Synthesis and evaluation of aryliden- and hetarylidenfuranone derivatives of usnic acid as highly potent Tdp1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.07.039

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for stalled DNA-topoisomerase 1 (Top 1) cleavage complexes and other 3′-end DNA lesions. Tdp1 is a promising target for anticancer therapy, since it can repair DNA lesions caused by Top1 inhibitors leading to drug resistance. Hence, Tdp1 inhibition should result in synergistic effect with Top1 inhibitors. Twenty nine derivatives of (+)-usnic acid were tested for in vitro Tdp1 inhibitory activity using a fluorescent-based assay. Excellent activity was obtained, with derivative 6m demonstrating the lowest IC₅₀ value of 25 nM. The established efficacy was verified using a gel-based assay, which gave close results to that of the fluorescent assay. In addition, molecular modeling in the Tdp1 substrate binding pocket suggested plausible binding modes for the active analogues. The synergistic effect of the Tdp1 inhibitors with topotecan, a Top1 poison in clinical use, was tested in two human cell lines, A-549 and HEK-293. Compounds 6k and 6x gave very promising results. In particular, 6x has a low cytotoxicity and an IC₅₀ value of 63 nM, making it a valuable lead compound for the development of potent Tdp1 inhibitors for clinical use.

Full text of DOI:10.1016/j.bmc.2018.07.039

Accepted Manuscript
Synthesis and evaluation of aryliden- and hetarylidenfuranone derivatives of
usnic acid as highly potent Tdp1 inhibitors
Olga Zakharova, Olga Luzina, Alexandra Zakharenko, Dmitry Sokolov,
Alexandr Filimonov, Nadezhda Dyrkheeva, Arina Chepanova, Ekaterina Ilina,
Anna Ilyina, Kristina Klabenkova, Boris Chelobanov, Dmitry Stetsenko,
Ayesha Zafar, Chatchakorn Eurtivong, Jóhannes Reynisson, Konstantin
Volcho, Nariman Salakhutdinov, Olga Lavrik
PII:
S0968-0896(18)30657-6
https://doi.org/10.1016/j.bmc.2018.07.039
BMC 14476
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2018
19 July 2018
22 July 2018
Please cite this article as: Zakharova, O., Luzina, O., Zakharenko, A., Sokolov, D., Filimonov, A., Dyrkheeva, N.,
Chepanova, A., Ilina, E., Ilyina, A., Klabenkova, K., Chelobanov, B., Stetsenko, D., Zafar, A., Eurtivong, C.,
Reynisson, J., Volcho, K., Salakhutdinov, N., Lavrik, O., Synthesis and evaluation of aryliden- and
hetarylidenfuranone derivatives of usnic acid as highly potent Tdp1 inhibitors, Bioorganic & Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.039
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Synthesis and evaluation of aryliden- and hetarylidenfuranone derivatives of
usnic acid as highly potent Tdp1 inhibitors
Olga Zakharovaa#, Olga Luzinab#, Alexandra Zakharenkoa, Dmitry Sokolovb, Alexandr
Filimonovb,c, Nadezhda Dyrkheevaa, Arina Chepanovaa, Ekaterina Ilinaa, Аnna Ilyinac, Kristina
Klabenkovac, Boris Chelobanova,c, Dmitry Stetsenkoa, Ayesha Zafard, Chatchakorn Eurtivongd,
Jóhannes Reynissond, Konstantin Volchob,c, Nariman Salakhutdinovb,c, Olga Lavrika,c,*
*E-mail: lavrik@niboch.nsc.ru
#Equal contribution
a Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the
Russian Academy of Sciences, Novosibirsk, 630090, Russian Federation
b N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian
Academy of Sciences, Novosibirsk, 630090, Russian Federation
c Novosibirsk State University, Novosibirsk, 630090, Russian Federation
d School of Chemical Sciences, University of Auckland, New Zealand
ABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for stalled DNA-
topoisomerase 1 (Top 1) cleavage complexes and other 3′-end DNA lesions. Tdp1 is a promising
target for anticancer therapy, since it can repair DNA lesions caused by Top1 inhibitors leading
to drug resistance. Hence, Tdp1 inhibition should result in synergistic effect with Top1
inhibitors. Twenty nine derivatives of (+)-usnic acid were tested for in vitro Tdp1 inhibitory
activity using a fluorescent-based assay. Excellent activity was obtained, with derivative 6m
demonstrating the lowest IC50 value of 25 nM. The established efficacy was verified using a gel-
based assay, which gave close results to that of the fluorescent assay. In addition, molecular
modeling in the Tdp1 substrate binding pocket suggested plausible binding modes for the active
analogues. The synergistic effect of the Tdp1 inhibitors with topotecan, a Top1 poison in clinical
use, was tested in two human cancer cell lines, A-549 and HEK-293. Compounds 6k and 6x
gave very promising results. In particular, 6x has a low cytotoxicity and an IC50 value of 63 nM,
making it a valuable lead compound for the development of potent Tdp1 inhibitors for clinical
use.
1

1. Introduction
Developing inhibitors of DNA repair enzymes is a promising approach to improve
anticancer therapy, particularly for drug-resistant tumors. Radiation and chemotherapy of cancer
usually induce DNA lesions; however, tumors can be resistant, or develop resistance, during
treatment. Tyrosyl-DNA phosphodiesterase (Tdp1) plays a crucial role in repair of DNA lesions
formed by antitumor drugs such as the Top1 inhibitors camptothecin, topotecan and irinotecan,
making Tdp1 a promising target for adjunctive cancer treatment.[1-3] A relatively small number
of Tdp1 inhibitors[4-13] have been reported in literature; most of them exhibit moderate inhibiting
activity, which makes discovering novel Tdp1 inhibitors a worthwhile undertaking. Natural
products are an important source of compounds for the development of novel medicines, due to
the high chemical diversity often lacking in libraries of synthetic molecules.[14,15] Indeed, a
number of effective Tdp1 inhibitors were identified from natural products.[16-19] It has been
previously demonstrated that selective inhibition of Tdp1 using terpene-coumarin based ligands
(see analogue 1, Fig. 1), has a synergistic effect with camptothecin on the viability of the MCF-7
breast cancer cell line.[18,20]
Fig. 1 Known Tdp1 coumarin based inhibitor 1, usnic acid (+)-2 and its derivative 3.
Usnic acid is a secondary metabolite commonly found in various types of lichens, which
has a broad range of biological activities: antiviral, antibiotic, analgesic, antimycotic and
insecticide.[21,22] Research conducted over the past few decades has demonstrated that targeted
synthesis of usnic acid derivatives yields various polycyclic compounds that exhibit potent
biological activity for a number of important diseases, such as malaria, influenza and
tuberculosis.[23-25]
It has been previously found that usnic acid derivatives, modified at ring C (general
structure formula 3, Fig. 1), are effective Tdp1 inhibitors with significant synergistic effect on
the viability of the MCF-7 breast cancer cell line.[20] However, the presence of the native
moieties on ring C is known to be important for the penetration of usnic acid derivatives through
biological membranes.[26] This study aimed to synthesize and test the Tdp1 inhibitory activity of
2

usnic acid derivatives containing aryl- or hetarylidenfuranone moieties at ring A, keeping the
triketone system in ring C of natural dibenzofurane intact.
2. Results and discussion
2.1. Chemistry
We have previously proposed the three-step synthesis scheme of benzylidenefuranone
derivatives from (+)-usnic acid that included the stage of electrophilic bromination with bromine
in dioxane giving bromoderivative 4; subsequent intramolecular cyclization of compound 4 into
furanone 5 in the presence of potassium hydroxide; and alkali-catalyzed condensation of 5 with
appropriate aldehydes, resulting in compounds 6a-d, 6h-k (Scheme 1).[27]
Scheme 1. The general reaction pathway for the furanones 6a-zz.
Compounds 6a-k, m-z (Table 1) as well as compound 6zz with aliphatic substituent for
structure activity relationship (SAR) purpose were successfully synthesized using this approach.
Synthesis and NMR data of compounds 6a-d, 6h-k, 6n, 6p used in this study have been
previously reported,[27,28] while, to the best of our knowledge, compounds 6e-g, 6l, 6m, 6o, 6q-zz
are new. Compounds 6e-g, 6m, 6o, 6q-zz were synthesized by the reaction between compound 5
and appropriate aldehydes and isolated with the yields 30-94%. In order to synthesize compound
6l, which contains a hydroxyl group at the para-position of the aromatic substituent, the
hydroxyl group in 4-hydroxybenzaldhyde 7l was first protected by reaction with 3,4-dihydro-2H-
pyran (Scheme 2). After the reaction between compound 8l and furanone 5, the protective group
in compound 9l was removed by exposure to K-10 clay.
3

Scheme 2. The reaction pathway to compound 6l.
In order to estimate the role of the alternate isomer of usnic acid in inhibitory activity,
two isomeric compounds 7m and 7x were synthesized from (-)-usnic acid, following the same
synthetic way as for the (+)-enantiomer (Scheme 3).
Scheme 3. The synthesis of compounds 7m,x.
The derivatives of usnic acid were evaluated for their Tdp1 inhibitory activity in vitro.
2.2 Biology
2.2.1 Inhibitory activity of compounds 6a-zz and 7m,x against Tdp1
We have recently designed an oligonucleotide biosensor for real-time detection of Tdp1
activity based on the ability of Tdp1 to remove fluorophore quenchers from the 3’-end of
DNA.[10] The hexadecameric oligonucleotide carried 5(6)-carboxyfluorescein (FAM) at the 5’-
end and the fluorophore quencher BHQ1 (Black Hole Quencher-1) at the 3’-end. Tdp1 inhibitors
prevent removal of fluorophore quenchers, thus reducing fluorescence intensity.
The results from the Tdp1 assay for derivatives 6a-zz and 7m,x using the FAM-BHQ1
biosensor are shown in the Table 1.
For the phenyl-substituted compound 6a, submicromolar activity (0.72 μM) was
demonstrated. Among the halogen derivatives 6b-g, compounds with one or more bromine
atoms were the most active (IC50 = 0.15-0.53 μM), while fluoro- (6b) and chloro- (6c)
derivatives appeared to be less active. The position of the bromine atom on the benzene ring did
not significantly influence the activity of the inhibitors.
4

The introduction of the nitrogen atom into the aromatic ring does not affect the ability of
the compounds to inhibit the enzyme (6n-p, IC50 values from 0.45 μM to 0.86 μM).
The addition of methoxy substituents in compounds 6h, 6i and 6k increased activity
approximately twofold (0.34-0.39 μM) in comparison with 6a, though compound 6j, with the
3,4-dimethoxyphenyl ring was less active. Replacement of the methoxy group by the hydroxyl
group, when proceeding from 6h to 6l, decreased the activity fivefold.
Table 1. Inhibition of compounds 6a-zz, 7m and 7x on Tdp1 activity.
R
IC50a μM
R
IC50a μM
Comp
ound
Compo
und
Phenyl
0.72±0.08
0.98±0.26
1.22±0.15
0.15 ± 0.03
0.23±0.06
0.232±0.004
0.53±0.28
0.39±0.13
0.34±0.04
1.02±0.18
0.34±0.06
1.59±0.07
0.025±0.002
>15
2-Pyridinyl
3-Pyridinyl
0.86±0.39
0.45±0.07
0.81±0.28
3.65±1.38
5.50±1.35
0.16±0.04
6.73±1.42
0.90±0.17
0.84±0.04
0.63±0.29
0.063±0.002
0.60±0.14
3.30±1.05
6a
6b
6c
6n
6o
6p
6q
6r
6s
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
2-Bromophenyl
3-Bromophenyl
3,5-Dibromophenyl
2-Methoxyphenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
4-Pyridinyl
Pyrrol-2-yl
6d
6e
Furan-2-yl
5-Methylfuran-2-yl
Furan-3-yl
6f
6g
6t
Thiophen-2-yl
6h
6i
6u
6v
6w
6x
6y
6z
5-Methylthiophen-
2-yl
3-Methylthiophen-
6j
2-yl
2,3,4-
Trimethoxyphenyl
5-Bromothiophen-2-
6k
6l
yl
4-Bromothiophen-2-
yl
4-Hydroxyphenyl
3,5-Di-tert-butyl-2-
Thiophen-3-yl
6m
6zz
7m
Furb
hydroxyphenyl
Cyclohexyl
3,5-Di-tert-butyl-2-
5-Bromothiophen-2-
yl
0.064±0.021
1.23±0.33
0.081±0.011
7x
hydroxyphenyl
a IC50 is the concentration that inhibits the activity of the enzyme by 50%.
b
Fur is furamidine, diamidine NSC 305831, a commercial Tdp1 inhibitor,[6] which is used as a
positive control.
5

At the same time, the most effective Tdp1 inhibitor from the compounds tested, with an
excellent IC50 value of 25 nM, was 6m, containing a large 3,5-di-tert-butyl-2-hydroxyphenyl
substituent.
Compounds with a five-membered unsubstituted aromatic heterocycle (6q, 6r, 6z) were
generally less potent Tdp1 inhibitors (IC50 values from 3.65 to 5.50 µM), excluding thiophen-2-
yl derivative 6u, with an IC50 of 0.9 µM. The insertion of the methyl group on the furan ring
resulted in 35-fold increase in inhibitory activity (6s against 6q). At the same time, the
introduction of a methyl group or a bromine atom to the thiophene residue of thiophene-2-yl
derivatives did not affect the inhibitory properties of the compounds (6v, 6w, 6y), with the
exception of compound 6x, which has a bromine atom at position 5. This modification led to a
tenfold increase in the activity (IC50 63 nM) in comparison with parent compound 6u (IC50 0.9
μM).
Compound 6zz, containing a cyclohexyl substituent at the furanone ring instead of aryl or
hetaryl substituents, was not active against Tdp1.
The influence of stereochemistry was studied using the most effective compounds, 6m
and 6x, as examples. Their enantiomers, 7m and 7x, were synthesized from (-)-usnic acid and
have the same effect on enzyme activity as found for the (+)-usnic acid derivatives (Table 1).
Thus, the absolute configuration of the asymmetric center of the usnic acid does not influence
inhibition potential.
Overall, most of the tested (het)arylidenfuranone derivatives of usnic acid demonstrated
potent anti-Tdp1 activity in submicromolar, or even nanomolar, concentration.
2.2.2 Validation of Tdp1 inhibition by gel-based assay
In order to validate the results from the fluorescence-based assay by means of standard
gel-based methods, we used two oligonucleotides of the same sequence 5’-d(AAC GTC AGG
GTC TTC C) radiolabeled with [32P] at the 5'-end, one containing fluorescence quencher BHQ1
and another containing a natural phosphotyrosine adduct at the 3’-end (Fig. 2). For the synthesis
of the latter, a CPG (controlled pore glass) solid support was prepared, which incorporated N-
Fmoc-tyrosine attached to an alkali-labile linker by its carboxyl group (Scheme 4). The
oligonucleotide synthesis was started from the phenolic group of support-bound N-Fmoc-
tyrosine.
6

Fig. 2. The structure of the oligodeoxynucleotide with 3’-attached phosphotyrosine residue used
as a Tdp1 substrate.
Scheme 4. The preparation of a CPG support with tyrosine attached to its carboxyl group by an
ester bond.
Compound 6g, which is active in submicromolar concentration, was chosen for this test.
In the absence of an inhibitor, the enzyme cleaves the tyrosine residue (Fig.3A, lane 1), and the
amount of product decreases with increasing concentration of inhibitor (Fig.3A, lanes 2-6). In
the case of BHQ1, similar products were observed (data not shown). Fig.3B shows the amount of
product as function of inhibitor concentration. The IC50 values were determined for compound
6g, by the gel electrophoresis method, to be 0.64±0.33 µM for the 3’-BHQ1 substrate and
4.0±1.1 µM for the 3’-tyrosine substrate. For the 3’-BHQ1 substrate, the IC50 value was virtually
identical to the one obtained using the fluorescence based assay (0.53±0.28 µM).
Both fluorescent and gel-based assays yield close results for 3’-BHQ1 substrate but differ
by an order of magnitude for the 3’-tyrosine substrate. Taking into account that different
substrates were used in the experiments, it can be concluded that very close results were
obtained.
80
70
1
2 3 4 5 6 7
60
50
40
30
20
10
0
Substrate
Product
Tdp1, 5 nM
I, µM
+ + + + + +
0 0.3 1 3 10 30
-
-
0
5
10
15
20
25
30
Inhibitor 6g concentration, M
A
B
Fig. 3 (A) Gel-electrophoresis autoradiogram of Tdp1 products on 3’-tyrosine substrate. 50 nM
5’-radiolabeled DNA substrate was reacted with 5 nM Tdp1 and indicated concentration of
inhibitor 6g at 25°C. “I” designates inhibitor (B). Plot of the amount of product as a function of
the inhibitor concentration.
7

2.2.3 Cytotoxicity
It is desirable that the inhibitors have low toxicity, which would simplify their use as a
component of "cocktails" with known anticancer drugs. The cytotoxicity of selected Tdp1
inhibitors was assessed using the A-549 adenocarcinomic human alveolar basal epithelial cells
and HEK-293 embryonic kidney cells. The cells were incubated in the presence of the inhibitors
at concentrations ranging from 1 to 100 µM. The diagrams of the survival rate of cells vs
inhibitor concentration are shown in Figure 4.
To study cytotoxicity on A-549 and HEK-293 cells, the most effective Tdp1 inhibitors
were selected (6d, 6e, 6f, 6i, 6k, 6m, 6s, and 6x). The compounds proved to be moderately toxic
for the cells (CC50 values in the range from 4.6 to 20 μM, see Table 2) except for compound 6s,
for which CC50 value could not be determined for both cell lines (>50 μM).
Topotecan (tpc) is a bioavailable derivative of camptothecin, a topoisomerase 1 (Top1)
inhibitor. Tpc is an important chemotherapeutic drug in the treatment of different cancer
types.[29-31] To check for synergy of Top1 and Tdp1 inhibition, the cytotoxic effect of tpc in the
presence of the Tdp1 inhibitors were tested. The nontoxic concentration of 1 μM was used in
conjunction with varied concentrations of tpc to derive CC50 values.
120
120
6i
6f
100
80
60
40
20
100
80
60
40
20
0
6i
6f
6x
6z
6s
6e
6k
6d
6x
6z
6d
6k
6e
6s
0
20
40
60
80
100
0
20
40
60
80
100
inhibitor concentration, M
inhibitor concentration, M
B
A
Fig. 4. The influence of selected Tdp1 inhibitors on A-549 (A) and HEK-293 (B) cell viability.
MTT tests showed a twofold reduction of CC50 values for tpc in A-549 cells with 6e, 6k,
6s, and 6x, and a two- to fourfold reduction of CC50 values for tpc in HEK-293 cells with 6d, 6f,
6k and 6x, as shown in Figure 5 and Table 2. Compounds 6d, 6f and 6i had minor influence on
tpc cytotoxicity for A-549 cells and compounds 6e, 6i, 6m and 6s had marginal effect on tpc
cytotoxicity for HEK-293 cells. Surprisingly, the most effective Tdp1 inhibitor, 6m, increased
the value of CC50 of tpc five-fold for A-549 cells, thereby protecting the cells from the action of
the drug. In addition, 6m barely affected the cytotoxicity of tpc on HEK-293 cells. Two
8

compounds enhanced tpc cytotoxicity on both cell lines: 6k and 6x. Compound 6k has quite a
low selectivity index (the CC50 / IC50 ratio) (~60), whereas the SI of 6x is 250 for the HEK-293
cell line. To summarize, we do not observe a correlation between the efficiency against the
purified enzyme, the intrinsic cytotoxicity of the compounds, and their ability to enhance the
effect of topotecan. Probably, it is related to the presence of other targets in the cells, such as
PARP1 or Tdp2. As compound 6x both enhances the effect of tpc and has a high SI, it is a
promising candidate for further development.
Table 2. The IC50 values of the usnic acid derivatives, their A-549 and HEK-293 cell viabilities
and selectivity indices (SI), as well as the cell viability in conjunction with tpc, a Top1 poison.
CC50
HEK-
tpc CC50
A-549
nMd
tpc CC50
HEK-293
nMd
a
R
IC50
CC50
Com-
pound
SIc
SIc
μM
A-549b μM
293b μM
111±27
162±66
48±18
24±1
10±3
21±6
10±2
22±6
tpc
6d
6e
6f
4-Bromophenyl
2-Bromophenyl
3-Bromophenyl
0.15
0.23
10.0±0.4
4.56±0.08
11.4±2.3
8.3±3.6
66.7
19.8
49.1
24.4
14.6±0.5
15.9±1.5
14.5±0.7
15.8±0.6
97.3
69.1
62.5
46.5
0.232
0.34
132±10
90±34
4-
Methoxyphenyl
2.3.4-
6i
Trimethoxyphen
yl
0.34
4.6±0.4
13.5
20±2
58.8
48±3
5.3±0.5
6k
3.5-Di-tert-
butyl-2-
hydroxyphenyl
5-Methylfuran-
2-yl
0.025
0.16
6.8±2.4
>50
272
>300
138
14.5±0.7
>50
180
>300
249
558±39
40±4
16±6
22±6
13±6
6m
6s
5-
Bromothiophen- 0.063
2-yl
8.7±1.0
15.7±0.5
47±5
6x
a IC50 - 50% inhibition concentration.
b CC50 - median cytotoxic concentration causing 50% cell death.
c SI selectivity index, the CC50 / IC50 ratio.
d In the presence of 1 μM of Tdp1 inhibitor
9

120
100
80
60
40
20
0
120
100
80
tpc
tpc
tpc + 6x
tpc + 6k
tpc + 6i
tpc + 6s
tpc + 6d
tpc + 6f
tpc + 6k
tpc + 6x
60
40
20
0
50
100
150
200
250
300
-20
0
20
40
60
80
100 120 140 160 180
Topotecan concentration, nM
Topotecan concentration, nM
B
A
Fig. 5. Dose-dependent action of tpc in combination with the furanone derivatives of usnic
acid on A-549 (A) and HEK-293 (B) cell viability.
2.3 Molecular modeling
Recently, Tdp1 was proposed to have two distinct binding sites: the substrate and an allosteric
binding pocket. This was based on molecular dynamic simulations for usnic acid derivatives,
including compound of general formula 3 (Fig. 1).[20] Until now, the substrate pocket has been
considered to be the binding site of Tdp1 inhibitors.[32] The molecular modeling work was aimed
to establish the preferential binding sites and modes of binding for the usnic acid derivatives
using docking (see experimental section for further detail). The derivatives were docked to both
the substrate pocket and the allosteric binding cavity. The scores and IC50 values are shown in
Table 3.
The computed affinity scores were based on four different algorithms: GoldScore
(GS),[33] ChemScore (CS),[34,35] Piecewise Linear Potential (ChemPLP)[36] and Astex Statistical
Potential (ASP).[37] There were no obvious correlations between the IC50 values and the scores.
The four algorithms predicted higher scores for the inhibitors docked to the substrate binding
pocket compared to the allosteric cavity on all occasions except one (derivative 6m for
ChemPLP). This suggests higher affinity and more preferential binding of the usnic acid
derivatives to the substrate binding pocket. Furthermore, pose predictions of the four scoring
functions was consistent for the substrate pocket, while more variation was seen for the allosteric
cavity.
10

Table 3. The scores predicted by docking the usnic acid derivatives to the allosteric and
substrate binding sites of Tdp1. Their IC50 values are also given.
Com-
IC50
GS
CS
ChemPLP
ASP
pound
(μM)
Allosteric Substrate Allosteric Substrate Allosteric Substrate Allosteric Substrate
0.72
0.98
1.22
0.15
0.23
0.232
0.53
0.39
0.34
1.02
0.34
1.59
0.025
0.86
0.45
0.81
3.65
5.5
0.16
6.73
0.90
0.84
0.63
0.063
0.60
3.30
> 15
0.064
0.081
37.7
37.5
39.2
43.7
40.2
42.3
47.2
45.0
42.9
46.3
43.2
41.4
48.8
45.1
40.8
43.5
45.1
43.0
46.3
37.9
40.6
44.1
43.1
46.6
45.1
45.8
44.1
45.6
49.9
52.5
52.1
53.3
52.8
53.4
55.4
56.5
52.0
54.1
53.6
57.9
51.7
63.1
50.1
51.2
49.8
49.1
48.0
49.1
48.2
50.2
53.1
55.1
51.9
52.1
51.1
50.0
53.1
55.4
20.4
19.9
21.4
20.8
21.0
21.4
22.3
21.3
19.6
19.7
19.8
18.4
22.2
20.1
21.8
20.4
21.8
21.7
20.6
19.5
20.7
21.1
20.4
21.9
21.8
21.3
21.3
20.4
22.6
29.1
29.1
30.6
29.8
30.6
30.5
31.8
31.0
29.5
29.4
28.4
27.1
28.3
30.8
33.1
30.6
30.5
30.6
31.2
32.4
30.6
31.7
30.4
31.1
31.2
30.8
30.3
28.6
31.4
44.6
47.0
49.0
48.6
48.6
49.1
49.4
49.9
48.9
51.5
50.2
52.4
59.6
51.5
52.9
52.2
48.1
51.5
49.8
51.4
49.9
51.3
49.6
51.3
50.6
52.6
43.4
49.7
53.1
61.6
58.1
58.0
59.5
63.6
58.2
54.2
59.5
57.7
61.5
56.7
57.4
52.2
61.3
63.4
62.4
60.1
58.4
61.1
63.1
60.7
61.9
62.2
61.8
61.4
62.8
59.4
55.1
62.5
25.8
26.6
27.7
25.5
25.6
24.1
24.8
27.3
25.9
26.4
29.1
28.1
28.9
26.8
26.6
28.5
25.7
25.8
27.2
25.4
24.9
25.2
25.1
26.3
26.6
24.6
24.1
26.5
26.2
37.3
37.3
38.3
37.3
36.2
37.2
33.5
35.9
38.5
38.7
34.5
36.6
38.6
39.2
39.3
38.2
38.2
37.9
36.7
39.9
37.2
37.2
36.2
36.4
36.1
37.1
35.1
36.3
39.9
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
6y
6z
6zz
7m
7x
Tdp1 catalyzes the hydrolysis of the Top1 tyrosine residue that is covalently linked by its
phenolic group to the 3’-phosphate of DNA.[9,38] The catalytic mechanism involves the formation
and hydrolysis of a Tdp1-DNA intermediate.[34] These catalytic steps are mediated by the
protonation and deprotonation of the His263 and His493 residues and are crucial for the function
of Tdp1.[9,38] Hence, a promising inhibition strategy is to disrupt the catalytic functions of His263
and His493. As an example, derivative 6m (Fig. 6) forms hydrogen bonds with His263 and
Asn516 residues. The heterocyclic ring system is buried in the hydrophobic groove of the
substrate binding pocket, depicted by the grey regions, and can form favorable hydrophobic
contacts with the Tyr204, Pro461 and His493 amino acid residue side chains.
11

Figure 6. (A) Docked configurations of derivative 6m to the allosteric and substrate binding
sites predicted by the ChemPLP algorithm. The protein surface is rendered with partial positive
charges colored as blue, negative charges are red and neutral regions are grey. The hydrogen
atoms are removed for clarity. (B) The hydrogen bond interactions are depicted as green dotted
lines between compound 6m and amino acids residues His263 and Asn516 in the substrate
binding pocket. Furthermore, lipophilic contacts (LC) are shown as purple dashed lines with
His493, Tyr204 and Pro461.
The 3,5-di-tert-butylphenol moiety is an extension from the heterocycle located near
Ile285. The kink shape of the usnic acids caused by the double bond allows the aryl moiety to fit
into the concave shape of the groove (Fig. 6A). In general, an excellent binding mode is
predicted for the investigated ligand.
According to the modeling, the methyl groups of 7m and 7x are easily accommodated by
the substrate binding pocket, which explains their similar IC50 values to the 6m, x enantiomers.
2.3.1. Chemical space
The calculated molecular descriptors (MW – molecular weight, Log P – water / 1-octanol
partition coefficient, HD – hydrogen bond donors, HA – hydrogen bond acceptors, PSA – polar
surface area and RB – rotatable bonds) for the twenty nine derivatives are given in SI (Table).
The ligands are relatively large, with molecular weight between 419.4 and 588.2. 6d, 6e, 6f, 6g,
6k, 6m, 6x and 6y derivatives lie in the Known Drug Space region while others are within the
boundaries of drug-like space (for definitions of lead-like, drug-like and Known Drug Space
regions see ref).[39] Also, 6j, 6k and 6l lie within the Known Drug Space. The log P values range
from 1.9 to 4.7 and the rest of the descriptors adhere to the definition of drug-like chemical
space.
12

3. Conclusion
Twenty nine usnic acid derivatives 6a-zz, 7m and 7x were synthesized, and evaluated for
their in vitro Tdp1 inhibitory activity using a fluorescent based assay, which was verified with a
gel-based assay for analogue 6g. It was found that the absolute configuration of the asymmetric
center in usnic acid derivatives does not influence inhibitory activity of the compounds.
Compounds 6a-z, 7m and 7x exhibited very high to moderate inhibitory activity (IC50 =
0.025 – 6.7 μM), compared to the presently known inhibitors. An excellent selectivity index was
obtained for 6x, which had a moderate cytotoxicity profile and potent inhibition of Tdp1.
Furthermore, 6x proved to be a tpc sensitizer for the A-549 and HEK-293 cell lines, with
twofold enhancement of cytotoxicity of topotecan in non-toxic concentration. It should be
mentioned that the lack of correlation between the efficiency of inhibition of the purified
enzyme, cytotoxicity and the ability to sensitize to the effect of topotecan may indicate the
presence of additional targets in a cell other than Tdp1.
Docking investigation predicted higher affinity and more preferential binding to the
substrate binding pocket for the usnic acid derivatives compared to a potential adjacent allosteric
cavity. This study identified a number of potential lead molecules that can aid in the
development of drug candidates that enhance the potency of the Top 1 poisons in clinical use.
4. Experimental section
4.1. Chemistry
The analytical and spectral studies were conducted in the Chemical Service Center for the
collective use of SB RAS.
The 1H and 13C NMR spectra for solutions of the compounds in CDCl3 were recorded on a
Bruker AV-400 spectrometer (400.13 and 100.61 MHz, respectively). The residual signals of the
solvent were used as references (δ 2.48, δ 39.52). The mass spectra (70 eV) were recorded on a
H
C
DFS Thermo Scientific high-resolution mass spectrometer. The melting points were measured
using a Kofler heating stage. The specific rotation was determined on a PolAAr 3005 and
provided in (deg×mL)×(g×dm)–1, where the concentration of the solutions is shown in
g×(100×mL)–1. Merck silica gel (63-200 μ) was used for the column chromatography. Thin-layer
chromatography was performed on Silufol plates (UV-254).
13

The atom numerations in the compound are provided for the assignment of signals in the
NMR spectra and are different from the atom numerations in the nomenclature name. The target
compounds reported in this manuscript have a purity of at least 97% (HPLC).
(R)-(+)-Usnic acid (+)-2 (αD +478 (c 0.1, CHCl3)) was isolated by the procedure of
Salakhutdinov et al.[40] from a mixture of lichens of the genus Usnea. (S)-(-)-Usnic acid (-)-2
([α]D -458 (c 0.1, CHCl3) was isolated by the same procedure from Cladonia stellaris.
Compounds 6a-d, 6h-k were synthesized by the procedure described in [27]
.
Compounds 6n and 6p were synthesized by the procedure described in [24]
.
4.1.1 Synthesis of compounds 6e-g, 6m, 6o, 6q-zz. General procedure.
Usnic acid 2 (1 mmol, 344 mg) was treated with the bromine–dioxane complex (2 mmol
Br2, 0.10 mL, dissolved in dioxane, 14 mL) and several drops of HBr then left for 7 d at room
temperature. The reaction mixture was concentrated in a rotary evaporator and chromatographed
over silica gel with elution by CH2Cl2 to yield compound 4 (67%).
A solution of 4 (1 mmol, 423 mg) in acetone (25 mL) was treated with KOAc (150 mg,
1.5 mmol), refluxed for 2 h, diluted with H2O (up to ~50–60 mL), acidified with HCl (1:4) to pH
3–4, and extracted with CH2Cl2 (3 - 10 mL). The extracts were dried over calcined MgSO4. The
solvent was removed. The residue was chromatographed over a column of silica gel with elution
by CH2Cl2 to yield compound 5 (90 %).
A solution of 5 (1 mmol, 342 mg) in MeOH (24 mL) was treated with the appropriate
aldehyde (1.1 mmol) and aqueous KOH (1 mL, 50%), heated at temperature 600C for 1.5 h,
cooled, diluted with H2O (up to ~50–60 mL), acidified with HCl (1:4) to pH 3–4, and extracted
with CH2Cl2 (3 - 10 mL). The extracts were dried over calcined MgSO4. The solvent was
removed. The residue was chromatographed over a column of silica gel with elution by CH2Cl2.
((4Z,10R)-12-Acetyl-4-[(2-bromophenyl)methylidene]-8,13-dihydroxy-7,10-dimethyl-
5,16-dioxatetracyclo[7.7.0.02.6.010.15]hexadeca-1,6,8,12,14-pentaene-3,11-dione (6e). Yellow
amorphous powder.
Yield 58%. M.p. 230 °C dec. Spectrum NMR 1H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-
15), 2.25 (s, 3H, H-10), 2.65 (s, 3H, H-12), 6.06 (s, 1H, H-4), 7.19 (s, 1H, H-16), 7.19 (t, J = 7.7,
1H, H-20), 7.39 (t, J = 7.7, 1H, H-21), 7.61 (d, J = 7.7, 1H, H-22), 8.23 (d, J = 7.7, 1H, H-19),
11.44 (s, 1H, OH-9), 18.85 (s, 1H, OH-3). NMR 13C (100.61 Hz, CDCl3): δ=7.49 (C-10), 27.90
(C-12), 31.95 (C-15), 58.65 (C-9b), 99.19 (C-4), 100.67 (C-6), 105.11 (C-2), 105.81 (C-8),
108.36 (C-9a), 109.63 (C-16), 126.35 (C-18), 131.92 (CC-17), 127.57, 130.49, 131.84 (C-20, C-
21 and C-22), 133.30 (C-19), 148.17 (C-14), 149.89 (C-5a), 159.49 (C-9), 165.63 (C-7), 179.15
14

(C-4a), 179.49 (C-13), 191.61 (C-3), 197.80 (C-1), 201.82 (C-11). HRMS m/z found: 508.0161
[M]+ C25H17O7Br1, calcd.: M = 508.0152.
(4Z,10R)-12-Acetyl-4-[(3-bromophenyl)methylidene]-8,13-dihydroxy-7,10-dimethyl-
5,16-dioxatetracyclo[7.7.0.02.6.010.15]hexadeca-1,6,8,12,14-pentaene-3,11-dione (6f). Yellow
amorphous powder.
1
Yield 30%. M.p. 218-219 °C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-15), 2.28
(s, 3H, H-10), 2.65 (s, 3H, H-12), 6.05 (s, 1H, H-4), 6.67 (s, 1H, H-16), 7.29 (dd, 1H, H-19),
7.49 (d, 1H, H-18), 7.72 (d, 1H, H-20), 8.02 (s, 1H, H-22), 11.44 (s, 1H, OH-9), 18.84 (s, 1H,
OH-3). NMR 13C (100.61 Hz, CDCl3): δ=7.45 (C-10), 27.84 (C-12), 31.93 (C-15), 58.64 (C-9b),
99.20 (C-4), 100.60 (C-6), 105.10 (C-2), 105.88 (C-8), 108.39 (C-9a), 109.97 (C-16), 122.78 (C-
21), 129.55, 130.19, 132.30 and 133.68 (C-18, C-19, C-20 and C-22), 134.17 (C-17), 147.94 (C-
14), 149.88 (C-5a), 159.61 (C-9), 165.62 (C-7), 179.21 (C-4a), 179.41 (C-13), 191.59 (C-3),
197.79 (C-1), 201.79 (C-11). HRMS m/z found: 508.0161 [M]+ C25H17O7Br1, calcd.: M =
508.0152.
(4Z,10R)-12-Acetyl-4-[(3,5-dibromophenyl)methylidene]-8,13-dihydroxy-7,10-
dimethyl-5,16-dioxatetracyclo[7.7.0.02.6.010.15]hexadeca-1,6,8,12,14-pentaene-3,11-dione
(6g). Yellow amorphous powder.
1
Yield 62%. M.p. 176-178 °C. NMR H (400.13 Hz, CDCl3): δ=1.79 (s, 3H, H-15), 2.32
(s, 3H, H-10), 2.68 (s, 3H, H-12), 6.08 (s, 1H, H-4), 6.62 (s, 1H, H-16), 7.66 (t, J = 1.7, 1H, H-
20), 7.94 (d, J = 1.7, 2H, H-18), 11.53 (s, 1H, OH-9), 18.84 (s, 1H, OH-3). NMR 13C (100.61 Hz,
CDCl3): δ =7.41 (C-10), 27.83 (C-12), 31.86 (C-15), 58.60 (C-9b), 99.31 (C-4), 100.40 (C-6),
105.12 (C-2), 105.94 (C-8), 108.31 (C-16), 108.55 (C-9a), 123.11 (2C-19), 132.24 (2C-18),
134.45 (C-20), 135.56 (C-17), 148.48 (C-14), 150.02 (C-5a), 159.75 (C-9), 165.54 (C-7), 179.04
(C-4a), 179.22 (C-13), 191.03 (C-3), 197.78 (C-1), 201.23 (C-11). HRMS m/z found: 585.9250
[M]+ C25H16O7Br2, calcd.: M = 585.9257.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-[(3,5-di-tert-butyl-2-
hydroxyphenyl)methylidene]-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-
1(9),2(6),7,12,14-pentaene-3,11-dione (6m). Yellow amorphous powder.
Yield 74%. mp 186 °C. [α]D25.9 +197 (c 0.1, CHCl3). NMR 1H (400.13 Hz, CDCl3): δ=1.34
(c, 9H, H-tert-Bu) and 1.46 (c, 9H, H-tert-Bu), 1.74 (s, 3H, H-15), 2.22 (s, 3H, H-10), 2.65 (s,
3H, H-12), 5.99 (s, 1H, H-4), 6.60 (bs, 1H, OH), 7.21 (c, 1H, H-16), 7.40 (c, 1H, H-20), 7.84 (c,
1H, H-22), 11.41 (s, 1H, OH-9), 18.86 (s, 1H, OH-3). NMR 13C (100.61 Hz, CDCl3): δ=7.27 (C-
10), 27.93 (C-12), 29.79 and 31.34 (6C-tert-Bu), 31.95 (C-15), 34.29 and 34.88 (2C- tert-Bu),
58.69 (C-9b), 99.14 (C-4), 101.10 (C-6), 105.09 (C-2), 105.64 (C-8), 108.31 (C-9a), 109.03 (C-
17), 119.21 (C-16), 126.51 and 126.92 (C-20 and C-22), 136.69 (C-19), 142.61 (C-21), 146.56
15

(C-14), 149.82 (C-5a), 152.41 (C-18), 159.21 (C-9), 164.96 (C-7), 178.93 (C-4a), 179.58 (C-13),
191.62 (C-3), 197.88 (C-1), 201.85 (C-11). HRMS, found: m/z 558.2246 [M]+ C33H34O8.
Calculated: M = 558.2248.
(4Z,10S)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-[(3,5-di-tert-butyl-2-
hydroxyphenyl)methylidene]-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-
25.9
1(9),2(6),7,12,14-pentaene-3,11-dione (7m). Yellow amorphous powder. [α]D
-191 (c 0.1,
CHCl3).
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(pyridin-3-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (6o). Yellow
amorphous powder.
0
1
Yield 66%, mp decomposition >200 C, NMR H (400.13 Hz, CDCl3): δ=1.75 (s, 3H, H-
15), 2.26 (s, 3H, H-10), 2.64 (s, 3H, H-12), 6.04 (s, 1H, H-4), 6.72 (s, 1H, H-16), 7.38 (dd, J =
3.4 and 7.8 Hz, 1H, H-19), 8.18 (d, J = 7.8 Hz, 1H, H-18,), 8.56 (d, J = 3.4 Hz, 1H, H-20), 8.99
(s, 1H, H-21), 11.47 (s, 1H, 9-OH), 18.85 (s, 1H, 3-OH). NMR 13C (100.61 Hz, CDCl3): =7.47
(C-10), 27.84 (C-12), 31.88 (C-15), 58.54 (C-9b), 99.24 (C-4), 100.46 (C-6), 105.05 (C-2),
105.87 (C-8), 107.80 (C-16), 108.45 (C-9a), 123.67 (C-18), 128.41 (C-17), 137.38 (C-19),
148.57 (C-14), 149.70 (C-20), 149.88 (C-5a), 151.80 (C-21), 159.69 (C-9), 165.52 (C-7), 178.97
and 179.25 (C-13, C-4a), 191.54 (C-3), 197.71 (C-1), 201.78 (C-11). HRMS, found: m/z
431.1006 [M]+C24H17NO7. Calculated: M = 431.1000.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-[(1H-pyrrol-2-yl)methylidene]-
5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione
(6q).
Yellow amorphous powder.
0
1
Yield 45%. mp 144-145 C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-15), 2.21 (s,
3H, H-10), 2.65 (s, 3H, H-12), 6.04 (s, 1H, H-4), 6.36 (m, 1H, H-19), 6.64 (m, 1H, H-18), 6.96
(s, 1H, H-16), 7.08 (m, 1H, H-20), 11.24 (s, 1H, OH-9), 12.91 (1H, NH), 18.84 (s, 1H, OH-3).
13
NMR C (100.61 Hz, CDCl3): =7.20 (C-10), 27.88 (C-12), 32.03 (C-15), 58.92 (C-9b), 98.96
(C-4), 102.91 (C-6), 105.10 (C-2), 105.24 (C-8), 107.81 (C-9a), 111.98 (C-18), 114.21 (C-19),
119.14 (C-16), 124.04 (C-20), 126.80 (C-17), 145.07 (C-14), 149.58 (C-5a), 158.39 (C-9),
164.43 (C-7), 177.45 (C-4a), 179.85 (C-13), 191.63 (C-3), 197.92 (C-1), 201.74 (C-11). HRMS,
found: m/z 419.1002 [M]+ C23H17NO7. Calculated: M = 419.1005.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(furan-2-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (6r). Yellow
amorphous powder.
1
Yield 65%. mp 189-190 °C. NMR H (400.13 Hz, CDCl3): =1.76 (s, 3H, H-15), 2.29 (s,
3H, H-10), 2.65 (s, 3H, H-12), 6.05 (s, 1H, H-4), 6.59 (s, 1H, H-16), 6.83 (m, 1H, H-19), 7.07
16

(m, 1H, H-18), 7.58 (m, 1H, H-20), 11.38 (s, 1H, OH-9), 18.83 (s, 1H, OH-3). NMR 13C (100.61
Hz, CDCl3): =7.41 (C-10), 27.89 (C-12), 31.96 (C-15), 58.72 (C-9b), 99.13 (C-4), 101.30 (C-
6), 100.84 (C-19), 105.13 (C-2), 105.80 (C-8), 108.19 (C-9a), 113.01 (C-18), 116.69 (C-16),
145.03 (C-20), 145.70 (C-14), 148.65 (C-17), 149.67 (C-5a), 159.21 (C-9), 165.30 (C-7), 178.78
(C-4a), 179.63 (C-13), 191.64 (C-3), 197.89 (C-1), 201.81 (C-11). HRMS, found: m/z 420.0839
[M]+ C23H16O8. Calculated: M = 420.0840.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(5-methylfuran-2-ylmethylidene)-
5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione
(6s).
Yellow amorphous powder.
0
1
Yield 63%. mp decomposition >220 C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-
15), 2.26 (s, 3H, H-10), 2.38 (s, 3H, H-21), 2.64 (s, 3H, H-12), 6.03 (s, 1H, H-4), 6.20 (d, 1H, H-
19), 6.78 (s, 1H, H-16), 6.98 (d, 1H, H-18), 11.34 (s, 1H, OH-9), 18.81 (s, 1H, OH-3). NMR 13C
(100.61 Hz, CDCl3): =7.05 (C-10), 13.56 (C-21), 27.54 (C-12), 31.63 (C-15), 58.44 (C-9b),
98.71 (C-4), 101.13 (C-19), 104.78 (C-6), 105.36 (C-2), 106.62 (C-8), 107.74 (C-9a), 109.55 (C-
18), 118.44 (C-16), 144.58 (C-17), 146.85 (C-14), 149.48 (C-5a), 155.77 (C-20), 158.62 (C-9),
164.80 (C-7), 178.40 (C-4a), 179.39 (C-13), 191.30 (C-3), 197.60 (C-1), 201.46 (C-11). HRMS,
found: m/z 434.0990 [M]+ C24H18O8. Calculated: M = 434.0996.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(furan-3-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (6t). Yellow
amorphous powder.
0
1
Yield 49%. mp decomposition >210 C 0C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H,
H-15), 2.26 (s, 3H, H-10), 2.64 (s, 3H, H-12), 6.04 (s, 1H, H-4), 6.76 (s, 1H, H-16), 6.87 (s, 1H,
H-20), 7.49 (s, 1H, H-19), 7.90 (s, 1H, H-18), 11.38 (s, 1H, OH-9), 18.84 (s, 1H, OH-3). NMR
13C (100.61 Hz, CDCl3): =7.28 (C-10), 27.74 (C-12), 31.80 (C-15), 58.55 (C-9b), 98.94 (C-4),
101.16 (C-6), 103.27 (C-20), 104.94 (C-2), 105.42 (C-8), 107.93 (C-9a), 110.59 (C-16), 118.54
(C-17), 143.79 and 145.64 (C-18 and C-19), 147.36 (C-14), 149.52 (C-5a), 159.03 (C-9), 165.16
(C-7), 178.69 (C-4a), 179.45 (C-13), 191.46 (C-3), 197.70 (C-1), 201.64 (C-11). HRMS, found:
m/z 420.0838 [M]+ C23H16O6. Calculated: M = 420.0840.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(thiofen-2-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (6u). Yellow
amorphous powder.
0
1
Yield 75%. mp decomposition >220 C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-
15), 2.31 (s, 3H, H-10), 2.65 (s, 3H, H-12), 6.05 (s, 1H, H-4), 7.10 (s, 1H, H-16), 7.12 (m, 1H,
H-19), 7.46 (d, J = 3.6, 1H, H-18), 7.58 (d, J = 5.0, 1H, H-20), 11.38 (s, 1H, OH-9), 18.84 (s,
1H, OH-3). NMR 13C (100.61 Hz, CDCl3): =7.53 (C-10), 27.92 (C-12), 31.98 (C-15), 58.74 (C-
17

9b), 99.12 (C-4), 101.51 (C-6), 105.13 (C-2), 105.90 (C-8), 106.25 (C-16), 108.21 (C-9a),
127.85 (C-19), 131.51 (C-18), 132.72 (C-20), 135.50 (C-17), 145.92 (C-14), 149.66 (C-5a),
159.22 (C-9), 165.28 (C-7), 178.70 (C-4a), 179.63 (C-13), 191.64 (C-3), 197.88 (C-1), 201.82
(C-11). HRMS, found: m/z 436.0608 [M]+ C23H16O7S. Calculated: M = 436.0611.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-[(5-methylthiofen-2-
yl)methylidene]-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-
3,11-dione (6v). Yellow amorphous powder.
0
1
Yield 90%. mp 154-155 C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-15), 2.30 (s,
3H, H-10), 2.55 (s, 3H, H-21), 2.65 (s, 3H, H-12), 6.04 (s, 1H, H-4), 6.78 (d, J = 4, 1H, H-19),
7.02 (s, 1H, H-16), 7.26 (d, J = 4, 1H, H-18), 11.34 (s, 1H, OH-9), 18.84 (s, 1H, OH-3). NMR
13C (100.61 Hz, CDCl3): =7.50 (C-10), 15.71 (C-21), 27.86 (C-12), 31.93 (C-15), 58.70 (C-9b),
98.97 (C-4), 101.61 (C-6), 105.04 (C-2), 105.74 (C-8), 106.90 (C-16), 107.99 (C-9a), 126.44 (C-
19), 133.40 (C-18), 133.47 (C-17), 145.16 (C-14), 147.45 (C-20), 149.48 (C-5a), 158.86 (C-9),
165.09 (C-7), 178.55 (C-4a), 179.66 (C-13), 191.56 (C-3), 197.83 (C-1), 201.73 (C-11). HRMS,
found: m/z 450.0769 [M]+ C24H18O7S. Calculated: M = 450.0768.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-[(3-methylthiofen-2-
yl)methylidene]-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-
3,11-dione (6w). Yellow amorphous powder.
0
1
Yield 90%. mp decomposition >220 C. NMR H (400.13 Hz, CDCl3): =1.76 (s, 3H, H-
15), 2.32 (s, 3H, H-10), 2.42 (s, 3H, H-21), 2.65 (s, 3H, H-12), 6.06 (s, 1H, H-4), 6.94 (d, J = 4,
1H, H-19), 7.16 (s, 1H, H-16), 7.51 (d, J = 4, 1H, H-20), 11.34 (s, 1H, OH-9), 18.82 (s, 1H, OH-
3). NMR 13C (100.61 Hz, CDCl3): =7.56 (C-10), 14.46 (C-21), 27.92 (C-12), 31.99 (C-15),
58.76 (C-9b), 99.07 (C-4), 101.69 (C-6), 105.00 (C-16), 105.12 (C-2), 105.91 (C-8), 108.11 (C-
9a), 130.12 (C-17), 130.31 (C-19), 130.69 (C-20), 142.75 (C-18), 145.45 (C-14), 149.59 (C-5a),
159.02 (C-9), 165.17 (C-7), 178.68 (C-4a), 179.73 (C-13), 191.64 (C-3), 197.91 (C-1), 201.81
(C-11). HRMS, found: m/z 450.0764 [M]+ C24H18O7S. Calculated: M = 450.0768.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(5-bromothiofen-2-
ylmethylidene)-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-
3,11-dione (6x). Yellow amorphous powder.
0
Yield 94%. mp 162-163 C. [α]D26 +259 (c 0.1, CHCl3). NMR 1H (400.13 Hz, DMSO-d6):
=1.74 (s, 3H, H-15), 2.21 (s, 3H, H-10), 2.58 (s, 3H, H-12), 6.31 (s, 1H, H-4), 7.21 (s, 1H, H-
19), 7.64 (s, 1H, H-16), 7.99 (s, 1H, H-18), 11.82 (s, 1H, OH-9). NMR 13C (100.61 Hz, DMSO-
d6): =7.38 (C-10), 27.99 (C-12), 31.35 (C-15), 58.06 (C-9b), 99.16 (C-4), 100.56 (C-6), 104.23
(C-16), 104.41 (C-8), 105.63 (C-2), 109.40 (C-9a), 110.06 (C-20), 129.71 (C-19), 134.65 (C-18),
136.56 (C-17), 146.00 (C-14), 149.55 (C-5a), 158.88 (C-9), 164.34 (C-7), 177.26 (C-4a), 178.95
18

(C-13), 190.99 (C-3), 197.21 (C-1), 200.98 (C-11). HRMS, found: m/z 513.9714 [M]+
C23H15O7BrS. Calculated: M = 513.9716.
(4Z,10S)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(5-bromothiofen-2-
ylmethylidene)-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-
3,11-dione (7x). Yellow amorphous powder. [α]D26 -259 (c 0.1, CHCl3).
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-[(4-bromothiofen-2-
yl)methylidene]-5,16-dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-
3,11-dione (6y). Yellow amorphous powder.
0
1
Yield 68%. mp decomposition >220 C. NMR H (400.13 Hz, CDCl3): =1.76 (s, 3H, H-
15), 2.29 (s, 3H, H-10), 2.65 (s, 3H, H-12), 6.05 (s, 1H, H-4), 6.97 (s, 1H, H-16), 7.34 (s, 1H, H-
20), 7.44 (s, 1H, H-18), 11.45 (s, 1H, OH-9), 18.86 (s, 1H, OH-3). NMR 13C (100.61 Hz,
CDCl3): =7.43 (C-10), 27.74 (C-12), 31.97 (C-15), 58.61 (C-9b), 99.16 (C-4), 101.11 (C-6),
104.23 (C-16), 105.07 (C-2), 105.95 (C-8), 108.39 (C-9a), 111.02 (C-19), 127.87 (C-18), 133.82
(C-20), 136.55 (C-17), 146.59 (C-14), 149.75 (C-5a), 159.49 (C-9), 165.21 (C-7), 178.36 (C-4a),
179.38 (C-13), 191.55 (C-3), 197.77 (C-1), 201.72 (C-11). HRMS, found: m/z 513.9713 [M]+
C23H15O7BrS. Calculated: M = 513.9716.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(thiofen-3-ylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (6z). Yellow
amorphous powder.
0
1
Yield 74%. mp 158-159 C. NMR H (400.13 Hz, CDCl3): =1.75 (s, 3H, H-15), 2.28 (s,
3H, H-10), 2.64 (s, 3H, H-12), 6.04 (s, 1H, H-4), 6.87 (s, 1H, H-16), 7.38 (s, 1H, H-20), 7.56 (s,
1H, H-19), 7.83 (s, 1H, H-18), 11.38 (s, 1H, OH-9), 18.84 (s, 1H, OH-3). NMR 13C (100.61 Hz,
CDCl3): =7.57 (C-10), 27.96 (C-12), 32.03 (C-15), 58.78 (C-9b), 99.17 (C-4), 101.31 (C-6),
105.16 (C-2), 105.70 (C-8), 106.36 (C-16), 108.19 (C-9a), 126.36, 129.01 and 130.08 (C-18, C-
19 and C-20), 133.65 (C-17), 146.74 (C-14), 149.74 (C-5a), 159.27 (C-9), 165.52 (C-7), 179.46
(C-4a), 179.66 (C-13), 191.68 (C-3), 197.92 (C-1), 201.86 (C-11). HRMS, found: m/z 436.0607
[M]+ C23H16O7S. Calculated: M = 436.0611.
(4Z,10R)-12-Acetyl-8,13-dihydroxy-7,10-dimethyl-4-(cyclohexylmethylidene)-5,16-
dioxatetracyclo[7.7.0.02,6.010,15]hexadeca-1(9),2(6),7,12,14-pentaene-3,11-dione (6zz). Yellow
amorphous powder.
Yield 46%. mp 110 °C. NMR 1H (400.13 Hz, CDCl3): =1.23-1.39 (m, 3H, H-19 and H-20
and H-21), 1.73 (s, 3H, H-15), 1.78 (m, 2H, H-18 and H-22), 2.20 (s, 3H, H-10), 2.64 (s, 3H, H-
12), 2.69 (m, 1H, H-17), 5.95 (d, J = 9.5, 1H, H-16), 6.04 (s, 1H, H-4), 11.30 (s, 1H, OH-9),
18.83 (s, 1H, OH-3). NMR 13C (100.61 Hz, CDCl3): =7.28 (C-10), 25.32 (C-19 and C-21),
25.33 (C-20), 27.87 (C-12), 31.94 (C-15), 31.97 (C-18 and C-22), 35.19 (C-17), 58.74 (C-9b),
19

99.01 (C-4), 101.56 (C-6), 105.08 (C-2), 105.49 (C-8), 107.61 (C-9a), 121.27 (C-16), 147.99 (C-
14), 149.70 (C-5a), 159.17 (C-9), 165.85 (C-7), 179.25 (C-4a), 179.76 (C-13), 191.62 (C-3),
197.91 (C-1), 201.75 (C-11). HRMS, found: m/z 436.1512 [M]+ C25H24O7. Calculated: M =
436.1517.
4.1.2 Synthesis of compound 6l
A solution of 3,4-dihydro-2H-pyran (0.67 ml, 7.4 mmol) in dichloromethane (5 mL) was
added dropwise into a well-stirred suspension of 4-hydroxybenzaldehyde (300 mg, 2.46 mmol)
and pyridinium p-toluene sulfonate (8 mg) in dichloromethane (5 mL). The mixture was stirred
at room temperature for 1.5 h, then extracted with brine and dried over MgSO4. The solvent was
removed under reduced pressure and the crude compound was purified by column
chromatography on silica gel using an elution of 20% EtOAc/hexane to give 6l as a yellow oil
(the yield of compound 8l 100%).
A solution of 5 (1 mmol, 342 mg) in MeOH (24 mL) was treated with the aldehyde 8l
(1.1 mmol) and aqueous KOH (1 mL, 50%), heated for 1.5 h, diluted with H2O (up to ~50–60
mL), acidified with HCl (1:4) to pH 3–4, and extracted with CH2Cl2 (3 - 10 mL). The extracts
were dried over calcined MgSO4. The solvent was removed and the crude compound was
dissolved in 10 ml MeOH/CH2Cl2. Then the clay K-10 (100 mg) was added and the mixture was
0
stirred at temperature 50 C for 2 h. The powder was filtered and the filtrate was evaporated
under reduced pressure. The residue was chromatographed over a column of silica gel with
elution by 3% MeOH/CH2Cl2. The yield of compound 6l was 78%.
(4Z,10R)-12-Acetyl-4-[(4-hydroxyphenyl)methylidene]-8,13-dihydroxy-7,10-dimethyl-
5,16-dioxatetracyclo[7.7.0.02.6.010.15]hexadeca-1,6,8,12,14-pentaene-3,11-dione (6l). Yellow
amorphous powder.
Yield 62%. M.p. 176-178 °C. NMR 1H (400.13 Hz, DMSO-d6): =1.71 (s, 3H, H-15), 2.17
(s, 3H, H-10), 2.61 (s, 3H, H-12), 6.31 (s, 1H, H-4), 6.67 (s, 1H, H-16), 6.84 (d, J = 8, 2H, H-19),
7.72 (d, J = 8, 2H, H-18), 10.19 (s, 1H, OH-20), 11.41 (s, 1H, OH-9), 18.73 (bs, 1H, OH-3).
NMR 13C (100.61 Hz, DMSO-d6): =7.49 (C-10), 27.78 (C-12), 31.51 (C-15), 58.53 (C-9b),
98.71 (C-4), 100.62 (C-6), 104.40 (C-2), 105.34 (C-8), 108.83 (C-9a), 112.19 (C-16), 116.13
(2C-19), 122.86 (C-17), 133.45 (2C-18), 145.33 (C-14), 149.282 (C-5a), 158.02 (C-20), 159.54
(C-9), 164.55 (C-7), 177.94 (C-4a), 179.72 (C-13), 191.48 (C-3), 197.88 (C-1), 201.42 (C-11).
HRMS m/z found: 446.0990 [M]+ C25H18O8, calcd.: M = 446.1002.
4.2 Biology
20

4.2.1 Tdp1 assay
The recombinant Tdp1 was purified to homogeneity by chromatography on Ni-chelating
resin and phosphocellulose P11 as described[41] using plasmid pET 16B-Tdp1 kindly provided by
Dr. K.W. Caldecott (University of Sussex, United Kingdom).
Real-Time Detection of Tdp1 Activity. The Tdp1 activity measurements were carried out
as described.[12] The Tdp1-biosensor with a final concentration of 50 nM was briefly incubated in
a volume of 200 μL containing Tdp1 buffer (50 mM Tris-HCl pH8.0, 50 mM NaCl, 7 mM β-
mercaptoethanol) supplemented with purified 1.3 nM Tdp1 and various concentrations of
intibitor. Fluorescence measurements (Ex485/Em520 nm) were carried out during the linear phase
of reaction (from 0 to 8 min) every 55 sec. The reactions were incubated at a constant
temperature of 26 °C in a POLARstar OPTIMA fluorimeter, BMG LABTECH, GmbH. The
influence of compounds was evaluated by comparing the fluorescence increase rate in the
presence of compounds to that of DMSO control wells. The data were imported into the MARS
Data Analysis 2.0 program (BMG LABTECH), and IC50 values (concentration of a compound
required to reduce the enzyme activity by 50%) were calculated. Tdp1-biosensor 5’-(5,6 FAM-
aac gtc agg gtc ttc c-BHQ1)-3’ was synthesized in the Laboratory of Biomedical Chemistry,
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia.
Gel-Based Enzyme Assay. Oligonucleotide 5’-([32P]-aac gtc agg gtc ttc c-X)-3’, with 5’-
radioactive label (X=tyrosine residue or fluorescence quencher BHQ1) was used. The reaction
mixture contained 50 nM DNA substrate, 5 nM Tdp1, 50 mM Tris-HCl, 50 mM NaCl, 7 mM β-
mercaptoethanol, and the inhibitor 6g in a concentration 0-30 µM. The reaction was conducted at
25°C for 20 min in the case of 3’-BHQ1 substrate and for 5 min in the case of 3’-tyrosine
substrate. The reaction products were separated by electrophoresis in a 20% denaturing
polyacrylamide gel with 7M urea. A Typhoon FLA 9500 phosphorimager (GE Healthcare) was
used for gel scanning and imaging, and the data were analyzed with QuantityOne 4.6.7 software.
IC50 values were determined using OriginPro 8.6.0 software.
Synthesis of oligodeoxynucleotide with 3’-attached phosphotyrosine residue. A standard
CPG 1000 support for DNA synthesis containing a DMTr-dT residue attached via its 3’-OH
group by succinate ester was detritylated, and the 5’-OH group was esterified to the carboxyl
group of Nα-Fmoc tyrosine t-butyl ether by diisopropyl carbodiimide (DCI) in the presence of 4-
dimethylaminopyridine (DMAP). The phenolic OH group was liberated by acid-catalyzed t-butyl
ether removal in the presence of a fluorinated alcohol as described previously[42,43] while the
21

Fmoc amino protecting group was retained until the end of solid-phase synthesis.
Oligonucleotide synthesis was carried out from the free phenol group of the attached tyrosine by
standard β-cyanoethyl phosphoramidite chemistry. The oligonucleotide with a 3’-
phosphotyrosine residue was cleaved from support by alkaline hydrolysis, deprotected by usual
ammonia treatment and isolated by standard reverse-phased (RP) HPLC using 5’-DMTr group as
a purification handle. The RP-HPLC trace of the crude reaction mixture for oligonucleotide
purification is shown in Fig. S1 in Supplementary. The identity of the isolated oligonucleotide
has been confirmed by mass spectrometry (Fig. S2 in Supplementary).
4.2.2 Cytotoxicity
Tumor cells from human alveolar basal epithelial cells A-549 and embryonic kidney cells
HEK-293 (∼2000 cells per well) were incubated for 24 h at 37 °C in IMDM medium containing
10% (v/v) fetal bovine serum, 50 U/ml penicillin, 50 mg/ml streptomycin in a humidified
atmosphere (5% CO2), and then they were treated with the tpc and/or UA derivatives in different
concentrations. After 72 h of incubation, the relative number of alive cells was determined using
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (standard colorimetric MTT
test),[44] and the drug concentration that causes 50% cell death (CC50) was determined. CC50
values were determined using OriginPro 8.6.0 software. All values were expressed as mean ±
standard deviation (SD) from triplicate experiments performed in a parallel manner. The CC50
was determined as the compound concentration required to decrease the A570 to 50% of the
control (no compound, DMSO), and was determined by interpolation from dose-response curve.
4.3 Molecular Modeling
The molecules were docked to the crystal structure of Tdp1 (PDB ID: 1MU7, resolution
2.0 Å),[45] which was obtained from the Protein Data Bank (PDB).[46] The Scigress Ultra version
v2.6[47] program was used to prepare the crystal structure for docking: hydrogen atoms were
added, the co-crystalized tungsten(VI) ion and crystallographic water molecules were removed.
The Scigress software suite was also used to build the inhibitors, and the MM2[48] force field was
used to optimize the structures. The centre of the substrate binding pocket was defined as the
position of the hydrogen atom of His263, as its nitrogen formed a coordination bond with the
tungsten ion (x = 8.312, y = 12.660, z = 35.452) with 10 Å radius. The centre of the allosteric
binding site was defined as the Hon Ala258 (x = -2.971, y = 24.833, z = 39.768) with 10 Å
radius. The molecules were docked at 100% efficiency with fifty docking runs per molecule. The
22

basic amino acids, lysine and arginine, were defined as protonated, while aspartic and glutamic
acids were assumed to be deprotonated. The GoldScore (GS),[33] ChemScore (CS),[34,35]
ChemPLP[36] and ASP[37] scoring functions were implemented to validate the predicted binding
modes and relative energies of the ligands using the GOLD v5.4 software suite.
The QikProp 3.2[49] software package was used to calculate the molecular descriptors of the
[50]
compounds. The reliability of QikProp is established for the molecular descriptors.
Acknowledgments
This work was supported by Russian Science Foundation (grant 16-13-10074), excluding
the synthesis of the tyrosine-containing solid support and oligonucleotide, which was funded by
Russian Science Foundation (grant 15-15-00121 to D. Stetsenko), and grant of Russian
Foundation for Basic Research 16-03-00374 to O. Luzina for synthesis of compound 6zz.
Supplementary data
Supplementary data (RP-HPLC elution profile, mass spectrum of oligodeoxynucleotide
1
with 3’-attached phosphotyrosine residue, and NMR H and 13C spectra of compounds 6a–zz)
associated with this article can be found in the online version.
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