Structure-activity relationships of triazole-benzodioxine inhibitors of cathepsin X

Article abstract of DOI:10.1016/j.ejmech.2020.112218

Cathepsin X is a cysteine carboxypeptidase that is involved in various physiological and pathological processes. In particular, highly elevated expression and activity of cathepsin X has been observed in cancers and neurodegenerative diseases. Previously, we identified compound Z9 (1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) as a potent and specific reversible cathepsin X inhibitor. Here, we have explored the effects of chemical variations to Z9 of either benzodioxine or triazol moieties, and the importance of the central ketomethylenethio linker. The ketomethylenethio linker was crucial for cathepsin X inhibition, whereas changes of the triazole heterocycle did not alter the inhibitory potencies to a greater extent. Replacement of benzodioxine moiety with substituted benzenes reduced cathepsin X inhibition. Overall, several synthesized compounds showed similar or improved inhibitory potencies against cathepsin X compared to Z9, with IC₅₀ values of 7.1 μM–13.6 μM. Additionally, 25 inhibited prostate cancer cell migration by 21%, which is under the control of cathepsin X.

Full text of DOI:10.1016/j.ejmech.2020.112218

Journal Pre-proof
Structure-activity relationships of triazole-benzodioxine inhibitors of cathepsin X
Urša Pečar Fonović, Damijan Knez, Martina Hrast, Nace Zidar, Matic Proj, Stanislav
Gobec, Janko Kos
PII:
S0223-5234(20)30185-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112218
EJMECH 112218
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2019
Revised Date: 23 February 2020
Accepted Date: 7 March 2020
Please cite this article as: U.P. Fonović, D. Knez, M. Hrast, N. Zidar, M. Proj, S. Gobec, J. Kos,
Structure-activity relationships of triazole-benzodioxine inhibitors of cathepsin X, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112218.
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Structure-activity relationships of triazole-benzodioxine inhibitors of cathepsin X
Urša Pečar Fonovića,*, Damijan Kneza, Martina Hrasta, Nace Zidara, Matic Proja, Stanislav
Gobeca, Janko Kosa,b
a University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
b Department of Biotechnology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
*Corresponding author:
Faculty of Pharmacy
University of Ljubljana
Aškerčeva 7
1000 Ljubljana, Slovenia
Tel: +386-1-4769-500
Email: ursa.pecarfonovic@ffa.uni-lj.si

Abstract
Cathepsin X is a cysteine carboxypeptidase that is involved in various physiological and
pathological processes. In particular, highly elevated expression and activity of cathepsin X
has been observed in cancers and neurodegenerative diseases. Previously, we identified
compound Z9 (1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-
yl)thio)ethan-1-one) as a potent and specific reversible cathepsin X inhibitor. Here, we have
explored the effects of chemical variations to Z9 of either benzodioxine or triazol moieties,
and the importance of the central ketomethylenethio linker. The ketomethylenethio linker was
crucial for cathepsin X inhibition, whereas changes of the triazole heterocycle did not alter the
inhibitory potencies to a greater extent. Replacement of benzodioxine moiety with substituted
benzenes reduced cathepsin X inhibition. Overall, several synthesized compounds showed
similar or improved inhibitory potencies against cathepsin X compared to Z9, with IC50
values of 7.1 µM to 13.6 µM. Additionally, 25 inhibited prostate cancer cell migration by
21%, which is under the control of cathepsin X.
Keywords: cathepsin X; triazole-benzodioxine inhibitors; 1,2,4-triazole; anticancer drugs

Introduction
Cathepsin X (CatX) is a lysosomal carboxypeptidase that is expressed predominantly in
immune and neuronal cells [1]. By cleaving diverse substrates, CatX can affect a number of
different physiological processes, including cell proliferation, migration and adhesion [2]. In
addition, it has also been implicated in various pathological processes, and particularly in
cancers and neurodegenerative diseases. CatX is highly up-regulated in many cancers, where
its proteolytic activity has a substantial role in the cleavage of different targets, such as
profilin 1 and the β2-chain of integrin receptors, which are involved in cytoskeleton
modulation and cell adhesion [3, 4]. Higher protein levels of CatX are associated with cancer
progression and have been related to poor survival of cancer patients [5, 6]. As CatX activity
cannot be regulated by endogenous cysteine peptidase inhibitors, it represents a promising
target for the development of novel anticancer drugs [7]. Up-regulation of CatX has also been
detected in several neurodegenerative diseases, including Alzheimer’s and Parkinson’s
diseases, amyotrophic lateral sclerosis, and Huntington’s disease (for review, see Pišlar and
Kos [8]). CatX is involved in the microglia-activation-mediated neurodegeneration [9] that
abolishes the neuroprotective activity of γ-enolase [10]. As for cancers, inhibition of CatX
activity during microglia activation might be a target to prevent and treat neurodegenerative
disorders [9].
AMS-36 was the first irreversible epoxysuccinyl-based inhibitor of the papain family
of cysteine proteases that showed some degree of specificity towards CatX in mice tumors
[11]. Later, we identified several triazole-based compounds that can act as selective CatX
inhibitors, and we revealed their reversible and competitive mechanism of action [12]. The
most potent inhibitor was 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-
triazol-3-yl)thio)ethan-1-one (Fig. 1, compound Z9), with Ki against CatX of 2.45 ± 0.05 µM.
Compound Z9 inhibited CatX by more than 75% in a kinetic assay using Abz-FEK(Dnp)-OH
as substrate. Z9 showed good selectivity in comparison to other related cysteine peptidases,
and was not cytotoxic to PC-3 or PC-12 cells at concentrations up to 10 µM. Importantly, Z9
significantly inhibited the migration of tumor cells and the outgrowth of neurites, which are
two processes under CatX control [12].
We report herein a concise series of Z9 variations to further explore the structure-activity
relationships of the triazole-2,3-dihydrobenzo[b][1,4]dioxine class of inhibitors. Selected
compounds showed improved inhibitory activities against CatX compared to Z9. The effects

of these new inhibitors were tested in cell-based assays, where compound 25 significantly
reduced the migration of PC-3 cells in vitro.
Results and Discussion
Design
The low molecular weight of the parent compound Z9 and its simple modular structure
allowed numerous structural modifications to explore the chemical space and assess the
importance of the fragments for the inhibition of CatX. In this design, three key modifications
to Z9 were pursued (Fig. 1).
First, several different heterocyclic rings were introduced to replace the 4-isopropyl-
4H-1,2,4-triazole-3-thio fragment. We established previously that this part of the triazole-2,3-
dihydrobenzo[b][1,4]dioxine-based CatX inhibitors allows major changes, which might
enhance the inhibitory potency [12]. We then investigated the importance of the
ketomethylenethio linker structure by changing the sulfur group for an oxygen or amino
group. The keto group was also transformed into secondary amino, hydroxyl, and
methoxyimino groups. By preparing ketone replacements, we wanted to examine whether this
fragment forms a hydrogen bond with the backbone NH of Gly73 of CatX, as indicated by
molecular modeling studies of Z9 in the procathepsin X with deleted proregion (PDB: 1DEU)
[12]. Finally, we prepared a variety of compounds with diverse substituents that replaced the
1,4-benzodioxan-6-yl moiety, which does not form any specific interactions in the S2 pocket
of CatX. The sequestering of additional interactions would potentially improve not only the
potency, but also the selectivity of inhibitors of CatX, compared to other structurally similar
cathepsin peptidases.
Chemistry
Analogues of Z9 were synthesized according to Schemes 1-4. First, a small library of
1,2,4-triazole-3-thiols (3a-h) and 1,2,4-triazole-5-thiols (5a-e) was prepared (Scheme 1). For
the synthesis of 3a-h, different isothiocyanates (1) were reacted with formylhydrazine to give
thiosemicarbazides 2a-h, which then underwent a cyclodehydration under basic conditions to
produce 3a-h [13]. For the synthesis of 5a-d, the reaction between different acyl chlorides and
thiosemicarbazide gave acylthiosemicarbazides 4a-c, which were then cyclized in alkaline
medium to yield their corresponding 1,2,4-triazole-5-thiols 5a-d. Compound 5e was prepared
in a reaction between isoniazid and ammonium thiocyanate in refluxing 1 M HCl.

Scheme 1. Reagents and conditions: (a) NH2NHCHO, EtOH, 90 °C, 18 h; (b) NaOH, H2O, EtOH, 90
°C, 2 h; (c) corresponding acid chloride or acid, various conditions; (d) 2 M NaOH(aq), reflux 4 h; (e)
NH4SCN, 1 M HCl, reflux, 18 h.
In the next step, commercially available 1,4-benzodioxan-6-yl methyl ketone was
selectively α-brominated with copper(II) bromide to yield α-bromo ketone 6, with no aromatic
ring bromination or dibromination of the α-methylene group, which are frequent side
reactions of bromination (Scheme 2). Compound 6 was then reacted with thiols 3a-h, 5a-e or
with various commercially available thiols in anhydrous ethanol to provide the desired
products Z9, 10–24. Compound 7a (Table S1) was obtained as the side product in the
synthesis of 7, where the nucleophilic N1-nitrogen of the 1,2,4-triazole reacted with the
carbonyl group, followed by elimination of water, to provide the bicyclic thiazolo[3,2-
b][1,2,4]triazole system [14]. Compound 25 was prepared from 19 by alkylating the N1-
nitrogen with chloroacetonitrile in the presence of potassium carbonate as base. The carbonyl
group of Z9 was transformed into an alcohol (26), an O-methyl oxime (27) or a secondary
amine (28), using established procedures, as shown in Scheme 2.

Scheme 2. Reagents and conditions: (a) CuBr2, CHCl3, 70 °C, 20 h; (b) anh EtOH, 90 °C, 20 h; (c)
chloroacetonitrile, K2CO3, MeCN, reflux, 18 h; (d) NaBH4, EtOH/THF (1/1), rt, 2 h (to obtain 26); (e)
O-methylhydroxylamine hydrochloride, pyridine, anh EtOH, 90 °C, 16 h (to obtain 27); (f) NH4OAc,
NaBH3CN, MeOH, 70 °C, 40 h (to obtain 28).
Analog 32, in which the thioether group in the linker is bioisosterically replaced with
the ether group, was prepared according to Scheme 3. First, 1,2,4-triazolin-5-one (29) was
synthesized from semicarbazide hydrochloride and trimethyl orthoformate, followed by
protection of N4-nitrogen with the Boc protecting group (30). Then, NaH was added to 30 to
form the enolate ion, which was alkylated with α-bromoketone 6 to yield 31. Finally, the
desired 32 was obtained by Boc-deprotection of 31 under anhydrous acidic conditions.
Replacement of the thioether group in the linker with the amino group was also
attempted. By reacting the unsubstituted 4H-1,2,4-triazol-3-amine with α-bromoketone 6, we
did not obtain the desired compound with 3-amino group substitution, alternatively, only the
N1-alkylated product 33 was obtained. To limit the possibility of alkylation of the ring
nitrogen, 1,2,4-triazol-3-amines with monomethylated ring nitrogens were prepared first,
according to the literature procedure [15]. According to this procedure, intermediates with
N,N-dimethylformimidamide group are produced first (34a-c, not shown in Scheme 3), which
are then hydrolyzed to methyl-1,2,4-triazol-3-amines. 1-Methyl-1H-1,2,4-triazol-3-amine

(35a) and 1-methyl-1H-1,2,4-triazol-5-amine (35b) were successfully isolated, whereas 4-
methyl-4H-1,2,4-triazol-3-amine (35c) was not produced in sufficient quantity to proceed
with the next reaction step. The isolated monomethylated 1,2,4-triazoles 35a and 35b were
reacted with α-bromoketone 6 in anhydrous EtOH under reflux. Despite introducing a methyl
group on the heterocyclic nitrogen, the primary aromatic amino group was not sufficiently
nucleophilic, and only the quaternary ammonium derivatives 36a and 36b were obtained
(Scheme 3).
Scheme 3. Reagents and conditions: (a) trimethyl orthoformate, MeOH, rt, 2 h; (b) (BOC)2O, DMAP,
DIPEA, DCM, rt, 18 h; (c) 1. 30, NaH, anh DMF, rt, 15 min; 2. 6, rt, 18 h; (d) TFA, DCM, rt, 2 h; (e)
K2CO3, anh EtOH, rt, 18 h; (f) 1. DMFDMA, 110 °C, 18 h; 2. 4 M NaOH(aq), 110 °C, 1 h; (g) anh
EtOH, 90 °C, 20 h.
Finally, a variety of compounds with diverse replacements of the 1,4-benzodioxan-6-
yl fragment were prepared by reacting commercially available α-haloketones with 4-
isopropyl-4H-1,2,4-triazole-3-thiol (3a) (Scheme 4). The 4-hydroxyl group in 50 was then

alkylated with small substituents to probe the chemical space (51–53). Compound 54 was
prepared from 53 by alkaline hydrolysis of ethyl ester group.
Scheme 4. Reagents and conditions: (a) anh EtOH, 90 °C, 20 h; (b) corresponding alkyl halogenide,
K2CO3, MeCN, 50 °C, 18 h; (c) 0.1 M NaOH(aq), MeOH, rt, 4 h.
Relative inhibition of cathepsin X and structure-activity relationships
Based on Z9 [12], 45 new analogs were designed and prepared. These compounds
were screened for their relative inhibition of CatX at 50 µM, using Abz-FEK(Dnp)-OH as the
specific CatX substrate [16]. Several of these compounds inhibited CatX by >75% (Tables 1,
2).
Based on the data presented in Tables 1 and 2 and in Table S1, the structural features
necessary for CatX inhibition can be deduced, and are summarized in Fig. 2. A wide variety
of substituents on position 4 of the 4H-1,2,4-triazole-3-yl ring in compounds 7–13 (i.e.,
hydrogen, methyl, ethyl, isopropyl, p-cyanophenyl, pyridine-3-yl) did not significantly alter
the inhibition, compared to Z9. The same was observed in our previous study, where
diversely substituted triazoles 1, 2, 12, 14, 17, 20 and 22 – compound Z9 (numbering of
compounds from reference [12]) displayed comparable inhibitory potencies. Similarly,
smaller (i.e, methyl, 14) or larger (e.g., phenyl, 16) fragments on position 3 of triazole did not

have significant impact on the inhibitory profile of these compounds (Table 1, residual
activities from 25% to 33%). Bioisosteric replacement of 4H-1,2,4-triazole (7) with imidazole
(19), 1-methyl-1H-tetrazole (21), and thiazole (24) resulted in active compounds versus CatX,
whereas introduction of phenyl (22) or thiophene (23) resulted in slightly decreased inhibition
of CatX. The presence of a nitrogen atom in the heterocycle thus appears to enhance the
inhibitory activity of these compounds. Transformation of the ketone to hydroxyl (26), amino
(28), and O-methylhydroxylamine (27) completely abolished the inhibitory potency. Also,
changing of the thioether group in the linker for an ether group resulted in an inactive
compound (32; residual activity, 71% at 50 µM). The preparation of compounds with an
amino group in the linker was also attempted, but as indicated above, only derivatives with
substituted ring nitrogens were obtained (Table S1). These derivatives were not active against
CatX, and together they show the pivotal role of the ketomethylenethio linker for potent CatX
inhibition. The fragments that comprise the Z9 molecule were also tested, but neither 1-(2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one (6ACBD), bromide (6), alcohol (55) nor 4-
isopropyl-4H-1,2,4-triazole-3-thiol (3a) alone were sufficient to inhibit CatX (Table S1).
Next, changing the left-hand side of the molecule (Scheme 4) to bicyclic systems such
as indole (37) and benzo[d][1,3]dioxole (39) retained relative inhibitions <50% (Table 2).
However, substituted phenyl derivatives (40-54) had significantly lower inhibitory activities
in comparison to Z9 (Table 2).
To rationalize the structure-activity relationships described above, molecular modeling
was performed. The modeling studies using ligand-free crystal structure of CatX (PDB: 1EF7)
suggested that compounds studied bind into the active site, where the best scores and
superpositions of ligands were achieved (Supplementary information, Fig. S1). Docking
studies did not indicate binding into potential allosteric binding sites as was seen in the case
of cathepsin K (PDB: 6ASH, 5JA7, 5J94). Keto group of the ketomethylenethio linker forms
a hydrogen bond with Gln22 side chain, which stabilizes inhibitors in the active site groove,
and explains why modifications of keto group resulted in inactive compounds. Moreover,
additional H-bonds between the Gly74 and Asn179, and nitrogens of the triazole and its
bioisosteres enhance inhibitory potencies, whereas phenyl (23) and thiophene (24) analogues
are inactive. The modelling studies did not show any specific interactions for the 1,4-
benzodioxan-6-yl moiety. In general, phenyl analogues (40–45) had higher docking score,
which is also in correlation with lower inhibitory activity obtained by the biochemical
evaluation.

The IC50 values were further determined for five compounds that demonstrated
improved or similar inhibitory potencies to the parent Z9 (Table 3). All of these derivatives
showed IC50 values in the low micromolar range, which were comparable to Z9. The dilution
assay confirmed the reversible binding of the compounds (Supplementary information, Fig.
S2). Compound 25 with cyanomethylene residue, which could form a covalent thioimidate
adduct with catalytic Cys31 [17], is a non-time dependent inhibitor of CatX (Supplementary
information, Fig. S3). The progress curves were identical as reported in our previous
communication, which indicates instantaneous competitive inhibition with preceding slow
inhibitor isomerization [12]. Moreover, inhibitory potencies towards cathepsins B (endo- and
exo-peptidase activities), L and S were determined. At 100 µM, no or lower than 30%
inhibition of the cathepsins’ activity was observed. The IC50 value of compound 25 against
cathepsin S was determined (241 ± 26.9 µM). These results show good selectivity profile of
studied compounds for CatX.
Cytotoxicity of compounds and their effects on migration of PC-3 cells
To distinguish the effects in migration assays that were caused by specific mechanisms from
those due to triggering of cell cytotoxicity, the cytotoxic effects of the selected compounds
were initially investigated for PC-3 cells, to select concentrations that did not decrease cell
viability. At 10 µM, 10 and 20 showed weak cytotoxic effects after 24 h and 48 h, while 10
was the only compound that showed statistically significant cytotoxicity at 20 µM, at both 24
h and 48 h. The other compounds tested did not affect cell viability after 24 h or 48 h (Fig. 3).
As CatX is involved in cell migration, we investigated how these compounds impeded this
process. Z9 was used as the positive control, as we have already shown that it has inhibitory
effects on PC-3 cell migration. Of the two compounds tested, 25 decreased cell migration by
21%, but at twice the concentration of Z9, while 8 had no effects (Fig. 4). The lower effect of
25 on cell migration in comparison with Z9 could be a result of lower permeation of cell
membrane of the former as also predicted by the QikProp prediction software (Supplementary
information, Table S2; Schrödinger Release 2019-3, Schrödinger, LLC, NY, 2019).
Conclusions
In summary, an extensive series of CatX inhibitors was developed based on the 1,2,4-triazole
parent compound Z9. Several of these compounds showed inhibitory activities against CatX

in the same concentration range as Z9, with IC50 values from 7.13 µM to 13.6 µM, thereby
providing important information about their structure-activity relationships. Compound 25,
the most potent of these CatX inhibitors, was not cytotoxic to PC-3 cells at ≤20 µM, and
decreased PC-3 cell migration. These data show that a possible approach for further
improvement of the inhibitory potency lies in the modification of the triazole substituents, and
highlight the therapeutic antitumor potential of this class of CatX inhibitors.
Experimental Methods
General synthetic chemistry experimental protocols
The reagents and solvents used were obtained from commercial sources (i.e., Acros Organics,
Sigma Aldrich, TCI Europe, Merck, Carlo Erba, Apollo Scientific, Alfa Aesar) and were used
as provided, unless otherwise indicated. Analytical thin-layer chromatography was performed
on silica gel aluminium sheets (60 F254, 0.20 mm; Merck), with visualization using UV light
(254 nm) and/or visualization reagents (i.e., ninhydrine, 2,4-dinitrophenylhydrazine). Flash
column chromatography was performed on silica gel 60 (particle size, 0.040–0.063 mm; 230–
400 mesh; Merck). 1H NMR and 13C NMR spectra were recorded at 400 MHz and 100 MHz,
respectively, on a nuclear magnetic resonance (NMR) spectrometer (Avance III; Bruker
Corporation, MA, USA), at 295 K. The chemical shifts (δ) are reported in parts per million
(ppm) and are referenced to the deuterated solvent used. High-resolution mass spectrometry
(HRMS) measurements were performed (Autospec Q Micromass mass spectrometer; Fisons,
VG Analytical, Manchester, UK) at the Jozef Stefan Institute, Ljubljana, Slovenia, and on a
liquid chromatography (LC)–tandem mass spectrometry (MS/MS) system (Q Executive Plus;
Thermo Scientific, MA, USA). The MS measurements were performed on a mass
spectrometer (Expression CMS; Advion, NY, USA). Analytical reversed-phase HPLC
analysis was performed on a modular system (Dionex UltiMate 3000; Thermo Fisher
Scientific Inc., MA, USA).
General method: C18 column (Acquity UPLC HSS; 1.8 µm, 2.1 × 50 mm; Waters), T = 40
°C; injection volume 1 µL, sample 0.2 mg/mL (4% DMSO in MeCN); flow rate, 0.4 mL/min;
detector λ = 254 nm; mobile phase A (0.1% TFA [v/v] in water), mobile phase B (MeCN).
Gradient (for mobile phase B): 0–7 min, 10%–90%; 7–10 min, 90%. Purities of the tested
compounds were established as ≥95% by HPLC, unless stated otherwise.

Synthesis
General procedure for synthesis of thiosemicarbazides (2a-h): To a suspension of
formylhydrazine (2.0 g, 33.5 mmol, 1.0 equiv.) in EtOH (100 mL), the corresponding
isothiocyanate (1.0 equiv.) was added. The reaction mixture was stirred at 90 °C for 20 h. The
volume of EtOH was reduced to half by evaporation under reduced pressure, and then the
reaction mixture was cooled to room temperature. The white precipitate was filtered off to
obtain the crude product, which was used in the next step without purification. As estimated
by NMR, the thiosemicarbazides were already partially cyclized to their corresponding 5-
mercapto-4-substituted-1,2,4-triazoles.
General procedure for synthesis of 1,2,4-triazole-3-thiols (3a-h): To a suspension of
thiosemicarbazides 2a-h (10 mmol, 1.0 equiv.) in EtOH, 4 M NaOH(aq) (5 mL, 1.5 equiv.)
was added, and the reaction mixture was refluxed at 90 °C for 4 h. The mixture was cooled to
room temperature, acidified with 2 M HCl(aq) to pH ~5, and the EtOH was evaporated off
under reduced pressure. The precipitate that formed was filtered off, yielding the pure
product, which was used in the next step.
1
4-Isopropyl-4H-1,2,4-triazole-3-thiol (3a). Yield, 80%. H NMR (400ꢀMHz, DMSO-d6) δ
1.34 (d, J = 6.9 Hz, 6H), 4.63 (hept, J = 6.8 Hz, 1 H), 8.60 (s, 1 H), 13.70 (bs, 1 H) ppm. 13C
NMR (100 MHz, DMSO-d6) δ 21.37, 46.88, 139.53, 164.97 ppm. ESI-MS for C5H8N3S [M–
H]– 142.11. HRMS (ESI) m/z calculated for C5H10O3S [Mꢀ+ꢀH]+ 144.0590, found 144.0587.
4H-1,2,4-Triazole-3-thiol (3b). Commercially available reagent was used.
1
4-Methyl-4H-1,2,4-triazole-3-thiol (3c). Yield, 25%. H NMR (400ꢀMHz, DMSO-d6) δ 3.43
(s, 3 H), 8.40 (s, 1 H), 13.65 (bs, 1 H) ppm. ESI-MS for C3H4N3S [M – H]– 144.13.
4-Ethyl-4H-1,2,4-triazole-3-thiol (3d). Yield, 60%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.27 (t,
J = 7.3 Hz, 3 H), 3.92 (q, J = 7.3 Hz, 2 H), 8.47 (d, J = 1.6 Hz, 1 H), 13.67 (bs, 1 H) ppm.
ESI-MS for C4H8N3S+MeOH [M+MeOH+H]+ 161.97.
1
4-(3-Mercapto-4H-1,2,4-triazol-4-yl)benzonitrile (3e). Yield, 77%. H NMR (400ꢀMHz,
DMSO-d6) δ 7.98 (d, Jꢀ=ꢀ8.7ꢀHz, 2ꢀH), 8.08 (d, Jꢀ=ꢀ8.7ꢀHz, 2ꢀH), 8.81 (d, Jꢀ=ꢀ1.6ꢀHz, 1ꢀH),
14.09 (bs, 1 H) ppm.

4-(3-Mercapto-4H-1,2,4-triazol-4-yl)benzamide (3f). Yield, 23%. 1H NMR (400ꢀMHz,
DMSO-d6) δ 7.54 (bs, 1 H), 7.78 (d, Jꢀ=ꢀ8.6ꢀHz, 2ꢀH), 8.02 (d, Jꢀ=ꢀ8.6ꢀHz, 2ꢀH), 8.12 (bs, 1 H),
8.77 (d, Jꢀ=ꢀ1.6ꢀHz, 1ꢀH), 14.01 (bs, 1H) ppm.
4-(4-(Dimethylamino)phenyl)-4H-1,2,4-triazole-3-thiol (3g). Yield, 91%. 1H NMR
(400ꢀMHz, DMSO-d6) δ 2.95 (s, 6 H), 6.81 (d, Jꢀ=ꢀ9.1ꢀHz, 2ꢀH), 7.37 (d, Jꢀ=ꢀ9.1ꢀHz, 2ꢀH), 8.57
(d, Jꢀ=ꢀ1.6ꢀHz, 1ꢀH), 13.82 (bs, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 39.93 (under
1
DMSO peak, confirmed by 2D NMR experiment H-13C HSQC), 111.86, 122.68, 126.49,
142.41, 150.27, 166.39 ppm. ESI-MS for C10H13N4S [M+H]+ 221.05.
4-(Pyridin-3-yl)-4H-1,2,4-triazole-3-thiol (3h). Yield, 73%. 1H NMR (400ꢀMHz, DMSO-d6) δ
7.62 (ddd, J = 8.2, 4.8, 0.8 Hz, 1 H), 8.16 (ddd, J = 8.2, 2.6, 1.5 Hz, 1 H), 8.68 (dd, J = 4.8,
13
1.5 Hz, 1 H), 8.79 (s, 1 H), 8.86 (dd, J = 2.6, 0.7 Hz, 1 H), 14.07 (bs, 1 H) ppm; C NMR
(100 MHz, DMSO-d6) δ 123.87, 131.15, 133.89, 142.07, 146.71, 149.70, 166.55 ppm. ESI-
MS for C7H7N4S [M + H]+ 178.91.
Synthesis of 2-acetylhydrazine-1-carbothioamide (4a): Acetic acid (20 mL) was added to
thiosemicarbazide (4,55 g, 50 mmol, 1.0 equiv.), and the mixture was stirred at reflux (130
°C) for 4 h. The reaction was cooled to room temperature, the solvent was evaporated under
reduced pressure, and the crude product was recrystallized from water. Yield, 65%. 1H NMR
(400ꢀMHz, DMSO-d6) δ 1.82 (s, 3 H), 7,48 (s, 1 H), 7.83 (s, 1 H), 9.15 (s, 1 H), 9.70 (s, 1 H)
ppm.
Synthesis of 2-(cyclohexanecarbonyl)hydrazine-1-carbothioamide (4b): To a solution of
cyclohexyl carboxylic acid (3.0 g, 23.4 mmol, 1.0 equiv) in DCM (20 mL), oxalyl chloride
(6.0 mL, 70.2 mmol, 3.0 equiv.) and a catalytic amount of DMF were added, and the reaction
mixture was stirred at 45 °C for 1 h. The mixture was cooled to room temperature, and the
solvent was evaporated under reduced pressure. The residue was dissolved in anhydrous THF
(20 mL), followed by addition of thiosemicarbazide (4.26 g, 46.8 mmol, 2 equiv.) and
pyridine (3.0 mL, 35.1 mmol, 1.5 equiv.). The mixture was stirred at room temperature for 18
h. EtOAc (100 mL) was then added and the organic phase was washed with water (2 × 50
mL) and brine (50 mL), and then dried over Na2SO4. The solvent was evaporated under
reduced pressure and the product was purified by flash chromatography using DCM/MeOH
(20:1, v/v) as mobile phase. The crude product was used in the next step without further
purification.

Synthesis of 2-benzoylhydrazine-1-carbothioamide (4c). To a cold solution (0 °C) of benzoyl
chloride (1.17 mL, 10.0 mmol, 1.0 equiv.) in THF (15 mL), thiosemicarbazide (1.0 g, 10.9
mmol, 1.1 equiv.) was added in aliquots, and the reaction was stirred at room temperature for
6 h. The solvent was evaporated under reduced pressure, the residue was dissolved in EtOAc
(30 mL), and the organic phase was washed with saturated NaHCO3(aq) (2 × 15 mL), then
dried over Na2SO4, filtered and evaporated. The product was recrystallized from EtOH and
1
used without further purification. Yield, 34%. H NMR (400ꢀMHz, DMSO-d6) δ 7.47–7.50
(m, 2 H), 7.55–7.59 (m, 1 H), 7.65 (bs, 1 H), 7.89 – 7.92 (m, 3 H), 9.36 (s, 1 H), 10.40 (s, 1
H) ppm. ESI-MS for C8H10N3OS [M + H]+ 196.02 [18].
General procedure for cyclization of carbothioamides (5a-c): A suspension of
acylthiosemicarbazides 4a-c (2.5 mmol, 1.0 equiv.) in 2 M NaOH(aq) (2 mL, 4 mmol, 1.6
equiv.) was stirred at reflux for 4 h. The mixture was cooled to room temperature and
acidified with concentrated HCl to pH ~3. The precipitate formed was filtered off,
recrystallized from MeOH, and used without further purification.
5-Methyl-4H-1,2,4-triazole-3-thiol (5a). Yield, 58%. 1H NMR (400ꢀMHz, DMSO-d6) δ 2.16
(s, 3 H), 13.05 (bs, 1 H), 13.15 (s, 1 H) ppm. ESI-MS for C3H6N3S [M + H]+ 116.02.
3-Cyclohexyl-1H-1,2,4-triazole-5-thiol (5b). Crude intermediate 4b was used as starting
compound. Yield, 44%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.13–1.46 (m, 5 H), 1.60–1.65 (m,
1 H), 1.71 (dt, J = 12.5, 3.5 Hz, 2 H), 1.84–1.88 (m, 2 H), 2.56 (tt, J = 11.3, 3.5 Hz, 1 H),
13.07 (s, 1 H), 13.19 (s, 1 H) ppm. ESI-MS for C8H14N3S [M + H]+ 183.99.
1
5-Phenyl-4H-1,2,4-triazole-3-thiol (5c). Yield, 61%. H NMR (400ꢀMHz, DMSO-d6) δ 7.50–
7.53 (m. 3 H), 7.89–7.92 (m, 2 H), 13.69 (s, 1 H), 13.86 (s, 1 H) ppm. ESI-MS for C8H8N3S
[M + H]+ 177.96 [19].
Synthesis of cyclohexyl(3-cyclohexyl-5-mercapto-1H-1,2,4-triazol-1-yl)methanone (5d): To a
solution of cyclohexyl carboxylic acid (3.0 g, 23.4 mmol, 1.0 equiv.) in DCM (20 mL), oxalyl
chloride (6.0 mL, 70.2 mmol, 3.0 equiv.) and a catalytic amount of DMF were added and
stirred at 45 °C for 1 h. The mixture was cooled to room temperature and the solvent was
evaporated under reduced pressure. The residue was dissolved in DCM (20 mL), followed by
addition of thiosemicarbazide (2.13 g, 23.4 mmol, 1 equiv.) and pyridine (3 mL, 35.1 mmol,
1.5 equiv.). The mixture was stirred at room temperature for 18 h to yield intermediate 4d

(1,2-di(cyclohexanecarbonyl)hydrazine-1-carbothioamide) The solvent was evaporated under
reduced pressure, and 2 M NaOH(aq) (5 mL) was added to the crude intermediate 4d, and
stirred at reflux for 4 h. The reaction was cooled to room temperature, the solvent was
evaporated off under reduced pressure, and the product was isolated by flash chromatography
using DCM/MeOH (20:1, v/v) as mobile phase. Yield, 3%. 1H NMR (400ꢀMHz, DMSO-d6) δ
1.28–1.38 (m, 2 H), 1.42–1.51 (m, 4 H), 1.55–1.67 (m, 4 H), 1.74–1.78 (m, 2 H), 1.83–1.91
(m, 4 H), 1.99–2.02 (m, 2 H), 2.14–2.18 (m, 2 H), 2.84–2.90 (m, 1 H), 3.04–3.10 (m, 1 H),
13.05 (s, 1 H) ppm. ESI-MS for C15H24N3OS [M + H]+ 294.08.
Synthesis of 3-(pyridin-4-yl)-1H-1,2,4-triazole-5-thiol (5e): To a solution of isoniazid (1.371
g, 20.0 mmol, 1.0 equiv.) in 1 M HCl(aq) (5 mL), NH4SCN (1.52 g, 20.0 mmol, 1.0 equiv.)
was added, and the reaction was stirred at reflux for 18 h. The mixture was cooled to room
temperature, and the forming yellow precipitate was filtered off, yielding the pure product.
1
Yield, 45%. H NMR (400ꢀMHz, DMSO-d6) δ 7.84 (dd, J = 4.4, 1.6 Hz, 2 H), 8.73 (dd, J =
4.4, 2.0 Hz, 2 H), 13.94 (s, 1 H), 14.11 (bs, 1 H) ppm. ESI-MS for C7H7N4S [M + H]+ 178.97.
Synthesis of 2-bromo-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one (6). To a solution
of 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one (5.0 g, 28.0 mmol, 1.0 equiv.) in
chloroform (100 mL), CuBr2 (10.7 g, 47.7 mmol, 1.7 equiv.) was added in one aliquot. The
reaction mixture was stirred at 70 °C for 20 h. After cooling the mixture to room temperature,
the excess CuBr2 and CuBr (formed as side product) were filtered off. The product was
1
purified by flash chromatograph using DCM/Hex (4:1, v/v) as mobile phase. Yield, 67%. H
NMR (400ꢀMHz, CDCl3) δ 4.27–4.29 (m, 2ꢀH), 4.31–4.34 (m, 2ꢀH), 4.37 (s, 2 H), 6.91–6.93
(m, 1ꢀH), 7.49–7.52 (m, 2ꢀH) ppm. 13C NMR (100 MHz, CDCl3) δ 30.89, 64.18, 64.85,
117.63, 118.53, 123.29, 127.73, 143.63, 148.91, 189.92 ppm. HRMS (ESI) m/z calculated for
C10H10O3Br [Mꢀ+ꢀH]+ 256.9808, found 256.9799.
General procedure for synthesis of final products (Z9, 7-24): The suspension of bromide 6
(8.0 mmol, 1.0 eq) and thiol (8.0 mmol, 1.0 equiv.) in anhydrous EtOH (30 mL) was stirred at
90 °C for 18 h. The reaction mixture was cooled to room temperature and the forming
precipitate was filtered off, yielding the pure product. If necessary, the product was purified
by flash chromatography.
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-
one (Z9). Yield, 81%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.50 (d, J = 6.7 Hz, 6 H), 4.28–4.30

(m, 2ꢀH), 4.33–4.35 (m, 2ꢀH), 4.58 (hept, J = 6.7 Hz, 1 H), 5.04 (s, 2 H), 7.01 (d, J = 8.5 Hz, 1
13
H), 7.52 (d, J = 2.1 Hz, 1 H), 7.55 (dd, J = 8.5, 2.2 Hz, 1 H), 9.79 (s, 1 H) ppm. C NMR
(100 MHz, DMSO-d6) δ 21.87, 41.29, 50.29, 63.99, 64.69, 117.40, 117.58, 122.61, 128.38,
142.99, 143.30, 148.65, 150.62, 190.77 ppm. HRMS (ESI) m/z calculated for C15H18N3O3S
[Mꢀ+ꢀH]+ 320.1069, found 320.1066. HPLC purity, 99.3% at 254 nm (tR = 3.57 min).
2-((4H-1,2,4-Triazol-3-yl)thio)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one
(7).
1
Yield, 23%. H NMR (400ꢀMHz, DMSO-d6) δ 4.28–4.30 (m, 2ꢀH), 4.33–4.35 (m, 2ꢀH), 4.68
(s, 2 H), 6.99 (d, J = 8.4 Hz, 1 H), 7.50 (d, J = 2.1 Hz, 1 H), 7.54 (dd, J = 8.4, 2.1 Hz, 1 H),
8.52 (s, 1H), 14.01 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 38.61, 63.97, 64.62,
117.23, 117.42, 122.43, 129.12, 143.22, 144.74, 148.19, 158.26, 192.15 ppm. HRMS (ESI)
m/z calculated for C12H12N3O3S [Mꢀ+ꢀH]+ 278.0599, found 278.0605. HPLC purity, 99.3% at
254 nm (tR = 2.77 min).
6-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)thiazolo[3,2-b][1,2,4]triazole (7a, Supplementary
information, Table S1). This compound was obtained as a side product in the synthesis of 7.
1
Yield, 37%. H NMR (400ꢀMHz, DMSO-d6) δ 4.31 (s, 4ꢀH), 7.03 (d, J = 8.5 Hz, 1 H), 7.69
(dd, J = 8.5, 2.2 Hz, 1 H), 7.79 (d, J = 2.2 Hz, 1 H), 7.83 (d, J = 1.5 Hz, 1 H), 8.42 (d, J = 1.5
Hz, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 64.14, 64.35, 109.27, 115.06, 117.53,
119.57, 120.87, 131.07, 143.44, 144.64, 156.07, 156.67 ppm. ESI-MS for C12H10N3O2S [M +
H]+ 260.16. HRMS (ESI) m/z calculated for C12H10N3O2S [Mꢀ+ꢀH]+ 260.0488, found
260.0483. HPLC purity, 100% at 254 nm (tR = 4.03 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-methyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one
1
(8). Yield, 86%. H NMR (400ꢀMHz, DMSO-d6) δ 3.69 (s, 3 H), 4.28–4.31 (m, 2ꢀH), 4.33–
4.35 (m, 2ꢀH), 4.89 (s, 2 H), 7.00 (d, J = 8.4 Hz, 1 H), 7.51 (d, J = 2.1 Hz, 1 H), 7.54 (dd, J =
13
8.4, 2.2 Hz, 1 H), 9.06 (s, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 31.79, 40.95, 63.96,
64.65, 117.32, 117.56, 122.56, 128.48, 143.25, 145.80, 148.52, 150.36, 191.22 ppm. HRMS
(ESI) m/z calculated for C13H14N3O3S [Mꢀ+ꢀH]+ 292.0756, found 292.0759. HPLC purity,
97.8% at 254 nm (tR = 2.90 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-ethyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one
1
(9). Yield, 40%. H NMR (400ꢀMHz, DMSO-d6) δ 1.42 (t, J = 7.3 Hz, 3 H), 4.14 (q, J = 7.3
Hz, 2 H), 4.28–4.30 (m, 2ꢀH), 4.33–4.35 (m, 2ꢀH), 5.02 (s, 2 H), 7.05 (d, J = 8.5 Hz, 1 H), 7.52
13
(d, J = 2.1 Hz, 1 H), 7.55 (dd, J = 8.4, 2.2 Hz, 1 H), 9.57 (s, 1H) ppm. C NMR (100 MHz,

DMSO-d6) δ 14.31, 41.26, 41.46, 64.00, 64.70, 117.40, 117.59, 122.61, 128.38, 143.30,
144.52, 148.64, 151.05, 190.80 ppm. HRMS (ESI) m/z calculated for C14H16N3O3S [Mꢀ+ꢀH]+
306.0907, found 306.0899. HPLC purity, 97.4% at 254 nm (tR = 3.27 min).
4-(3-((2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-oxoethyl)thio)-4H-1,2,4-triazol-4-
yl)benzonitrile (10). Yield, 54%. 1H NMR (400ꢀMHz, DMSO-d6) δ 4.28–4.31 (m, 2ꢀH), 4.33–
4.35 (m, 2ꢀH), 4.82 (s, 2 H), 6.98 (d, Jꢀ=ꢀ8.4ꢀHz, 1ꢀH), 7.45 (d, Jꢀ=ꢀ2.1ꢀHz, 1ꢀH), 7.51 (dd,
Jꢀ=ꢀ8.5,ꢀ2.2ꢀHz, 1ꢀH), 7.81 (d, Jꢀ=ꢀ8.6ꢀHz, 2ꢀH), 8.10 (d, Jꢀ=ꢀ8.7ꢀHz, 2ꢀH), 8.96 (s, 1H) ppm. 13C
NMR (100 MHz, DMSO-d6) δ 40.68, 63.91, 64.58, 111.91, 117.19, 117.42, 117.95, 122.47,
126.10, 128.54, 133.94, 143.15, 145.13, 148.36, 148.36, 191.22 ppm. HRMS (ESI) m/z
calculated for C19H14N4O3S [Mꢀ+ꢀH]+ 379.0865, found 379.0865. HPLC purity, 95.4% at 254
nm (tR = 3.93 min).
4-(3-((2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-oxoethyl)thio)-4H-1,2,4-triazol-4-
1
yl)benzamide (11). Yield, 14%. H NMR (400ꢀMHz, DMSO-d6) δ 4.29–4.31 (m, 2ꢀH), 4.33–
4.36 (m, 2ꢀH), 4.85 (s, 2 H), 7.00 (d, Jꢀ=ꢀ8.5ꢀHz, 1ꢀH), 7.49 (d, Jꢀ=ꢀ2.1ꢀHz, 1ꢀH), 7.53 (dd,
Jꢀ=ꢀ8.5, 2.1ꢀHz, 1ꢀH), 7.58 (bs, 1 H), 7.64 (d, Jꢀ=ꢀ8.5ꢀHz, 2ꢀH), 8.06 (d, Jꢀ=ꢀ8.5ꢀHz, 2ꢀH), 8.16
13
(bs, 1 H), 8.92 (s, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 40.40, 63.92, 64.59, 117.23,
117.43, 122.46, 124.93, 128.67, 128.94, 134.88, 135.46, 143.19, 145.18, 148.36, 148.60,
166.70, 191.27 ppm. HRMS (ESI) m/z calculated for C19H16N4O4S [Mꢀ+ꢀH]+ 397.0971, found
397.0974. HPLC purity, 99.0% at 254 nm (tR = 3.05 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-(4-(dimethylamino)phenyl)-4H-1,2,4-triazol-
3-yl)thio)ethan-1-one (12). Yield, 65%. 1H NMR (400ꢀMHz, DMSO-d6) δ 2.98 (s, 6 H), 4.29–
4.31 (m, 2ꢀH), 4.33–4.35 (m, 2ꢀH), 4.84 (s, 2 H), 6.86 (d, Jꢀ=ꢀ9.0 Hz, 2ꢀH), 7.00 (d, Jꢀ=ꢀ8.5ꢀHz,
1ꢀH), 7.31 (d, Jꢀ=ꢀ9.0ꢀHz, 2 H), 7.50 (d, Jꢀ=ꢀ2.0ꢀHz, 1ꢀH), 7.54 (dd, Jꢀ=ꢀ8.5,ꢀ2.0ꢀHz, 1ꢀH), 8.90
13
(s, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 38.84, 40.10, 63.92, 64.59, 112.44, 117.24,
117.45, 121.33, 122.46, 126.12, 128.69, 143.19, 145.50, 148.37, 149.88, 150.60, 191.28 ppm.
HRMS (ESI) m/z calculated for C20H20N4O3S [Mꢀ+ꢀH]+ 397.1334, found 397.1329. HPLC
purity, 98.2% at 254 nm (tR = 3.97 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-(pyridin-3-yl)-4H-1,2,4-triazol-3-
1
yl)thio)ethan-1-one (13). Yield, 25%. H NMR (400ꢀMHz, DMSO-d6) δ 4.29–4.31 (m, 2ꢀH),
4.33–4.35 (m, 2ꢀH), 4.82 (s, 2 H), 6.99 (d, Jꢀ=ꢀ8.4ꢀHz, 1ꢀH), 7.47 (d, Jꢀ=ꢀ2.2ꢀHz, 1ꢀH), 7.51 (dd,
Jꢀ=ꢀ8.4,ꢀ2.2ꢀHz, 1ꢀH), 7.66 (dd, J ꢀ=ꢀ8.4, 4.8ꢀHz, 1ꢀH), 8.06 (ddd, Jꢀ=ꢀ8.1,ꢀ2.6,ꢀ1.5ꢀHz, 1ꢀH), 8.74

13
(dd, Jꢀ=ꢀ8.4,ꢀ1.5ꢀHz, 1ꢀH), 8.78 (d, Jꢀ=ꢀ2.6ꢀHz, 1ꢀH), 8.94 (s, 1H) ppm. C NMR (100 MHz,
DMSO-d6) δ 40.74, 63.92, 64.59, 117.22, 117.45, 122.47, 124.38, 128.57, 130.33, 133.39,
143.18, 145.37, 146.09, 148.37, 148.83, 150.28, 191.28 ppm. HRMS (ESI) m/z calculated for
C17H14N4O3S [Mꢀ+ꢀH]+ 355.0865, found 355.0862. HPLC purity, 97.2% at 254 nm (tR = 3.32
min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((5-methyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one
(14). Yield, 79%. 1H NMR (400ꢀMHz, DMSO-d6) δ 2.45 (s, 3 H), 4.27–4.29 (m, 2 H), 4.32–
4.34 (m, 2 H), 4.86 (s, 2 H), 6.99 (d, J = 8.4 Hz, 1 H), 7.49 (s, J = 2.1 Hz, 1 H), 7.53 (dd, J =
13
8.4, 2.1 Hz, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 10.99, 39.79 (under DMSO peak,
1
confirmed by 2D NMR experiment H-13C HSQC), 64.01, 64.68, 117.32, 117.50, 122.54,
1
128.64, 143.26, 148.47, 153.78 (2 × Ar-C, confirmed by 2D NMR experiment H-13C
HMBC), 191.28. HRMS (ESI) m/z calculated for C13H14N3O3S [Mꢀ+ꢀH]+ 292.0750, found
292.0745. HPLC purity, 98.1% at 254 nm (tR = 2.82 min).
2-((3-Cyclohexyl-1H-1,2,4-triazol-5-yl)thio)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-
one (15). Yield, 26%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.17–1.24 (m, 1 H), 1.27–1.48 (m, 4
H), 1.62–1.69 (m, 1 H), 1.70–1.75 (m, 2 H), 1.87–1.91 (m, 2 H), 2.67–2.75 (m, 1 H), 4.28–
4.30 (m, 2 H), 4.32–4.35 (m, 2 H), 4.68 (s, 2 H), 6.99 (d, J = 8.3 Hz, 1 H), 7.50 (d, J = 2.1 Hz,
13
1 H), 7.53 (dd, J = 8.4, 2.1 Hz, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 25.09, 25.25,
30.58, 35.15, 38.61, 63.92, 64.57, 117.16, 117.44, 122.39, 129.01, 143.14, 148.18, 155.88,
161.81, 192.09 ppm. HRMS (ESI) m/z calculated for C18H22N3O3S [Mꢀ+ꢀH]+ 360.1376, found
360.1373. HPLC purity, 98.5% at 254 nm (tR = 4.08 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((5-phenyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one
(16). Yield, 61%. 1H NMR (400ꢀMHz, DMSO-d6) δ 4.29–4.31 (m, 2 H), 4.33–4.35 (m, 2 H),
4.78 (s, 2 H), 7.01 (d, J = 8.4 Hz, 1 H), 7.46–7.50 (m, 3 H), 7.55–7.59 (m, 2 H), 7.89–7.91 (m,
13
2 H) ppm. C NMR (100 MHz, DMSO-d6) δ 38.91 (under DMSO peak, confirmed by 2D
1
NMR experiment H-13C HSQC) 63.93, 64.58, 117.20, 117.50, 122.42, 125.88 (2 × Ar-C,
1
confirmed 2D NMR experiment H-13C HSQC), 128.94 (3 × Ar-C, confirmed 2D NMR
1
experiment H-13C HSQC), 126.00, 129.08, 129.93, 143.17, 148.21, 153.93, 192.14 ppm.
HRMS (ESI) m/z calculated for C18H16N3O3S [Mꢀ+ꢀH]+ 354.0907, found 354.0900. HPLC
purity, 97.5% at 254 nm (tR = 4.16 min).

2-((1-(Cyclohexanecarbonyl)-3-cyclohexyl-1H-1,2,4-triazol-5-yl)thio)-1-(2,3-
1
dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one (17). Yield, 42%. H NMR (400ꢀMHz, DMSO-
d6) δ 1.23–1.35 (m, 6 H), 1.47–1.57 (m, 4 H), 1.63–1.66 (m, 1 H), 1.74–1.78 (m, 3 H), 1.86–
1.89 (m, 2 H), 1.96–2.06 (m, 4 H), 2.42–2.49 (m, 1 H), 2.73–2.81 (m, 1 H), 4.29–4.32 (m, 2
H), 4.35–4.37 (m, 2 H), 5.66 (s, 2 H), 6.98 (d, J = 9.1 Hz, 1 H), 7.50–7.53 (m, 2 H) ppm. 13C
NMR (100 MHz, CDCl3) δ 25.20, 25.83, 26.19, 26.33, 30.08, 33.63, 39.68, 47.66, 58.40,
64.20, 64.94, 117.94, 118.10, 122.74, 127.30, 144.01, 149.84, 167.16, 170.63, 186.38, 186.66
ppm. HRMS (ESI) m/z calculated for C25H32N3O4S [Mꢀ+ꢀH]+ 470.2108, found 470.2101.
HPLC purity, 99.5% at 254 nm (tR = 2.52 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((3-(pyridin-4-yl)-1H-1,2,4-triazol-5-
1
yl)thio)ethan-1-one (18). Yield, 47%. H NMR (400ꢀMHz, DMSO-d6) δ 4.30–4.32 (m, 2 H),
4.35–4.37 (m, 2 H), 4.96 (s, 2 H), 7.03 (d, J = 8.4 Hz, 1 H), 7.56–7.60 (m, 2 H), 8.31 (d, J =
13
5.7 Hz, 2 H), 8.9 (d, J = 6.6 Hz, 2 H) ppm. C NMR (100 MHz, DMSO-d6) δ 39.94 (under
1
DMSO peak, confirmed by 2D NMR experiment H-13C HSQC), 63.95, 64.62, 117.30,
1
117.55, 122.32 (2 × Ar-C, pyridine, confirmed 2D NMR experiment H-13C HSQC), 122.47,
1
128.68, 143.20, 143.64 (2 × Ar-C, pyridine, confirmed 2D NMR experiment H-13C HSQC),
1
148.42, 157.00 (Ar-C, pyridine, confirmed 2D NMR experiment H-13C HMBC), 191.40
ppm. HRMS (ESI) m/z calculated for C17H15N4O3S [Mꢀ+ꢀH]+ 355.0859, found 355.0860.
HPLC purity, 99.2% at 254 nm (tR = 2.79 min).
2-((1H-Imidazol-2-yl)thio)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-one (19). Yield,
74%. 1H NMR (400ꢀMHz, DMSO-d6) δ 4.29–4.31 (m, 2ꢀH), 4.34–4.36 (m, 2ꢀH), 4.99 (s, 2 H),
7.04 (dd, J = 8.1, 0.7 Hz, 1 H), 7.51 (d, J = 2.0 Hz, 1 H), 7.53 (dd, J = 8.5, 2.1 Hz, 1 H), 7.72
(s, 2 H), 14.43 (bs, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 41.64, 64.01, 64.71, 117.44,
1
117.67, 121.14 (2 × C, confirmed 2D NMR experiment H-13C HSQC), 122.64, 128.08,
139.92, 143.31, 148.75, 191.12 ppm. HRMS (ESI) m/z calculated for C13H13N2O3S [Mꢀ+ꢀH]+
277.0647, found 277.0646. HPLC purity, 100% at 254 nm (tR = 2.14 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((1-methyl-1H-imidazol-2-yl)thio)ethan-1-one
1
(20). Yield, 93%. H NMR (400ꢀMHz, DMSO-d6) δ 3.83 (s, 3 H), 4.28–4.31 (m, 2ꢀH), 4.34–
4.36 (m, 2ꢀH), 4.91 (s, 2 H), 7.02 (dd, J = 8.2, 0.5 Hz, 1 H), 7.48–7.53 (m, 2 H), 7.75 (d, J =
2.0 Hz, 1 H), 7.82 (d, J = 2.0 Hz, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 35.20, 42.32,
63.98, 64.69, 117.42, 117.65, 120.95, 122.63, 125.28, 128.02, 139.68, 143.30, 148.76, 191.44

ppm. HRMS (ESI) m/z calculated for C14H15N2O3S [Mꢀ+ꢀH]+ 291.0803, found 291.0799.
HPLC purity, 100% at 254 nm (tR = 2.51 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((1-methyl-1H-tetrazol-5-yl)thio)ethan-1-one
1
(21). Yield, 79%. H NMR (400ꢀMHz, DMSO-d6) δ 4.00 (s, 3 H), 4.29–4.31 (m, 2ꢀH), 4.34–
4.36 (m, 2ꢀH), 5.01 (s, 2 H), 7.01 (d, J = 8.4 Hz, 1 H), 7.53 (d, J = 2.0 Hz, 1 H), 7.56 (dd, J =
8.5, 2.1 Hz, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 33.70, 41.09, 63.96, 64.65, 117.34,
117.53, 122.56, 128.52, 143.28, 148.56, 153.33, 190.91 ppm. HRMS (ESI) m/z calculated for
C12H13N4O3S [Mꢀ+ꢀH]+ 293.0708, found 293.0703. HPLC purity, 99.3% at 254 nm (tR = 3.43
min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(phenylthio)ethan-1-one (22). The product was
synthesized according to the general procedure for synthesis of the final compounds, and then
purified by flash chromatography using EtOAc/hex (1:3, v/v) as mobile phase. Yield, 12%. 1H
NMR (400ꢀMHz, CDCl3) δ 4.19 (s, 2 H), 4.25–4.27 (m, 2ꢀH), 4.29–4.31 (m, 2ꢀH), 6.89 (dd, J
= 8.4, 0.5 Hz, 1 H), 7.16–7.22 (m, 1 H), 7.24–7.31 (m, 2 H), 7.35–7.39 (m, 2 H), 7.47 (dd, J =
8.4, 2.1 Hz, 1 H), 7.49 (dd, J = 2.1, 0.4 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3) δ 41.09,
64.20, 64.83, 117.45, 118.32, 123.00, 127.07, 129.15, 129.26, 130.42, 135.12, 143.50, 148.51,
192.68 ppm. HRMS (ESI) m/z calculated for C16H15O3S [Mꢀ+ꢀH]+ 287.0742, found 287.0747.
HPLC purity, 85.2% at 254 nm (tR = 4.88 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(thiophen-2-ylthio)ethan-1-one (23). The product
was synthesized according to the general procedure for synthesis of the final compounds, and
then purified by flash chromatography using EtOAc/hex (1:3, v/v) as mobile phase. Yield,
7%. 1H NMR (400ꢀMHz, CDCl3) δ 4.09 (s, 2 H), 4.27–4.29 (m, 2ꢀH), 4.31–4.33 (m, 2ꢀH), 6.89
(d, J = 8.4 Hz, 1 H), 6.95 (dd, J = 5.4, 3.6 Hz, 1 H), 7.12 (dd, J = 3.6, 1.3 Hz, 1 H), 7.36 (dd, J
= 5.4, 1.3 Hz, 1 H), 7.43 (dd, J = 8.4, 2.2 Hz, 1 H), 7.46 (d, J = 2.1 Hz, 1 H) ppm. 13C NMR
(100 MHz, CDCl3) δ 45.24, 64.21, 64.85, 117.45, 118.29, 122.99, 127.80, 129.31, 130.63,
132.49, 135.34, 143.52, 148.52, 192.53 ppm. HRMS (ESI) m/z calculated for C14H13O3S2
[Mꢀ+ꢀH]+ 293.0306, found 293.0303. HPLC purity, 95.3% at 254 nm (tR = 4.77 min).
1-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-(thiazol-2-ylthio)ethan-1-one (24). Yield, 55%. 1H
NMR (400ꢀMHz, DMSO-d6) δ 4.29–4.31 (m, 2ꢀH), 4.34–4.36 (m, 2ꢀH), 4.90 (s, 2 H), 7.00 (d,
Jꢀ=ꢀ8.4ꢀHz, 1ꢀH), 7.53 (d, Jꢀ=ꢀ2.1ꢀHz, 1ꢀH), 7.56 (dd, Jꢀ=ꢀ8.4,ꢀ2.2ꢀHz, 1ꢀH), 7.63 (d, Jꢀ=ꢀ3.4ꢀHz,
13
1ꢀH), 7.67 (d, Jꢀ=ꢀ3.4ꢀHz, 1ꢀH) ppm. C NMR (100 MHz, DMSO-d6) δ 41.03, 63.91, 64.58,

117.24, 117.49, 120.43, 122.50, 128.76, 142.58, 143.20, 148.35, 162.97, 191.32 ppm. HRMS
(ESI) m/z calculated for C13H11NO3S2 [Mꢀ+ꢀH]+ 294.0259, found 294.0262. HPLC purity,
98.8% at 254 nm (tR = 4.17 min).
Synthesis of 2-(2-((2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-oxoethyl)thio)-1H-imidazol-1-
yl)acetonitrile (25): To a suspension of 19 (0.080 g, 0.29 mmol, 1.0 equiv.) in MeCN (10
mL), K2CO3 (0.120 g, 0.87 mmol, 3.0 equiv.) and chloroacetonitrile (0.055 mL, 0.87 mmol,
3.0 equiv.) were added, and the reaction mixture was stirred at reflux for 18 h. The solvent
was evaporated under reduced pressure, and the residue was dissolved in a mixture of DCM
(50 mL) and saturated NaHCO3(aq) (50 mL), and transferred into a separating funnel. The
organic phase was washed with saturated brine (50 mL), dried over Na2SO4, filtered and
evaporated under reduced pressure. The product was purified by flash chromatography using
1
DCM/MeOH (50:1, v/v) as mobile phase. Yield, 19%. H NMR (400ꢀMHz, CDCl3) δ 4.25–
4.28 (m, 2ꢀH), 4.30–4.32 (m, 2ꢀH), 4.45 (s, 2 H), 5.16 (s, 2 H), 6.88 (d, J = 8.4 Hz, 1 H), 7.12
(d, J = 1.4 Hz, 1 H), 7.15 (d, J = 1.4 Hz, 1 H), 7.40 (dd, J = 8.5, 2.2 Hz, 1 H), 7.44 (d, J = 1.9
Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3) δ 34.67, 43.09, 64.18, 64.85, 114.13, 117.63,
118.02, 121.44, 122.77, 128.87, 131.37, 140.70, 143.68, 148.97, 191.91 ppm. HRMS (ESI)
m/z calculated for C15H14O3N3S [Mꢀ+ꢀH]+ 316.0750, found 316.0739. HPLC purity, 99.5% at
254 nm (tR = 2.52 min).
Synthesis of 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-
yl)thio)ethan-1-ol (26): To a solution of Z9 (0.225 g, 0.704 mmol, 1.0 equiv.) in a mixture of
anhydrous EtOH/THF (25 mL + 25 mL), NaBH4 (0.040 g, 1.057 mmol, 1.5 equiv.) was
added, and the reaction mixture was stirred at room temperature for 2 h. Water (2 mL) was
added and the mixture was stirred for 5 min before the solvent was evaporated off under
reduced pressure. The residue was dissolved in EtOAc (50 mL) and washed with water (50
mL). The water phase was extracted with EtOAc (40 mL). The combined organic phases were
washed with saturated brine (50 mL), dried over Na2SO4, filtered and evaporated under
reduced pressure. The product was purified by flash chromatography using DCM/MeOH
(20:1, v/v) as mobile phase. Yield, 87%. 1H NMR (400ꢀMHz, CDCl3) δ 1.49 (dd, J = 6.7, 3.4
Hz, 6 H), 3.43 (dd, J = 14.4, 8.0 Hz, 1 H), 3.53 (dd, J = 14.4, 3.2 Hz, 1 H), 4.25 (s, 4ꢀH), 4.34
(hept, J = 6.7 Hz, 1 H), 5.06 (dd, J = 8.0, 3.1 Hz, 1 H), 5.15 (bs, 1 H), 6.84 (d, J = 8.3 Hz, 1
13
H), 6.91 (ddd, J = 8.3, 2.0, 0.6 Hz, 1 H), 6.97 (d, J = 2.0 Hz, 1 H), 8.19 (s, 1 H) ppm. C
NMR (100 MHz, CDCl3) δ 22.93, 23.05, 41.89, 48.41, 64.42, 64.43, 73.31, 115.01, 117.22,

119.05, 136.48, 141.42, 143.12, 143.52, 150.78 ppm. HRMS (ESI) m/z calculated for
C15H20N3O3S [Mꢀ+ꢀH]+ 322.1225, found 322.1222. HPLC purity, 98.3% at 254 nm (tR = 2.82
min).
Synthesis of (Z)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-
yl)thio)ethan-1-one O-methyl oxime (27): To a solution of Z9 (0.110 g, 0.376 mmol, 1.0
equiv.) in absolute EtOH (10 mL), pyridine (0.45 mL, 5.64 mmol, 15.0 equiv.) and O-
methylhydroxylamine hydrochloride (0.094 g, 1.128 mmol, 3.0 equiv.) were added. The
reaction mixture was stirred at 90 °C for 16 h. The solvent was evaporated off under reduced
pressure, and the residue was dissolved in DCM (50 mL) and washed with 1 M HCl (30 mL),
then saturated brine (30 mL), dried over Na2SO4, filtered and evaporated under reduced
pressure. The product was purified by flash chromatography using EtOAc as mobile phase.
Yield, 66%. 1H NMR (400ꢀMHz, CDCl3) δ 1.36 (d, J = 6.8 Hz, 6 H), 3.92 (s, 3 H), 4.19–4.25
(m, 4ꢀH), 4.28 (hept, J = 6.7 Hz, 1 H), 4.38 (s, 2 H), 6.79 (d, J = 8.5 Hz, 1 H), 7.11 (dd, J =
8.5, 2.2 Hz, 1 H), 7.21 (d, J = 2.2 Hz, 1 H), 8.17 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3) δ
23.10, 27.32, 48.06, 62.47, 64.25, 64.53, 115.22, 117.40, 119.68, 126.98, 141.57, 143.60,
145.08, 149.13, 152.62 ppm. HRMS (ESI) m/z calculated for C16H21N4O3S [Mꢀ+ꢀH]+
349.1329, found 349.1323. HPLC purity, 95.3% at 254 nm (tR = 4.10 min).
Synthesis of 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-
yl)thio)ethan-1-amine (28): A solution of Z9 (0.150 g, 0.47 mmol, 1.0 equiv.) and NH4OAc
(0.404 g, 4.70 mmol, 10.0 equiv.) in MeOH (15 mL) was stirred at room temperature for 10
min, and NaBH3CN (0.030 g, 0.47 mmol, 1.0 equiv.) was added. The reaction mixture was
stirred at 60 °C for 20 h. TLC analysis showed the presence of the starting compound,
therefore more NH4OAc (0.404 g, 4.70 mmol, 10.0 equiv.) and NaBH3CN (0.030 g, 0.47
mmol, 1.0 equiv.) were added, and the reaction mixture was stirred at 70 °C for 20 h. The
solvent was evaporated under reduced pressure, and the residue was dissolved in a mixture of
DCM (30 mL) and saturated NaHCO3(aq) (30 mL), and transferred into a separating funnel.
The organic phase was washed with saturated brine (50 mL), dried over Na2SO4, and filtered
and evaporated under reduced pressure. The product was purified by flash chromatography
1
using DCM/MeOH (9:1, v/v) as mobile phase. Yield, 50%. H NMR (400ꢀMHz, CDCl3) δ
1.45 (dd, J = 6.7, 2.1 Hz, 6 H), 3.32 (dd, J = 13.3, 8.4 Hz, 1 H), 3.55 (dd, J = 13.3, 5.0 Hz, 1
H), 4.22 (s, 4ꢀH), 4.25 (dd, J = 8.3, 5.0 Hz, 1 H), 4.32 (hept, J = 6.7 Hz, 1 H), 6.80 (d, J = 8.3
Hz, 1 H), 6.85 (ddd, J = 8.3, 2.1, 0.4 Hz, 1 H), 6.90 (d, J = 2.1 Hz, 1 H), 8.19 (s, 1 H) ppm,

resonance for NH2 missing. 13C NMR (100 MHz, CDCl3) δ 23.04, 41.98, 48.00, 54.52, 64.41
(2 × C), 115.32, 117.34, 119.55, 137.01, 141.41, 143.02, 143.57, 149.64 ppm. HRMS (ESI)
m/z calculated for C15H21N4O2S [Mꢀ+ꢀH]+ 321.1380, found 321.1373. HPLC purity, 96.02%
at 254 nm (tR = 2.23 min).
Synthesis of 2,4-dihydro-3H-1,2,4-triazol-3-one (29): A mixture of semicarbazide
hydrochloride (10.0 g, 89.6 mmol. 1.0 eq) and trimethyl orthofromate (28.5 g, 269.0 mmol,
3.0 eq) in MeOH (100 mL) was stirred at room temperature for 2 h. The reaction mixture was
concentrated under reduced pressure, toluene (100 mL) was added, and the mixture was
cooled to 0 °C. The forming white precipitate was filtered off to obtain the pure product.
Yield, 100%. 1H NMR (400ꢀMHz, DMSO-d6) δ 7.90 (bs, 2 H), 11.45 (s, 1 H) [20].
Synthesis of tert-butyl 5-oxo-1,5-dihydro-4H-1,2,4-triazole-4-carboxylate (30): To a solution
of 29 (2.0 g, 23.5 mmol, 1.0 equiv.) in DCM (20 mL), (BOC)2O (5.1 g, 23.5 mmol, 1.0
equiv.), DMAP (0.287 g, 2.35 mmol, 0.1 equiv.) and DIPEA (10.0 mL, 58.7 mmol, 2.5
equiv.) were added. The reaction was stirred at room temperature for 18 h. The reaction
mixture was concentrated under reduced pressure and purified by flash chromatography using
DCM/MeOH (20:1, v/v) as mobile phase. Yield, 14%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.50
13
(s, 9 H), 7.98 (s, 1H) ppm. C NMR (100 MHz, DMSO-d6) δ 27.97, 85.83, 136.53, 147.15,
153.68 ppm. ESI-MS for C7H11N3O [M–H]– 184.12.
Synthesis of tert-butyl 3-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-oxoethoxy)-4H-1,2,4-
triazole-4-carboxylate (31): To a solution of 30 (0.172 g, 0.934 mmol, 1.2 equiv.) in
anhydrous DMF (3 mL) under an argon atmosphere, NaH (0.040 mg, 1.01 mmol, 1.3 equiv.)
was added and stirred at room temperature for 15 min, followed by addition of bromide 6
(0.200 g, 0.778 mmol, 1.0 equiv.). The reaction mixture was stirred at room temperature for
18 h. EtOAc (60 mL) was then added, and the organic phase was washed with water (2 × 30
mL) and brine (30 mL), and dried over Na2SO4, and the solvent was evaporated under
reduced pressure. The product was purified by flash chromatography using DCM/MeOH
1
(50:1, v/v) as mobile phase. Yield, 47%. H NMR (400ꢀMHz, CDCl3) δ 1.47 (s, 9 H), 4.29–
4.36 (m, 4 H), 5.04 (s, 2 H), 6.96 (d, J = 9.1 Hz, 1 H), 7.50–7.52 (m, 2 H), 7.62 (s, 1 H) ppm.
ESI-MS for C17H19N3O6 [M + Na]+ 384.31; [M – H]– 360.37.
Synthesis of 2-((4H-1,2,4-triazol-3-yl)oxy)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-1-
one (32): To a solution of 31 (0.100 g, 0.276 mmol, 1.0 equiv.) in DCM (5 mL), TFA (1 mL)

was added drop-wise. The mixture was stirred at room temperature for 2 h, then the solvent
was evaporated off under reduced pressure. Further, 1 mL of 2 M HCl solution in ether was
added, forming of precipitate, followed by addition of saturated NaOH solution in MeOH.
The solvent was evaporated under reduced pressure, and the product was purified by flash
chromatography using DCM/MeOH (20:1, v/v) as mobile phase. Additionally, the product
1
was recrystallized from DCM. Yield, 58%. H NMR (400ꢀMHz, DMSO-d6) δ 4.29–4.31 (m,
2ꢀH), 4.34–4.36 (m, 2ꢀH), 5.12 (s, 2 H), 7.03 (d, Jꢀ=ꢀ8.4ꢀHz, 1ꢀH), 7.54 (d, Jꢀ=ꢀ2.1ꢀHz, 1ꢀH),
13
7.58 (dd, Jꢀ=ꢀ8.4,ꢀ2.1ꢀHz, 1ꢀH), 7.79 (d, Jꢀ=ꢀ1.4ꢀHz, 1ꢀH), 11.70 (s, 1 H) ppm. C NMR (100
MHz, DMSO-d6) δ 47.39, 63.95, 64.64, 117.03, 117.45, 122.08, 127.67, 138.63, 143.39,
148.62, 154.59, 191.19 ppm. ESI-MS for C12H11N3O4 [M + H]+ 262.3, [M – H]– 260.23.
HRMS (ESI) m/z calculated for C12H12N3O4 [Mꢀ+ꢀH]+ 262.0828, found 262.0821. HPLC
purity, 98.8% at 254 nm (tR = 2.06 min).
Synthesis of 2-(5-amino-1H-1,2,4-triazol-1-yl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)ethan-
1-one (33): To a suspension of bromide 6 (0.200 g, 0.778 mmol, 1.0 equiv.) and 4H-1,2,4-
triazol-3-amine (0.068 g, 0.778 mmol, 1.0 equiv.) in anhydrous EtOH (15 mL), K2CO3 (0.161
g, 1.167 mml, 1.5 equiv.) was added, and the reaction was stirred at room temperature for 18
h. The solvent was evaporated under reduced pressure, the residue was dissolved in EtOAc
(50 mL), and the organic phase was washed with water (3 × 25 mL). The organic phase was
evaporated under reduced pressure and the product was purified by flash chromatography
using DCM/MeOH (9:1, v/v) as mobile phase. Yield, 20%. 1H NMR (400ꢀMHz, DMSO-d6) δ
4.30–4.32 (m, 2ꢀH), 4.34–4.36 (m, 2ꢀH), 5.45 (s, 2 H), 6.16 (s, 2 H), 7.03 (d, Jꢀ=ꢀ8.4ꢀHz, 1ꢀH),
7.34 (s, 1H), 7.52‒7.55 (m, 2 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 52.50, 63.93, 64.59,
117.10, 117.29, 122.01, 128.06, 143.25, 148.32, 148.49, 156.26, 190.92 ppm. HRMS (ESI)
m/z calculated for C12H12N4O3 [Mꢀ+ꢀH]+ 261.0988, found 261.0985. HPLC purity, 97.1% at
254 nm (tR = 2.04 min).
General procedure for the synthesis of compounds 34a-c [15]: 3-Aminotriazole (3.36 g, 40.0
mmol, 1.0 equiv.) was suspended in DMFDMA reagent (20 mL), and the mixture was stirred
at 110 °C for 18 h. The solvent was evaporated under reduced pressure, the isomers 34a-c
were separated and purified by flash chromatography using DCM/MeOH = 9/1 (V/V) as a
mobile phase.

(E)-N,N-Dimethyl-N'-(1-methyl-1H-1,2,4-triazol-3-yl)formimidamide (34a). Yield (isolated),
1
15%. H NMR (400ꢀMHz, DMSO-d6) δ 2.90 (s, 3 H), 3.01 (s, 3 H), 3.59 (s, 3 H), 7.51 (s, 1
H), 8.37 (s, 1 H) ppm. ESI-MS for C6H11N5 [M+H]+ 153.9; [M + Na]+ 175.9.
(E)-N,N-Dimethyl-N'-(1-methyl-1H-1,2,4-triazol-5-yl)formimidamide (34b). Yield (isolated),
50%. 1H NMR (400ꢀMHz, DMSO-d6) δ 2.99 (d, J = 0.4 Hz, 3 H), 3.09 (s, 3 H), 3.68 (s, 3 H),
8.07 (s, 1 H), 8.26 (s, 1 H) ppm. ESI-MS for C6H11N5 [M + H]+ 153.9.
(E)-N,N-Dimethyl-N'-(4-methyl-4H-1,2,4-triazol-3-yl)formimidamide (34c). Yield (isolated),
2%. 1H NMR (400ꢀMHz, DMSO-d6) δ 2.97 (s, 3 H), 3.08 (s, 3 H), 3.39 (s, 3 H), 8.10 (s, 1 H),
8.33 (s, 1 H) ppm. ESI-MS for C6H11N5 [M+H]+ 154.1. 34c was not used in the next reaction
steps due to low amounts of the product yielded after purification.
General procedure for the synthesis of compounds 35a-b [15]: Compound 34a or 34b (1.0
equiv.) was suspended in 4 M NaOH(aq) (50 mL) and heated under reflux for 1 h. After
cooling, the solution was neutralized with 37% HCl(aq) and extracted with EtOAc (3 × 50
mL). The combined organic phases were washed with saturated brine (50 mL), dried over
Na2SO4, and filtered and evaporated under reduced pressure, to obtain pure products.
1-Methyl-1H-1,2,4-triazol-3-amine (35a). Yield, 66%. 1H NMR (400ꢀMHz, DMSO-d6) δ 3.59
(d, J = 0.5 Hz, 3 H), 5.19 (bs, 2 H), 7.87 (s, 1 H) ppm. ESI-MS for C3H6N4 [M+MeOH+H]+
130.9.
1-Methyl-1H-1,2,4-triazol-5-amine (35b). Yield, 36%. 1H NMR (400ꢀMHz, DMSO-d6) δ 3.48
(s, 3 H), 6.11 (bs, 2 H), 7.27 (s, 1 H) ppm. ESI-MS for C3H6N4 [M+MeOH+H]+ 130.9.
3-Amino-4-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-oxoethyl)-1-methyl-1H-1,2,4-triazol-
4-ium bromide (36a). Synthesized following the general procedure for the synthesis of thiols.
The product was purified by flash chromatography using DCM/MeOH (9:1, v/v) as mobile
phase. Yield, 41%. 1H NMR (400ꢀMHz, DMSO-d6) δ 3.90 (d, J = 0.9 Hz, 3 H), 4.31–4.33 (m,
2ꢀH), 4.36–4.38 (m, 2ꢀH), 5.71 (s, 2 H), 7.09 (d, J = 8.4 Hz, 1 H), 7.31 (bs, 2 H), 7.54 (d, J =
13
2.0 Hz, 1 H), 7.56 (dd, J = 8.4, 2.1 Hz, 1 H), 9.32 (d, J = 1.0 Hz, 1 H) ppm. C NMR (100
MHz, DMSO-d6) δ 38.26, 51.01, 64.03, 64.75, 117.33, 117.53, 122.40, 127.12, 139.82,
143.38, 148.91, 154.96, 188.90 ppm. HRMS (ESI) m/z calculated for C13H15O3N4 [M + H]+
275.1139, found 275.1127. HPLC purity, 96.7% at 254 nm (tR = 1.79 min).

5-Amino-4-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-oxoethyl)-1-methyl-1H-1,2,4-triazol-
4-ium bromide (36b). Synthesized following the general procedure for the synthesis of thiols.
The product was purified by flash chromatography using DCM/MeOH (9:1, v/v) as mobile
phase. Yield, 33%. 1H NMR (400ꢀMHz, DMSO-d6) δ 3.71 (s, 3 H), 4.31–4.33 (m, 2ꢀH), 4.36–
4.38 (m, 2ꢀH), 5.64 (s, 2 H), 7.09 (d, J = 8.4 Hz, 1 H), 7.53 (d, J = 2.0 Hz, 1 H), 7.56 (dd, J =
8.4, 2.2 Hz, 1 H), 8.36 (s, 1 H), 8.54 (bs, 2 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 35.14,
50.99, 64.01, 64.73, 117.25, 117.52, 122.34, 127.11, 140.26, 143.38, 148.87, 149.53, 188.98
ppm. HRMS (ESI) m/z calculated for C13H15O3N4 [M + H]+ 275.1139, found 275.1128.
HPLC purity, 98.0% at 254 nm (tR = 1.96 min).
1-(1H-Indol-3-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethanone (37). Synthesized
following the general procedure for the synthesis of the final products. Yield, 10%. 1H NMR
(400ꢀMHz, DMSO-d6) δ 1.46 (d, J = 6.7 Hz, 6 H), 4.54 (hept, J = 6.7 Hz, 1 H), 4.81 (s, 2 H),
7.22 (pd, J = 7.1, 1.4 Hz, 2 H), 7.49–7.51 (m, 1 H), 8.11 (dd, J = 7.0, 1.7 Hz, 1 H), 8.48 (d, J
13
= 3.2 Hz, 1 H), 9.37 (s, 1 H), 12.16 (s, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 22.10,
40.97, 49.44, 112.39, 114.49, 121.11, 122.17, 123.18, 125.30, 135.17, 136.59, 142.84
1
(confirmed by 2D NMR experiment H-13C HSQC), 150.08 (confirmed by 2D NMR
1
experiment H-13C HMBC), 187.00 ppm. HRMS (ESI) m/z calculated for C15H17ON4S [M +
H]+ 301.1118, found 301.1121. HPLC purity, 96.4% at 254 nm (tR = 3.46 min).
2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)-1-(2-methyl-1H-indol-3-yl)ethanone
(38).
Synthesized following the general procedure for the synthesis of the final products. Yield,
62%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.52 (d, J = 6.7 Hz, 6 H), 2.75 (s, 3 H), 4.61 (hept, J =
6.7 Hz, 1 H), 5.11 (s, 2 H), 7.15–7.20 (m, 2 H), 7.40–7.44 (m, 1 H), 7.98–8.03 (m, 1 H), 9.68
13
(s, 1 H), 12.38 (s, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 15.26, 21.90, 45.68, 50.15,
111.33, 111.59, 120.52, 121.86, 122.26, 126.55, 134.86, 142.68, 146.04, 150.93, 186.75 ppm.
HRMS (ESI) m/z calculated for C16H19ON4S [M + H]+ 315.1274, found 315.1276. HPLC
purity, 98.7% at 254 nm (tR = 3.61 min).
1-(Benzo[d][1,3]dioxol-5-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethanone
(39).
Synthesized following the general procedure for the synthesis of the final products. Yield,
66%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.50 (d, J = 6.7 Hz, 6 H), 4.57 (hept, J = 6.7 Hz, 1 H),
5.02 (s, 2 H), 6.17 (s, 2 H), 7.09 (d, J = 8.2 Hz, 1 H), 7.50 (d, J = 1.8 Hz, 1 H), 7,69 (dd, J =
13
8.2, 1.8 Hz, 1 H), 9.68 (s, 1 H) ppm. C NMR (100 MHz, DMSO-d6) δ 21.81, 41.26, 50.12,
102.25, 107.66, 108.24, 125.26, 129.34, 142.88, 147.88, 150.41, 152.11, 190.44 ppm. HRMS

(ESI) m/z calculated for C14H16O3N3S [M + H]+ 306.0907, found 306.0909. HPLC purity,
97.9% at 254 nm (tR = 3.58 min).
2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)-1-(4-methoxyphenyl)ethanone (40). Synthesized
following the general procedure for the synthesis of the final products. Yield, 60%. 1H NMR
(400ꢀMHz, DMSO-d6) δ 1.49 (d, J = 6.7 Hz, 6 H), 3.86 (s, 3 H), 4.55 (hept, J = 6.7 Hz, 1 H),
5.03 (s, 2 H), 7.09 (d, J = 8.9 Hz, 2 H), 8.00 (d, J = 8.9 Hz, 2 H), 9.57 (s, 1 H) ppm. 13C NMR
(100 MHz, DMSO-d6) δ 21.87, 41.44, 50.37, 55.77, 114.20, 127.75, 130.98, 142.97, 150.78,
163.84, 190.78 ppm. HRMS (ESI) m/z calculated for C14H18O2N3S [M + H]+ 292.1114, found
292.1116. HPLC purity, 99.6% at 254 nm (tR = 3.71 min).
2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)-1-(3-methoxyphenyl)ethanone (41). Synthesized
following the general procedure for the synthesis of the final products. Yield, 60%. 1H NMR
(400ꢀMHz, DMSO-d6) δ 1.50 (d, J = 6.7 Hz, 6 H), 3.83 (s, 3 H), 4.57 (hept, J = 6.7 Hz, 1 H),
5.10 (s, 2 H), 7.28 (ddd, J = 8.2, 2.6, 1.0 Hz, 1 H), 7.49 (t, J = 7.9 Hz, 1 H), 7.50 (dd, J = 2.6,
1.5 Hz, 1 H), 7.63 (dt, J = 7.8, 1.2 Hz, 1 H), 9.64 (s, 1 H) ppm. 13C NMR (100 MHz, DMSO-
d6) δ 21.91, 41.53, 50.15, 55.52, 113.01, 120.07, 120.99, 130.17, 136.30, 142.98, 150.37,
159.48, 192.40 ppm. HRMS (ESI) m/z calculated for C14H18O2N3S [M + H]+ 292.1114, found
292.1117. HPLC purity, 97.7% at 254 nm (tR = 3.77 min).
2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)-1-(2-methoxyphenyl)ethanone (42). Synthesized
following the general procedure for the synthesis of the final products. Yield, 70%. 1H NMR
(400ꢀMHz, CDCl3) δ 1.47 (d, J = 6.8 Hz, 6 H), 3.93 (s, 3 H), 4.46 (hept, J = 6.7 Hz, 1 H), 4.89
(s, 2 H), 6.96–7.02 (m, 2 H), 7.50 (ddd, J = 8.4, 7.3, 1.8 Hz, 1 H), 7.82 (dd, J = 7.8, 1.9 Hz, 1
H), 8.18 (s, 1 H) ppm. 13C NMR (100 MHz, CDCl3) δ 23.11, 46.64, 48.16, 55.84, 111.69,
120.92, 125.49, 131.19, 134.95, 141.37, 149.45, 159.30, 194.11 ppm. HRMS (ESI) m/z
calculated for C14H18O2N3S [M + H]+ 292.1114, found 292.1116. HPLC purity, 99.5% at 254
nm (tR = 3.79 min).
1-(4-Bromophenyl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethanone (43). Synthesized
following the general procedure for the synthesis of the final products. Yield, 89%. 1H NMR
(400ꢀMHz, DMSO-d6) δ 1.49 (d, J = 6.7 Hz, 6 H), 4.56 (hept, J = 6.7 Hz, 1 H), 5.08 (s, 2 H),
7.79 (d, J = 8.6 Hz, 2 H), 7.96 (d, J = 8.6 Hz, 2 H), 9.63 (s, 1 H) ppm. 13C NMR (100 MHz,
DMSO-d6) δ 21.93, 41.31, 50.05, 128.22, 130.49, 132.02, 134.00, 143.00, 150.13, 191.96

ppm. HRMS (ESI) m/z calculated for C13H15ON3SBr [M + H]+ 340.0114, found 340.0118.
HPLC purity, 99.3% at 254 nm (tR = 4.25 min).
2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)-1-(p-tolyl)ethanone (44). Synthesized following
the general procedure for the synthesis of the final products. Yield, 68%. 1H NMR (400ꢀMHz,
DMSO-d6) δ 1.50 (d, J = 6.7 Hz, 6 H), 2.39 (s, 3 H) 4.57 (hept, J = 6.6 Hz, 1 H), 5.09 (s, 2 H),
7.38 (d, J = 8.0 Hz, 2 H), 7.93 (d, J = 8.3 Hz, 2 H), 9.73 (s, 1 H) ppm. 13C NMR (100 MHz,
DMSO-d6) δ 21.30, 21.88, 41.52, 50.25, 128.63, 129.49, 132.44, 142.98, 144.70, 150.57,
192.01 ppm. HRMS (ESI) m/z calculated for C14H18ON3S [M + H]+ 276.1165, found
276.1168. HPLC purity, 98.0% at 254 nm (tR = 3.99 min).
2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)-1-phenylethanone (45). Synthesized following the
1
general procedure for the synthesis of the final products. Yield, 48%. H NMR (400ꢀMHz,
DMSO-d6) δ 1.49 (d, J = 6.7 Hz, 6 H), 4.56 (hept, J = 6.7 Hz, 1 H), 5.10 (s, 2 H), 7.55–7.60
13
(m, 2 H), 7.69–7.73 (m, 1 H), 8.02–8.05 (m, 2 H), 9.59 (s, 1 H) ppm. C NMR (100 MHz,
DMSO-d6) δ 21.94, 41.39, 49.92, 128.48, 128.93, 134.04, 134.96, 142.95, 150.11, 192.59
ppm. HRMS (ESI) m/z calculated for C13H16ON3 [M + H]+ 262.1009, found 262.1009. HPLC
purity, 97.1% at 254 nm (tR = 3.56 min).
1-(4-(Dimethylamino)phenyl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethanone
(46).
Synthesized following the general procedure for the synthesis of the final products. Yield,
52%. 1H NMR (400ꢀMHz, DMSO-d6) δ 1.49 (d, J = 6.7 Hz, 6 H), 3.04 (s, 6 H), 4.56 (hept, J =
6.7 Hz, 1 H), 4.97 (s, 2 H), 6.75 (d, J = 9.2 Hz, 2 H), 7.84 (d, J = 9.1 Hz, 2 H), 9.63 (s, 1 H)
13
ppm. C NMR (100 MHz, DMSO-d6) δ 21.90, 39.66, 41.29, 50.08, 110.78, 122.01, 130.62,
142.87, 150.69, 153.76, 189.42 ppm. HRMS (ESI) m/z calculated for C15H21ON4S [M + H]+
305.1431, found 305.1432. HPLC purity, 95.2% at 254 nm (tR = 3.88 min).
4-(2-((4-Isopropyl-4H-1,2,4-triazol-3-yl)thio)acetyl)benzonitrile (47). Synthesized following
the general procedure for the synthesis of the final products. Yield, 90%. 1H NMR (400ꢀMHz,
DMSO-d6) δ 1.49 (d, J = 6.7 Hz, 6 H), 4.56 (hept, J = 6.6 Hz, 1 H), 5.13 (s, 2 H), 8.07 (d, J =
8.6 Hz, 2 H), 8.18 (d, J = 8.7 Hz, 2 H), 9.66 (s, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ
21.89, 41.54, 50.29, 115.78, 118.08, 129.13, 132.96, 138.19, 143.08, 150.26, 192.15 ppm.
HRMS (ESI) m/z calculated for C14H15ON4S [M + H]+ 287.0961, found 287.0964. HPLC
purity, 93.6% at 254 nm (tR = 3.41 min).