1,2,4-Thiadiazole acyclic nucleoside phosphonates as inhibitors of cysteine dependent enzymes cathepsin K and GSK-3β

Article abstract of DOI:10.1016/j.bmc.2021.115998

In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has be

Full text of DOI:10.1016/j.bmc.2021.115998

Bioorg. Med. Chem. 32 (2021) 115998
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,2,4-Thiadiazole acyclic nucleoside phosphonates as inhibitors of cysteine
dependent enzymes cathepsin K and GSK-3β
a,
b
c
b
b
b
b
´
´
ˇ
´
ˇ
ˇ
d
Alice Pomeislova , Miroslav Otmar , Petra Rubesova , Jakub Benýsek , Marika Matousova ,
b
b
´
´
ˇ
´
ˇ
´
Helena Mertlíkova-Kaiserova , Radek Pohl , Lenka Postova Slavetínska , Karel Pomeisl ,
b,*
´
ˇ
Marcela Krecmerova
a Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, 128 43 Prague 2, Czech Republic
b
´
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
c
´
´
Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovskeho nam. 2, 162 06 Prague 6, Czech Republic
d Institute of Physics, Czech Academy of Sciences, Na Slovance 1999/2, 182 21 Prague 8, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords:
In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones
bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the
position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have
been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc iso-
thiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-
amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved
with bromotrimethylsilane, yielding N5-protected phosphonic acids. The subsequent attempts to remove the
protecting group from N5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring.
Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only
in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were
explored as potential inhibitors of cysteine-dependent enzymes – human cathepsin K (CatK) and glycogen syn-
thase kinase 3β (GSK-3β). Several compounds exhibited an inhibitory activity toward both enzymes in the low
micromolar range. The inhibitory potency of some of them toward GSK-3β was similar to that of the thiadiazole
GSK-3β inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good
inhibitory activity.
Thiadiazoles
5-Amino-1,2,4-thiadiazol-3-(2H)-one
Acyclic nucleoside phosphonates
Epigenetic
Cathepsin K, Glycogen synthase kinase 3β
1. Introduction
osteoporosis treatment, and a number of CatK inhibitors are being
developed or tested in clinical trials (for reviews, see e.g.2–4). Most of
It has been reported that 1,2,4-thiadiazole compounds are capable of
inhibiting cysteine proteases, which contain a catalytic cysteine residue
in the active site, as demonstrated for a selective inhibitor of cathepsin B
acting in the micromolar range.1 Among cathepsin-type cysteine pro-
teases, human cathepsin K (CatK) has been one of the most investigated
medically important members in the last decade. This lysosomal enzyme
is predominantly expressed in osteoclasts and plays a critical role in the
bone resorption process. It degrades the protein components of the bone
matrix, including its major constituent, type I collagen. The hyperac-
tivity of osteoclasts and an imbalance between bone formation and
resorption lead to the loss bone tissue, which is associated with osteo-
porosis.2 CatK has been evaluated as the main drug target for
these inhibitors contain an electrophilic warhead, such as ketone or
nitrile groups, for covalent binding to the catalytic cysteine of CatK.5–8
This work describes for the first time CatK inhibitors based on 1,2,4-thia-
diazole employed as a thiol-reactive warhead targeting the catalytic site.
The interaction of 1,2,4-thiadiazole derivatives with the thiol group of
the cysteine residue of the enzyme target is outlined in Fig. 1.
The nucleophilic attack of the cysteine thiol results in the formation
of a covalent adduct and the loss of enzymatic activity. Human cathepsin
K (CatK) is proposed to be inhibited by the modification of catalytic
Cys25 in the enzyme active site, while glycogen synthase kinase 3β
(GSK3β) is modified on Cys199, located in the ATP cofactor binding site.
A great effort has been made in the investigation of 1,2,4-
* Corresponding author.
ˇ
´
E-mail address: marcela.krecmerova@uochb.cas.cz (M. Krecmerova).
https://doi.org/10.1016/j.bmc.2021.115998
Received 7 December 2020; Accepted 31 December 2020
Available online 6 January 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

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A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
use 5-amino-1,2,4-thiadiazol-3(2H)-one as a cytosine surrogate in the
synthesis of nucleosides and their acyclic analogs.19,20 5-Amino-1,2,4-
thiadiazol-3(2H)-one itself has exhibited slight antibacterial activities.
The fact that the antibacterial activity of 5-amino-1,2,4-thiadiazol-3
(2H)-one is not reversed by the presence of natural pyrimidines implies
that in this case, it does not behave as a pyrimidine antimetabolite but
rather interferes with bacterial proteins and thus inhibits the growth of
bacteria.21 In order to identify the active form for biological in-
teractions, a systematic study of the tautomerism in 5-amino-1,2,4-
thiadiazol-3(2H)-one has been performed. In aqueous solutions, a sig-
nificant stabilization of the amino tautomer has been found, with the
equilibrium shifted to favor the amino form.22
Fig. 1. Scheme of the interaction of 1,2,4-thiadiazole-based inhibitors with the
thiol group of the functionally important cysteine residue of the enzyme target.
thiadiazolidine-3,5-diones (TDZD) as inhibitors of glycogen synthase
kinase-3β (GSK-3), a key enzyme of glycogen metabolism.9,10 In cancer
cells, aberrant nuclear accumulation of GSK-3 positively regulates the
binding of the transcription factor NF-kB to its target gene promoters
and its further transcriptional activity. In this way, the NF-kB activation
promotes the progression of human cancer and also causes resistance to
chemotherapeutic drugs.9,10 So far, the most promising inhibitor of GSK-
The present study focuses on the synthesis of compounds in which
the 5-amino-1,2,4-thiadiazol-3(2H)-one moiety (as the cytosine mimic)
is attached to a 2-(phosphonomethoxy)ethyl (PME) or 3-hydroxy-2-
(phosphonomethoxy)propyl (HPMP) group analogously to acyclic
nucleoside phosphonates (ANPs). ANPs are known for their large spec-
trum of biological activities, especially antiviral. Three of them have
already become successful commercial drugs: cidofovir, approved for
the treatment of human cytomegalovirus retinitis in AIDS patients but
used off-label also against many other DNA viruses, adefovir for the
treatment of hepatitis B (HBV), and tenofovir for the treatment of HIV
and HBV infections.23–25 The possibility that 5-amino-1,2,4-thiadiazol-3
(2H)-one could really work as a cytosine mimic together with the ability
3
is 4-benzyl-2-(1-naphthyl)-1,2,4-thiadiazolidine-3,5-dione (tide-
glusib). Regarding its ability to influence gene expression, its function
can be considered epigenetic. GSK-3 also plays a crucial role in Alz-
heimer’s disease and other neurodegenerative processes. Clinical studies
of tideglusib for the treatment of Alzheimer’s disease11–13 and myotonic
muscular dystrophy14 are currently being conducted in Phase II. In
dental medicine, tideglusib is a topic of extensive research aimed at
natural tooth repair by GSK3 antagonists. The ability of the promotion of
dentine reinforcement has been successfully proven in vivo.15
–
of the reactive N S bond to attack strong nucleophiles such as the
cysteine thiol group of enzymes have provided the impetus to synthesize
5-amino-1,2,4-thiadiazol-3(2H)-one APNs and to subject them to a
detailed biological activity screening.
A completely different mechanism of action has been reported in a
series of neuroprotective 1,2,4-thiadiazole derivatives influencing ion-
otropic transmembrane receptors for glutamate (NMDA and AMPA re-
ceptors). These receptors are important for controlling synaptic
plasticity and memory function and mediate fast synaptic transmission
in the central nervous system.16–18 A general structural motif of these
derivatives is outlined in Fig. 2.
2. Chemistry
This work is mainly focused on the synthesis of 5-amino-1,2,4-thia-
diazol-3(2H)-one derivatives with the HPMP arrangement, compounds
structurally analogous to the antiviral agents cidofovir (HPMPC)23–25
and its 5-azacytosine analog (HPMP-azaC), both depicted in Fig. 3.26,27
The HPMP series was also extended to the PME derivatives with respect
to the antiviral activity of PMEC and PME-azaC (see also Fig. 3), which,
even if it is lower, can be increased via transformation to appropriate
prodrugs.28
Besides, there are many 1,2,4-thiadiazol structures whose biological
properties still deserve further exploration. Our team of nucleoside
chemistry has focused on 5-amino-1,2,4-thiadiazol-3-(2H)-one, which
could be understood as a sulfur-containing mimic of cytosine in which
–
–
–
the CH CH group between the positions 5 and 6 of the pyrimidine
–
5-Amino-1,2,4-thiadiazol-3(2H)-one (1) was prepared by oxidative
cyclization of thiobiuret under basic conditions19 (Scheme 1). The direct
alkylation of 1 with the synthon iPr2-PME-Cl (2) or (S)-glycidyl trityl
ether (4) led to a mixture of compounds, whose TLC analysis profile did
not correspond to the desired products. Therefore, the 5-amino group of
ring is replaced by a divalent sulfur (–S–) as reported in several pa-
pers.19,20 5-Amino-1,2,4-thiadiazol-3(2H)-one is considered a bio-
isostere of cytosine because of the similar electronic and spatial
–
–
–
properties of the CH CH group in cytosine and the bivalent sul-
–
fur.19,20 Due to the proposed bioisosterism, there has been an effort to
Fig. 2. Examples of 1,2,4-thiadiazoles with diverse types of biological activities.
2

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Bioorganic & Medicinal Chemistry 32 (2021) 115998
Fig. 3. Acyclic nucleoside phosphonates with cytosine and 5-azacytosine base moieties and their 5-amino-1,2,4-thiadiazol-3(2H)-one analogues.
Scheme 1. The synthesis of 5-amino-1,2,4-thiadiazol-3(2H)-one (1). The reagents and conditions: (a) 2 M NaOH, H2O2, MeOH, 0 ◦C, 2 h; (b) HCl; (c) Cs2CO3, DMF,
reflux, 4.5 h; (d) Cs2CO3, DMF, reflux, 24 h.
1 was protected by dimethylaminomethylene (DMA) to form 6, which
was subsequently alkylated with the synthons (Scheme 2). However, in
the case of the iPr2-PME-Cl (2), only the product of the O3-alkylation of 7
was formed, and with the synthon 4, a 4:1 mixture of the O3-10a and the
N2-alkylated 10b product was obtained. The deprotection of 7 by hy-
drazine and the subsequent removal of phosphonate ester groups from
the resulting compound 8 by bromotrimethylsilane afforded the free
phosphonate 9. Like in the case of the iPr2-PME-Cl (2), the alkylation of
the isomers 10a and 10b with the tosylate 11 did not provide the desired
HPMP derivatives 12a and 12b.
methods. In the first one, iPr2-PME-Cl (2) was converted to 16 by re-
action with potassium phthalimide, and the subsequent removal of the
phthalic moiety by hydrazine released iPr2-PME-NH2 (17). In the second
method, 2-aminoethanol was N-tritylated to 18, then O-alkylated with
the tosylated synthon 11 in the presence of magnesium tert-butoxide in
N-methyl-2-pyrrolidone29 to form 19, and after that deprotected to
obtain iPr2-PME-NH2 (17).
The synthon iPr2-Tr-HPMP-NH2 (23) was prepared in three steps
from (S)-glycidyl trityl ether (4) (Scheme 5). In the first step, the oxirane
ring of 4 was opened with sodium azide at 45 ◦C to form 20. To minimize
the formation of undesired side products, it was necessary to increase
the temperature only slightly and to use a 40-fold excess of sodium azide
in the reaction. Compound 20 was alkylated with the bromomethyl-
phosphonate 21 to obtain the corresponding ether 22. The azide func-
tion in 22 was then reduced to an amino group by the Staudinger
reaction with triphenylphosphine, yielding iPr2-Tr-HPMP-NH2 (23).
The starting synthons, iPr2-PME-NH2 (17) and iPr2-Tr-HPMP-NH2
(23), on which the stepwise construction of the 5-amino-1,2,4-thiadia-
zol-3(2H)-one moiety, mimicking the cytosine nucleobase, was per-
formed, were converted to the corresponding N-substituted ureas 24 and
25 (Scheme 6). The urea 24, with the iPr2-PME substituent, was pre-
pared by the reaction of the amine 17 with potassium cyanate under
acidic conditions. In the case of the amine 23, bearing the iPr2-Tr-HPMP
substituent, the acidic treatment could not be used, because the trityl
In a further attempt to achieve N2 alkylation of 1 with the synthons 2
and 4, the DMA protecting group was replaced by acyl (Scheme 3). The
N5-acetyl- (14a) and N5-benzoyl- (14b) derivatives were thus prepared
by the reaction of acetyl or benzoyl isothiocyanate with urea, yielding
the formation of the corresponding 1-acetyl- (13a) and 1-benzoyl-2-thi-
obiuret (13b), followed by oxidative cyclization with bromine or NBS.
The N5-benzoyl derivative 14b was alkylated with 4 to yield only the
undesired N4-substituted product 15. Therefore, this synthetic strategy
based on the N5-acyl protection was abandoned.
In order to obtain the desired N2-substituted derivatives of 1, a
stepwise construction of the 5-amino-1,2,4-thiadiazol-3(2H)-one ring on
PME and HPMP scaffolds was performed. The starting synthetic pre-
cursors were iPr2-PME-NH2 (17) and iPr2-Tr-HPMP-NH2 (23). Com-
pound 17 was synthesized according to Scheme 4 by two different
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Bioorganic & Medicinal Chemistry 32 (2021) 115998
Scheme 2. Alkylation of the DMA protected 5-amino-1,2,4-thiadiazol-3(2H)-ones with PME a HPMP synthons.. The reagents and conditions: (a) DMF DMA,
acetonitrile, 24 h; (b) Cs2CO3, DMF, 95 ◦C, 4.5 h; (c) NH2NH2, MeOH, 1 h; (d) TMSBr, CH3CN, 24 h; (e) H2O; (f) NaH, THF, 24 h.
Scheme 3. The synthesis of N5-acyl-5-amino-1,2,4-thiadiazol-3(2H)-ones and their N4-alkylation. The reagents and conditions: (a) acetone, 60 ◦C, 5 h; (b) Br2/CHCl3
(1 M), EtOH, 35 ◦C, 20 min; (c) NBS, MeOH, reflux, 1 h; (d) Cs2CO3, DMF, reflux, 24 h.
Scheme 4. The synthesis of iPr2-PME-NH2 (17). The reagents and conditions: (a) DMF, 120 ◦C, 3.5 h; (b) 1 M NH2NH2/THF, MeOH, 2 h; (c) TrCl, Et3N, CH2Cl2, 2 h;
(d) Mg(OtBu)2, NMP, 75 ◦C, 2 h; (e) 35% HCl (aq.), dioxane, 1 h.
4

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Bioorganic & Medicinal Chemistry 32 (2021) 115998
Scheme 5. The synthesis of iPr2-Tr-HPMP-NH2 (23). The reagents and conditions: (a) NaN3, DMF, 45 ◦C, 1 week; (b) NaH, DMF, 60 ◦C, 24 h; (c) Ph3P, H2O, THF,
0 ◦C to RT, 24 h.
Scheme 6. The synthesis of N5-protected 5-amino-1,2,4-thiadiazol-3(2H)-ones substituted on N2 with the PME and HPMP moiety. The reagents and conditions: (a)
KOCN, 35% HCl, 70 ◦C, 24 h; (b) CDI, CH2Cl2, 3 h; (c) NH3/MeOH, 24 h; (d) acetone, 60 ◦C, 24 h; (e) 0.5 M Br2/EtOAc, 4 ◦C, 24 h; (f) NBS, MeOH, 80 ◦C, 30 min; (g)
TMSBr, CH3CN, 24 h; (h) H2O.
protecting group would be removed. Instead, the amine 23 was treated
with carbonyldiimidazole (CDI) and then with methanolic ammonia to
form the urea 25 with an intact trityl group. In the next step, the ureas
24 and 25 were reacted with benzoyl, ethoxycarbonyl (Eoc), and Fmoc
isothiocyanate with the formation of the corresponding thiobiurets
26–31.
The oxidative cyclization of compounds 26–28 with bromine or NBS
led to the formation of the N5-protected 5-amino-1,2,4-thiadiazol-3
(2H)-ones 32–34 alkylated on the N2-position with the iPr2-PME syn-
thon. In the compounds 29–31 the oxidative cyclization also caused the
Scheme 7. Attempts for the removal of protecting groups from the amino
removal of the trityl protecting group, yielding the iPr-HPMP derivatives
group in position 5. The reagents and conditions: (a) 35% aq.HCl/dioxane 1:1,
35–37. In the next step, the phosphonate ester groups of the compounds
reflux, 8 h; (b) 2 M KOH, reflux, 8 h.
32–37 were cleaved with trimethylsilyl bromide to give the N5-pro-
tected phosphonic acids 38–43.
(2H)-one 44 with bromine was unsuccessful.
The attempts to remove the protecting groups with sodium hydrox-
The remaining synthetic path to obtain the desired N2-substituted
ide or hydrochloric acid at elevated temperature were unsuccessful
derivatives of 1 consists in the oxidative cyclization of the deprotected 2-
(Scheme 7). The use of pyrrolidine and ethanolamine for the depro-
thiobiurets bearing at the N5 position the PME or HPMP group with or
tection of the Fmoc derivatives 40 and 43 according to the literature30
without ester groups (46–49, Scheme 9).
only led to the open-ring phosphonates 46 and 47 (Scheme 8). Similarly,
The compounds 28 and 31 were thus deprotected with piperidine,
the removal of Fmoc from the PME derivative 40 with hydrochloric acid
yielding the 5-subtituted biurets 48 and 49. The isopropyl ester and
in harsh conditions under microwave irradiation resulted in the for-
trityl groups were then cleaved with trimethylsilyl bromide to give the
mation of the open-ring phosphonate 46. The same product was ob-
free phosphonic acids 46 and 47. The attempts to cyclize the compounds
tained by the deprotection of 40 with sodium azide.31 The attempt for
46 and 47 oxidatively with bromine to the 1,2,4-thiadiazol-3(2H)-ones
the oxidative recyclization of the compound 46 to the 1,2,4-thiadiazol-3
44 and 45 were unsuccessful, only recovering the starting material.
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Bioorganic & Medicinal Chemistry 32 (2021) 115998
Scheme 8. The formation of open-ring derivatives during the deprotection of the 5-amino group. The reagents and conditions: (a) pyrrolidine, CH2Cl2, 24 h; (a)
ethanolamine, CH2Cl2, 24 h; (b) pyrrolidine, CH2Cl2, 24 h; (c) 0.5 M HCl, M-W, 150 ◦C, 16 min; (d) NaN3, DMF, 50 ◦C, 3.5 h; (e) Br2/EtOAc.
–
Scheme 9. Cyclization and ring-opening of 5-amino-1,2,4-thiadiazol-3(2H)-ones caused by a redox character of S N bond. The reagents and conditions: (a)
piperidine, CH2Cl2, 24 h; (b) TMSBr, CH3CN, 24 h; (c) H2O, 1 h; (d) 0.5 M Br2/EtOAc, H2O, 4 ◦C.
Interestingly, the phosphonate diester 48 was successfully cyclized with
bromine to the 1,2,4-thiadiazol-3(2H)-one 50. However, the subsequent
ester cleavage with trimethylsilyl bromide gave only the open-ring
product 46 instead, which, as mentioned above, cannot be recyclized.
47 with 0.17% hydrogen peroxide in D2O. The process was monitored at
one-hour intervals by 31P NMR (Scheme 10). Hydrogen peroxide was
chosen for its easy decomposition to water not disturbing the mea-
surement. The reaction exhibited equilibrium between the linear com-
pound 47 and the cyclization product 45. The ratio of the peaks in 31
P
–
The redox character of the S N bond was studied in the reaction of
NMR spectra for 47 and 45 was 1:1. The addition of anotherr equivalent
of hydrogen peroxide did not lead to a change in the equilibrium.
This equilibrium illustrates the above-mentioned tendency to ring
opening of 5-amino-1,2,4-thiadiazol-3(2H)-ones bearing at the N2 po-
sition a PME or HPMP substituent with a free phosphonate function and
the impossibility to recyclize the resulted open-ring derivatives suc-
cessfully. If the amino group at position 5 of the 1,2,4-thiadiazol-3(2H)-
ones is protected and thus the tautomerism is prevented, the stability of
the ring is much higher. In order to explain that the simultaneous
presence of the unprotected 1,2,4-thiadiazol-3(2H)-one ring and the free
phosphonate function is not a stable arrangement, it is necessary to look
Scheme 10. The cyclization of 47 by hydrogen peroxide; the experiment was
performed in a NMR tube in D2O.
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Bioorganic & Medicinal Chemistry 32 (2021) 115998
for an analogy with the formation of cyclic HPMPC (51) under the
cleavage of HPMPC diisopropyl ester (52) (Scheme 11).32 The sponta-
neous origination of cyclic HPMPC (51) is conditioned by the possibility
of the formation of the six-membered ring by the nucleophilic attack of
the primary hydroxy group on the intermediate reactive trimethylsilyl
phosphonate ester. A similar situation occurred in the compound 50,
where even the more nucleophilic N2 nitrogen atom is in an analogous
position (enabling the formation of a six-membered ring) to that of the
primary hydroxy group of HPMPC. Contrary to the cyclic HPMPC (51),
the cyclic phosphonoamide 53 is more easily cleavable to the open-ring
Table 1
The inhibition of human cathepsin K and GSK-3β by 5-amino-1,2,4-thiadiazol-3
(2H)-ones applied at the concentration of 10 µM. Residual enzyme activities
were measured as described in the Experimental section and expressed relative
to the uninhibited control (100%). The compounds are ordered according to
decreasing inhibitory potency toward CatK.
Compound name
CatK residual activity (%)
GSK3 residual activity (%)
10a
5.3 ± 1.5
16.9 ± 1.0
28.3 ± 1.7
34.1 ± 2.4
34.2 ± 3.0
39.1 ± 1.0
52.9 ± 3.8
67.0 ± 5.8
67.0 ± 0.6
76.6 ± 3.8
78.3 ± 2.6
80.6 ± 5.0
83.4 ± 8.6
85.0 ± 3.5
85.5 ± 5.3
86.8 ± 2.7
90.0 ± 3.5
90.3 ± 6.0
92.3 ± 4.5
92.9 ± 2.8
97.5 ± 2.8
N/A*
72 ± 16.7
78 ± 7.3
1
10b
64 ± 14.1
76 ± 2.4
37
–
phosphonate 46 due to the inherent reactivity of the P N bond than the
15
59 ± 13.6
87 ± 4.1
phosphonate cyclic ester 51.
39
To summarize the results of the above-described syntheses, only the
N5-substituted 1,2,4-thiadiazol-3(2H)-one analogs of ANPs were suffi-
ciently stable and isolable in the form of free phosphonic acids. Com-
pounds with the unprotected amino group of the 5-amino-1,2,4-
thiadiazol-3(2H)-one base moiety were stable only in the form of
phosphonate esters. These findings correspond to our previous obser-
vations, when the esterification of the phosphonic acid residue increased
the stability of a labile base moiety, e.g. the 5-azacytosine ring in 5-aza-
cytosine ANPs.33,34
7
102 ± 8.8
97 ± 12.3
85 ± 4.8
6
40
14a
82 ± 9.7
8
113 ± 15.5
87 ± 25.0
110 ± 9.1
85 ± 18.5
98 ± 12.5
154 ± 0.5
106 ± 6.2
75 ± 7.4
32
36
14b
43
33
9
Most of the newly prepared 5-amino-1,2,4-thiadiazol-3(2H)-ones
have exhibited interesting biological activities concerning inhibitory
effects to cysteine-dependent enzymes.
38
41
106 ± 12.3
81 ± 3.5
35
42
101 ± 10.0
70 ± 7.2
Tideglusib
CHIR99021
3. Results and Discussion
N/A
10 ± 5.2
*
not applicable for CatK
3.1. The inhibition of cathepsin K by 5-amino-1,2,4-thiadiazol-3(2H)-
ones
1,2,4-thiadiazol-3(2H)-one (1) (IC50 of 6.34 ± 0.63), (ii) derivatives
protected on N5 bearing 2-hydroxy-3-trityloxypropyl group: 10a (2.63
± 0.31 µM), 10b (10.08 ± 0.95 µM), and 15 (14.9 ± 0.75 µM), and (iii)
derivatives protected on N5 bearing a phosphonate group on HPMP or
PME aliphatic chain, with: 37 (9.25 ± 1.46) or without: 39 (10.15 ±
1.48) esterification. The whole series tested revealed several general
trends in SAR. The esterification of the phosphonate function resulted in
more potent inhibitors of CatK in comparison with free phosphonic acids
(8, 32, and 37 versus 9, 38, and 43, respectively). The presence of the
polar hydroxymethyl group (HPMP) decreased inhibition in contrast to
its absence (PME) (35, 41, 43, and 42 versus 32, 38, 40, and 39,
Twenty-one derivatives of 5-amino-1,2,4-thiadiazol-3(2H)-one de-
rivatives have been explored as potential inhibitors of human CatK. The
compounds were screened in vitro, at the concentration of 10 µM, against
the recombinant enzyme using a kinetic inhibition assay with the fluo-
rogenic substrate Z-Gly-Pro-Arg-AMC. They exhibited a wide range of
inhibitory activities, with the six most potent inhibitors showing higher
inhibition than 50% (residual enzyme activity below 50%, Table 1). The
determined IC50 values of the effective compounds ranged from of 2.6 to
14.9 µM. According to structural features, the active compounds can be
divided into three categories: (i) the parental unsubstituted 5-amino-
Scheme 11. An explanation of the tendency to ring opening of 5-amino-1,2,4-thiadiazol-3(2H)-ones bearing at the N2 position a PME or HPMP substituent with the
free phosphonate function.
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Bioorganic & Medicinal Chemistry 32 (2021) 115998
respectively). At the amino group in position 5, the Fmoc protecting
group effects a higher inhibition than that of benzoyl (40, 37, and 43
versus 38, 35, and 41, respectively). In conclusion, the screened series of
1,2,4-thiadiazoles provided several inhibitors of CatK effective in the
low micromolar range.
4. Conclusions
In this work, we describe for the first time CatK and GSK-3β in-
hibitors based on the 1,2,4-thiadiazole scaffold, employed as a thiol-
reactive warhead targeting the thiol group located in the catalytic
(cofactor binding) site of these cysteine-dependent enzymes. We have
screened a set of 5-amino-1,2,4-thiadiazol-3(2H)-one compounds;
among them, we have identified several inhibitors of CatK effective in
the low micromolar range. The inhibitors are comparable in efficacy to
previously published cathepsin B inhibitors.1 These compounds have
also been shown to inhibit GSK-3β with similar potency as the estab-
lished 1,2,4-thiadiazole GSK-3β inhibitor tideglusib, with some of them
(e.g. 37) exhibiting even a more favorable toxicity profile while
retaining good inhibitory activity.
3.2. The inhibition of GSK-3β and the cytotoxicity of 5-amino-1,2,4-
thiadiazol-3(2H)-ones
The prepared 5-amino-1,2,4-thiadiazol-3(2H)-ones have also been
screened for their ability to inhibit GSK-3β (human, recombinant).
Interestingly, the inhibition by 10-µM 1,2,4-thiadiazole derivatives
(including the reference inhibitor tideglusib) was relatively weak in
comparison with previously published data,35 with the IC50 values > 10
µM, which was not the case for another non-thiadiazole inhibitor,
CHIR99021 (IC50 = 9.9 ± 0.65 nM). Since we have employed a validated
commercial GSK-3β kit (Promega), there might be some unknown in-
terferences of assay components with the 1,2,4-thiadiazole moiety,
resulting in the apparently low inhibition effects. Therefore, we have set
tideglusib as a benchmark compound when evaluating the activity of the
compounds toward GSK-3β. Most compounds that have exhibited ac-
tivity similar to tideglusib (i.e. the inhibition of approx. 30%) under the
conditions of the assay fall into the cluster of compounds that have also
been identified as CatK inhibitors (10a, 1, 10b, 37, 15) (Table 1) and
therefore probably follow a similar SAR pattern to that described above.
The cytotoxicity of the compounds intended for use in the treatment
of human diseases is, of course, of concern. This paper shows that the
hereby presented thiadiazole derivatives are generally less toxic than
tideglusib, which exhibited cytotoxicity in two leukemic cell lines
(CCRF-CEM, HL-60) but, more importantly, also to normal human
dermal fibroblasts (NHDF) at 10 µM (Table 2). With the exception of
compounds 10a, 10b and 15, which all include the triphenylmethyl
moiety, and unsubstituted 5-amino-1,2,4-thiadiazol-3(2H)-ones (1), the
compounds have proven to be safe at concentrations up to 100 µM. The
presence of the trityl group increased the inhibitory activity of the
compound toward Cys-containing enzymes CatK and GSK-3β, but at the
expense of undesired toxicity.
The future research of 1,2,4-thiadiazole inhibitors should take more
into account the interior of the enzyme recognition site and design the
new molecules accordingly, similarly as it was published in the case of
peptidomimetic inhibitors of CatK with ketone or nitrile warheads.5–8
5. Experimental
5.1. General
The melting points were determined on a Kofler melting point
apparatus with a microscope KBT 300 (Santiago) and are uncorrected.
The analytical TLC was performed on silica gel pre-coated aluminum
plates with a fluorescent indicator (Merck 5554, 60 F254). The spots were
visualized with UV light (254 nm) or by spraying with ninhydrine (1%
solution in ethanol) followed by short heating to 300–400 ◦C. The col-
umn chromatography was performed on silica gel (Sigma S-0507, 40–63
μ
m). Mass spectra were measured on a Q-Tof micro (Waters) and the HR
MS were taken on a LTQ Orbitrap XL (Thermo Fisher Scientific) spec-
trometer. 1H and 13C NMR spectra were recorded on a Bruker Avance
400 (1H at 400.0 MHz, 13C at 100.6 MHz and 31P at 162.0 MHz), Bruker
Avance 500 (1H at 500.0 MHz, 13C at 125.7 MHz and 31P at 202.4 MHz),
and Bruker Avance 600 (1H at 600.1 MHz and 13C at 150.9 MHz) in
CDCl3 (referenced to TMS as an internal standard), in DMSO‑d6
Table 2
The cytotoxicity of 5-amino-1,2,4-thiadiazol-3(2H)-ones to tumor and non-tumor cell lines following the 72-h incubation of the cells with the compounds (10 and 100
µM). The data are expressed as the percentage of viable cells vs. untreated control.
Compound name
Cell viability [%]
CCRF-CEM
HL-60
HeLa S3
HepG2
SH-SY5Y
NHDF
10 µM
100 µM
10 µM
100 µM
10 µM
100 µM
10 µM
100 µM
10 µM
100 µM
10 µM
100 µM
1
120
122
106
114
113
101
15
4
98
95
100
97
87
87
54
85
99
56
75
101
105
101
104
105
97
49
6
84
102
97
105
98
112
89
97
100
101
98
107
99
7
93
101
97
99
90
87
8
103
112
2
103
103
86
108
100
1
98
100
97
99
55
114
96
9
101
91
102
2
93
101
99
99
10a
92
1
2
1
10b
1
64
2
80
1
87
1
67
3
77
1
14a
104
102
81
102
96
105
101
93
108
110
9
97
102
101
3
103
99
88
102
103
96
99
99
105
100
79
14b
99
77
90
104
98
15
5
45
101
100
97
91
37
101
100
99
34
101
104
97
32
100
92
94
104
102
106
97
115
111
118
101
110
113
100
106
106
99
100
101
105
87
103
104
109
101
102
100
98
104
105
100
104
102
104
103
102
101
100
99
103
101
103
104
98
101
98
33
98
35
86
97
104
100
100
102
97
36
67
45
82
90
93
104
85
37
82
103
102
96
99
100
101
96
92
38
98
100
77
101
94
100
97
105
105
99
103
94
39
74
40
85
90
102
103
95
97
102
101
94
105
101
99
103
100
89
93
84
41
72
61
97
103
99
105
101
98
98
42
94
114
119
n.a.*
n.a. *
97
99
43
88
93
100
n.a.
n.a.
98
99
106
92
88
93
95
Tideglusib
30
34
69
n.a.
n.a.
n.a.
n.a.
80
n.a.
n.a.
57
n.a.
n.a.
CHIR99021
28
119
67
86
77
53
*
n.a. – not available.
8

´
A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
(referenced to the solvent signal δ(1H) = 2.50 ppm and δ(13C) = 39.70
ppm), in CD3OD (referenced to the solvent signal δ(1H) = 3.31 ppm and
δ(13C) = 49.0 ppm), or in D2O (referenced to 1,4-dioxane as an internal
standard, δ(1H) = 3.75 ppm and δ(13C) = 69.3 ppm). The complete
assignment was based on H,H-COSY, H,C-HSQC and H,C-HMBC corre-
lation experiments. The chemical shifts (δ) are given in ppm and the
coupling constants (J) in Hz. The IR spectra were obtained on an FT IR
NICOLET 6700 spectrometer in CCl4 or KBr pellets. Optical rotations
were measured on an Autopol IV polarimeter (Rudolph Research
5.2.4. Diisopropyl ((2-((5-amino-1,2,4-thiadiazol-3-yl)oxy)ethoxy)
methyl)phosphonate (8)
Hydrazine (8 mL; 35% aqueous solution) was added to a solution of 7
(4.10 g; 10.4 mmol) in MeOH (15 mL) and the mixture was stirred at RT
for 1 h. The solvent was evaporated and the residue was chromato-
graphed on a silica gel column in a gradient of 0–5% MeOH in CHCl3.
Yield: 2.7 g (75%) of a colorless amorphous solid. MS (ESI) m/z (%): 362
(100, M+Na+), 340 (23, M+H+). HRMS (ESI) calcd. for C11H22O5N3PS
(M+Na+): 362.0910, found: 362.0909. 1H NMR (DMSO‑d6): δ 7.94 (s,
2H, NH2), 4.59 (dsept, 2H, J(H,P) = 7.8 Hz, Jvic = 6.2 Hz, CH-i-Pr), 4.30
(m, 2H, H-1′), 3.79 (d, 2H, J(3′,P) = 8.3 Hz, H-3′), 3.78 (m, 2H, H-2′),
1.24 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.23 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr).
13C NMR (DMSO‑d6): δ 182.76 (C-5), 167.37 (C-3), 70.72 (d, J(2′,P) =
12.1 Hz, CH2-2′), 70.38 (d, J(C,P) = 6.3 Hz, CH-i-Pr), 67.47 (CH2-1′),
65.00 (d, J(3′,P) = 164.4 Hz, CH2-3′), 24.03 (d, J(C,P) = 3.8 Hz, CH3-i-
Pr), 23.89 (d, J(C,P) = 4.5 Hz, CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ
Analytical, U.S.A.) at 20 ◦C, [
α
]D values are given in 10ꢀ 1 deg cm2 gꢀ 1
.
The concentrations for specific rotation measurements are in the units of
g/100 mL.
The structure numbering for NMR assignment and the NMR spectra
of all compounds are available in the Supporting Information.
5.2. The synthesis of compounds
20.38. FTIR (KBr, cmꢀ 1
) νmax: 3298, 3162, 2980, 2935, 2882, 1631,
5.2.1. 5-Amino-1,2,4-thiadiazol-3(2H)-one hydrochloride (1)
1548, 1507, 1465, 1454, 1409, 1387, 1375, 1337, 1237, 1222, 1178,
1142, 1105, 1013, 995, 891.
30% Hydrogen peroxide (12 mL) was added dropwise to a 0 ◦C cold
solution of thiobiuret (10.0 g; 83.9 mmol) in 2 M NaOH (12 mL) and the
mixture was stirred at 0 ◦C for 3 h. Upon successful conversion, the
mixture was acidified to pH 4.5 by the addition of 35% HCl. The
resulting precipitate was filtered off and recrystallized from boiling
water. Yield: 6.19 g (48%) of white crystals, mp 219–220 ◦C. The
spectral data are in accordance with Ref.19
5.2.5. ((2-((5-Amino-1,2,4-thiadiazol-3-yl)oxy)ethoxy)methyl)
phosphonic acid (9)
Bromotrimethylsilane (21.6 mL; 0.164 mol) was added to a solution
of 8 (2.78 g; 8.19 mmol) in acetonitrile (50 mL) and the mixture was
stirred at RT overnight. The volatiles were removed in vacuo, the residue
was co-evaporated with acetonitrile (3x) and then with 25% aqueous
ammonia. The crude product was purified on a column of Dowex 50 × 8
(H+ form). Impurities and salts were eluted first with water and the
product was than eluted with 2.5% aqueous ammonia. The compound
was crystallized from the MeOH/diethyl ether mixture. Yield: 1.52 g
(73%) of a white crystalline solid, mp 161–163 ◦C. MS (ESI) m/z (%):
276 (30, Mꢀ 2H++Na+), 254 (100, Mꢀ H+). HRMS (ESI) calcd. for
C5H9O5N3PS (Mꢀ H+): 254.0006, found: 254.0006. 1H NMR (DMSO‑d6):
δ 8.00 (s, 2H, NH2), 7.47 (vbs, NH+4 ), 4.26 (m, 2H, H-1′), 3.71 (m, 2H, H-
2′), 3.37 (d, 2H, J(3′,P) = 8.7 Hz, H-3′). 13C NMR (DMSO‑d6): δ 182.74
(C-5), 167.44 (C-3), 70.07 (d, J(2′,P) = 10.0 Hz, CH2-2′), 69.08 (d, J(3′,
P) = 155.0 Hz, CH2-3′), 67.88 (CH2-1′). 31P {1H} NMR (DMSO‑d6): δ
5.2.2. (E)-N,N-Dimethyl-N′-(3-oxo-2,3-dihydro-1,2,4-thiadiazol-5-yl)
formimidamide (6)
A solution of 1 (5.450 g; 35.5 mmol) and N,N-dimethylformamide
dimethyl acetal (12.4 mL; 93.3 mmol) in acetonitrile (50 mL) was stirred
at RT overnight. An excess of diethyl ether was added, the resulting
precipitate was filtered off and washed with diethyl ether. Yield: 4.7 g
(77%) of white crystals, mp 197–198 ◦C. MS (ESI) m/z (%): 195 (100,
M+Na+), 173 (15, M+H+). HRMS (ESI) calcd. for C5H9ON4S (M+H+):
173.0492, found: 173.0492. 1H NMR (DMSO‑d6): δ 10.56 (bs, 1H, NH),
8.45 (s, 1H, CH-7), 3.17 (s, 3H, 9b), 3.02 (s, 3H, 9a). 13C NMR
(DMSO‑d6): δ 187.02 (C-5), 168.22 (C-3), 157.98 (CH-7), 40.95 (CH3-
9b), 35.04 (CH3-9a).
12.88. FTIR (KBr, cmꢀ 1
) νmax: 3290, 3179, 2855, 1635, 1547, 1513,
1455, 1414, 1390, 1339, 1105, 1063, 975, 922.
5.2.3. Diisopropyl (E)-((2-((5-(((dimethylamino)methylene)amino)-
1,2,4-thiadiazol-3-yl)oxy)ethoxy)methyl)phosphonate (7)
5.2.6. The reaction of compound 6 with (S)-glycidyl trityl ether
Diisopropyl 2-(phosphonomethoxy)ethyl chloride (6.28 g; 24.3
mmol) and caesium carbonate (11.89 g; 36.5 mmol) were added to a
solution of 6 (4.59 g; 26.7 mmol) in DMF (150 mL) and the mixture was
stirred at 95 ◦C for 5 h. After cooling to RT, the mixture was diluted with
EtOAc (300 mL), washed with aqueous NaHCO3 (2x 250 mL) and then
with brine (1x 250 mL). An organic layer was dried over MgSO4 and
evaporated. The residue was chromatographed on a silica gel column in
a gradient of 0–5% MeOH in CHCl3. Yield: 4.15 g (39%) as yellow oil.
MS (ESI) m/z (%): 417 (90, M+Na+), 395 (100, M+H+). HRMS (ESI)
calcd. for C14H27O5N4NaPS (M+Na+): 417.1332, found: 417.1337;
HRMS (ESI) calcd. for C14H28O5N4PS (M+H+): 395.1513, found:
395.1511. 1H NMR (DMSO‑d6): δ 8.40 (m, 1H, CH-7), 4.59 (dsept, 2H, J
(H,P) = 7.8 Hz, Jvic = 6.2 Hz, CH-i-Pr), 4.37 (m, 2H, H-1′), 3.81 (m, 2H,
H-2′), 3.80 (d, 2H, J(3′,P) = 8.3 Hz, H-3′), 3.16 (d, 3H, J(9b,7) = 0.5 Hz,
9b), 3.02 (d, 3H, J(9a,7) = 0.8 Hz, 9a), 1.24 (d, 6H, Jvic = 6.2 Hz, CH3-i-
Pr), 1.23 (d, 6H, Jvic = 6.1 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 191.16
(C-5), 168.05 (C-3), 157.93 (CH-7), 70.71 (d, J(2′,P) = 12.0 Hz, C-2′),
70.35 (d, J(C,P) = 6.3 Hz, CH-i-Pr), 67.59 (C-1′), 65.08 (d, J(3′,P) =
164.6 Hz, C-3′), 40.83 (9b), 34.99 (9a), 24.01 (d, J(C,P) = 3.8 Hz, CH3-i-
Pr), 23.87 (d, J(C,P) = 4.6 Hz, CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ
Compound 6 (2.50 g; 14.5 mmol), (S)-glycidyl trityl ether (4.18 g;
13.2 mmol) and caesium carbonate (0.860 g; 2.64 mmol) were dissolved
in DMF (50 mL) and the mixture was stirred at 95 ◦C for 4 h. Diethyl
ether (200 mL) was added and the mixture was washed with an aqueous
solution of NaHCO3 (2x 150 mL) and then with brine (150 mL). The
organic phase was dried over MgSO4, evaporated, and the residue was
purified on a silica gel column in a gradient of 0–2% MeOH in CHCl3.
Compound 10a was eluted first, followed by compound 10b.
5.2.6.1. (R,E)-N′-(3-(2-Hydroxy-3-(trityloxy)propoxy)-1,2,4-thiadiazol-
5-yl)-N,N-dimethylformimidamide (10a). Yield: 2.5 g (35%) as a white
crystalline solid, mp 72–74 ◦C. [
α] ꢀ 2.5 (c 0.360 g/100 mL, MeOH).
D
MS (ESI) m/z (%): 511 (100, M+Na+), 999 (18, 2M+Na+). HRMS (ESI)
calcd. for C27H28O3N4NaS (M+Na+): 511.1774, found: 511.1773. 1H
NMR (DMSO‑d6): δ 8.39 (m, 1H, H-7), 7.37–7.42 (m, 6H, H-o-Tr),
7.29–7.35 (m, 6H, H-m-Tr), 7.23–7.28 (m, 3H, H-p-Tr), 5.23 (d, 1H, J
(OH,2′) = 5.6 Hz, OH), 4.33 (dd, 1H, Jgem = 10.8 Hz, J(1′b,2′) = 4.4 Hz,
H-1′b), 4.26 (dd, 1H, Jgem = 10.8 Hz, J(1′a,2′) = 5.8 Hz, H-1′a), 3.97
(pentd, 1H, J(2′,1′a) = J(2′,3′a) = J(2′,3′b) = J(2′,OH) = 5.7 Hz, J
(2′,1′b) = 4.4 Hz, H-2′), 3.15 (bs, 3H, 9b), 3.04 (dd, 1H, Jgem = 9.2 Hz, J
(3′b,2′) = 5.6 Hz, H-3′b), 3.02 (d, 3H, J(9a,7) = 0.8 Hz, 9a), 3.00 (dd,
1H, Jgem = 9.2 Hz, J(3′a,2′) = 5.7 Hz, H-3′a). 13C NMR (DMSO‑d6): δ
191.07 (C-5, determined by HSQC and HMBC), 168.24 (C-3), 157.87
(CH-7), 143.98 (C-i-Tr), 128.47 (CH-o-Tr), 128.19 (CH-m-Tr), 127.19
20.40. FTIR (KBr, cmꢀ 1
) ν: 2980, 2881, 2820, 1705, 1624, 1468, 1375,
1253, 1193, 1107, 1060, 1010, 985, 950, 889.
9

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A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
(CH-p-Tr), 86.00 (C-Tr), 70.32 (C-1′), 67.91 (C-2′), 64.99 (C-3′), 40.83
gradient of 0–2% MeOH in CHCl3 and the product was recrystallized
(9b), 35.00 (9a). FTIR (KBr, cmꢀ 1
)
νmax: 3415, 3086, 3057, 3032, 3023,
from ethyl acetate/hexane. Yield: 1.02 g (21%) as white crystals, mp
3001, 2928, 2815, 1750, 1685, 1623, 1597, 1583, 1523, 1497, 1449,
1337, 1323, 1154, 1107, 1076, 1033, 1002, 946, 938, 765, 707, 700,
633, 316.
176–177 ◦C. [
α]D ꢀ 23.3 (c 0.313 g/100 mL, MeOH). MS (ESI) m/z (%):
560 (100, M+Na+). HRMS (ESI) calcd. for C31H27O4N3NaS (M+Na+):
560.1615, found: 560.1609. 1H NMR (DMSO‑d6): δ 10.24 (bs, 1H, NH,
determined by HMBC), 8.16 (m, 2H, H-o-Ph), 7.63 (m, 1H, H-p-Ph), 7.46
(m, 2H, H-m-Ph), 7.35–7.41 (m, 6H, H-o-Tr), 7.24–7.30 (m, 6H, H-m-Tr),
7.19–7.24 (m, 3H, H-p-Tr), 5.30 (d, 1H, J(OH,2′) = 5.8 Hz, OH), 4.31
(bdpent, 1H, J(2′,1′a) = 7.8 Hz, J(2′,1′b) = J(2′,3′) = J(2′,OH) = 5.7 Hz,
H-2′), 4.09 (dd, 1H, Jgem = 13.2 Hz, J(1′b,2′) = 5.5 Hz, H-1′b), 4.02 (dd,
1H, Jgem = 13.2 Hz, J(1′a,2′) = 7.9 Hz, H-1′a), 3.12 (dd, 1H, Jgem = 9.5
Hz, J(3′b,2′) = 5.1 Hz, H-3′b), 2.98 (dd, 1H, Jgem = 9.5 Hz, J(3′a,2′) =
6.3 Hz, H-3′a). 13C NMR (DMSO‑d6): δ 176.97 (C-7), 171.52 (C-5),
153.51 (C-3), 143.91 (C-i-Tr), 133.97 (C-i-Ph), 133.23 (CH-p-Ph),
129.25 (CH-o-Ph), 128.80 (CH-m-Ph), 128.34 (CH-o-Tr), 128.02 (CH-m-
Tr), 127.17 (CH-p-Tr), 86.27 (C-Tr), 66.70 (CH2-3′), 66.33 (CH-2′),
48.34 (CH2-1′).
5.2.6.2. (S,E)-N′-(2-(2-Hydroxy-3-(trityloxy)propyl)-3-oxo-2,3-dihydro-
1,2,4-thiadiazol-5-yl)-N,N-dimethylformimidamide (10b). Yield: 0.62 g
(9%) of a white crystalline solid, mp 200–202 ◦C. [
α]D ꢀ 3.4 (c 0.295 g/
100 mL, MeOH). MS (ESI) m/z (%): 511 (100, M+Na+), 999 (19,
2M+Na+). HRMS (ESI) calcd. for C27H28O3N4Na4S (M+Na+): 511.1774,
found: 511.1773. 1H NMR (DMSO‑d6): δ 8.55 (s, 1H, H-7), 7.38–7.42 (m,
6H, H-o-Tr), 7.31–7.36 (m, 6H, H-m-Tr), 7.23–7.28 (m, 3H, H-p-Tr), 5.41
(d, 1H, J(OH,2′) = 5.4 Hz, OH), 3.86 (dd, 1H, Jgem = 13.8 Hz, J(1′b,2′) =
3.4 Hz, H-1′b), 3.81 (m, 1H, H-2′), 3.52 (dd, 1H, Jgem = 13.8 Hz, J(1′a,2′)
= 7.2 Hz, H-1′a), 3.20 (s, 3H, 9b), 3.03 (s, 3H, 9a), 2.97 (dd, 1H, Jgem
=
9.3 Hz, J(3′b,2′) = 5.2 Hz, H-3′b), 2.88 (dd, 1H, Jgem = 9.4 Hz, J(3′a,2′)
= 6.2 Hz, H-3′a). 13C NMR (DMSO‑d6): δ 181.53 (C-5), 165.94 (C-3),
158.01 (CH-7), 143.88 (C-i-Tr), 128.48 (CH-o-Tr), 128.07 (CH-m-Tr),
127.20 (CH-p-Tr), 86.11 (C-Tr), 68.67 (C-2′), 65.34 (C-3′), 47.11 (C-1′),
5.2.11. Diisopropyl ((2-(N-phthalimido)ethoxy)methyl)phosphonate (16)
A solution of diisopropyl 2-(phosphonomethoxy)ethyl chloride
(20.28 g; 78.4 mmol) and potassium phthalimide (14.52 g; 78.4 mmol)
in DMF (200 mL) was stirred at 120 ◦C for 3.5 h and then evaporated.
The residue was co-evaporated with toluene and purified on a silica gel
column in a gradient of 0–5% MeOH in CHCl3. Yield: 24.7 g (85%) of a
white amorphous solid. MS (ESI) m/z (%): 392 (100, M+Na+). HRMS
(ESI) calcd. for C17H24O6NNaP (M+Na+): 392.1234, found: 392.1235.
1H NMR (CDCl3): δ 7.77 (m, 2H, CH-4,7-Ar), 7.67 (m, 2H, CH-5,6-Ar),
4.63 (m, 2H, CH-i-Pr), 3.85 (m, 2H, H-1′), 3.80 (m, 2H, H-2′), 3.71 (d,
2H, J(3′,P) = 8.3 Hz, H-3′), 1.19–1.22 (m, 12H, CH3-i-Pr). 13C NMR
(CDCl3): δ 167.95 (C-2, C-9), 133.85 (CAr-3,4), 131.93 (CAr-1,6), 123.09
(CH-4,7-Ar), 70.96 (d, J(C,P) = 6.6 Hz, CH-i-Pr), 69.55 (d, J(2′,P) =
10.8 Hz, CH2-2′), 65.28 (d, J(3′,P) = 167.2 Hz, CH2-3′), 36.90 (CH2-1′),
23.87 (d, J(C,P) = 3.8 Hz, CH3-i-Pr), 23.76 (d, J(C,P) = 4.6 Hz, CH3-i-Pr).
41.12 (9b), 35.17 (9a). FTIR (KBr, cmꢀ 1
) νmax: 3288, 3085, 3057, 3032,
3022, 3001, 2926, 2810, 1678, 1623, 1596, 1585, 1491, 1448, 1418,
1339, 1307, 1153, 1113, 1077, 1033, 1002, 950, 935, 762, 709, 699,
633, 617.
5.2.7. The general procedure for the synthesis of N1-substituted thiobiurets
Potassium thiocyanate (48.0 g; 0.49 mol) was dissolved in acetone
(400 mL) and the appropriate acyl chloride (0.41 mol) was added
dropwise. The mixture was stirred at 50 ◦C for 3.5 h. After cooling to RT,
the precipitated potassium chloride was filtered off over Celite and the
volume of the filtrate was reduced in vacuo. Urea (24.6 g; 0.41 mol) was
◦
added to the solution and the mixture stirred at 65 C for 5 h. After
cooling to RT, the flask was cooled in an ice bath to initiate crystalli-
zation and the solution was then left at RT overnight. Crystals were
collected by filtration and recrystallized from methanol.
31P {1H} NMR (CDCl3): δ 18.84. FTIR (CCl4, cmꢀ 1
) νmax: 3088, 3061,
2981, 2938, 2875, 1778, 1720, 1617, 1469, 1393, 1375, 1241, 1179,
1142, 1107, 1009, 990, 889, 718, 529.
5.2.7.1. N-(Carbamoylcarbamothioyl)acetamide (13a). Yield: 9.4
g
◦
5.2.12. 2-(Tritylamino)ethan-1-ol (18)
(14%) of yellowish crystals, mp 196–198 C. The spectral data are in
agreement with Ref.36
Triethylamine (60 mL; 0.431 mol) was added to a solution of 2-ami-
noethan-1-ol (13.0 g; 0.21 mol) in CH2Cl2 (250 mL). Trityl chloride
(59.1 g; 0.212 mol) was added under cooling in an ice bath and the
mixture was stirred at RT for 2 h. The solvent was evaporated and the
residue was partitioned between ethyl acetate (300 mL) and aqueous
NaHCO3 (2x 250 mL) and brine (250 mL). The organic layer was dried
over MgSO4 and evaporated. The residue was crystallized from the
mixture of ethyl acetate/hexane. Yield: 52.4 g (82%) as a white crys-
talline solid. The spectral data are in agreement with Ref.38
5.2.7.2. N-(Carbamoylcarbamothioyl)benzamide (13b). Yield: 36.18 g
(40%) of yellow crystals, mp 174–175 ◦C. The spectral data are in
agreement with Ref.19
5.2.8. N-(3-Oxo-2,3-dihydro-1,2,4-thiadiazol-5-yl)acetamide (14a)
1 M Br2 in CHCl3 (43.8 mL; 43.8 mmol) was added dropwise to a
solution of 13a (5.00 g; 31.0 mmol) in EtOH (40 mL) at 35 ◦C and the
mixture was stirred at the same temperature for 20 min. After cooling to
RT, the crystals formed were filtered off, washed with diethyl ether and
recrystallized from water. Yield: 2.95 g (60%) as white crystals, mp
236–237 ◦C. The spectral data are in agreement with Ref.36
5.2.13. Diisopropyl ((2-(tritylamino)ethoxy)methyl)phosphonate (19)
The synthetic methodology described in Ref.29 was utilized. Mag-
nesium di-tert-butoxide (5.63 g; 33.0 mmol) and diisopropyl tosylox-
ymethanephosphonate (6.94 g; 19.8 mmol) were added to a solution of
18 (5.0 g; 16.5 mmol) in N-methylpyrrolidone (30 mL). The mixture was
5.2.9. N-(3-Oxo-2,3-dihydro-1,2,4-thiadiazol-5-yl)benzamide (14b)
Compound 13b (7.46 g, 33.4 mmol) and N-bromosuccinimide (7.43
g; 41.8 mmol) were dissolved in MeOH (200 mL) and the mixture was
◦
stirred at 75–78 C for 4.5 h. After cooling to RT, water (30 mL) was
added and the mixture was extracted with diethyl ether (50 mL). An
organic layer was washed with aqueous NaHCO3 (2x 50 mL), then with
brine (1x 50 mL), and it was dried over MgSO4. The solvent was evap-
orated and the residue was chromatographed on a silica gel column in
the CHCl3/hexane system (1:9 to 1:1). Yield: 7.2 g (91%) of a colorless
oil. MS (ESI) m/z (%): 504 (90, M+Na). HRMS (ESI) calcd. for
◦
stirred at 70 C for 6 h. After cooling to RT, the crystals formed were
filtered off and washed with diethyl ether. Yield: 4.15 g (56%) as white
crystals. The spectral data are in agreement with Ref.19,37
5.2.10. (S,Z)-N-(4-(2-Hydroxy-3-(trityloxy)propyl)-3-oxo-1,2,4-
thiadiazolidin-5-ylidene)benzamide (15)
C
28H36O4NNaP (M+Na+): 504.2274, found: 504.2273; HRMS (ESI)
calcd. for C28H37O4NP (M+H+): 482.2455, found: 482.2455. H NMR
(DMSO‑d6): δ 7.41 (m, 6H, H-o-Tr), 7.29 (m, 6H, H-m-Tr), 7.19 (m, 3H,
H-p-Tr), 4.59 (dsept, 2H, J(H,P) = 7.8 Hz, Jvic = 6.2 Hz, CH-i-Pr), 3.68
(d, 2H, J(3,P) = 8.4 Hz, H-3), 3.62 (t, 2H, J(2,1) = 5.6 Hz, H-2), 2.59 (t,
1H, J(NH,1) = 7.9 Hz, NH), 2.16 (dt, 2H, J(1,NH) = 7.8 Hz, J(1,2) = 5.6
1
Compound 14b (2.00 g; 9.04 mmol), (S)-glycidyl trityl ether (2.62 g;
8.28 mmol) and caesium carbonate (0.553 g; 1.70 mmol) were dissolved
in DMF (70 mL), the mixture was stirred at 110 ◦C for 24 h and evap-
orated. The residue was chromatographed on a silica gel column in a
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A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
Hz, H-1), 1.23 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.21 (d, 6H, Jvic = 6.2 Hz,
CH3-i-Pr). 13C NMR (DMSO‑d6): δ 146.09 (C-i-Tr), 128.47 (CH-o-Tr),
127.88 (CH-m-Tr), 126.29 (CH-p-Tr), 72.56 (d, J(2,P) = 12.1 Hz, CH2-2),
70.27 (C-Tr), 70.22 (d, J(C,P) = 6.5 Hz, CH-i-Pr), 64.96 (d, J(3,P) =
165.0 Hz, CH2-3), 43.09 (CH2-1), 23.96 (d, J(C,P) = 3.9 Hz, CH3-i-Pr),
23.88 (d, J(C,P) = 4.5 Hz, CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 21.81.
mixture was stirred for 5 min and evaporated. The residue was extracted
between water (300 mL) and ethyl acetate (2x 450 mL). Combined
organic layers were dried over Na2SO4 and evaporated. The residue was
purified on a silica gel column, first in CHCl3/hexane (40:60), followed
by a gradient of MeOH in CHCl3 (0–15%). Yield: 7.85 g (70%) of a
colorless oil. [α]D ꢀ 7.4 (c 0.298 g/100 mL, MeOH). MS (ESI) m/z (%):
FTIR (CCl4, cmꢀ 1
)
νmax: 3335, 3085, 3062, 3033, 2980, 2934, 2873,
560 (100, M+Na+). HRMS (ESI) calcd. for C29H36O5N3NaP (M+Na+):
560.2285, found: 560.2284. 1H NMR (DMSO‑d6): δ 7.36–7.43 (m, 6H, H-
o-Tr), 7.31–7.38 (m, 6H, H-m-Tr), 7.25–7.29 (m, 3H, H-p-Tr), 4.53–4.64
(m, 2H, CH-i-Pr), 3.84 (dd, 1H, Jgem = 13.9 Hz, J(3a,P) = 9.0 Hz, H-3a),
3.81 (dd, 1H, Jgem = 13.9 Hz, J(3b,P) = 9.0 Hz, H-3b), 3.74 (dddd, 1H, J
(2,1b) = 5.7 Hz, J(2,4b) = 5.3 Hz, J(2,4a) = 4.9 Hz, J(2,1a) = 4.1 Hz, H-
2), 3.56 (dd, 1H, Jgem = 13.1 Hz, J(1a,2) = 4.1 Hz, H-1a), 3.45 (dd, 1H,
Jgem = 13.1 Hz, J(1b,2) = 5.7 Hz, H-1b), 3.10 (dd, 1H, Jgem = 10.2 Hz, J
(4a,2) = 4.9 Hz, H-4a), 3.08 (dd, 1H, Jgem = 10.2 Hz, J(4b,2) = 5.3 Hz,
H-4b), 1.18–1.28 (m, 12H, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 143.70 (C-
i-Tr), 128.44 (CH-o-Tr), 128.12 (CH-m-Tr), 127.30 (CH-p-Tr), 86.36 (C-
Tr), 79.35 (d, J(2,P) = 12.2 Hz, CH-2), 70.38 (d, J(C,P) = 6.3 Hz, CH-i-
Pr), 65.86 (CH2-4), 64.07 (d, J(3,P) = 165.2 Hz, CH2-3), 50.93 (CH2-1),
23.72–24.06 (CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 18.82. FTIR (CCl4,
1597, 1489, 1466, 1449, 1386, 1375, 1318, 1242, 1216, 1191, 1180,
1142, 1107, 1033, 1008, 990, 940, 902, 706, 698, 648, 625.
5.2.14. Diisopropyl ((2-aminoethoxy)methyl)phosphonate (17)
Method A. Hydrazine (1 M in THF; 140 mL, 0.141 mol) was added to
a solution of 16 (5.2 g; 14.1 mmol) in EtOH (50 mL) and the mixture was
stirred at RT for 2 h. The precipitated phthalhydrazide was filtered off.
The filtrate was evaporated and the residue was purified on a silica gel
column under basic conditions in the system of 2–10% methanolic
ammonia (2.35 M NH3) in CHCl3. Yield: 3.07 g (91%) of a colorless oil.
Method B. 35% Hydrochloric acid (30 mL) was added to a 0 ◦C cold
solution of 19 (7.0 g; 4.5 mmol) in dioxane (40 mL). The solution was
then stirred at RT for 1.5 h, washed with diethyl ether, and the aqueous
phase was concentrated in vacuo. The residue was purified on a silica gel
column under basic conditions (5% TEA in toluene/CHCl3 2:1 to TEA/
CHCl3 5:95). Yield: 2.26 g (65%) of a colorless oil. MS (ESI) m/z (%): 262
(27, M+Na+), 240 (100, M+H+). HRMS (ESI) calcd. for C9H22O4NNaP
(M+Na+): 262.1179, found: 262.1179; HRMS1 (ESI) calcd. for
C9H23O4NP (M+H+): 240.1359, found: 240.1360. H NMR (CDCl3): δ
4.73 (dsept, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz, CH-i-Pr), 3.73 (d, 2H, J
(3,P) = 8.5 Hz, H-3), 3.68 (t, 2H, J(2,1) = 5.1 Hz, H-2), 2.97 (t, 2H, J
(1,2) = 4.9 Hz, H-1), 1.33 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.32 (d, 6H,
Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR (CDCl3): δ 75.34 (d, J(C,P) = 11.2 Hz,
CH2-2), 70.89 (d, J(C,P) = 6.7 Hz, CH-i-Pr), 65.68 (d, J(3,P) = 168.2 Hz,
CH2-3), 41.38 (CH2-1), 24.02 (d, J(C,P) = 3.9 Hz, CH3-i-Pr), 23.95 (d, J
(C,P) = 4.6 Hz, CH3-i-Pr). 31P {1H} NMR (CDCl3): δ 20.15. FTIR (CCl4,
cmꢀ 1
) νmax: 3088, 3062, 3035, 3024, 2981, 2934, 2874, 2103, 1598,
1491, 1467, 1449, 1386, 1375, 1310, 1260, 1244, 1178, 1142, 1159,
1153, 1107, 1088, 1080, 1035, 1008, 991, 936, 889, 705, 697, 633, 555.
5.2.17. Diisopropyl (S)-(((1-amino-3-(trityloxy)propan-2-yl)oxy)methyl)
phosphonate (23)
Triphenylphosphine (4.19 g; 16.0 mmol) and water (2.0 mL) were
added while stirring to a 0 ◦C cold solution of 22 (7.80 g; 14.5 mmol) in
THF (200 mL) and the mixture left stirring overnight to reach RT. The
solvent was evaporated and the residue was chromatographed on a silica
gel column (a gradient of 0–5% MeOH in CHCl3 containing 1% TEA).
Yield: 4.8 g (65%) of a colorless oil. [α] ꢀ 8.8 (c 0.656 g/100 mL,
MeOH). MS (ESI) m/z (%): 1024 (88, 2M+DH+), 512 (85, M+H+). HRMS
cmꢀ 1
)
νmax: 3334, 2981, 2934, 2876, 1467, 1387, 1376, 1241, 1179,
(ESI) calcd. for C29H39O5NP (M+H+): 512.2560, found: 512.2557. H
1
1142, 1106, 1010, 993, 889.
NMR (DMSO‑d6): δ 7.38–7.42 (m, 6H, H-o-Tr), 7.31–7.36 (m, 6H, H-m-
Tr), 7.23–7.28 (m, 3H, H-p-Tr), 4.55–4.65 (m, 2H, CH-i-Pr), 3.85 (dd,
1H, Jgem = 13.3 Hz, J(3a,P) = 8.6 Hz, H-3a), 3.81 (dd, 1H, Jgem = 13.3
Hz, J(3b,P) = 8.6 Hz, H-3b), 3.50 (dddd, 1H, J(2,1b) = 6.1 Hz, J(2,4b) =
5.2.15. (S)-1-Azido-3-(trityloxy)propan-2-ol (20)
Sodium azide (41.1 g; 632 mmol) was added to a solution of (S)-
glycidyl trityl ether (10.0 g; 31.6 mmol) in DMF (500 mL) and the
mixture was stirred at 45 ◦C for 7 days. Water (5.0 mL) was added and
the reaction mixture was stirred for 10 min. The precipitated salts were
filtered off on a Büchner funnel and the filtrate was evaporated. The
residue was partitioned between water (350 mL) and ethyl acetate (2x
350 mL). An organic layer was dried over Na2SO4, evaporated, and the
residue was purified on a silica gel column in CHCl3/hexane (15:85).
5.7 Hz, J(2,1a) = 5.2 Hz, J(2,4a) = 3.9 Hz, H-2), 3.11 (dd, 1H, Jgem
=
10.0 Hz, J(4a,2) = 3.9 Hz, H-4a), 3.04 (dd, 1H, Jgem = 10.0 Hz, J(4b,2)
= 5.7 Hz, H-4b), 2.66 (dd, 1H, Jgem = 13.2 Hz, J(1a,2) = 5.2 Hz, H-1a),
2.62 (dd, 1H, Jgem = 13.2 Hz, J(1b,2) = 6.1 Hz, H-1b), 1.24 (d, 3H, Jvic
=
6.2 Hz, CH3-i-Pr), 1.23 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.22 (d, 3H, Jvic
= 6.2 Hz, CH3-i-Pr), 1.21 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR
(DMSO‑d6): δ 143.94 (C-i-Tr), 128.45 (CH-o-Tr), 128.06 (CH-m-Tr),
127.20 (CH-p-Tr), 86.15 (C-Tr), 82.78 (d, J(2,P) = 11.8 Hz, CH-2), 70.30
(d, J(C,P) = 6.2 Hz, CH-i-Pr), 70.26 (d, J(C,P) = 6.2 Hz, CH-i-Pr), 64.34
(d, J(3,P) = 165.3 Hz, CH2-3), 63.83 (CH2-4), 42.59 (CH2-1), 24.02 (d, J
(C,P) = 3.6 Hz, CH3-i-Pr), 24.01 (d, J(C,P) = 3.6 Hz, CH3-i-Pr), 23.93 (d,
J(C,P) = 4.5 Hz, CH3-i-Pr), 23.91 (d, J(C,P) = 4.5 Hz, CH3-i-Pr). 31P {1H}
NMR (DMSO‑d6): δ 19.55. 31P {1H} NMR (DMSO‑d6): δ 19.55. FTIR
Yield: 7.53 g (66%) of a colorless oil. [α] ꢀ 17.6 (c 0.250 g/100 mL,
D
MeOH). MS (ESI) m/z (%): 382 (100, M+Na+). HRMS (ESI) calcd. for
C
22H21O2N3Na (M+Na+): 382.1526, found: 382.1523. 1H NMR
(DMSO‑d6): δ 7.36–7.42 (m, 6H, H-o-Tr), 7.30–7.38 (m, 6H, H-m-Tr),
7.23–7.30 (m, 3H, H-p-Tr), 5.35 (d, 1H, J(OH,2) = 5.3 Hz, OH), 3.83
(ttd, 1H, J(2,1b) = J(2,3b) = 6.5 Hz, J(2,OH) = J(2,3a) = 5.3 Hz, J
(2,1a) = 3.6 Hz, H-2), 3.36 (dd, 1H, Jgem = 12.6 Hz, J(1a,2) = 3.6 Hz, H-
1a), 3.30 (dd, 1H, Jgem = 12.6 Hz, J(1b,2) = 6.5 Hz, H-1b), 3.01 (dd, 1H,
Jgem = 9.2 Hz, J(3a,2) = 5.3 Hz, H-3a), 2.89 (dd, 1H, Jgem = 9.2 Hz, J
(3b,2) = 6.5 Hz, H-3b). 13C NMR (DMSO‑d6): δ 143.90 (C-i-Tr), 128.45
(CH-o-Tr), 128.08 (CH-m-Tr), 127.22 (CH-p-Tr), 86.11 (C-Tr), 69.14
(CCl4, cmꢀ 1
) νmax: 3088, 3061, 3035, 3024, 2980, 2874, 1598, 1491,
1466, 1449, 1386, 1375, 1241, 1179, 1159, 1153, 1142, 1106, 1089,
1078, 992, 936, 890, 787, 764, 706, 633.
5.2.18. Diisopropyl ((2-ureidoethoxy)methyl)phosphonate (24)
(CH-2), 65.27 (CH2-3), 53.80 (CH2-1). FTIR (CCl4, cmꢀ 1
2928, 2858, 2105, 1737, 1467, 1393, 1087, 1077, 529.
)
νmax: 3600,
35% hydrochloric acid (1.65 mL) was added dropwise to a 10 ◦C cold
solution of 17 (5.12 g; 21.4 mmol) in H2O (50 mL). Potassium cyanate
(3.47 g; 42.8 mmol) was added and the reaction mixture was stirred at
70 ◦C overnight. The mixture was extracted with CHCl3 (4x 70 mL), an
organic layer was dried over MgSO4 and concentrated in vacuo. The
residue was purified on a silica gel column (0–5% MeOH in CHCl3).
Yield: 4.33 g (72%) of a colorless oil. MS (ESI) m/z (%): 305 (100,
M+Na+). HRMS (ESI) calcd. for C10H23O5N2NaP (M+Na+): 305.1237,
found: 305.1237. 1H NMR (DMSO‑d6): δ 5.95 (bt, 1H, J(H,1′) = 5.8 Hz,
NH), 5.48 (s, 2H, NH2), 4.60 (dsept, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz,
5.2.16. (Diisopropyl (S)-(((1-azido-3-(trityloxy)propan-2-yl)oxy)methyl)
phosphonate (22)
Sodium hydride (60% suspension in mineral oil; 2.095 g; 52.3 mmol)
was added to a solution of 20 (7.5 g; 20.9 mmol) in DMF (300 mL) and
the mixture was stirred at RT for 15 min. Diisopropyl (bromomethyl)
phosphonate 21 (8.12 g; 31.4 mmol) was added and the mixture stirred
at 60 ◦C overnight. The reaction was quenched with water (30 mL), the
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Bioorganic & Medicinal Chemistry 32 (2021) 115998
CH-i-Pr), 3.72 (d, 2H, J(3′,P) = 8.2 Hz, H-3′), 3.47 (t, 2H, J(2′,1′) = 5.8
Hz, H-2′), 3.12 (q, 2H, J(1′,2′) = J(1′,NH) = 5.8 Hz, H-1′), 1.25 (d, 6H,
Jvic = 6.2 Hz, CH3-i-Pr), 1.24 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR
(DMSO‑d6): δ 158.86 (C-1), 72.41 (d, J(2′,P) = 11.2 Hz, CH2-2′), 70.42
(d, J(C,P) = 6.4 Hz, CH-i-Pr), 64.94 (d, J(C,P) = 164.4 Hz, CH2-3′), 39.05
(CH2-1′), 24.09 (d, J(C,P) = 3.7 Hz, CH3-i-Pr), 23.98 (d, J(C,P) = 4.6 Hz,
153.60 (C-1; determined by HMBC), 133.56 (CH-p-Ph), 131.42 (C-i-Ph),
129.16 (CH-m-Ph), 128.09 (CH-o-Ph), 71.08 (d, J(2′,P) = 11.4 Hz, CH2-
2′), 70.38 (d, J(C,P) = 6.4 Hz, CH-i-Pr), 64.97 (d, J(3′,P) = 164.5 Hz,
CH2-3′), 39.20 (CH2-1′), 24.03 (d, J(C,P) = 3.8 Hz, CH3-i-Pr), 23.92 (d, J
(C,P) = 4.6 Hz, CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 19.34. FTIR
(CCl4, cmꢀ 1
) νmax: 3497, 3423, 3330, 3237, 3068, 3035, 2981, 2937,
CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 19.67. FTIR (CCl4, cmꢀ 1
)
νmax
:
2875, 1723, 1689, 1675, 1603, 1582, 1540, 1502, 1491, 1467, 1451,
1386, 1375, 1316, 1238, 1179, 1143, 1106, 1070, 1013, 994, 890, 704,
686, 644.
3362, 3210, 2982, 2935, 2876, 1690, 1665, 1612, 1555, 1467, 1387,
1376, 1240, 1179, 1142, 1105, 1014, 995, 890.
5.2.19. Diisopropyl (S)-(((1-(trityloxy)-3-ureidopropan-2-yl)oxy)methyl)
phosphonate (25)
5.2.20.2. Diisopropyl
(6,10-dioxo-8-thioxo-2,11-dioxa-5,7,9-triaza-
tridecyl)phosphonate (27). Prepared from 24 (11.22 g; 39.7 mmol) and
ethoxycarbonyl isothiocyanate (5.21 g; 39.7 mmol) in acetone (400 mL);
reaction time: 4 h. Yield: 10.3 g (63%) of a yellowish oil. MS (ESI) m/z
(%):436 (100, M+Na+), 414 (47, M+H+). HRMS (ESI) calcd. for
C14H28O7N3NaPS (M+Na+): 436.1278, found: 436.1281; HRMS (ESI)
calcd. for C14H29O7N3PS (M+H+): 414.1458, found: 414.1451. 1H NMR
(DMSO‑d6): δ 12.21 (bs, 1H, NH), 11.05 (bs, 1H, NH), 8.34 (bs, 1H, NH),
4.55–4.65 (m, 2H, CH-i-Pr), 4.18 (q, 2H, J(4,5) = 7.1 Hz, H-4), 3.76 (d,
2H, J(3′,P) = 8.3 Hz, H-3′), 3.59 (bt, 2H, J(2′,1′) = 5.4 Hz, H-2′), 3.35
(bq, 2H, J(1′,2′) = 5.4 Hz, H-1′), 1.24 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr),
1.23 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.229 (t, 3H, J(5,4) = 7.1 Hz, H-5).
13C NMR (DMSO‑d6): δ 178.93 (C-2), 153.13 (C-1), 152.00 (C-3), 71.04
(d, J(2′,P) = 12.1 Hz, CH2-2′), 70.36 (d, J(C,P) = 6.4 Hz, CH-i-Pr), 64.97
(d, J(3′,P) = 164.4 Hz, CH2-3′), 62.39 (CH2-4), 39.22 (CH2-1′), 24.00 (d,
J(C,P) = 3.7 Hz, CH3-i-Pr), 23.90 (d, J(C,P) = 4.5 Hz, CH3-i-Pr), 14.18
Compound 23 (4.75 g; 9.29 mmol) and 1,1′-carbonyldiimidazole
(1.81 g; 11.2 mmol) were dissolved in dichloromethane (200 mL) and
the reaction mixture was stirred at RT for 3 h. Afterwards, 7 M meth-
anolic ammonia (50 mL; 0.350 mol) was added and the mixture was
stirred at RT overnight. The solvent was evaporated and the residue was
purified on a silica gel column (0–5% MeOH in CHCl3). Yield: 3.71 g
(72%) of a colorless oil. [α]D ꢀ 3.7 (c 0.216 g/100 mL, MeOH). MS (ESI)
m/z (%): 577 (100, M+Na+). HRMS (ESI) calcd. for C30H39O6N2NaP
(M+Na+): 577.2438, found: 577.2439; HRMS (ESI) calcd. for
C
30H40O6N2P (M+H+): 555.2619, found: 555.2619. 1H NMR (CDCl3): δ
7.43 (m, 6H, H-o-Tr), 7.30 (m, 6H, H-m-Tr), 7.24 (m, 3H, H-p-Tr), 6.26
(bs, 1H, NH), 4.77 (dsept, 1H, J(H,P) = 7.6 Hz, Jvic = 6.2 Hz, CH-i-Pr),
4.72 (dsept, 1H, J(H,P) = 7.6 Hz, Jvic = 6.2 Hz, CH-i-Pr), 4.52 (bs, 2H,
NH2), 4.13 (dd, 1H, Jgem = 14.1 Hz, J(3′a,P) = 6.4 Hz, H-3′a), 3.76 (dd,
1H, Jgem = 14.1 Hz, J(3′b,P) = 8.7 Hz, H-3′b), 3.62 (m, 1H, H-2′), 3.50
(ddd, 1H, Jgem = 13.8 Hz, J(1′a,2′) = 7.8 Hz, J(1′a,NH) = 3.1 Hz, H-1′a),
3.17 (dd, 1H, Jgem = 10.2 Hz, J(4′a,2′) = 6.1 Hz, H-4′a), 3.13 (dd, 1H,
Jgem = 10.2 Hz, J(4′b,2′) = 4.0 Hz, H-4′b), 3.01 (ddd, 1H, Jgem = 13.8
(CH3-5). 31P {1H} NMR (DMSO‑d6): δ 20.34. FTIR (CCl4, cmꢀ 1) νmax
:
3252, 3186, 2980, 2935, 2875, 1705, 1537, 1508, 1468, 1454, 1386,
1374, 1337, 1261, 1227, 1177, 1142, 1104, 1012, 993, 890, 794.
Hz, J(1′b,2′) = 9.3 Hz, J(1′b,NH) = 3.0 Hz, H-1′b), 1.356 (d, 3H, Jvic
=
5.2.20.3. Diisopropyl
(1-(9H-fluoren-9-yl)-3,7-dioxo-5-thioxo-2,11-
6.2 Hz, CH3-i-Pr), 1.349 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.340 (d, 3H,
Jvic = 6.2 Hz, CH3-i-Pr), 1.336 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR
(CDCl3): δ 159.02 (C-1), 143.65 (C-i-Tr), 128.61 (CH-o-Tr), 127.85 (CH-
m-Tr), 127.10 (CH-p-Tr), 86.83 (C-Tr), 83.25 (d, J(2′,P) = Hz, CH-2′),
71.82 (d, J(C,P) = 6.6 Hz, CH-i-Pr), 71.24 (d, J(C,P) = 7.1 Hz, CH-i-Pr),
66.99 (d, J(3′,P) = 168.1 Hz, CH2-3′), 64.42 (CH2-4′), 42.16 (CH2-1′),
24.15 (d, J(C,P) = 3.7 Hz, CH3-i-Pr), 24.09 (d, J(C,P) = 4.6 Hz, CH3-i-Pr),
24.02 (d, J(C,P) = 3.9 Hz, CH3-i-Pr), 23.93 (d, J(C,P) = 4.5 Hz, CH3-i-Pr).
31P {1H} NMR (CDCl3): δ 21.09. FTIR (CHCl3, cmꢀ 1) νmax: 3518, 3413,
3331, 3222, 3089, 3062, 3035, 2985, 2934, 2875, 1675, 1598, 1554,
1491, 1467, 1449, 1443, 1388, 1377, 1305, 1238, 1179, 1159, 1153,
1142, 1103, 1088, 1079, 1035, 1015, 1000, 890, 707, 701, 642, 633,
617.
dioxa-4,6,8-triazadodecan-12-yl)phosphonate (28). Prepared from 24
(1.97 g; 6.96 mmol) and 9-fluorenylmethoxycarbonyl isothiocyanate
(2.43 g; 6.96 mmol) in acetone (100 mL); reaction time: 6 h. Yield: 2.13
g (54%) of a white amorphous solid. MS (ESI) m/z (%): 586 (95,
M+Na+), 564 (12, M+H+). HRMS (ESI) calcd. for C26H34O7N3NaPS
(M+Na+): 586.1747, found: 586.1748. 1H NMR (DMSO‑d6): δ 12.37 (bs,
1H, NH), 11.13 (bs, 1H, NH), 8.52 (bs, 1H, NH), 7.90 (dt, 2H, J
(10a,9a&10b,9b) = 7.5 Hz, J(10a,8a&10b,8b) = J(10a,7a&10b,7b) =
1.1 Hz, H-10a,b), 7.76 (bd, 2H, J(7a,8a&7b,8b) = 7.5 Hz, H-7a,b), 7.43
(tdd, 2H, J(9a,10a&9b,10b)
= J(9a,8a&9b,8b) = 7.5 Hz, J
(9a,7a&9b,7b) = 1.2 Hz, J(9a,5&9b,5) = 0.7 Hz, H-9a,b), 7.35 (td, 2H, J
(8a,9a&8b,9b) = J(8a,7a&8b,7b) = 7.5 Hz, J(8a,10a&8b,10b) = 1.1 Hz,
H-8a,b), 4.60 (dsep, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz, CH-i-Pr), 4.46 (d,
2H, J(4,5) = 7.1 Hz, H-4), 4.31 (bt, 1H, J(5,4) = 7.1 Hz, H-5), 3.77 (d,
2H, J(3′,P) = 8.2 Hz, H-3′), 3.61 (t, 2H, J(2′,1′) = 5.3 Hz, H-2′), 3.37 (q,
2H, J(1′,2′) = 5.3 Hz, H-1′), 1.25 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr) 1.24 (d,
6H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 178.94 (C-2), 152.93
(C-1), 152.17 (C-3), 143.43 (C-6a,b), 140.94 (C-11a,b), 128.04 (CH-9a,
b), 127.37 (CH-8a,b), 125.50 (CH-7a,b), 120.40 (CH-10a,b), 71.03 (d, J
(2′,P) = 11.5 Hz, CH2-2′), 70.37 (d, J(C,P) = 6.4 Hz, CH-i-Pr), 67.66
(CH2-4), 64.98 (d, J(3′,P) = 164.3 Hz, CH2-3′), 46.22 (CH-5), 39.30
(CH2-1′), 24.02 (d, J(C,P) = 3.7 Hz, CH3-i-Pr), 23.92 (d, J(C,P) = 4.5 Hz,
5.2.20. N1-Substituted thiobiurets with N5-[(diisopropoxy)phosphoryl]
methoxyalkyl arrangement (26–31). The general procedure
Compound 24 or 25 was dissolved in acetone and the appropriate
◦
isothiocyanate was added dropwise. The solution was stirred at 60 C
until quantitative conversion (4–24 h). The solvent was removed in
vacuo and the residue was purified on a silica gel column (a gradient of
40–100% EtOAc/hexane). For specific conditions, see below.
CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 20.32. FTIR (KBr, cmꢀ 1
)
νmax:
5.2.20.1. Diisopropyl
(1,5-dioxo-1-phenyl-3-thioxo-9-oxa-2,4,6-tri-
azadecan-10-yl)phosphonate (26). Prepared from 24 (7.88 g; 27.9
mmol) and benzoyl isothiocyanate (4.56 g; 27.9 mmol) in acetone (300
mL); reaction time: 4 h. Yield: 7.8 g (63%) of a yellowish oil. MS (ESI) m/
z (%): 468 (100, M+Na+), 446 (15, M+H+). HRMS (ESI) calcd. for
3322, 3243, 3186, 3066, 3040, 2979, 1721, 1705, 1609, 1538, 1509,
1466, 1451, 1386, 1360, 1338, 1323, 1224, 1209, 1172, 1142, 1105,
1084, 994, 840, 760, 741, 726, 621.
C
18H28O6N3NaPS (M+Na+): 468.1329, found: 468.1328; HRMS (ESI)
5.2.20.4. Diisopropyl
(S)-(1,5-dioxo-1-phenyl-3-thioxo-8-((trityloxy)
calcd. for C18H29O6N3PS (M+H+): 446.1509, found: 446.1509. 1H NMR
(DMSO‑d6): δ 7.90 (m, 2H, H-o-Ph), 7.68 (m, 1H, H-p-Ph), 7.58 (m, 2H,
H-m-Ph), 4.60 (dsept, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz, CH-i-Pr), 3.78
(d, 2H, J(3′,P) = 8.5 Hz, H-3′), 3.61 (t, 2H, J(2′,1′) = 5.3 Hz, H-2′), 3.38
methyl)-9-oxa-2,4,6-triazadecan-10-yl)phosphonate (29). Prepared from
25 (1.50 g; 2.71 mmol) and benzoyl isothiocyanate (0.53 g; 3.25 mmol)
in acetone (100 mL); reaction time: 24 h. Yield: 0.59 g (30%) of a yellow
amorphous solid. [α] ꢀ 3.4 (c 0.205 g/100 mL, MeOH). MS (ESI) m/z
(%): 740 (100, M+DNa+). HRMS (ESI) calcd. for C38H44O7N3NaPS
(m, 2H, H-1′), 1.24 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.23 (d, 6H, Jvic
=
6.2 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 178.96 (C-2), 168.03 (C-3),
(M+Na+): 740.2530, found: 740.2524. 1H NMR (DMSO‑d6): δ 7.86–7.89
12

´
A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
(m, 2H, H-o-Ph), 7.67 (m, 1H, H-p-Ph), 7.54–7.58 (m, 2H, H-m-Ph),
7.38–7.41 (m, 6H, H-o-Tr), 7.30–7.35 (m, 6H, H-m-Tr), 7.25 (m, 3H, H-
p-Tr), 4.53–4.63 (m, 2H, CH-i-Pr), 3.84 (dd, 1H, Jgem = 13.7 Hz, J(3′a,P)
= 8.8 Hz, H-3′a), 3.79 (dd, 1H, Jgem = 13.7 Hz, J(3′b,P) = 9.3 Hz, H-3′b),
3.75 (p, 1H, J(2′,1′) = J(2′,3′) = 5.2 Hz, H-2′), 3.42–3.47 (bm, 2H, H-1′),
3.09 (dd, 1H, Jgem = 10.1 Hz, J(4′a,2′) = 4.9 Hz, H-4′a), 3.06 (dd, 1H,
Jgem = 10.1 Hz, J(4′b,2′) = 5.3 Hz, H-4′b), 1.22 (d, 3H, Jvic = 6.2 Hz,
CH3-i-Pr), 1.21 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.20 (d, 3H, Jvic = 6.2 Hz,
CH3-i-Pr), 1.19 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ
168.05 (C-3), 143.72 (C-i-Tr), 133.47 (Ch-p-Ph), 133.10 (C-i-Ph), 129.09
(CH-m-Ph), 128.44 (CH-o-Tr), 128.07 (CH-o-Ph, CH-m-Tr), 127.26 (CH-
p-Tr), 86.33 (C-Tr), 78.91 (d, J(2′,P) = 12.0 Hz, CH-2′), 70.39 (d, J(C,P)
= 6.3 Hz, CH-i-Pr), 70.38 (d, J(C,P) = 6.3 Hz, CH-i-Pr), 64.02 (d, J(3′,P)
= 165.2 Hz, CH2-3′), 62.89 (CH2-4′), 39.87 (CH2-1′), 24.00 (d, J(C,P) =
3.7 Hz, CH3-i-Pr), 23.90 (d, J(C,P) = 4.4 Hz, CH3-i-Pr), 23.88 (d, J(C,P)
= 4.4 Hz, CH3-i-Pr); C-1 and C-2 not detected. 31P {1H} NMR (DMSO‑d6):
3.74 (p, 1H, J(2′,1′) = J(2′,3′) = 5.2 Hz, H-2′), 3.39–3.51 (bm, 2H, H-1′),
3.08 (dd, 1H, Jgem = 10.8 Hz, J(4′a,2′) = 5.2 Hz, H-4′a), 3.06 (dd, 1H,
Jgem = 10.8 Hz, J(4′b,2′) = 5.2 Hz, H-4′b), 1.22 (d, 3H, Jvic = 6.2 Hz,
CH3-i-Pr), 1.21 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.19 (d, 3H, Jvic = 6.2 Hz,
CH3-i-Pr), 1.18 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ
178.90 (C-2), 152.94 (C-1), 152.18 (C-3), 143.74 (C-i-Tr), 143.43 (C-6a,
b), 140.96 (C-11a,b), 128.46 (CH-o-Tr), 128.12 (CH-m-Tr), 128.08 (CH-
9a,b), 127.39 (CH-8a,b), 127.29 (CH-p-Tr), 125.54 (CH-7a,b), 120.45
(CH-10a,b), 86.34 (C-Tr), 78.80 (d, J(2′,P) = 12.2 Hz, CH-2′), 70.42 (d, J
(C,P) = 6.3 Hz, CH-i-Pr), 67.70 (CH2-4), 64.00 (d, J(3′,P) = 165.1 Hz,
CH2-3′), 62.82 (CH2-4′), 46.23 (CH-5), 39.70 (CH2-1′), 24.05 (d, J(C,P)
= 3.7 Hz, CH3-i-Pr), 23.95 (d, J(C,P) = 4.2 Hz, CH3-i-Pr), 23.92 (d, J(C,
P) = 4.2 Hz, CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 20.11. FTIR (CHCl3,
cmꢀ 1
) νmax: 3395, 3263, 3208, 3088, 3066, 2985, 1768, 1742, 1712,
1607, 1598, 1538, 1505, 1478, 1450, 1387, 1377, 1305, 1249, 1172,
1104, 1056, 999, 890, 707, 642, 545, 427.
δ 20.11. FTIR (CCl4, cmꢀ 1
) νmax: 3423, 3225, 3063, 3035, 2981, 2934,
1722, 1687, 1678, 1603, 1578, 1541, 1491, 1468, 1449, 1386, 1375,
1258, 1238, 1179, 1159, 1143, 1105, 1087, 1079, 1026, 1009, 993, 881,
706, 696, 648, 633.
5.2.21. N5-Protected thiadiazolones with N2-[(diisopropoxy)phosphoryl]
methoxyalkyl arrangement. The general procedure
An appropriate starting compound (26, 27, 29, 30 or 31) and N-
bromosuccinimide (1.25 eq.) were dissolved in MeOH and stirred at
80 ◦C for 30 min. After cooling to RT, the solvent was evaporated and the
residue was chromatographed on a silica gel column (0–3% MeOH/
CHCl3).
5.2.20.5. Diisopropyl (S)-(6,10-dioxo-8-thioxo-3-(trityloxymethyl)-2,11-
dioxa-5,7,9-triazatridecyl)phosphonate (30). Prepared from 25 (1.00 g;
1.80 mmol) and ethoxycarbonyl isothiocyanate (0.355 g; 2.70 mmol) in
acetone (80 mL); reaction time: 4 h. Yield: 0.76 g (62%) of a yellowish
crystalline solid, mp 61–62 ◦C. [
α]
D
ꢀ 0.7 (c 0.430 g/100 mL, MeOH).
5.2.21.1. Diisopropyl ((2-(5-benzamido-3-oxo-1,2,4-thiadiazol-2(3H)-yl)
ethoxy)methyl)phosphonate (32). Prepared from 26 (7.78 g; 17.5 mmol)
and N-bromosuccinimide (3.90 g; 21.9 mmol) in MeOH (200 mL). Yield:
MS (ESI) m/z (%): 708 (100, M+Na+). HRMS (ESI) calcd. for
C
34H44O8N3NaPS (M+Na+): 708.2479, found: 708.2477. 1H NMR
◦
(DMSO‑d6): δ 12.18 (bs, 1H, NH), 11.04 (bs, 1H, NH), 8.24 (bs, 1H, NH),
7.37–7.41 (m, 6H, H-o-Tr), 7.30–7.35 (m, 6H, H-m-Tr), 7.26 (m, 3H, H-
p-Tr), 4.52–4.63 (m, 2H, CH-i-Pr), 4.18 (q, 2H, J(4,5) = 7.1 Hz, H-4),
3.83 (dd, 1H, Jgem = 13.7 Hz, J(3′a,P) = 8.8 Hz, H-3′a), 3.78 (dd, 1H,
Jgem = 13.7 Hz, J(3′b,P) = 9.3 Hz, H-3′b), 3.72 (p, 1H, J(2′,1′) = J(2′,4′)
= 5.0, H-2′), 3.35–3.48 (bm, 2H, H-1′), 3.08 (dd, 1H, Jgem = 10.4 Hz, J
(4′a,2′) = 4.8 Hz, H-4′a), 3.05 (dd, 1H, Jgem = 10.4 Hz, J(4′b,2′) = 5.3
Hz, H-4′b), 1.16–1.26 (m, 15H, CH3-i-Pr, H-5). 13C NMR (DMSO‑d6): δ
178.86 (C-2), 153.13 (C-1), 152.03 (C-3), 143.72 (C-i-Tr), 128.43 (CH-o-
Tr), 128.08 (CH-m-Tr), 127.26 (CH-p-Tr), 86.33 (C-Tr), 78.84 (d, J(2′,P)
= 12.2 Hz, CH-2′), 70.38 (d, J(C,P) = 6.3 Hz, CH-i-Pr), 64.01 (d, J(3′,P)
= 165.1 Hz, CH2-3′), 62.90 (CH2-4′), 62.40 (CH2-4), 39.70 (CH2-1′),
24.00 (d, J(C,P) = 3.7 Hz, CH3-i-Pr), 23.90 (d, J(C,P) = 4.4 Hz, CH3-i-Pr),
23.87 (d, J(C,P) = 4.4 Hz, CH3-i-Pr), 14.19 (CH3-5). 31P {1H} NMR
3.74 g (48%) of a white crystalline solid, mp 83–84 C. MS (ESI) m/z
(%): 466 (100, M+Na+), 444 (9, M+H+). HRMS (ESI) calcd. for
C
18H26O6N3NaPS (M+Na+): 466.1172, found: 466.1171; HRMS (ESI)
calcd. for C18H27O6N3PS (M+H+): 444.1353, found: 444.1352. 1H NMR
(DMSO‑d6): δ 13.42 (vbs, 1H, NH), 8.12 (m, 2H, H-o-Ph), 7.66 (m, 1H, H-
p-Ph), 7.56 (m, 2H, H-m-Ph), 4.59 (dsept, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2
Hz, CH-i-Pr), 3.80 (d, 2H, J(3′,P) = 8.2 Hz, H-3′), 3.71–3.79 (m, 4H, H-
1′, H-2′), 1.23 (bt, 12H, Jvic = 5.9 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ
174.52 (C-7, determined by HMBC), 170.86 (C-5, determined by
HMBC), 161.53 (C-3), 133.57 (CH-p-Ph), 130.56 (C-i-Ph), 129.04 (CH-
m-Ph), 128.94 (CH-o-Ph), 70.74 (d, J(2′,P) = 11.5 Hz, CH2-2′), 70.40 (d,
J(C,P) = 6.4 Hz, CH-i-Pr), 64.98 (d, J(3′,P) = 163.9 Hz, CH2-3′), 42.47
(CH2-1′), 23.98 (d, J(C,P) = 3.8 Hz, CH3-i-Pr), 23.88 (d, J(C,P) = 4.5 Hz,
CH3-i-Pr). 31P {1H} NMR (DMSO‑d6): δ 19.01. FTIR (CCl4, cmꢀ 1
) νmax:
(DMSO‑d6): δ 18.91. FTIR (CCl4, cmꢀ 1
)
νmax: 3413, 3255, 3201, 3088,
3503, 3430, 3123, 3068, 3032, 2981, 2930, 2877, 1714, 1706, 1684,
1676, 1653, 1601, 1570, 1559, 1488, 1468, 1449, 1387, 1375, 1336,
1302, 1231, 1177, 1143, 1106, 1069, 1017, 999, 890, 687, 639, 529.
3062, 3035, 3024, 2981, 2934, 2874, 1777, 1742, 1717, 1707, 1597,
1535, 1503, 1478, 1467, 1449, 1442, 1386, 1374, 1304, 1223, 1174,
1170, 1142, 1121, 1105, 1096, 1077, 1035, 1011, 993, 936, 890, 706,
696, 642, 633.
5.2.21.2. Ethyl
(2-(2-((diisopropoxyphosphoryl)methoxy)ethyl)-3-oxo-
2,3-dihydro-1,2,4-thiadiazol-5-yl)carbamate (33). Prepared from 27
(10.20 g; 24.7 mmol) and N-bromosuccinimide (5.50 g; 30.9 mmol) in
MeOH (200 mL). Yield: 2.42 g (19%) of a white crystalline solid, mp
84–86 ◦C. MS (ESI) m/z (%): 434 (100, M+Na+), 412 (20, M+H+).
HRMS (ESI) calcd. for C14H26O7N3NaPS (M+Na+): 434.1121, found:
434.1121; HRMS (ESI) calcd. for C14H27O7N3PS (M+H+): 412.1302,
found: 412.1302. 1H NMR (DMSO‑d6): δ 12.79 (bs, 1H, NH), 4.59 (dsept,
2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz, CH-i-Pr), 4.23 (q, 2H, J(9,10) = 7.1
Hz, CH2-9), 3.77 (d, 2H, J(3′,P) = 8.2 Hz, H-3′), 3.76 (m, 2H, H-1′), 3.69
5.2.20.6. Diisopropyl
(S)-(1-(9H-fluoren-9-yl)-3,7-dioxo-5-thioxo-10-
(trityloxymethyl)-2,11-dioxa-4,6,8-triazadodecan-12-yl)phosphonate
(31). Prepared from 25 (1.50 g; 2.71 mmol) and 9-fluorenylmethoxy-
carbonyl isothiocyanate (0.92 g; 3.25 mmol) in acetone (100 mL); re-
action time: 18 h. Yield: 1.14 g (51%) of a white crystalline solid, mp
89–90 ◦C. [
α]D ꢀ 2.8 (c 0.109 g/100 mL, MeOH). MS (ESI) m/z (%): 859
(100, M+Na+). HRMS (ESI) calcd. for C46H50O8N3NaPS (M+Na+):
858.2948, found: 858.2949; HRMS (ESI) calcd. for C46H51O8N3PS
(M+H+): 836.3129, found: 836.3134. 1H NMR (DMSO‑d6): δ 12.36 (bs,
1H, NH), 11.11 (bs, 1H, NH), 8.48 (bs, 1H, NH), 7.90 (dt, 2H, J
(10a,9a&10b,9b) = 7.5 Hz, J(10a,8a&10b,8b) = J(10a,7a&10b,7b) =
0.9 Hz, H-10a,b), 7.76 (bd, 2H, J(7a,8a&7b,8b) = 7.5 Hz, H-7a,b), 7.43
(bt, 2H, J(9a,10a&9b,10b) = J(9a,8a&9b,8b) = 7.5 Hz, H-9a,b), 7.39
(m, 6H, H-o-Tr), 7.30–7.35 (m, 8H, H-8a,b, H-m-Tr), 7.25 (m, 3H, H-p-
Tr), 4.53–4.63 (m, 2H, CH-i-Pr), 4.47 (d, 2H, J(4,5) = 7.1 Hz, H-4), 4.31
(t, 1H, J(5,4) = 7.1 Hz, H-5), 3.83 (dd, 1H, Jgem = 13.7 Hz, J(3′a,P) =
8.8 Hz, H-3′a), 3.79 (dd, 1H, Jgem = 13.7 Hz, J(3′b,P) = 9.3 Hz, H-3′b),
(m, 2H, H-2′), 1.25 (t, 3H, J(10,9) = 7.1 Hz, H-10), 1.23 (d, 6H, Jvic
=
6.2 Hz, CH3-i-Pr), 1.21 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR
(DMSO‑d6): δ 170.54 (C-7), 162.95 (C-3), 157.49 (C-5), 71.94 (d, J(C,P)
= 6.4 Hz, CH-i-Pr), 71.76 (d, J(2′,P) = 11.2 Hz, CH2-2′), 65.66 (d, J(3′,P)
= 164.7 Hz, CH2-3′), 64.53 (CH2-9), 43.70 (CH2-1′), 24.85 (d, J(C,P) =
3.8 Hz, CH3-i-Pr), 24.75 (d, J(C,P) = 4.6 Hz, CH3-i-Pr), 15.23 (CH3-10).
31P {1H} NMR (DMSO‑d6): δ 19.02. FTIR (CCl4, cmꢀ 1
) νmax: 3426, 3145,
3076, 2982, 2933, 2876, 1716, 1689, 1635, 1576, 1467, 1386, 1375,
1320, 1306, 1252, 1233, 1178, 1144, 1106, 1014, 996, 890.
13

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Bioorganic & Medicinal Chemistry 32 (2021) 115998
5.2.21.3. Diisopropyl
(S)-(((1-(5-benzamido-3-oxo-1,2,4-thiadiazol-2
H-10), 3.89 (dd, 1H, Jgem = 14.0 Hz, J(3′a,P) = 8.8 Hz, H-3′a), 3.78–3.85
(m, 2H, H-1′a, H-3′b), 3.58–3.65 (m, 2H, H-2′, H-1′b), 3.40–3.52 (m, 2H,
H-4′), 1.219 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.214 (d, 3H, Jvic = 6.2 Hz,
CH3-i-Pr), 1.208 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.199 (d, 3H, Jvic = 6.2
Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 143.41 (C-11a,b), 140.99 (C-16a,
b), 128.03 (CH-14a,b), 127.37 (CH-13a,b), 125.34 (CH-12a,b), 120.44
(CH-15a,b), 80.64 (d, J(2′,P) = 10.5 Hz, CH2-2′), 70.41 (d, J(C,P) = 6.4
Hz, CH-i-Pr), 70.34, (d, J(C,P) = 6.4 Hz, CH-i-Pr), 68.09 (CH2-9), 63.85
(d, J(3′,P) = 164.8 Hz, CH2-3′), 60.16 (CH2-4′), 46.36 (CH-10), 43.99
(CH2-1′), 23.97 (d, J(C,P) = 3.5 Hz, CH3-i-Pr), 23.94 (d, J(C,P) = 3.5 Hz,
CH3-i-Pr), 23.87 (d, J(C,P) = 4.5 Hz, CH3-i-Pr), 23.85 (d, J(C,P) = 4.5 Hz,
CH3-i-Pr); C-3, C-5, and C-7 not detected. 31P {1H} NMR (DMSO‑d6): δ
(3H)-yl)-3-hydroxypropan-2-yl)oxy)methyl)phosphonate (35). Prepared
from 29 (0.58 g; 0.802 mmol) and N-bromosuccinimide (0.18 g; 1.01
mmol) in MeOH (40 mL). Yield: 0.34 g (89%) of a white amorphous
solid. [α]D ꢀ 4.2 (c 0.169 g/100 mL, MeOH). MS (ESI) m/z (%): 969 (50,
2M+Na+), 496 (100, M+Na+), 474 (44, M+H+). HRMS (ESI) calcd. for
C
19H28O7N3NaPS (M+Na+): 496.1278, found: 496.1275; HRMS (ESI)
calcd. for C19H29O7N3PS (M+H+): 474.1458, found: 474.1458. 1H NMR
(DMSO‑d6): δ 13.40 (bs, 1H, NH), 8.10–8.14 (m, 2H, H-o-Ph), 7.66 (m,
1H, H-p-Ph), 7.54–7.58 (m, 2H, H-m-Ph), 4.86 (t, 1H, J(OH,4′) = 5.3 Hz,
OH), 4.54–4.65 (m, 2H, CH-i-Pr), 3.92 (dd, 1H, Jgem = 14.0 Hz, J(3′a,P)
= 8.7 Hz, H-3′a), 3.84 (dd, 1H, Jgem = 14.0 Hz, J(3′b,P) = 8.7 Hz, H-3′b),
3.81 (dd, 1H, Jgem = 14.5 Hz, J(1′a,2′) = 3.1 Hz, H-1′a), 3.67 (m, 1H, H-
2′), 3.61 (dd, 1H, Jgem = 14.5 Hz, J(1′b,2′) = 7.1 Hz, H-1′b), 3.45–3.54
20.34. FTIR (CCl4, cmꢀ 1
) νmax: 3371, 3068, 3042, 3018, 2981, 2931,
2874, 2856, 2742, 1717, 1668, 1571, 1478, 1466, 1452, 1387, 1375,
1341, 1294, 1243, 1179, 1143, 1105, 1015, 997, 891, 729, 689, 621,
428.
(m, 2H, H-3′), 1.23 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.22 (d, 3H, Jvic
=
6.2 Hz, CH3-i-Pr), 1.21 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.20 (d, 3H, Jvic
= 6.2 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 133.46 (CH-p-Ph), 129.01
(CH-m-Ph), 128.91 (CH-o-Ph), 80.53 (d, J(2′,P) = 10.7 Hz, CH-2′), 70.41
(d, J(C,P) = 6.4 Hz, CH-i-Pr), 70.34 (d, J(C,P) = 6.4 Hz, CH-i-Pr), 63.86
(d, J(3′,P) = 164.7 Hz, CH2-3′), 60.22 (CH2-4′), 43.75 (CH2-1′), 23.97 (d,
J(C,P) = 3.5 Hz, CH3-i-Pr), 23.94 (d, J(C,P) = 3.5 Hz, CH3-i-Pr), 23.88 (d,
J(C,P) = 4.5 Hz, CH3-i-Pr), 23.85 (d, J(C,P) = 4.5 Hz, CH3-i-Pr); C-3, C-5,
C-7, and C-i-Ph not detected. 31P {1H} NMR (DMSO‑d6): δ 20.38. FTIR
5.2.22. (9H-Fluoren-9-yl)methyl (2-(2-((diisopropoxyphosphoryl)
methoxy)ethyl)-3-oxo-2,3-dihydro-1,2,4-thiadiazol-5-yl)carbamate (34)
0.5 M bromine solution in ethyl acetate (19.0 mL; 9.5 mmol) was
added at 0 ◦C to a solution of 28 (2.54 g; 4.51 mmol) in ethyl acetate
(150 mL). The mixture was stirred at 4 ◦C for 17 h and evaporated. The
residue was purified on a silica gel column (a gradient of 0–5% MeOH/
CHCl3). Yield: 2.03 g (80%) of a colorless oil. MS (ESI) m/z (%): 584
(100, M+Na+), 562 (7, M+H+). HRMS (ESI) calcd. for C26H32O7N3NaPS
(M+Na+): 584.1591, found: 584.1592. 1H NMR (DMSO‑d6): δ 7.90 (bd,
2H, J(15a,14a&15b,14b) = 7.5 Hz, H-15a,b), 7.71 (bd, 2H, J
(KBr, cmꢀ 1
) νmax: 3424, 3253, 2981, 1703, 1648, 1600, 1579, 1569,
1555, 1487, 1465, 1449, 1387, 1376, 1257, 1240, 1143, 1104, 1081,
1012, 1001, 994, 893, 690.
(12a,13a&12b,13b)
=
7.5 Hz, H-12a,b), 7.43 (bt, 2H,
J
5.2.21.4. Ethyl (S)-(2-(2-((diisopropoxyphosphoryl)methoxy)-3-hydrox-
(14a,13a&14b,13b) = J(14a,15a&14b,15b) = 7.5 Hz, H-14a,b), 7.34
(td, 2H, J(13a,12a&13b,12b) = J(13a,14a&13b,14b) = 7.5 Hz, H-13a,
b), 4.57 (dsep, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz, CH-i-Pr), 4.56 (d, 2H, J
(9,10) = 6.8 Hz, H-9), 4.33 (bt, 1H, J(10,9) = 6.8 Hz, H-10), 3.77 (d, 2H,
J(3′,P) = 8.1 Hz, H-3′), 3.76 (bt, 2H, J(1′,2′) = 4.8 Hz, H-1′), 3.69 (bt,
2H, J(2′,1′) = 4.8 Hz, H-2′), 1.21 (d, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.20 (d,
6H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR (DMSO‑d6): δ 169.90 (C-5), 160.40
(C-3), 157.55 (C-7), 143.39 (C-11a,b), 140.99 (C-16a,b), 128.03 (CH-
14a,b), 127.37 (CH-13a,b), 125.34 (CH-12a,b), 120.44 (CH-15a,b),
70.84 (d, J(2′,P) = 11.2 Hz, CH2-2′), 70.39 (d, J(C,P) = 6.4 Hz, CH-i-Pr),
68.12 (CH-10), 64.94 (d, J(3′,P) = 164.0 Hz, CH2-3′), 46.35 (CH-10),
42.70 (CH2-1′), 23.97 (d, J(C,P) = 3.7 Hz, CH3-i-Pr), 23.86 (d, J(C,P) =
ypropyl)-3-oxo-2,3-dihydro-1,2,4-thiadiazol-5-yl)carbamate
(36). Pre-
pared from 30 (0.70 g; 1.02 mmol) and N-bromosuccinimide (0.23 g;
1.28 mmol) in MeOH (50 mL). Yield: 0.34 g (75%) of a white amorphous
solid. [α] ꢀ 5.6 (c 0.197 g/100 mL, MeOH). MS (ESI) m/z (%): 464
D
(100, M+Na+), 442 (18, M+H+). HRMS (ESI) calcd. for C15H28O8N3
-
NaPS (M+Na+): 464.1227, found: 464.1226. 1H NMR (CD3OD): δ
4.69–4.78 (m, 2H, CH-i-Pr), 4.33 (q, 2H, J(9,10) = 7.1 Hz, H-9), 4.02
(dd, 1H, Jgem = 13.9 Hz, J(3′a,P) = 9.0 Hz, H-3′a), 3.96 (dd, 1H, Jgem
=
14.9 Hz, J(1′a,2′) = 3.4 Hz, H-1′a), 3.93 (dd, 1H, Jgem = 13.9 Hz, J(3′b,
P) = 9.3 Hz, H-3′b), 3.82 (dd, 1H, Jgem = 14.9 Hz, J(1′b,2′) = 6.8 Hz, H-
1′b), 3.73 (m, 1H, H-2′), 3.68 (dd, 1H, Jgem = 12.1 Hz, J(4′a,2′) = 4.7 Hz,
H-4′a), 3.62 (dd, 1H, Jgem = 12.1 Hz, J(4′b,2′) = 4.9 Hz, H-4′b),
1.31–1.37 (m, 15H, CH3-i-Pr, H-10). 13C NMR (CD3OD): δ 172.10 (C-5),
166.20 (C-3), 157.19 (C-7), 82.50 (d, J(2′,P) = 11.6 Hz, CH-2′), 73.34 (d,
J(C,P) = 6.8 Hz, CH-i-Pr), 73.25 (d, J(C,P) = 6.8 Hz, CH-i-Pr), 65.28 (d, J
(3′,P) = 168.6 Hz, CH2-3′), 64.84 (CH2-9), 61.82 (CH2-4′), 45.40 (CH2-
1′), 24.33 (d, J(C,P) = 3.6 Hz, CH3-i-Pr), 24.32 (d, J(C,P) = 3.6 Hz, CH3-
i-Pr), 24.29 (d, J(C,P) = 4.5 Hz, CH3-i-Pr), 24.28 (d, J(C,P) = 4.5 Hz,
CH3-i-Pr), 14.55 (CH3-10). 31P {1H} NMR (CD3OD): δ 19.76. FTIR (CCl4,
4.5 Hz, CH3-i-Pr). 31P NMR {1H} (DMSO‑d6): δ 20.04. FTIR (KBr, cmꢀ 1
)
νmax: 3435, 3250, 2980, 2927, 1716, 1636, 1568, 1478, 1465, 1451,
1232, 1254, 1180, 1145, 1105, 1003, 761, 741, 621.
5.2.23. N5-Substituted N2-[2-(phosphonomethoxy)ethyl]thiadiazolones
(38–40). The general procedure
Bromotrimethylsilane (2.7 mL; 20 mmol) was added to the appro-
priate phosphonate ester 32–34 (1 mmol) dissolved in acetonitrile (30
mL) and the mixture was left at RT overnight. The volatiles were
removed in vacuo and the residue was co-evaporated three times with
acetonitrile, then with 25% aqueous ammonia and finally three times
with ethanol. The product was recrystallized from ethanol.
cmꢀ 1
) νmax: 3385, 2981, 2934, 2875, 2737, 1715, 1670, 1635, 1570,
1467, 1387, 1375, 1245, 1178, 1143, 1105, 1062, 1012, 996, 890, 690.
5.2.21.5. (9H-Fluoren-9-yl)methyl (S)-(2-(2-((diisopropoxyphosphoryl)
methoxy)-3-hydroxypropyl)-3-oxo-2,3-dihydro-1,2,4-thiadiazol-5-yl)
carbamate (37). Prepared from 31 (1.04 g; 1.25 mmol) and N-bromo-
succinimide (0.28 g; 1.56 mmol) in MeOH (60 mL). Yield: 0.53 g (72%)
5.2.23.1. ((2-(5-Benzamido-3-oxo-1,2,4-thiadiazol-2(3H)-yl)ethoxy)
methyl)phosphonic acid (38). Prepared from the starting compound 32
(0.44 g; 1 mmol). Yield: 0.34 g (96%) of a white crystalline solid, mp
219–221 ◦C. MS (ESI) m/z (%): 380 (13, Mꢀ 2H++Na+), 358 (100,
Mꢀ H+). HRMS (ESI) calcd. for C12H13O6N3PS (Mꢀ H+): 358.0268,
found: 358.0268. 1H NMR (DMSO‑d6): δ 8.09 (m, 2H, H-o-Ph),
7.40–7.48 (m, 3H, H-m-Ph, H-p-Ph), 6.97 (vbs, NH4+), 3.68 (m, 2H, H-
1′), 3.60 (m, 2H, H-2′), 3.36 (d, 2H, J(H,P) = 8.8 Hz, H-3′). 13C NMR
(DMSO‑d6): δ 176.89 (C-5), 175.28 (C-7), 166.16 (C-3), 136.88 (C-i-Ph),
130.96 (CH-p-Ph), 128.42 (CH-o-Ph), 128.22 (CH-m-Ph), 71.13 (d, J(C,
P) = 10.7 Hz, CH2-2′), 69.17 (d, J(C,P) = 154.4 Hz, CH2-3′), 42.10 (CH2-
of a white crystalline solid, mp 68–70 ◦C. [
α]D ꢀ 2.8 (c 0.212 g/100 mL,
MeOH). MS (ESI) m/z (%): 1181 (8, 2Mꢀ H+), 590 (100, Mꢀ H+). HRMS
(ESI) calcd. for C27H33O8N3PS (Mꢀ H+): 590.1732, found: 590.1729. 1H
NMR (DMSO‑d6): δ 7.91 (ddd, 2H, J(15a,14a&15b,14b) = 7.5 Hz, J
(15a,13a&15b,13b) = 1.2 Hz, J(15a,12a&15b,12b) = 0.7 Hz, H-15a,b),
7.71 (bd, 2H, J(12a,13a&12b,13b) = 7.5 Hz, H-12a,b), 7.43 (tdd, 2H, J
(14a,13a&14b,13b)
=
J(14a,15a&14b,15b)
=
7.5 Hz,
J
(14a,12a&14b,12b) = 1.2 Hz, J(14a,11a&14b,11b) = 0.7 Hz, H-14a,b),
7.34 (td, 2H, J(13a,12a&13b,12b) = J(13a,14a&13b,14b) = 7.5 Hz, J
(13a,12a&13b,12b) = 1.2 Hz, H-13a,b), 4.84 (t, 1H, J(OH,4′) = 5.4 Hz,
OH), 4.52–4.63 (m, 4H, CH2-9, CH-i-Pr), 4.34 (t, 1H, J(10,9) = 6.8 Hz,
1′). 31P {1H} NMR (DMSO‑d6): δ 12.45. FTIR (KBr, cmꢀ 1
) νmax: 3127,
14

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A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
3010, 2930, 1669, 1655, 1617, 1603, 1582, 1564, 1536, 1496, 1448,
1399, 1334, 1302, 1145, 1113, 1070, 1015, 995, 952, 811, 772, 704,
687, 641, 529.
(MeOD-d4): δ 173.57 (C-7), 171.68 (C-5), 162.56 (C-3), 134.74 (CH-p-
Ph), 132.54 (C-i-Ph), 129.94 (CH-m-Ph), 129.82 (CH-o-Ph), 82.41 (d, J
(2′,P) = 10.4 Hz, CH-2′), 68.28 (d, J(3′,P) = 157.6 Hz, CH2-3′), 62.21
(CH2-4′), 44.96 (CH2-1′). 31P NMR {1H} (MeOD-d4): δ 16.50. FTIR (KBr,
cmꢀ 1
) νmax: 3390, 3200, 3136, 2893, 2516, 2270, 1753, 1705, 1687,
5.2.23.2. ((2-(5-((Ethoxycarbonyl)amino)-3-oxo-1,2,4-thiadiazol-2(3H)-
yl)ethoxy)methyl)phosphonic acid (39). Prepared from the starting
compound 33 (0.41 g; 1 mmol). Yield: 0.30 g (91%) of a white crys-
talline solid, mp 184–186 ◦C. MS (ESI) m/z (%): 348 (26, Mꢀ 2H++Na+),
326 (100, Mꢀ H+). HRMS (ESI) calcd. for C8H13O7N3PS (Mꢀ H+):
326.0217, found: 326.0213. 1H NMR (D2O): δ 4.23 (q, 2H, J(9,10) = 7.1
Hz, H-9), 3.88 (m, 2H, H-1′), 3.82 (m, 2H, H-2′), 3.62 (d, 2H, J(3′,P) =
8.5 Hz, H-3′), 1.29 (t, 3H, J(10,9) = 7.1 Hz, H-10). 13C NMR (D2O): δ
177.80 (C-5), 166.86 (C-3), 163.26 (C-7), 71.39 (d, J(2′,P) = 10.4 Hz,
CH2-2′), 68.32 (d, J(3′,P) = 154.2 Hz, CH2-3′), 63.94 (CH2-9), 43.70
(CH2-1′), 14.41 (CH3-10). 31P {1H} NMR (D2O): δ 15.56. FTIR (KBr,
1652, 1600, 1589, 1547, 1487, 1472, 1449, 1303, 1261, 1185, 1157,
1107, 1057, 1027, 1013, 838, 709, 686, 632.
5.2.24.2. (S)-(((1-(5-((Ethoxycarbonyl)amino)-3-oxo-1,2,4-thiadiazol-2
(3H)-yl)-3-hydroxypropan-2-yl)-oxy)methyl)phosphonic acid (42). Pre-
pared from the starting compound 36 (0.44 g; 1 mmol). Yield: 0.26 g
(73%) as a white lyophilizate. [α]D ꢀ 2.6 (c 0.272 g/100 mL, H2O). MS
(ESI) m/z (%): 735 (12, 2Mꢀ 2H++Na+), 713 (15, 2Mꢀ H+), 378 (5,
Mꢀ 2H++Na+), 356 (100, Mꢀ H+). HRMS (ESI) calcd. for C9H15O8N3PS
1
(Mꢀ H+): 356.0323, found: 356.0322. H NMR (MeOD-d4): δ 4.33 (q,
cmꢀ 1
)
νmax: 3138, 2978, 2934, 2884, 1706, 1665, 1500, 1483, 1403,
2H, J(9,10) = 7.1 Hz, H-9), 3.95 (dd, 1H, Jgem = 14.8 Hz, J(1′a,2′) = 3.9
Hz, H-1′a), 3.78–3.90 (m, 3H, H-1′b, 2H-4′), 3.68–3.74 (m, 2H, H-2′, H-
3′a), 3.60 (dd, 1H, Jgem = 13.5 Hz, J(3′b,2′) = 6.3 Hz, H-3′b), 1.35 (t, 3H,
J(10,9) = 7.1 Hz, H-10). 13C NMR (MeOD-d4): δ 172.09 (C-5), 166.32 (C-
3), 156.97 (C-7), 82.50 (d, J(2′,P) = 10.7 Hz, CH-2′), 66.81 (d, J(3′,P) =
162.1 Hz, CH2-3′), 64.86 (CH2-9), 62.02 (CH2-4′), 45.14 (CH2-1′), 14.53
1339, 1312, 1218, 1107, 1027, 1011, 956, 937.
5.2.23.3. ((2-(5-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-oxo-
1,2,4-thiadiazol-2(3H)-yl)ethoxy)methyl)phosphonic acid (40). Prepared
from the starting compound 34 (0.56 g; 1 mmol). Yield: 0.39 g (82%) of
a yellowish amorphous solid. MS (ESI) m/z (%): 476 (43, Mꢀ H+). HRMS
(ESI) calcd. for C20H19O7N3PS (Mꢀ H+): 476.0684, found: 476.0684. 1H
NMR (MeOD-d4): δ 7.78 (dt, 2H, J(15a,14a&15b,14b) = 7.5 Hz, J
(15a,13a&15b,13b) = J(15a,12a&15b,12b) = 1.1 Hz, H-15a,b), 7.64
(dq, 2H, J(12a,13a&12b,13b) = 7.5 Hz, J(12a,14a&12b,14b) = J
(12a,15a&12b,15b) = J(12a,10&12b,10) = 1.1 Hz, H-12a,b), 7.38 (tdd,
(CH3-10). 31P NMR {1H} (MeOD-d4): δ 19.45. FTIR (KBr, cmꢀ 1
) νmax:
3259, 2281, 2137, 1758, 1714, 1689, 1563, 1513, 1370, 1363, 1323,
1276, 1257, 1231, 1034, 1010, 988, 942, 883, 701, 552.
5.2.24.3. (S)-(((1-(5-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-
oxo-1,2,4-thiadiazol-2(3H)-yl)-3-hydroxypropan-2-yl)oxy)methyl)phos-
phonic acid (43). Prepared from the starting compound 37 (0.59 g; 1
2H, J(14a,13a&14b,13b)
= J(14a,15a&14b,15b) = 7.5 Hz, J
(14a,12a&14b,12b) = 1.1 Hz, J(14a,11a&14b,11b) = 0.7 Hz, H-14a,b),
7.31 (td, 2H, J(13a,12a&13b,12b) = J(13a,14a&13b,14b) = 7.5 Hz, J
(13a,15a&13b,15b) = 1.1 Hz, H-13a,b), 4.59 (d, 2H, J(9,10) = 6.5 Hz,
H-9), 4.27 (t, 1H, J(10,9) = 6.5 Hz, H-10), 3.87 (m, 2H, H-1′), 3.79 (m,
2H, H-2′), 3.76 (d, 2H, J(3′,P) = 8.3 Hz, H-3′). 13C NMR (MeOD-d4): δ
171.87 (C-5), 165.70 (C-3), 156.99 (C-7), 144.47 (C-11a,b), 142.63 (C-
16a,b), 129.04 (CH-14a,b), 128.29 (CH-13a,b), 126.04 (CH-12a,b),
121.08 (CH-15a,b), 72.34 (d, J(2′,P) = 10.3 Hz, CH2-2′), 69.91 (CH2-9),
67.33 (d, J(3′,P) = 162.9 Hz, CH2-3′), 47.90 (CH-10), 44.44 (CH2-1′).
mmol). Yield: 0.44 g (86%) as a white lyophilizate. [α]D ꢀ 1.8 (c 0.221
g/100 mL, H2O). MS (ESI) m/z (%): 528 (10, Mꢀ 2H++Na+), 506 (60,
Mꢀ H+). HRMS (ESI) calcd. for C21H20O8N3NaPS (Mꢀ 2H++Na+):
528.0612, found: 528.0608; HRMS (ESI) calcd. for C21H21O8N3PS
(Mꢀ H+): 506.0793, found: 506.0789. 1H NMR (MeOD-d4): δ 7.82 (ddd,
2H, J(15a,14a&15b,14b) = 7.6 Hz, J(15a,13a&15b,13b) = 1.3 Hz, J
(15a,12a&15b,12b)
=
0.7 Hz, H-15a,b), 7.67 (ddt, 2H,
J
(12a,13a&12b,13b) = 7.5 Hz, J(12a,14a&12b,14b) = 1.3 Hz, J
(12a,13a&12b,13b) = J(12a,10&12b,10) = 0.7 Hz, H-12a,b), 7.41 (tdd,
31P NMR {1H} (MeOD-d4): δ 19.15. FTIR (KBr, cmꢀ 1
)
νmax: 3431, 3065,
2H, J(14a,13a&14b,13b) = J(14a,15a&14b,15b) = 7.5 Hz, J
3039, 3015, 2925, 2720, 2320, 1731, 1713, 1643, 1566, 1478, 1450,
1295, 1256, 1230, 1107, 1033, 1005, 942, 761, 741, 621, 428.
(14a,12a&14b,12b) = 1.3 Hz, J(14a,10&14b,10) = 0.7 Hz, H-14a,b),
7.33 (td, 2H, J(13a,12a&13b,12b) = J(13a,14a&13b,14b) = 7.5 Hz, J
(13a,15a&13b,15b) = 1.3 Hz, H-13a,b), 4.65 (d, 2H, J(9,10) = 6.5 Hz,
H-9), 4.32 (t, 1H, J(10,9) = 6.5 Hz, H-10), 3.94 (dd, 1H, Jgem = 14.9 Hz,
J(1′a,2′) = 3.8 Hz, H-1′a), 3.87 (dd, 1H, Jgem = 13.4 Hz, J(3′a,P) = 9.1
Hz, H-3′a), 3.83 (dd, 1H, Jgem = 13.4 Hz, J(3′b,P) = 9.5 Hz, H-3′b), 3.81
(dd, 1H, Jgem = 14.9 Hz, J(1′b,2′) = 6.5 Hz, H-1′b), 3.65–3.72 (m, 2H, H-
2′, H-3′a), 3.60 (m, 1H, H-3′b). 13C NMR (MeOD-d4): δ 172.11 (C-5),
166.11 (C-3), 157.00 (C-7), 144.53 (C-11a,b), 142.70 (C-16a,b), 129.07
(CH-14a,b), 128.32 (CH-13a,b), 126.07 (CH-12a,b), 121.10 (CH-15a,b),
82.52 (d, J(2′,P) = 11.0 Hz, CH-2′), 69.93 (CH2-9), 66.49 (d, J(3′,P) =
163.0 Hz, CH2-3′), 61.99 (CH2-4′), 47.96 (CH-10), 45.16 (CH2-1′). 31P
5.2.24. N5-Substituted N2-[3-hydroxy-2-(phosphonomethoxy)propyl]
thiadiazolones (41–43)
The general procedure. Bromotrimethylsilane (2.7 mL; 20 mmol)
was added to a solution of the appropriate phosphonate ester 35–37 (1
mmol) in acetonitrile (30 mL) and the mixture was left at RT overnight.
The volatiles were removed in vacuo and the residue was co-evaporated
three times with acetonitrile. Water (5 mL) was added and the mixture
was stirred at RT for 1 h. The mixture was evaporated, the residue was
co-evaporated three times with ethanol and the crude product was pu-
rified on a reverse-phase (C18) column in a gradient of 0–60% acetoni-
trile/water. Product-containing fractions were combined and
lyophilized.
NMR {1H} (MeOD-d4): δ 20.15. FTIR (KBr, cmꢀ 1
) νmax: 3427, 3203,
3069, 3000, 1706, 1626, 1568, 1545, 1496, 1477, 1449, 1247, 1223,
1116, 1057, 1024, 1009, 621, 430.
5.2.25. N5-{[(Diisopropoxy)phosphoryl]methoxyalkyl}thiobiurets (48,
49)
5.2.24.1. (S)-(((1-(5-Benzamido-3-oxo-1,2,4-thiadiazol-2(3H)-yl)-3-
hydroxypropan-2-yl)oxy)methyl)phosphonic acid (41). Prepared from the
starting compound 35 (0.47 g; 1 mmol). Yield: 0.31 g (80%) as a white
The general procedure. Piperidine (0.15 mL; 1.5 mmol) was added
to a solution of Fmoc derivative 28 or 31 (1 mmol) in dichloromethane
(40 mL). The mixture was stirred at RT overnight, evaporated, and the
residue was chromatographed on a silica gel column (0–3% MeOH/
CHCl3).
lyophilizate. [α]D ꢀ 1.2 (c 0.332 g/100 mL, H2O). MS (ESI) m/z (%): 410
(32, Mꢀ 2H++Na+), 388 (Mꢀ H+). HRMS (ESI) calcd. for C13H15O7N3PS
(Mꢀ H+): 388.0374, found: 388.0370. 1H NMR (MeOD-d4): δ 8.10–8.14
(m, 2H, H-o-Ph), 7.66 (m, 1H, H-p-Ph), 7.52–7.59 (m, 2H, H-m-Ph), 3.90
(dd, 1H, Jgem = 14.7 Hz, J(1′a,2′) = 4.6 Hz, H-1′a), 3.78–3.86 (m, 2H, H-
1′b, H-3′a), 3.76 (dd, 1H, Jgem = 12.2 Hz, J(4′a,2′) = 3.6 Hz, H-4′a), 3.71
(m, 1H, H-2′), 3.67 (dd, 1H, Jgem = 12.8 Hz, J(3′b,P) = 9.9 Hz, H-3′b),
3.58 (dd, 1H, Jgem = 12.2 Hz, J(4′b,2′) = 4.8 Hz, H-4′b). 13C NMR
5.2.25.1. Diisopropyl
((2-(3-carbamothioylureido)ethoxy)methyl)phos-
phonate (48). Prepared from starting compound 28 (0.56 g; 1 mmol).
Yield: 92% (0.31 g) of white crystals, mp 125–126 ◦C. MS (ESI) m/z (%):
15

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A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
705 (35, 2M+Na+), 364 (100, M+Na+), 342 (38, M+H+). HRMS (ESI)
calcd. for C11H24O5N3NaPS (M+Na+): 364.1067, found: 364.1068;
HRMS (ESI) calcd. for C11H25O5N3PS (M+H+): 342.1247, found:
342.1248. 1H NMR (CDCl3): δ 9.75 (bs, 1H, NH), 7.22 (bs, 1H, NH), 7.12
(bs, 2H, NH2), 4.76–4.89 (m, 2H, CH-i-Pr), 3.79 (d, 2H, J(3′,P) = 8.8 Hz,
H-3′), 3.66 (m, 2H, H-2′), 3.39 (m, 2H, H-1′), 1.35 (d, 6H, Jvic = 6.3 Hz,
CH3-i-Pr), 1.33 (d, 6H, Jvic = 6.3 Hz, CH3-i-Pr). 13C NMR (CDCl3): δ
182.43 (C-2), 154.35 (C-1), 72.39 (d, J(2′,P) = 12.1 Hz, CH2-2′), 71.99
(d, J(C,P) = 6.8 Hz, CH-i-Pr), 66.23 (d, J(3′,P) = 170.3 Hz, CH2-3′),
39.54 (CH2-1′), 24.07 (d, J(C,P) = 3.8 Hz, CH3-i-Pr), 24.00 (d, J(C,P) =
5.2.26.2. (S)-(((1-(3-Carbamothioylureido)-3-hydroxypropan-2-yl)oxy)
methyl)phosphonic acid (47). Prepared from the starting compound 49
(0.61 g; 1 mmol). Yield: 0.26 g (92%) of a colorless oil. [α] ꢀ 11.7 (c
D
0.214 g/100 mL, MeOH). MS (ESI) m/z (%): 595 (9, 2Mꢀ 2H++Na+),
308 (7, Mꢀ 2H++Na+), 286 (100, Mꢀ H+). HRMS (ESI) calcd. for
C6H13O6N3PS (Mꢀ H+): 286.0268, found: 286.0267. 1H NMR (CD3OD):
δ 3.85–3.91 (m, 2H, CH2-3′), 3.65 (dd, 1H, Jgem = 11.8 Hz, J(4′a,2′) =
4.5 Hz, H-4′a), 3.59 (dd, 1H, Jgem = 11.8 Hz, J(4′b,2′) = 5.3 Hz, H-4′b),
3.55 (m, 1H, H-2′), 3.46 (dd, 1H, Jgem = 14.2 Hz, J(1′a,2′) = 4.5 Hz, H-
1′a), 3.27 (dd, 1H, Jgem = 14.2 Hz, J(1′b,2′) = 6.1 Hz, H-1′b). 13C NMR
(CD3OD): δ 183.74 (C-2), 155.89 (C-1), 82.72 (d, J(2′,P) = 11.5 Hz, CH-
2′), 66.04 (d, J(3′,P) = 164.2 Hz, CH2-3′), 62.51 (CH2-4′), 40.64 (CH2-
4.7 Hz, CH3-i-Pr). 31P {1H} NMR (CDCl3): δ 19.98. FTIR (CHCl3, cmꢀ 1
)
νmax: 3488, 3340, 3296, 3270, 3190, 2984, 2939, 2876, 1704, 1580,
1537, 1466, 1415, 1388, 1377, 1232, 1180, 1143, 1117, 1103, 1018,
1002, 891.
1′). 31P {1H} NMR (CD3OD): δ 21.13. FTIR (KBr, cmꢀ 1
) νmax: 3375, 3280,
2945, 2306, 1730, 1711, 1643, 1599, 1546, 1437, 1222, 1120, 1040,
1002, 943.
5.2.25.2. Diisopropyl
(S)-(((1-(3-carbamothioylureido)-3-(trityloxy)
5.2.27. Diisopropyl ((2-(5-amino-3-oxo-1,2,4-thiadiazol-2(3H)-yl)
ethoxy)methyl)phosphonate (50)
propan-2-yl)oxy)methyl)phosphonate (49). Prepared from the starting
compound 31 (0.83 g; 1 mmol). Yield: 0.43 g (69%) of white crystals,
mp 77–78 ◦C. [
α]D ꢀ 4.6 (c 0.240 g/100 mL, MeOH). MS (ESI) m/z (%):
0.5 M Bromine solution in ethyl acetate (2.5 mL; 12.5 mmol) was
added to a 4 ◦C cold solution of 48 (0.20 g; 0.59 mmol) in ethyl acetate
1250 (14, 2M+Na+), 636 (100, M+Na+). HRMS (ESI) calcd. for
◦
31H40O6N3NaPS (M+Na+): 636.2268, found: 636.2269. 1H NMR
(10 mL). The mixture was stirred overnight at 4 C for 16.5 h (until
C
(DMSO‑d6): δ 9.75 (d, 1H, 4J = 1.6 Hz, CSNH), 9.36 (d, 1H, Jgem = 3.3
Hz, NH2), 8.95 (d, 1H, Jgem = 3.3 Hz, NH2), 7.37–7.41 (m, 6H, H-o-Tr),
7.31–7.36 (m, 6H, H-m-Tr), 7.26 (m, 3H, H-p-Tr), 6.82 (bt, 1H, J(NH,1′)
= 5.3 Hz, CONH), 4.53–4.63 (m, 2H, CH-i-Pr), 3.81 (dd, 1H, Jgem = 13.7
Hz, J(3′a,P) = 8.7 Hz, H-3′a), 3.76 (dd, 1H, Jgem = 13.7 Hz, J(3′b,P) =
9.3 Hz, H-3′b), 3.65 (p, 1H, J(2′,1′) = J(2′,4′) = 5.1 Hz, H-2′), 3.23–3.35
(m, 2H, H-1′), 3.05 (dd, 1H, Jgem = 10.4 Hz, J(4′a,2′) = 4.6 Hz, H-4′a),
3.02 (dd, 1H, Jgem = 10.4 Hz, J(4′b,2′) = 5.4 Hz, H-4′b), 1.23 (d, 3H, Jvic
= 6.2 Hz, CH3-i-Pr), 1.22 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr), 1.20 (d, 3H,
Jvic = 6.2 Hz, CH3-i-Pr), 1.19 (d, 3H, Jvic = 6.2 Hz, CH3-i-Pr). 13C NMR
(DMSO‑d6): δ 181.51 (C-2), 154.31 (C-1), 143.77 (C-i-Tr), 128.44 (CH-o-
Tr), 128.09 (CH-m-Tr), 127.28 (CH-p-Tr), 86.32 (C-Tr), 79.17 (d, J(2′,P)
= 12.0 Hz, CH-2′), 70.42 (d, J(C,P) = 6.3 Hz, CH-i-Pr), 70.39 (d, J(C,P)
= 6.3 Hz, CH-i-Pr), 64.00 (d, J(3′,P) = 165.1 Hz, CH2-3′), 63.01 (CH2-4′),
39.48 (CH2-1′), 24.02 (d, J(C,P) = 3.6 Hz, CH3-i-Pr), 23.91 (d, J(C,P) =
4.4 Hz, CH3-i-Pr), 23.87 (d, J(C,P) = 4.4 Hz, CH3-i-Pr). 31P {1H} NMR
quantitative conversion). The solvent was removed in vacuo and the
residue was purified on a preparative TLC plate (5% MeOH/CHCl3).
Yield: 0.18 g (90%) of a colorless oil. MS (ESI) m/z (%): 362 (100,
M+Na+), 340 (M+H+). HRMS (ESI) calcd. for C11H22O5N3NaPS
(M+Na+): 362.0910, found: 362.0910. 1H NMR (DMSO‑d6): δ 8.33 (bs,
2H, NH2), 4.59 (dsep, 2H, J(H,P) = 7.7 Hz, Jvic = 6.2 Hz, CH-i-Pr), 3.76
(d, 2H, J(3′,P) = 8.2 Hz, H-3′), 3.66 (m, 2H, H-1′), 3.64 (m, 2H, H-2′),
1.24 (dd, 6H, Jvic = 6.2 Hz, CH3-i-Pr), 1.23 (dd, 6H, Jvic = 6.2 Hz, CH3-i-
Pr). 13C NMR (DMSO‑d6): δ 172.14 (C-5), 164.86 (C-3), 71.22 (d, J(2′,P)
= 11.3 Hz, CH2-2′), 70.46 (d, J(C,P) = 6.4 Hz, CH-i-Pr), 64.95 (d, J(3′,P)
= 164.1 Hz, CH2-3′), 43.16 (CH2-1′), 24.07 (d, J(C,P) = 3.6 Hz, CH3-i-
Pr), 23.95 (d, J(C,P) = 4.5 Hz, CH3-i-Pr). 31P NMR {1H} (MeOD-d4): δ
19.08. FTIR (CHCl3, cmꢀ 1
) νmax: 3464, 3398, 3274, 3126, 2983, 2877,
1668, 1630, 1608, 1544, 1388, 1377, 1237, 1179, 1143, 1103, 1000.
5.3. Biological assays
(DMSO‑d6): δ 18.99. FTIR (CHCl3, cmꢀ 1
) νmax: 3488, 3390, 3340, 3295,
5.3.1. Recombinant expression, purification, and activation of CatK
3264, 3088, 3063, 3035, 2984, 2935, 2875, 1705, 1599, 1578, 1519,
1491, 1466, 1449, 1442, 1412, 1388, 1377, 1228, 1180, 1159, 1152,
1143, 1117, 1101, 1090, 1080, 1036, 1018, 1002, 891, 707, 700, 643,
633, 617.
The synthetic gene of human CatK (GenScript) was cloned into the
expression vector pPICZ
α and expressed in the X-33 strain of the
methylotrophic yeast Pichia pastoris.39 The medium was lyophilized and
desalted over a Sephadex G25 column. The recombinant protein was
purified over a Mono S HR 5/5 column (GE Healthcare Life Sciences)
equilibrated in 50 mM sodium acetate pH 5.5 and eluted using a linear
gradient of 0–1 M NaCl. The purified CatK in the zymogen form was
concentrated to 1 mg/ml by an Amicon Ultracel-10 k ultrafiltration
device (Millipore) and activated to the mature form by autocatalytic
processing at acidic pH as described previously.40 The activation process
was monitored by a CatK activity assay and SDS-PAGE. The concentra-
tion of mature CatK was determined by active site titration with E-64
inhibitor.
5.2.26. N5-[(Phosphonomethoxy)alkyl]thiobiurets (46, 47). The general
procedure
Bromotrimethylsilane (2 mL; 15 mmol) was added to the appropriate
phosphonate ester 48 or 49 (1 mmol) in acetonitrile (30 mL) and the
mixture was left at RT overnight. The volatiles were evaporated and the
residue was co-evaporated with acetonitrile. After the addition of water,
the mixture was stirred for 1 h and evaporated (for compound 46) or
extracted with diethyl ether (for compound 47 in order to remove trityl
alcohol) and then evaporated.
5.3.2. CatK inhibition assay
5.2.26.1. ((2-(3-Carbamothioylureido)ethoxy)methyl)phosphonic
acid
Inhibition measurements were performed in a microplate format
(100-µl assay volume) at 30 ◦C. The reaction mixture contained 0.7 nM
CatK, 20 µM fluorogenic substrate Z-Gly-Pro-Arg-AMC (Bachem), 0.1 M
sodium acetate pH 5.5, 150 mM NaCl, 50 µM dithiothreitol, 0.1% PEG
6000, and 1 mM EDTA. CatK was preincubated with 0–100 µM inhibitor
(stock solution in DMSO) at 30 ◦C for 10 min followed by the addition of
substrate. The kinetics of the release of the fluorescent product were
monitored continuously for 30 min in an Infinite M200 microplate
reader (Tecan) at excitation and emission wavelengths of 360 and 465
nm, respectively. The IC50 values were determined using nonlinear
regression (GraFit software; Erithacus Software). The final concentra-
tion of DMSO did not exceed 5%. The assay concentration of
(46). Prepared from the starting compound 48 (0.34 g; 1 mmol). Yield:
0.24 g (93%) of a yellowish crystalline solid, mp 139–140 ◦C. MS (ESI)
m/z (%): 513 (25, 2Mꢀ H+), 256 (100, Mꢀ H+). HRMS (ESI) calcd. for
C5H11O5N3PS (Mꢀ H+): 256.0163, found: 256.0161. 1H NMR (CD3OD):
δ 3.76 (d, 2H, J(3′,P) = 9.0 Hz, H-3′), 3.64–3.67 (m, 2H, H-2′), 3.36–3.39
(m, 2H, H-1′). 13C NMR (CD3OD): δ 183.67 (C-2), 155.73 (C-1), 72.84 (d,
J(2′,P) = 11.9 Hz, CH2-2′), 67.08 (d, J(3′,P) = 163.7 Hz, CH2-3′), 40.16
(CH2-1′). 31P {1H} NMR (CD3OD): δ 19.57. FTIR (KBr, cmꢀ 1
) νmax: 3336,
3247, 3199, 3139, 2675, 2258, 1723, 1714, 1626, 1604, 1563, 1535,
1223, 1120, 1102.
16

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A. Pomeislova et al.
Bioorganic & Medicinal Chemistry 32 (2021) 115998
dithiothreitol was set as low as possible to maintain sufficient CatK ac-
tivity and avoid interference with 1,2,4-thiadiazoles. The measurements
were performed in triplicate.
Acknowledgements
This work was supported by the Subvention for Development of
Research Organization RVO 61388963, by the MEYS, project Czech
National Node to the European Infrastructure for Translational Medicine
EATRIS-CZ, grant No. LM2018133 and by the project ChemBioDrug
CZ.02.1.01/0.0/0.0/16_019/0000729 from the European Regional
Development Fund (OP RDE).
5.3.3. GSK-3 inhibition assay
Derivatives of 1,2,4-thiadiazole were screened for their potential
inhibition effect toward GSK3β at the concentration of 10 µM using the
GSK3β kinase assay plus ADP-Glo® assay (Promega) according to the
manufacturer’s instructions with minor modifications. Briefly, the re-
action was performed in 384-well white plates (NUNC) in a total volume
of 5 µL. The reaction contained commercial GSK3β kinase buffer with 2
ng of GSK3β kinase recombinant protein, 1 µg of a GSK3 substrate
peptide (GSM) and 10 µM of the respective inhibitors added to the re-
action using the ECHO 550 liquid handler. Following a 15 min pre-
incubation, the reaction was started by 25 µM ATP and incubated for 90
min at RT. The kinase reaction was stopped by adding 5 µL of the Kinase
Glo® reagent and incubated at RT for 40 min (ATP depletion). Lumi-
nescence was recorded 40 min after the addition of 10 µL of the kinase
detection reagent (ADP to ATP conversion) on a Cytation 3 imaging
reader (BioTek). Tideglusib and CHIR99021 were used as reference
GSK3 inhibitors, while staurosporin represented a non-specific pan-ki-
nase inhibitor (all 1 µM).
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