Design, synthesis, and molecular docking study of new tyrosyl-dna phosphodiesterase 1 (Tdp1) inhibitors combining resin acids and adamantane moieties

Article abstract of DOI:10.3390/ph14050422

In this paper, a series of novel abietyl and dehydroabietyl ureas, thioureas, amides, and thioamides bearing adamantane moieties were designed, synthesized, and evaluated for their in-hibitory activities against tyrosil-DNA-phosphodiesterase 1 (TDP1). The synthesized compounds were able to inhibit TDP1 at micromolar concentrations (0.19–2.3 μM) and demonstrated low cytotox-icity in the T98G glioma cell line. The effect of the terpene fragment, the linker structure, and the adamantane residue on the biological properties of the new compounds was investigated. Based on molecular docking results, we suppose that adamantane derivatives of resin acids bind to the TDP1 covalent intermediate, forming a hydrogen bond with Ser463 and hydrophobic contacts with the Phe259 and Trp590 residues and the oligonucleotide fragment of the substrate.

Full text of DOI:10.3390/ph14050422

pharmaceuticals
Article
Design, Synthesis, and Molecular Docking Study of New
Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Inhibitors
Combining Resin Acids and Adamantane Moieties
Kseniya Kovaleva 1 , Olga Yarovaya 1,2,
* ,
, Konstantin Ponomarev 1 , Sergey Cheresiz 2
Amirhossein Azimirad 2, Irina Chernyshova 2, Alexandra Zakharenko 3, Vasily Konev 4, Tatiana Khlebnikova 4,
Evgenii Mozhaytsev 1, Evgenii Suslov 1 , Dmitry Nilov 5 , Vytas Švedas 5,6 , Andrey Pokrovsky 2,
Olga Lavrik 2,3 and Nariman Salakhutdinov 1
1
N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, Lavrentiev Ave. 9, 630090 Novosibirsk, Russia; kseniya.kovaleva3103@yandex.ru (K.K.);
ponomarev@nioch.nsc.ru (K.P.); mozh@nioch.nsc.ru (E.M.); suslov@nioch.nsc.ru (E.S.);
anvar@nioch.nsc.ru (N.S.)
2
V. Zelman Institute for the Medicine and Psychology, Novosibirsk State University, Pirogova St. 1,
630090 Novosibirsk, Russia; cheresiz@yandex.ru (S.C.); azimirad@mail.ru (A.A.);
chernyshova0305@gmail.com (I.C.); agpok@inbox.ru (A.P.); lavrik@niboch.nsc.ru (O.L.)
Novosibirsk Institute of Chemical Biology and Fundamental Medicine,
Siberian Branch of the Russian Academy of Sciences, Lavrentiev Ave. 8, 630090 Novosibirsk, Russia;
a.zakharenko73@gmail.com
Boreskov Institute of Catalysis, Siberian Branch of the Russian Academy of Sciences, Lavrentiev Ave. 5,
630090 Novosibirsk, Russia; konvasnik@gmail.com (V.K.); khleb@catalysis.nsk.su (T.K.)
Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Lenin Hills 1, Bldg. 40,
119991 Moscow, Russia; nilovdm@gmail.com (D.N.); vytas@belozersky.msu.ru (V.Š.)
Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Lenin Hills 1, Bldg. 73,
119991 Moscow, Russia
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
3
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kovaleva, K.; Yarovaya, O.;
Ponomarev, K.; Cheresiz, S.;
4
5
6
Azimirad, A.; Chernyshova, I.;
Zakharenko, A.; Konev, V.;
Khlebnikova, T.; Mozhaytsev, E.; et al.
Design, Synthesis, and Molecular
Docking Study of New Tyrosyl-DNA
Phosphodiesterase 1 (TDP1)
*
Correspondence: ooo@nioch.nsc.ru
Inhibitors Combining Resin Acids
and Adamantane Moieties.
Abstract: In this paper, a series of novel abietyl and dehydroabietyl ureas, thioureas, amides, and
thioamides bearing adamantane moieties were designed, synthesized, and evaluated for their in-
hibitory activities against tyrosil-DNA-phosphodiesterase 1 (TDP1). The synthesized compounds
Pharmaceuticals 2021, 14, 422.
https://doi.org/10.3390/ph14050422
were able to inhibit TDP1 at micromolar concentrations (0.19–2.3 µM) and demonstrated low cytotox-
icity in the T98G glioma cell line. The effect of the terpene fragment, the linker structure, and the
adamantane residue on the biological properties of the new compounds was investigated. Based on
molecular docking results, we suppose that adamantane derivatives of resin acids bind to the TDP1
covalent intermediate, forming a hydrogen bond with Ser463 and hydrophobic contacts with the
Phe259 and Trp590 residues and the oligonucleotide fragment of the substrate.
Academic Editors: Mary Meegan and
Niamh M O’Boyle
Received: 26 March 2021
Accepted: 23 April 2021
Published: 1 May 2021
Keywords: tyrosil-DNA-phosphodiesterase 1; adamantane; resin acid; TDP1
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
DNA in living organisms is constantly exposed to a variety of physical and chem-
ical stresses, and damage occurs as a result. Bulk DNA damage is caused by UV light
and environmental mutagens, and X-rays cause DNA double-strand breaks. Defects in
the repair of DNA damage are implicated in a variety of diseases, many of which are
typified by neurological dysfunction and/or increased genetic instability and cancer [1].
Traditional cancer chemotherapy is aimed at damaging the DNA of malignant cells, and
the results depend on the effectiveness of their repair systems. Recently, compounds that
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
act as DNA repair inhibitors have been considered as potential drugs [
2
,3]. The enzyme
tyrosil-DNA-phosphodiesterase 1 (TDP1) is one of the promising ones [
4]. This enzyme
is an important supplementary target for anticancer therapies based on topoisomerase
Pharmaceuticals 2021, 14, 422. https://doi.org/10.3390/ph14050422
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 422
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inhibitors 1 (TOP1), since it plays a key role in the removal of TOP1-DNA adducts stabilized
by TOP1 inhibitors such as camptothecine [5] and its clinical derivatives [6]. TDP1 is also
capable of hydrolysing apurinic sites, and thus leading to their repair. This may be the
key activity needed for the repair of DNA damage caused by antitumour alkylating drugs
such as temozolomide (TMZ), as well as ionising radiation [7]. Thus, the inhibition of
TDP1 activity may significantly enhance the therapeutic effect of some anticancer agents.
SCAN1 is a natural mutant of TDP1 where His493 is replaced with Arg493 in the binding
pocket [8]. The mutation changes the geometry of the enzyme active site, and the enzyme
remains covalently bound to DNA. This mutation leads to a severe neurodegenerative
disease spinocerebellar ataxia syndrome with axonal neuropathy (SCAN1). It is currently
suggested that the pathology is caused by the accumulation of the SCAN1-DNA covalent
cleavage complexes [9]. It is assumed that nerve cells especially suffer from the accu-
mulation of such adducts due to their nonproliferative nature leading to the progressive
accumulation of unrepaired DNA lesions [10]. Therefore, suppression of SCAN1 activity
could potentially improve the SCAN1 patients’ condition and prevent the progression of
the disease. The search for inhibitors of key DNA repair enzymes is a promising area of
medical chemistry, as it represents one of the ways to design effective therapies for cancer,
as well as cardiovascular and neurodegenerative diseases. Recently, a number of TDP1
inhibitor structural classes have been studied, including pyrimidine nucleosides [11], fu-
ramidine [12], compounds with benzopentathiepine moiety [13], indenoisoquinolines [14],
and 5-arylidenethioxothiazolidinones [15] (Figure 1).
Figure 1. Structures of known TDP1 inhibitors.
Hybrid molecules created from different pharmacophores of natural and synthetic
equivalents are successfully used in pharmaceutical practice [16]. New hybrid compounds
have been synthesised starting from the pharmacophoric natural compounds with in-
hibitory properties against TDP1. These include phenolic usnic acid derivatives A [17],
7-hydroxycoumarins B [18], and 4-arylcoumarins C [19], derivatives of deoxycholic acid
D [20] and adamantanecarboxylic acid monoterpene esters E [21] (Figure 2).
Our group previously obtained a set of ureas and thioureas based on the natural
terpenoid dehydroabietylamine [22]. These compounds are able to inhibit TDP1 in the
submicromolar range. They also lack toxicity against different cell lines in concentra-
tions up to 100
inhibitors in combination with TMZ demonstrate a better cytotoxic effect on glioblas-
toma cells than TMZ alone, taken at the same concentration. Compound (Figure 3),
µM. For the first time, we have shown that dehydroabietylamine TDP1
1
which has a fragment of resin acid and adamantane, was an efficient inhibitor of TDP1
activity in vitro, and enhanced the cytotoxic effect of TMZ on glioblastoma cells. We syn-
thesised a series of dehydroabietylamine derivatives containing the heterocyclic fragment
2-thioxoimidazolidin-4-ones and studied their activity against TDP1 [23]. It is important
to note that not all of the synthesized heterocyclic derivatives are suitable for studying
inhibitory activity, as some of the substances proved to be extremely insoluble. In addition,

Pharmaceuticals 2021, 14, 422
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the combination of an adamantane fragment with terpenes of various structures has been
shown to be successful in identifying new inhibitors of the TDP1 repair enzyme [23
Since the combination of a terpene resin acid backbone with an adamantane fragment in
compound proved to be most successful and the compound had the most important bio-
logical properties, we set out in the present study to synthesise analogues of compound to
–27].
1
1
examine their structural activity. The design of the target derivatives is shown in Figure 3.
It includes the variations of the linker type and length, diterpene, and adamantane moieties.
Understanding which structural blocks are most important for the target biological activity
and whether more active agents can be obtained by available synthetic methods is the main
goal of the presented work.
Figure 2. Structures of natural-based TDP1 inhibitors and their IC50 values.
Figure 3. Design strategy for new abietylamine-based compounds.
2. Results and Discussion
2.1. Chemistry
Dehydroabietylamine (DHAm) is a diterpenic primary amine obtained from dehydroabietic
acid (DHA). Dehydroabietic and abietic acids are components of resins of coniferous plants; for
example, the high acid content is found in the resin of Picea obovata [27]. Dehydroabietylamine
can be directly obtained from the resin by the reduction of dehydroabietyl nitrile.

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The synthetic route for obtaining compounds
1
–
12 is shown in Scheme 1. A set of
ureas and thioureas 1–4 was obtained by the interaction of dehydroabietylamine hy-
drochloride with 1- and 2- adamantyl isocyanates and isothiocyanates. The starting
isocyanates and isothiocyanates were synthesized using the methods described earlier.
In particular, 1-adamantyl isocyanate was obtained by the Curtius rearrangement of 1-
adamantyl acyl azide formed in situ by interaction of corresponding acyl chloride with
sodium azide [28]. Isomeric 2-adamantyl isocyanate was synthesized by the reaction of
2-adamantylamine hydrochloride with triphosgene in the presence of sodium hydrocar-
bonate, with dichloromethane used as a solvent [29]. Refluxing of 1-adamantaneamine
with phenyl isothiocyanate in dry toluene resulted in 1-adamantane isothiocyanate [30].
To obtain 2-adamantyl isothiocyanate, 2-adamantaneamine hydrochloride was treated by
triethylamine followed by carbon disulfide and DMAP/Boc2O subsequently [31]. For
the present study, we re-synthesised compound
inhibitory characteristics and the ability to enhance the cytostatic properties of TMZ. Mono-
substituted urea was prepared by treating dehydroabietylamine hydrochloride with
potassium cyanate. Amide and thioamide groups were considered as another variant of
linker structurally similar to the ureas. The target amides and were obtained from
dehydroabietylamine hydrochloride and 1- and 2-adamantanecarbonyl chlorides. The
amide group of compound was converted to thioamide using Lawesson’s reagent. The
reaction proceeded under harsh conditions. Refluxing in toluene led to the formation of
thioamide . Compound was isolated individually with a small yield. When the reaction
1, which previously showed the best
5
6
7
6
8
8
was carried out in lower boiling solvents, the target product was not detected, even after a
long period.
O
X =
O
R = Yield, %
1-Ad
2-Ad 46
R =
Yield, %
70
Ad
Cl
Ad
N C X
6
7
50
1
2
3
4
1-Ad
2-Ad
1-Ad
2-Ad
CHCl3, Et3N
24 h, 25 o
CH3CN, Et3N
24 h, 25 o
С
O
H
72
H
H
С
NH2 . HCl
NH
NH
S
70
O
X
dehydroabietylamine
hydrochloride
S
75
R
NH
R
KNCO
Lawesson's reagent
o-xylene, 3 h, 140 o
С
H2O/EtOH
6 h, 80 o
С
R = Yield, %
Yield 46%
5
8
1-Ad 10
H
H
NH
NH
S
O
R
NH2
1. SOCl2, CH2Cl2
2. NaN3, H2O
3. Toluene, 110 o
R = Yield, %
С
Ad-NH2 . HCl
9
1-Ad 85
CHCl3/EtOH, Et3N
H
H
N
H
NH
10 2-Ad 80
24 h, 25 o
С
HN
HO
O
C
O
O
R
dehydroabietic
acid
nordehydroabietyl
isocyanate
1. SOCl2, CH2Cl2
2. NaN3, H2O
3. Toluene, 110 oС
R = Yield, %
Ad-NH2 . HCl
11 1-Ad 90
12 2-Ad 80
CHCl3/EtOH, Et3N
H
H
N
H
24 h, 25 o
С
HN
HO
O
C
NH
O
O
R
abietic acid
norabietyl
isocyanate
Scheme 1. General procedure for the synthesis of compounds 1–12.

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To obtain the target compounds
9
–
12, starting with the dehydroabietic and abietic
acids, the following synthetic route was taken. Following the three-step procedure de-
scribed previously [32], nordehydroabietyl and norabietyl isocyanates were prepared.
According to this method, treatment of the resin acids with SOCl2 afforded the correspond-
ing chlorides, which were then converted to azides by interaction with NaN3. The azides
underwent Curtius rearrangement by refluxing in toluene, resulting in decarboxylation
and the formation of the corresponding isocyanates. Reaction of the obtained resin acid iso-
cyanates with 1- and 2-adamantylamine hydrochlorides in the presence of a base provided
good yields of the ureas 9–12 (80–90%).
As a result of this work, compounds
ized using physico-chemical methods. The ureas
from one another by the position of the adamantane fragment (1 and 2 respectively). Mono-
substituted urea , without the adamantane fragment in its structure, was prepared to
clarify the contribution of this moiety to the studied compounds. Substances have
linkers of a different type. Ureas 10 differed from the leading compound by lacking
12, in addition to the above, differed in the
2–
12 (Scheme 1) were synthesised and character-
1
and (and thioureas and ) differed
2
3
4
5
6–8
9–
1
a CH2 group in the linker, while ureas 11
–
terpene part.
2.2. TDP1 Assay and Cytotoxicity Studies
The primary screening of the inhibitory activities against TDP1 was performed using
an in vitro cell-free system involving the recombinant TDP1 and a fluorescent reporter
probe as previously described [13]. The IC50 values were found for derivatives 2–12 (and
are presented in Table 1), a commercially available TDP inhibitor Furamidine was used
as a reference drug [12]. We show here that the obtained compounds have the capacity to
inhibit TDP1 in vitro within a 0.19–2.3
µM range. As can be seen from the data in the table,
only compound —monosubstituted urea (lacking the adamantane moiety)—showed no
5
activity against TDP1.
All the compounds with the exception of 5 were able to inhibit TDP1 at micromolar
concentrations (0.19–2.3
of the compounds synthesized. After considering the effect of the diterpene fragment
on the inhibitory characteristics, we conclude that ureas with dehydroabietyl and
nordehydroabietyl 10 backbone work in lower concentrations than with norabietyl 11 12
For ureas and 10, the IC50 values were in the 0.19–0.8 M range, and for ureas
11 12, the IC50 values were higher—1.4–1.7 M. However, ureas 12, lacking a CH2
µM). We studied the structure–activity relationship for a number
1–2
9–
–
.
1–
2
9–
µ
–
µ
9–
group in the terpene part, demonstrated extremely low solubility in water and almost
all organic solvents, which does not make them promising for further study. The choice
of 1-adamantane or 2-adamantane substituent did not significantly affect the inhibitory
characteristics, but their absence negatively affected them. Monosubstituted urea
5 (without
any bulky fragment) showed no activity at concentrations up to 15 M. In a previous
µ
study [22], we showed that a decrease in the size of the substituent led to a decrease in
activity, and the date obtain herein consistent with this. Among the compounds belonging
to the urea, thiourea, amide, and thioamide classes, compound
8 with the thioamide linker
inhibited TDP1 in the highest concentrations (IC50 = 2.3 µM).

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Table 1. Inhibitory activity of compounds
1
–12 against TDP1 and predicted LogP (main determinant
of brain tissue binding).
IC50 (TDP1),
LogP
Compound
µM
1
2
DGAAm
DGAAm
DGAAm
DGAAm
DGAAm
DGAAm
DGAAm
DGAAm
nor-DGAA
nor-DGAA
nor-AA
1-Ad
2-Ad
1-Ad
2-Ad
-
0.19 ± 0.03
0.80 ± 0.20
1.50 ± 0.50
0.67 ± 0.19
>15
7.50
7.31
7.60
7.38
5.45
7.48
7.34
8.28
7.19
7.27
6.72
3
4
5
6
1-Ad
2-Ad
1-Ad
1-Ad
2-Ad
1-Ad
0.94 ± 0.18
1.80 ± 0.40
2.30 ± 0.80
0.57 ± 0.16
0.59 ± 0.15
1.70 ± 0.60
7
8
9
10
11
12
nor-AA
-
2-Ad
-
1.40 ± 0.30
1.20 ± 0.30
6.30
-
Furamidine
IC50: 50% inhibiting concentration.
-
QSAR prediction methods offer a useful tool to identify drug-like compounds [33
and therefore we have calculated LogP values for synthesized inhibitors as main determi-
nant of brain tissue binding. Octanol/water LogP predicted with GALAS algorithm [35
,34],
]
and QSAR software ACD/Percepta (www.acdlabs.com accessed on 29 April 2021) indi-
cate that the obtained adamantane derivatives have similar lipophilicity (Table 1). The
corresponding rate of brain penetration LogPS and extent of brain penetration LogBB,
calculated using LogP, molecular size, and H-bonding parameters as inputs, are suitable
for penetration into the central nervous system (see Table S1).
Since dehydroabietylamine and its derivatives are known to possess high cytotoxicity
against several cancer cells lines [36–38], the ureas, thioureas, amides, and thioamide (1–12)
synthesised in the present study were tested against the T98G glioma cells. Since we used
the T98G glioblastoma cell line for the first time for our experiments, we first attempted to
perform the cytotoxicity study in a range of concentrations from 10 to 100
previously with the TDP1 inhibitory compounds when working with the other cell lines.
However, the cytotoxicity at 50 and 100 M of our compounds turned out to be rather high
in the T98G cell line. We then measured the cytotoxicity of individual TDP1 inhibitors at 2.5,
5, 10, and 25 M. The studied compounds were prepared as 50 mM stock solutions in DMSO
µM, as we did
µ
µ

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and added to T98G glioma cells at 2.5
combination with 1000 or 2000
studied compounds varied in their solubility in DMSO. Compounds
to dissolve either at 50 M or at 10 M concentrations. They were therefore discarded from
the later cytotoxicity studies.
µM to 25
µM concentrations, either individually or in
µM of TMZ. Preparation of stock solutions showed that the
9,
10, 11, and 12 failed
µ
µ
When individual compounds were added to T98G glioma cells at 2.5, 5, 10, and 25
concentrations, they demonstrated moderate toxicity. The cell viabilities at 2.5 and 5
lay within the 90–100% range (Figure 4).
µM
µM
Figure 4. Individual cytotoxicity of studied compounds in the T98G and SNB19 glioma cell lines. * Compounds 7 and 8
formed visible micelles when stock solutions were dissolved in a cell growth medium, as shown by light microscopy. The
cytotoxicity of these compounds was studied; however, the concentrations of their solutions may be significantly different
from those indicated in dilutions.
To investigate the cytotoxicity of combinations of the studied compounds with TMZ,
we combined 5 M concentrations of each with 1000 M or 2000 M of TMZ and compared
their toxicity with 1000 M and 2000 M of TMZ alone. At 1000 M, TMZ was almost
non-toxic to our cell culture, with ~95% of cells surviving the treatment. Adding 5 M of
µ
µ
µ
µ
µ
µ
µ

Pharmaceuticals 2021, 14, 422
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the compounds to 1000
µM of TMZ did not increase the cytotoxicity to the glioma cells.
Higher cytotoxicity was obtained with 2000
µM of TMZ, which inhibited cell viability
by ~40%. Combining the compounds with 2000
µM of TMZ resulted in considerably
higher toxicity (i.e., an increase of 5–15%) compared with TMZ alone, which indicated the
additive profile of the action of TDP1 inhibitors with TMZ. The effects of combinations of
dehydroabietylamine derivatives
Figure 5.
1–4 and 6–8 with TMZ on T98G viability are shown in
Figure 5. Cytotoxicities of combinations of studied compounds with temozolomide in T98G glioma
cell line.
2.3. Molecular Docking Studies
The reaction catalyzed by TDP1 proceeds in two steps: the nucleophile His263 residue
attacks the 30-phosphotyrosyl bond of the substrate, and the His493 residue activates
the water molecule to cleave the covalent intermediate [39–41]. This offers two potential
therapeutic strategies: (1) the inhibition of the first step to prevent the formation of the
30-phosphohistidine intermediate; and (2) the inhibition of the second step to prevent the
intermediate hydrolysis [4,42]. Using molecular docking, we tested the discussed resin
acid derivatives against both the molecular model of the apo form and that of the covalent
intermediate. The inhibitors were found to bind preferentially to the intermediate structure,
as demonstrated in Figure 6.
A resin acid fragment occupies part of the peptide binding site—peptide is released
upon the intermediate formation—and forms hydrophobic contacts with the side chain of
Trp590. An adamantane fragment interacts with both the Phe259 residue of the oligonu-
cleotide binding site and with methylene (ribose) and the methyl (nucleobase) groups of
the oligonucleotide. A carbamide linker forms a hydrogen bond with the Ser463 side chain,
whilst its NH groups are orientated towards the solvent.
We conclude that adamantane derivatives of resin acids stabilize the TDP1 inter-
mediate (covalent complex of TDP1 with DNA) in a manner that is analogous with the
stabilisation of topoisomerase–DNA covalent complexes by camptothecins [43
,44]. It is
worth noting that compound , which lacked the adamantane fragment, failed to inhibit
5
TDP1. Using our proposed model, this can be explained as follows: monosubstituted urea
has an additional hydrogen bond donor, the –NH2 group, which is orientated towards a
hydrophobic adamantane-binding region. This may result in the unfavourable interaction
of compound 5 with the TDP1 intermediate.

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Figure 6. Position of the resin acid derivative
Gcalc = –9.0 kcal/mol. (
1
in the molecular model of human TDP1 intermediate,
∆
A
) Inhibitor’s interactions with a covalently bound DNA fragment and
TDP1 residues. The oligonucleotide is shown in orange, His263 in green, and hydrophobic Phe259
and Trp590 in yellow. The dotted line indicates a hydrogen bond. (B) Van der Waal’s representation
of the modeled TDP1–inhibitor complex.
3. Materials and Methods
3.1. Chemistry
All reagents and solvents were purchased from commercial sources and were used as
received without further purification. Reactions were monitored by thin-layer chromatogra-
phy (TLC) in silica gel. The TLC plates were visualised by exposure to ultraviolet light (254
and 365 nm). Merck (Merck KGaA, Darmstadt, Germany) silica gel (63–200 µm) was used
for column chromatography. The 1H and 13C NMR spectra in CDCl3, CD3OD, and DMSO-
d6 were recorded on a Bruker AV-400 spectrometer (400.13 and 100.61 MHz, respectively,
Bruker, Billerica, MA, USA). The residual signals of the solvent were used as references
(δH 7.24, δC 76.90 for CDCl3; δH 2.50, δC 39.50 for DMSO-d6). High-resolution mass
spectra were recorded on a Thermo Scientific DFS instrument (Thermo Fisher Scientific
Inc., Waltham, MA, USA) in full scan mode over the m/z range of 0–500 by ionisation with
an electron impact of 70 eV, and direct introduction of samples. IR spectra were recorded
on a Vector22 spectrometer (KBr, Bruker, Billerica, MA, USA). Thin-layer chromatography
was performed on Silufol plates (UV-254, Merck KGaA, Darmstadt, Germany). The atomic
numbering in the compounds is provided for the assignment of signals in the NMR spectra
and is different from the atomic numbering in the systematic name. The analytical and
spectroscopic studies were conducted at the Chemical Service Center for the collective use
of the Siberian Branch of the Russian Academy of Sciences (SB RAS).

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3.1.1. General Procedure for the Synthesis of Ureas and Thioureas 1–4
Dehydroabietylamine hydrochloride (0.5 g, 1.55 mmol) and triethylamine (0.28 mL,
2.0 mmol) were dissolved in CHCl3 (25 mL) and an equimolar amount of the appropriate
adamantyl isocyanate or isothiocyanate was added. The reaction mixture was stirred on a
magnetic stirrer for 24 h at room temperature. Conversion was monitored by TLC. The
reaction mixture was washed with 10 mL of distilled water. The organic layer was dried
over anhydrous Na2SO4 and filtered. The resultant liquid was evaporated under vacuum.
The residue was purified using column chromatography on silica gel with CHCl3 as an
eluent and a MeOH gradient from 0 to 100%.
N-abieta-8,11,13-trien-18-yl-N’-1-adamantylurea
pound 1 has been described previously [22].
(
1
). The spectral data for the com-
N-abieta-8,11,13-trien-18-yl-N’-2-adamantylurea
(
2
). Yield 72%, white powder. M.p.
1
150 ◦C. IR (KBr)
ν
max 3361, 2908, 1629, 1562 cm−1. H NMR (400MHz, CDCl3,
δ, ppm,
J/Hz): 6.86 (1H, s, H–14), 6.96 (1H, d, J11, 12 = 8.2, H–12), 7.14 (1H, d, J11, 12 = 8.2, H–11),
5.06 and 5.26 (1H both, s, NH), 0.87 (3H, s, Me–19), 1.20 (6H, d, J16, 15 = 6.9, Me–16 and
Me–17), 1.18 (3H, s, Me–20), 2.80 (1H, sept, J15, 16 = 6.9, H–15), 2.82–2.95 (2H, m, 2H–7),
2.24 (1H, d, 2J = 12.3, H–1e), 3.72–3.83 (1H, m, H–23), 2.95–3.13 (2H, m, H–18), 1.65–1.91
(15H, m, H–22, H–26, H-27, H-28, 2H-25, 2H-24, 2H-30, 2H-29, H-3e, H-3a, H-6e), 1.27-1.65
(7H, m, 2H-31, H-5a, H-6a, H-2a, H-2e, H–1a). 13C NMR (100MHz, CDCl3,
δ, ppm): 158.10
(C-20), 147.21 (C-9), 145.31 (C-13), 134.77 (C-8), 126.68 (C-14), 124.05 (C-11), 123.59 (C-12),
18.43 (Me-19), 23.85 (Me-17 and Me-16), 25.16 (Me-20), 27.19 and 27.04 (C-22, C-26), 32.48
and 32.43 (C-27, C-28), 33.28 (C-15), 45.03 (C-5), 53.92 (C-23), 18.58 (C-2), 18.70 (C-6), 30.05
(C-7), 31.60 and 31.57 (C-25, C-30), 50.61 (C-18), 37.30 and 37.27 (C-23, C-28), 38.29 (C-4),
37.51 (C-3), 37.16 (C-10), 37.06 (C-1), 38.36 (C–30). Found, m/z: 462.3613 [M]+. C31H46ON2.
Calculated, m/z: 462.3605.
N-abieta-8,11,13-trien-18-yl-N’-1-adamantylthiourea
(
3
). Yield 70%, white powder.
o
M.p. 103 C. IR (KBr)
ν
max 3265, 2908, 1538 cm–1 1H NMR (400MHz, CDCl3,
δ, ppm,
J/Hz): 6.88 (1H, s, H–14), 6.95 (1H, d, J11, 12 = 8.1, H–12), 7.13 (1H, d, J11, 12 = 8.1, H–11), 0.98
(3H, s, Me–19), 1.18 (6H, d, J16, 15 =6.9, Me–16 and Me–17), 1.20 (3H, s, Me–20), 2.79 (1H,
sept, J15, 16 = 6.9, H–15), 2.82–2.98 (2H, m, 2H–7), 2.29 (1H, d, 2J = 13.0, H–1e), 2.00–2.21 (4H,
m, H–26, H–27, H–28, H–6a), 1.83–1.99 (8H, m, 2H-23, 2H-24, 2H-25, H-6e, H-2a), 3.23-3.40
2
and 3.65-3.84 (2H, m, H-18), 1.36–1.44 (1H, d, J = 13.0, H-1a), 1.45-1.81 (9H, m, 2H-29,
2H-30, 2H-31, H-3, H-2e, H–5a). 13C NMR (100MHz, CDCl3 + CD3OD,
δ, ppm): 181.35
(C-21), 146.72 (C-9), 145.42 (C-13), 134.17 (C-8), 126.48 (C-14), 123.75 (C–11), 123.51 (C-12),
18.26 (Me-19), 23.60 and 23.69 (Me-17 and Me-16), 24.66 (Me-20), 33.15 (C-15), 46.00 (C-5),
29.04 (C-26, C–27, C-28), 18.29 (C-2), 18.72 (C-6), 29.66 (C-7), 38.21 (C-4), 37.28 (C-1), 37.14
(C–10), 36.54 (C-3), 53.66 (C-18), 35.66 (C-29, C-30, C-31), 42.00 (C-23, C-24, C-25), 56.15
(C–22). Found, m/z: 478.3380 [M]+. C31H46N2S. Calculated, m/z: 478.3376.
N-abieta-8,11,13-trien-18-yl-N’-2-adamantylthiourea
(
4
). Yield 75%, white powder.
1
M.p. 116 oC. IR (KBr)
ν
max 3278, 2908, 1537 cm−1. H NMR (400MHz, CDCl3,
δ
, ppm,
J/Hz): 6.86 (1H, s, H–14), 6.95 (1H, d, J11, 12 = 8.1, H–12), 7.12 (1H, d, J11, 12 = 8.1, H–11),
6.94 and 5.21 (1H both, s, NH), 0.95 (3H, s, Me–18), 1.19 (6H, d, J16,15 = 6.9, Me–16 and
Me–17), 1.19 (3H, s, Me–19), 2.79 (1H, sept, J15,16 =6.9, H–15), 2.92–2.92 (2H, m, 2H–7), 4.05
(1H, br s, H–23), 3.09-3.62 (2H, m, 2H-18), 2.26 (1H, 2J = 12.6, H–1e), 1.94-2.10 (2H, m, H-22,
H-26), 1.26–1.51 (4H, m, H-6, 2H-2, H–1a), 1.51-1.93 (16H, m, 2H-24, 2H-29, 2H-25, 2H-31,
2H-30, H–27, H-28, H-6, 2H-3, H–5). 13C NMR (100MHz, CDCl3,
δ, ppm): 181.06 (C-21),
146.72 (C-9), 145.47 (C-13), 134.37 (C-8), 126.67 (C-14), 123.89 (C-11), 123.65 (C-12), 18.44
(Me-19), 23.87 and 23.82 (Me-17 and Me-16), 25.02 (Me-20), 33.24 (C-15), 45.76 (C-5), 26.78
(C-27, C-28), 31.58 (C-22, C-26), 57.83 (C-23), 55.31 (C-18), 18.40 (C-2), 18.93 (C-6), 29.86
(C-7), 31.72 (C-25, C-31), 38.11 (C-4), 37.57 (C-1), 36.57 (C-30), 36.78 (C-24, C-29), 37.26 and
37.16 (C-10 and C–3). Found, m/z: 478.3368 [M]+. C31H46N2S. Calculated, m/z: 478.3376.

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3.1.2. Synthesis of Urea 5
Dehydroabietylamine hydrochloride (0.34 g, 1.06 mmol) was dissolved in EtOH
(30 mL), and an aqueous solution of potassium cyanate (0.1 g of KNCO in 5 mL of water)
was added. The mixture was refluxed for 6 h, then cooled to room temperature. The
solvent was evaporated under vacuum. The solid residue was dissolved in CHCl3 (20 mL)
and washed with water (10 mL) and 5% aqueous NaOH solution (10 mL). The urea was
purified using column chromatography on silica gel with CHCl3 as an eluent and a MeOH
gradient from 0 to 20%.
N-abieta-8,11,13-trien-18-ylurea
(5
). Yield 46%, white powder. M.p. 108 oC. IR (KBr)
, ppm, J/Hz): 6.86 (1H, s, H–14),
1
ν
max 3430, 2927, 1652 cm−1. H NMR (400MHz, CDCl3,
δ
6.96 (1H, d, J11, 12 = 8.2, H–12), 7.14 (1H, d, J11, 12 = 8.2, H–11), 0.89 (3H, s, Me–19), 1.19 (6H,
d, J16, 15 = 6.9, Me–16 and Me–17), 1.18 (3H, s, Me–20), 2.25 (1H, d, 2J = 12.3, H–1e), 2.79
(1H, sept, J15, 16 = 6.9, H–15), 2.93–3.00 and 3.02–3.11 (1H both, m, H–18), 2.80–2.95 (2H, m,
H–7), 1.77–1.94 (2H, m, H–6e, H–3a), 1.52–1.76 (3H, m, H–2e, H–3e, H–1a), 1.26–1.50 (3H,
m, H–6e, H–2a H–5a), 4.53 (2H, s, NH2), 4.96 (1H, s, NH). 13C NMR (100MHz, CDCl3,
δ,
ppm): 159.18 (C–21), 147.17 (C–9), 145.47 (C–13), 134.72 (C–8), 126.72 (C–14), 124.02 (C–11),
123.65 (C–12), 23.85 (Me–17 and Me–16), 25.05 (Me–20), 33.29 (C–15), 44.87 (C–5), 29.95
(C–7), 38.28 (C–4), 37.27 (C–1, C–10), 35.89 (C–3), 50.73 (C-18), 18.76 (Me–19, C–2, C–6).
Found, m/z: 328.2503 [M]+. C21H32ON2. Calculated, m/z: 328.2509.
−1
Norabietyl isocyanate. Yield 56%, light-yellow oil. IR (KBr)
1H NMR (400MHz, CDCl3,
and 0.99 (3H both,
νmax 2933, 2250, 1459 cm
.
δ, ppm, J/Hz): 5.77 (1H, s, H-14), 5.39-5.44 (1H, m, H-7), 1.00
д
, J16, 15=6.9, Me-16 and Me-17), 0.75 (3H, s, Me-18), 1.34 (3H, s, Me-19),
2.21 (1H, sept, J15, 16=6.9, H-15), 2.27 (1H, d, J=18.2, H-5), 1.08 (1H, dt, J=3.8, J=13.3, H-11a),
0.81-0.88 (1H, m, H-1a), 1.15-1.29 (2H, m, H-2a, H-2e), 1.42-1.48 (1H, m, H-3), 1.54-1.70
(3H, m, H-1e, H-11e, H-9), 1.74-1.80 (1H, m, H-3e), 1.80-1.86 (1H, m, H-6a), 1.86-2.03 (3H,
m, H-6e, H-12a, H-12e). 13C NMR (100MHz, CDCl3,
δ, ppm): 145.18 (C-13), 135.22 (C-8),
122.13 (C-14), 119.97 (C-7), 21.17 and 20.62 (Me-17 and Me-16), 23.66 (Me-18), 13.32 (Me-19),
34.66 (C-15), 50.60 (C-9), 51.30 (C-5), 19.18 (C-2), 27.20 (C-12), 22.54 (C-11), 23.86 (C-6),
35.59 (C-10), 38.00 (C-1), 43.14 (C-3), 61.24 (C-4), 121.92 (C-20). Found, m/z: 299.2240 [M]+.
C20H29ON. Calculated, m/z: 299.2244.
3.1.3. General Procedure for the Synthesis of Amides 6–7
Dehydroabietylamine hydrochloride (1.0 g, 3.1 mmol) was mixed with an equimolar
amount of 1- or 2-adamantanecarbonyl chloride (0.62 g, 3.1 mmol) in 30 mL of CH3CN with
the addition of Et3N (0.56 mL, 4.0 mmol). The reaction mixture was stirred on a magnetic
stirrer for 24 h at room temperature. Upon completion, the solvent was evaporated under
vacuum. The solid residue was dissolved in CHCl3 (20 mL) and washed with water (15 mL).
The organic layer was dried over anhydrous Na2SO4 and filtered. The resultant liquid was
evaporated under vacuum. The residue was purified using column chromatography on
silica gel with hexane/ethyl acetate system, with a concentration gradient (EtOAc 0–25%)
as an eluent.
N-abieta-8,11,13-trien-18-yladamantan-1-carboxamide
(6
). Yield 50%, white powder.
o
M.p. 90 C. IR (KBr)
ν
max 3363, 2906, 1639, 1525 cm−1
.
1H NMR (400MHz, CDCl3,
δ,
ppm, J/Hz): 6.88 (1H, d, J12,14=1.7, H-14), 6.98 (1H, dd, J11, 12=8.2, J12,14=1.7, H-12), 7.16
(1H, d, J11, 12=8.2, H-11), 0.91 (3H, s, Me-19), 1.21 (6H, d, J16, 15=6.9, Me-16 and Me-17), 1.20
(3H, s, Me-20), 2.81 (1H, sept, J15, 16=6.9, H-15), 2.28 (1H, d, 2J=12.3, H-1e), 3.17-3.22 and
3.08-3.13 (1H both, m, H-18), 2.85-2.91 and 2.73-2.79 (1H both, m, H-7), 1.99-2.02 (3H, m,
H-26, H-27, H-28), 1.80-1.83 (6H, m, H-23, H-24, H-25), 1.84-1.89 (1H, m, H-6e), 1.42 (1H,
d, 2J=13.0, H-3e), 1.32-1.39 (2H, m, H-5a, H-1a), 1.62-1.75 (8H, m, H-29, H-30, H-31, H-3a,
H-2e), 1.22-1.30 (2H, m, H-6e, H-2a). 13C NMR (100MHz, CDCl3,
δ, ppm): 177.7 (C-21),
146.9 (C-9), 145.4 (C-13), 134.6 (C-8), 126.8 (C-14), 124.1 (C-11), 123.7 (C-12), 18.4 (Me-19),
23.84 and 23.80 (Me-17 and Me-16), 25.4 (Me-20), 33.3 (C-15), 46.2 (C-5), 28.0 (C-26, C-27,
C-28), 18.5 (C-2), 18.9 (C-6), 30.4 (C-7), 36.4 (C-29, C-30, C-31), 39.3 (C-23, C-24, C-25), 38.3

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(C-4), 37.5 (C-1), 37.3 (C-10), 36.2 (C-3), 49.5 (C-18), 40.8 (C-22). Found, m/z: 447.3490 [M]+.
C31H45ON. Calculated, m/z: 447.3496.
N-abieta-8,11,13-trien-18-yladamantan-2-carboxamide
(7
). Yield 46%, white powder.
o
M.p. 94 C. IR (KBr)
ν
max 3311, 2904, 1642, 1542 cm−1
.
1H NMR (400MHz, CDCl3,
δ,
ppm, J/Hz):6.87 (1H, s, H-14), 6.97 (1H, d, J11, 12=8.1, H-12), 7.15 (1H, d, J11, 12=8.1, H-11),
0.92 (3H, s, Me-19), 1.20 (6H, d, J16, 15=6.9, Me-16 and Me-17), 1.19 (3H, s, Me-20), 2.80 (1H,
sept, J15, 16=6.9, H-15), 2.27 (1H, d, 2J=12.7, H-1e), 2.73-2.94 (2H, m, H-7), 5.59 (1H, s, NH),
3.13-3.26 (2H, m, H-18), 2.39-2.47 (1H, m, H-23), 2.16-2.23 (2H, m, H-22, H-26), 1.63-1.80 (7H,
m, H-24, H-29, H-25, H-31, H-30, H-3, H-6), 1.52-1.63 (3H, m, H-5a, H-1e, H-3), 1.80-2.00
(6H, m, H-24, H-29, H-25, H-31, H-28, H-27), 1.29-1.46 (4H, m, H-6, 2H-2, H-1a). 13C NMR
(100MHz, CDCl3, δ, ppm): 173.9 (C-21), 147.0 (C-9), 145.5 (C-13), 134.7 (C-8), 126.8 (C-14),
124.0 (C-11), 123.7 (C-12), 18.5 (Me-19), 23.8 and 23.9 (Me-17 and Me-16), 25.2 (Me-20), 33.3
(C-15), 45.6 (C-5), 50.0 (C-23), 29.96 and 30.04 (C-22, C-26), 27.24 and 27.36 (C-27, C-28),
18.5 (C-2), 18.9 (C-6), 30.2 (C-7), 49.5 (C-18), 33.16 and 33.21 (C-25, C-31), 38.20 and 38.26
(C-25, C-31, C-4), 36.3 (C-3), 37.22, 37.31, 37.33 (C-10, C-1, C-30). Found, m/z: 447.3503
[M]+. C31H45ON. Calculated, m/z: 447.3500.
3.1.4. Synthesis of Thioamide 8
Amide 6 (0.4 g, 0.9 mmol) and Lawesson’s reagent (0.18 g, 0.45 mmol) were refluxed
in o-xylene (20 mL) for 3 h. Conversion was monitored by TLC. The solvent was removed
under vacuum. The residue was purified using column chromatography on silica gel with
CHCl3 as an eluent and a MeOH gradient from 0 to 20%.
N-abieta-8,11,13-trien-18-yladamantan-1-carbothioamide (8). Yield 10%, light-yellow
1
powder. M.p. 166 oC. IR (KBr)
ν
max 3386, 2904, 1525 cm−1. H NMR (400MHz, CDCl3,
δ, ppm, J/Hz): 6.89 (1H, s, H-14), 6.99 (1H, d, J11, 12=8.2, H-12), 7.15 (1H, d, J11, 12=8.2,
H-11), 7.42 (1H, s, NH), 0.99 (3H, s, Me-19), 1.21 (6H, d, J16, 15=6.9, Me-16 and Me-17),
1.22 (3H, s, Me-20), 2.81 (1H, sept, J15, 16=6.9, H-15), 2.72-2.93 (2H, m, 2H-7), 2.31 (1H, m,
H-1e), 3.75-3.85 (1H, m, H-18), 3.48-3.56 (1H, m, H-18), 2.04-2.14 (3H, m, H-26, H-27, H-28),
1.50-1.56 (1H, m, H-3a), 1.91-2.03 (7H, m, 2H-23, 2H-24, 2H-25, H-6e), 1.28-1.47 (3H, m, H-5a,
H-1a, H-3e), 1.60-1.91 (9H, m, 2H-28, 2H-29, 2H-30, H-6a, H-2a, H-2e). 13C NMR (100MHz,
CDCl3, δ, ppm): 212.88 (C-21), 146.27 (C-9), 145.28 (C-13), 134.11 (C-8), 126.56 (C-14), 123.84
(C-11), 123.56 (C-12), 18.33 (Me-19), 23.54 and 23.49 (Me-17 and Me-16), 25.08 (Me-20), 32.97
(C-15), 46.75 (C-5), 28.14 (C-26, C-27, C-28), 18.18 (C-2), 18.79 (C-6), 30.04 (C-7), 35.93 (C-29,
C-30, C-31), 41.53 (C-23, C-24, C-25), 37.85 (C-4), 37.27 (C-1), 37.18 (C-10), 36.56 (C-3), 56.12
(C-18), 46.08 (C-22). Found, m/z: 463.3264 [M]+. C31H45NS. Calculated, m/z: 463.3267.
3.1.5. General Procedure for the Synthesis of Norabietyl and Nordehydroabietyl Ureas
Norabietyl or nordehydroabietyl isocyanate (0.3 g, 1.0 mmol) was dissolved in CHCl3
(15 mL). An equimolar amount (0.19 g, 1.0 mmol) of 1- or 2-adamantylamine hydrochloride
with triethylamine (0.17 mL, 1.2 mmol) was dissolved in EtOH (15 mL) and added to
isocyanate solution. The reaction mixture was stirred on a magnetic stirrer for 24 h at room
temperature. The precipitated norabietyl ureas were filtered off and were not additionally
purified. The nordehydroabietyl urea solutions were washed with water (15 mL) and dried
over Na2SO4. The solvent was removed in vacuo. The solid residues were recrystallized
from acetonitrile.
N-1-adamantyl-N’-[(1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
o
octahydrophenanthren-1-yl]urea
ν
(
9
). Yield 85%, white powder. M.p. 235 C. IR (KBr)
1
max 3357, 2906, 1629, 1554 cm−1. H NMR (400MHz, CDCl3 + CD3OD,
δ
, ppm, J/Hz):
6.76 (1H, s, H–14), 6.87 (1H, d, J11, 12 = 8.2, H–12), 7.05 (1H, d, J11, 12 = 8.2, H–11), 1.07 (3H,
s, Me–18), 1.11 (6H, d, J16, 15 = 6.9, Me–16 and Me–17), 1.10 (3H, s, Me–19), 2.71 (1H, sept,
J15, 16 = 6.9, H–15), 2.74-2.83 (2H, m, 2H–7), 2.10 (2H, m, H–1e, H–5a), 1.88–2.01 (4H, m,
H–25, H–26, H–27, H–6e), 1.73–1.86 (8H, m, 2H–22, 2H–23, 2H–24, H–3e, H–3a), 1.47–1.66
(9H, m, 2H–28, 2H–29, 2H–30, H–6a, H–2a, H–2e), 1.30-1.40 (1H, m, H–1a). 13C NMR
(100MHz, CDCl3 + CD3OD, δ, ppm):157.17 (C–20), 146.74 (C–9), 145.16 (C–13), 134.44 (C-8),

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126.44 (C–14), 124.08 (C–11), 123.49 (C–12), 20.76 (Me–18), 23.62 and 23.66 (Me–17 and
Me–16), 24.77 (Me–19), 33.15 (C–15), 46.93 (C–5), 29.25 (C–25, C-26, C-27), 18.59 (C–2), 19.46
(C–6), 30.11 (C–7), 36.18 (C–28, C-29, C-30), 42.13 (C–22, C-23, C-24), 55.77 (C–4), 37.89 (C–1),
37.50 and 37.46 (C–10 and C-3), 50.04 (C-21). Found, m/z: 448.3445 [M]+. C30H44ON2.
Calculated, m/z: 448.3448.
N-2-adamantyl-N’-[(1R,4aS,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-
octahydrophenanthren-1-yl]urea
ν
(
10). Yield 80%, white powder. M.p. 233 oC. IR (KBr)
1
max 3357, 2912, 1623, 1556 cm−1. H NMR (400MHz, CDCl3,
δ, ppm, J/Hz): 6.78 (1H, s,
H–14), 6.89 (1H, d, J11, 12 = 8.2, H–12), 7.07 (1H, d, J11, 12 = 8.2, H–11), 1.20 (3H, s, Me–18),
1.11 (6H, d, J16, 15 = 6.9, Me–16 and Me–17), 1.10 (3H, s, Me–19), 2.73 (1H, sept, J15, 16 = 6.9,
H–15), 2.74–2.87 (2H, m, 2H–7), 2.15 (1H, d, 2J = 12.3, H–1e), 3.64 (1H, s, H–22), 1.29–1.41
(1H, m, H–1a), 1.42–1.54 (2H, m, H–24, H–29), 1.94–2.08 (2H, m, H–23, H–28), 1.78–1.93 (2H,
m, H–24, H–29), 1.55–1.65 (4H, m, H–26, H–27, 2H–30), 1.65–1.78 (11H, m, 2H–2, 2H–3,
2H–6, H–5, H–21, H–25, H–23, H–28). 13C NMR (100MHz, DMSO-d6,
δ, ppm, J/Hz): 157.25
(C–20), 147.74 (C–9), 145.54 (C–13), 134.99 (C-8), 127.00 (C–14), 124.69 (C–11), 124.16 (C–12),
21.91 (Me–18), 24.14 and 24.48 (Me–17 and Me–16), 25.25 (Me–19), 46.53 (C–5), 30.43 (C–7),
18.89 (C–2), 20.06 (C–6), 55.76 (C–4), 53.25 (C-22), 27.46 and 27.52 (C-21, C-25), 31.80 and
31.92 (C-24, C-29), 32.86 and 32.94 (C-26, C-27), 33.43 (C–15), 37.57 (C-3), 37.73 (C–10), 38.32
and 38.36 (C–1, C-30), 37.97 and 37.92 (C-23, C-28). Found, m/z: 448.3449 [M]+. C30H44ON2.
Calculated, m/z: 448.3448.
N-1-adamantyl-N’-[(1R,4aR,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,4b,5,6,10,10a-
decahydrophenanthren-1-yl]urea
ν
(
11). Yield 90%, white powder. M.p. 224 oC. IR (KBr)
1
max 3346, 2906, 1633, 1560 cm−1. H NMR (400MHz, CDCl3 + CD3OD,
δ
, ppm, J/Hz):
5.65 (1H, s, H–14), 5.30 (1H, s, H–7), 0.88 and 0.89 (3H both, d, J16, 15=6.9, Me–16 and Me–17),
0.67 (3H, s, Me–18), 1.12 (3H, s, Me–19), 2.10 (1H, sept, J15, 16 = 6.9, H–15), 0.95–1.10 (2H, m,
H–1a, H–11a), 1.32–1.48 (2H, m, H–2a, H–2e), 1.63–1.73 (2H, m, H–3a, H–11e), 1.48–1.57
(6H, m, 2H–29, 2H–30, 2H–28), 1.72–1.87 (9H, m, 2H–22, 2H–23, 2H–24, H–1e, H–3e, H–9),
1.87–2.05 (8H, m, H-25, H-26, H–27, H–5, H–6a, H–6e, 2H–12). 13C NMR (100MHz, CDCl3
+ CD3OD, δ, ppm): 156.99 (C–20), 145.02 (C–13), 135.33 (C-8), 122.19 (C–14), 120.64 (C–7),
29.32 (C–25, C-26, C-27), 36.22 (C–28, C-29, C-30), 42.21 (C–22, C–23, C–24), 21.10 and
20.53 (Me–17 and Me–16), 21.38 (Me–18), 13.61 (Me–19), 34.65 (C–15), 50.65 (C–9), 47.00
(C–5), 19.15 (C–2), 27.20 (C–12), 35.44 (C–10), 38.14 (C–1), 37.92 (C–3), 22.49 (C–11), 23.50
(C–6), 55.45 (C–4), 50.17 (C–21). Found, m/z: 450.3600 [M]+. C30H46ON2. Calculated,
m/z: 450.3605.
N-2-adamantyl-N’-[(1R,4aR,10aR)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,4b,5,6,10,10a-
decahydrophenanthren-1-yl]urea
ν
(
12). Yield 80%, white powder. M.p. 213 oC. IR (KBr)
1
max 3395, 2908, 1629, 1556 cm−1. H NMR (400MHz, CDCl3 + CD3OD,
δ
, ppm, J/Hz):
5.62 (1H, s, H–14), 5.27 (1H, s, H–7), 0.88 and 0.87 (3H both, d, J16, 15 = 6.9, Me–16 and
Me–17), 0.67 (3H, s, Me–18), 1.13 (3H, s, Me–19), 2.08 (1H, sept, J15, 16 = 6.9, H–15), 0.93–1.10
(2H, m, H–1a, H–11a), 1.32–1.48 (4H, m, H–2a, H–2e, H–24, H–29), 1.54–1.61 (2H, m, 2H–30),
1.89–2.05 (5H, m, H–5, H–6a, H–6e, 2H–12), 1.73–1.89 (3H, m, H–1e, H–3e, H–9), 1.60–1.75
(10H, m, H–3a, H–11e, H–26, H–27, H–21, H–25, 2H–23, 2H–28, H–24, H–29), 3.59 (1H, s,
H–22). 13C NMR (100MHz, CDCl3 + CD3OD,
δ, ppm): 157.29 (C–20), 144.82 (C–13), 135.25
(C–8), 122.11 (C–14), 120.52 (C–7), 13.53 (Me–19), 21.03 and 20.47 (Me–17 and Me–16), 21.28
(Me–18), 22.42 (C–11), 23.40 (C–6), 27.13 (C–12), 27.05 and 26.90 (C–21, C-25), 31.37 (C–24,
C-29), 32.38 and 32.40 (C–26, C–27), 34.55 (C–15), 35.35 (C–10), 37.05 and 37.03 (C–23, C–28),
38.10 (C–1), 37.92 (C–3), 37.35 (C–30), 46.87 (C–5), 50.68 (C–9), 55.33 (C–4), 53.10 (C–22).
Found, m/z: 450.3604 [M]+. C30H46ON2. Calculated, m/z: 450.3605.
3.2. TDP1 Assay
The recombinant TDP1 was purified to homogeneity by chromatography on Ni-
chelating resin and phosphocellulose P11 as previously described [45], using plasmid pET
16B-TDP1, kindly provided by Dr. K.W. Caldecott (University of Sussex, United Kingdom).
The TDP1 activity measurements were carried out as described [13]. Briefly, TDP1-biosensor

Pharmaceuticals 2021, 14, 422
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with fluorofore (FAM) at the 50-end and a fluorescence quencher (BHQ1) at the 30-end at a
final concentration of 50 nM were incubated in a 200
(50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 7 mM
µ
L volume that contained TDP1 buffer
β
-mercaptoethanol) supplemented with
purified 1.5 nM TDP1 and various concentrations of inhibitor. Fluorescence measurements
(Ex485/Em520 nm) were carried out during the linear phase of the reaction (from 0 to 8 min
◦
for TDP1) 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
with that of DMSO control wells. The data were imported into the MARS Data Analysis
2.0 program (BMG LABTECH), and the IC50 values (the concentration of a compound
required to reduce the enzyme activity0by 50%) were calculated. The TDP1-biosensor
0
5 -(5,6 FAM-aac gtc agg gtc ttc c-BHQ1)-3 was synthesised in the Laboratory of Biomedical
Chemistry, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia.
3.3. Cytotoxicity Experiments
Individual TDP1 inhibitors were prepared as 50 mM stock solutions in DMSO and
were added to the cells at 2.5, 5, 10, or 25 µM. Temozolomide was prepared as 200 mM
stock solution in DMSO and was added at 1 mM or 2 mM concentrations, either alone
or in combination with TDP1 inhibitors. T98G and SNB19 glioma cells were maintained
in DMem/F12 medium supplemented with 10% foetal bovine serum, l-glutamine, and
penicillin/streptomycin and were split at 10,000 cells/well into the 96-well plates for
cytotoxicity experiments. The drugs or drug combinations were incubated with cells for
72 h, then the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
was added for 4 h. The produced purple formazan dye reporting the activity of cellular
oxireductases was dissolved overnight in 10% solution of acidified SDS and the absorbance
was measured using a Tecan plate reader. Each concentration of individual substance or
each drug combination was tested in triplicate. The data obtained were processed using
MS Excel software and presented as histogram plots.
3.4. Molecular Docking
The models of the apo form and covalent intermediate of human TDP1 were based on
the 1NOP crystal structure [46] and constructed as reported in our previous work [47,48]
.
Molecular docking of inhibitors was performed with Lead Finder 1.1.15 [49 50]. An en-
,
ergy grid box with edges of 35 Å was centred on the Nε2 atom of the catalytic residue
His263 and overlapped the active site and adjacent cavities. In our previous study, the
TDP1 substrate-binding groove was identified and mapped based on the 1NOP structure
(covalent complex with substrate analogue), and successfully tested in docking runs with
diazaadamantane derivatives as reference TDP1 inhibitors [48]. Docking was done using
a genetic algorithm in ‘extra precision’ mode. The protein structure was rigid, whereas
rotating functional/rotatable groups of ligands was allowed. VMD 1.9.2 was used to
visualise molecular structures [51].
4. Conclusions
As a result of this study, we synthesized a set of compounds containing the diterpene
fragment. The effect of the terpene structural blocks, the length and structure of the ureide
linker, and the site of attachment of the adamantane residue on the biological properties of
the new abietylamine-based compounds were investigated, and in particular their ability
both to inhibit the DNA repair enzyme TDP1 and to enhance the cytotoxic effect of TMZ.
In this library of compounds, we studied the structure of compounds with demonstrated
biological activity. The choice of 1-adamantane or 2-adamantane substituent did not
significantly affect the inhibitory characteristics, but their absence negatively affected them.
Ureas on nordehydroabietyl and norabietyl isocyanates lacking a CH2 group in the terpene
part, demonstrated extremely low solubility in water and almost all organic solvents,
which does not make them promising for further study. The starting compound
1 was

Pharmaceuticals 2021, 14, 422
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the most effective in the inhibition of TDP1 and compound
2
was the most effective in the
sensitization of glioma T98G cells to TMZ. We found some synergistic effects on cells T98G
when using repair enzyme inhibitors, but they are not as high as we expected. At the same
time, the substances we described could be of considerable interest when studied on other
cancer cell lines or simultaneously with other cytostatics.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/ph14050422/s1.
Author Contributions: Conceptualization, K.K., O.Y.; methodology, K.K., O.Y., S.C., A.Z.; investi-
gation, K.K., K.P., A.A., I.C., V.K., T.K., E.M., D.N.; writing—original draft preparation, K.K., O.Y.,
S.C., A.Z.; supervision, E.S., V.Š., A.P., O.L., N.S. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by a grant from the Russian Science Foundation 19-73-00051.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to acknowledge the Multi-Access Chemical Service
Centre SB RAS for spectral and analytical measurements.
Conflicts of Interest: The authors declare no conflict of interest.
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