (s, C(8)); 101.80 (d, C(9)); 30.28 (t, C(10)); 22.48 (t,
C(11)); 31.95 (t, C(12)); 70.35 (t, C(13)); 135.88 (s, C(14));
127.41 (d, C(15), C(19)); 128.63 (d, C(16), C(18)); 128.21 (d,
C(17)). HRMS: 292.1098 ([M]+ , m/z calcd for C19H16O3
292.1094).
7-((3-
Methoxybenzyl)
oxy)-4-methyl-2H-chromen-2-on
1
(26a). Yield 46%, method a. M.p. 87 °C. H-NMR (CDCl3, δH):
2.36 (d, J(10,3)=1.2, 3H-C(10)); 3.80 (s, 3H-C(18)); 5.08 (s, 2H-
C(11)); 6.10 (q, 1H, J(3,10)=1.2, H-C(3)); 6.847 (d, 1H,
J(9,7)=2.5, H-C(9)); 6.854 (ddd, 1H, J(15,16)=8.0, J(15,13)=2.6,
J(15,17)=1.0, 1H-C(15)); 6.91 (dd, J(7,6)=8.8, J(7,9)=2.5, H-
C(7)), 6.95 (dd, 1H, J(13,15)=2.6, J(13,17)=1.6, H-C(13));
6.98(ddd, 1H, J(17,16)=7.5, J(17,13)=1.6, J(17,15)=1.0, H-
C(17)), 7.28 (dd, 1H, J(16,15)=8.0, J(16,17)=7.5, H-C(16)); 7.47
(d, 1H, J(6,7)=8.8, H-C(6)). 13C-NMR (CDCl3, δC): 155.05 (s,
C(1)); 161.07 (s, C(2)); 111.91 (d, C(3)); 152.35 (s, C(4)); 113.65
(s, C(5)); 125.42 (d, C(6)); 112.74 (d, C(7)); 161.51 (s, C(8));
101.84 (d, C(9)); 18.46 (q, C(10)); 70.18 (t, C(11)); 137.29 (s,
C(12)); 112.84(d, C(13)); 159.80 (s, C(14)); 113.59 (d, C(15));
129.67 (d, C(16)); 119.44 (d, C(17)); 55.11 (q, C(18)). HRMS
296.1045 ([M]+, m/z calcd for C18H16O4 296.1043).
7-(((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
yl)methoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one ((-)-
25c). Yield 40%, method a. M.p. 145 °C. []3580.98 =-20.5, (c=0.5,
1
EtOH). H-NMR (CDCl3, δH): 0.80 (s, 3H-C(22)); 1.16 (d, 1H,
2J=8.6, H-C(20a)); 1.28 (s, 3H-C(21)); 2.10 (ddddd, 1H,
J(17,19)=J(17,20s)=5.6, J(17,16a)=J(17,16s)=2.9, J(17,15)=1.3,
H-C(17)); 2.13-2.20 (m, 2H, 2H-C(11)); 2.20 (ddd, 1H,
J(19,17)=J(19,20s)=5.6, J(19,15)=1.4, H-C(19)); 2.25 (dm, 1H,
2
2J=18.0, H-C(16a)); 2.32 (d m, 1H, J=18.0, H-C(16s)); 2.40
2
(ddd, 1H, J=8.6, J(20s,17)=J(20s,19)=5.6, H-C(20s)); 2.84-2.88
(m, 2H, 2H-C(10)); 2.99-3.03 (m, 2H, 2H-C(12)); 4.42 (dm, 1H,
2
2J=12.4, other J≤2.0, H-C(13)); 4.44 (dm, 1H, J=12.4, other
J≤2.0, H-C(13’)); 5.60-5.63 (m, 1H, H-C(15)), 6.82 (dd, 1H,
J(7,6)=8.6, J(7,9)=2.4, H-C(7)); 6.85 (d, 1H, J(9,7)=2.4, H-C(9));
7.29 (d, 1H, J(6,7)=8.6, H-C(6)). 13C-NMR (CDCl3, δC): 155.60
(s, C(1)); 160.48 (s, C(2)); 124.28 (s, C(3)); 156.23 (s, C(4));
112.21 (s, C(5)); 125.28 (d, C(6)); 112.85 (d, C(7)); 161.29 (s,
C(8)); 101.67 (d, C(9)); 30.24 (t, C(10)); 22.47 (t, C(11)); 31.93
(t, C(12)); 71.00 (t, C(13)); 143.07 (s, C(14)); 121.22 (d, C(15));
31.17 (t, C(16)); 40.69 (d, C(17)); 37.99 (s, C(18)); 43.07 (d,
C(19)); 31.39 (t, C(20)); 26.01 (q, C(21)); 20.97 (q, C(22)).
HRMS: 336.1722 ([M]+ , m/z calcd for C22H24O3 336.1720).
7-(((1S,5R)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
7-(Benzyloxy)-4-methyl-2H-chromen-2-on (26b). Yield 78%,
method b. M.p. 115°C. The 1H NMR spectrum of 26b is
coincided with the corresponding spectra published in literature.38
7-(((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
yl)methoxy)-4-methyl-2H-chromen-2-one ((-)-26c). Yield 54%,
31.7
1
method b. M.p. 70 °C. []589 =-36 (c=0.8, EtOH). H-NMR
2
(CDCl3, δH): 0.80 (s, 3H-C(20)); 1.17 (d, 1H, J=8.7, H-C(18a));
yl)methoxy)-2,3-dihydrocyclopenta[c]chromen-4(1H)-one ((+)-
30.7
1.28 (s, 3H-C(19)); 2.08-2.13 (m, 1H, H-C(15)); 2.20 (ddd, 1H,
25c). Yield 55%, method a. M.p. 140 °C. [] =+27.6
589
J(17,15)=J(17,18s)=5.6, J(17,13)=1.4, H-C(17)); 2.25 (dm, 1H,
1
(c=0.65, EtOH). The H and 13C NMR spectra of ((+)-25c)
2
2J=18.0, H-C(14a)); 2.33 (dm, 1H, J=18.0, H-C(14s)); 2.35 (d,
3H, J(10,3)=1.2, H-C(10)); 2.40 (ddd, 1H, 2J=8.7,
J(18s,15)=J(18s,17)=5.6, H-C(18s)); 4.43 (dm, 1H, 2J=12.4,
correspond to the spectra of the enantiomer ((-)-25c). HRMS
336.1718 ([M]+ , m/z calcd for C22H24O3 336.1720).
2
7-((3,4,5-Trimethoxybenzyl)oxy)-2,3-
other J≤2.0, H-C(11)); 4.45 (dm, 1H, J=12.4, other J≤2.0, H-
dihydrocyclopenta[c]chromen-4(1H)-one (25d). Yield 40%,
method b. M.p. 179 °C. 1H-NMR (CDCl3, δH): 2.14-2.22 (m, 2H,
H-C(11)); 2.85-2.90 (m, 2H, H-C(10)); 3.00-3.05 (m, 2H, H-
C(12)); 3.84 (s, 3H, H-C(21)); 3.86 (s, 6H, 3H-C(20), 3H-C(22));
5.02 (s, 2H, H-C(13)); 6.64 (s, 2H, H-C(15), H-C(19)); 6.91 (dd,
J(7,6)=8.8, J(7,9)=2.5, 1H, H-C(7); 6.92 (d, J(9,7)=2.5, 1H, H-
C(9)); 7.34 (br. d, J(6,7)=8.8, 1H, H-C(6)). 13C-NMR (CDCl3,
δC): 155.63 (s, C(1)); 160.35 (s, C(2)); 124.68 (s, C(3)); 156.14
(s, C(4)); 112.64 (s, C(5)); 125.54 (d, C(6)); 112.73 (d, C(7));
C(11’)); 5.60-5.63 (m, 1H, H-C(13)), 6.10 (q, 1H, J(3,10)=1.2,
H-C(3)); 6.80 (d, 1H, J(9,7)=2.4, H-C(9)); 6.83 (dd, 1H,
J(7,6)=8.7, J(7,9)=2.4, H-C(7)); 7.44 (d, 1H, J(6,7)=8.7, H-C(6)).
13C-NMR (CDCl3, δC): 155.10 (s, C(1)); 161.28 (s, C(2)); 111.74
(d, C(3)); 152.44 (s, C(4)); 113.37 (s, C(5)); 125.21 (d, C(6));
112.91 (d, C(7)); 161.96 (s, C(8)); 101.75 (d, C(9)); 18.51 (q,
C(10)); 71.06 (t, C(11)); 142.96 (s, C(12)); 121.30 (d, C(13));
31.18 (t, C(14)); 40.70 (d, C(15)); 37.99 (s, C(16)); 43.10 (d,

C(17)); 31.39 (t, C(18)); 26.01 (q, C(19)); 20.96 (q, C(20)).
HRMS: 310.1564 ([M]+ , m/z calcd for C20H22O3 310.1563).
7-(((1S,5R)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-
determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide (a standard colorimetric MTT-test).40
yl)methoxy)-4-methyl-2H-chromen-2-one ((+)-26c). Yield 30%,
3.3. Molecular Modelling and virtual screening
method b. M.p. 85 °C. []30.7 =+36 (c=0.8, EtOH). The 1H and
589
The compounds were docked to the crystal structure of Tdp1
(PDB ID: 1MU7, resolution 2.0 Å),23 which was obtained from
the Protein Data Bank (PDB).41,42 This structure can be found
here: http://www.rcsb.org/pdb/explore.do?structureId=1mu7. The
Scigress Ultra version 7.7.0.47 program43 was used to prepare the
crystal structure for docking, i.e., hydrogen atoms were added,
the co-crystallised tungsten(VI)ion was removed as well as
crystallographic water molecules. The Scigress software suite
was also used to build the inhibitors and the MM244 force field
was used to optimise the structures. The centre of the binding
pocket was defined as the position of the hydrogen atom of
HIS263, which nitrogen formed a coordination bond with the
tungsten ion (x = 8.312, y = 12.660, z = 35.452) with 10 Å
radius. For the initial screen 30% search efficiency was used
(virtual screen) with ten runs per compound. For the second
phase (re-dock) and the molecular modelling 100% efficiency
was used in conjunction with fifty docking runs. The basic amino
acids lysine and arginine were defined as protonated.
Furthermore, aspartic and glutamic acids were assumed to be
deprotonated. The GoldScore (GS),24 ChemScore (CS),25,26
ChemPLP27 and ASP28 scoring functions were implemented to
validate the predicted binding modes and relative energies of the
ligands using the GOLD v5.2 software suite. The virtual screen
was conducted with the InterBioScreen natural product
collection.
13C NMR spectra of ((+)-26c) correspond to the spectra of the
enantiomer (-)-26c. HRMS: 310.1559 ([M]+ , m/z calcd for
C20H22O3 310.1563).
4-Methyl-7-((3,4,5-trimethoxybenzyl)oxy)-2H-chromen-2-one
(26d). Yield 34%, method a. M.p. 145 °C. 1H-NMR (CDCl3, δH):
2.38 (d, 3H, J(10,3)=1.2, H-C(10)); 3.83 (s, 3H, H-C(19)); 3.86
(s, 6H, 3H-C(18), 3H-C(20)); 5.03 (s, 2H, H-C(11)); 6.12 (q, 1H,
J(3,10)=1.2, H-C(3)); 6.63 (s, 2H, H-C(13), H-C(17)); 6.87 (d,
1H, J(9,7)=2.5, H-C(9)); 6.92 (dd, 1H, J(7,6)=8.8, J(7,9)=2.5, H-
C(7)); 7.49 (d, 1H, J(6,7)=8.8, H-C(6)). 13C-NMR (CDCl3, δC):
155.11 (s, C(1)); 161.06 (s, C(2)); 112.04 (d, C(3)); 152.33 (s,
C(4)); 113.76 (s, C(5)); 125.47 (d, C(6)); 112.78 (d, C(7));
161.50 (s, C(8)); 101.80 (d, C(9)); 18.52 (q, C(10)); 70.64 (t,
C(11)); 131.22 (s, C(12)); 104.57 (d, C(13), C(17)); 153.46 (s,
C(14), C(16)); 137.96 (s, C(15)); 56.07 (q, C(18), C(20)); 60.72
(q, C(19)). HRMS 353.1363 ([M]+ m/z calcd for C20H20O6
353.1365).
3.2. General biological methods
The recombinant Tdp1 was purified to homogeneity by the
chromatography on Ni-chelating resin and phosphocellulose P11
as described1,39 using plasmid pET 16B-Tdp1 kindly provided by
Dr. K.W. Caldecott (University of Sussex, United Kingdom).
The QikProp 3.245 software package was used to calculate the
molecular descriptors of the compounds. The reliability of
QikProp is established for the molecular descriptors.46
Tdp1-biosensor 5’-(5,6 FAM-aac gtc agg gtc ttc c-BHQ1)-3’
was synthesized in Laboratory of Medicinal Chemistry, Institute
of Chemical Biology and Fundamental Medicine, Novosibirsk,
Russia.
Acknowledgments
Real-time detection of Tdp1 activity
This work was supported by Russian Scientific Foundation
(grant 16-13-10074).
The Tdp1 activity measurements were carried out as
described.13 Briefly, Tdp1-biosensor with final concentration of
50 nM was incubated in a volume of 200 uL containing Tdp1
buffer (50 mM Tris–HCl pH 8.0, 50 mM NaCl, 7 mM β-
mercaptoethanol) supplemented with a purified 1.3 nM Tdp1.
The reaction mixtures were incubated at a constant temperature
of 26 °C in a POLARstar OPTIMA fluorimeter, BMG
LABTECH, GmbH, to measure fluorescence every 1 min
(Ex485/Em520 nm). The efficiency of Tdp1 inhibition was
calculated by comparing the rate of increase in fluorescence of
biosensor in the presence of compound to that of DMSO control
wells. Values IC50 were determined using an eleven-point
concentration response curve. The data were imported into
MARS Data Analysis 2.0 program (BMG LABTECH) and the
slope during the linear phase (here data from 0-7 min) was
calculated.
Supplementary Material
Supplementary information (the results of the scoring
functions for compounds 6, 24b-e, 25a-e, 26a-d; the calculated
molecular descriptors for compounds 6, 24b-e, 25a-e, 26a-d; and
1
NMR H and 13C spectra) associated with this article can be
found, in the online version.
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