Promising new inhibitors of tyrosyl-DNA phosphodiesterase I (Tdp 1) combining 4- arylcoumarin and monoterpenoid moieties as components of complex antitumor therapy

Article abstract of DOI:10.3390/ijms21010126

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 μM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.

Full text of DOI:10.3390/ijms21010126

International Journal of
Molecular Sciences
Article
Promising New Inhibitors of Tyrosyl-DNA
Phosphodiesterase I (Tdp 1) Combining
4-Arylcoumarin and Monoterpenoid Moieties as
Components of Complex Antitumor Therapy
Tatyana M. Khomenko 1, Alexandra L. Zakharenko 2, Arina A. Chepanova 2, Ekaterina S. Ilina 2,
Olga D. Zakharova 2, Vasily I. Kaledin 3, Valeriy P. Nikolin 3, Nelly A. Popova 3,4
,
Dina V. Korchagina 1, Jóhannes Reynisson 5 , Raina Chand 6, Daniel M. Ayine-Tora 6,
Jinal Patel 6, Ivanhoe K. H. Leung 6 , Konstantin P. Volcho 1,4,
and Olga I. Lavrik 2,4,7
*
, Nariman F. Salakhutdinov 1,4
1
N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, 9 acad. Lavrentjev ave., 630090 Novosibirsk,
Russia; chomenko@nioch.nsc.ru (T.M.K.); korchaga@nioch.nsc.ru (D.V.K.); anvar@nioch.nsc.ru (N.F.S.)
Novosibirsk Institute of Chemical Biology and Fundamental Medicine, 8, acad. Lavrentjev ave.,
630090 Novosibirsk, Russia; sashaz@niboch.nsc.ru (A.L.Z.); arinachepanova@mail.ru (A.A.C.);
katya.plekhanova@gmail.com (E.S.I.); garonna3@mail.ru (O.D.Z.); lavrik@niboch.nsc.ru (O.I.L.)
Institute of Cytology and Genetics, 10, acad. Lavrentjev Ave., 630090 Novosibirsk, Russian;
kaledin@bionet.nsc.ru (V.I.K.); nikolin@bionet.nsc.ru (V.P.N.); nelly@bionet.nsc.ru (N.A.P.)
Novosibirsk State University, V. Zelman Institute for Medicine and Psychology and Department of Natural
Sciences, 2, Pirogova str., 630090 Novosibirsk, Russia
School of Pharmacy and Bioengineering, Keele University, Hornbeam Building, Staffordshire ST5 5BG, UK;
j.reynisson@keele.ac.uk
School of Chemical Sciences, The University of Auckland, Private Bag 92019, 1142 Auckland, New Zealand;
rcha387@aucklanduni.ac.nz (R.C.); dayi479@aucklanduni.ac.nz (D.M.A.-T.); jpat649@aucklanduni.ac.nz (J.P.);
i.leung@auckland.ac.nz (I.K.H.L.)
2
3
4
5
6
7
Department of Physical and Chemical Biology and Biotechnology, Altai State University, 61, Lenina Ave.,
656049 Barnaul, Russia
*
Correspondence: volcho@nioch.nsc.ru
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 3 December 2019; Accepted: 20 December 2019; Published: 23 December 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans,
and a current and promising inhibition target for the development of new chemosensitizing agents
due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan
and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1
inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results
showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo
experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in
the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen
the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent
adjunct therapy in conjunction with Top1 poisons.
Keywords: coumarin; neoflavone; DNA repair enzymes; Tdp1 inhibitor; cancer; tumor; topotecan;
topoisomerase 1 inhibitors; molecular modeling; chemical space
1. Introduction
Natural and synthetic coumarins (2H-chromen-2-one) demonstrate diverse biological activities,
and are often considered as a privileged scaffold [
1–
6]. In particular, a large number of coumarin
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derivatives with high antitumor activity have been found in recent years [
7
–18]. Natural derivatives of
7-hydroxycoumarin containing terpene fragments have also attracted the attention of the medicinal
chemistry community [19 21]. The best-known compound of this structural type is auraptene (Figure 1),
–
for which a variety of biological activities are known, including antitumor properties [22].
One of the current approaches to increase the efficacy of clinically-established antitumor therapy
is the inhibition of DNA repair enzymes that counteract the effect of DNA-damaging chemotherapy
agents [23–25]. One of these important enzymes is tyrosyl-DNA phosphodiesterase 1 (Tdp1) [26]. Tdp1
is involved in the repairing of damaged DNA, including the removal of lesions caused by topoisomerase
1 (Top1) inhibitors. Top1 inhibitors such as the camptothecin derivatives (CPTs), topotecan (tpc), and
irinotecan are well-established antitumor agents [27] that are widely used [28]. Thus, Tdp1 reduces the
impact of Top1 poisons, resulting in diminished DNA damage and reduced efficacy of this class of
chemotherapeutic drugs.
Because Tdp1 repairs Top1/DNA cleavage complexes induced by CPTs, inhibitors of Tdp1 can
enhance the sensitivity of cancer cells to CPT analogues [29]. Furthermore, increased Tdp1 expression
counteracts the cytotoxicity of CPTs [30
therapy [31 33]. Convincing evidence exists from preclinical studies that the ratio of Tdp1/Top1 activity
influences cellular sensitivity to Top1 inhibitors [34 35], and that the suppression of Tdp1 activity
leads to an increase in the sensitivity of tumor cells to CPTs [32 36 39]. It is believed that targeted
,31], and is frequently observed in cancers resistant to CPT
–
,
,
–
short-term treatment with a potent Tdp1 inhibitor will not lead to serious poisoning in normal cells.
Indeed, it was shown that Tdp1-/- knockout mice were fertile and had a normal life expectancy, with
no signs of premature aging [40]. Until now, no inhibitors of the Tdp1 enzyme have reached human
clinical testing.
To date, many Tdp1 inhibitors have been identified. A major class of Tdp1 inhibitors
comprises those based on natural products including usnic acid derivatives [41
adamantanes [47 49], nucleoside analogs [50], dehydroabietylamine derivatives [51], chromenes [52],
bile acids derivatives [53], and fungal products [54 56]. There are also early reports of Tdp1 inhibition
based on diamidines [57], antibiotics [58 59], steroids [60], and other compounds [61]. Nevertheless,
–45], coumarins [46],
–
–
,
only a few Tdp1 inhibitors have been tested in cell- or cancer-models. Synergy with tpc has been
demonstrated in vivo for just two Tdp1 inhibitors, both of which were derivatives of usnic acid,
a natural product [43,44].
Previously, virtual screening of the InterBioScreen natural product library [62] and subsequent
testing identified that 3-methoxybenzyl, a derivative of 7-hydroxycoumarin, annelated with the
cyclohexane ring
1 (Figure 1) as a new structural type of Tdp1 inhibitors [46]. Further optimization of
the inhibitor, including the replacement of the aromatic substituent in the phenolic group with bulky
monoterpenoid substituents, allowed us to increase the potency by almost an order of magnitude,
thereby reaching nanomolar activity [46]. Most importantly, the use of compound
significantly increased the cytotoxic activity of CPT in human cancer cells [46].
2 in non-toxic doses
Figure 1. Examples of biologically-active coumarins: auraptene and compounds 1–3.

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Based on molecular modeling, it was predicted that the attachment of an aromatic substituent at
the fourth position of coumarin would be promising for enhanced binding. Note that 4-arylcoumarins
are often considered a separate group of natural products, called neoflavones. Natural and synthetic
neoflavones have low toxicities and exhibit a broad spectrum of biological activity, in particular against
tumors [7]. The aim of this work was to synthesize neoflavone derivatives of structural type 3 (Figure 1)
by varying both aromatic and monoterpene substituents to determine their inhibitory activity against
Tdp1, and to study the synergistic effect with tpc, a clinically-important Top1 inhibitor, in in vivo
experiments. As a result, it was discovered that arylcoumarins containing monoterpenoid substituents
are indeed potent Tdp1 inhibitors and, most importantly, are able to enhance the antitumor activity of
tpc in animal models.
2. Results and Discussion
2.1. Chemistry
The main approach to producing 4-arylcoumarins unsubstituted at the hydroxy group is
acid-catalyzed Pechmann condensation between resorcinol
(Scheme 1). Using this approach, we synthesized 7-hydroxy-4-arylcoumarins 6a–d with yields of
63-81% by interaction of resorcinol with esters 5a–d (Scheme 1). Ester 5a is commercially available,
4 and ester of β-keto-carboxylic acids 5 [63]
4
while compounds 5b–d were obtained by the reaction of substituted acetophenones 5b–d with diethyl
carbonate in the presence of sodium hydride.
Scheme 1. Synthesis of 7-hydroxy-4-arylcoumarins 6a–d.
Monoterpenoid bromides 8a–d were obtained from the corresponding alcohols (geraniol, (
)- and (+)-myrtenols) by interaction with PBr3 according to the procedure [46] (Scheme 2). Geraniol
and ( )-nopol were purchased from commercial sources, while ( )- and (+)-myrtenols were synthesized
from ( )-myrtenal and (+)- -pinene in accordance with the methods [46]. The choice of monoterpenoids
−
)-nopol,
(−
−
−
−
α
was based on the results we obtained previously, i.e., when both the absolute configuration of the
pinane cycle and the length of the bridge played important roles [46]. A further consideration was
the desire to compare the activity data obtained with bulky bicyclic substituents with corresponding
data for products with acyclic monoterpene fragments. Note that coumarin-containing derivatives
of geraniol can be considered as analogues of the natural coumarin auraptene, having the same
monoterpenoid fragment (Figure 1).

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Scheme 2. Synthesis of monoterpenoid bromides 8a–d.
The target monoterpenoid-arylcoumarin hybrids
3
were synthesized by the reaction of
7-hydroxycoumarins 6a–d with monoterpenoid bromides 8a–d using DBU in DMF (Scheme 3).
To compare and identify the importance of the monoterpenoid fragment, compounds 10a, c, d
containing a benzyl substituent were also synthesized. The products were purified by recrystallization
or column chromatography, and obtained with yields of 12–65%. In the case of the synthesis of nopol
derivatives, a low conversion was observed, and double purification on SiO2 was required, for example,
for compounds 3ab or 3cb with yields less than 20%).
Scheme 3. Synthesis of monoterpenoid-arylcoumarin hybrids.

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2.2. Biology
A previously designed [64], real-time, hexadecameric oligonucleotide biosensor with
5(6)-carboxyfluorescein (FAM) at the 50 end and fluorophore quencher BHQ1 (Black Hole Quencher-1)
at the 30-end was used to determine the inhibitory properties of the new compounds.
The results of the Tdp1 assay for the arylcoumarin derivatives are shown in Figure 2 and
Supplementary Table S1. All arylcoumarin derivatives containing a geraniol residue (3aa–3da) showed
high inhibitory activity, with IC50 values in the submicromolar range; compound 3ac with a bromine
atom in the aromatic ring was slightly less active. Among the derivatives of nopol 3ab–3db, only the
fluorine derivative 3bb showed a markedly lower activity; the remaining compounds were comparable
in activity with the derivatives of geraniol. Since it was necessary to use column chromatography to
obtain nopol-arylcoumarin hybrids, which complicates and increases the cost of the synthesis process,
geraniol-containing inhibitors are more promising for further studies.
Almost all derivatives of (
similar inhibitory activity with IC50 values in the 0.4-1.0
−
)- and (+)-myrtenols (3ac–3dc and 3ad–3dd, respectively) showed
M range, except compound 3cc. Interestingly,
µ
compounds 10a, c, d containing a benzyl substituent instead of monoterpenoid fragments were
significantly less active than most of their monoterpenoid-containing analogues, with an IC50 in the
micromolar range.
Figure 2. The Tdp1 inhibitory activities of compounds 3aa–3dd and 10a, c, d. Furamidine (Fur) was
used as a positive control.
An analysis of the cytotoxicity of the synthesized compounds was performed on cell lines of
human breast adenocarcinoma MCF-7 and human cervical cancer HeLa. It turned out that cytotoxicity
is absent or insignificant in the entire range of studied concentrations (up to 100 µM) for all the tested
compounds, which makes it possible to use them as tumor sensitizers for currently-used antitumor
drugs without introducing additional toxic burden (Figure 3).

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Figure 3. The effect of compounds 3 on the survival of cells of the lines MCF-7 (a) and HeLa (b).
Since most monoterpene-arylcoumarin hybrids showed comparable inhibitory activity against
Tdp1 (~0.5 M) and no or limited cytotoxicity, we selected a candidate for subsequent studies based on
µ
the following considerations. Derivatives of nopol were the most complex compounds to synthesize
and purify. Therefore, they were excluded from further consideration. Since we previously obtained
contradictory results in in vivo experiments with the myrtenol derivative
2 (unpublished data), in this
work, we decided to focus on geraniol derivatives for the in vivo studies. Among the three derivatives
of geraniol that showed similar inhibitory activity (Figure 2), we selected compound 3ba containing a
fluorine atom in the para position of the aryl substituent, which can contribute to greater metabolic
stability of the inhibitor [65]. In addition to the activity assay that we reported above, we wanted
to confirm the interactions between compound 3ba and Tdp1 before progressing to in vivo studies.
Using an intrinsic tryptophan fluorescence quenching assay that we previously applied to study the
binding interactions of Tdp1 and its inhibitors [43,45,48,49,52], we evaluated the binding of compound
3ba to recombinant Tdp1. Clear quenching of the Tdp1 intrinsic fluorescence was observed upon the
addition of 3ba (Supplementary Figure S1). Titration experiments were then performed to determine
the dissociation constant (KD) of compound 3ba with Tdp1. A KD value of 63.0
±
11 µM was obtained,
indicating that it is a reasonable binder to the enzyme. This confirmed that the inhibition efficacy
of compound 3ba was due to binding to the enzyme, and gave us confidence to progress with this
compound towards in vivo studies.
A study of the influence of 3ba on the antitumor effect of tpc (topotecan) was performed using a
murine Krebs-2 carcinoma model. An ascitic tumor model combines the advantages of in vitro and
in vivo approaches in studying the cytotoxic effect of compounds, since ascitic cells grow in the context
of the organism (in vivo), and the intraperitoneal administration of drug ensures its direct contact
with tumor cells (in vitro). The experiments were performed using female C57BL/6 mice, which were
injected intraperitoneally with 2
×
105 ascitic cells on day zero. The mice were divided into six groups
of ten animals each. Control group 1 did not receive treatment; group 2 received tpc in a single dose of
0.5 mg/kg of body weight intraperitoneally after 2 days; group 3 received tpc as described above, and
3ba at a dose of 80 mg/kg intraperitoneally; group 4 received tpc and 3ba 40 mg/kg; group 5 received
tpc and 3ba 20 mg/kg; and finally, group 6 received 3ba 80 mg/kg only.
The combined use of 3ba at a maximum concentration of 80 mg/kg with tpc (group 3) led to a
significant decrease in the weight of the ascitic tumor compared to the use of only tpc (Figure 4). The
3ba dose of 40 mg/kg also caused a decrease in ascites weight, although the difference between groups
2 and 4 was not significant. The dose of 3ba 20 mg/kg co-administered with tpc, as well as the use of
3ba (80 mg/kg) in the absence of tpc, did not affect tumor growth.

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12
10
8
6
4
2
0
-2
Median
25%-75%
Non-Outlier Range
Outliers
Extremes
Figure 4. Box plot of the 3ba influence on the antitumor effect of tpc against Krebs-2 carcinoma with
intraperitoneal administration. P1–2 = 0.002; P1–3 = 0.00013; P2–3 = 0.04. The differences between group
2 and groups 4–6 are not significant.
The number of tumor cells in ascites (Figure 5) in the control group (1750 million per mouse) and
in group 2, which received only tpc (950 mln), was significantly different, i.e., by half, p = 0.005. The
size of ascites in group 3 (tpc + 80 mg/kg 3ba) was very small; we managed to extract ascitic fluid to
determine the number of cells in only one mouse; the number of tumor cells in this mouse was 250 mln.
Differences in other groups are not significant.
2400
2200
2000
1800
1600
1400
1200
1000
800
600
400
200
0
Median
25%-75%
Non-Outlier Range
Outliers
Extremes
Figure 5. A box plot of the 3ba influence on the number of tumor cells in ascites.
We then examined the effect of 3ba in combination with tpc on the lifespan of mice. C57BL/6
mice were intraperitoneally inoculated with the ascites variant of Lewis carcinoma. Group 1: control

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without treatment; group 2: tpc 0.5 mg/kg intraperitoneally; group 3: tpc and 3ba intraperitoneally
120 mg/kg; and group 4: 3ba 120 mg/kg only. The results are given in Figure 6.
16
15
14
13.4±0.4
13
10.6±0.5
11.2±0.2
12
11
10
9
9.4±0.5
Median
8
25%-75%
Non-Outlier Range
Outliers
7
Extremes
1. control
2. tрс
3. tрс +3ba
4. 3ba
Figure 6. The influence of 3ba in combination with tpc on the lifespan of mice. The numbers above the
boxes indicate the average lifespan in the group.
When using a combination of tpc and 3ba, a significant increase in lifespan was noted by 26%
(p = 0.0065) compared with mice receiving only tpc, and by 42% compared with the control group
(p = 0.0002). Monotherapy with tpc or 3ba at selected doses prolonged the life of experimental animals
unreliably, i.e., by 13–19%, р > 0.05.
2.3. In Silico
2.3.1. Molecular Modeling
The 19 compounds were docked into the binding site of Tdp1 (PDB ID: 6DIE, resolution 1.78 Å) [66
]
with three water molecules (HOH 814, 821 and 1078). It has been shown that keeping these crystalline
water molecules improves the prediction quality of the docking scaffold [45]. The modeling shows that
all the ligands have a plausible binding mode and good scores with the four scoring functions used, i.e.,
Astex Statistical Potential (ASP) [67], improved Piecewise Linear Potential (ChemPLP) [68], ChemScore
(CS) [69,70] and GoldScore (GS) [71]; the results are given in Table S2, Supplementary Information.
Considering 3ba, one of the most active compounds, the coumarin moiety occupies the hydrophilic
binding region, which contains amino acids such as threonine and glutamic acid, whilst the alkene
side chain occupies the hydrophobic region formed by isoleucine, leucine, and phenylalanine. The
carbonyl on the benzopyrone group forms hydrogen bonds with the amine side chain groups of Lys495
and Asn516. The predicted binding mode of 3ba is shown in Figure 7.

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Figure 7. The docked configuration of 3ba in the binding site of Tdp1 as predicted using the ChemPLP
scoring function. ( ) The protein surface is rendered. The ligand occupies the binding pocket. Blue
depicts a hydrophilic region with a partial positive charge on the surface; brown depicts hydrophobic
region with a partial negative charge and grey shows neutral areas. ( ) Hydrogen bonds are shown
a
b
as green lines between the ligand and residues Lys495 and Asn516. The water molecules also form
hydrogen bonds with Ser514 and Lys459.
2.3.2. Chemical Space
The calculated molecular descriptors (MW (molecular weight), log P (water-octanol partition
coefficient), HD (hydrogen bond donors), HA (hydrogen bond acceptors), PSA (polar surface area),
and RB (rotatable bonds)) are given in Table S3. The log P values range from 4.4 and 6.3, lying between
the drug-like and Known Drug Space (KDS), while the HD and PSA values are within the lead-like
space (for the definition of lead-like, drug-like, and KDS regions, see [72] and Table S4). The molecular
weight of the ligands is between 326.4 and 453.4 g
mol−1, falling in the drug-like chemical space. The
·
main issue with these ligands is their relatively high lipophilicity, with Log P values reaching into
the KDS.
The Known Drug Indexes (KDIs) of each ligand were calculated to gauge the balance of the
molecular descriptor of the ligands (Table S5, Supplementary Information). This method is based on
the statistical analysis of drugs in clinical use (KDS) and a weighted index for each of the six molecular
descriptors used; both the summation (KDI2a) and multiplication (KDI2b) methods were used [73].
The KDI2a values range from 4.7 to 5.3, with a theoretical maximum of 6 and an average of 4.08 for
known drugs. KDI2b ranges from 0.2 to 0.5, with a theoretical maximum of 1 and a KDS average of
0.18. This indicates that the majority of the ligands are well balanced. The most potent ligand 3ba has
KDI2a of 4.90 and KDI2b of 0.27, while drugs with high bioavailability (>50%) have average KDI2a of
4.43 and KDI2b of 0.21, which shows that 3ba has a very good balance of physicochemical properties
for bioavailability.
3. Materials and Methods
3.1. Chemistry Section
General Information. 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 µ
m. Optical rotation: polAAr 3005 spectrometer. 1H and 13C NMR: Bruker
DRX-500 apparatus at 500.13 MHz (1H) and 125.76 MHz (13C) and Bruker Avance—III 600 apparatus
1
at 600.30 MHz (1H) and 150.95 MHz (13C), J in Hz; structure determinations by analyzing the H
NMR spectra, including 1H–1H double resonance spectra and 1H–1H 2D homonuclear correlation,

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J-modulated 13C NMR spectra (JMOD), and 13C–1H 2D heteronuclear correlation with one-bond (C–H
1
1
COSY, J(C,H) = 160 Hz, HSQC, J(C,H) = 145 Hz) and long-range spin-spin coupling constants
(COLOC, 2,3J(C,H) = 10 Hz, HMBC, 2,3J(C,H) = 7 Hz). HR-MS: DFS Thermo Scientific spectrometer in
a full scan mode (15–500 m/z, 70 eV electron impact ionization, direct sample administration).
Spectral and analytical investigations were carried out at the Multi-Access Chemical Research
Center of Siberian Branch of Russian Academy of Sciences. All product yields are given for pure
compounds purified by recrystallization from ethanol or isolated by column chromatography (SiO2;
60–200 µ; Macherey-Nagel). The purity of the target compounds was determined by GC-MS methods.
All of the target compounds reported in this paper have purities of no less than 95%.
3.1.1. Synthesis of Compounds 5b–d
General procedure. To a stirred mixture of sodium hydride (3 mol equiv), washed with hexane
(3 × 15 mL), and diethyl carbonate (4 mol equiv) in 50 mL of tetrahydrofuran (THF), the corresponding
substituted acetophenone (1 mol equiv) was added dropwise over 30 min. The reaction mixture was
refluxed for 4 h, and then poured into ice water, acidified with 5 mL of glacial acetic acid, and extracted
with EtOAc (3
×
100 mL). The combined organic phase was washed with saturated sodium bicarbonate,
brine, and water, and then dried over anhydrous Na2SO4 and evaporated in vacuo. The crude products
were purified by silica gel column chromatography eluting with dichloromethane to afford 5b–d. The
yields of 5b, 5c, and 5d were 80%, 89%, and 92%, respectively.
3.1.2. Synthesis of Compounds 6a–d
Syntheses were carried out from resorcinol
with [46]. Conc. H2SO4 (2 mL, 37.6 mmol) was added dropwise to cooled (0–5 ◦C) solution of resorcinol
(2.3 g, 21 mmol) and appropriate -keto esters (5a–d) (21 mmol) in dry ethanol (5 mL) with vigorously
4 and appropriate β-keto esters (5a–d) in accordance
4
β
stirring. The reaction mixture was stirred at room temperature for 15 min, and then heated at 60 ◦C
until it congealed. It was then left overnight at room temperature. Finally, it was poured into ice water
(50 mL). The resulting solid was filtered off and crystallized from ethanol–water. The yields of 6a, 6b,
6c, and 6d were 81%, 79%, 73%, and 63% respectively.
3.1.3. Synthesis of Compounds 8a–d
(+)-Myrtenal was synthesized according to the procedure [46] by the oxidation of (+)-
α-pinene
using t-BuOOH/SeO2 system with a 57% yield. ( )- The (+)-myrtenols were synthesized from the
−
corresponding aldehydes via reduction to alcohols with NaBH4, as described above. NaBH4 (10.3
mmol) was added to a cooled (0–5 ◦C) solution of 10.3 mmol of the appropriate aldehyde in methanol
(20 mL), and the reaction mixture was stirred for 3 h at room temperature. Then, 5% aqueous HCl was
added to reach a pH of 4–5. The solvent was distilled off and the product was extracted using ether
and dried with Na2SO4. The solvent was evaporated; the resulting alcohols (58% and 54% yields) were
used in the synthesis without purification.
Bromides 8a–d were synthesized from geraniol, (
−
)-nopol, and ( )- and (+)-myrtenols via the
−
aforementioned reaction with PBr3. PBr3 (8.9 mmol) was added to a cooled (0–5 ◦C) solution of the
corresponding monoterpenoid alcohols (26.7 mmol) in dry ether (30 mL), and the reaction mixture
was stirred for 2 h at room temperature. Saturated aqueous NaHCO3 was added, and the product
was extracted with ether. The extracts were washed with brine, dried with Na2SO4, and evaporated.
Compounds 8a, 8c, and 8d (with yields of 91%, 55%, and 60%, respectively) were sufficiently pure and
used for the next step without purification. The compound 8b was purified by column chromatography
on SiO2, eluent–hexane (yield 24%).
3.1.4. Synthesis of Compounds 3aa–3da, 3ab–3db, 3ac–3dc, 3ad–3dd, and 10a, c, d
Compounds 3aa–3da, 3ab–3db, 3ac–3dc, 3ad–3dd, and 10a, c, d were synthesized from coumarins
6a–d and the corresponding bromides 8a–d, 9 using DBU and DMF.

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DBU (1.0 mmol) and corresponding bromide 8a–d, 9 (0.75 mmol) were added to compound 6a–d
(0.5 mmol) in dry DMF (5 mL) at room temperature under stirring. The reaction mixture was stirred
at room temperature for 15 min and then heated at 60 ◦C for 5 h. H2O (15 mL) was added and the
product was extracted with ethyl acetate. The extracts were washed with brine, dried with Na2SO4,
and evaporated. The products 3aa–3da, 3ab–3db, 3ac–3dc, 3ad–3dd, and 10a, c, d were isolated in the
individual form
a) by recrystallization from ethanol; or b) by column chromatography on silica gel
using eluent–hexane, a solution containing from 25 to 100% ethyl acetate in hexane, and ethanol.
(E)-7-(3,7-Dimethylocta-2,6-dienyloxy)-4-phenyl-2H-chromen-2-one 3aa. Yield 56%, method
a
. M.p.
1
54 ◦C. HRMS: 374.1879 [M]+; calcd. 374.1877 (C25H26O3)+. H NMR (CDCl3,
δ
ppm, J, Hz): 1.58 (br.s,
3H, CH3-24), 1.64 (m, 3H, all J < 1.5, CH3-23), 1.74 (m, 3H, all J < 1.5, CH3-25), 2.03-2.15 (m, 4H, 2H-19,
2H-20), 4.60 (d, 2H, J16,17 = 6.6, 2H-16), 5.06 (tm, 1H, J21,20 = 6.8, other J < 1.5, H-21), 5.45 (tm, 1H, J17,16
= 6.6, other J < 1.5, H-17), 6.18 (s, 1H, H-3), 6.77 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.87 (d, 1H, J9,7 = 2.5,
H-9), 7.34 (d, 1H, J6,7 = 8.9, H-6), 7.39-7.43 (m, 2H, H-11, H-15), 7.46-7.51 (m, 3H, H-12, H-13, H-14). 13C
NMR (δ ppm, CDCl3): 155.85 (s, C-1), 161.12 (s, C-2), 111.61 (d, C-3), 155.70 (s, C-4), 112.28 (s, C-5),
127.75 (d, C-6), 112.81 (d, C-7), 161.99 (s, C-8), 101.75 (d, C-9), 135.52 (s, C-10), 128.23 (d, C-11, C-15),
128.66 (d, C-12, C-14), 129.40 (d, C-13), 65.38 (t, C-16), 118.34 (d, C-17), 142.19 (s, C-18), 39.37 (t, C-19),
26.11 (t, C-20), 123.49 (d, C-21), 131.78 (s, C-22), 25.50 (k, C-23), 17.56 (k, C-24), 16.63 (k, C-25).
(E)-7-(3,7-Dimethylocta-2,6-dienyloxy)-4-(4-fluorophenyl)-2H-chromen-2-one 3ba. Yield 35%, method
1
a
. M.p. 72 ◦C. HRMS: 392.1778 [M]+; calcd. 392.1782 (C25H25FO3)+. H NMR (CDCl3,
δ
ppm, J, Hz):
1.58 (s, 3H, CH3-24), 1.64 (s, 3H, CH3-23), 1.74 (s, 3H, CH3-25), 2.02-2.16 (m, 4H, 2H-19, 2H-20), 4.60 (d,
2H, J16,17 = 6.5, 2H-16), 5.06 (tm, 1H, J21,20 = 6.8, other J < 2, H-21), 5.45 (tm, 1H, J17,16 = 6.5, other J < 2,
H-17), 6.16 (s, 1H, H-3), 6.78 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.87 (d, 1H, J9,7 = 2.5, H-9), 7.19 (dd, 2H,
J12,11
=
J
14,15 = 8.7, J12(14),F = 8.7, H-12, H-14), 7.30 (d, 1H, J6,7 = 8.9, H-6), 7.41 (dd, 2H, J11,12 =J15,14
=
8.7, J11(15),F = 5.3, H-11, H-15). 13C NMR (
δ
ppm, CDCl3, C,F, Hz): 155.86 (s, C-1), 160.98 (s, C-2), 111.77
J
(d, C-3), 154.66 (s, C-4), 112.17 (s, C-5), 127.51 (d, C-6), 112.95 (d, C-7), 162.12 (d, C-8), 101.84 (d, C-9),
131.52 (s, 4J = 3.4, C-10), 130.18 (d, 3J = 8.3, C-11, C-15), 115.89 (d, 2J = 21.4, C-12, C-14), 163.35 (s, 1J =
250.0, C-13), 65.43 (t, C-16), 118.29 (d, C-17), 142.30 (s, C-18), 39.39 (t, C-19), 26.13 (t, C-20), 123.49 (d,
C-21), 131.82 (s, C-22), 25.51 (k, C-23), 17.57 (k, C-24), 16.64 (k, C-25).
(E)-4-(4-Bromophenyl)-7-(3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one 3ca. Yield 40%, method
1
a
. M.p. 80 ◦C. HRMS: 452.0979 [M]+; calcd. 452.0982 (C25H25BrO3)+. H NMR (CDCl3,
δ
ppm, J, Hz):
1.58 (br.s, 3H, CH3-24), 1.64 (m, 3H, all J < 1.5, CH3-23), 1.74 (br.s, 3H, CH3-25), 2.03-2.14 (m, 4H, 2H-19,
2H-20), 4.59 (d, 2H, J16,17 = 6.6, 2H-16), 5.06 (tm, 1H, J21,20 = 6.7, other J 1.5, H-21), 5.44 (tm, 1H, J17,16
≤
= 6.6, other J < 1.5, H-17), 6.16 (s, 1H, H-3), 6.78 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.87 (d, 1H, J9,7 = 2.5,
H-9), 7.28 (d, 1H, J6,7 = 8.9, H-6), 7.29 (br.d, 2H, J11,12 =J15,14 = 8.5, H-11, H-15), 7.63 (br.d, 2H, J12,11
= J14,15 = 8.5, H-12, H-14). 13C NMR (
δ ppm, CDCl3): 155.82 (s, C-1), 160.92 (s, C-2), 111.65 (d, C-3),
154.52 (s, C-4), 111.83 (s, C-5), 127.43 (d, C-6), 112.99 (d, C-7), 162.13 (c, C-8), 101.78 (d, C-9), 134.31 (s,
C-10), 129.84 (d, C-11, C-15), 131.98 (d, C-12, C-14), 123.87 (s, C-13), 65.39 (t, C-16), 118.16 (d, C-17),
142.38 (s, C-18), 39.37 (t, C-19), 26.08 (t, C-20), 123.45 (d, C-21), 131.84 (s, C-22), 25.54 (k, C-23), 17.59 (k,
C-24), 16.65 (k, C-25).
(E)-7-(3,7-Dimethylocta-2,6-dienyloxy)-4-(4-methoxyphenyl)-2H-chromen-2-one 3da. Yield 29%,
1
method
b
. HRMS: 404.1980 [M]+; calcd. 404.1982 (C26H28O4)+. H NMR (CDCl3,δ ppm, J, Hz): 1.58 (s,
3H, CH3-24), 1.64 (m, 3H, all J < 2.0, CH3-23), 1.74 (m, 3H, all J < 1.5, CH3-25), 2.03-2.15 (m, 4H, 2H-19,
2H-20), 3.86 (s, 3H, CH3-26), 4.59 (d, 2H, J16,17 = 6.6, 2H-16), 5.06 (tm, 1H, J21,20 = 6.8, other J < 1.5,
H-21), 5.45 (tm, 1H, J17,16 = 6.6, other J < 1.5, H-17), 6.16 (s, 1H, H-3), 6.78 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5,
H-7), 6.86 (d, 1H, J9,7 = 2.5, H-9), 7.00 (br.d, 2H, J12,11 = J14,15 = 8.7, H-12, H-14), 7.37 (br.d, 2H, J11,12
=J15,14 = 8.7, H-11, H-15), 7.41 (d, 1H, J6,7 = 8.9, H-6). 13C NMR (
δ ppm, CDCl3): 155.85 (s, C-1), 161.34
(s, C-2), 111.08 (d, C-3), 155.40 (s, C-4), 112.39 (s, C-5), 127.78 (d, C-6), 112.74 (d, C-7), 161.87 (s, C-8),
101.70 (d, C-9), 127.74 (s, C-10), 129.73 (d, C-11, C-15), 114.12 (d, C-12, C-14), 160.60 (s, C-13), 65.34 (t,
C-16), 118.31 (d, C-17), 142.21 (s, C-18), 39.38 (t, C-19), 26.10 (t, C-20), 123.48 (d, C-21), 131.80 (s, C-22),
25.52 (k, C-23), 17.57 (k, C-24), 16.63 (k, C-25), 55.28 (k, C-26).

Int. J. Mol. Sci. 2020, 21, 126
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7-(2-((1R,5S)-6,6-Dimethylbicyclo[3 1.1]hept-2-en-2-yl)ethoxy)-4-phenyl-2H-chromen-2-one 3ab. Yield
22
12%, method
b
. HRMS: 386.1872 [M]+; calcd. 386.1877 (C26H26O3)+. [α]
= 20.0 (c = 0.53, CHCl3).
−
589
1H NMR (CDCl3,
δ
ppm, J, Hz): 0.81 (c, 3H, CH3-26), 1.16 (d, 1H, J24a,24s = 8.6, H-24a), 1.26 (s, 3H,
0
CH3-25), 2.05-2.11 (m, 2H, H-21, H-23), 2.20 (dm, 1H, J20,20 = 17.6, other J < 3.5, H-20), 2.26 (dm, 1H,
J20 ,20 = 17.6, other J < 3.5, H-200), 2.37 (ddd, 1H, J24s,24a = 8.6, J24s,21 = J24s,23 = 5.6, H-24s), 2.40-2.52 (m,
0
2H, 2H-17), 3.98-4.06 (m, 2H, 2H-16), 5.32-5.36 (m, 1H, H-19), 6.18 (s, 1H, H-3), 6.74 (dd, 1H, J7,6 = 8.9,
J
7,9 = 2.5, H-7), 6.85 (d, 1H, J9,7 = 2.5, H-9), 7.34 (d, 1H, J6,7 = 8.9, H-6), 7.39-7.44 (m, 2H, H-11, H-15),
7.46-7.51 (m, 3H, H-12, H-13, H-14). 13C NMR (
δ
ppm, CDCl3): 155.91 (s, C-1), 161.11 (s, C-2), 111.63 (d,
C-3), 155.71 (s, C-4), 112.30 (s, C-5), 127.80 (d, C-6), 112.63 (d, C-7), 162.07 (s, C-8), 101.50 (d, C-9), 135.54
(s, C-10), 128.25 (d, C-11, C-15), 128.68 (d, C-12, C-14), 129.42 (d, C-13), 66.90 (t, C-16), 36.07 (t, C-17),
143.89 (s, C-18), 118.99 (d, C-19), 31.27 (t, C-20), 40.64 (d, C-21), 37.97 (c, C-22), 45.79 (d, C-23), 31.54 (t,
C-24), 26.18 (k, C-25), 21.08 (k, C-26).
7-(2-((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethoxy)-4-(4-fluorophenyl)-2H-chromen-2-one
22
3bb. Yield 37%, method
0.78, CHCl3). H NMR (CDCl3,
b
. HRMS: 404.1774 [M]+; calcd. 404.1782 (C26H25FO3)+. [α]
= 17.4 (c =
−
589
1
δ
ppm, J, Hz): 0.81 (s, 3H, CH3-26), 1.16 (d, 1H, J24a,24s = 8.6, H-24a),
0
1.26 (s, 3H, CH3-25), 2.06-2.10 (m, 2H, H-21, H-23), 2.20 (dm, 1H, J20,20 = 17.7, H-20), 2.27 (dm, 1H,
J20 ,20 = 17.7, H-200), 2.36 (ddd, 1H, J24s,24a = 8.6, J24s,21 = J24s,23 = 5.6, H-24s), 2.40-2.51 (m, 2H, 2H-17),
0
4.02 (t, 2H, J16,17 = 7.0, 2H-16), 5.32-5.36 (m, 1H, H-19), 6.16 (s, 1H, H-3), 6.75 (dd, 1H, J7,6 = 8.9, J7,9
2.5, H-7), 6.84 (d, 1H, J9,7 = 2.5, H-9), 7.16-7.21 (m, 2H, J12,11 = J14,15 = 8.7, J12(14),F =8.7, H-12, H-14),
7.30 (d, 1H, J6,7 = 8.9, H-6), 7.38-7.43 (m, 2H, J11,12 = J15,14 = 8.7, J11(15),F = 5.3, H-11, H-15). 13C NMR (
=
δ
ppm, CDCl3, JC,F, Hz): 155.90 (s, C-1), 160.94 (s, C-2), 111.76 (d, C-3), 154.64 (s, C-4), 112.16 (s, C-5),
127.54 (d, C-6), 112.74 (d, C-7), 162.18 (s, C-8), 101.57 (d, C-9), 131.51 (s, 4J = 3.5, C-10), 130.18 (d, 3J =
8.3, C-11, C-15), 115.88 (d, 2J = 21.8, C-12, C-14), 66.93 (t, C-16), 36.05 (t, C-17), 143.86 (s, C-18), 119.02
(d, C-19), 31.27 (t, C-20), 40.64 (d, C-21), 37.97 (s, C-22), 45.79 (d, C-23), 31.54 (t, C-24), 26.17 (k, C-25),
21.08 (k, C-26).
4-(4-Bromophenyl)-7-(2-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethoxy)-2H-chromen-2-one
26.6
3cb. Yield 15%, method
b.
[α]
=
17.9 (c = 0.58, CHCl3). HRMS: 464.0988 [M]+; calcd. 450.0982
−
589
(C26H25BrO3)+. H NMR (CDCl3,δ ppm, J, Hz): 0.80 (s, 3H, CH3-26), 1.15 (d, 1H, J24a,24s = 8.6, H-24a),
1
0
1.25 (s, 3H, CH3-25), 2.06-2.09 (m, 2H, H-21, H-23), 2.19 (dm, 1H, J20,20 = 17.6, H-20), 2.26 (dm, 1H,
J20 ,20 = 17.6, H-200), 2.36 (ddd, 1H, J24s,24a = 8.6, J24s,21 = J24s,23 = 5.6, H-24s), 2.40-2.51 (m, 2H, 2H-17),
0
3.98-4.04 (m, 2H, 2H-16), 5.33-5.36 (m, 1H, H-19), 6.16 (s, 1H, H-3), 6.75 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5,
H-7), 6.84 (d, 1H, J9,7 = 2.5, H-9), 7.28 (d, 1H, J6,7 = 8.9, H-6), 7.28-7.31 (m, 2H, J11,12 = J15,14 = 8.4, H-11,
H-15), 7.61-7.64 (m, 2H, J12,11 = J14,15 = 8.4, H-12, H-14). 13C NMR (
δ ppm, CDCl3): 155.86 (s, C-1),
160.87 (s, C-2), 111.65 (d, C-3), 154.49 (s, C-4), 111.81 (s, C-5), 127.44 (d, C-6), 112.79 (d, C-7), 162.19 (s,
C-8), 101.50 (d, C-9), 134.30 (s, C-10), 129.84 (d, C-11, C-15), 131.97 (d, C-12, C-14), 123.86 (s, C-13), 66.87
(t, C-16), 36.01 (t, C-17), 143.78 (s, C-18), 119.03 (d, C-19), 31.23 (t, C-20), 40.54 (d, C-21), 37.95 (s, C-22),
45.65 (d, C-23), 31.51 (t, C-24), 26.14 (k, C-25), 21.08 (k, C-26).
7-(2-((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethoxy)-4-(4-methoxyphenyl)-2H-chromen-2-one
3db. Yield 65%, method b. HRMS: 415.1906 [M-H]+; calcd. 415.1904 (C27H27O4)+. [α]52829 = −15.7 (c =
1
0.75, CHCl3). H NMR (CDCl3,
δ
ppm, J, Hz): 0.81 (s, 3H, CH3-27), 1.16 (d, 1H, J25a,25s = 8.6, H-25a),
1.26 (s, 3H, CH3-26), 2.05-2.11 (m, 2H, H-21, H-24), 2.19 (dm, 1H, 2J = 17.6, H-21), 2.26 (dm, 1H, 2J =
17.6, H-210), 2.36 (ddd, 1H, J25s,25a = 8.6, J25s,22 = J25s,24 = 5.6, H-25s), 2.40-2.51 (m, 2H, 2H-18), 3.86 (s,
3H, CH3-16), 4.02 (t, 2H, J17,18 = 7.0, H-17), 5.32-5.36 (m, 1H, H-20), 6.15 (s, 1H, H-3), 6.75 (dd, 1H, J7,6
8.9, J7,9 = 2.5, H-7), 6.83 (d, 1H, J9,7 = 2.5, H-9), 7.01 (br.d, 2H, J12,11 = J14,15 = 8.7, H-12, H-14), 7.37 (br.d,
2H, J11,12 = J15,14 = 8.7, H-11, H-15), 7.40 (d, 1H, J6,7 = 8.9, H-6). 13C NMR (
ppm, CDCl3): 155.93 (s,
=
δ
C-1), 161.28 (s, C-2), 111.12 (d, C-3), 155.38 (s, C-4), 112.43 (s, C-5), 127.81 (d, C-6), 112.54 (d, C-7), 161.97
(s, C-8), 101.49 (d, C-9), 127.79 (s, C-10), 129.74 (d, C-11, C-15), 114.16 (d, C-12, C-14), 160.64 (s, C-13),
55.30 (k, C-16), 66.88 (t, C-17), 36.07 (t, C-18), 143.90 (s, C-19), 118.97 (d, C-20), 31.27 (t, C-21), 40.64 (d,
C-22), 37.97 (s, C-23), 45.79 (d, C-24), 31.54 (t, C-25), 26.17 (k, C-26), 21.08 (k, C-27).

Int. J. Mol. Sci. 2020, 21, 126
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7-(((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-4-phenyl-2H-chromen-2-one 3ac. Yield
27.3
40%, method
a
. M.p. 106 ◦C. HRMS: 372.1717 [M]+; calcd. 372.1720 (C25H24O3)+. [α]
= 25.33 (c =
−
589
1.02, EtOH). 1H NMR (CDCl3,
δ
ppm, J, Hz): 0.82 (s, 3H, CH3-25), 1.18 (d, 1H, 2J = 8.7, H-23a), 1.28 (s,
3H, CH3-24), 2.08-2.14 (m, 1H, H-20), 2.20 (ddd, 1H, J22,20 = J22,23s = 5.6, J22,18 = 1.4, H-22), 2.26 (dm,
1H, 2J = 18.1, H-19), 2.33 (dm, 1H, 2J = 18.1, H-190), 2.40 (ddd, 1H, 2J = 8.7, J23s,20= J23s,22 = 5.6, H-23s),
4.44 (dm, 1H, 2J = 12.4, other J
≤
2.0, H-16), 4.47 (dm, 1H, 2J = 12.4, other J
≤
2.0, H-160), 5.61-5.64 (m,
1H, H-18), 6.19 (s, 1H, H-3), 6.77 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.89 (d, 1H, J9,7 = 2.5, H-9), 7.33 (d,
1H, J6,7 = 8.9, H-6), 7.40-7.43 (m, 2H, H-11, H-15), 7.47-7.51 (m, 3H, H-12, H-13, H-14). 13C NMR (
δ
ppm, CDCl3): 155.81 (s, C-1), 161.24 (s, C-2), 111.65 (d, C-3), 155.76 (s, C-4), 112.32 (s, C-5), 127.68 (d,
C-6), 112.94 (d, C-7), 162.14 (s, C-8), 102.00 (d, C-9), 135.55 (s, C-10), 128.27 (d, C-11, C-15), 128.68 (d,
C-12, C-14), 129.42 (d, C-13), 71.12 (t, C-16), 142.92 (s, C-17), 121.34 (d, C-18), 31.18 (t, C-19), 40.71 (d,
C-20), 38.00 (s, C-21), 43.11 (d, C-22), 31.40 (t, C-23), 26.02 (k, C-24), 20.97 (k, C-25).
7-(((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-4-(4-fluorophenyl)-2H-chromen-2-one
22.0
3bc. Yield 53%, method
b.
[α]
=
15.6 (c = 0.68, CHCl3). HRMS: 390.1624 [M]+; calcd. 390.1626
ppm, J, Hz): 0.81 (s, 3H, CH3-25), 1.17 (d, 1H, J23a,23s = 8.7, H-23a),
−
589
(C25H23FO3)+. H NMR (CDCl3,
δ
1
1.28 (s, 3H, CH3-24), 2.11 (ddtd, 1H, J20,22 = J20,23s = 5.6, J20,19 = 2.9, J20,18 = 1.3, H-20), 2.20 (ddd, 1H,
,
J22,20 = J22,23s = 5.6, J22,18 = 1.4, H-22), 2.26 (dm, 1H, J19,19 = 18.0, other J < 3.5, H-19), 2.33 (dm, 1H,
J19 ,19 = 18.0, other J < 3.5, H-190), 2.40 (ddd, 1H, J23s,23a = 8.7, J23s,20= J23s,22 = 5.6, H-23s), 4.41-4.49 (m,
0
2H, 2H-16), 5.61-5.64 (m, 1H, H-18), 6.16 (s, 1H, H-3), 6.78 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.88 (d, 1H,
J9,7 = 2.5, H-9), 7.16-7.21 (m, 2H, J12,11 = J14,15 = 8.8, J12(14),F = 8.6, H-12, H-14), 7.29 (d, 1H, J6,7 = 8.9,
H-6), 7.38-7.43 (m, 2H, J11,12 = J15,14 = 8.8, J11(15),F = 5.3, H-11, H-15). 13C NMR (
δ ppm, CDCl3, JC,F,
Hz): 155.80 (s, C-1), 161.02 (s, C-2), 111.78 (d, C-3), 154.66 (s, C-4), 112.18 (s, C-5), 127.42 (d, C-6), 113.03
(d, C-7), 162.23 (s, C-8), 102.07 (d, C-9), 131.52 (s, 4J = 3.5, C-10), 130.19 (d, 3J = 8.3, C-11, C-15), 115.88
(d, 2J = 21.8, C-12, C-14), 71.14 (t, C-16), 142.86 (s, C-17), 121.39 (d, C-18), 31.18 (t, C-19), 40.69 (d, C-20),
38.00 (s, C-21), 43.09 (d, C-22), 31.39 (t, C-23), 26.00 (k, C-24), 20.97 (k, C-25).
4-(4-Bromophenyl)-7-(((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-2H-chromen-2-one 3cc
.
26.4
Yield 36%, method
13.9 (c = 0.52, CHCl3). H NMR (CDCl3,
8.7, H-23a), 1.28 (s, 3H, CH3-24), 2.08-2.12 (m,1H, H-20), 2.19 (ddd, 1H, J22,20 = J22,23s = 5.6, J22,18
1.3, H-22), 2.25 (dm, 1H, J19,19 = 18.0, other J < 3.5, H-19), 2.33 (dm, 1H, J19 ,19 = 18.0, other J < 3.5,
H-190), 2.40 (ddd, 1H, J23s,23a = 8.7, J23s,20= J23s,22 = 5.6, H-23s), 4.43-4.48 (m, 2H, 2H-16), 5.60-5.63 (m,
1H, H-18), 6.16 (s, 1H, H-3), 6.78 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.88 (d, 1H, J9,7 = 2.5, H-9), 7.27 (d,
1H, J6,7 = 8.9, H-6), 7.30 (d, 2H, J11,12 = J15,14 = 8.5, H-11, H-15), 7.63 (d, 2H, J12,11 = J14,15 = 8.5, H-12,
a
. M.p. 130 ◦C. HRMS: 450.0826 [M]+; calcd. 450.0825 (C25H23O3Br)+. [α]
=
589
1
−
δ
ppm, J, Hz): 0.80 (s, 3H, CH3-25), 1.16 (d, 1H, J23a,23s
=
=
,
,
H-14). 13C NMR (
δ ppm, CDCl3): 155.76 (s, C-1), 160.97 (s, C-2), 111.67 (d, C-3), 154.53 (s, C-4), 111.85
(s, C-5), 127.33 (d, C-6), 113.10 (d, C-7), 162.25 (s, C-8), 102.02 (d, C-9), 134.32 (s, C-10), 129.85 (d, C-11,
C-15), 131.98 (d, C-12, C-14), 123.87 (s, C-13), 71.12 (t, C-16), 142.77 (s, C-17), 121.43 (d, C-18), 31.15 (t,
C-19), 40.60 (d, C-20), 37.99 (s, C-21), 43.00 (d, C-22), 31.37 (t, C-23), 25.98 (k, C-24), 20.96 (k, C-25).
7-(((1R,5S)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-4-(4-methoxyphenyl)-2H-chromen-2-one
30.6
3dc. Yield 55%, method
b
. M.p. 124 ◦C. [α]
=
17.3 (c = 0.82, CHCl3). HRMS: 402.1820 [M]+; calcd.
−
589
402.1827 (C26H26O4)+. H NMR (CDCl3,
δ ppm, J, Hz): 0.81 (s, 3H, CH3-26), 1.17 (d, 1H, J24a,24s = 8.7,
1
H-24a), 1.28 (s, 3H, CH3-25), 2.10 (ddtd, 1H, J21,23 = J21,24s = 5.6, J21,20 = 2.8, J21,19 = 1.3, H-21), 2.20
(ddd, 1H, J23,21 = J23,24s = 5.6, J23,19 = 1.4, H-23), 2.25 (dm, 1H, J20,20, = 18.0, other J < 3.0, H-20), 2.33
(dm, 1H, J20 ,20 = 18.0, other J < 3.5, H-200), 2.40 (ddd, 1H, J24s,24a = 8.7, J24s,21= J24s,23 = 5.6, H-24s), 3.85
0
(s, 3H, CH3-16), 4.41-4.49 (m, 2H, 2H-17), 5.60-5.63 (m, 1H, H-19), 6.14 (s, 1H, H-3), 6.77 (dd, 1H, J7,6
8.9, J7,9 = 2.5, H-7), 6.86 (d, 1H, J9,7 = 2.5, H-9), 7.00 (br.d, 2H, J12,11 = J14,15 = 8.8, H-12, H-14), 7.36 (br.d,
2H, J11,12 = J15,14 = 8.8, H-11, H-15), 7.39 (d, 1H, J6,7 = 8.9, H-6). 13C NMR (
ppm, CDCl3): 155.80 (s,
=
δ
C-1), 161.30 (s, C-2), 111.11 (d, C-3), 155.36 (s, C-4), 112.41 (s, C-5), 127.67 (d, C-6), 112.79 (d, C-7), 161.99
(s, C-8), 101.96 (d, C-9), 127.77 (s, C-10), 129.72 (d, C-11, C-15), 114.13 (d, C-12, C-14), 160.62 (s, C-13),
55.27 (k, C-16), 71.06 (t, C-17), 142.91 (s, C-18), 121.26 (d, C-19), 31.15 (t, C-20), 40.67 (d, C-21), 37.97 (s,
C-22), 43.07 (d, C-23), 31.37 (t, C-24), 25.99 (k, C-25), 20.94 (k, C-26).

Int. J. Mol. Sci. 2020, 21, 126
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7-(((1S,5R)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-4-phenyl-2H-chromen-2-one 3ad. Yield
26.6
46%, method b.
[α] = +23.33 (c=1.02, EtOH). HRMS: 372.1718 [M]+; calcd. 372.1720 (C25H24O3)+.
589
The 1H and 13C NMR spectra of 3ad correspond to the spectra of the enantiomer 3ac.
7-(((1S,5R)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-4-(4-fluorophenyl)-2H-chromen-2-one
22.0
3bd. Yield 35%, method
a
. M.p. 129 ◦C. HRMS: 390.1628 [M]+; calcd. 390.1626 (C25H23FO3)+. [α]
589
= +22.3 (CHCl3, c = 0.53). The H and 13C NMR spectra of 3bd correspond to the spectra of the
1
enantiomer 3bc.
4-(4-Bromophenyl)-7-(((1S,5R)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-2H-chromen-2-one
26.7
3cd. Yield 39%, method a. M.p. 138 ◦C. HRMS: 450.0825 [M]+; calcd. 450.0820 (C25H23BrO3)+. [α]
589
= +22.4 (c = 0.58, CHCl3). The H and 13C NMR spectra of 3cd correspond to the spectra of the
1
enantiomer 3cc.
7-(((1S,5R)-6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl)methoxy)-4-(4-methoxyphenyl)-2H-chromen-2-one
22.0
3dd. Yield 33%, method
a
. M.p. 112 ◦C. HRMS: 402.1823 [M]+; calcd. 402.1827 (C26H26O4)+. [α]
589
= +20.8 (c = 0.72, CHCl3). The H and 13C NMR spectra of 3dd correspond to the spectra of the
1
enantiomer 3dc.
7-(Benzyloxy)-4-phenyl-2H-chromen-2-one 10a. Yield 42%, method
b
. M.p. 92 ◦C. HRMS: 328.1093
[M]+; calcd. 328.1094 (C22H16O3)+. H NMR (CDCl3,δ ppm, J, Hz): 5.13 (s, 2H, 2H-16), 6.19 (s, 1H, H-3),
1
6.85 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.94 (d, 1H, J9,7 = 2.5, H-9), 7.31-7.35 (m, 1H, H-20), 7.35–7.44 (m,
7H, H-6, H-11, H-15, H-18, H-19, H-21, H-22), 7.46-7.52 (m, 3H, H-12, H-13, H-14). 13C NMR (
δ ppm,
CDCl3): 155.80 (s, C-1), 161.00 (s, C-2), 111.86 (d, C-3), 155.62 (s, C-4), 112.62 (s, C-5), 127.88 (d, C-6),
112.80 (d, C-7), 161.71 (s, C-8), 102.09 (d, C-9), 135.44 (s, C-10), 128.23 (d, C-11, C-15), 128.68 (d, C-12,
C-14), 129.44 (d, C-13), 70.39 (t, C-16), 135.67 (s, C-17), 127.35 (d, C-18, C-22), 128.62 (d, C-19, C-21),
128.23 (d, C-20).
7-(Benzyloxy)-4-(4-bromophenyl)-2H-chromen-2-one 10c. Yield 25%, method
a
. M.p. 128 ◦C. HRMS:
1
406.0204 [M]+; calcd. 406.0199 (C22H15BrO3)+. H NMR (CDCl3,δ ppm, J, Hz): 5.13 (s, 2H, 2H-16), 6.17
(s, 1H, H-3), 6.86 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.94 (d, 1H, J9,7 = 2.5, H-9), 7.29 (dm, 2H, J11,12
J15,14 = 8.5, H-11, H-15), 7.31 (d, 1H, J6,7 = 8.9, H-6), 7.32-7.35 (m, 1H, H-20), 7.37-7.43 (m, 4H, H-18,
H-19, H-21, H-22), 7.63 (dm, 2H, J12,11 = J14,15 = 8.5, H-12, H-14). 13C NMR (
ppm, CDCl3): 155.79 (s,
=
δ
C-1), 160.81 (s, C-2), 111.92 (d, C-3), 154.46 (s, C-4), 112.18 (s, C-5), 127.56 (d, C-6), 113.02 (d, C-7), 161.86
(s, C-8), 102.14 (d, C-9), 134.24 (s, C-10), 129.83 (d, C-11, C-15), 132.01 (d, C-12, C-14), 123.92 (s, C-13),
70.42 (t, C-16), 135.54 (s, C-17), 127.38 (d, C-18, C-22), 128.66 (d, C-19, C-21), 128.30 (d, C-20).
◦
7-(Benzyloxy)-4-(4-methoxyphenyl)-2H-chromen-2-one 10d. Yield 34%, method
HRMS: 358.1198 [M]+; calcd. 358.1200 (C23H18O4)+. 1H NMR (CDCl3,
CH3-16), 5.12 (s, 2H, 2H-17), 6.16 (s, 1H, H-3), 6.85 (dd, 1H, J7,6 = 8.9, J7,9 = 2.5, H-7), 6.93 (d, 1H, J9,7
2.5, H-9), 7.01 (dm, 2H, J12,11 = J14,15 = 8.7, H-12, H-14), 7.30-7.35 (m, 1H, H-21), 7.35–7.45 (m, 7H, H-6,
H-11, H-15, H-19, H-20, H-22, H-23). 13C NMR (
ppm, CDCl3): 155.82 (s, C-1), 161.18 (s, C-2), 111.34
b
. M.p. 143 C.
δ
ppm, J, Hz): 3.86 (s, 3H,
=
δ
(d, C-3), 155.30 (s, C-4), 112.75 (s, C-5), 127.90 (d, C-6), 112.72 (d, C-7), 161.61 (s, C-8), 102.07 (d, C-9),
127.68 (s, C-10), 129.72 (d, C-11, C-15), 114.16 (d, C-12, C-14), 160.65 (s, C-13), 55.29 (k, C-16), 70.37 (t,
C-17), 135.70 (s, C-18), 127.36 (d, C-19, C-23), 128.61 (d, C-20, C-22), 128.22 (d, C-21).
3.2. Biology Section
Real-Time Detection of Tdp1 Activity. The Tdp1 activity measurements were carried out as
described [64]. Briefly, Tdp1-biosensor with a final concentration of 50 nM was incubated in a
volume of 200 µL containing buffer (50 mM Tris-HCl pH8.0, 50 mM NaCl, 7 mM β-mercaptoethanol)
supplemented with purified 1.3 nM Tdp1. The reactions were incubated in a POLARstar OPTIMA
fluorimeter, BMG LABTECH, GmbH, to measure fluorescence every 1 min (Ex485/Em520 nm). Tdp1
inhibition was calculated by comparing the rate of increase in fluorescence in the presence of the
compound to that of DMSO control wells. IC50 values were determined using a 6-point concentration
response curve. The data were imported into the MARS Data Analysis 2.0 program (BMG LABTECH),
and the slope during the linear phase (here data from 0 to 7 min) was calculated.

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Cell Culture Assays. Tumor cells from human mammary adenocarcinoma cell line MCF-7 and
cervical cancer cell line HeLa (~2000 cells per well) were incubated for 24 h at 37 ◦C in IMDM medium
(5% CO2), and then treated with the synthesized derivatives. After 72 h of cell incubation, the
relative amount of alive cells was determined using standard colorimetric MTT test [74] or EZ4U Cell
Proliferation and Cytotoxicity Assay (Biomedica, Austria), as per the manufacturer’s protocols.
Binding Assay. Synthetic DNA encoding human Tdp1 (residues 149-608) was cloned into pET-28a
(+) (GenScript), which was then transformed into Escherichia coli BL21 (DE3) for recombinant protein
production. Protein production was induced with 1 mM IPTG at 28 ◦C with overnight incubation.
Purification of Tdp1 was performed using affinity and size exclusion chromatography. Intrinsic protein
fluorescence was measured using PerkinElmer EnSpire Multimode Reader. The Tdp1 concentration
was 10
M. The buffer was composed of 20 mM Tris and 250 mM NaCl, pH 8. The excitation wavelength was
280 nm and the intrinsic fluorescence was measured at 350 nm. Compound control was performed
using the buffer and compound only. The total volume per well was 30 L. Dissociation constants (KD)
µM, and the compound concentrations were 25 µM, 50 µM, 75 µM, 100 µM, 150 µM, and 250
µ
µ
were calculated using the following formula, that takes nonspecific binding into account.
Imax × [LT]
I =
+ Ns[LT]
KD + [LT]
In this formula, I indicates changes in fluorescence intensity from the titration, Imax indicates
the maximum fluorescence intensity change, [ T] is the titration ligand concentration, and Ns is the
L
non-specific term. Non-linear curve fitting was conducted using SigmaPlot 13.0 (Systat Software, San
Jose, CA, USA). Experiments were conducted in triplicate and the errors shown are standard derivations.
Lab animals. Thre-e to four-month-old male and female C57Bl/6 mice from the breeding colony of the
Institute of Cytology and Genetics, SB RAS, were used in the study. The animals were kept on sawdust
in plastic cages with 5–7 mice per cage, with free access to ground food (“Laboratorkorm”, Moskow,
Russia) and tap water. All experiments were performed in accordance with protocols approved by the
Animal Care and Use Committee of the Institute of Cytology and Genetics. Also, all experimental
procedures were performed in accordance with the Directive 2010/63/EU for animal experiments.
Tumor models. The experimental tumor used was Lewis Lung Carcinoma (LLC) and Krebs-2. The
animals were treated with tpc and the Tdp1 inhibitor 3ba two days after tumor transplantation. The
tumor was transplanted into the muscles of the thigh by 0.2 million cells per mouse. Tpc (Sindan
Pharma SRL, Romania) was administered intraperitoneally at a single dose of 0.5 mg/kg; Tdp1 inhibitor
3ba was administrated intraperitoneally at a single dose of 20, 40, or 80 mg/kg (for Krebs-2), or 120
mg/kg (for LLC) in 15% dimethyl sulfoxide (DMSO)–10% Tween-80 suspension in water (0.2 mL of
suspension per mouse) simultaneously with tpc. Control mice were injected with a DMSO-Tween-80
mixture into the stomach.
The antitumor effect was assessed by the size and weight of the solid tumors at 18 days after
transplantation. For estimations of daily gain in volume, the tumor nodules were periodically measured
with a caliper.
Statistical analysis. The experimenter measuring and calculating the primary animal data (tumor
size, lifespan) was blinded. After unblinding, the animal data were statistically processed using oneway
ANOVA. Post-hoc testing was completed using Turkey’s Honestly Significant Difference (HSD). p <
0.05 was considered to be statistically significant. The statistical package STATISTICA version 12.5 was
used for analysis. All results are expressed as mean ± SEM.
3.3. Modeling Section
Molecular modeling and chemical space. The compounds were docked against the crystal structure
of Tdp1 (PDB ID: 6DIE, resolution 1.78 Å) [75], which was obtained from the Protein Data Bank
(PDB) [76
,77]. The Scigress version FJ 2.6 program [78] was used to prepare the crystal structure for
docking, i.e., the hydrogen atoms were added, and the cocrystallized ligand benzene-1,2,4-tricarboxylic

Int. J. Mol. Sci. 2020, 21, 126
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acid was removed, as well as crystallographic water molecules, except HOH 814, 821, and 1078. The
Scigress software suite was also used to build the inhibitors, and the MM2 [77] force field was used
to optimize the structures. The docking centre was defined as the position of a carbon on the ring
of benzene-1, 2, 4-tricarboxylic acid (x =
docking runs were allowed for each ligand with the default search efficiency (100%). The basic amino
−
6.052, y = 14.428, z = 33.998) with 10 Å radius. Fifty
−
acids lysine and arginine were defined as protonated. Furthermore, aspartic and glutamic acids
were assumed to be deprotonated. The GoldScore(GS) [71] and ChemScore (CS) [69,70], ChemPLP
(Piecewise Linear Potential) [68], and ASP (AstexStatistical Potential) [67] scoring functions were
implemented to validate the predicted binding modes and relative energies of the ligands using the
GOLD v5.4.1 software suite (The Cambridge Crystallographic Data Centre, Cambridge, UK). The
QikProp 3.2 [79] software package (Schrödinger, New York, USA) was used to calculate the molecular
descriptors of the molecules; the reliability of this method has been established for the calculated
descriptors [80].
4. Conclusions
Overall, we reported the synthesis and evaluation of novel Tdp1 inhibitors that combine the
arylcoumarin and monoterpenoid moieties. Our results found that these compounds are good
Tdp1 inhibitors with IC50 in the submicromolar or low submicromolar ranges. Compound 3ba
showed a significant increase in the antitumor effect of tpc on Krebs-2 ascites in an in vivo tumor
model. In addition, these compounds presented the good physicochemical properties required
for oral bioavailability, making them good candidates for further development. Thus, this type of
arylcoumarin-monoterpenoid hybrids represents an excellent starting point for the further development
of adjuvant therapy against cancer in combination with Top 1 poisons.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/1/126/s1.
Author Contributions: Chemistry investigation, T.M.K., D.V.K. and K.P.V.; In vitro investigation, A.L.Z.; A.A.C.,
E.S.I., O.D.Z., J.P., I.K.H.L.; In vivo investigation, V.I.K., V.P.N., N.A.P., Modeling, J.R., R.C., D.M.A.-T.; Methodology,
N.F.S. and O.I.L.; Project administration, K.P.V.; Supervision, K.P.V.; Writing—original draft, A.L.Z. and T.M.K.;
Writing—review & editing, K.P.V., J.R., I.K.H.L., N.F.S., O.I.L. All authors have read and agreed to the published
version of the manuscript.
Funding: This study was funded by the Russian Science Foundation grant N◦ 19-13-00040. A.A. Chepanova is
grateful to Russian State funded budget project of ICBFM SB RAS No AAAA-A17-117020210022-4 for financial
support for Tdp1 purification.
Acknowledgments: Authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS for
spectral and analytical measurements.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
Abbreviations
Tdp1
Top1
CPTs
tpc
Tyrosyl-DNA phosphodiesterase 1
topoisomerase 1
camptothecin derivatives
topotecan
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