Novel tdp1 inhibitors based on adamantane connected with monoterpene moieties via heterocyclic fragments

Article abstract of DOI:10.3390/molecules26113128

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising target for anticancer therapy due to its ability to counter the effects topoisomerase 1 (Top1) poison, such as topotecan, thus, decreasing their efficacy. Compounds containing adamantane and monoterpenoid residues connected via 1,2,4-triazole or 1,3,4-thiadiazole linkers were synthesized and tested against Tdp1. All the derivatives exhibited inhibition at low micromolar or nanomolar concentrations with the most potent inhibitors having IC₅₀ values in the 0.35–0.57 μM range. The cytotoxicity was determined in the HeLa, HCT-116 and SW837 cancer cell lines; moderate CC₅₀ (μM) values were seen from the mid-teens to no effect at 100 μM. Furthermore, citral derivative 20c, α-pinene-derived compounds 20f, 20g and 25c, and the citronellic acid derivative 25b were found to have a sensitizing effect in conjunction with topotecan in the HeLa cervical cancer and colon adenocarcinoma HCT-116 cell lines. The ligands are predicted to bind in the catalytic pocket of Tdp1 and have favorable physicochemical properties for further development as a potential adjunct therapy with Top1 poisons.

Full text of DOI:10.3390/molecules26113128

molecules
Article
Novel Tdp1 Inhibitors Based on Adamantane Connected with
Monoterpene Moieties via Heterocyclic Fragments
Aldar A. Munkuev 1 , Evgenii S. Mozhaitsev 1, Arina A. Chepanova 2, Evgeniy V. Suslov 1 , Dina V. Korchagina 1,
Olga D. Zakharova 2, Ekaterina S. Ilina 2, Nadezhda S. Dyrkheeva 2, Alexandra L. Zakharenko 2,
Jóhannes Reynisson 3,
*
, Konstantin P. Volcho 1 , Nariman F. Salakhutdinov 1 and Olga I. Lavrik 2
1
N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, 9, Akademika Lavrentieva Ave., 630090 Novosibirsk, Russia; amunkuev@nioch.nsc.ru (A.A.M.);
mozh@nioch.nsc.ru (E.S.M.); suslov@nioch.nsc.ru (E.V.S.); korchaga@nioch.nsc.ru (D.V.K.);
volcho@nioch.nsc.ru (K.P.V.); anvar@nioch.nsc.ru (N.F.S.)
2
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8,
Lavrentiev Ave., 630090 Novosibirsk, Russia; arinachepanova@mail.ru (A.A.C.); garonna3@mail.ru (O.D.Z.);
katya.plekhanova@gmail.com (E.S.I.); elpida80@mail.ru (N.S.D.); a.zakharenko73@gmail.com (A.L.Z.);
lavrik@niboch.nsc.ru (O.I.L.)
3
School of Pharmacy and Bioengineering, Keele University, Hornbeam Building, Staffordshire ST5 5BG, UK
*
Correspondence: j.reynisson@keele.ac.uk
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Abstract: Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a promising target for anticancer therapy due
to its ability to counter the effects topoisomerase 1 (Top1) poison, such as topotecan, thus, decreasing
their efficacy. Compounds containing adamantane and monoterpenoid residues connected via 1,2,4-
triazole or 1,3,4-thiadiazole linkers were synthesized and tested against Tdp1. All the derivatives
exhibited inhibition at low micromolar or nanomolar concentrations with the most potent inhibitors
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Munkuev, A.A.;
Mozhaitsev, E.S.; Chepanova, A.A.;
Suslov, E.V.; Korchagina, D.V.;
Zakharova, O.D.; Ilina, E.S.;
having IC50 values in the 0.35–0.57
and SW837 cancer cell lines; moderate CC50
100 M. Furthermore, citral derivative 20c
citronellic acid derivative 25b were found to have a sensitizing effect in conjunction with topotecan
in the HeLa cervical cancer and colon adenocarcinoma HCT-116 cell lines. The ligands are predicted
to bind in the catalytic pocket of Tdp1 and have favorable physicochemical properties for further
development as a potential adjunct therapy with Top1 poisons.
µ
M range. The cytotoxicity was determined in the HeLa, HCT-116
M) values were seen from the mid-teens to no effect at
-pinene-derived compounds 20f 20g and 25c, and the
Dyrkheeva, N.S.; Zakharenko, A.L.;
Reynisson, J.; et al. Novel Tdp1
Inhibitors Based on Adamantane
Connected with Monoterpene
(
α
µ
µ
,
,
Moieties via Heterocyclic Fragments.
Molecules 2021, 26, 3128. https://
doi.org/10.3390/molecules26113128
Academic Editor: María
Ángeles Castro
Keywords: topoisomerase 1; monoterpenoid; cancer; DNA repair enzyme; SAR; molecular modeling;
chemical space
Received: 21 April 2021
Accepted: 21 May 2021
Published: 24 May 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Cancer is one of the leading causes of death worldwide; according to the International
Agency for Research on Cancer (IARC), in 2018 new cancer cases were estimated to be
approximately 18.1 million, with the number of fatal outcomes being almost 10 million
worldwide [1].
The camptothecin derivatives irinotecan and topotecan are frontline cancer drugs used
for the treatment of breast, small-cell lung, lymphomas, cervical, colorectal and ovarian
cancers [
of relieving the torsional DNA strain during replication, transcription and chromatin
remodeling [ ] by generating a reversible single-stranded break and covalently attaching
to the 30-end, followed by religation and releasing of DNA. The camptothecins, or Top1
poisons, stabilize the Top1 DNA complex [ ], thereby inhibiting the religation process
that eventually leads to cell death [ ]. A major disadvantage of Top1 poisons-based therapy
2–5]. Their target is Topoisomerase 1 (Top1), an enzyme that catalyzes the process
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/).
6
−
7,8
6
is the ability of the cancer cells’ DNA repair systems to remove the lesions, preventing cell
death. One of the DNA repair enzymes is tyrosyl-DNA-phosphodiesterase 1 (Tdp1), which
Molecules 2021, 26, 3128. https://doi.org/10.3390/molecules26113128
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3128
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is capable of cleaving different adducts from the DNA 30-end restoring its integrity [
].
9
On the one hand, Tdp1 is overexpressed in some types of cancer, such as non-small lung
cancer [10] and glioblastoma [11]. On the other hand, it has been shown that Tdp1 knockout
mice and Tdp1-deficient cells are hypersensitive to Top1 inhibitors [12–14]. Therefore, the
inhibition of Tdp1 could increase the efficacy of Top1 poisons, making it a promising
anticancer target [15].
Monoterpenoid is a useful moiety in potent Tdp1 inhibitors. e.g., the aniline deriva-
tive
(Figure 1)
1
with a myrtenal moiety has inhibitory activity at submicromolar concentrations
16]; coumarin-based (-)-myrtenal derivative potentiated the cytotoxicity of
[
2
camptothecin in cancer cells [17]; it has also been shown that 3, comprising diazaadaman-
tane and citronellal residues, inhibits Tdp1 with IC50 (half maximal inhibitory concentra-
tion) values of ~15
in in vitro and in vivo tumor models [19]; octahydro-2H-chromene-derived compound
was found to be an effective Tdp1 inhibitor with an IC50 value of 2 M [20]. Finally, isoben-
zofuran , containing a thiophene fragment, has a synergistic effect with topotecan in wild
µM [18]; geranyl derivative 4 enhances the topotecan antitumor effect
5
µ
6
type cells, but not in Tdp1 knockout cells [21].
Figure 1. The molecular structures of known monoterpenoid-derived Tdp1 inhibitors.
Adamantane derivatives enact many types of biological activity, including antiviral,
actoprotective, antituberculosis and cannabimimetic properties, with some in clinical
use [22]. Furthermore, the adamantane moiety is associated with the enhanced lipophilicity
and stability of the drugs [22].
Terpenoids linked to an adamantane moiety were also found to exhibit Tdp1 activity;
the dehydroabietyl amine derivative with a 1-adamantyl fragment
inhibitory activity against Tdp1, but also enhanced the antitumor effect of temozolomid
in glioblastoma cells [23]; adamantyl-derived compounds containing citronellyl and (+)-
myrtenyl fragments inhibited Tdp1 activity in the low micromolar range and citronellol
derivative potentiated the cytotoxicity of topotecan in the human colorectal carcinoma
7 (Figure 2) not only had
8
9
8
cell line HCT-116 [24]; moderate activity against Tdp1 was found for thioamides having
adamantane and 3,7-dimethyloctyl 10 and citronellyl 11 moieties, with compound 10
showing synergetic effect with topotecan [25].
1,2,4-Triazole derivatives are broadly used as antifungal, antimigraine and antiviral
agents (see Figure S1 in the Supplementary Materials). Additionally, drugs with a 1,3,4-
thiadiazole fragment are used mainly as antibacterial agents, such as acetazolamide, a
carbonic anhydrase inhibitor, which is used for the treatment of glaucoma [26] and high-
altitude illness [27].
There are few examples of Tdp1 inhibitors having a five-membered heterocyclic core.
The 1,3-thiazole moiety is as a linker between a geranyl fragment and usnic acid moiety
in compound 12 (Figure 3), with the hybrid molecule demonstrating synergetic effects
in combination with topotecan [28]. Another example of using a heterocyclic core as a

Molecules 2021, 26, 3128
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linker is compound 13, where a thiophene residue connects an octahydro-2H-chromen-4-ol
scaffold with an adamantyl residue with an IC50 value of 1.2 µM [29].
Figure 2. The molecular structures of Tdp1 inhibitors containing adamantane and terpene moieties.
Figure 3. The molecular structures of Tdp1 inhibitors with a heterocyclic linker.
In this study, we aimed at synthesizing several conjugates linking adamantane and
monoterpenoid residues via 1,2,4-triazole and 1,3,4-thiadiazole linkers, to derive their
structure–activity relationship for Tdp1 activity. This study is a continuation of our previous
work on adamantyl conjugates with monoterpenoid residues capable of inhibiting Tdp1 to
expand and improve the chemical matter of potent Tdp1 inhibitors.
2. Results and Discussion
2.1. Chemistry
According to Scheme 1, we synthesized starting compound 15 by reacting commer-
cially available adamantane-1-carbonyl chloride 14 with thiosemicarbazide, which was
used in a double excess for capturing HCl. This method allows the synthesis of the desired
compound at an excellent yield. Synthesis of 1,2,4-triazole 16 was performed by the cyclo-
condensation of compound 15 in an aqueous solution of sodium hydroxide under refluxing
conditions with 80% yield, as described previously [30]. 2-Amino-1,3,4-thiadiazole 17 was
synthesized with a 71% yield after recrystallization from EtOH following a procedure
previously described [31], which involved the treatment of starting compound 15 with
concentrated sulfuric acid at room temperature.
Scheme 1. Synthesis of starting heterocyclic compounds 16 and 17. Reagents and conditions: (
0 C, then r.t., (b) NaOH, H2O, ∆, (c) H2SO4 conc., r.t.
a
) THF,
◦
For the attachment of monoterpenoid residues to compound 16, we obtained bromides
using the previously reported methods [32 34]. The treatment of alcohols 18c with
19a–
g
–
–f

Molecules 2021, 26, 3128
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PBr3 in Et2O or THF resulted in the bromides 19c
18a
–
f
. The reaction of monoterpene alcohols
,
18b and 18g with NBS and PPh3 resulted in compounds 19a
, 19b and 19g (Scheme 2).
The further alkylation of compound 16 with bromides in the presence of MeONa in
MeOH led to the formation of the desired adamantane derivatives of 1,2,4-triazole bearing
monoterpenoid fragments 20a–g. All the reactions proceeded smoothly with moderate to
very good yields. Interestingly, the interaction of 15 with geranyl bromide 19c, with the
formation of 20c, led to its (Z)-isomer 20d as an admixture with a ratio of 9:1 (20c:20d), as
assessed by NMR. A similar situation was observed for the reaction of 15 with bromide
19d; in this case the ratio between 20d and 20c was 9.5:0.5 (Scheme 2). This ratio can be
explained by a partial isomerization process of bromides or target products as described
previously [35].
Scheme 2. Synthesis of bromides 19a
conditions: ( ) NBS, PPh3, CH2Cl2, r.t., (
(c) MeONa, MeOH, 60 C.
–
g
and target 1,2,4-triazole derivatives 20a
–
g
. Reagents and
◦
a
b
) PBr3, 0 C, (Et2O for 18b
,
18e
,
18f and THF for 18d),
◦
In order to modify amine 17, carboxylic acids 23a
previously reported procedures [36 38]. 3,7-Dimethyloctan-1-ol 18a was oxidized by Jones
reagent to obtain carboxylic acid 23a 36]. Acids 23c and 23d were synthesized using
Pinnick oxidation [37 38], which involved the treatment of aldehydes 21 and 22 with
,c,d were obtained by applying
–
[
,
sodium chlorite at mild acidic conditions. The yields of compounds 23c and 23d were 85%
and 50%, respectively (Scheme 3).
One of the most common methods to activate a carboxylic acid functional group is
converting it to the corresponding carbonyl chloride. Utilizing this protocol, we synthesized
acid chlorides 24a
–d from acids 23a, 23c and 23d and commercially available citronellic
acid 23b using SOCl2, which were all used in further reactions without purification. The
reaction of acid chlorides with amine 17 resulted in 1,3,4-thiadiazole derivatives 25a–d
containing 1-adamantyl moiety and monoterpenoid residues at acceptable yields.
Propylphosphonic acid anhydride T3P is a useful coupling reagent that generates
water-soluble by-products easily separated from reaction mixtures [39]. Applying T3P
allowed us to synthesize amides 25a
was observed after 24 h at room temperature.
–
d
at good yields and with high purity. Full conversion
-Unsaturated acids 23c and 23d were
α,β
found to react too slowly with amine 17 at room temperature. Carrying out the reaction
under refluxing conditions for 24 h in EtOAc followed by silica gel column chromatography
gave compounds 25c and 25d in very good yields (Scheme 3). Compound 26a was obtained
at a good yield using the same procedure. The rest of the amides containing citronellic and
(-)-myrtenic acid moieties were synthesized as previously reported [40].
Thus, starting from compound 15, we synthesized a series of conjugates, with 1,2,4-
triazole and 1,3,4-thiadiazole as linkers. 1,2,4-Triazol-2-thione 16 was modified by alkyla-
tion with the corresponding bromides in good yields. To obtain 1,3,4-thiadiazole amides
25a–d, two methods were utilized: (1) converting carboxylic acids into corresponding acid

Molecules 2021, 26, 3128
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chlorides followed by the acylation of amine 17; (2) direct interaction of carboxylic acids
with 1,3,4-thiadiazole 17 in the presence of coupling reagent T3P. The second approach
proved to be more convenient due to the expedient work-up of the reaction mixtures, the
high target compound yields, and the high purity of the amides.
Scheme 3. Synthesis of desired amides 25a
acetone, H2O; ( ) NaClO2, KH2PO4, DMSO, H2O, r.t.; (
r.t.; ( ) SOCl2, benzene, ; ( ) toluene, Et3N, r.t.; ( ) T3P, Py, EtOAc (r.t. for 25a,b, reflux for 25c,d);
–
d
and 26a. Reagents and conditions: (a) CrO3, H2SO4,
b
c) NaClO2, KH2PO4, H2O2, CH3CN, H2O,
d
∆
e
f
(g) 1-aminoadamantane, T3P, Py, EtOAc, r.t.
2.2. Biological Assays
In order to assess the inhibitory activity of the synthesized compounds, a fluorophore
quencher-coupled DNA-biosensor for the real-time measurement of Tdp1 cleavage activity
was used [41]. Furamidine was used as a positive control, which is a commercially available
Tdp1 inhibitor (IC50 1.23
µM) [42], and the results are presented in Table 1. 25d could not
be measured due to its poor solubility.
Table 1. The results for the Tdp1 activity inhibition at 50% (IC50) values of the derivatives. Additionally, the CC50 (50%
cytotoxic concentration) values are given for cervical cancer (HeLa) and colorectal (HCT-116, SW837) human cancer cell lines.
Structure
IC50, µM
CC50, µM HeLa
CC50, µM HCT-116
CC50, µM SW837
1,2,4-Triazole derivatives
20a
20b
20c
0.54 ± 0.09
85 ± 23
>100
27 ± 6
>100
52 ± 6
41 ± 4
41 ± 6
1.5 ± 0.3
5.3 ± 1.7
64 ± 6
16 ± 7
20d
20e
20f
5.6 ± 0.6
6.2 ± 2.2
7.5 ± 1.8
31± 11
24 ± 16
15 ± 2
20 ± 9
15 ± 5
54 ± 5
17 ± 5
40 ± 4
46 ± 6

Molecules 2021, 26, 3128
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Table 1. Cont.
Structure
IC50, µM
CC50, µM HeLa
CC50, µM HCT-116
CC50, µM SW837
1,3,4-Thiadiazole derivatives
20g
0.57 ± 0.14
74 ± 17
34 ± 6
40 ± 4
25a
25b
0.35 ± 0.05
2.59 ± 0.48
>100
>100
>100
>100
66 ± 6
83 ± 8
25c
0.45 ± 0.09
>100
>100
>100
According to Table 1, of the triazoles 20a–d with aliphatic chains, the saturated 3,7-
dimethyloctane derivative 20a was the most active as compared to its unsaturated coun-
terparts. The thio-derivatives 20c and 20d, cis-trans isomers, showed practically the same
IC50 values (~5
µM). Interestingly, the bicyclic derivative 20f had an IC50 value of 7.50
µM,
whilst 20g substituted with a (-)-nopol moiety was in the submicromolar range (0.57
µM).
The same pattern of saturated and unsaturated derivatives could be observed for the
amides 25a and 25b as for the triazoles. Additionally, 25c with a (-)-myrtenic moiety had
activity comparable to 20g, but much better activity than 20f, its triazole counterpart.
The 1,3,4-thiadiazole ring plays an important role in the inhibition of Tdp1, i.e., the
amide derivatives 26 (see Scheme 4), composed of
1-aminoadamantane [25 40], were less active, whereas their structural analogues, the
thiadiazoles (25), showed good activity. Furthermore, by comparing 20a with the pre-
viously reported amides 28a 40] and thioamide 29a 25] (Scheme 4), also made up by
a monoterpenoid and
,
–
g
–
g
[
[
1-adamantane and monoterpenoid fragments, we see that the replacement of the amide or
thioamide functional groups with a 1,2,4-triazole linker increased the potency for Tdp1.
Scheme 4. The synthesis of amides 26b
28e–g were reported previously [40].
, 28a, 28d, and 29a was previously described [25]. Compounds 26c, 28b, 28c, and

Molecules 2021, 26, 3128
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The cytotoxicity of compounds was investigated using HeLa (cervical cancer), SW837
and HCT-116 (colon cancers) cell lines, and the results are shown in Table 1 and Figure S2 in
the Supplementary Materials. The 1,3,4-thiadiazole derivatives (25) had a minor effect on
the viability of the cell lines, with only two measured CC50 values under 100
µM, whereas
the triazole series (20) had a considerable impact, with CC50 values as low as 15
µM. In
general, the HeLa cell line was less sensitive to the inhibitors than the HCT-116 and SW837
cells. Leading compounds 20c and 20g were also tested on the noncancerous cell line
HEK293A; they had no effect on cell viability (data not shown) at concentrations up to
30 µM.
The potential for sensitizing the cancer cell lines to topotecan using the Tdp1 inhibitors
was investigated using the MTT assay. Neutralizing the enzymatic activity of Tdp1 by
blocking access to its catalytic site using a small molecule should sensitize the cytotoxic
potential of topotecan, since the complex of Top1 with DNA cannot be unraveled. Interest-
ingly, other repair mechanisms than Tdp1 may also be at play in the cancer cells mitigating
the cytotoxicity of topotecan. Nontoxic concentrations of the adamantane derivatives
(5 µM) were used for the HeLa cell line; the dose response for topotecan was measured
and the corresponding CC50 values were derived, and the results are shown in Figure 4 for
20c and in Table 2 for all the derivatives.
For HeLa cells, increased cytotoxicity was seen when the adamantanes and topotecan
were combined. The most effective sensitizer was 20c, followed by all the derivatives
containing bicyclic fragments. Compound 25b, with an acyclic substituent, exhibited a
moderate sensitizing effect, but due to its poor solubility it was not considered further. The
combination index (CI) for different concentrations of topotecan and 20c at 5
µM was in the
range 0.49–0.82, and at 10 M of 20c, it was 0.23–0.78. This indicates a synergistic effect of
µ
topotecan and 20c. The CI values were calculated with the CompuSyn version 1.0 software.
For the HCT-116 cells, the synergistic effects of the compounds were different than for the
HeLa cells. The most potent compound was 20g, followed by 20c
and 25b also showed moderate sensitization. The CI plot at a different fraction affected
(Fa) was determined for topotecan (5, 15, 150, 500 and 1500 nM) and 20g (1, 3, 10, 20 M).
, 20e, 25a and 25c. 20b
µ
The CI values for most of the combinations correspond to a synergistic effect (CI < 1, see
Figure S3 in the Supplementary Materials).
Figure 4. Sensitizing effect of 20c at 5 µM non-toxic concentration on the viability of HeLa cells.

Molecules 2021, 26, 3128
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Table 2. The effect of the Tdp1 inhibitors at 5
µM on the 50% cytotoxic concentration (CC50) value of
topotecan for HeLa cancer cells. The concentrations of the Tdp1 inhibitors are 5 µM for HeLa cells and
ranged from 15 to 45
µM (concentration is given in parenthesis in
µM) for the HCT-116 cancer cell line.
Compound
HeLa—CC50, µM
HCT116—CC50, nM
Topotecan
20a
6.0
4.5
ND *
1.9
5.4
ND
2.8
2.6
4.9
2.9
3.6
715.0
650.0 (20)
575.3 (20)
460.0 (15)
689.0 (15)
392.0 (15)
715.0 (25)
235.8 (20)
455.7 (45)
611.9 (20)
486.0 (25)
20b
20c
20d
20e
20f
20g
25a
25b
25c
* Not determined due to poor solubility.
2.3. Molecular Modeling
Thirteen adamantane–monoterpene derivatives were docked into the binding site
of the Tdp1 structure (PDB ID: 6DIE, resolution 1.78 Å) [43] with three water molecules
retained (HOH814, 821 and 1078). It has been shown that keeping these crystalline water
molecules improves the prediction quality of the docking scaffold (see the Methodology
section for further information) [44]. The binding predictions of the four scoring functions
used are given in Table S1 in the Supplementary Materials; all the ligands show reasonable
scores, but no linear correlation was observed with the measured IC50 values. Nevertheless,
when the scores of the ligands with IC50 values >15 µM were calculated, a clear statistical
difference was seen between their average and that of the active compounds, with the
exception of ChemScore (CS) (see Table S1). When the docked poses were analyzed, it
emerged that the adamantane and monoterpene moieties were driving the binding, i.e.,
adamantane occupied the pocket containing both the catalytic histidine amino acid residues
(His263 and 493) and the monoterpene fit into a lipophilic cleft. No hydrogen bonding
interactions were predicted, indicating that the interaction was dominated by lipophilic
contacts. The predicted configuration of 20a is shown in Figure 5 as an example of the
dominant binding mode.
Figure 5. The docked pose of 20a in the binding site of Tdp1 as predicted by the ChemPLP scoring function. (A) The
predicted configuration is shown in the ball-and-stick format and the adjacent amino acid residues are depicted in the
stick format; the hydrogen atoms are not shown for clarity. The amino acids forming the pocket accommodating the
adamantine cage are Tyr204, His263, Lys269, Asn283, Ser399, His493 and Lys495; the lipophilic cleft occupied by the
aliphatic monoterpene is made up by Pro461, Gly498 and Try590. (B) The protein surface is rendered; blue depicts regions
with a partial positive charge on the surface; red depicts regions with a partial negative charge and grey shows neutral
areas. The ligand occupies the catalytic binding pocket, blocking access to it.

Molecules 2021, 26, 3128
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A subset of ligands with varied affinity to Tdp1 is 20a (0.54
µM), 20b (1.5
µM) and 20c
(5.30 µM). These have similar structures; only the saturation state of the aliphatic chain is
different, and thus an interesting trend is seen. The binding of a ligand to an enzyme is
governed by Equation (1).
∆GBind = ∆GDehydration + ∆GCon f iguration + ∆GIntramol.
(1)
binding
The three ligands were all predicted to bind to Tdp1 in the same manner, i.e., the
configuration and intramolecular binding terms would be the same. When the log p
values are considered, since log P correlates with ∆GDehydration [45], an excellent negative
correlation is seen (R2—0.894, see Figure S4A). It is therefore clear that the interaction of
these ligands is governed by an entropic push from the water phase and lipophilic contacts
with the binding pocket’s surface.
The same trend was seen when structurally near-identical ligands 20f (7.50
20g (0.57 M) were compared; they had the same predicted binding, but 20g had a higher
log p value than 20f (6.1 vs. 5.8). This trend was repeated for the 20a (0.54 M) and 28a
(>15 M) pair, with the same binding pose but substantially different log p values (6.6 vs.
5.1). For 20g (0.57 M) and 28g (>15 M), a structurally similar pair, the latter was not
docked into the catalytic binding pocket, i.e., it did not fit into it and therefore did not
fulfill the Configuration term in Equation (1). Interestingly, 28g docked into the same site
as our previously reported adamantine–monoterpene series [25]. Finally, the difference
in the 25a (0.45 M) and 26a (>15 M) pair can be explained by the low molecular weight
µM) and
µ
µ
µ
µ
µ
∆
G
µ
µ
of the latter (383.6 vs. 299.5 g mol−1), as relatively small ligands have decreased binding
affinity compared to their much bigger counterparts (see Chemical Space section and
Figure S4B). This can be explained in terms of the smaller molecules having relatively few
intramolecular interactions with the enzyme, leading to the third term in Equation (1) being
small. In conclusion, the activity, or inactivity, of the ligands depends on their lipophilicity,
as well as their fitting into the binding pocket (configuration) and finally having sufficient
intramolecular interactions with the enzyme.
2.4. 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 S2 in the Supplementary
Materials. Interestingly, when the molecular descriptor numbers were correlated with
the IC50 values, MW showed good correlation with R2—0.5638, and HA (R2—0.2267) and
PSA (R2—0.2242) also had reasonable correlations (see Figure S4 in the Supplementary
Materials). A correlation between the molecular descriptor values and their corresponding
binding efficacy to Tdp1 has been previously seen for deoxycholic acid derivatives, with
MW having an R2 of 0.452, and 0.316 for RB [46]. The values of the molecular descriptors
all lie within the lead- and drug-like chemical space, except for log P, which ranged from
4.7 to 6.6, thus reaching into the known drug space (for the definition of lead-like, drug-like
and known drug space regions, see [47] and Table S3).
The known drug indexes (KDIs) for the ligands were calculated to gauge the balance
of the molecular descriptors (MW, log P, HD, HA, PSA and RB). This method is based on
the analysis of drugs in clinical use, i.e., the statistical distribution of each descriptor is
fitted to a Gaussian function and normalized to 1, resulting in a weighted index. Both the
summation of the indexes (KDI2a) and multiplication (KDI2b) methods were used [48], as
shown for KDI2a in Equation (2) and for KDI2b in Equation (3); the numerical results are
given in Table S2 in the Supplementary Materials.
KDI2a = IMW + Ilog P + IHD + IHA + IRB + IPSA
(2)
(3)
KDI2b = IMW × Ilog P × IHD × IHA × IRB × IPSA

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The KDI2a values for the ligands range from 4.82 to 5.59, with a theoretical maximum
of 6 and an average of 4.08 ( 1.27) for known drugs. These values are very good, since most
±
of the descriptors lie within the lead- and drug-like boundaries of chemical space, except
log P. The KDI2b ranges from 0.23 to 0.64, with a theoretical maximum of 1 and with a KDS
average of 0.18 ( 0.20). Again, good values were obtained for the ligands even though
±
the KDI2b index is more sensitive than KDI2a to outliers, since the multiplication of small
numbers leads to smaller numbers. It can be concluded that the ligands are biocompatible
as compared to drugs in clinical use.
3. Materials and Methods
3.1. Chemistry
All chemicals were purchased from commercial sources (Sigma Aldrich, Acros Or-
1
ganics) and used without further purification. H and 13C NMR spectra were recorded
on a Bruker AV-300 spectrometer (300.13 MHz and 75.46 MHz, respectively), a Bruker
AV-400 (400.13 MHz and 100.61 MHz), a Bruker DRX-500 (500.13 MHz and 125.76 MHz)
and on a Bruker Advance—III 600 (600.30 MHz and 150.95 MHz). Residual chloroform
signals were used as references (δH 7.24, δC 76.90 ppm). Compound structures were deter-
mined by analyzing the 1H-NMR spectra and 1H–1H 2D homonuclear correlation (COSY,
NOESY), J-modulated 13C NMR spectra (JMOD), and 13C–1H 2D heteronuclear correlation
with one-bond (HSQC, 1J = 145 Hz) and long-range spin–spin coupling constants (HMBC,
2,3J = 7 Hz). Mass spectra (70 eV) were recorded on a DFS Thermo Scientific high-resolution
mass spectrometer. The conversion of starting materials was analyzed by thin-layer chro-
matography, which was performed on Merck plates (UV-254). A PolAAr 3005 polarimeter
was used to measure optical rotations ([α]D). Target compounds were isolated by column
chromatography (SiO2; 63–200 m; Macherey-Nagel). Melting points were measured on
µ
a Mettler Toledo FP900 Thermosystem apparatus. Spectral and analytical measurements
were carried out at the Multi-Access Chemical Service Center of Siberian Branch of Russian
Academy of Sciences (SB RAS). The atom numeration of the substances is provided for the
assignment of signals into the NMR spectra, and differs from that in IUPAC. The spectra
are shown in the Supplementary Materials, Figures S5–S40.
Synthesis of 2-(adamantane-1-carbonyl)hydrazine-1-carbothioamide 15
A solution of◦adamantane-1-carbonyl chloride (10.0 g, 50.4 mmol) in 30 mL of THF
was added to a 0 C suspension of thiosemicarbazide (10.08 g, 110.8 mmol) in 200 mL
of THF while stirring. The resulting mixture was stirred at room temperature overnight
followed by evaporating the solvent on a rotary evaporator. To the reaction mixture was
added water; the solid mass was filtered off, washed with water thoroughly and dried. The
yield of the compound 15 was 11.48 g (90%); the product was isolated as a white solid.
Synthesis of 5-(adamantan-1-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione 16
A mixture containing 5.0 g (8.0 mmol) of 2-(adamantane-1-carbonyl) hydrazine-1-
carbothioamide 15 and 1 M solution of NaOH (25 mL) was refluxed for 3 h. After cooling
to room temperature, the mixture was neutralized using concentrated HCl. The precipitate
was collected by filtration, washed with water, and recrystallized from MeOH to give the
product as a white solid (1.5 g, 80%).
General procedure for obtaining 1,2,4-triazole derivatives 20a–20g
To a suspension of compound 16 (0.25 g, 1.06 mmol) in 1 mL of MeOH was added
0.304 mL of a 3.5 M solution of MeONa in MeOH. The solution obtained was stirred at
room temperature for 30 min, and then corresponding bromide was added. The reaction
mixture was stirred at 60 ◦C for 12 h and then the solvent was evaporated under reduced
pressure. The product was extracted with Et2O. The desired compound was purified using
column chromatography using a n-hexane to ethyl acetate gradient.

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5-(Adamantan-1-yl)-3-((3,7-dimethyloctyl)thio)-1H-1,2,4-triazole 20a
Colorless oil, yield 75%.
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.82 (d, J(23, 22) = J(24, 22) = 6.7 Hz, 6Н, 3Н-23,
3Н-24), 0.84 (d, J(25, 18) = 6.5 Hz, 3Н, Н-25), 1.03–1.12 (m, 3Н, Н-19b, 2Н-21), 1.15–1.29 (m,
3Н, Н-19a, 2Н-20), 1.42–1.55 (m, 3Н, Н-17b, Н-18, Н-22), 1.62–1.69 (m, 1Н, Н-17a), 1.70–1.77
(m, 6Н, 2Н-4, 2Н-6, 2Н-10), 1.96 (d, 3J = 3.0 Hz, 6Н, 2Н-2, 2Н-8, 2Н-9), 2.02–2.06 (m, 3Н,
Н-3, Н-5, Н-7), 3.02 (ddd, J(16b, 16a) = 12.5 Hz, J(16b, 17b) = 9.5 Hz, J(16b, 17a) = 6.3 Hz,
1Н, Н-16b), 3.10 (ddd, J(16a, 16b) = 12.5 Hz, J(16a, 17a) = 9.8 Hz, J(16a, 17b) = 5.4 Hz, 1Н,
H-16a). 13C-NMR (150 MHz, CDCl3) (ppm)
δ: 34.12 (s, C-1), 40.68 (t, C-2, C-8,C-9), 27.91 (d,
C-3, C-5, C-7), 36.25 (t, C-4, C-6, C-10), 166.49 (s, C-11), 159.05 (s, C-13), 30.24 (t, C-16), 36.58
(t, C-17), 32.10 (d, C-18) 36.76 (t, C-19), 24.49 (t, C-20), 39.08 (t, C-21), 27.81 (d, C-22), 22.45,
22.55 (2q, C-23, C-24), 19.17 (q, C-25). HR MS: 375.2705 (M+, C22H37N3S1+; calc. 375.2703).
5-(Adamantan-1-yl)-3-(((S)-3,7-dimethyloct-6-en-1-yl)thio)-1H-1,2,4-triazole 20b
Colorless oil, yield 77%.
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.84 (d, J(25, 18) = 6.5 Hz, 3Н, Н-25), 1.07–1.14 (m,
1Н, Н-19b), 1.25–1.32 (m, 1Н, Н-19a), 1.42–1.55 (m, 2Н, Н-17b, Н-18), 1.54 (br.s, 3Н, Н-24),
1.62 (m, 3Н, Н-23), 1.61–1.68 (m, 1Н, Н-17a), 1.68–1.75 (m, 6Н, 2Н-4. 2Н-6, 2Н-10), 1.84–1.98
(m, 2Н, Н-20), 1.94 (d, 3J = 3.0 Hz, 6Н, 2Н-2, 2Н-8, 2Н-9), 2.00–2.04 (m, 3Н, Н-3, Н-5, Н-7),
3.01 (ddd, J(16b, 16a) = 12.4 Hz, J(16b, 17) = 9.4 Hz, J(16b, 17a) = 6.2 Hz, 1Н, Н-16b), 3.08
(1Н, ddd, J(16a, 16b) = 12.4 Hz, J(16a, 17a) = 9.8 Hz, J(16a, 17b) = 5.4 Hz, H-16a), 5.03 (t.m,
J(21, 20) = 7.1 Hz, 1Н, Н-21). 13C-NMR (150 MHz, CDCl3) (ppm)
δ: 34.11 (s, C-1), 40.65 (t,
C-2, C-8, C-9), 27.90 (d, C-3, C-5, C-7), 36.22 (t, C-4, C-6, C-10), 166.45 (s, C-11), 158.99 (s,
C-13), 30.15 (t, C-16), 36.47 (t, C-17), 31.73 (d, C-18), 36.54 (t, C-19), 25.23 (t, C-20), 124.47 (d,
C-21), 131.02 (s, C-22), 25.52 (q, C-23), 17.47 (q, C-24), 19.02 (q, C-25). HR MS: 373.2542 (M+,
C22H35N3S1+; calc. 373.2546). [α]2D4.5 = +7 (c 0.48 in МеОН).

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5-(Adamantan-1-yl)-3-(((E)-3,7-dimethylocta-2,6-dien-1-yl)thio)-1H-1,2,4-triazole 20c
White solid, mp = 106.6–109.8 ◦C, yield 67%.
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 1.56 (br.s, 3Н, Н-24), 1.62 (m, 3Н, Н-25), 1.64 (m, 3Н,
Н-23), 1.71–1.79 (m, 6Н, 2Н-4, 2Н-6, 2Н-10), 1.93–2.05 (m, 4Н, 2Н-19, 2Н-20), 1.99 (d, 3J =
3.0 Hz, 6Н, 2Н-2, 2Н-8, 2Н-9), 2.04–2.08 (m, 3Н, Н-3, Н-5, Н-7), 3.72 (d, J(16, 17) = 7.8 Hz,
2Н, Н-16), 5.02 (t.m, J(20, 21) = 6.9 Hz, 1Н, Н-21), 5.33 (t.m, J(17, 16) = 7.8 Hz, 1Н, Н-17).
13C-NMR (150 MHz, CDCl3) (ppm)
δ: 34.18 (s, C-1), 40.78 (t, C-2, C-8, C-9), 27.95 (d, C-3,
C-5, C-7), 36.30 (t, C-4, C-6, C-10), 167.05 (s, C-11), 158.32 (s, C-13), 30.69 (t, C-16), 118.91
(d, C-17), 140.41 (s, C-18), 39.43 (t, C-19), 26.27 (t, C-20), 123.74 (d, C-21), 131.64 (s, C-22),
25.55 (q, C-23), 17.59 (q, C-24), 15.98 (q, C-25). HR MS: 371.2389 (M+, C22H33N3S1+; calc.
371.2390).
5-(Adamantan-1-yl)-3-(((Z)-3,7-dimethylocta-2,6-dien-1-yl)thio)-1H-1,2,4-triazole 20d
White solid, mp = 110.9–113.5 ◦C, yield 60%.
1H-NMR (600 MHz, CDCl3) (ppm)
Н-25), 1.70–1.77 (m, 6Н, 2Н-4, 2Н-6, 2Н-10), 1.96 (d, J = 3.0 Hz, 6Н, 2Н-2, 2Н-8, 2Н-9),
1.98–2.06 (m, 7Н, Н-3, Н-5, Н-7, 2Н-19, 2Н-20), 3.70 (d.m, J(16, 17) = 8.0 Hz, 2Н, Н-16),
5.06 (t.m, J(21, 20) = 6.7 Hz, 1Н, Н-21), 5.32 (t.m, J(17, 16) = 8.0 Hz, 1Н, Н-17). 13C-NMR
δ
: 1.56 (m, 3Н, Н-24), 1.64 (br.s, 3Н, Н-23), 1.67 (m, 3Н,
3
(150 MHz, CDCl3) (ppm) δ: 34.14 (s, C-1), 40.71 (t, C-2, C-8, C-9), 27.93 (d, C-3, C-5, C-7),
36.26 (t, C-4, C-6, C-10), 166.70 (s, C-11), 158.63 (s, C-13), 30.40 (t, C-16), 119.60 (d, C-17),
140.32 (s, C-18), 31.71 (t, C-19), 26.48 (t, C-20), 123.72 (d, C-21), 131.79 (s, C-22), 25.53 (q,
C-23), 17.54 (q, C-24), 23.27 (q, C-25). HR MS: 371.2393 (M+, C22H33N3S1+; calc. 371.2390).
5-(Adamantan-1-yl)-3-((((S)-4-(prop-1-en-2-yl)cyclohex-1-en-1-yl)methyl)thio)-1H-1,2,4-
triazole 20e
Colorless oil, yield 65%.

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1H-NMR (600 MHz, CDCl3) (ppm)
δ
: 1.38–1.47 (m, 1Н, Н-21b), 1.68 (br.s, 3Н, Н-25), 1.71–
1.81 (m, 7Н, 2Н-4, 2Н-6, 2Н-10, Н-21a), 1.82–1.90 (m, 1Н, Н-19b), 1.99 (d, 3J = 3.0 Hz, 6Н,
2Н-2, 2Н-8, 2Н-9), 2.01–2.10 (m, 5Н, Н-3, Н-5, Н-7, Н-19a, Н-20), 2.13–2.18 (m, 2Н, Н-22),
3.65 (br.d, J(16b, 16a) = 12.9 Hz, 1Н, Н-16b), 3.69 (br.d, J(16a, 16b) = 12.9 Hz, 1Н, Н-16a),
4.64 (m, 1Н, Н-24b), 4.67 (m, 1Н, Н-24a), 5.61–5.65 (m, 1Н, Н-18). 13C-NMR (150 MHz,
CDCl3) (ppm) δ: 34.18 (s, C-1), 40.77 (t, C-2, C-8, C-9), 27.94 (d, C-3, C-5, C-7), 36.28 (t, C-4,
C-6, C-10), 166.96 (s, C-11), 158.19 (s, C-13), 39.90 (t, C-16), 133.01 (s, C-17), 125.29 (d, C-18),
30.63 (t, C-19), 40.56 (d, C-20), 27.38, 27.43 (2t, C-21, C-22), 149.50 (s, C-23), 108.56 (t, C-24),
20.64 (q, C-25). HR MS: 369.2235 (M+, C22H33N3S1+; calc. 369.2233). [α]D24
in МеОН).
= ć243 (c 0.24
5-(Adamantan-1-yl)-3-((((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)methyl)thio)-1H-
1,2,4-triazole 20f
White solid, mp = 121.1–121.5 ◦C, yield 70%.
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.72 (s, 3Н, Н-25), 1.07 (d, J(23b, 23a) = 8.7 Hz, 1Н,
Н-23b), 1.23 (s, 3Н, Н-24), 1.70–1.78 (m, 6Н, 2Н-4, 2Н-6, 2Н-10), 1.97 (d, 3J = 3.0 Hz, 6Н,
2Н-2, 2Н-8, 2Н-9), 2.00–2.09 (m, 4Н, Н-3, Н-5,Н-7, Н-20), 2.14 (d.m, J(19b, 19a) = 17.8 Hz,
1Н, Н-19b), 2.17–2.23 (m, 2Н, Н-19a, H-22), 2.34 (1Н, ddd, J(23a, 23b) = 8.7 Hz, J(23a, 20)
= J(23a, 22) = 5.6 Hz, Н-23a), 3.66–3.72 (m, 2Н, Н-16), 5.45–5.48 (m, 1Н, Н-18). 13C-NMR
(150 MHz, CDCl3) (ppm) δ: 34.17 (s, C-1), 40.73 (t, C-2, C-8, C-9), 21.93 (d, C-3, C-5, C-7),
36.29 (t, C-4, C-6, C-10), 166.83 (s, C-11), 158.17 (s, C-13), 38.88 (t, C-16), 142.70 (s, C-17),
121.11 (d, C-18), 31.20 (t, C-19), 40.31 (d, C-20), 37.97 (s, C-21), 44.97 (d, C-22), 31.56 (t, C-23),
25.96 (q, C-24), 20.95 (q, C-25). HR MS: 369.2233 (M+, C22H31N3S1+; calc. 369.2234). [α]2D4.5
= −14 (c 0.53 in МеОН).
5-(Adamantan-1-yl)-3-((2-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethyl)thio)-1H-
1,2,4-triazole 20g
Colorless oil, yield 85%.

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1H-NMR (600 MHz, CDCl3) (ppm)
δ
: 0.78 (s, 3Н, Н-26), 1.10 (d, J(24b, 24a) = 8.6 Hz, 1Н,
Н-24b), 1.22 (s, 3Н, Н-25), 1.69–1.76 (m, 6Н, 2Н-4, 2Н-6, 2Н-10), 1.95 (d, 3J = 3.0 Hz, 6Н,
2Н-2, 2Н-8, 2Н-9), 1.99–2.05 (m, 5Н, Н-3, Н-5, Н-7, Н-21, Н-23), 2.14 (dm, J(20b, 20a) = 17.6
Hz, 1H, H-20b), 2.20 (dm, J(20a, 20b) = 17.6 Hz, 1H, H-20a), 2.25–2.32 (m, 3Н, 2Н-17, Н-24a),
3.02–3.11 (m, 2Н, Н-16), 5.19–5.22 (m, 1Н, Н-19). 13C-NMR (150 MHz, CDCl3) (ppm)
δ:
34.12 (s, C-1), 40.67 (t, C-2, C-8, C-9), 27.90 (d, C-3, C-5, C-7), 36.24 (t, C-4, C-6, C-10), 166.45
(s, C-11), 159.01 (s, C-13), 30.25 (t, C-16), 36.89 (t, C-17), 146.03 (s, C-18), 117.65 (d, C-19),
31.11 (t, C-20), 40.58 (d, C-21), 37.83 (s, C-22), 45.39 (d, C-23), 31.47 (t, C-24), 26.12 (q, C-25),
21.03 (q, C-26). HR MS: 383.2388 (M+, C23H33N3S1+; calc. 383.2390). [α]D24.5
in МеОН).
=
−
21 (c 0.51
General procedure for synthesis of bromides
To a solution of the corresponding monoterpenoid alcohol (6.5 mmol) in 30 mL of Et2O
cooled to 0 ◦C was added 0.3 mL (3.1 mmol) of PBr3 dropwise. The resulting mixture was
stirred at the same temperature for 4 h. After the reaction was completed, the solution was
poured onto crushed ice and extracted with Et2O. The combined organic layer was washed
with saturated aqueous NaHCO3 then brine and dried over anhydrous sodium sulfate.
The solvent was removed under reduced pressure; the resulting crude was used without
1
further purification. H NMR spectra were consistent with previously reported data.
(E)-1-Bromo-3,7-dimethylocta-2,6-diene 19c, yield 84%. The spectral data were consis-
tent with those in the literature [49].
(S)-1-(Bromomethyl)-4-(prop-1-en-2-yl)cyclohex-1-ene 19e, yield 77%. The spectral
data were consistent with those in the literature [50].
2-(Bromomethyl)-6,6-dimethylbicyclo[3.1.1]hept-2-ene 19f, yield 72%. The spectral
data were consistent with those in the literature [51].
A solution of PBr3 (0.247 mL, 2.6 mmol) in 4 mL of dry THF was added dropwise to
a solution of nerol (1.0 g, 6.5 mmol) in 7 mL of THF under argon at
−
10 ◦C. The reaction
mixture was stirred for 1.5 h and the solvent was evaporated under reduced pressure.
The residue was dissolved in a mixture of n-hexane/diethyl ether (1:1). The solution was
washed with a saturated aqueous solution of NaHCO3, then with water and brine. The
organic phase was dried over sodium sulfate, and the solvent was evaporated. The product
was used directly without further purification.
(Z)-1-Bromo-3,7-dimethylocta-2,6-diene 19d, yield 68%. The spectral data were consis-
tent with those in the literature [33].
To a solution of PPh3 (12.2 g, 46 mmol) in dry DCM (46 mL) was added N-bromosuccin-
imide (NBS) (8.4 g, 46 mmol) in small portions under an ice-water bath. The mixture was
cooled to r.t. and was stirred for 30 min. Then, pyridine (2 mL) was added, followed by
the corresponding alcohol (24 mmol). The reaction mixture was stirred overnight at room
temperature. Then, the mixture was diluted with n-hexane (60 mL) and filtered through a
silica gel plug. The reaction flask was stirred three times with EtOAc: n-hexane (6:6 mL)
for around 1 h and filtered through the silica gel plug. The solution was concentrated
in vacuo. The crude residue was purified by flash chromatography (n-hexane) to obtain
the corresponding bromide as a colorless oil. The 1H NMR spectra were consistent with
previously reported data.

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1-Bromo-3,7-dimethyloctane 19a, yield 90%. The spectral data were consistent with
those in the literature [52].
(S)-8-Bromo-2,6-dimethyloct-2-ene 19b, yield 85%. The spectral data were consistent
with those in the literature [53].
2-(2-Bromoethyl)-6,6-dimethylbicyclo[3.1.1]hept-2-ene 19g, yield 88%. The spectral
data were consistent with those in the literature [34].
Synthesis of 5-(adamantan-1-yl)-1,3,4-thiadiazol-2-amine 17
A solution of 2.2 g (8.7 mmol) of 2-(adamantane-1-carbonyl) hydrazine-1-
carbothioamide in 20 mL of concentrated sulfuric acid was kept at room temperature
overnight, and then the solution was neutralized with aqueous ammonia solution while
cooling with ice until pH 7 was reached. The resulting solid was filtered off, washed with
water, dried and recrystallized from EtOH to yield 5-(adamantan-1-yl)-1,3,4-thiadiazol-2-
amine as a pale yellow solid (1.46 g, 71%).
Synthesis of 3,7-dimethyloctanoic acid 23a
A solution of 3,7-dimethyloctan-1-ol (5.0 g, 31.6 mmol) in acetone (17 mL) was added
dropwise to a stirred solution of chromium trioxide (7.0 g, 70 mmol) in water (11 mL)
and concentrated sulfuric acid (2 mL) cooled to 0 ◦C for 30 min. The mixture was then
diluted with water and extracted with ether. The organic layer was washed with 10%
sodium hydroxide solution and the water fraction was separated followed by acidification
of the alkaline solution by 10% HCl. The product was extracted with ether to afford
3,7-dimethyloctanoic acid (3.06 g, 55%) as a colorless oil.
Synthesis of (-)-myrtenic acid 23c
A solution of NaClO2 (8.0 g, 70 mmol) in H2O (70 mL) was added slowly for 2 h to a
stirred mixture of (-)-myrtenal (7.7 g, 51 mmol) in CH3CN (50 mL), KH2PO4 (1.8 g) in water
◦
(20 mL), H2O2 (37%, 5 mL, 52 mmol) and polyethylene glycol (PEG-400, 3.0 g) at 10 C with
ice-water cooling. The reaction was stirred overnight at room temperature. Then, Na2SO3
(0.5 g) was added. The resulting mixture was acidified with 10% aqueous HCl to pH 3 and
extracted several times with diethyl ether. The separated organic phase was washed with
saturated sodium bisulfite and water, respectively, and then dried over anhydrous sodium
sulfate. Evaporation of the solvent gave (-)-myrtenic acid as a yellow solid (7.67 g, 90%).
Synthesis of (S)-4-(prop-1-en-2-yl)cyclohex-1-ene-1-carboxylic acid 23d
A solution of 1.12 g (10 mmol) of 80% sodium chlorite in 10 mL of distilled water
was added dropwise over 30 min at ambient temperature to a stirred mixture of 1.0 g
(6.7 mmol) of (S)-(-)-perillaldehyde in 7 mL of DMSO and 0.28 g of KH2PO4 in 3 mL of
water. The mixture was stirred at ambient temperature overnight. The reaction mixture
was quenched with 30 mL of 10% sodium hydroxide solution followed by extraction of non-
acidic compounds with 80 mL of diethyl ether three times. After acidifying the aqueous
phase with concentrated HCl, the acid was extracted with 30 mL of diethyl ether three
times. The ether layers were combined and dried over sodium sulfate. The evaporation of
the solvent under reduced pressure gave 0.5 g (50%) of a slightly yellow solid.
General procedure for acid chloride synthesis
To a solution of carboxylic acid (1.8 mmol) in 10 mL of benzene was added SOCl2
(5.4 mmol). The solution was refluxed for 3 h and the solvent was evaporated under
reduced pressure. The crude material was used with no further purification.
General procedure for obtaining 25a–d and 26a
Method A. A solution of corresponding carboxylic acid chloride (1.2 mmol) in dry
toluene (1 mL) was added to a suspension of amine 17 (1.06 mmol) and triethylamine
(0.177 mL, 1.2 mmol) in 10 mL of dry toluene. The reaction mixture was stirred overnight
at room temperature and the solvent was evaporated under reduced pressure. To the
residue was added the aqueous solution of KOH, followed by extraction with EtOAc. The

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product was purified using silica gel column chromatography using an n-hexane to ethyl
acetate gradient.
Method B. To a mixture of a carboxylic acid (0.9 mmol), amine 17 (1.06 mmol), pyridine
(0.264 mL) and EtOAc (0.528 mL) were added T3P (50 wt. % in EtOAc, 2 mmol) while
stirring. The reaction mixture was stirred overnight, then water was added. The precipitate
formed was washed with water and dried. The product obtained was purified using silica
gel column chromatography using an n-hexane to ethyl acetate gradient.
N-(5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl)-3,7-dimethyloctanamide 25a
White solid, mp = 148.6 ◦C followed by decomposition, yield 61% (method A), 81%
(method B).
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.81 (d, J(23, 22) = J(24, 22) = 6.7 Hz, 6Н, Н-23, Н-24),
0.97 (d, J(25, 18) = 6.7 Hz, 3Н, Н-25), 1.08–1.16 (m, 2Н, Н-21), 1.20–1.43 (m, 4Н, 2Н-19,
2Н-20), 1.43–1.51 (m, 1Н, Н-22), 1.73–1.82 (m, 6Н, 2Н-4, 2Н-6, 2Н-10), 2.06 (d, 3J = 2.8 Hz,
6Н, 2Н-2, 2Н-8, 2Н-9), 2.07–2.15 (m, 4Н, Н-3, Н-5, Н-7, Н-18), 2.58 (dd, 2J = 14.1 Hz, J(17b,
18) = 8.5 Hz, 1Н, Н-17b), 2.72 (dd, 2J = 14.1 Hz, J(17a, 18) = 6.1 Hz, 1Н, Н-17a). 13C-NMR
(150 MHz, CDCl3) (ppm) δ: 37.71 (s, C-1), 43.07 (t, C-2, C-8, C-9), 28.29 (d, C-3, C-5, C-7),
36.29 (t, C-4, C-6, C-10), 174.22 (s, C-11), 159.87 (s, C-14), 171.69 (s, C-16), 43.72 (t, C-17),
30.95 (d, C-18), 36.88 (t, C-19), 24.37 (t, C-20), 39.04 (t, C-21), 27.76 (d, C-22), 22.41 (q, C-23),
22.56 (q, C-24), 19.28 (q, C-25). HR MS: 389.2495 (M+, C22H35О1N3S1+; calc. 389.2496).
N-(5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl)-3,7-dimethyloct-6-enamide 25b
White solid, mp = 136.7 ◦C followed by decomposition, yield 52% (method A), 85%
(method B).
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.99 (d, J(25, 18) = 6.8 Hz, 3Н, Н-25), 1.26–1.33 (m,
1Н, Н-19b), 1.43–1.50 (m, 1Н, Н-19a), 1.55 (s, 3Н, Н-24), 1.63 (m, 3Н, Н-23), 1.73–1.82 (m,
6Н, 2Н-4, 2Н-6, 2Н-10), 1.92–2.07 (m, 2Н, Н-20), 2.05 (d, 3J = 2.8 Hz, 6Н, 2Н-2, 2Н-8, 2Н-9),
2.07–2.17 (m, 4Н, Н-3, Н-5, Н-7, Н-18), 2.59 (dd, 2J = 14.0 Hz, J(17b, 18) = 8.5 Hz, 1Н, Н-17b),
2.73 (dd, 2J = 14.0 Hz, J(17a, 18) = 6.1 Hz, 1Н, Н-17a), 5.06 (t.m, J(21, 20) = 7.0 Hz, 1Н, Н-21).
13C-NMR (150 MHz, CDCl3) (ppm)
δ: 37.74 (s, C-1), 43.04 (t, C-2, C-8, C-9), 28.29 (d, C-3,
C-5, C-7), 36.26 (t, C-4, C-6, C-10), 174.27 (s, C-11), 159.90 (s, C-14), 171.59 (s, C-16), 43.58

Molecules 2021, 26, 3128
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(t, C-17), 30.66 (d, C-18), 36.68 (t, C-19), 25.20 (t, C-20), 124.27 (d, C-21), 131.25 (s, C-22),
25.56 (q, C-23), 17.50 (q, C-24), 19.21 (q, C-25). HR MS: 387.2339 (M+, C22H33О1N3S1+; calc.
387.2337).
(1R,5S)-N-(5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl)-6,6-dimethylbicyclo[3.1.1]hept-2-ene-
2-carboxamide 25c
White solid, mp = 240.2 ◦C followed by decomposition, yield 50% (method A), 70%
(method B).
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.84 (s, 3Н, Н-25), 1.21 (d, J(23b, 23a) = 9.2 Hz, 1Н,
Н-23b), 1.34 (s, 3Н, Н-24), 1.72–1.81 (m, 6Н, 2Н-4, 2Н-6, 2Н-10), 2.05 (d, 3J = 2.7 Hz, 6Н,
2Н-2, 2Н-8, 2Н-9), 2.07–2.11 (m, 3Н, Н-3, Н-5, Н-7), 2.14–2.19 (m, 1Н, Н-20), 2.47–2.53 (2Н,
m, Н-19b, Н-23a), 2.56 (ddd, 2J = 19.7 Hz, J(19a, 18) = J(19a, 20) = 3.0 Hz, 1Н, Н-19a), 2.91
(ddd, J(22, 20) = J(22, 23a) = 5.6 Hz, J(22, 18) = 1.5 Hz, 1Н, Н-22), 7.13–7.16 (m, 1Н, Н-18).
13C-NMR (150 MHz, CDCl3) (ppm)
δ: 37.68 (s, C-1), 43.07 (t, C-2, C-8, C-9), 28.28 (d, C-3,
C-5, C-7), 36.29 (t, C-4, C-6, C-10), 173.99 (s, C-11), 160.51 (s, C-14), 164.92 (s, C-16), 140.86
(s, C-17), 136.01 (d, C-18), 32.33 (t, C-19), 40.16 (d, C-20), 37.83 (s, C-21), 41.17 (d, C-22),
31.23 (t, C-23), 25.76 (q, C-24), 20.93 (q, C-25). HR MS: 383.2026 (M+, C22H29О1N3S1+; calc.
383.2024). [α]2D6 = −11 (c 0.84 in CHCl3).
(S)-N-(5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl)-4-(prop-1-en-2-yl)cyclohex-1-ene-1-
carboxamide 25d
White solid, mp = 265.6 ◦C followed by decomposition, yield 55% (method A), 75%
(method B).
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 1.49–1.57 (m, 1Н, Н-21b), 1.72–1.82 (m, 6Н, 2Н-4,
2Н-6, 2Н-10) 1.75 (s, 3Н, Н-25), 1.92–1.98 (m, 1Н, Н-21a), 2.06 (d, 3J = 3.0 Hz, 6Н, 2Н-2, 2Н-8,
2Н-9), 2.07–2.12 (m, 3Н, Н-3, Н-5, Н-7), 2.18–2.27 (m, 2Н, Н-19b, H-20), 2.35–2.44 (m, 1H,
H-22b), 2.45–2.52 (m, 1H, H-19a), 2.58–2.65 (m, 1H, H-22a), 4.72 (br.s, 1Н, Н-24b), 4.75–4.77
(m, 1Н, Н-24a), 7.23–7.26 (m, 1Н, Н-18). 13C-NMR (150 MHz, CDCl3) (ppm)
δ: 37.81 (s,

Molecules 2021, 26, 3128
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C-1), 42.99 (t, C-2, C-8, C-9), 28.23 (d, C-3, C-5, C-7), 36.21 (t, C-4, C-6, C-10), 174.17 (s, C-11),
160.77 (s, C-14), 166.07 (s, C-16), 130.80 (s, C-17), 139.50 (d, C-18), 31.21 (t, C-19), 39.83 (d,
C-20), 26.80 (t, C-21), 24.30 (t, C-22), 148.45 (s, C-23), 109.25 (t, C-24), 20.60 (q, C-25). HR
MS: 383.2026 (M+, C22H29О1N3S1+; calc. 383.2022) [α]D27 = −32 (c 0.74 in CHCl3).
N-(Adamantan-1-yl)-3,7-dimethyloctanamide 26a
White solid, mp = 97.9 ◦C followed by decomposition, yield 52% (method B).
1H-NMR (600 MHz, CDCl3) (ppm)
δ: 0.827 and 0.829 (2d, J(19, 18) = J(20, 18) = 6.6 Hz, 3H
each, H-19, H-20), 0.88 (d, J(21, 14) = 6.6 Hz, 3H, H-21), 1.05–1.16 (m, 3H, H-15b, 2H-17),
1.17–1.33 (m, 3H, H-15a, 2H-16), 1.43–1.53 (m, 1H, H-18), 1.60–1.68 (m, 6H, 2H-4, 2H-6,
2
2H-10), 1.78 (dd, J = 13.6 Hz, J(13, 14) = 8.3 Hz, 1H, H-13b), 1.84–1.93 (m, 1H, H-14),
1.94–1.99 (m, 6H, 2H-2, 2H-8, 2H-9), 2.01–2.06 (m, 3H, H-3, H-5, H-7), 2.05 (dd, 2J = 13.6 Hz,
J(13a, 14) = 6.0 Hz, 1H, H-13a), 5.08 (br. s, 1H, H-11). 13C-NMR (150 MHz, CDCl3) (ppm)
δ:
51.65 (s, C-1), 41.60 (t, C-2, C-8, C-9), 29.33 (d, C-3, C-5, C-7), 36.27 (t, C-4, C-6, C-10), 171.69
(s, C-12), 45.56 (t, C-13), 30.72 (d, C-14), 36.90 (t, C-15), 24.57 (t, C-16), 38.99 (t, C-17), 27.81
+
(d, C-18), 22.45, 22.54 (2q, C-19, C-20), 19.46 (q, C-21). HR MS: 305.2715 (M+, C20H35O1N1
calc. 305.2713).
;
3.2. Biology
3.2.1. Detection of Tdp1 Activity
The recombinant Tdp1 was purified to homogeneity by chromatography on Ni-
chelating resin and phosphocellulose P11 as described [54], using plasmid pET 16B-Tdp1,
kindly provided by Dr. K.W. Caldecott (University of Sussex, United Kingdom). Tdp1
activity was detected as described in the work [41]. Briefly, we measured the fluorescence
intensity after quencher removal from a fluorophore quencher–coupled DNA oligonu-
cleotide catalyzed by Tdp1. The reaction was carried out at different concentrations of
inhibitors. The control samples contained 1% DMSO. The reaction mixtures contained
Tdp1 buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 7 mM β-mercaptoethanol), 50 nM
biosensor, and an inhibitor being tested. The reaction was started by the addition of puri-
fied Tdp1 (1.5 nM). The biosensor (50-[FAM] AAC GTC AGGGTC TTC C [BHQ]-30) was
synthesized in the Laboratory of Biomedical Chemistry at the Institute of Chemical Biology
and Fundamental Medicine (Novosibirsk, Russia).
The reactions were incubated on a POLARstar OPTIMA fluorimeter (BMG LAB-TECH,
GmbH) to measure fluorescence every 55 s (ex. 485 nm/em. 520 nm) during the linear
phase (here, data are from minute 0 to minute 8). The values of IC50 were determined using
a six-point concentration response curve in a minimum of three independent experiments,
and were calculated using MARS Data Analysis 2.0 (BMG LABTECH).
3.2.2. Cytotoxicity Assays
The human cervical cancer HeLa cells were grown in Iscove’s modified Dulbecco’s
medium (IMDM) with 40 µg/mL gentamicin, 50 IU/mL penicillin, 50 µg/mL streptomycin
(MP Biomedicals), and 10% of fetal bovine serum (Biolot) in a 5% CO2 atmosphere. Human
colon adenocarcinoma HCT-116, rectum adenocarcinoma SW837, and human embryo
kidney HEK293A cell lines were cultured in DMEM medium (Invitrogen) supplemented
with 10% fetal bovine serum (FBS) (Invitrogen), penicillin (100 units/mL), and streptomycin
(100 µ
g/mL), at 37 ◦C, 5% CO2 in a humid atmosphere. After the formation of a 30–50%
monolayer, the tested compounds were added to the medium. The volume of the added
reagents was 1/100 of the total volume of the culture medium, and the amount of DMSO

Molecules 2021, 26, 3128
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was 1% of the final volume. Control cells were grown in the presence of 1% DMSO. The
cell culture was monitored for 3 days.
The cytotoxicity of the compounds to HeLa and HEK293A cell lines was examined
using the EZ4U Cell Proliferation and Cytotoxicity Assay (Biomedica, Austria), according
to the manufacturer’s protocols.
The inhibition of HCT-116 and SW837 cell growth was assessed by the MTT test, based
on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
into formazan by mitochondrial NAD(P)H-dependent oxidoreductase enzymes [55]. Cells
in the exponential growth phase were seeded in 96-well plates (2000 cells per well). The
cells were allowed to attach for 24 h and were treated with compounds with concentrations
◦
ranging from 1 to 100
µ
M for 72 h at 37 C. MTT solution was added to each well at a
◦
final concentration of 0.5 mg/mL, and the plates were incubated at 37 C for 2 h. The
medium was removed, and the dark blue crystals of formazan were dissolved in 100 µL
of isopropanol. The absorbance at 570 nm (peak) and 620 nm (baseline) was determined
using a microplate reader Multiscan FC (Thermo Fisher Scientific Corporation). All concen-
trations were performed in triplicate. The values are given as mean ± standard deviation
(S.D.) values, and all measurements were repeated three times.
3.3. Molecular Modeling and Screening
The compounds were docked against the crystal structure of Tdp1 (PDB ID: 6DIE,
resolution 1.78 Å) [43], which was obtained from the Protein Data Bank (PDB) [56,57].
The Scigress version FJ 2.6 program [58] was used to prepare the crystal structure for
docking, i.e., the hydrogen atoms were added, and the co-crystallized ligand benzene-1,2,4-
tricarboxylic acid was removed, as well were the crystallographic water molecules except
HOH 814, 821 and 1078. The waters were toggle-bound or were displaced by the ligand
during docking, and spin–automatic optimization of the orientation of the hydrogen atoms
was performed. The Scigress software suite was also used to build the inhibitors and the
MM2 [59] force field was applied to identify the global minimum using the CONFLEX
method [60] followed by structural optimization. The docking center was defined as the
position of a carbon on the ring of the co-crystallized benzene-1, 2, 4-tricarboxylic acid
(
x = −6.052, y =
−
14.428, z = 33.998) with 10 Å radius. Fifty docking runs were allowed for
each ligand with default search efficiency (100%). The basic amino acids lysine and arginine
were defined as protonated. Furthermore, aspartic and glutamic acids were assumed to be
deprotonated. The GoldScore(GS) [61] and ChemScore (CS) [62,63] ChemPLP (piecewise
linear potential) [64] and ASP (Astex statistical potential) [65] scoring functions were
implemented to predict the binding modes and relative energies of the ligands using the
GOLD v5.4.1 software suite.
The QikProp 6.2 [66] software package was used to calculate the molecular descriptors
of the molecules. The reliability was established by QikProp for the calculated descrip-
tors [67]. The known drug indexes (KDI) were calculated from the molecular descriptors
as described by Eurtivong and Reynisson [48]. For application in Excel, columns for each
property were created and the following equations used to derive the KDI numbers for
each descriptor: KDI MW: = EXP(
EXP( ((LogP 2.82)ˆ2)/((2 2.21)ˆ2)); KDI HD: = EXP(
KDI HA: = EXP( ((HA 5.72)ˆ2)/((2
3.55)ˆ2)), and KDI PSA: = EXP( ((PSA
simply be copied into Excel and the descriptor name (e.g., MW) substituted with the value
in the relevant column. In order to derive KDI2A, this equation was used: = (KDI MW +
KDI LogP + KDI HD + KDI HA + KDI RB + KDI PSA) and for KDI2B we used = (KDI MW
× KDI LogP × KDI HD × KDI HA × KDI RB × KDI PSA).
−
((MW
−
371.76)ˆ2)/((2
((HD
2.86)ˆ2)); KDI RB = EXP(
79.4)ˆ2)/((2 54.16)ˆ2)). These equations could
×
112.76)ˆ2)); KDI Log P: =
1.88)ˆ2)/((2 1.7)ˆ2));
((RB 4.44)ˆ2)/((2
−
−
×
−
−
×
−
−
×
−
−
−
×
−
×
4. Conclusions
New hybrid molecules consisting of adamantane, monoterpene and heterocyclic frag-
ments were synthesized and tested for their Tdp1-inhibitory properties. All the compounds

Molecules 2021, 26, 3128
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were found to exhibit an inhibitory activity, and some had submicromolar potency, which
was supported by the plausible binding modes in the catalytic pocket predicted by molecu-
lar modeling. The triazole and thiadiazole linkers showed marked improvement in potency
as compared to the amides and thioamides previously reported [25
thiadiazole derivatives showed comparable IC50 values and the saturated aliphatic chain,
3,7-dimethyloctane, was the best substituent for both series (20a 25a). It was found that
some of the new ligands potentiated the cytotoxic potential of topotecan in vitro 20c being
the most effective sensitizer, tripling (3 ) the potency. The comparison of substances
synthesized with structurally similar compounds 26a
the incorporation of a 1,3,4-thiadiazole core into amides 26a
properties. The replacement of the amide bond in 28a
,40]. The triazole and
,
,
×
–c
,
28a
–
g
and 29a demonstrated that
–c
increased Tdp1 inhibitory
–c or the thioamide bond in 29a with
a 1,2,4-triazole linker also led to enhanced activity against Tdp1.
Supplementary Materials: The following are available online. Figure S1: The molecular structures
of pharmaceuticals containing 1,2,4-triazole or 1,3,4-thiadiazole moieties, Figure S2: Dose-dependent
impact of the Tdp1 inhibitors on the viability of HeLa (
Combination index plot determined for combined action of 20g and topotecan on HCT-116 cells,
Figure S4a: The correlation of the IC50 values of the ligands 20a 20b and 20c with log P, Figure S4b:
a), HCT-116 (b), and SW837 (c) cells, Figure S3:
,
The correlation of the IC50 values of the ligands with MW, Figures S5–S40: NMR and mass spectra of
the ligands, Table S1: The binding affinities as predicted by the scoring functions used, Table S2: The
molecular descriptors and their corresponding Known Drug Indexes 2a and 2b (KDI2a/2b). Table S3:
Definition of lead-like, drug-like and Known Drug Space (KDS) in terms of molecular descriptors.
Author Contributions: Chemistry investigation, A.A.M., E.S.M., D.V.K., E.V.S., K.P.V.; in vitro in-
vestigation, A.A.C., O.D.Z., E.S.I., N.S.D., A.L.Z.; molecular modeling, J.R.; methodology, N.F.S.
and O.I.L.; project administration, K.P.V., E.V.S.; supervision, K.P.V., E.V.S.; writing—original draft
preparation, A.A.M., writing—review and editing, K.P.V., J.R., N.F.S., O.I.L. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was supported by the Russian Scientific Foundation (grant 19-13-00040).
Authors would like to acknowledge the Multi-Access Chemical Research Center SB RAS for spectral
and analytical measurements.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Authors would like to acknowledge the Multi-Access Chemical Research Center
SB RAS for spectral and analytical measurements.
Conflicts 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.
Sample Availability: Samples of the compounds are available from the authors.
Abbreviations
Tdp1
Tyrosyl-DNA phosphodiesterase 1
DMSO Dimethyl sulfoxide
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