Developing novel classes of protein kinase CK1δ inhibitors by fusing [1,2,4]triazole with different bicyclic heteroaromatic systems

Article abstract of DOI:10.1016/j.ejmech.2021.113331

Protein kinase CK1δ expression and activity is involved in different pathological situations that include neuroinflammatory and neurodegenerative diseases. For this reason, protein kinase CK1δ has become a possible therapeutic target for these conditions. 5,6-fused bicyclic heteroaromatic systems that resemble adenine of ATP represent optimal scaffolds for the development of a new class of ATP competitive CK1δ inhibitors. In particular, a new series of [1,2,4]triazolo[1,5-c]pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]triazines was developed. Some crucial interactors have been identified, such as the presence of a free amino group able to interact with the residues of the hinge region at the 5- and 7- positions of the [1,2,4]triazolo[1,5-c]pyrimidine and [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffolds, respectively; or the presence of a 3-hydroxyphenyl or 3,5-dihydroxyphenyl moiety at the 2- position of both nuclei. Molecular modeling studies identified the key interactions involved in the inhibitor-protein recognition process that appropriately fit with the outlined structure-activity relationship. Considering the fact that the CK1 protein kinase is involved in various pathologies in particular of the central nervous system, the interest in the development of new inhibitors permeable to the blood-brain barrier represents today an important goal in the pharmaceutical field. The best potent compound of the series is the 5-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)benzen-1,3-diol (compound 51, IC₅₀ = 0.18 μM) that was predicted to have an intermediate ability to cross the membrane in our in vitro assay and represents an optimal starting point to both studies the therapeutic value of protein kinase CK1δ inhibition and to develop new more potent derivatives.

Full text of DOI:10.1016/j.ejmech.2021.113331

European Journal of Medicinal Chemistry 216 (2021) 113331
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Developing novel classes of protein kinase CK1
d
inhibitors by fusing
[1,2,4]triazole with different bicyclic heteroaromatic systems
Ilenia Grieco a, Maicol Bissaro b, Davide Benedetto Tiz a, Daniel I. Perez c,
,
Conception Perez d, Ana Martinez c e, Sara Redenti a, Elena Mariotto f,
,
Roberta Bortolozzi g, Giampietro Viola f g, Giorgio Cozza h, Giampiero Spalluto a,
,
*
Stefano Moro b, Stephanie Federico a
a
ꢀ
Dipartimento di Scienze Chimiche e Farmaceutiche, Universita degli Studi di Trieste, Via Licio Giorgieri 1, 34127, Trieste, Italy
b
ꢀ
Molecular Modeling Section (MMS), Dipartimento di Scienze del Farmaco, Universita degli Studi di Padova, via Marzolo 5, 35131, Padova, Italy
c Centro de Investigaciones Biologicas, CSIC, Ramiro de Maetzu 9, 28040, Madrid, Spain
d Instituto de Quimica Medica, CSIC, Juan de la Cierva 3, 28006, Madrid, Spain
e Centro de Investigacion Biomedica en Red en Enfermedades Neurodegenerativas (CIBERNED), Instituto Carlos III, 28031, Madrid, Spain
f
ꢀ
Dipartimento di Salute della Donna e del Bambino, Laboratorio di Oncoematologia, Universita di Padova, 35131, Padova, Italy
g Istituto di Ricerca Pediatrica (IRP), Corso Stati Uniti 4, 35128, Padova, Italy
h
ꢀ
Dipartimento di Medicina Molecolare, Universita degli Studi di Padova, Via U. Bassi 58/B, 35131, Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein kinase CK1
neuroinflammatory and neurodegenerative diseases. For this reason, protein kinase CK1
possible therapeutic target for these conditions. 5,6-fused bicyclic heteroaromatic systems that resemble
adenine of ATP represent optimal scaffolds for the development of a new class of ATP competitive CK1
d
expression and activity is involved in different pathological situations that include
Received 31 December 2020
Received in revised form
21 February 2021
Accepted 21 February 2021
Available online 2 March 2021
d
has become a
d
inhibitors. In particular, a new series of [1,2,4]triazolo[1,5-c]pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]
triazines was developed. Some crucial interactors have been identified, such as the presence of a free
amino group able to interact with the residues of the hinge region at the 5- and 7- positions of the [1,2,4]
triazolo[1,5-c]pyrimidine and [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffolds, respectively; or the presence
of a 3-hydroxyphenyl or 3,5-dihydroxyphenyl moiety at the 2- position of both nuclei. Molecular
modeling studies identified the key interactions involved in the inhibitor-protein recognition process
that appropriately fit with the outlined structure-activity relationship. Considering the fact that the CK1
protein kinase is involved in various pathologies in particular of the central nervous system, the interest
in the development of new inhibitors permeable to the blood-brain barrier represents today an
important goal in the pharmaceutical field. The best potent compound of the series is the 5-(7-amino-5-
Keywords:
Protein kinase CK1 delta
[1,2,4]triazolo[1,5-c]pyrimidine
[1,2,4]triazolo[1,5-a][1,3,5]triazine
BBB-PAMPA assay
Kinase molecular modeling
Neurodegenerative diseases
(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)benzen-1,3-diol (compound 51, IC50 ¼ 0.18
m
M)
that was predicted to have an intermediate ability to cross the membrane in our in vitro assay and
represents an optimal starting point to both studies the therapeutic value of protein kinase CK1
bition and to develop new more potent derivatives.
d
inhi-
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Protein kinase CK1d is a member of the protein kinase CK1
family of serine-threonine kinases. This family gives also the name
to the homonym group of kinases which, in humans, beyond the
other five CK1 isoforms, comprises Tau Tubulin Kinase (TTBK) and
Vaccinia Related Kinase (VRK) families [1,2]. Deregulation of CK1d is
involved in the pathogenesis of several diseases [3], including sleep
Abbreviations: CK1d, protein kinase CK1 delta; BM, binding mode; HB, hydrogen
bond; IE, interaction energy; MD, molecular dynamics; TM, transmembrane; TP,
[1,2,4]triazolo[1,5-c]pyrimidine; TT, [1,2,4]triazolo[1,5-a][1,3,5]triazine; vdW, van
der Waals.
* Corresponding author.
E-mail address: sfederico@units.it (S. Federico).
disorders [4,5], cancer [6e10] and infections [11,12]. In particular,
https://doi.org/10.1016/j.ejmech.2021.113331
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
CK1
d
is nowadays considered an appealing target in different
was that in the circadian rhythm [41e43]. Pfizer developed also the
neurodegenerative diseases [13e15], i.e. Alzheimer’s disease (AD)
[16e19], amyotrophic lateral sclerosis (ALS) [20e23], and Parkin-
son’s disease [24e26] where it co-localizes with mis-
phosphorylated proteins that are typical hallmarks of these dis-
eases (tau protein; TDP-43, transactive response DNA-binding
brain penetrant PF-5006739 (II, IC50 CK1
CK1ε ¼ 17 nM) that was found to attenuate the opioid-seeking
behavior in rodents [34]. Several CK1 ATP competitive inhibitors
have an adenine scaffold [9,35,36,44,45]. Among them, (R)-DRF053
(III, IC50 ¼ 14 nM) [35] SR-2890 (IV, IC50 ¼ 4 nM) and SR-3029 (V,
IC50 ¼ 44 nM) [36] display potent inhibitor properties against ki-
nase. SR-3029 (V) demonstrated to inhibit growth of specific breast
d
¼
3.9 nM, IC50
d
protein 43 KDa; and
a-synuclein, respectively). Consequently,
different CK1 inhibitors were developed over the years and some
d
of them are currently studied at the preclinical phase [27]. Firstly
reported inhibitors showed low potency and non-optimal phar-
macological properties, i.e. CK1-7 [28], D4476 [29], IC261 [30],
while now different potent inhibitors are available but most of
cancer cell lines where CK1
[45,46]. Finally, the benzothiazole IGS-2.7 (VI, IC50 ¼ 23 nM) [20]
developed by Martinez’s group was used to evaluate CK1 inhibi-
tion as strategy for the treatment of ALS and frontotemporal de-
mentia by targeting TDP-43 phosphorylation [21,22].
Over the years, we have developed [1,2,4]triazolo[1,5-c]pyrimi-
dines (TP, VII) [47,48] and [1,2,4]triazolo[1,5-a][1,3,5]triazines (TT,
VIII) [49e52] (Fig. 2) as adenosine receptor (AR) antagonists, Due to
the obvious similarity between adenosine (the endogenous agonist
of AR) and ATP (one of the substrates of kinases), herein reported a
series of TP (compounds 1e36) and TT (compounds 37e57) with
d resulted overexpressed or amplified
d
them lack of selectivity against CK1
of 6 different CK1 isoforms in humans (e.g. CK1
CK1 3, CK1 and CK1ε) is the biggest challenge in development of
d
isoform. In fact, the existence
a
, CK1 1, CK1 2,
g
g
g
d
inhibitors selective towards only the desired isoform, because they
are highly conserved especially in their catalytic domain. In addi-
tion, isoforms are also present as different splice variants that
mediate different activities but are structurally very similar each
other [31]. Finally, CK1 activities were regulated also by their
different localization, even at subcellular level [32]. For all these
the aim of obtaining new ATP-competitive inhibitors for CK1
kinase.
d
reasons, development of new CK1
challenge. The more interesting CK1
d
inhibitors is still an interesting
pharmacological tools are PF-
d
2. Chemistry
670462 (I) [33], PF-5006739 (II) [34], (R)-DRF053 (III) [35], SR-2890
(IV), SR-3029 (V) [36] and IGS-2.7 (VI) [20] (Fig. 1). In particular, PF-
670462 (I), a 2-aminopyrimidine, was intensively used as phar-
Synthesis
of
2,5,8-trisubstituted
[1,2,4]triazolo[1,5-c]
pyrimidines.
macological tool for CK1d/ε inhibition (IC50s of 14 and 7.7 nM,
All the 2,5,8-trisubstituted TP derivatives (1e29) have been
synthesized following the strategy of Kyowa Hakko Kogyo Co., [53],
adjusting it to our synthetic purposes as summarized below
(Schemes 1e3).
respectively [33]). Noteworthy, it demonstrated to attenuate
fibrosis in idiopathic pulmonary fibrosis [37] and epithelial-to-
mesenchymal transition in chronic obstructive pulmonary disease
[38], both via inhibition of tumor growth factor
b
. The same com-
implication
Condensation between S-methylisothiourea (58) and diethyl
ethoxymethylene malonate (59) in basic conditions led to the py-
rimidine sodium salt 60, which after treatment with phosphorous
oxychloride under reflux afforded chloro derivative 61 that was
immediately coupled with the required hydrazides (62e76) in the
presence of DBU, to obtain derivatives 77e91. The intramolecular
cyclization that led to the [1,2,4]triazolo[1,5-c]pyrimidine system
(92e106) was conducted using the trimethylsilyl ester of poly-
phosphoric acid as a dehydrating agent [54]. For those compounds
presenting at the 2- position of the triazolo-pyrimidine scaffold
polar groups susceptible to secondary reactions (e.g. OH for com-
pounds 17e21 and NH2 for compound 22), a protective group was
required in the initial steps of the synthetic pathway. So we decided
to protect the hydroxyl groups with a benzyl group due to the mild
conditions involved in its removal by catalytic hydrogenolysis
(Scheme 2, reaction II). The same conditions were applied also in
the palladium-catalyzed reduction of the nitro group (9) used to
protect the amino group in compound 22 (Scheme 2, reaction III).
Nucleophilic substitutions of methylthio group with primary
amines (obtaining compounds 11e14,110) were conducted heating
the mixture in a sealed tube (Scheme 1). To obtain a free amino
pound was used by different groups to investigate CK1
d
in AD mouse models [16,39] and cancer (i.e. bladder cancer [9] and
chronic lymphocytic leukemia [40]) even if the most studied effect
Fig. 2. [1,2,4]Triazolo[1,5-c]pyrimidine (VII) and [1,2,4]triazolo[1,5-a][1,3,5]triazine
Fig. 1. Representative CK1
d
inhibitors.
(VIII) nuclei developed in this work to find new CK1d inhibitors.
2

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Scheme 1. Synthesis of the 2,5,8-trisubstituted [1,2,4]triazolo[1,5-c]pyrimidine nucleus and of final compounds 1e14. Reagents and conditions. a: NaOH, H2O, ethanol, rt, 24 h; b:
POCl3, rfx, 5 h; c: DBU, THF, rt, 12 h; d: P2O5, HMDSO, xylene, from rt to 90 ꢀC, 2.5 h, rfx, 12 h; e: ammonia 7 N in methanol, ethanol, 70 ꢀC, sealed tube, 3e12 h; f: R2-NH2, ethanol,
90 ꢀC, sealed tube, 3e12 h.
Scheme 2. Synthesis of final compounds 15e22. Reagents and conditions. I) Deprotection of amino group at the 5-position, a: CF3COOH, trifluoromethanesulfonic acid, anisole, rt,
overnight; II) Deprotection of hydroxy groups and reduction of nitro group, b: Pd/C, ammonium formate, methanol, rfx, 3e12 h.
3

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Scheme 3. Synthesis of final compounds 23e31. Reagents and conditions. a: BBr3 1 M in DCM, DCM, from ꢁ78 ꢀC to rt, 12 h; b: SOCl2, DMF, chloroform, rfx, 5 h; c: ethanol, rt, 12 h; d:
SOCl2, ethylamine, pyridine, THF, from 0 ꢀC to rt, 12 h; e: NH2(CH2)2NHBOC, Et3N, THF, rt, 12 h; f: CF3COOH 10% in DCM, rt, 2 h.
group at the 5-position, the 2-phenyl (15) and 2-(4-fluoro)phenyl
(16) derivatives were obtained following the two-step strategy
reported in the literature [47,53] which consists of the synthesis of
the 3,4-dimethoxybenzylamino derivatives (11,12) and the subse-
quent deprotection with trifluoromethanesulfonic acid, anisole,
and trifluoroacetic acid, leading to the desired derivatives 15 an 16
(Scheme 2, reaction I). This strategy allowed to avoid the possible
ammonolysis of the ethyl ester [55], that could occur proceeding
with a direct replacement of methylthio group with ammonia.
Since the overall yields were not high, we tried to directly substi-
tute the 5-methylthio derivatives 92e101 and 104e106 with
ammonia using mild conditions (temperature not above 70 ꢀC),
obtaining the desired 5-amino derivatives 1e10,107e109 in a single
step with acceptable yields most of the time (Scheme 1).
The final 5-benzylamino derivatives 24e25 were obtained from
the 3-methoxyphenyl derivatives 13,14 as an alternative to the
benzyl as hydroxyl protecting group that could have led to the
undesired loss of the benzyl and 4-phenylbenzyl groups present at
the 5-position during the deprotection step. Demethylation was
achieved in 3e12 h using boron tribromide (BBr3), the classical but
harsh reagent used for this purpose. Unfortunately, the reactivity of
BBr3 led also to the hydrolysis of the ethyl ester at the 8-position,
even using the minimum excess of reagent (1.1 equivalents) and
maintaining the temperature below the 0 ꢀC. So we decided to force
the reaction using an excess of BBr3 that ensured the complete
consumption of starting material. Therefore, compounds 24e25
were obtained after re-esterification of 112e113, via acyl chloride
formation (Scheme 3). The same strategy was used to obtain 8-
amido derivatives 26e29, from compounds 7 and 10, hydroxyl
groups deprotection and ester hydrolysis were obtained in one step
leading to compounds 23 and 111. The reaction of the carboxylic
acids with thionyl chloride and the desired amine afforded amido
derivatives 26e29. Finally, the tert-butyloxycarbonyl (BOC) group
was removed by classical trifluoroacetic acid deprotection leading
to the final compounds 30e31.
Synthesis
pyrimidines.
of
2,5,7-trisubstituted
[1,2,4]triazolo[1,5-c]
2,5,7-trisubstituted [1,2,4]triazolo[1,5-c]pyrimidines were ob-
tained following the approach used by Schering Co. (Scheme 4) [56].
The commercially available 2-amino-4,6-dihydroxypyrimidine 114
was converted to the dichloride derivative 115 with phosphorus
oxychloride and triethylamine in refluxing acetonitrile, which was,
in turn, reacted with desired hydrazide (68,71) and dia-
zabicycloundecene (DBU) to obtain N’-(2-amino-6-chloropyrimidin-
4-yl)-3-hydrazide derivatives 116e117. These compounds were then
cyclized in dehydrating conditions by N,O-bis(trimethylsilyl)acet-
amide (BSA) leading to the 5-amino-7-chloro-[1,2,4]triazolo[1,5-c]-
pyrimidines 118e119. [57] Nucleophilic substitution of chlorine at
the 7-position of the TP 118 with benzylamine in ethanol in the
presence of base yielded derivative 32. On the contrary, compound
119 was not soluble in ethanol and DMF was used as co-solvent, but
instead, to obtain the desired compound 33, the reaction afforded
dimethylamino derivative 36. Finally, compound 33 was achieved
performing the reaction in butanol. All compounds (32,33,36) were
then demethylated with boron tribromide to afford derivatives 34,35
and 37.
Synthesis of 2,5,7-trisubstituted [1,2,4]triazolo[1,5-a][1,3,5]
triazines.
In addition to 2,5,7-trisubstituted-[1,2,4]triazolo[1,5-c]pyrimi-
dines also a series of [1,2,4]triazolo[1,5-a][1,3,5]triazines has been
developed bearing the same or similar substitutions at equivalent
positions. The synthesis started by reacting the commercially
available 2,4,6-trichloro[1,3,5]triazine (120) with phenol under
reflux for 5 h. The 2,4,6-triphenoxy[1,3,5]triazine 121 was reacted
with the required arylhydrazides (65,68,71,72,122) and DBU to give
4,6-diphenoxy-[1,3,5]triazin-2-yl-hydrazides 123e127, that suc-
cessively underwent dehydrative cyclization with phosphorus
pentoxide and HMDSO in xylene at reflux, yielding the 2-
substituted
5,7-diphenoxy[1,2,4]triazolo[1,5-a][1,3,5]triazines
128e132 (Scheme 5) [54,58]. Addition of methanolic ammonia led
4

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Scheme 4. Synthesis of the 2,5,7-trisubstituted [1,2,4]triazolo[1,5-c]pyrimidine nucleus and final compounds 32e37. Reagents and conditions. a: POCl3, Et3N, acetonitrile, rfx, 1 h; b:
hydrazide (68 or 71), DBU, THF, rt, 12 h; c: BSA, rfx, 12 h; d: for 34 benzylamine, K2CO3, ethanol, 95 ꢀC, sealed tube, 4 days; for 35 benzylamine, K2CO3, butanol, 100 ꢀC, sealed tube,
36 h; e: BBr3 1 M in DCM, DCM, from ꢁ78 ꢀC to rt, 12h; f: benzylamine, K2CO3, ethanol, DMF, 100 ꢀC, sealed tube, 3 days.
Scheme 5. Synthesis of the 2,5,7-trisubstituted [1,2,4]triazolo[1,5-a][1,3,5]triazine nucleus and final compounds 38e57. Reagents and conditions. a: phenol, rfx, 5 h; b: DBU, THF, rt,
12 h; c: P2O5, HMDSO, xylene, from rt to 90 ꢀC, 2.5 h, rfx, 2e4 h; d: ammonia 7 N in methanol, methanol, rt, 2 h; e: Method A. desired amine, ethanol, 95e100 ꢀC, sealed tube,
24e72 h. Method B. butanol, MW, 170 ꢀC, 100W, 2 h (compounds 42e45); f: BBr3 1 M in DCM, DCM, from ꢁ78 ꢀC to rt, 12 h.
to the corresponding 7-amino derivatives 133e137, while nucleo-
philic substitution at the 5-position with benzylamines occurred
after heating the mixture in ethanol in a sealed tube at 95e100 ꢀ
for days (compounds 38e47) [52]. During the synthesis of the 3-
C
5

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
methoxy derivative 39, also the N5,N7-bisbenzylamino derivative
40 was isolated and characterized. Finally, removal of the methoxy
group with an excess of boron tribromide afforded the hydrox-
yphenyl derivatives 48e57.
performed in duplicate; cdata represent the % of inhibition of kinase
activity expressed as a mean SD of 2 independent experiments
performed in duplicate at 40 m
M concentration; dinhibition curve
could not be obtained for this compound, thus it was a false posi-
tive. n.d. not determined.
A simple phenyl ring, compound 15, led to an IC50 of 8.67
Substitution with a 4-pyridyl ring led to a detrimental effect in
M). Different groups
were then introduced at the para or meta positions of the phenyl
ring. The most interesting results were obtained with 3-
mM.
3. Structure-activity relationship (SAR) studies
terms of inhibitory activity (5, IC50 ¼ 20.19
m
Investigations at the 2 position of the [1,2,4]triazolo[1,5-c]
pyrimidine nucleus [1,2,4]Triazolo[1,5-c]pyrimidine nucleus is an
adenine-like nucleus that represents a good starting point to
develop new ATP-competitive CK1d inhibitors. Thus, different ethyl
5-amino-[1,2,4]triazolo[1,5-c]pyrimidines-8-carboxylates
(1e10,15e20,22) with substituents at the 2 positions of different
size and nature were synthetized and tested at a fixed dose of
40 mM on truncated CK1d (aa 1e294) using a luminescent kinase
assay (Table 1) that uses luciferase to give a luminescence signal
proportional to the unreacted amount of ATP in the enzymatic re-
action. Because kinase was truncated at residue 294, all CK1
aminophenyl derivative 22 (IC50 ¼ 16.59
m
M) and especially with
3-hydroxyphenyl compound 18 which exhibited an IC50 of 2.06
mM.
These results provided the first information about the requirement
of a polar H-bond donor group with a specific orientation to gain an
affinity towards the kinase. Hydroxyl group at the para position led
to a compound with a residual kinase activity of only 39.3%.
Focusing our attention on the hydroxyl group, disubstituted
derivatives were explored: the meta,para-disubstituted derivative
20 resulted less active than the meta-derivative 18 (20,
d
autophosphorylation sites are not present in this protein, thus only
the peptide substrate could be phosphorylated. A threshold of
IC50 ¼ 4.75
m
M vs. 18, IC50 ¼ 2.06
mM), while more effective than the
para- (17) and the not substituted phenyl derivative (15). Notably,
with the 3,5-dihydroxy-substituted derivative 19, we reached the
40
mM allows to include also not so potent inhibitors that is
essential at this stage where no SAR is available. IC50s were deter-
mined only for compounds that displayed an inhibition of kinase
activity more than 50% at that concentration.
As reported in Table 1, all compounds can inhibit CK1
extent. A large aromatic moiety such indole seems to not give
favorable interactions with kinase (22.4% inhib. at 40 M) but
submicromolar range of inhibiting concentrations (IC50 ¼ 0.48
with a 4-fold increase in inhibition potency if compared with the
mono-meta- substituted derivative 18 (IC50 ¼ 2.06 M).
mM),
m
d to some
To rationalize the molecular basis responsible for such a
different inhibitory profile shown by this first series of 2-
substituted TP derivates, molecular docking calculations were
performed, providing an atomistic description of the ligand
possible bound states. In detail, as accurately described in the
Experimental Section of the manuscript, for each docking-
predicted binding pose, the ligand-protein electrostatic and hy-
drophobic contributions to binding were computed and graphically
represented in the form of heatmaps. Two colorimetric scales were
exploited to quantitatively represent the magnitude of these two
different interactions (i.e. IEele and IEhyd), computed between the
kinase binding site residues (x-axis) and each of the TP derivates
taken into consideration (y-axis). As reported in Fig. 3-A, com-
m
smaller moieties, both aromatic and aliphatic, displayed higher
kinase activity inhibition.
aParallel artificial membrane permeability assay-blood brain
barrier (PAMPA- BBB), central nervous system (CNS), CNS þ means
that compound was predicted able to cross the BBB by passive
permeation, CNS- means that compound was predicted unable to
cross the BBB by passive permeation, CNS means that perme-
ability value was lower than limit for CNSþ and upper than limit for
CNS-; bdata represent the mean SD of 3 independent experiments
pounds able to significantly inhibit the enzymatic activity of CK1
d
Table 1
are all characterized by the presence of strong electrostatic in-
teractions with the residues belonging to the kinase hinge region
(E83, L84, and L85) and with the two negative charged amino acids
located in the inner portion of the catalytic cleft (E52 and D149).
The aliphatic residues defining the ceil and the floor of the binding
site (respectively I15, I23 and L135, I148) mediate instead the
greatest hydrophobic stabilizing contacts. For the most potent de-
rivative of this series, specifically, compound 19, the docking pre-
dicted binding mode was reported in Fig. 3-B, highlighting how the
TP scaffold is tightly anchored to the backbone of the kinase hinge
region by a bidentate hydrogen bond pattern, closely mimicking
the interactivity of the physiological substrate ATP. The 3,5-
dihydroxyl functional groups introduced on the phenyl ring at the
2-position mediate in turn a double hydrogen bond interaction,
respectively with residue E52 and D149.
Inhibitory activity of 2-substituted TP (1e10,15e20,22) towards CK1d.
Cmpd
R1
PAMPA
BBBa
IC50 mMb (% inhib. at 40 M)c
m
1
2
3
15
16
5
6
4
-CH3
-cC5H9
-2-indolyl
-Ph
-(4-F)Ph
CNS-
CNSþ
CNSþ
CNSþ
n.d.
CNS
n.d.
CNS
CNS
15.17 5.70 (66.6% 1.1)
12.32 5.17 (76.8% 6.8)
n.d. (22.4% 3.5)
8.67 4.73 (62.2% 9.2)
n.d. (44.7% 12.3)
To better understand the ligands recognition mechanism,
however, the pivotal role played by solvent molecules must be
taken into consideration and integrated into traditional in-silico
tools, for example molecular docking protocols, which otherwise
would poorly treat this aspect. For this reason, an all-atoms mo-
lecular dynamics (MD) simulation has been collected starting from
-4-pyr
20.19 5.57 (59.7% 6.6)
n.d. (39.7% 7.4)
-(4-OBn)Ph
-(4-OCH3)Ph
-(4-OH)Ph
-(3-OCH3)Ph
-(3-F)Ph
-(3-NO2)Ph
-(3-NH2)Ph
-(3-OH)Ph
-(3,4-(OH)2)Ph
-(3,5-(OCH3)2)Ph
-(3,5-(OH)2)Ph
n.d.d (50.3% 9.35)
17
7
8
n.d. (39.3% 19.1)
n.d.
25.00 5.04 (52.0% 9.2)
15.24 5.00 (53.7% 4.4)
n.d. (21.6% 10.3)
16.59 5.03 (66.8% 12.2)
2.06 0.28 (88.9% 0.3)
4.75 0.35 (98.0% 0.3)
n.d. (18.8% 15.1)
CNSþ
9
n.d.
22
18
20
10
19
CNS-
CNS-
CNS-
n.d.
the unliganded form of CK1d protein kinase (PDB ID: 5IH4) and it
has been subsequently analyzed with AquaMMapS, an in-house
developed algorithm able to characterize the hydrodynamic
behavior of water molecules within the target binding site. Aqua-
MMapS results, graphically reported on the rounded box present in
CNS
0.48 0.20 (97.8% 5.3)
6

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Fig. 3. Panel A. Interaction Energy (IE) fingerprints shown as heat maps reporting the electrostatic (IEele) and hydrophobic (IEhyd) interaction between each 2-substituted TP
derivate (y-axis) and each CK1 binding site residue (x-axis). The intensity of the electrostatic interactions is rendered by a colorimetric scale going from blue to red (for negative to
positive values) while for the hydrophobic interactions a scale going from white to dark green (for low to high values) is exploited. Panel B. Representation of the docking-predicted
structure of the complex between protein kinase CK1 (grey) and compound 19 (orange). Hydrogen bond interactions are depicted as cyan dotted lines. The rounded box contains a
d
d
zoomed view within the inner portion of the kinase binding site, showing the stable hydration sites predicted using AquaMMapS analysis. The solvent stationary behavior with each
of these regions is rendered by a colorimetric scale going from blue to red (for high to low % ORMSF values).
Fig. 3-B, have highlighted the presence in the inner portion of CK1
d
aminoethyl 30e31) to explore if stabilizing interactions could be
reached in that part of the kinase pocket (Table 2). Also the car-
boxylic acid 23 (R1 ¼ -(3OH)Ph) has been assayed but resulted less
catalytic pocket of two important hydration sites populated by
stationary solvent molecules, respectively W1 (% ORMSF ¼ 74) and
W2 (% ORMSF ¼ 21). Intriguingly, the two polar phenolic function-
alities introduced in the TP derivative 19 perfectly occupy regions
W1 and W2, allowing us to hypothesize how the displacement of
stationary water molecules could contribute, along with the tight
network of hydrogen bond interactions described above, to the
submicromolar potency of the compound.
active than the corresponding ethyl ester (23, IC50 ¼ 26.75
mM vs.
18, IC50 ¼ 2.06
led to improved inhibition potencies of both ethylamido derivative
26 (IC50 0.32 M) and aminethylamido derivative 30
(IC50 ¼ 1.26 M), while BOC protected compound 28 did not affect
mM). Instead, the introduction of an amido moiety
¼
m
m
enzyme activity at low concentrations. Interestingly, a different
behavior was observed for 3,5-dihydroxyphenyl derivatives
(27,29,31): aminethylamido derivative was more potent than the
Investigations at the 8 position of the [1,2,4]triazolo[1,5-c]py-
rimidine nucleus.
Exploration at the 8-position was performed maintaining the
best substitutions at the 2-position of the TP scaffold highlighted in
the previous series, that is the 3-hydroxyphenyl and 3,5-
dihydroxyphenyl moieties.
The ethyl ester of compounds 7 and 10 was hydrolyzed to the
corresponding carboxylic acid and then three different amides
were synthesized (ethyl 26e27, N-BOC-aminoethyl 28e29,
corresponding ethylamido compounds (27, IC50 ¼ 0.99
m
M vs. 31,
M) but showed a similar potency to the ester 19
M). Notably, an IC50 of 5.32 M was determined for
IC50 ¼ 0.21
(IC50 ¼ 0.48
m
m
m
BOC protected compound 29, denoting that very different in-
teractions occurred when this substitution is coupled with 3,5-
dihydroxyphenyl moiety at the 2-position with respect to a single
meta hydroxyl group (28, % inhib. at 40
mM ¼ 16.8).
Table 2
Inhibitory activity of 8-substituted TP (23,26e31) towards CK1d.
Cmpd
R1
R2
PAMPABBBa
IC50 m m
Mb (% inhib. at 40 M)c
23
26
27
28
29
30
31
-(3-OH)Ph
-(3-OH)Ph
-(3,5-(OH)2)Ph
-(3-OH)Ph
-(3,5-(OH)2)Ph
-(3-OH)Ph
-COOH
-CONHC2H5
-CONHC2H5
-CONH(CH5)2NHBOC
-CONH(CH5)2NHBOC
-CONH(CH5)2NH2
-CONH(CH5)2NH2
CNS-
CNS
CNS-
n.d.
CNS-
CNSþ
CNS-
26.75 6.03 (75.8% 1.8)
0.32 0.10 (90.4% 4.6)
0.99 0.12 (86.1% 4.7)
n.d. (16.8% 25.9)
5.32 1.02 (84.2% 0.4)
1.26 0.19 (93.0% 4.6)
0.21 0.09 (93.6% 11.5)
-(3,5-(OH)2)Ph
aCNS þ means that compound was predicted able to cross the BBB by passive permeation, CNS- means that compound was predicted unable to cross the BBB by passive
permeation, CNS means that permeability value was lower than limit for CNSþ and upper than limit for CNS-; bdata represent the mean SD of 3 independent experiments
performed in duplicate; cdata represent the % of inhibition of kinase activity expressed as a mean SD of 2 independent experiments performed in duplicate at 40
concentration. n.d.: not determined.
mM
7

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Fig. 4. Panel A. Interaction Energy (IE) fingerprints shown as heat maps reporting the electrostatic (IEele) and hydrophobic (IEhyd) interaction between each 8-substituted TP
derivate (y-axis) and each CK1 binding site residue (x-axis). The intensity of the electrostatic interactions is rendered by a colorimetric scale going from blue to red (for negative to
positive values) while for the hydrophobic interactions a scale going from white to dark green (for low to high values) is exploited. Panel B. Representation of the docking-predicted
structure of the complex between protein kinase CK1 (grey) and compound 31 (orange). Hydrogen bond interactions are depicted as cyan dotted lines. The rounded box contains a
d
d
zoomed view within the vestibular region of the kinase binding site, showing the stable hydration sites predicted using AquaMMapS analysis. The solvent stationary behavior with
each of these regions is rendered by a colorimetric scale going from blue to red (for high to low % ORMSF values).
In-silico studies have confirmed how modification at the 8-
position of the TP scaffold only marginally affects the binding
modes of the analyzed derivatives. As shown on the IE maps re-
ported in Fig. 4-A, the main electrostatic (IEele) and hydrophobic
(IEhyd) interactions with the protein kinase binding site residues, as
discussed in the previous paragraph, are all conserved. The reason
for this lies in the fact that the substituents in position 8 protrude
towards the external region of the catalytic site, partially exposed to
the solvent, and therefore they do not alter the crucial interactivity
with the kinase hinge region. The predicted binding mode for
compound 31, the most potent derivative of this series, has been
reported in Fig. 4-B, highlighting a salt bridge interaction taking
place between the positively charged aminoethylamido moiety
introduced at the 8-position of the TP scaffold and the negative
the proximity to the kinase hinge region, respectively W3 (%
ORMSF ¼ 57), W4 (% ORMSF ¼ 18), W5 (% ORMSF ¼ 26) and W6 (%
ORMSF ¼ 18). The polar free amino group at the 5-position of the TP
scaffold, similarly to what happens with the physiological substrate
ATP, perfectly occupies region W3, the one characterized by the
more stationary water behavior. Moreover, the polar atoms
belonging to the amide functionality inserted at the 8-position
respectively perturb the other two regions W5 and W6, although
it is reasonable to hypothesize how the contribution to binding
brought by their displacement could be less impacting, considering
the greater solvent turbulence experienced within these spots.
Investigations at the 5 position of the [1,2,4]triazolo[1,5-c]py-
rimidine nucleus.
To assess if a substitution at the 5-position of the TP scaffold is
tolerated, the free amino group was substituted with methyl (21),
benzyl (13,24), and 4-phenyl-benzyl groups (14,25), while veratryl
compounds 11e12 are intermediates for the synthesis of com-
pounds 15e16 that were purified and assayed. From what can be
seen from Table 3, the substitution at the 5-position led to inactive
charged residue D91, located in the vestibular region of CK1
d
binding site. The role of solvent molecules during binding was
taken into consideration also in this case and, as reported on the
rounded box present in Fig. 4-B, four regions populated by sta-
tionary water molecules were identified by AquaMMapS analysis in
compounds at 40 mM, even when one of the best substituents is
present at the 2-position (e.g. R1 ¼ -(3-OH)Ph) such as in com-
pounds 21, 24e25.
Table 3
Molecular docking calculations were then exploited to assess
the impact of modification at the 5-position of the TP scaffold. As
noticeable in Fig. 5-A, the IE fingerprint depicts a completely dis-
rupted electrostatic (IEele) and hydrophobic (IEhyd) interactions
Inhibitory activity of 5-substituted TP (11e14,21,24e25) towards CK1d..
pattern with the residues composing CK1d binding site. The reason
for this is observable in Fig. 4-B, showing how even the smallest
methyl substituent, present in derivative 21, prevents the crucial
hydrogen bond interactions with the hinge region residues E83 and
L85. Moreover, modification at this specific position impairs the
ability of the compound to effectively displace stationary water
molecules populating regions W3, W5, and W6.
Cmpd
R1
R2
% inhib. at 40 m
Ma
Investigations at the 7 position of the [1,2,4]triazolo[1,5-c]py-
rimidine nucleus.
11
12
13
14
21
24
25
-Ph
-(4-F)Ph
-CH2(3,4-(OCH3)2)Ph
-CH2(3,4-(OCH3)2)Ph
-Bn
-CH2(4-Ph)Ph
-CH3
35.7% 9.3
11.3% 8.1
10.9% 2.3
17.5% 19.7
7.1% 7.1
-(3-OCH3)Ph
-(3-OCH3)Ph
-(3-OH)Ph
-(3-OH)Ph
-(3-OH)Ph
Another opportunity with the [1,2,4]triazolo[1,5-c]pyrimidine
scaffold is to investigate the 7-position, thus, few compounds have
been synthesized bearing the free amino group at the 5-position
and the meta or 3,5-di- hydroxyl substitutions on the phenyl ring
at the 2-position. At the 7-position benzylamino or dimethylamino
moieties were introduced (34e35,37). Again, also the protected
-Bn
-CH2(4-Ph)Ph
1.2% 3.5
9.0% 11.6
a
Data represent the % of inhibition of kinase activity expressed as a mean SD of
M concentration.
2 independent experiments performed in duplicate at 40
m
8

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Fig. 5. Panel A. Interaction Energy (IE) fingerprints shown as heat maps reporting the electrostatic (IEele) and hydrophobic (IEhyd) interaction between each 8-substituted TP
derivate (y-axis) and each CK1 binding site residue (x-axis). The intensity of the electrostatic interactions is rendered by a colorimetric scale going from blue to red (for negative to
positive values) while for the hydrophobic interactions a scale going from white to dark green (for low to high values) is exploited. Panel B. Representation of the docking-predicted
structure of the complex between protein kinase CK1 (grey) and compound 21 (orange). Hydrogen bond interactions are depicted as cyan dotted lines. The rounded box contains a
d
d
zoomed view within the vestibular region of the kinase binding site, showing the stable hydration sites predicted using AquaMMapS analysis. The solvent stationary behavior with
each of these regions is rendered by a colorimetric scale going from blue to red (for high to low % ORMSF values).
Table 4
is easier to develop 7-substituted TP (5 synthetic steps) than 8-
substituted TP (7 synthetic steps).
Inhibitory activity of 7-substituted TP (32e37) towards CK1d..
In-silico investigations, as reported in Fig. 6-A, confirmed how
only benzylamino functionality is tolerated at the 7-position of the
TP scaffold, while dimethylamino derivates deeply perturb the
previously discussed IE fingerprint, especially as regards the IEele
component. Also, in this case, compounds interactivity with CK1
d
hinge region residues has proved to be an essential pharmaco-
phoric feature. As it is possible to appreciate in Fig. 6-B, by the
docking-predicted binding mode for derivative 35, the mono-
substituted amino group at the 7 position allows enriching the
hinge region recognition motif of a further hydrogen bond inter-
action, mediated with the residue L85 carbonyl group. On the
contrary, a di-substituted amino group at the same position not
only prevents this specific interaction but also disrupts the ca-
nonical hinge region hydrogen bond network, due to a steric
repulsion mediated by the dimethylamino group. As regards
AquaMMapS analysis, the rounded box depicted in Fig. 6-B high-
lights a different pattern of stationary water molecules displace-
ment for this series, if compared to the previously discussed 8-
substituted TP derivates. In particular, the amino moiety intro-
duced at the 7-position closely occupy the W4 region, which is
located in proximity to the key residue L85, while W5, and W6
stationary solvent spots are completely unperturbed.
Cmpd R1
R2
PAMPA IC50
BBBa
m m
Mb (% inhib. at 40 M)c
32
33
36
34
35
37
-(3-OCH3)Ph
-(3,5-(OCH3)2)Ph -NHBn
-(3,5-(OCH3)2)Ph -N(CH3)2 n.d.
-(3-OH)Ph
-(3,5-(OH)2)Ph
-(3,5-(OH)2)Ph
-NHBn
n.d.
n.d.
n.d. (ꢁ12.7% 4.5)
n.d. (19.4% 15.5)
n.d. (33.5% 9.9)
-NHBn
-NHBn
-N(CH3)2 n.d.
CNSþ
0.30 0.07 (75.2% 7.6)
0.74 0.08 (80.2% 5.6)
n.d. (ꢁ22.1% 2.5)
CNS
a
CNS þ means that compound was predicted able to cross the BBB by passive
permeation, CNS- means that compound was predicted unable to cross the BBB by
passive permeation, CNS means that permeability value was lower than limit for
CNSþ and upper than limit for CNS-.
b
Data represent the mean
SD of 3 independent experiments performed in
duplicate.
c
Data represent the % of inhibition of kinase activity expressed as a mean SD of
2 independent experiments performed in duplicate at 40
not determined.
mM concentration. n.d.:
Investigations on the [1,2,4]triazolo[1,5-a][1,3,5]triazine
nucleus.
Finally, due to the good results obtained with the 7-substituted
TP, we decided to explore another scaffold, which bears an addi-
tional nitrogen atom at the 8 position of TP, leading to the [1,2,4]
triazolo[1,5-a][1,3,5]triazine nucleus. There is good expertise on the
chemistry of this nucleus in our group [49e52,59], thus we decided
to perform investigations at the 5-position of the TT ring that is
equivalent to the 7-position in TP.
Table 5 shows that the simple phenyl ring at the 2-position of
the TT scaffold was not sufficient to confer potency against CK1d. As
we can observe, the presence of a hydroxyl group on the phenyl
ring at the 2-position has been confirmed to confer affinity to the
kinase (compounds 49,51e57). In particular, again we can see in the
5-benzyl series that the best results were obtained with the meta-
methoxy derivatives (32e33,36) were tested for their ability to
inhibit CK1
As expected (Table 4), compounds with masked hydroxyl groups
at the 2-position (32e33,36) were inactive at a concentration of
d.
40
droxyl groups are present, compounds displayed a submicromolar
potency towards CK1 , and monosubstitution on the phenyl ring
seems to be favorable with respect to 3,5-dihydroxy substitution
M). When a dimethylamino
mM (% inhib. at 40 mM 33.5e12.7). Interestingly, when free hy-
d
(34, IC50 ¼ 0.30
m
M vs. 35, IC50 ¼ 0.74
m
moiety, instead of a benzylamino moiety, was introduced at the 7-
position, the compound remains inactive also when free hydroxyl
groups were present at the phenyl ring at the 2-position (37, %
inhib. at 40
m
M ¼ ꢁ22.1). These results revealed that 7-position
(49, IC50
IC50 ¼ 0.18
¼
0.46
mM) and 3,5-disubstituted derivatives (51,
gives a valuable opportunity to develop new compounds in order
to draw SAR for this series. In fact, from a synthetic point of view, it
mM), probably due to the formation of important
9

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Fig. 6. Panel A. Interaction Energy (IE) fingerprints shown as heat maps reporting the electrostatic (IEele) and hydrophobic (IEhyd) interaction between each 7-substituted TP
derivate (y-axis) and each CK1 binding site residue (x-axis). The intensity of the electrostatic interactions is rendered by a colorimetric scale going from blue to red (for negative to
positive values) while for the hydrophobic interactions a scale going from white to dark green (for low to high values) is exploited. Panel B. Representation of the docking-predicted
structure of the complex between protein kinase CK1 (grey) and compound 35 (orange). Hydrogen bond interactions are depicted as cyan dotted lines. The rounded box contains a
d
d
zoomed view within the vestibular region of the kinase binding site, showing the stable hydration sites predicted using AquaMMapS analysis. The solvent stationary behavior with
each of these regions is rendered by a colorimetric scale going from blue to red (for high to low % ORMSF values).
interactions between the hydroxyl group and an amino acid residue
of the ATP-binding pocket. Potency towards kinase raise from 4-OH
assay (40
rivative 47 (IC50 ¼ 5.65
m
M), with the only exception of the 3,4-dimethoxy de-
mM). We can also notice that even in this
(48, IC50 ¼ 1.32
m
M), 3,4-(OH)2 (57, IC50 ¼ 0.91
m
M).
M), 3-OH (49,
case the introduction of substituents on the amino group at the 7-
position of TT (that corresponds to the 5-position in TP) have a
detrimental effect in terms of potency: in fact, both derivative 40,
isolated as side-product of the nucleophilic substitution at the 5-
IC50 ¼ 0.46
m
M) and 3,5-(OH)2 (51, IC50 ¼ 0.18
m
It is worth noticing that all the methoxy derivatives (38e45)
resulted nearly inactive at the concentration used in the screening
Table 5
Inhibitory activity of 5-substituted TT (38e57) towards CK1
d
..
Cmpd
R1
R2
R3
PAMPABBBa
IC50 mMb (% inhib. at 40 M)c
m
46
38
48
39
40
49
50
47
57
41
42
43
44
45
51
52
53
54
55
56
Ph
-H
-H
-H
-H
-Bn
-H
-Bn
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Bn
-Bn
-Bn
-Bn
-Bn
-Bn
-Bn
-Bn
CNSþ
n.d.
CNS-
n.d.
n.d. (39.7% 14.4)
n.d. (15.6% 21.9)<
1.32 0.51 (70.4% 11.2)
n.d. (18.1% 7.7)
-(4-OCH3)Ph
-(4-OH)Ph
-(3-OCH3)Ph
-(3-OCH3)Ph
-(3-OH)Ph
-(3-OH)Ph
-(3,4-(OCH3)2)Ph
-(3,4-(OH)2)Ph
-(3,5-(OCH3)2)Ph
-(3,5-(OCH3)2)Ph
-(3,5-(OCH3)2)Ph
-(3,5-(OCH3)2)Ph
-(3,5-(OCH3)2)Ph
-(3,5-(OH)2)Ph
-(3,5-(OH)2)Ph
-(3,5-(OH)2)Ph
-(3,5-(OH)2)Ph
-(3,5-(OH)2)Ph
- (3-OCH3)(5-OH)Ph
n.d.
n.d. (ꢁ10.3% 14.7)
0.46 0.15 (84.2% 9.6)
n.d. (ꢁ8.5% 2.9)
CNSþ
n.d.
CNSþ
CNSþ
n.d.
n.d.
n.d.
5.65 1.34 (72.4% 2.4)
0.91 0.30 (93.8% 10.5)
n.d. (4.5% 2.3)
-Bn
-Bn
-CH2(4-Cl)Ph
-CH2(4-F)Ph
-CH2(3-CH3)Ph
-cC6H11
-Bn
-CH2(4-Cl)Ph
-CH2(4-F)Ph
-CH2(3-CH3)Ph
-cC6H11
-cC6H11
n.d. (9.7% 10.4)
n.d. (10.5% 13.8)
n.d. (13.9% 3.9)
n.d. (41.9% 23.5)
0.18 0.04 (85.3% 4.4)
0.75 0.06 (95.8% 2.5)
0.47 0.10 (87.0% 12.2)
0.53 0.11 (79.7% 9.3)
0.67 0.16 (89.3% 5.1)
3.30 1.17 (68.6% 28.0)
n.d.
n.d.
CNS
CNSþ
CNS
CNS
CNS
CNSþ
a
CNS þ means that compound was predicted able to cross the BBB by passive permeation, CNS- means that compound was predicted unable to cross the BBB by passive
permeation, CNS means that permeability value was lower than limit for CNSþ and upper than limit for CNS-.
b
Data represent the mean SD of 3 independent experiments performed in duplicate.
Data represent the % of inhibition of kinase activity expressed as a mean SD of 2 independent experiments performed in duplicate at 40 mM concentration. n.d.: not
c
determined.
10

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Fig. 7. Panel A. Interaction Energy (IE) fingerprints shown as heat maps reporting the electrostatic (IEele) and hydrophobic (IEhyd) interaction between each 5-substituted TT derivate
(y-axis) and each CK1 binding site residue (x-axis). The intensity of the electrostatic interactions is rendered by a colorimetric scale going from blue to red (for negative to positive
values) while for the hydrophobic interactions a scale going from white to dark green (for low to high values) is exploited. Panel B. Representation of the docking-predicted structure
of the complex between protein kinase CK1 (grey) and compound 51 (orange). Hydrogen bond interactions are depicted as cyan dotted lines. The rounded box contains a zoomed
d
d
view within the vestibular region of the kinase binding site, showing the stable hydration sites predicted using AquaMMapS analysis. The solvent stationary behavior with each of
these regions is rendered by a colorimetric scale going from blue to red (for high to low % ORMSF values).
position, and the corresponding hydroxyl-derivative 50, presenting
a benzylamino group also at the 7-position, have no effects on CK1
activity at 40 M. Finally, the introduction of substituents on the
phenyl ring of the benzylamino group at the 5-position, led to
compounds 52e54 still active in the submicromolar range
inhibition, both as regards the IEele and the IEele components. The
binding mode for compound 51, as reported in Fig. 7-B, appears
almost completely superimposable to that previously described for
compound 31. In detail, a tridentate hydrogen bond network tightly
anchors the TT heterocyclic scaffold to the protein kinase hinge
domain (residues E83 and L85), while the resorcinol-like substit-
uent at the 2-position engaged a double hydrogen bond interaction
with the negatively charged amino acids buried within the catalytic
cleft (residues E52 and D91). Moreover, the benzylamino group
inserted at the 5-position of the TT scaffold and protruding towards
the binding site vestibular regions, adopts a conformation nearly
indistinguishable from that described for the same substituent,
d
m
(IC50s ¼ 0.75, 0.47 and 0.53
mM for compounds 52,53 and 54,
respectively), also, an aromatic ring seems to not be essential
because the introduction of a cyclohexylamino moiety in com-
pound 55 displayed an IC50 of 0.67
mM, comparable to that of
benzylamino derivatives 52e54. During deprotection of compound
45 to 55, mono-deprotected derivative 56 was isolated: as ex-
pected, it was remarkably more potent than the corresponding fully
protected compound 45 and less potent than the completely
when inserted at the 7-position of the TP scaffold. As
a
deprotected compound 55 (45, % inhib. at 40
M).
m
M ¼ 41.9 vs. 56,
IC50 ¼ 3.30
m
M vs. 55, IC50 ¼ 0.67
m
Table 6
Molecular docking studies were finally exploited to investigate
Competition binding assay KINOMEscan™ [60]. Data are reported as % of binding to
kinases with respect to a positive control at a concentration of compound 51 of
10 mM.
the impact of TP scaffold replacement with the closely related TT
nucleus, in terms of CK1d binding mechanism. The IE fingerprints
depicted in Fig. 7-A clearly show how most of the TT derivates
herein reported conserving the key interactions which have been
previously validated as crucial for the kinase recognition and
Cmpd
51
CK1a1
CK1a1L
CK1g1
CK1g2
CK1g3
CK1
14
d
CK1ε
3.2
14
32
20
14
1.4
Finally, compound 51 displayed to be not cytotoxic against three different types of
non-tumoral cells (IC50 > 10 M, Table 7).
m
Fig. 8. Panel A. Concentrationꢁinhibition curve of compound 51 at full-length CK1
d
. Panel B. Truncated CK1
11
d activity at different concentrations of ATP (e.g. 2, 20 and 200 mM).

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Table 7
many of the tested 2,5,8-trisubstituted TPs (18e20 and 26e31),
presenting one or more hydroxyl groups on the scaffold, found
difficult to pass the BBB in the in vitro assay, excepted for compound
30, that even if bears an additional polar moiety at the 8-position (a
free amino group) resulted to passively permeate the BBB in this
assay. However, the same substitutions at the 2-position on the TT
scaffold (48e57) or the 2,5,7-trisubstituted TPs (34,35) are better
tolerated, probably for the presence of the benzylamino moiety at
the 5 (TT) or 7 (TP) positions, leading to more BBB-permeable de-
rivatives (i.e. 34,49,52) and, thus, representing a valuable starting
point for further structural optimizations and biological
evaluations.
Cytotoxicity of compound 51 in non-tumoral cells.
IC50 (m
M) a
b
c
PBLresting
>10
PBLPHA
>10
Human fibroblast
>10
51
a
Compound concentration required to inhibit cell growth by 50%.
PBL not stimulated with PHA.
PBL stimulated with PHA.
b
c
consequence, also the solvent displacement profile for this last
series, reported on the rounded box of Fig. 7-B, doesn’t show a
remarkable difference with respect to the previous one.
The binding mechanism of ligand 51, the most potent derivative
identified through this medicinal chemistry optimization
campaign, was further investigated using MD simulations, in such a
way to fully address the ligand-receptor conformational flexibility
while explicitly accounting for the solvent contribution during the
binding process. In detail, supervised molecular dynamics (SuMD),
an in-house developed computational protocol, was exploited to
collect a MD trajectory describing compound 51 recognition
mechanism, from the unbound to the bound state, in a very
competitive timescale.
5. Conclusions
In summary, in this work a new series of [1,2,4]triazolo[1,5-c]
pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]triazines have been
developed as protein kinase CK1d inhibitors. Substitutions on all
positions have been investigated: 2,5,7 and 8 for the [1,2,4]triazolo
[1,5-c]pyrimidine scaffold and 2,5,and 7 for the [1,2,4]triazolo[1,5-
a][1,3,5]triazine one. Structure-activity relationship for these
compounds revealed that a free amino group is required at the 5
position of the [1,2,4]triazolo[1,5-c]pyrimidine nucleus (and at the
correspondent position in the triazolo-triazine ring, position 7)
while at the 2 position an opportunely substituted phenyl ring can
tune kinase inhibitory activity. In particular, the insertion of hy-
droxy groups at the 3 and 5 positions gave optimal interactions in
the catalytic binding cleft, confirmed by molecular modeling
studies. Prediction of passive blood-brain barrier permeability of
most potent compounds highlighted some promising compounds
The SuMD trajectory collected, along with all the geometric and
energetic analysis performed, has been graphically summarized in
a
synchronized video, reported on supplementary materials
(Video1). As noticeable from the two upper panels, few ns are
sufficient for the TT derivative to approach the vestibular region of
CK1d binding site, where it engages the first interaction with the
residues located on the kinase N-lobe. On the contrary, ligand ac-
commodation within the catalytic pocket required a long negotia-
tion, as evincible by the important repulsive interactions occurring
in the first 40 ns of the SuMD simulation. Once this important
energy barrier has been overcome, the ligand experiences the first
contact with the kinase hinge region residues and quickly con-
verges towards molecular docking anticipated bound state, repro-
ducing both the hydrogen bond network as well as the interactivity
with AquaMMapS-predicted hydration sites.
which could be used as tools for the study of protein kinase CK1
implications in neuroinflammatory and neurodegenerative disease,
as also as hit compounds for
optimization.
d
a more focused structural
6. Experimental Section
6.1. Chemistry
Supplementary video related to this article can be found at
https://doi.org/10.1016/j.ejmech.2021.113331Video 1.
end, considering the promising nature characterizing derivative 51
it was firstly tested against full length CK1 confirming the high
inhibitor activity showing an IC50 of 0.30 M (Fig. 8-A). Then its
mechanism of action was investigated by determining truncated
CK1 activity at different ATP concentrations. In Fig. 8-B it is
possible to observe an increase in kinase activity with increasing
concentrations of ATP, demonstrating that compound 51 has an ATP
competitive behavior. In order to asses compound 51 selectivity it
In the
6.1.1. General chemistry
Reagents were obtained from commercial suppliers and used
without further purification. Reactions were monitored by TLC, on
precoated silica gel plates (Macherey-Nagel, 60FUV254) or
aluminum oxide plates (Macherey-Nagel, 60NUV254). Final com-
pounds and intermediates were purified by flash chromatography
using stationary phases silica gel (Macherey-Nagel, silica 60,
240e400 mesh) or (Macherey-Nagel, aluminum oxide 90 neutral,
d
m
d
50e200 mm). When used, light petroleum ether refers to the frac-
tions boiling at 40e60 ꢀC. Melting points were determined with a
Stuart SMP10 melting point apparatus and were not corrected. The
1H NMR and 13C NMR spectra were determined in CDCl3, DMSO‑d6,
D2O or CD3OD solutions and recorded on Jeol GX 270 MHz, Jeol
400 MHz, Varian 400 MHz or Varian 500 MHz spectrometers;
was tested at a fixed dose of 10 mM against a panel of 452 kinases
(Table S1) comprised all other CK1 isoforms (Table 6). Compound
51 displayed a quite good selectivity, only 17 off-target kinases,
excepted for CK1 isoforms, were found by this screening, but un-
fortunately as showed in Table 6, the compound binds all CK1
isoforms, denoting its ability to interact with common features of
these family.
chemical shifts (d scale) are reported in parts per million (ppm) and
referenced to residual solvent peak, with splitting patterns abbre-
viated to: s (singlet), d (doublet), dd (doublet of doublets), dt
(doublet of triplets), t (triplet), m (multiplet) and bs (broad signal).
Coupling constants (J) are given in Hz. MS-ESI analysis were per-
formed using ESI Bruker 4000 Esquire spectrometer while for the
accurate mass the instrument microTOF-Q e Bruker have been
used. Compound purities were determined by two different
methods: method A) HPLC-DAD (Waters 515 HPLC pump; Waters
PDA 2998 Detector) using Luna C8(2) HPLC column (150 ꢂ 4.6 mm,
4. Prediction of blood-brain barrier (BBB) permeability
As mentioned before, since CK1d is considered a potential target
for therapeutic intervention in CNS diseases and one of the main
obstacles for the treatment of the diseases of the CNS is the drug’s
penetration into the BBB at therapeutic concentrations [61], we
decided to apply the Parallel Artificial Membrane Permeability
Assay (PAMPA) to predict the BBB-permeability of most active
developed triazolo derivatives [62]. As we can see from Tables 1e5,
particle size 3
mm). Isocratic elution was performed for 40 min at a
flow rate of 300
mL/min in water-methanol 30:70. UV absorption
12

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
was detected from 220 to 400 nm using a diode array detector;
purity was determined at the maximum absorption wavelength of
the compound and at 254 nm. Method B) HPLC (Agilent Technol-
ogies 1260 Infinity; UV-VIS detector) using column (Zorbax Eclipse
ethyl 4-(2-(4-methoxybenzoyl)hydrazinyl)-2-(methylthio)pyrimi-
dine-5-carboxylate (80): flash chromatography eluent: light
petroleum-EtOAc 6:4. Yield 57% (8.68 g); white solid; mp 186 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz, CDCl3):
d 10.57 (s, 1H),
Plus, C18, 95 Å, 3,5
m
m, 4,6 ꢂ 100 mm). Elution gradient was per-
9.71 (s, 1H), 8.66 (s, 1H), 7.88 (d, J ¼ 8.8 Hz, 2H), 7.04 (d, J ¼ 8.8 Hz,
2H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.81 (s, 3H), 2.31 (s, 3H), 1.32 (t,
J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 361.0 [M ꢁ H]-.
formed for 20 min at a flow rate of 1 mL/min from water-methanol
40:60 to 20:80, detection was determined at 254 nm.
Synthesis of 2,5,8-trisubstituted [1,2,4]triazolo[1,5-c]pyrimi-
dines (1e31).
Synthesis of 4-hydroxy-2-methylthio-5-carbethoxypyrimidine
sodium salt (60).
ethyl 4-(2-isonicotinoylhydrazinyl)-2-(methylthio)pyrimidine-5-
carboxylate (81): flash chromatography eluent: EtOAc-light petro-
leum 8:2. Yield 24% (4.12 g); white solid; mp 203e206 ꢀC (EtOEt-
light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 11.04 (bs, 1H),
To a solution of 34.4 g (0.860 mol) of sodium hydroxide in
215 mL of water at 20 ꢀC were added 59.8 g of 2-methyl-2-thio-
pseudourea hemisulfate (58, 0.430 mol). The mixture was stirred
for 5 min, by which time most of the pseudothiourea had dissolved.
Diethyl ethoxymethylenemalonate (59, 0.430 mol, 92.9 g), dis-
solved in 500 mL of ethanol, was added under stirring for 1 h. After
the addition was completed, stirring was continued for 3 h and then
the solid mixture was allowed to stand for 24 h. Finally, the mixture
was filtered and washed using the minimum amount of water to
give a pink solid (81.2 g). Yield 40%; mp > 300 ꢀC. 1H NMR
9.85 (bs, 1H), 8.80 (d, J ¼ 4.7 Hz, 2H), 8.70 (s, 1H), 7.81 (d, J ¼ 4.7 Hz,
2H), 4.35 (q, J ¼ 7.1 Hz, 2H), 2.34 (s, 3H), 1.34 (t, J ¼ 7.1 Hz, 3H). ES-
MS (methanol) m/z: 333.9 [MþH]þ, 355.9 [MþNa]þ.
ethyl 4-(2-(4-(benzyloxy)benzoyl)hydrazinyl)-2-(methylthio)py-
rimidine-5-carboxylate (82): flash chromatography eluent: DCM-
EtOAc 9:1. Yield 40% (7.46 g); yellow solid; mp 202e205 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 10.58 (bs,
1H), 9.75 (bs, 1H), 8.67 (s, 1H), 7.89 (d, J ¼ 7.9 Hz, 2H), 7.47e7.35 (m,
5H), 7.14 (d, J ¼ 7.9 Hz, 2H), 5.19 (s, 2H), 4.34 (q, J ¼ 6.6 Hz, 2H), 2.33
(s, 3H), 1.33 (t, J ¼ 6.6 Hz, 3H). ES-MS (methanol) m/z: 439.2
[MþH]þ, 461.1 [MþNa]þ, 477.1 [MþK]þ.
(400 MHz, D2O):
1.20 (t, J ¼ 7.1 Hz, 3H).
d
8.35 (s, 1H), 4.16 (q, J ¼ 7.1 Hz, 2H), 2.33 (s, 3H),
ethyl 4-(2-(3-methoxybenzoyl)hydrazinyl)-2-(methylthio)pyrimi-
dine-5-carboxylate (83): flash chromatography eluent: from DCM-
EtOAc 95:5 to DCM-MeOH 9:1.Yield 43% (6.54 g); white solid; mp
145e150 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, CDCl3):
General procedure for the synthesis of ethyl 4-(2-hydrazido)-2-
(methylthio)pyrimidine-5-carbocarboxylate derivatives (77e91).
To 10.0 g (42 mmol) of dried 60 were added 30 mL of phosphoryl
oxychloride dropwise at 0 ꢀC. After the addition was completed, the
mixture was refluxed for 5 h. The excess of phosphoryl oxychloride
was then removed under reduced pressure and ice and cold water
were added to the solid residue (150 mL). The mixture was
extracted with diethylether (3 ꢂ 50 mL), dried over sodium sulfate
anhydrous and the solvent was removed by evaporation under
reduced pressure to give the 4-chloro-2-methylthio-5-
carbethoxypyrimidine (61), which was readily used in the next
step without purification. The chloropyrimidine obtained (61) and
74 mmol of the required hydrazide (62e76) were dissolved in
60 mL THF, while 74 mmol of DBU were added dropwise at 0 ꢀC. The
mixture was stirred at room temperature overnight. The solvent
was then removed, the residue was dissolved in dichloromethane
(300 mL) and the resulting solution was washed with water
(3 ꢂ 100 mL). The organic layer was dried over anhydrous sodium
sulfate, concentrated under reduced pressure, and purified by flash
chromatography.
d
10.34 (bs, 1H), 8.88 (bs, 1H), 8.71 (s, 1H), 7.44e7.33 (m, 3H), 7.10
(bs, 1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 3.87 (s, 3H), 2.49 (s, 3H), 1.41 (t,
J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 363.1 [MþH]þ, 385.1
[MþNa]þ.
ethyl
4-(2-(3-fluorobenzoyl)hydrazinyl)-2-(methylthio)pyrimi-
dine-5-carboxylate (84): flash chromatography eluent: DCM-EtOAc
9:1. Yield 66% (9.7 g); white solid; mp 175 ꢀC (EtOEt-light petro-
leum). 1H NMR (400 MHz, CDCl3):
d 10.82 (s, 1H), 9.78 (s, 1H), 8.67
(s, 1H), 7.75 (d, J ¼ 7.8 Hz, 1H), 7.67 (dt, J ¼ 8.0 Hz, J ¼ 2.4 Hz, 1H),
7.61e7.55 (m, 1H), 7.45 (td, J ¼ 8.0, J ¼ 2.4 Hz, 1H), 4.33 (q, J ¼ 7.1 Hz,
2H), 2.33 (s, 3H), 1.32 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z:
351.0 [MþH]þ, 373.0 [MþNa]þ.
ethyl 4-(2-(3-nitrobenzoyl)hydrazinyl)-2-(methylthio)pyrimidine-
5-carboxylate (85): flash chromatography eluent: from light
petroleum-EtOAc 9:1 to 4:6. Yield 63% (10.0 g); white solid; mp
204e208 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
DMSO‑d6):
d 11.14 (s, 1H), 9.90 (s, 1H), 8.74 (s, 1H), 8.70 (s, 1H),
ethyl
4-(2-acetylhydrazinyl)-2-(methylthio)pyrimidine-5-
8.49e8.44 (m, 1H), 8.36e8.31 (m, 1H), 7.86 (t, J ¼ 8.0 Hz, 1H), 4.36
(q, J ¼ 7.1 Hz, 2H), 2.35 (s, 3H), 1.34 (t, J ¼ 7.1 Hz, 3H).). ES-MS
(methanol) m/z: 378.3 [MþH]þ, 400.3 [MþNa]þ.
carboxylate (77): flash chromatography eluent: light petroleum-
EtOAc 6:4. Yield 42% (4.76 g); pale yellow solid; mp 155 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz; CDCl3): 10.17 (s, 1H),
9.60 (s, 1H), 8.62 (s, 1H), 4.30 (q, J ¼ 7.0 Hz, 2H), 2.46 (s, 3H), 1.92 (s,
3H), 1.29 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 293.0 [MþNa]þ.
ethyl 4-[2-(cyclopentancarbonyl)hydrazinyl]-2-(methylthio)py-
rimidine-5-carboxylate (78): flash chromatography eluent: light
petroleum-EtOAc 8:2. Yield 24% (3.27 g); pale yellow solid; mp
ethyl 4-(2-(3,5-dimethoxybenzoyl)hydrazinyl)-2-(methylthio)py-
rimidine-5-carboxylate (86): flash chromatography eluent: from
DCM-EtOAc 9.8:0.2 to DCM-EtOAc 9:1. Yield 21% (5,76 g, reaction
performed on 70 mmol of 60); white solid; mp 194 ꢀC (EtOAc-light
petroleum). 1H NMR (400 MHz, CDCl3)
d
10.28 (d, J ¼ 5.1 Hz, 1H),
8.77 (d, J ¼ 5.1 Hz, 1H), 8.72 (s, 1H), 6.97 (d, J ¼ 2.2 Hz, 2H), 6.63 (t,
J ¼ 2.2 Hz, 1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 3.84 (s, 6H), 2.49 (s, 3H), 1.41
(t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 393.2 [MþH]þ.
127 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, CDCl3):
d 10.05
(bs, 1H), 8.67 (s, 1H), 8.20 (bs, 1H), 4.35 (q, J ¼ 7.0 Hz, 2H), 2.80e2.57
(m, 1H), 2.52 (s, 3H), 2.06e1.49 (m, 8H), 1.37 (t, J ¼ 7.0 Hz, 3H). ES-
MS (methanol) m/z: 323.0 [M ꢁ H]-.
ethyl
4-(2-benzoylhydrazinyl)-2-(methylthio)pyrimidine-5-
carboxylate (87): flash chromatography eluent: DCM-EtOAc 95:5.
ethyl
4-[2-(1H-indol-2-carbonyl)hydrazinyl]-2-(methylthio)py-
Yield 34% (4.75 g); white solid; mp 168 ꢀC (EtOEt-light petroleum).
rimidine-5-carboxylate (79): flash chromatography eluent: light
petroleum-EtOAc 8:2. Yield 46% (7.18 g); pale yellow solid; mp
253e255 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
1H NMR (270 MHz, CDCl3):
d 10.43 (bs, 1H), 8.96 (bs, 1H), 8.75 (s,
1H), 7.91 (d, J ¼ 7.5 Hz, 2H), 7.67e7.41 (m, 3H), 4.43 (q, J ¼ 7.2 Hz,
2H), 2.52 (s, 3H), 1.44 (t, J ¼ 7.2 Hz, 3H). ES-MS (methanol) m/z:
331.0 [M ꢁ H]-.
DMSO‑d6): d 11.71 (s, 1H), 10.69 (s, 1H), 9.76 (s, 1H), 8.68 (s, 1H), 7.65
(d, J ¼ 8.0 Hz,1H), 7.43 (d, J ¼ 8.3 Hz,1H), 7.27 (d, J ¼ 1.3 Hz,1H), 7.20
(t, J ¼ 7.6 Hz, 1H), 7.05 (t, J ¼ 7.3 Hz, 1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 2.29
(s, 3H), 1.33 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 370.0
[M ꢁ H]-.
ethyl
4-(2-(4-fluorobenzoyl)hydrazinyl)-2-(methylthio)pyrimi-
dine-5-carboxylate (88): flash chromatography eluent: DCM-EtOAc
9:1. Yield 28%; white solid; mp 200 ꢀC (EtOEt-light petroleum).
1H NMR (270 MHz, DMSO‑d6):
d 10.77 (bs, 1H), 9.78 (bs, 1H), 8.68 (s,
13

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
1H), 7.98 (dd, JH ¼ 8.8, JHF ¼ 5.6 Hz, 2H), 7.37 (t, JH,HF ¼ 8.8 Hz, 2H),
4.34 (q, J ¼ 7.0 Hz, 2H), 2.33 (s, 3H), 1.33 (t, J ¼ 7.0 Hz, 3H). ES-MS
(methanol) m/z: 348.9 [M ꢁ H]-.
ethyl 2-(1-H-indol-2-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (94): Method A. Flash chromatography
eluent: light petroleum-EtOAc 7:3. Yield 28% (0.989 g); yellow
solid; mp 187e190 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
ethyl 4-(2-(3-(benzyloxy)benzoyl)hydrazinyl)-2-(methylthio)py-
rimidine-5-carboxylate (89): flash chromatography eluent: DCM-
EtOAc 9:1. Yield 34% (6.17 g); white solid; mp 117 ꢀC (EtOEt-light
CDCl3):
d
9.39 (s, 1H), 8.80 (s, 1H), 7.71 (d, J ¼ 8.0 Hz, 1H), 7.50e7.45
(m, 2H), 7.31e7.27 (m, 1H), 7.17e7.13 (m, 1H), 4.54 (q, J ¼ 7.1 Hz, 2H),
2.82 (s, 3H), 1.49 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 354.1
[MþH]þ, 376.0 [MþNa]þ.
petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 10.72 (bs, 1H), 9.77 (bs,
1H), 8.68 (s, 1H), 7.71e7.11 (m, 9H), 5.17 (s, 2H), 4.34 (q, J ¼ 7.1 Hz,
2H), 2.33 (s, 3H), 1.33 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z:
438.9 [MþH]þ, 460.8 [MþH]þ.
ethyl 2-(4-methoxyphenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]
pyrimidine-8-carboxylate (95): Method A. Flash chromatography
eluent: DCM (stationary phase: aluminium oxide). Yield 57%
(1.96 g); white solid; mp 182e184 ꢀC (EtOEt-light petroleum). 1H
ethyl 4-(2-(3,4-bis(benzyloxy)benzoyl)hydrazinyl)-2-(methylthio)
pyrimidine-5-carboxylate (90): flash chromatography eluent: from
DCM-light petroleum 9.5:0.5 to DCM-EtOAc 8:2. Yield 30% (6.86 g);
white solid; mp 186 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz,
NMR (400 MHz, CDCl3):
d
8.78 (s, 1H), 8.35 (d, J ¼ 8.9 Hz, 2H), 7.01
(d, J ¼ 8.9 Hz, 2H), 4.52 (q, J ¼ 7.1 Hz, 2H), 3.88 (s, 3H), 2.80 (s, 3H),
1.49 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 345.2 [MþH]þ, 367.1
[MþNa]þ.
DMSO‑d6):
d 10.59 (bs, 1H), 9.74 (bs, 1H), 8.68 (s, 1H), 7.62 (s, 1H),
7.60e7.26 (m, 11H), 7.20 (d, J ¼ 8.4 Hz, 1H), 5.21 (d, J ¼ 9.9 Hz, 4H),
4.35 (q, J ¼ 6.7 Hz, 2H), 2.31 (s, 3H), 1.33 (t, J ¼ 6.7 Hz, 3H). ES-MS
(methanol) m/z: 544.9 [MþH]þ, 566.9 [MþNa]þ.
ethyl 2-(pyridin-4-yl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8-carboxylate (96): Method B1. Flash chromatography eluent:
DCM-MeOH 97:3. Yield 8% (0.252 g); white solid; mp 183e185 ꢀC
ethyl 4-(2-(3,5-bis(benzyloxy)benzoyl)hydrazinyl)-2-(methylthio)
pyrimidine-5-carboxylate (91): flash chromatography eluent: from
DCM-EtOAc 9:1 to DCM-EtOAc 8.5:1.5. Yield 54% (12.35 g); white
solid; mp 184e188 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 8.83e8.81
(m, 3H), 8.14 (dd, J ¼ 4.4, 1.6 Hz, 2H), 4.43 (q, J ¼ 7.1 Hz, 2H), 2.80 (s,
3H), 1.40 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 316.4 [MþH]þ,
338.3 [MþNa]þ.
DMSO‑d6):
d 10.69 (s, 1H), 9.77 (s, 1H), 8.68 (s, 1H), 7.49e7.33 (m,
10H), 7.16 (s, 2H), 6.91 (s, 1H), 5.16 (s, 4H), 4.35 (q, J ¼ 7.0 Hz, 2H),
2.33 (s, 3H), 1.34 (t, J ¼ 7.0 Hz, 3H). ES-MS (methanol) m/z: 545.2
[MþH]þ, 567.3 [MþNa]þ. MW 544.62.
ethyl 2-(4-(benzyloxy)phenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-
c]pyrimidine-8-carboxylate (97): Method B1. Flash chromatography
eluent: DCM. Yield 7% (0.294 g); white solid; mp 190e192 ꢀC
General procedure for the synthesis of 2-substituted 5-(meth-
ylthio)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate derivatives
(92e106).
(EtOEt-light petroleum). 1H NMR (400 MHz, CDCl3):
d 8.79 (s, 1H),
8.37 (d, J ¼ 8.7 Hz, 2H), 7.56e7.30 (m, 5H), 7.09 (d, J ¼ 8.7 Hz, 2H),
5.14 (s, 2H), 4.53 (q, J ¼ 7.1 Hz, 2H), 2.80 (s, 3H), 1.49 (t, J ¼ 7.1 Hz,
3H). ES-MS (methanol) m/z: 421.0 [MþH]þ, 443.0 [MþNa]þ, 458.9
[MþK]þ.
Method A: To 150 mL of dry xylene were added 0.030 mol of
phosphorus pentoxide and hexamethyldisiloxane, and the mixture
was heated at 90 ꢀC for 2 h. Pyrimidine hydrazides (0.010 mol) were
then added, and the reaction was refluxed for 2 days. After that, the
solvent was removed under reduced pressure, and the residue was
dissolved in EtOAc (200 mL) and washed with water (3 ꢂ 70 mL)).
The organic layer was then dried over anhydrous sodium sulfate,
concentrated under reduced pressure, and purified by flash
chromatography.
Method B: The mixture of phosphorous pentoxyde [0.045 mol
(Method B1) or 0.075 mol (Method B2)] and hexamethyldisiloxane
[0.045 mol (Method B1) or 0.075 mol (Method B2)] in anhydrous
xylene (150 mL) was heated to 90 ꢀC over 1.5 h and then stirred for
1 h at 90 ꢀC, under argon atmosphere. The well-dried hydrazides
compounds (62e76) (0.015 mol, 1 equivalent) were then added to
the clear solution and the temperature is increased to reflux and
stirred for 2e3 h. The solvent was then removed, the residue dis-
solved in DCM (300 mL) and the resulting solution was washed
with water (3 ꢂ 100 mL). The organic layer was concentrated, dried
over anhydrous sodium sulfate and purified to afford the desired
compounds.
ethyl 2-(3-methoxyphenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]
pyrimidine-8-carboxylate (98): Method B2. Flash chromatography
eluent: light petroleum-EtOAc 8:2. Yield 59% (2.03 g)); white solid;
mp 154 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, CDCl3):
d
8.80 (s, 1H), 8.06e7.87 (m, 2H), 7.41 (t, J ¼ 7.8 Hz, 1H), 7.06 (dd,
J ¼ 7.8, 2.7 Hz, 1H), 4.53 (q, J ¼ 7.1 Hz, 2H), 3.93 (s, 3H), 2.81 (s, 3H),
1.49 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 345.4 [MþH]þ, 367.3
[MþNa]þ.
ethyl 2-(3-fluorophenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (99): Method B2. Flash chromatography
eluent: light petroleum-EtOAc 95:5 (stationary phase: aluminium
oxide). Yield 14% (0.465 g); white solid; mp 153e156 ꢀC (EtOEt-
light petroleum). 1H NMR (400 MHz, CDCl3): 8.81 (s, 1H), 8.20 (m,
1H), 8.12e8.09 (m, 1H), 7.47 (td, J ¼ 8.0, JHF ¼ 5.7 Hz, 1H), 7.23e7.17
(m, 1H), 4.53 (q, J ¼ 7.1 Hz, 2H), 2.81 (s, 3H), 1.49 (t, J ¼ 7.1 Hz, 3H).
ES-MS (methanol) m/z: 333.0 [MþH]þ.
ethyl
2-(3-nitrophenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (100): Method B2. Flash chromatography
eluent: DCM-light petroleum 8:2 (stationary phase: aluminium
oxide). Yield 7% (0.252 g); white solid; mp 210e215 ꢀC (EtOEt-light
ethyl 2-methyl-5-(methylthio)-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxylate (92): Method A. Flash chromatography eluent: light
petroleum-EtOAc 6:4. Yield 32% (0.807 g); pale yellow solid; mp
petroleum). 1H NMR (400 MHz, CDCl3):
d 9.23 (s, 1H), 8.84 (s, 1H),
8.77e8.72 (m, 1H), 8.39e8.33 (m, 1H), 7.70 (t, J ¼ 8.0 Hz, 1H), 4.54
(q, J ¼ 7.1 Hz, 2H), 2.83 (s, 3H), 1.49 (t, J ¼ 7.1 Hz, 3H). ES-MS
(methanol) m/z: 332.3 [M þ H-(CH2CH3)]þ, 360.3 [MþH]þ, 382.3
[MþNa]þ.
120 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, CDCl3):
d 8.76 (s,
1H), 4.50 (q, J ¼ 7.1 Hz, 2H), 2.78 (s, 3H), 2.70 (s, 3H), 1.44 (t,
J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 225.0 [M þ H-(CH2CH3)]þ,
253.0 [MþH]þ, 275.0 [MþNa]þ.
ethyl 2-(3,5-dimethoxyphenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-
c]pyrimidine-8-carboxylate (101): Method B2. Flash chromatog-
raphy eluent: light petroleum-EtOAc from 8:2 to 6:4. Yield: 12%
(0.43 g, starting from 10 mmol of 86); white solid; mp 206e208 ꢀC
ethyl
2-cyclopentyl-5-(methylthio)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8-carboxylate (93): Method A. Flash chromatography eluent:
light petroleum-EtOAc 8:2. Yield 28% (0.858 g); white solid; mp
95 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, CDCl3):
d
8.74 (s,
(EtOAc-light petroleum). 1H NMR (400 MHz, CDCl3)
d 8.80 (s, 1H),
1H), 4.49 (q, J ¼ 7.1 Hz, 2H), 3.48 (p, J ¼ 8.3 Hz, 1H), 2.77 (s, 3H),
2.21e2.13 (m, 2H), 2.09e1.99 (m, 2H), 1.90e1.82 (m, 2H), 1.76e1.67
(m, 2H), 1.44 (d, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 307.1
[MþH]þ, 329.1 [MþNa]þ.
7.57 (d, J ¼ 2.1 Hz, 2H), 6.61 (t, 1H), 4.53 (q, J ¼ 7.1 Hz, 2H), 3.90 (s,
6H), 2.81 (s, 3H), 1.49 (t, J ¼ 7.1 Hz, 3H).13C NMR (101 MHz, CDCl3)
d
166.24, 163.26, 161.09, 158.13, 150.59, 148.10, 131.45, 110.72,
106.08, 104.18, 62.03, 55.78, 14.47, 13.80. ES-MS (methanol) m/z:
14

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
375.1 [MþH]þ, 397.1 [MþNa]þ.
1.31 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6):
d 171.6, 163.4,
ethyl 2-phenyl-5-(methylthio)-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxylate (102): Method B1. Flash chromatography eluent: light
petroleum-EtOAc from 9:1 to 4:6 (stationary phase: aluminium
oxide). Yield 16% (0.503 g); white solid; mp 169e171 ꢀC (EtOEt-light
152.0, 151.7, 150.2, 103.1, 60.5, 39.0, 32.4, 25.8, 14.7. ES-MS (meth-
anol) m/z: 276.1 [MþH]þ, 298.1 [MþNa]þ. HRMS (ESI-TOF) m/z:
C
13H17N5O2 experimental 298.1271 [MþNa]þ, theoretical 298.1274
[MþNa]þ,
D
¼ 0.0003. Purity HPLC (Method A): 99.0%.
petroleum). 1H NMR (270 MHz, CDCl3):
d
8.80 (s, 1H), 8.41 (m, 2H),
ethyl 5-amino-2-(1H-indol-2-yl)-[1,2,4]triazolo[1,5-c]pyrimidine-
8-carboxylate (3): flash chromatography eluent: light petroleum-
EtOAc 6:4. Yield 16% (26 mg); white solid; mp 296e299 ꢀC
7.51 (m, 3H), 4.53 (q, J ¼ 6.5 Hz, 2H), 2.81 (s, 3H), 1.49 (t, J ¼ 6.5 Hz,
3H). ES-MS (methanol) m/z: 315.2 [MþH]þ, 337.2 [MþNa]þ, 353.1
[MþK]þ.
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 11.80 (s,
ethyl 2-(4-fluorophenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (103): Method B1. Flash chromatography
eluent: DCM-EtOAc from 95:5. Yield 6% (0.199 g); white solid; mp
188e190 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, CDCl3):
1H), 8.95e8.30 (m, 3H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7.55 (d, J ¼ 8.1 Hz,
1H), 7.19e7.15 (m, 2H), 7.06 (t, J ¼ 7.3 Hz,1H), 4.35 (q, J ¼ 7.1 Hz, 2H),
1.36 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6):
d 163.2, 159.4,
152.4, 152.2, 150.4, 137.8, 128.7, 128.2, 123.3, 121.4, 120.2, 112.8,
110.0, 103.4, 60.6, 14.8. ES-MS (methanol) m/z: 323.2 [MþH]þ, 345.1
[MþNa]þ. HRMS (ESI-TOF) m/z: C16H14N6O2 experimental 345.1072
d
8.80 (s, 1H), 8.41 (dd, JH ¼ 8.2 Hz, JHF ¼ 5.7 Hz, 2H), 7.18 (t,
JH,HF ¼ 8.2Hz, 2H), 4.53 (q, J ¼ 7.1 Hz, 2H), 2.80 (s, 3H) 1.48 (t,
J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 332.9 [MþH]þ, 354.9
[MþNa]þ, 370.8 [MþK]þ.
[MþNa]þ, theoretical 345.1070 [MþNa]þ,
D
¼ 0.0002. Purity HPLC
(Method A): 96.2%.
ethyl 2-(3-(benzyloxy)phenyl)-5-(methylthio)-[1,2,4]triazolo[1,5-
c]pyrimidine-8-carboxylate (104): Method B1. Flash chromatog-
raphy eluent: light petroleum-EtOAc 8:2. Yield 7% (0.294 g); white
solid; mp 167e169 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz,
ethyl 5-amino-2-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8-carboxylate (4): flash chromatography eluent: DCM-MeOH
98:2. Yield 40% (63 mg); white solid; mp 232 ꢀC (EtOEt-light pe-
troleum). 1H NMR (270 MHz, DMSO‑d6):
d 8.90 (s, 1H), 8.65e8.28
CDCl3):
d
8.81 (s, 1H), 8.03 (d, J ¼ 8.0 Hz, 2H), 7.57e7.30 (m, 6H), 7.12
(m, 2H), 8.18 (d, J ¼ 8.8 Hz, 2H), 7.13 (d, J ¼ 8.8 Hz, 2H), 4.33 (q,
(d, J ¼ 6.6 Hz, 1H), 5.18 (s, 2H), 4.53 (q, J ¼ 7.1 Hz, 2H), 2.81 (s, 3H),
1.49 (t, J ¼ 7.1 Hz, 3H). ES-MS (methanol) m/z: 421.4 [MþH]þ, 443.3
[MþNa]þ.
J ¼ 7.1 Hz, 2H), 3.85 (s, 3H), 1.35 (t, J ¼ 7.1 Hz, 3H). 13C NMR (68 MHz,
DMSO‑d6):
d 163.86, 163.23, 161.43, 152.07, 151.97, 150.19, 128.99,
122.53, 114.46, 102.98, 60.18, 55.36, 14.25. ES-MS (methanol) m/z:
314.1 [MþH]þ, 336.1 [MþNa]þ. HRMS (ESI-TOF) m/z: C15H15N5O3
experimental 314.1247 [MþH]þ, theoretical 314.1247 [MþH]þ,
ethyl 2-(3,4-bis(benzyloxy)phenyl)5-(methylthio)-[1,2,4]triazolo
[1,5-c]pyrimidine-8 carboxylate (105): Method B1. Flash chroma-
tography eluent: DCM-EtOAc 99:1. Yield 6% (0.342 g); white solid;
mp 170e172 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz; CDCl3):
D
¼ 0.0000. Purity HPLC (Method A): 98.3%.
ethyl 5-amino-2-(piridin-4-yl)-[1,2,4]triazolo[1,5-c]pyrimidine-8-
d
8.80 (s, 1H), 8.08e7.97 (m, 2H), 7.57e7.28 (m, 10H), 7.08e6.99 (m,
carboxylate (5): flash chromatography eluent: EtOAc-MeOH 99:1.
1H), 5.34e5.17 (m, 4H), 4.59e4.48 (q, J ¼ 6.5 Hz, 2H), 2.81 (s, 3H),
1.55e1.43 (t, J ¼ 6.5 Hz, 3H). ES-MS (methanol) m/z: 527.2 [MþH]þ,
549.2 [MþNa]þ, 565.1 [MþK]þ.
Yield 58% (83 mg); white solid; mp 268e270 ꢀC (EtOEt-light pe-
troleum). 1H NMR (400 MHz, DMSO‑d6):
d 9.08 (bs, 1H), 8.82 (d,
J ¼ 5.9 Hz, 2H), 8.71e8.47 (m, 2H), 8.12 (d, J ¼ 5.9 Hz, 2H), 4.34 (q,
ethyl 2-(3,5-bis(benzyloxy)phenyl)5-(methylthio)-[1,2,4]triazolo
[1,5-c]pyrimidine-8 carboxylate (106): Method B2. Flash chroma-
tography eluent: light petroleum-EtOAc 8:2. Yield 5% (0.263 g);
white solid; mp 170e175 ꢀC (EtOEt-light petroleum). 1H NMR
J ¼ 7.1 Hz, 2H), 1.36 (t, J ¼ 7.1 Hz, 3H). 13C NMR (270 MHz, CDCl3-
CF3COOD):
d 161.88, 152.73, 149.16, 146.08, 143.03, 142.45, 125.69,
123.71, 106.72, 63.09, 14.11. ES-MS (methanol) m/z: 284.9 [MþH]þ,
306.9 [MþNa]þ. HRMS (ESI-TOF) m/z: C13H12N6O2 experimental
(400 MHz, CDCl3):
d
8.80 (s, 1H), 7.69 (s, 2H), 7.53 7.30 (m, 10H), 6.76
285.1095 [MþH]þ, theoretical 285.1094 [MþH]þ,
D
¼ 0.0001. Purity
(s, 1H), 5.14 (s, 4H), 4.53 (q, J ¼ 7.0 Hz, 2H), 2.80 (s, 3H), 1.48 (t,
J ¼ 7.0 Hz, 3H). ES-MS (methanol) m/z: 527.3 [MþH]þ, 549.2
[MþNa]þ.
HPLC (Method A): 96.3%.
ethyl 5-amino-2-(4-(benzyloxy)phenyl)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (6): flash chromatography eluent: DCM-
methanol 95:5. Yield 93% (185 mg); white solid; mp 277e280 ꢀC
General procedure for the preparation of 2-substituted 5-
amino-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate derivatives
(1e10, 107e109).
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 8.86 (bs,
1H), 8.65e8.32 (m, 2H), 8.18 (d, J ¼ 8.4 Hz, 2H), 7.44 (m, 5H), 7.22 (d,
1 mL of methanolic ammonia 7 N and 4 mL of ethanol were
added to 0.5 mmol of 92e101,104e106 and the mixture was heated
at 70 ꢀC in a sealed tube for 3e12 h. The solvent was then removed
under vacuum and the solid purified by flash chromatography.
J ¼ 8.4 Hz, 2H), 5.20 (s, 2H), 4.33 (q, J ¼ 6.9 Hz, 2H),1.35 (t, J ¼ 6.9 Hz,
3H). 13C NMR (68 MHz, CDCl3):
d 163.26, 161.81, 160.57, 152.11,
151.99, 150.21, 136.92, 129.00, 128.66, 128.16, 128.04, 115.28, 102.98,
69.48, 60.18, 14.26. ES-MS (methanol) m/z: 390.2 [MþH]þ, 412.1
[MþNa]þ, 428.1 [MþK]þ. HRMS (ESI-TOF) m/z: C21H19N5O3 exper-
imental 390.1557 [MþH]þ, theoretical 390.1560 [MþH]þ,
ethyl
5-amino-2-methyl-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxylate (1): flash chromatography eluent: light petroleum-
EtOAc 8:2. Yield 52% (57 mg); white solid; mp 238e241 ꢀC
D
¼ 0.0003. Purity HPLC (Method A): 93.2%.
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d
8.90e8.35
ethyl 5-amino-2-(3-methoxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
(m, 3H), 4.30 (q, J ¼ 7.1 Hz, 2H), 2.51 (s, 3H),1.31 (t, J ¼ 7.1 Hz, 3H). 1H
dine-8-carboxylate (7): flash chromatography eluent: light
petroleum-EtOAc 8:2. Yield 81% (127 mg); white solid; mp
200e204 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
NMR (270 MHz, DMSO‑d6/D2O) d: 8.44 (s, 1H), 4.27 (bs, 2H), 2.46 (s,
3H), 1.27 (bs, 3H). 13C NMR (101 MHz, DMSO‑d6):
d 164.5, 163.5,
152.2, 151.6, 150.2, 103.0, 60.5, 14.80, 14.70. ES-MS (methanol) m/z:
222.4 [MþH]þ, 244.4 [MþNa]þ. HRMS (ESI-TOF) m/z: C9H11N5O2
experimental 244.0803 [MþNa]þ, theoretical 244.0804 [MþNa]þ,
DMSO‑d6): d 8.99 (s, 1H), 8.52 (bs, 2H), 7.89e7.74 (m, 2H), 7.50 (t,
J ¼ 7.9 Hz, 1H), 7.13 (d, J ¼ 7.9 Hz, 1H), 4.34 (q, J ¼ 7.0 Hz, 2H), 3.87 (s,
3H), 1.36 (t, J ¼ 7.0 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 163.84,
D
¼ 0.0001. Purity HPLC (Method A): 98.1%.
163.18, 159.81, 152.24, 152.06, 150.31, 131.61, 130.35, 119.80, 116.57,
112.44, 103.15, 60.27, 55.31, 14.33. ES-MS (methanol) m/z: 286.3
[Mþ2H-(CH2CH3)]þ, 314.4 [MþH]þ, 336.3 [MþNa]þ. HRMS (ESI-
TOF) m/z: C15H15N5O3 experimental 314.1245 [MþH]þ, theoretical
ethyl 5-amino-2-cyclopentyl-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxylate (2): flash chromatography eluent: light petroleum-
EtOAc 1:1. Yield 27% (37 mg); white solid; mp 179e181 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz, CDCl3):
d
8.60 (s, 1H),
314.1247 [MþH]þ,
D
¼ 0.0002. Purity HPLC (Method A): 98.0%.
6.74 (bs, 2H), 4.29 (q, J ¼ 7.1 Hz, 2H), 3.36e3.28 (m, 1H), 2.09e2.00
ethyl
5-amino-2-(3-fluorophenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
(m, 2H), 1.98e1.85 (m, 2H), 1.83e1.73 (m, 2H), 1.73e1.61 (m, 2H),
dine-8-carboxylate (8): flash chromatography eluent: DCM-MeOH
15

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
96:4. Yield 40% (60 mg); white solid; mp 223e225 ꢀC (EtOEt-light
aryl-5-amino-8-ethoxycarbonyl-[1,2,4]triazolo[1,5-c]pyrimidine
derivatives (11e14,110).
petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 9.17e8.34 (m, 3H),
8.09 (d, J ¼ 7.0 Hz, 1H), 7.95 (m, 1H), 7.70e7.59 (m, 1H), 7.42 (m, 1H),
Compounds 98,102e104 (0.5 mmol) were dissolved in 4 mL of
ethanol and reacted with 1.5 mmol of the required amine, heating
at 90 ꢀC for 3e12 h in a sealed tube. Once the reaction was
completed, the solvent was evaporated and the crude products
purified through flash chromatography.
4.34 (q, J ¼ 7.0 Hz, 2H), 1.35 (t, J ¼ 7.0 Hz, 3H). 13C NMR (68 MHz,
DMSO‑d6):
d
163.1, 162.8, 162.6 (d, JCF ¼ 244.1 Hz), 152.4, 152.2,
150.3, 132.5 (d, JCF ¼ 8.8 Hz), 131.4 (d, JCF ¼ 8.4 Hz), 123.5 (d,
JCF ¼ 2.8 Hz), 117.7 (d, JCF ¼ 21.0 Hz), 113.75 (d, JCF ¼ 23.1 Hz), 103.2,
60.3, 14.25. ES-MS (methanol) m/z: 274.4 [Mþ2H-(CH2CH3)]þ,
302.5 [MþH]þ, 324.4 [MþNa]þ, 340.3 [MþK]þ. HRMS (ESI-TOF) m/
ethyl
5-((3,4-dimethoxybenzyl)amino)-2-phenyl-[1,2,4]triazolo
[1,5-c]pyrimidine-8-carboxylate (11): flash chromatography eluent:
light petroleum-EtOAc 8:2. Yield 38% (82 mg); white solid; mp
163e166 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
z:
C
14H12FN5O2 experimental 302.1046 [MþH]þ, theoretical
302.1047 [MþH]þ,
D
¼ 0.0001. Purity HPLC (Method A): 95.6%.
ethyl 5-amino-2-(3-nitrophenyl)-[1,2,4]triazolo[1,5-c]pyrimidine-
8-carboxylate (9): flash chromatography eluent: light petroleum-
EtOAc 6:4. Yield 91% (149 mg); white solid; mp 275e280 ꢀC
d
9.41 (t, J ¼ 6.1 Hz, 1H), 8.58 (s, 1H), 8.39e8.13 (m, 2H), 7.70e7.42
(m, 3H), 7.08 (s, 1H), 6.95 (d, J ¼ 7.9Hz, 1H), 6.88 (d, J ¼ 7.9 Hz, 1H),
4.75 (d, J ¼ 6.1 Hz, 2H), 4.34 (q, J ¼ 6.8 Hz, 2H), 3.73 (s, 3H), 3.71 (s,
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
9.24e8.91
3H), 1.35 (t, J ¼ 6.8 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 163.9,
(m, 2H), 8.83e8.47 (m, 3H), 8.41 (d, J ¼ 7.8 Hz,1H), 7.91 (t, J ¼ 7.9 Hz,
1H), 4.35 (q, J ¼ 6.7 Hz, 2H), 1.36 (t, J ¼ 6.7 Hz, 3H). 13C NMR
(68 MHz, DMSO‑d6): 163.06, 161.98, 152.63, 152.41, 150.38, 148.46,
133.40, 131.75, 131.06, 125.37, 121.70, 103.19, 60.42, 14.30. ES-MS
(methanol) m/z: 329.4 [MþH]þ, 351.4 [MþNa]þ. HRMS (ESI-TOF)
163.2, 151.87, 151.7, 148.8, 148.5, 148.3, 130.9, 130.1, 129.1, 127.4,
120.0, 111.9, 103.4, 60.3, 55.6, 55.51, 43.9, 14.25. ES-MS (methanol)
m/z: 434.1 [MþH]þ, 456.1 [MþNa]þ, 472.0 [MþK]þ. MW 433.46.
C
23H23N5O4. HRMS (ESI-TOF) m/z: C23H23N5O4 experimental
434.1825 [MþH]þ, theoretical 434.1822 [MþH]þ,
D
¼ 0.0003. Purity
m/z:
C
14H12N6O4 experimental 302.0996 [MþH]þ, theoretical
HPLC (Method A): 99.4%.
329.0992 [MþH]þ,
D
¼ 0.0004. Purity HPLC: 95.1%.
ethyl 5-((3,4-dimethoxybenzyl)amino)-2-(4-fluorophenyl)-[1,2,4]
triazolo[1,5-c]-8-carboxylate (12): flash chromatography eluent:
light petroleum-EtOAc 8:2. Yield 73% (165 mg); white solid; mp
156 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
ethyl 5-amino-2-(3,5-dimethoxyphenyl)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (10): flash chromatography eluent: from
EtOAc-light petroleum 4:6 to EtOAc-light petroleum 8:2. Yield 87%
(150 mg); white solid; mp 222e223 ꢀC (EtOEt-light petroleum). 1H
d
9.42 (bs, 1H), 8.58 (s, 1H), 8.29 (m, 2H), 7.43 (t, JH,HF ¼ 8.4 Hz, 2H),
NMR (400 MHz, DMSO)
d
8.94 (bs, 1H), 8.66e8.32 (m, 2H), 7.39 (d,
7.07 (s, 1H), 6.94 (d, J ¼ 8.0 Hz, 1H), 6.88 (d, J ¼ 8.0 Hz, 1H), 4.74 (d,
J ¼ 5.5 Hz, 2H), 4.34 (q, J ¼ 6.8 Hz, 2H), 3.73 (s, 3H), 3.71 (s, 3H), 1.35
J ¼ 2.3 Hz, 2H), 6.67 (t, J ¼ 2.3 Hz,1H), 4.31 (q, J ¼ 7.1 Hz, 2H), 3.83 (s,
6H), 1.34 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO)
d
163.41,
(t, J ¼ 6.8 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 163.85 (d,
162.90, 160.75, 151.97, 151.76 (CH), 150.04, 131.85, 105.11 (2xCH),
102.99 (CH), 102.56, 60.19 (CH2), 55.42 (2xCH3), 14.27 (CH3).
MW ¼ 343.13. ES-MS (methanol) m/z 344.2 [MþH]þ, 366.1
[MþNa]þ. HRMS (ESI-TOF) m/z: C16H17N5O4 experimental 344.1352
JCF ¼ 247.3 Hz), 163.16, 163.05, 151.95, 151.81, 148.85, 148.45, 148.29,
130.82, 129.72 (d, JCF ¼ 8.7 Hz), 126.70 (d, JCF ¼ 2.7 Hz), 119.96,
116.21 (d, JCF ¼ 22.1 Hz), 111.92, 111.82, 103.40, 60.29, 55.56, 55.51,
43.91, 14.24. ES-MS (methanol) m/z: 452.1 [MþH]þ, 474.1 [MþNa]þ,
490.0 [MþK]þ. HRMS (ESI-TOF) m/z: C23H22FN5O4 experimental
[MþH]þ, theoretical 344.1353 [MþH]þ,
466.1175 [MþNa]þ, 366.1172 [MþNa]þ,
(Method A): 97.9%.
D
D
¼ 0.0001; experimental
¼ 0.0003. Purity HPLC
452.1722 [MþH]þ, theoretical 452.1728 [MþH]þ,
D
¼ 0.0006. Purity
HPLC (Method A): 97.3%.
ethyl 5-amino-2-(3-(benzyloxy)phenyl)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxylate (107): flash chromatography eluent: EtOAc-
light petroleum 6:4. Yield 45% (88 mg); white solid; mp 196e198 ꢀC
ethyl 5-(benzylamino)-2-(3-methoxyphenyl)-[1,2,4]triazolo[1,5-
c]-8-carboxylate (13): flash chromatography eluent: DCM-
methanol 98.5:1.5. Yield 74% (149 mg); white solid; mp
159e160 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
9.26e8.35
(m, 3H), 7.88 (m, 2H), 7.71e7.28 (m, 6H), 7.21 (s, 1H), 5.21 (s, 2H),
4.33 (bs, 2H), 1.35 (bs, 3H). 13C NMR (68 MHz, DMSO‑d6):
163.4,
d
9.48 (s, 1H), 8.54 (s, 1H), 7.97e7.73 (m, 2H), 7.58e7.23 (m, 6H),
d
7.19e7.05 (m, 1H), 4.81 (s, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 3.84 (s, 3H),
162.9, 158.6, 151.95, 151.78, 150.0, 136.9, 131.3, 130.1, 128.5, 127.9,
127.7, 119.8, 117.1, 113.4, 103.0, 69.3, 60.2, 14.3. ES-MS (methanol) m/
z: 390.0 [MþH]þ, 412.0 [MþNa]þ. HPLC: 96.4%.
1.33 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6):
d 164.03,
163.36, 159.99, 152.05, 151.91, 148.81, 138.82, 131.70, 130.62, 128.79,
127.86, 127.56, 120.07, 116.97, 112.62, 103.87, 60.82, 55.69, 44.50,
14.68. ES-MS (methanol) m/z: 404.2 [MþH]þ, 426.1 [MþNa]þ.
HRMS (ESI-TOF) m/z: C22H21N5O3 experimental 404.1716 [MþH]þ,
ethyl 5-amino-2-(3,4-bis(benzyloxy)phenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8 carboxylate (108): Flash chromatography eluent:
DCM-MeOH 99:1. Yield 65% (161 mg); white solid; mp 200e202 ꢀC
theoretical 404.1717 [MþH]þ,
D
¼ 0.0001. Purity HPLC (Method A):
(EtOEt-light petroleum). 1H NMR (270 MHz; DMSO‑d6):
d
9.01e8.84
98.5%.
(m, 1H), 8.56e8.36 (m, 2H), 7.94e7.80 (m, 2H), 7.56e7.24 (m, 11H),
ethyl
methoxyphenyl)-[1,2,4]triazolo[1,5-c]-8-carboxylate
5-(([1,10-biphenyl]-4-ylmethyl)amino)-2-(3-
(14): flash
5.29e5.16 (m, 4H), 4.33 (q, J ¼ 7.0 Hz, 2H), 1.36 (t, J ¼ 7.0 Hz, 3H). 13
C
NMR (68 MHz, DMSO‑d6):
d
163.5, 163.0, 151.9, 151.7, 150.5, 150.0,
chromatography eluent: DCM-methanol 99:1. Yield 68% (163 mg);
white solid; mp 160 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz,
DMSO‑d6): 9.52 (s, 1H), 8.56 (s, 1H), 7.98e7.73 (m, 2H), 7.71e7.29
(m, 10H), 7.21e7.05 (m, 1H), 4.85 (s, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 3.85
(s, 3H), 1.33 (t, J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6):
148.2, 137.0, 136.9, 128.4, 127.9, 127.6, 122.9, 121.0, 114.1, 113.0, 102.9,
70.3, 70.0, 60.2, 14.3. ES-MS (methanol) m/z: 496.2 [MþH]þ, 518.2
[MþNa]þ, 534.1 [MþK]þ.
ethyl 5-amino-2-(3,5-bis(benzyloxy)phenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8 carboxylate (109): Flash chromatography eluent:
DCM-MeOH 99:1. Yield 70% (173 mg); white solid; mp 245e248 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz; DMSO‑d6):
d
164.03, 163.39, 162.69, 160.25, 157.51, 151.93, 148.75, 138.07,
131.77, 131.19, 129.35, 128.52, 127.13, 127.03, 121.74, 117.03, 114.32,
112.60, 110.08, 104.00, 60.74, 55.57, 49.29, 14.82. ES-MS (methanol)
m/z: 480.2 [MþH] þ, 502.2 [MþNa] þ. HRMS (ESI-TOF) m/z:
d
9.00e8.52 (m, 3H), 7.68e7.24 (m, 12H), 6.89 (s, 1H), 5.20 (s, 4H),
4.34 (q, J ¼ 7.0 Hz, 2H), 1.35 (t, J ¼ 7.0 Hz, 3H). 13C NMR (68 MHz,
C
28H25N5O3 experimental 502.1852 [MþNa]þ, theoretical 502.1849
DMSO‑d6):
d
163.6, 163.2, 160.05, 152.2, 152.0, 150.3, 137.0, 132.1,
[MþNa]þ,
D
¼ 0.0003. Purity HPLC (Method A): 99.0%.
128.7, 128.1, 127.9, 106.4, 104.2, 103.2, 69.55, 60.2, 14.3. ES-MS
(methanol) m/z: 496.2 [MþH]þ, 518.2 [MþNa]þ, 534.2 [MþK]þ.
General procedure for the synthesis of the N5-substituted 2-
ethyl
5-(methylamino)-2-(3-(benzyloxy)phenyl)-[1,2,4]triazolo
[1,5-c]-8-carboxylate (110): flash chromatography eluent: light
petroleum-EtOAc 8:2. Yield 29% (58 mg); white solid; mp
16

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
174e175 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
8.85 (s, 1H), 8.57 (s, 1H), 7.96e7.76 (m, 2H), 7.61e7.26 (m, 6H),
7.26e7.12 (m, 1H), 5.20 (s, 2H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.10 (s, 3H),
ethyl 5-amino-2-(3-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8-carboxylate (18): flash chromatography eluent: DCM-
methanol 98:2. Yield 51% (30 mg); white solid; mp 282e284 ꢀC
1.34 (t, J ¼ 7.1 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d
163.4, 163.1,
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 9.76 (s,
158.7, 151.7, 151.5, 148.6, 136.9, 131.4, 130.2, 128.5, 128.0, 127.7, 119.8,
1H), 8.97 (bs, 1H), 8.64e8.25 (m, 2H), 7.76e7.63 (m, 2H), 7.36 (t,
J ¼ 8.1 Hz, 1H), 7.02e6.85 (m, 1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 1.36 (t,
117.2, 113.4, 102.9, 69.4, 60.3, þ27.9, 14.4. ES-MS (methanol) m/z:
404.2 [MþH] þ, 426.1 [MþNa]
.
J ¼ 7.1 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 164.0, 163.2, 157.9,
General procedure for the removal of the 3,4-dimethoxybenzyl
group (15e16).
152.1, 152.0, 150.3, 131.4, 130.1, 118.2, 117.8, 114.25, 103.1, 60.2, 14.3.
ES-MS (methanol) m/z: 299.8 [MþH]þ, 321.8 [MþNa]þ, 337.8
[MþK]þ. HRMS (ESI-TOF) m/z: C14H13N5O3 experimental 300.1098
To 0.2 mol of compounds 11 and 12, TFA (1 mL), anisole
(0.8 mmol) and trifluoromethanesulfonic acid (HOTf) (0.8 mmol)
were added at 0 ꢀC and the reaction proceeded for 12 h at room
temperature. The solvent was evaporated, DCM added (15 mL), and
washed with three aliquots of water (5 mL). After drying the
organic phase over anhydrous sodium sulfate, the solvent is evap-
orated and the residue purified by flash chromatography.
[MþH]þ, theoretical 300.1091 [MþH]þ,
D
¼ 0.0007. Purity HPLC
(Method A): 86.4%.
ethyl
5-amino-2-(3,5-bis(hydroxy)phenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8 carboxylate (19): yield 54% (34 mg); white solid; mp
255e260 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
9.55 (bs, 2H), 8.50 (m, 3H), 7.20e7.11 (m, 2H), 6.36 (m, 1H), 4.33 (q,
J ¼ 7.1 Hz, 2H), 1.35 (t, J ¼ 7.1 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
ethyl
5-amino-2-phenyl-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxylate (15): flash chromatography eluent: DCM-EtOAc from
d 164.3, 163.2, 158.95, 152.1, 151.8, 150.2, 131.7, 105.7, 104.95, 103.1,
9:1 to 8:2. Yield 22% (12 mg); white solid; mp 195e198 ꢀC (EtOEt-
60.2, 14.3. ES-MS (methanol) m/z: 316.1 [MþH]þ, 338.0 [MþNa]þ.
light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 8.98 (bs, 1H),
HRMS (ESI-TOF) m/z: C14H13N5O4 experimental 338.0857 [MþNa]þ,
8.52e8.33 (m, 2H), 8.25 (m, 2H), 7.58 (m, 3H), 4.34 (q, J ¼ 7.1 Hz,
theoretical 338.0859 [MþNa]þ,
D
¼ 0.0002. Purity HPLC (Method
2H), 1.35 (t, J ¼ 7.1 Hz, 3H). 13C NMR (68 MHz, CDCl3):
d
163.90,
A): 98.0%.
163.20, 152.20, 152.07, 150.29, 130.84, 130.17, 129.08, 127.39, 103.14,
60.22, 14.26. ES-MS (methanol) m/z: 284.0 [MþH]þ, 305.9
[MþNa]þ, 321.9 [MþK] þ. HRMS (ESI-TOF) m/z: C14H13N5O2 exper-
imental 309.0962 [MþNa]þ, theoretical 306.0961 [MþNa]þ,
ethyl
5-amino-2-(3,4-bis(hydroxy)phenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8 carboxylate (20): yield 64% (40 mg); white solid; mp
270e272 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
9.50e8.80 (m, 3H), 8.50e8.25 (m, 2H), 7.68 (s, 1H), 7.60e7.53 (m,
1H), 6.88 (d, J ¼ 8.2 Hz, 1H), 4.33 (q, J ¼ 7.1 Hz, 2H), 1.35 (t, J ¼ 7.1 Hz,
3H). 13C NMR (68 MHz, DMSO‑d6):
164.1, 163.0, 151.68, 151.57,
D
¼ 0.0001. Purity HPLC (Method A): 99.0%.
ethyl
5-amino-2-(4-fluorophenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
d
dine-8-carboxylate (16): flash chromatography eluent: DCM-EtOAc
149.9, 148.0, 145.4, 121.1, 119.2, 115.8, 114.8, 102.8, 60.1, 14.3. ES-MS
(methanol) m/z: 316.1 [MþH]þ, 338.1 [MþNa]þ. HRMS (ESI-TOF) m/
z: C14H13N5O4 experimental 316.1036 [MþH]þ, theoretical 316.1040
from 9:1 to 8:2. Yield 73% (44.0 mg); pale pink solid; mp 263 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 8.99 (bs,
1H), 8.61e8.37 (m, 2H), 8.28 (dd, J ¼ 8.8 Hz, JHF ¼ 5.6 Hz, 2H), 7.43 (t,
[MþH]þ,
D
¼ 0.0004. Purity HPLC (Method A): 99.4%.
JH,HF ¼ 8.8 Hz, 2H), 4.33 (q, J ¼ 7.1 Hz, 2H), 1.35 (t, J ¼ 7.1 Hz, 3H). 13
C
ethyl 5-methylamino-2-(3-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8-carboxylate (21): yield 66%; white solid (41 mg); mp
234 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
NMR (68 MHz, DMSO‑d6):
d
163.84 (d, JCF ¼ 247.5 Hz), 163.19,
163.06, 152.29, 152.14, 150.29, 129.70 (d, JCF ¼ 8.8 Hz), 126.76 (d,
JCF ¼ 1.2 Hz), 116.19 (d, JCF ¼ 22.0 Hz), 103.12, 60.22, 14.26. ES-MS
(methanol) m/z: 323.9 [MþNa]þ. HRMS (ESI-TOF) m/z:
d
9.73 (s, 1H), 8.81 (bs, 1H), 8.56 (s, 1H), 7.75e7.61 (m, 2H), 7.34 (t,
J ¼ 8.1 Hz, 1H), 6.98e6.84 (m, 1H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.10 (s,
C
14H12FN5O2 experimental 302.1043 [MþH]þ, theoretical 302.1047
3H), 1.34 (t, J ¼ 7.1 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6)
d 163.0,
[MþH]þ,
D
¼ 0.0004. Purity HPLC (Method A): 95.0%.
157.7, 153.9, 153.1, 151.5, 148.6, 131.1, 129.9, 117.9, 117.6, 114.0, 102.9,
60.2, 27.9, 14.3. ES-MS (methanol) m/z: 314.1 [MþH]þ, 336.1
[MþNa]þ. HRMS (ESI-TOF) m/z: C15H15N5O3 experimental 314.1251
General procedure for the removal of benzyl group (17e21) and
the reduction of nitro group to amine (22).
To a stirred suspension of compounds 6,9,107e110 (0.2 mmol)
and an equal weight of 10% PdeC in dry methanol (10 mL),
ammonium formate (2 mmol) was added in a single portion. The
resulting reaction mixture was stirred under argon atmosphere at
60 ꢀC for 3e12 h. Once completed, the catalyst was removed by
filtration through a Celite pad, which was then washed with
methanol. The solvent was evaporated under reduced pressure and
the crude product dissolved in EtOAc (50 mL) and washed with
water (3 ꢂ 15 mL). Finally, the organic phase was dried over
anhydrous sodium sulfate, the solvent evaporated under vacuum
and, if necessary, the residue purified by flash chromatography or
precipitated with EtOEt and light petroleum to afford the desired
derivatives.
[MþH]þ, theoretical 314.1247 [MþH]þ,
D
¼ 0.0004. Purity HPLC
(Method A): 97.6%.
ethyl
5-amino-2-(3-aminophenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8 carboxylate (22): flash chromatography eluent: DCM-
methanol 95:5. Yield 65% (39 mg); white solid; mp 245e250 ꢀC
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 8.97e8.50
(m, 3H), 7.49 (s, 1H), 7.41 (d, J ¼ 7.3 Hz, 1H), 7.18 (t, J ¼ 7.9 Hz, 1H),
6.76e6.72 (m, 1H), 5.37 (s, 2H), 4.33 (q, J ¼ 7.0 Hz, 2H), 1.35 (t,
J ¼ 7.0 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 164.74, 163.21,
151.99, 151.83, 150.24, 149.28, 130.67, 129.42, 116.20, 115.01, 112.83,
103.08, 60.18, 14.30. ES-MS (methanol) m/z: 299.1 [MþH]þ, 321.1
[MþNa]þ. HRMS (ESI-TOF) m/z: C14H14N6O2 experimental 299.1252
[MþH]þ, theoretical 299.1251 [MþH]þ,
D
¼ 0.0001. Purity HPLC
ethyl 5-amino-2-(4-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8-carboxylate (17): flash chromatography eluent: DCM-
methanol from 98:2 to 95:5. Yield 72% (43 mg); white solid;
(Method A): 98.8%.
General procedure for the synthesis of 2,5-disubstitited-[1,2,4]
triazolo[1,5-c]pyrimidine-8-carboxylic acids (23,111e113).
mp
>
300 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
A solution of boron tribromide (1 M in DCM, 12 equivalents) was
slowly added to a cooled (ꢁ78 ꢀC) solution of methoxy-derivatives
7,10,13e14 (1 equivalent) in DCM (5 mL/mmol of methoxy-
derivatives) under argon atmosphere. The cooling bath was
removed and the suspension was slowly warmed up to room
temperature and stirred for 12 h. Once completed, an equal volume
of cool methanol was added dropwise and the solution stirred for
15 min. Then the volatile species were removed under vacuum, thus
DMSO‑d6):
d
10.01 (bs, 1H), 8.89 (bs, 1H), 8.50e8.19 (m, 2H), 8.07 (d,
J ¼ 7.7 Hz, 2H), 6.93 (d, J ¼ 7.7 Hz, 2H), 4.33 (q, J ¼ 6.3 Hz, 2H),1.35 (t,
J ¼ 6.3 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 164.2, 163.3, 160.0,
152.00, 151.9, 150.2, 129.15, 120.9, 115.8, 102.9, 60.2, 14.3. ES-MS
(methanol) m/z: 322.1 [MþNa]þ, 338.0 [MþK]þ. HRMS (ESI-TOF)
m/z:
C
14H13N5O3 experimental 300.1088 [MþH]þ, theoretical
300.1091 [MþH]þ,
D
¼ 0.0003. Purity HPLC (Method A): 95.1%.
17

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
giving the corresponding carboxylic acids derivatives (23,111e113)
that were suspended in methanol and filtered. Compounds
112e113 were obtained starting from 0.25 mmol of compounds 13
and 14 and immediately used in the next step without any
characterization.
under reduced pressure, water (10 mL) and a saturated solution of
sodium carbonate (10 mL) were added to the mixture and the
product was extracted with EtOAc (5 ꢂ 5 mL). The organic layers
were dried over anhydrous sodium sulfate and the compounds
were concentrated under vacuum. The residues were purified with
flash chromatography (ethyl acetate for compound 26 and ethyl
acetate and dichloromethane 10% for compound 27). Finally, the
product was filtered to obtain white solids.
5-amino-2-(3-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxylic acid (23):. Yield 92% (62 mg from 0.25 mmol of 7); white
solid; mp 290e295 ꢀC. 1H NMR (400 MHz, DMSO-D6)
d 12.69 (bs,
1H), 9.81 (s,1H), 8.58 (bm, 3H), 7.89e7.53 (m, 2H), 7.34 (t, J ¼ 7.9 Hz,
5-amino-N-ethyl-2-(3-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]py-
rimidine-8-carboxamide (26):Yield 9% (10 mg); white solid; mp
170e175 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
1H), 6.94 (d, J ¼ 8.0 Hz, 1H). 13C NMR (68 MHz, DMSO)
d 164.63,
163.85, 157.97, 152.20, 152.17, 150.15, 131.25, 130.17, 118.21, 117.88,
114.32, 103.65. ES-MS (methanol) m/z: 272.0 [MþH]þ, 294.0
[MþNa]þ. HRMS (ESI-TOF) m/z: C12H9N5O3 experimental 272.0776
d
9.80 (s, 1H), 8.99e8.13 (m, 4H), 7.79e7.74 (m, 2H), 7.38 (t,
J ¼ 7.6 Hz, 1H), 6.96 (d, J ¼ 7.6 Hz, 1H), 3.48e3.41 (m, 2H), 1.23 (t,
[MþH]þ, theoretical 272.0778 [MþH]þ,
D
¼ 0.0002. Purity HPLC
J ¼ 6.5 Hz, 3H). 13C NMR (68 MHz, DMSO‑d6):
d 174.4, 163.9, 162.9,
(Method A): 91.5%.
161.9, 157.9, 151.9, 149.41, 130.7, 118.3, 114.2, 108.3, 105.1, 33.7, 15.0.
ES-MS (methanol) m/z: 299.1 [MþH]þ, 321.1 [MþNa]þ. HRMS (ESI-
TOF) m/z: C14H14N6O2 experimental 321.1069 [MþNa]þ, theoretical
5-amino-2-(3,5-dihydroxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimi-
dine-8-carboxylic acid (111): Yield: 98% (320 mg from 391 mg of 10);
grey solid. 1H NMR (400 MHz, DMSO)
d
9.19e8.05 (m, 3H), 7.16 (d,
321.1070 [MþNa]þ,
D
¼ 0.0001. Purity HPLC (Method A): 97.2%.
J ¼ 2.1 Hz, 2H), 6.35 (t, J ¼ 2.1 Hz, 1H). ES-MS negative (methanol)
5-amino-2-(3,5-dihydroxyphenyl)-N-ethyl-[1,2,4]triazolo[1,5-c]
m/z 286.0 [M ꢁ H]-.
pyrimidine-8-carboxamide (27): Yield: 2,9% (3,2 mg); white solid;
Synthesis of N5-substituted ethyl 2-(3-hydroxyphenyl)-5-
amino-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylates 24e25.
Carboxylic acids 112e113 (0.25 mmol) were reacted with thionyl
chloride (1 mmol) and DMF (catalytic) in chloroform at reflux for
5 h. Then, the volatile species were removed under reduced pres-
sure, the crude product was suspended in ethanol, and triethyl-
amine was added (0.375 mmol), then the reaction was stirred at
room temperature overnight. When completed, the solvent was
removed under reduced pressure and the product purified by flash
chromatography.
mp 258 d ꢀC (EtOEt-methanol). 1H NMR (400 MHz, DMSO‑d6)
d 9.57
(s, 2H), 8.98e8.00 (m, 4H), 7.16 (d, J ¼ 2.2 Hz, 2H), 6.40 (t, J ¼ 2.2 Hz,
1H), 3.48e3.38 (m, 2H), 1.23 (t, J ¼ 7.2 Hz, 3H). 13C NMR (101 MHz,
CD3OD)
d 163.97, 163.39, 158.59, 151.72, 149.64, 149.37, 131.10,
105.84, 105.01, 104.69, 33.99, 13.71. ES-MS (methanol) m/z 315.2
[MþH]þ, 337.1 [MþNa]þ. ES-MS negative (methanol) m/z 313.1
[M ꢁ H]-. HRMS (ESI-TOF) m/z: C14H14N6O3 experimental 337.1017
[MþNa]þ, theoretical 337.1019 [MþNa]þ,
D
¼ 0.0002.
General procedure for the synthesis of tert-butyl (2-(5-amino-2-
substituted-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxamido)ethyl)
carbamate (28e29).
ethyl 5-(benzylamino)-2-(3-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8-carboxylate (24): flash chromatography eluent: DCM-
methanol 98.5:1.5. Yield 13% (12 mg); white solid; mp 219e220 ꢀC
To carboxylic acid derivatives 23,111 (0.37 mmol) in chloroform
(3 mL), and a catalytic amount of DMF (20 mL), thionyl chloride
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
9.72 (s,
(0.107 mL, 1.48 mmol) was added at 0 ꢀC. The mixture was heated
under reflux for 5 h and under an argon atmosphere. The solvent
was removed under reduced pressure to yield the corresponding
chloride derivative (yellowish oil) which was immediately dis-
solved in THF (3 mL). The mixture was then cooled to 0 ꢀC and an
equimolar amount of triethylamine (0.051 mL, 0.37 mmol) was
1H), 9.44 (s, 1H), 8.54 (s, 1H), 7.77e7.63 (m, 2H), 7.44e7.18 (m, 6H),
6.99e6.84 (m, 1H), 4.81 (s, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 1.33 (t,
J ¼ 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6):
d 164.3, 163.3, 158.1,
151.97, 151.82, 148.8, 138.8, 131.6, 130.4, 128.8, 127.8, 127.5, 118.4,
118.1, 114.6, 103.9, 60.7, 14.7. ES-MS (methanol) m/z: 390.1 [MþH]þ,
412.1 [MþNa]þ. HRMS (ESI-TOF) m/z: C21H19N5O3 experimental
added
to
the
solution;
afterward,
tert-butyl
2-
390.1563 [MþH]þ, theoretical 390.1560 [MþH]þ,
D
¼ 0.0003. Purity
aminoethylcarbamate (118 mg, 0.74 mmol) was added slowly. The
solution was allowed to warm to room temperature and stirred
overnight. Once completed, the solvent was then removed under
reduced pressure and the residue was suspended in EtOAc (30 mL)
and washed with water (3 ꢂ 10 mL). The organic phase was then
dried over anhydrous sodium sulfate and concentrated at reduced
pressure. The crude residue was purified with flash chromatog-
raphy with methanol 2% in ethyl acetate affording compounds
28e29 as solids.
HPLC (Method A): 99.7%.
ethyl
5-(([1,10-biphenyl]-4-ylmethyl)amino)-2-(3-
hydroxyphenyl)-[1,2,4]triazolo[1,5-c] pyrimidine-8 carboxylate (25):
flash chromatography eluent: DCM-methanol 99.5:0.5. Yield 20%
(23 mg); white solid; mp 227e229 ꢀC (EtOEt-light petroleum). 1H
NMR (400 MHz, DMSO‑d6):
d 9.72 (s, 1H), 9.48 (s, 1H), 8.55 (s, 1H),
7.69 (s, 2H), 7.66e7.57 (m, 4H), 7.54e7.30 (m, 6H), 6.97e6.88 (m,
1H), 4.85 (s, 2H), 4.32 (q, J ¼ 7.1 Hz, 2H), 1.33 (t, J ¼ 7.1 Hz, 3H). 13
C
NMR (68 MHz, DMSO‑d6):
d
163.8, 162.9, 157.7, 151.5, 151.35, 139.9,
tert-butyl (2-(5-amino-2-(3-hydroxyphenyl)-[1,2,4]triazolo[1,5-c]
pyrimidine-8-carboxamido)ethyl)carbamate (28): Yield 19% (29 mg);
white solid; mp 220e223 ꢀC (EtOEt-light petroleum). 1H NMR
139.8, 139.0, 137.6, 131.1, 129.9, 128.9, 128.0, 126.7, 126.6, 118.0, 117.7,
115.9, 114.1, 103.4, 60.2, 14.3. ES-MS (methanol) m/z: 465.9 [MþH]þ,
487.9 [MþNa]þ. HRMS (ESI-TOF) m/z: C27H23N5O3 experimental
(400 MHz, DMSO‑d6): d 9.78 (s, 1H), 8.79 (s, 1H), 8.46 (s, 1H), 7.77 (d,
488.1696 [MþNa]þ, theoretical 488.1693 [MþNa]þ,
D
¼ 0.0003.
J ¼ 7.8 Hz,1H), 7.70 (s, 1H), 7.36 (t, J ¼ 7.9 Hz,1H), 7.03e6.94 (m, 2H),
3.47 (d, J ¼ 5.4 Hz, 2H), 3.16 (d, J ¼ 5.6 Hz, 2H), 1.31 (m, 11H). ES-MS
(methanol) m/z: 436.1 [MþNa]þ. HRMS (ESI-TOF) m/z: C19H23N7O4
experimental 436.1707 [MþNa]þ, theoretical 436.1703 [MþNa]þ,
Purity HPLC (Method A): 95.6%.
General procedure for the synthesis of 5-amino-N-ethyl-2-
substituted-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxamides
(26e27).
The mixture of 100 mg of carboxylic acids 23,111 (0.37 mmol)
dissolved in 2 mL of dry pyridine and 2 mL of dry THF was cooled to
D
¼ 0.0004. Purity HPLC (Method A): 91.2%.
tert-butyl
(2-(5-amino-2-(3,5-dihydroxyphenyl)-[1,2,4]triazolo
[1,5-c]pyrimidine-8-carboxamido)ethyl)carbamate (29): Yield 10%
0
ꢀC and 0.405 mL (5.55 mmol) of thionyl chloride were added
(16 mg); white solid; mp 232 d ꢀC (EtOEt-methanol). 1H NMR
dropwise. The reaction was then allowed to warm up to 10 ꢀC, and
2.78 mL of ethylamine 2 M in THF were added (5.55 mmol). The
mixture was left to reach room temperature and stirred for 24 h.
When the reaction was terminated, the pyridine was removed
(400 MHz, CD3OD)
J ¼ 2.2 Hz, 1H), 3.60 (t, J ¼ 6.0 Hz, 2H), 3.40e3.30 (m, 2H), 1.34 (s,
9H); 13C NMR (101 MHz, CD3OD)
197.78, 172.98, 166.01, 163.92,
160.02, 153.14, 151.11 (CH), 150.92, 107.43 (CH), 106.47, 106.09 (CH),
d
8.55 (s, 1H), 7.28 (d, J ¼ 2.2 Hz, 2H), 6.43 (t,
d
18

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
61.53, 41.10 (2xCH2), 28.63 (3xCH3). ES-MS (methanol) m/z 452.1
[M þ Na]þ. HRMS (ESI-TOF) (negative) m/z: C19H23N7O5 experi-
mental 428.1685 [M ꢁ H]þ, theoretical 428.1687 [M ꢁ H]þ,
2H), 6.70 (s, 1H), 6.62 (bs, 2H), 5.79 (bs, 1H), 3.80 (s, 6H). ES-MS
(methanol) m/z 324.1 [MþH]þ.
General procedure for the synthesis of 7-chloro-2-substituted-
[1,2,4]triazolo[1,5-c]pyrimidine-5-amines (118e119).
D
¼ 0.0003. Purity HPLC (Method A): 97.4%.
General procedure for the synthesis of 5-amino-N-(2-
7
mmol of N’-(2-amino-6-chloropyrimidin-4-yl)-benzohy-
aminoethyl)-2-substituted-[1,2,4]triazolo[1,5-c]pyrimidine-8-
carboxamide, trifluoroacetate salts (30e31).
drazide (116e117) in 270 mmol of BSA (bis(trimethylsilyl)acet-
amide, (69.5 mL) are refluxed under argon atmosphere for 12 h.
When the starting material was consumed, 40 mL of ice methanol
were added slowly keeping the temperature near 0 ꢀC, and then the
volatile species were removed under vacuum. The crude material,
dissolved in dichloromethane (200 mL), was washed with water
(3 ꢂ 65 mL). The organic phase was dried over anhydrous sodium
sulfate, concentrated under reduced pressure and the mixture
purified by flash chromatography.
7-chloro-2-(3-methoxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimidine-5-
amine (118): flash chromatography eluent: dichloromethane-
methanol 99:1; solid phase: aluminum oxide. Yield 50% (0.96 g);
white solid; mp 249e250 ꢀC (EtOEt-light petroleum). 1H NMR
The N-BOC derivatives 28e29 were suspended in a 10% solution
of trifluoroacetic acid in DCM (2 mL every 0.5 mmol) and stirred at
room temperature for 2 h. Once completed, the solvent was
removed under reduced pressure and the residue precipitated from
EtOAc-light petroleum to give the desired trifluoroacetate salts
30e31.
5-amino-N-(2-aminoethyl)-2-(3-hydroxyphenyl)-[1,2,4]triazolo
[1,5-c]pyrimidine-8-carboxamide, trifluoroacetate salt (30): Yield
95% (20 mg from 0.05 mol of 28); white solid; mp 210e212 ꢀC
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 9.84 (bs,
1H), 8.93e8.40 (m, 4H), 7.80 (m, 5H), 7.38 (m, 1H), 6.98 (s, 1H),
3.78e3.55 (m, 2H), 3.17e2.92 (m, 2H). 13C NMR (68 MHz,
(400 MHz, DMSO‑d6):
d
8.43 (bs, 2H), 7.90e7.58 (m, 2H), 7.48 (s,
163.99,
DMSO‑d6):
d
163.0, 162.8, 157.7, 151.6, 150.0, 149.3, 130.5, 129.9,
1H), 7.11 (s, 2H), 3.85 (s, 3H). 13C NMR (68 MHz, DMSO‑d6):
d
118.4, 118.0, 114.3, 104.7, 56.4, 37.0. ES-MS (methanol) m/z: 314.1
[MþH]þ, 336.0 [MþNa]þ. HRMS (ESI-TOF) m/z: C14H15N7O2 exper-
imental 314.1356 [MþH]þ, theoretical 314.1359 [MþH]þ,
159.78, 154.71, 148.28, 147.27, 131.47, 130.31, 119.58, 116.76, 112.12,
96.92, 55.30. ES-MS (methanol) m/z: 276.0 [MþH]þ.
7-chloro-2-(3,5-dimethoxyphenyl)-[1,2,4]triazolo[1,5-c]pyr-
imidin-5-amine (119): flash chromatography eluent: EtOAc-light
petroleum 1:1. Yield: 42% (0.88 g); white solid; mp 251 ꢀC
D
¼ 0.0003. Purity HPLC (Method A): 98.5%.
5-amino-N-(2-aminoethyl)-2-(3,5-dihydroxyphenyl)-[1,2,4]tri-
azolo[1,5-c]pyrimidine-8-carboxamide, trifluoroacetate salt (31):
(EtOAc-light petroleum). 1H NMR (400 MHz, DMSO)
d
8.40 (bs, 2H),
7.35 (d, J ¼ 2.3 Hz, 2H), 7.09 (s, 1H), 6.67 (t, J ¼ 2.3 Hz, 1H), 3.83 (s,
6H). 13C NMR (101 MHz, DMSO)
163.65, 160.74, 154.41, 148.05,
Yield: 34%; (8 mg from 0.07 mol of 29) white solid. 1H NMR
(400 MHz, dmso)
7.20 (d, J ¼ 2.1 Hz, 2H), 6.44 (t, J ¼ 2.1 Hz, 1H), 3.76e3.57 (m, 2H),
3.14e2.98 (m, 2H). 13C NMR (101 MHz, DMSO)
163.45, 159.10,
d
9.59 (s, 2H), 9.05e8.12 (m, 4H), 7.83 (bs, 3H),
d
147.02, 131.80, 104.86, 102.73, 96.78, 55.42. ES-MS (methanol) m/z
d
306,1 [MþH]þ.
151.75, 149.76, 144.73, 134.33, 131.28, 106.11, 105.15, 39.34, 37.46.
General synthesis of N7-benzyl-2-substituted-[1,2,4]triazolo
[1,5-c]pyrimidine-5,7-diamine (32,33,36).
ES-MS (methanol) m/z 330.1 [MþH]þ. HRMS (ESI-TOF) m/z:
C
14H15N7O3 experimental 330.1306 [MþH]þ, theoretical 330.1309
Method A: 1.5 mmol of derivatives 118e119 were reacted with
3 mmol of benzylamine and 4.5 mmol of potassium carbonate in
8 mL of ethanol in a sealed tube at 95 ꢀC for 4 days. When the re-
action was completed, the solvent was evaporated and the crude
material purified by chromatography.
[MþH]þ,
D
¼ 0.0003. Purity HPLC (Method A): 95.1%.
Synthesis of 2,5,7-trisubstituted [1,2,4]triazolo[1,5-c]pyrimidines
(32e37).
Synthesis of 2-amino-4,6-dichloro-pyrimidine (115).
To 10 g of 2-amino-4,6-dihydroxy-pyrimidine 114 (79 mmol) in
40 mL of acetonitrile, 22 mL of triethylamine were added
(158 mmol) followed by the dropwise addition of 14.7 mL of
phosphorous oxychloride (158 mmol). The solution was refluxed
for 1 h, cooled to room temperature and ice was added. The
resulting brown solid was filtered, washed with cold water to afford
12.9 g of a brown solid directly reacted in the subsequent step.
Synthesis of N’-(2-amino-6-chloropyrimidin-4-yl)- benzohy-
drazides (116e117).
4 g of 2-amino-4,6-dichloro-pyrimidine (115, 25 mmol) were
reacted with appropriated benzohydrazides (68,71, 29 mmol) and
DBU (5.05 mL, 34 mmol) in 40 mL of THF. The solution was stirred at
room temperature overnight. After evaporation of the solvent un-
der vacuum, the mixture was suspended with 300 mL of EtOAc and
washed with three aliquots of water (3 ꢂ 100 mL). Then, the organic
phase was dried over anhydrous sodium sulfate, concentrated and
purified by flash chromatography, and the product obtained as a
white solid (1.32 g).
in a gradient from DCM-MeOH 99.5:0.5 to 99:1.
Method B: 0.33 mmol of 7-chloro-2-(3,5-dimethoxyphenyl)-
[1,2,4]triazolo[1,5-c]pyrimidin-5-amine (119, 100 mg) were added
to 0.64 mmol (70 mg) of benzylamine, 0.98 mmol (135 mg) of
potassium carbonate and 2 mL of butanol. The mixture was stirred
for 36 h at the temperature of 100 ꢀC. The solvent was removed
under reduced pressure and the residue was purified with flash
chromatography (light petroleum/ethyl acetate 40%). The com-
pound was filtered with ethyl acetate and light petroleum to give a
pale yellow solid.
N7-benzyl-2-(3-methoxyphenyl)-[1,2,4]triazolo[1,5-c]pyrimidine-
5,7-diamine (32): Method A. Yield 28% (131 mg); white solid; mp
207 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d
7.75e7.56 (m, 2H), 7.56e7.15 (m, 9H), 7.07e6.99 (m, 1H), 5.66 (s,
1H), 4.42 (d, J ¼ 6.0 Hz, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, dmso)
d
162.71, 159.35,157.51,156.07, 146.51, 139.81, 132.35,129.71, 128.28,
127.10, 126.67, 119.15, 115.79, 111.74, 73.75, 55.14, 44.74. ES-MS
(methanol) m/z: 347.1 [MþH]þ. HRMS (ESI-TOF) m/z: C19H18N6O
experimental 347.1614 [MþH]þ, theoretical 347.1614 [MþH]þ,
N’-(2-amino-6-chloropyrimidin-4-yl)-3-methoxybenzohydrazide
(116): flash chromatography eluent: dichloromethane-methanol
from 98:2 to 95:5). Yield 18% (1.32 g); white solid; mp 253 ꢀC
D
¼ 0.0000. Purity HPLC (Method A): 99.4%.
N7-benzil-2-(3,5-dimethoxyphenyl)[1,2,4]triazolo[1,5-c]pyr-
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d
10.45 (s,
imidin-5,7-diamine (33): Method B. Yield: 17% (22 mg); pale yellow
1H), 9.09 (s, 1H), 7.45 (m, 3H), 7.15 (d, J ¼ 8.0 Hz, 1H), 6.64 (s, 2H),
5.80 (bs, 1H), 3.82 (s, 3H). ES-MS (methanol) m/z: 294.1 [MþH]þ.
N’-(2-amino-6-chloropyrimidin-4-yl)-3,5-
solid; mp 208 ꢀC (EtOAc-light petroleum). 1H NMR (400 MHz,
CD3OD)
4.3 Hz, 1H), 6.57 (t, J ¼ 2.3 Hz, 1H), 5.73 (s, 1H), 4.49 (s, 2H), 3.84 (s,
6H). 13C NMR (101 MHz, CD3OD)
164.57, 162.48, 159.84, 157.70,
d
7.40e7.35 (m, 2H), 7.34e7.28 (m, 4H), 7.23 (dt, J ¼ 9.3,
dimethoxybenzohydrazide (117): flash chromatography eluent:
d
EtOAc. Yield: 44% (3.6 g); white solid; mp 200 ꢀC (EtOAc). 1H NMR
148.26, 140.65, 133.55, 129.51 (CH), 128.21 (CH), 128.01 (CH), 106.09
(CH), 103.59 (CH), 74.98 (CH), 55.92 (CH3), 46.62 (CH2). ES-MS
(400 MHz, DMSO)
d
10.41 (bs, 1H), 9.07 (bs, 1H), 7.07 (d, J ¼ 2.1 Hz,
19

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
(methanol) m/z 377.2 [MþH]þ. HRMS (ESI-TOF) m/z: C20H20N6O2
experimental 377.1721 [MþH]þ, theoretical 377.1720 [MþH]þ,
reaction was extracted with methanol and a white solid (253.7 g)
was obtained. Yield 71%; mp 235 ꢀC (EtOAc-light petroleum); 1H
D
¼ 0.0001. Purity HPLC (Method A): 97.7%.
NMR (270 MHz, CDCl3):
6H).
d 7.39 (m, 6H), 7.32e7.22 (m, 3H), 7.17 (m,
2-(3,5-dimethoxyphenyl)-N7,N7-dimethyl-[1,2,4]triazolo[1,5-c]py-
rimidine-5,7-diamine (36): during synthesis of compound 33 using
method A, 5 drops of DMF were added to the reaction mixture to
solubilize the starting material and reaction was heated at 100 ꢀC
for 3 days. Compound 33 was not detected while the only product
obtained was compound 36. Yield: 30% (0.141 g), white solid; mp
General procedure for the synthesis of substituted N’-(4,6-
diphenoxy-1,3,5-triazin-2-yl)hydrazides (123e127).
2,4,6-triphenoxy-[1,3,5]triazine (121, 10.0 g, 0.028 mol) and the
required hydrazide (65, 68, 71e72, 122) (0.0476 mmol) was dis-
solved in anhydrous THF (200 mL) while DBU (7.1 mL,
0.0476 mmol) was added dropwise at 0 ꢀC. The mixture was stirred
at room temperature for 12 h. The solvent was then removed, the
residue was dissolved in DCM (300 mL), and the resulting solution
was washed with water (3 ꢂ 100 mL). The organic layer was
concentrated, dried over anhydrous sodium sulfate, and purified by
flash chromatography.
242 ꢀC (EtOEt-methanol). 1H NMR (400 MHz, DMSO)
d
7.53 (bs, 2H),
7.30 (d, J ¼ 2.4 Hz, 2H), 6.60 (t, J ¼ 2.4 Hz, 1H), 5.84 (s, 1H), 3.81 (s,
6H), 3.03 (s, 6H). 13C NMR (101 MHz, DMSO)
162.78, 160.54,
d
157.52, 156.24, 145.80, 132.95, 104.63 (CHx2), 102.03 (CH), 73.70
(CH), 55.32 (CH3x2), 37.61 (CH3x2). ES-MS (methanol) m/z 315.1
[MþH]þ. HRMS (ESI-TOF) m/z: C15H18N6O2 experimental 315.1559
[MþH]þ, theoretical 315.1564 [MþH]þ,
D
¼ 0.0005. Purity HPLC
N’-(4,6-diphenoxy-1,3,5-triazin-2-yl)-4-methoxybenzohydrazide
(123): flash chromatography eluent: DCM-EtOAc 98:2. Yield 48%
(5.77 g); white solid; mp 220e221 ꢀC (EtOEt-light petroleum). 1H
(Method A): 96.0%.
General procedure for deprotection of 7-substituted [1,2,4]tri-
azolo[1,5-c]pyrimidines (34,35e37).
NMR (270 MHz, CDCl3):
d
8.59 (bs, 1H), 7.68 (d, J ¼ 8.7 Hz, 2H),
12 equivalents of a 10 M solution of boron tribromide in DCM
were slowly added to a cooled (ꢁ78 ꢀC) solution of methoxy-
derivatives (32,33,361 equivalent) in dichloromethane (10 mL/
mmol of methoxy derivatives) under argon atmosphere. The cool-
ing bath was removed and the suspension was slowly warmed up
to room temperature and stirred for 12 h. Once completed, at least
an equal volume of cool methanol was added dropwise and the
solution stirred for 15 minutes-1 hour. Then the volatile species
were removed under reduced pressure, thus giving the corre-
sponding deprotected derivatives (34,35e37).
7.42e7.32 (m, 2H), 7.30e7.19 (m, 4H), 7.21e7.06 (m, 5H), 6.87 (d,
J ¼ 8.7 Hz, 2H), 3.84 (s, 3H). ES-MS (methanol) m/z: 430.2 [MþH]þ,
452.2 [MþNa]þ.
N’-(4,6-diphenoxy-1,3,5-triazin-2-yl)-3-methoxybenzohydrazide
(124): flash chromatography eluent: DCM-EtOAc 95:5. Yield 39%
(4.69 g); white solid; mp 182e184 ꢀC (EtOEt-light petroleum). 1H
NMR (400 MHz, DMSO‑d6):
d 10.45 (s, 1H), 10.05 (s, 1H), 7.49e7.21
(m, 10H), 7.18e7.09 (m, 4H), 3.80 (s, 3H). ES-MS (methanol) m/z:
430.1 [MþH]þ, 452.1 [MþNa]þ.
N’-(4,6-diphenoxy-1,3,5-triazin-2-yl)-3,5-
3-(5-amino-7-(benzylamino)-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)
phenol (34): Flash chromatography eluent: DCM-MeOH 98.5:1.5.
Yield 39% (38 mg from 0.29 mmol of 32); white solid; mp 267 ꢀC
dimethoxybenzohydrazide (125): flash chromatography eluent:
DCM-EtOAc 90:10. Yield 38% (4.89 g); white solid; mp 212e213 ꢀC
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 10.45 (s,
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6)
d
9.59 (s,
1H), 10.05 (s, 1H), 7.53e7.35 (m, 2H), 7.35e7.04 (m, 8H), 7.04e6.89
(m, 2H), 6.73e6.68 (m, 1H), 3.79 (s, 6H). ES-MS (methanol) m/z:
460.2 [MþH]þ, 482.1 [MþNa]þ.
1H), 7.63e7.51 (m, 2H), 7.45 (bs, 2H), 7.41e7.12 (m, 7H), 6.95e6.72
(m, 1H), 5.66 (s, 1H), 4.42 (d, J ¼ 6.0 Hz, 2H). 13C NMR (101 MHz,
dmso)
d
162.97, 157.47, 157.44, 156.00, 146.49, 139.85, 132.20,
N’-(4,6-diphenoxy-1,3,5-triazin-2-yl)benzohydrazide (126): flash
chromatography eluent: DCM-EtOAc 95:5. Yield 62% (4.95 g); white
solid; mp 204e207 ꢀC (EtOEt-light petroleum); 1H NMR (270 MHz,
129.55, 128.28, 127.08, 126.67, 117.66, 116.86, 113.70, 73.75, 44.73.
ES-MS (methanol) m/z: 333.2 [MþH]þ. HRMS (ESI-TOF) m/z:
C
18H16N6O experimental 333.1459 [MþH]þ, theoretical 333.1458
CDCl3):
d
8.54 (s, 1H), 7.66 (d, J ¼ 7.6 Hz, 2H), 7.52 (q, J ¼ 7.0 Hz, 1H),
[MþH]þ,
D
¼ 0.0001. Purity HPLC (Method A): 95.7%.
7.46e7.31 (m, 4H), 7.31e7.19 (m, 4H), 7.11 (dd, J ¼ 12.8, 7.8 Hz, 5H).
ES-MS (methanol) m/z: 400.3 [MþH]þ, 422.2 [MþNa]þ, 438.2
[MþK]þ.
5-(5-amino-7-(benzylamino)-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl)
benzene-1,3-diol (35): Yield 30% (6.6 mg from 0.058 mmol of 33);
white solid; mp 250-256 d ꢀC (EtOEt-methanol). 1H NMR (400 MHz,
N’-(4,6-diphenoxy-1,3,5-triazin-2-yl)-3,4-
DMSO‑d6)
d
9.38 (s, 2H), 7.52e7.27 (m, 6H), 7.27e7.09 (m, 2H), 6.99
dimethoxybenzohydrazide (127): flash chromatography eluent:
(s, 2H), 6.28 (s, 1H), 5.64 (s, 1H), 4.42 (d, J ¼ 5.7 Hz, 2H). 13C NMR
DCM-EtOAc 90:10. Yield 25% (3.22 g); white solid; mp 146e149 ꢀC
(101 MHz, CD3OD)
d
163.48, 158.41, 158.29, 156.14, 146.77, 139.24,
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 10.34 (s,
132.03, 128.08, 126.77, 126.57, 105.49, 104.06, 72.21, 45.20. ES-MS
(methanol) m/z 349.2 [MþH]þ. HRMS (ESI-TOF) m/z: C18H16N6O2
experimental 349.1404 [MþH]þ, theoretical 349.1407 [MþH]þ,
1H), 9.99 (s, 1H), 7.55e6.97 (m, 13H), 3.83 (s, 3H), 3.79 (s, 3H). ES-
MS (methanol) m/z: 460.2 [MþH]þ, 482.2 [MþNa]þ.
General procedure for the synthesis of 5,7-diphenoxy-[1,2,4]
triazolo[1,5-a][1,3,5]triazines (128e132) The mixture of phospho-
rous pentoxyde (0.045 mol, Method A or 0.075 mol, Method B) and
hexamethyldisiloxane (0.045 mol, Method A or 0.075 mol Method
B) in anhydrous xylene (150 mL) was heated to 90 ꢀC over 1.5 h and
then stirred for 1 h at 90 ꢀC, under argon atmosphere. The well-
dried hydrazides compounds (123e127) (0.015 mol, 1 equivalent)
were then added to the clear solution and the temperature is
increased to reflux and stirred for 2e4 h. The solvent was then
removed, the residue dissolved in DCM (300 mL) and the resulting
solution was washed with water (3 ꢂ 100 mL). The organic layer
was concentrated, dried over anhydrous sodium sulfate, and puri-
fied to afford the desired compounds.
D
¼ 0.0003. Purity HPLC (Method A): 98.9%.
5-(5-amino-7-(dimethylamino)-[1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)benzene-1,3-diol (37): Yield 82% (122 mg from 0.45 mmol of 36);
white solid; mp 268 d ꢀC (EtOEt-methanol). 1H NMR (400 MHz,
DMSO‑d6)
d
9.80 (s, 2H), 8.16 (s, 2H), 6.94 (d, J ¼ 2.0 Hz, 2H), 6.51 (t,
J ¼ 2.0 Hz, 1H), 5.89 (s, 1H), 3.12 (s, 6H). 13C NMR (101 MHz, DMSO)
d
159.55, 159.42, 146.73, 146.45, 106.59, 105.80, 71.50, 38.22 (some
signals were not detected). ES-MS (methanol) m/z 287.1 [MþH]þ,
309.1 [MþNa]þ. HRMS (ESI-TOF) m/z: C13H14N6O2 experimental
287.1253 [MþH]þ, theoretical 287.1251 [MþH]þ,
D
¼ 0.0002. Purity
HPLC (Method A): 98.2%.
Synthesis of 2,5,7-trisubstituted [1,2,4]triazolo[1,5-a][1,3,5]tri-
azines (38e57).
Synthesis of 2,4,6-triphenoxy-[1,3,5]triazine (121).
A mixture of 2,4,6-trichloro-[1,3,5]triazine (120, 184.4 g, 1.0 mol)
was dissolved in phenol (3.0e4.0 mol) and refluxed for 5h. The hot
2-(4-methoxyphenyl)-5,7-diphenoxy-[1,2,4]triazolo[1,5-a][1,3,5]
triazine (128): Method A. Flash chromatography eluent: DCM. Yield
39% (2.41 g); white solid; mp 216 ꢀC (EtOEt-light petroleum). 1H
NMR (270 MHz, CDCl3):
d
8.28 (d, J ¼ 8.8 Hz, 2H), 7.56e7.47 (m, 2H),
20

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
7.44e7.34 (m, 5H), 7.24e7.15 (m, 3H), 7.00 (d, J ¼ 8.8 Hz, 2H), 3.88 (s,
triazin-7-amine (135): yield 95% (0.692 mg); white solid; mp 274 ꢀC
3H).
ES-MS
(acetonitrile)
m/z:
412.2
[MþH]þ0.2-(3-
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
9.12 (s,
1H), 8.78 (s, 1H), 7.46 (t, J ¼ 7.8 Hz, 2H), 7.36e7.20 (m, 5H), 6.65 (s,
1H), 3.81 (s, 6H). 13C NMR (101 MHz, DMSO‑d6):
165.3,164.0, 161.1,
methoxyphenyl)-5,7-diphenoxy-[1,2,4]triazolo[1,5-a][1,3,5]triazine
(129):Method B. Flash chromatography eluent: DCM. Yield 71%
(4.38 g); white solid; mp 235 ꢀC (EtOEt-light petroleum). 1H NMR
d
159.4,152.8, 152.4, 132.5, 130.0, 125.9,122.3, 110.0, 105.0, 103.1, 55.8.
(400 MHz, DMSO‑d6):
7.65e7.39 (m, 8H), 7.32 (t, J ¼ 7.3 Hz, 1H), 7.28e7.19 (m, 2H),
7.18e7.11 (m, 1H), 3.85 (s, 3H). 13C NMR (68 MHz, CDCl3):
167.51,
d
7.80 (d, J ¼ 7.6 Hz, 1H), 7.70 (s, 1H),
ES-MS (methanol) m/z: 365.1 [MþH]þ, 387.1 [MþNa]þ. MW 364.36.
C
18H16N6O3.
d
5-phenoxy-2-phenyl-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-amine
164.98, 160.68, 160.18, 155.03, 152.06, 150.78, 130.91, 130.35, 130.05,
129.90, 127.79, 126.47, 121.73, 121.42, 120.44, 118.42, 112.20, 55.59.
ES-MS (acetonitrile) m/z: 412.1 [MþH]þ, 434.1 [MþNa]þ.
(136): flash chromatography eluent: gradient from DCM-EtOAc
99:1 to 98:2. Yield 89% (0.133 g from 0.5 mmol of 131); white
solid. 1H NMR (270 MHz, DMSO)
d 9.11 (s, 1H), 8.79 (s, 1H), 8.13 (d,
2-(3,5-dimethoxyphenyl)-5,7-diphenoxy-[1,2,4]triazolo[1,5-a]
[1,3,5]triazine (130): Method B. Flash chromatography eluent: DCM.
Yield 25% (1.65 g); white solid; mp 211 ꢀC (EtOEt-light petroleum).
J ¼ 3.8 Hz, 2H), 7.74e7.38 (m, 5H), 7.38e7.03 (m, 3H). ES-MS
(methanol) m/z: 305.2 [MþH]þ, 327.1 [MþNa]þ.
2-(3,4-dimethoxyphenyl)-5-phenoxy-[1,2,4]triazolo[1,5-a][1,3,5]
triazin-7-amine (137): flash chromatography eluent: DCM-MeOH
99:1. Yield 89.5% (0.652 g); white solid; mp 276e278 ꢀC (EtOEt-
1H NMR (270 MHz, CDCl3):
d
7.59e7.47 (m, 4H), 7.47e7.34 (m, 5H),
7.34e7.12 (m, 3H), 6.68e6.55 (m, 1H), 3.86 (s, 6H). 13C NMR
(101 MHz, CDCl3): 167.18, 164.67, 161.00, 160.32, 154.72, 151.77,
d
light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 9.02 (bs, 1H), 8.83
150.51, 131.19, 130.09, 129.64, 127.53, 126.22, 121.49,121.18, 105.27,
104.65, 55.64. ES-MS (acetonitrile) m/z: 442.2 [MþH]þ, 464.1
[MþNa]þ.
(bs, 1H), 7.76e7.73 (m, 2H), 7.53e7.41 (m, 2H), 7.34e7.21 (m, 3H),
7.17e7.08 (m, 1H), 3.83 (s, 6H). 13C NMR (101 MHz, DMSO‑d6):
d
165.2, 164.3, 159.4, 152.9, 152.3, 151.3, 149.2, 130.0, 125.8, 123.1,
2-phenyl-5,7-diphenoxy-[1,2,4]triazolo[1,5-a][1,3,5]triazine (131):
Method A. Flash chromatography eluent: DCM. Yield 75% (4.3 g);
white solid; mp 246e250 ꢀC (EtOEt-light petroleum); 1H NMR
122.3, 120.4, 112.2, 110.2, 56.0, 55.9. ES-MS (methanol) m/z: 365.2
[MþH]þ, 387.1 [MþNa]þ.
General procedure for the synthesis of N5-benzyl-[1,2,4]triazolo
[1,5-a][1,3,5]triazin-5,7-diamine (38,39,41e47) and N7,N5-diben-
zyl-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5,7-diamine (40).
(270 MHz, CDCl3)
d
: 8.38e8.28 (m, 2H), 7.65e7.34 (m, 9H),
167.36, 164.72,
7.34e7.10 (m, 4H). 13C NMR (68 MHz, CDCl3):
d
160.49, 154.80, 151.83, 150.55, 131.24, 130.08, 129.62, 129.41, 128.73,
127.81, 127.52, 126.20, 121.47, 121.17. ES-MS (methanol) m/z: 382.2
[MþH]þ, 404.2 [MþNa]þ, 420.1 [MþK]þ.
Method A: A solution of 7-amino-5phenoxy-[1,2,4]triazolo[1,5-
a][1,3,5]triazine derivatives (133e134, 136e137) (2 mmol) with
6 mmol of the required amine in ethanol (20 mL) was stirred at
95e100 ꢀC in a sealed tube for 24e72 h. The solvent was removed
under reduced pressure and the residue was purified, when
necessary, by flash chromatography or directly crystallized from
EtOEt-light petroleum and washed with a small amount of meth-
anol to afford the desired compounds as solids.
2-(3,4-dimethoxyphenyl)-5,7-diphenoxy-[1,2,4]triazolo[1,5-a]
[1,3,5]triazine (132): Method B. Flash chromatography eluent: DCM-
MeOH 99:1. Yield 51.5% (3.41 g); white solid; mp 248e249 ꢀC
(EtOEt-light petroleum). 1H NMR (270 MHz, CDCl3):
d 8.06e7.88 (m,
1H), 7.84 (m, 1H), 7.58e7.47 (m, 2H), 7.47e7.32 (m, 5H), 7.32e7.13
(m, 3H), 6.98 (m, 1H), 3.98 (s, 3H), 3.95 (s, 3H). 13C NMR (68 MHz,
Method B: 0.750 mmol of 2-(3,5-dimethoxyphenyl)-5-phenoxy-
[1,2,4]triazolo[1,5-a][1,3,5]triazine-7-amine (135) were dissolved
in 5 mL of butanol, then 2.25 mmol of the desired amine were
added. The reaction was allowed to react in a microwave (100 W) at
170 ꢀC for 2 h. Once the reaction was completed, the solvent was
removed under reduced pressure and the residue was purified by
flash chromatography.
DMSO‑d6):
d 165.5, 164.4, 160.4, 155.1, 152.0, 151.5, 150.7, 149.1,
130.2, 129.8127.4, 126.2, 121.9, 121.6, 121.4, 120.5, 112.0, 109.7, 55.6,
55.5. ES-MS (acetonitrile) m/z: 442.2 [MþH]þ, 464.2 [MþNa]þ.
General procedure for the synthesis of 5-phenoxy-[1,2,4]tri-
azolo[1,5-a][1,3,5]triazin-7-amines (133e137).
A
solution of the 5,7-diphenoxy-[1,2,4]triazolo[1,5-a][1,3,5]
triazine derivative (2 mmol) in methanol (20 mL) with methanolic
ammonia 7 N (12 mmol) was stirred for 2 h at room temperature.
The solvent was removed under reduced pressure and the residue
purified by flash chromatography, when required, or directly
crystallized from EtOEt-light petroleum to afford the desired
compounds as solids.
N5-benzyl-2-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a][1,3,5]
triazine-5,7-diamine (38): Method A. Flash chromatography eluent:
DCM-MeOH 99:1. Yield 12% (21 mg); white solid; mp 266e269 ꢀC
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
8.51e7.75
(m, 5H), 7.49e7.14 (m, 5H), 7.14e6.92 (m, 2H), 4.50 (d, J ¼ 6.9 Hz,
2H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSO‑d6):
161.2, 160.1, 154.5,
d
2-(4-methoxyphenyl)-5-phenoxy-[1,2,4]triazolo[1,5-a][1,3,5]tri-
azin-7-amine (133): flash chromatography eluent: DCM-MeOH
99:1. Yield 86% (0.575 g); white solid; mp > 300 ꢀC (EtOEt-light
140.4, 136.2, 132.9, 128.6, 127.5, 125.2, 123.7, 122.6, 114.5, 55.7, 44.2.
ES-MS (methanol) m/z: 348.1 [MþH]þ, 370.1 [MþNa]þ. HRMS (ESI-
TOF) m/z: C18H17N7O experimental 348.1570 [MþH]þ, theoretical
petroleum).1H NMR (400 MHz, DMSO‑d6):
d
9.07 (s, 1H), 8.72 (s,
348.1567 [MþH]þ,
D
¼ 0.0003. Purity HPLC (Method A): 96.4%.
1H), 8.06 (d, J ¼ 8.8 Hz, 2H), 7.46 (t, J ¼ 7.9 Hz, 2H), 7.39e7.20 (m,
N5-benzyl-2-(3-methoxyphenyl)-[1,2,4]triazolo[1,5-a][1,3,5]
triazine-5,7-diamine (39): Method A. Flash chromatography eluent:
DCM-MeOH 97:3. Yield 46% (80 mg); white solid; mp 271 ꢀC
3H), 7.09 (d, J ¼ 8.8 Hz, 2H), 3.83 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6):
d 165.2, 164.2, 161.6, 159.4, 152.9, 152.3, 130.0, 128.8,
125.8, 123.1, 122.3, 114.7, 55.8. ES-MS (methanol) m/z: 335.1
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 8.54e8.01
[MþH]þ, 357.1 [MþNa]þ.
(m, 2H), 7.98 (t, J ¼ 6.3 Hz, 1H) 7.73e7.61 (m, 2H), 7.42 (t, J ¼ 7.9 Hz,
1H), 7.37e7.28 (m, 4H), 7.28e7.19 (m, 1H), 7.09e7.01 (m, 1H),
4.57e4.45 (m, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO‑d6):
2-(3-methoxyphenyl)-5-phenoxy-[1,2,4]triazolo[1,5-a][1,3,5]tri-
azin-7-amine (134): flash chromatography eluent: DCM-MeOH
99:1. Yield 91% (0.608 g); white solid; mp 226e229 ꢀC (EtOEt-
d
162.94, 161.6, 159.8, 151.1, 150.6, 140.4, 132.6, 130.3, 128.6, 127.5,
light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d
9.13 (s, 1H), 8.80
127.0, 119.4, 116.5, 112.0, 55.6, 44.2. ES-MS (methanol) m/z: 348,1
[MþH]þ, 370,1 [MþNa]þ. HRMS (ESI-TOF) m/z: C18H17N7O experi-
mental 348.1568 [MþH]þ, theoretical 348.1567 [MþH]þ,
(s, 1H), 7.73 (d, J ¼ 7.7 Hz, 1H), 7.67 (s, 1H), 7.57e7.40 (m, 3H),
7.37e7.19 (m, 3H), 7.10 (d, J ¼ 7.7, 1H), 3.83 (s, 3H). 13C NMR
(101 MHz, DMSO‑d6):
d
165.3, 164.1, 159.9, 159.4, 152.9, 152.4, 132.0,
D
¼ 0.0001. Purity HPLC (Method A): 98.8%.
130.5, 130.0, 125.9, 122.3, 119.6, 116.9, 112.2, 55.7. ES-MS (methanol)
In this case, also the N5,N7-dibenzyl-2-(3-methoxyphenyl)-[1,2,4]
m/z: 335.1 [MþH]þ, 357.1 [MþNa]þ, 373.0 [MþK]þ.
triazolo[1,5-a][1,3,5]triazine-5,7-diamine was isolated (40): Yield
2-(3,5-dimethoxyphenyl)-5-phenoxy-[1,2,4]triazolo[1,5-a][1,3,5]
15% (33 mg); white solid; mp 169e170 ꢀC (EtOEt-light petroleum).
21

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
1H NMR (400 MHz, DMSO‑d6):
d
9.22 þ 9.07 (m, 1H), 8.20-8-09 (m,
230e232 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6)
1H), 7.70 (d, J ¼ 7.6 Hz, 1H), 7.64 (s, 1H), 7.51e7.16 (m, 11H),
d
8.48e7.82 (m, 2H), 7.39e7.18 (m, 2H), 6.61 (t, J ¼ 2.4 Hz, 1H), 3.81
(s, 7H), 1.95e1.82 (m, 2H), 1.79e1.68 (m, 2H), 1.65e1.54 (m, 1H),
1.38e1.04 (m, 5H).13C NMR (101 MHz, DMSO‑d6)
163.56, 160.57,
7.10e7.03 (m, 1H), 4.72e4.56 (m, 2H), 4.50 (d, J ¼ 6.2 Hz, 2H), 3.82
(s, 3H). 13C NMR (101 MHz, DMSO‑d6):
d
163.0, 161.4, 159.9, 149.2,
d
140.2, 138.9, 132.6, 130.3, 128.8, 128.6, 128.2, 127.9, 127.6, 127.5,
127.1, 119.4, 116.6, 111.9, 55.6, 44.3, 43.7. ES-MS (methanol) m/z:
438.2 [MþH]þ, 460.2 [MþNa]þ. HRMS (ESI-TOF) m/z: C25H23N7O
experimental 438.2032 [MþH]þ, theoretical 438.2036 [MþH]þ,
160.20, 159.35, 132.81, 132.19, 104.38 (2 x CH2), 101.03 (CH), 78.33
(CH), 55.33 (2 x CH3), 32.25 (2 x CH2), 25.31 (CH2), 24.92 (2 x CH2).
HRMS (ESI-TOF) m/z: C18H23N7O2 experimental 370.1988 [MþH]þ,
theoretical 370.1987 [MþH]þ,
[MþNa]þ, 392.1805 [MþNa]þ,
98.3%.
D
¼ 0.0001; experimental 392.1807
¼ 0.0002. Purity HPLC (Method B):
D
¼ 0.0004. Purity HPLC (Method A): 95.1%.
D
N5-benzyl-2-(3,5-dimethoxyphenyl)-[1,2,4]triazolo[1,5-a][1,3,5]
triazine-5,7-diamine (41): Method A. Flash chromatography eluent:
EtOAc-light petroleum 7:3. Yield 10% (19 mg); white solid; mp
279e281 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
N5-benzyl-2-phenyl-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-
diamine (46): Method A. Flash chromatography eluent: DCM-MeOH
99:1. Yield 27% (12 mg from 0.141 mmol of 136); white solid. 1H
DMSO‑d6):
d
8.46e7.84 (m, 3H), 7.43e7.13 (m, 7H), 6.59 (bs, 1H),
NMR (200 MHz, DMSO) d 8.73e7.78 (m, 5H), 7.65e7.42 (m, 3H),
4.51 (bs, 2H), 3.79 (s, 6H). ES-MS (methanol) m/z: 378.1 [MþH]þ,
400.1 [MþNa]þ, 416.1 [MþK]þ. HRMS (ESI-TOF) m/z: C19H19N7O2
experimental 378.1672 [MþH]þ, theoretical 378.1672 [MþH]þ,
7.40e7.01 (m, 5H), 4.51 (d, J ¼ 5.7 Hz, 2H). ES-MS (methanol) m/z:
318.2 [MþH]þ, 340.1 [MþNa]þ.
N5-benzyl-2-(3,4-dimethoxyphenyl)-[1,2,4]triazolo[1,5-a][1,3,5]
triazine-5,7-diamine (47): Method A. Flash chromatography eluent:
D
¼ 0.0000. Purity HPLC (Method A): 97.7%.
N5-(4-chlorobenzyl)-2-(3,5-dimethoxyphenyl)-[1,2,4]-triazolo-
DCM-MeOH 97:3. Yield 33% (62 mg); white solid; mp 234e235 ꢀ
C
[1,5-a]-[1,3,5]-triazine-5,7-diamine (42): Method B. flash chroma-
tography eluent: DCM-MeOH 98:2. Yield: 39% (0.110 g); white
solid; mp 285e287 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
(EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
d 8.42e7.81
(m, 3H), 7.72e7.55 (m, 2H), 7.44e7.18 (m, 6H), 7.08 (d, J ¼ 8.4 Hz,
1H), 4.50 (m, 1H), 3.82 (s, 6H). 13C NMR (101 MHz, DMSO‑d6):
DMSO‑d6)
J ¼ 2.4 Hz, 2H), 6.61 (t, J ¼ 2.4 Hz, 1H), 4.48 (d, J ¼ 6.4 Hz, 2H), 3.80
(s, 6H). 13C NMR (101 MHz, DMSO‑d6)
167.55, 161.59, 161.02,
d
8.48e7.90 (m, 3H), 7.42e7.34 (m, 4H), 7.26 (d,
d 160.0, 159.6, 150.9, 150.5, 149.1, 140.5, 136.1, 130.7, 128.6, 127.5,
127.0, 120.1, 112.1, 110.2, 55.99, 55.81, 44.24. ES-MS (methanol) m/z:
d
378.2 [MþH]þ, 400.1 [MþNa]þ, 416.1 [MþK]þ. HRMS (ESI-TOF) m/z:
159.65, 150.60, 139.49, 133.14, 131.53, 129.36 (2 x CH), 128.58 (2 x
CH), 104.85 (2 x CH), 102.73 (CH), 55.79 (2 x CH3), 43.64 (CH2).
MW ¼ 411.1211. ES-MS (methanol) m/z: 412,1 [MþH]þ. HRMS (ESI-
TOF) m/z: C19H18ClN7O2 experimental 412.2282 [MþH]þ, theoret-
C
19H19N7O2 experimental 378.1669 [MþH]þ, theoretical 378.1672
[MþH]þ,
D
¼ 0.0003. Purity HPLC (Method B): 99.7%.
General procedure for deprotection of the hydroxyl group of
2,5,7-disubstituted [1,2,4]triazolo[1,5-a][1,3,5]triazines (48e57).
3 equivalents of a 1 M solution of boron tribromide in DCM were
slowly added to a cooled (ꢁ78 ꢀC) solution of methoxy-derivatives
(38e45,47, 1 equivalent) in dichloromethane (10 mL/mmol of
methoxy derivatives) under argon atmosphere. The cooling bath
was removed and the suspension was slowly warmed up to room
temperature and stirred for 12 h. Once completed, at least an equal
volume of cool methanol was added dropwise and the solution
stirred for 15 minutes-1 hour. Then the volatile species were
removed under reduced pressure, thus giving the corresponding
deprotected derivatives (48e57) that were purified by column
chromatography or preparative TLC.
ical 412.1283 [MþH]þ,
D
¼
0.0999; experimental 434.1101
37
[MþNa]þ, 434.1103 [MþNa]þ,
D
¼ 0.0002; C19
H ClN7O2 experi-
18
mental 414.1260 [MþH]þ, theoretical 414.1253 [MþH]þ,
D
D
¼ 0.0007; experimental 436.1084 [MþNa]þ, 436.1073 [MþNa]þ,
¼ 0.0011. Purity HPLC (Method B): 96.1%
N5-(4-fluorobenzyl)-2-(3,5-dimetoxyphenyl)-[1,2,4]-triazolo-[1,5-
a]-[1,3,5]-triazine-5,7-diamine (43): Method B. flash chromatog-
raphy eluent: DCM-MeOH 98:2. Yield: 41% (0.112 g); white solid;
mp 264e266 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
DMSO‑d6)
d 8.27e7.91 (m, 3H), 7.40e7.34 (m, 2H), 7.26 (d,
J ¼ 2.3 Hz, 2H), 7.17e7.09 (m, 2H), 6.61 (t, J ¼ 2.3 Hz, 1H), 4.48 (d,
J ¼ 6.5 Hz, 2H), 3.80 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
d
170.93,
4-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-
2-yl)phenol (48): flash chromatography eluent: DCM-MeOH 95:5.
Yield 30% (25 mg from 0.25 mmol of 38); white solid; mp 295 ꢀC
1
161.14, 160.59, 159.24, 150.16, 136.11, 131.92 (d, JCF ¼ 160.6 Hz),
131.11, 129.00 (d, 2JCF ¼ 8.0 Hz, 2 x CH), 114.89 (d, 3JCF ¼ 21.0 Hz, 2 x
CH), 104.43 (2 x CH), 102.29 (CH), 55.34 (2 x CH3), 43.12 (CH2).
HRMS (ESI-TOF) m/z: C19H18FN7O2 experimental 396.1580
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
d 9.87 (s,
1H), 8.51e7.80 (m, 5H), 7.41e7.15 (m, 5H), 6.86 (d, J ¼ 8.2 Hz, 2H),
[MþH]þ, theoretical 396.1579 [MþH]þ,
418.1395 [MþNa]þ, 418.1398 [MþNa]þ,
(Method B): 98.1%
D
D
¼ 0.0001; experimental
4.50 (d, J ¼ 6.7 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6):
d 163.0,161.6,
¼ 0.0003. Purity HPLC
159.7, 150.5, 140.4, 138.5, 133.8, 128.7, 128.6, 127.5, 127.0, 115.8, 44.2.
ES-MS (methanol) m/z: 334.1 [MþH]þ, 356.1 [MþNa]þ, 372.0
[MþK]þ. HRMS (ESI-TOF) m/z: C17H15N7O experimental 334.1409
N5-(3-methylbenzyl)-2-(3,5-dimethoxyphenyl)-[1,2,4]-triazolo-
[1,5-a]-[1,3,5]-triazine-5,7-diamine (44): Method B. flash chroma-
tography eluent: DCM-MeOH 99:1. Yield: 13% (35.2 mg); white
solid; mp 262e264 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
[MþH]þ, theoretical 334.1410 [MþH]þ,
D
¼ 0.0001. Purity HPLC
(Method A): 95.1%.
3-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-
2-yl)phenol (49): flash chromatography eluent: DCM-MeOH 96:4.
Yield 30% (25 mg from 0.25 mmol of 39); white solid; mp
237e238 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6):
DMSO‑d6)
d
8.16e7.89 (m, 3H), 7.27 (d, J ¼ 2.4 Hz, 2H), 7.20 (t,
J ¼ 7.4 Hz, 1H), 7.15e7.10 (m, 2H), 7.04 (d, J ¼ 7.4 Hz, 1H), 6.61 (t,
J ¼ 2.4 Hz, 1H), 4.47 (d, J ¼ 6.4 Hz, 2H), 3.81 (s, 6H), 2.28 (s, 3H).13
C
NMR (101 MHz, DMSO‑d6)
d
162.87, 161.60, 161.02, 159.71, 150.55,
d 9.65 (s, 1H), 8.50e7.82 (m, 3H), 7.61e7.45 (m, 2H), 7.45e7.17 (m,
140.33, 137.64, 133.17, 128.5 (CH), 127.99 (CH), 127.65 (CH), 124.56
(CH),104.86 (2 x CH),102.70 (CH), 55.77 (2 x CH3), 44.17 (CH2), 21.51
(CH3). HRMS (ESI-TOF) m/z: C20H21N7O2 experimental 392.1828
6H), 6.86 (d, J ¼ 7.1 Hz, 1H), 4.51 (d, J ¼ 6.5 Hz, 2H). 13C NMR
(101 MHz, DMSO‑d6): d 163.6, 163.2, 161.6, 157.9, 150.6, 140.4, 132.5,
130.1, 128.6, 127.5, 127.0, 117.9, 117.5, 114.0, 44.2. ES-MS (methanol)
[MþH]þ, theoretical 392.1829 [MþH]þ,
D
¼ 0.0001; experimental
m/z: 334.1 [MþH]þ, 356.1 [MþNa]þ. HRMS (ESI-TOF) m/z:
414.1650 [MþNa]þ, theoretical 414.1649 [MþNa]þ,
D
¼ 0.0001.
C
17H15N7O experimental 334.1412 [MþH]þ, theoretical 334.1411
Purity HPLC (Method B): 96.7%.
[MþH]þ,
D
¼ 0.0001. Purity HPLC (Method A): 99.7%.
N5-cyclohexyl-2-(3,5-dimethoxyphenyl)-[1,2,4]-triazolo-[1,5-a]-
[1,3,5]-triazine-5,7-diamine (45): Method B. flash chromatography
eluent: DCM-MeOH 99:1. Yield: 16% (44.1 mg); white solid, mp
3-(5,7-bis(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)
phenol (50): flash chromatography eluent: DCM-MeOH 98:2 (sta-
tionary phase: alumina). Yield 15% (16 mg from 0.25 mmol of 40);
22

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
white solid; mp 275e276 ꢀC (EtOEt-light petroleum). 1H NMR
(Method B): 98.7%.
(400 MHz, DMSO‑d6):
d
9.65 (s, 1H), 9.09 (m, 1H), 8.12 (s, 1H),
4-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-
2-yl)benzen-1,2-diol (57): flash chromatography eluent: EtOAc-
MeOH 8:2. Yield 33% (29 mg from 0.5 mmol of 47); white solid;
mp 233 ꢀC (EtOEt-light petroleum). 1H NMR (270 MHz, DMSO‑d6):
7.69e7.08 (m, 13H), 6.87 (d, J ¼ 8.2 Hz, 1H), 4.77e4.40 (m, 4H). 13
C
NMR (101 MHz, DMSO‑d6):
d 161.4, 157.9, 151.7, 140.2, 138.9, 132.4,
130.1, 128.76, 128.72, 128.64, 128.1, 127.9, 127.64, 127.50, 127.1, 117.9,
117.6, 114.0, 44.3, 44.1. ES-MS (methanol) m/z: 424.2 [MþH]þ, 346.2
[MþNa]þ. MW 423.47. C24H21N7O. HRMS (ESI-TOF) m/z: C24H21N7O
experimental 424.1884 [MþH]þ, theoretical 424.1880 [MþH]þ,
d
9.32 (s, 1H), 9.19 (s,1H), 8.36e7.77 (m, 4H), 7.65e7.16 (m, 6H), 6.81
(d, J ¼ 8.2 Hz, 1H), 4.56e4.41 (m, 2H). 13C NMR (101 MHz,
DMSO‑d6):
d 163.4, 161.6, 159.6, 158.9, 150.5, 143.2, 140.4, 132.9,
D
¼ 0.0004. Purity HPLC (Method A): 99.7%.
128.6, 127.5, 127.0, 105.5, 104.7, 44.2. ES-MS (methanol) m/z: 350.1
5-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-
[MþH]þ. HRMS (ESI-TOF) m/z: C17H15N7O2 experimental 350.1355
2-yl)benzen-1,3-diol (51): flash chromatography eluent: DCM-
MeOH 96:4. Yield 40% (35 mg from 0.25 mmol of 41); white
solid; mp > 300 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
[MþH]þ, theoretical 350.1359 [MþH]þ,
D
¼ 0.0004. Purity HPLC:
99.1%.
DMSO‑d6):
7.00e6.92 (m, 2H), 6.28 (bs, 1H), 4.57e4.43 (m, 2H). 13C NMR
(101 MHz, DMSO‑d6): 163.5, 161.5, 159.6, 158.9, 150.5, 140.5, 132.9,
d
9.39 (s, 2H), 7.93e7.82 (m, 2H), 7.39e7.14 (m, 6H),
7. Biochemistry
d
7.1. CK1d activity assays
128.6, 127.5, 127.0, 105.5, 104.7, 44.2. ES-MS (methanol) m/z: 350.1
[MþH]þ. HRMS (ESI-TOF) m/z: C17H15N7O2 experimental 350.1360
Assay with truncated CK1
d. Compounds were evaluated towards
[MþH]þ, theoretical 350.1359 [MþH]þ,
D
¼ 0.0001. Purity HPLC
CK1 (Merck Millipore, recombinant human, amino acids 1e294,
d
(Method A): 99.4%.
with N-terminal GST-tagged) with the KinaseGlo® luminescence
assay (Promega) following procedures reported in literature
[20,59]. In detail, luminescent assays were performed in black 96-
well plates, using the following buffer: 50 mM HEPES (pH 7.5),
1 mM EDTA, 1 mM EGTA, and 15 mM magnesium acetate. Com-
N5-(4-chlorobenzyl)-2-(3,5-dihydroxyphenyl)-[1,2,4]-triazolo-
[1,5-a][1,3,5]-triazine-5,7-diamine (52): flash chromatography
eluent: EtOAc-MeOH 90:10. Yield 80% (35 mg from 0.11 mmol of 42
using 12 equivalents of boron tribromide); white solid; mp 260 ꢀ
C
(EtOEt-light petroleum). 1H NMR (400 MHz, DMSO‑d6)
d
9.43 (s,
pound CR8 (IC50 ¼ 0.4
[44] while DMSO/buffer solution was used as a negative control. In
a typical assay, 10 L of inhibitor solution (dissolved in DMSO at
10 mM concentration and diluted in assay buffer to the desired
concentration) and 10 L (16 ng of CK1 ) of enzyme solution were
added to the well, followed by 20 L of assay buffer containing 0.1%
casein substrate and 4 M ATP (or 40 M and 400 M for ATP
mM) was used as a positive control for CK1d
2H), 8.58e7.75 (m, 3H), 7.43e7.29 (m, 4H), 6.98 (s, 2H), 6.31 (t,
J ¼ 2.2 Hz,1H), 4.49 (d, J ¼ 5.9 Hz, 2H) 13C NMR (101 MHz, DMSO‑d6)
m
d
162.96, 161.09, 158.47 (2CH), 150.14, 139.06, 132.37, 131.10 (2CH),
128.93 (2CH), 128.13, 105.03 (2CH), 104.29 (CH), 43.17 (CH2). ES- MS
(methanol) m/z: 384.1 [MþH]þ. Purity HPLC (Method B): 97.7%.
N5-(4-fluorobenzyl)-2-(3,5-dihydroxyphenyl)-[1,2,4]-triazolo-
[1,5-a][1,3,5]-triazine-5,7-diamine (53): preparative TLC eluent:
DCM-MeOH 97:3. Yield 8% (6.2 mg from 0.2 mmol of 43); light
brown solid; mp 269e271 ꢀC (EtOEt-light petroleum). 1H NMR
m
d
m
m
m
m
competition experiment). The final DMSO concentration in the
reaction mixture did not exceed 1e2%. After 60 min of incubation at
30 ꢀC for CK1
d the enzymatic reactions were stopped with 40 mL of
(400 MHz, CD3OD)
d
7.51e7.28 (m, 2H), 7.07 (d, J ¼ 2.2 Hz, 2H), 7.02
Kinase-Glo reagent (Promega). Luminescence signal (relative light
unit, RLU) was recorded after 10 min at 25 ꢀC using Tecan Infinite
M100 or FLUOstar Optima multimode readers. The activity is pro-
portional to the difference of the total and consumed ATP. The
inhibitory activities were calculated on the basis of maximal ac-
tivities measured in the absence of inhibitor [63].
(t, J ¼ 8.8 Hz, 2H), 6.37 (t, J ¼ 2.2 Hz, 1H), 4.59 (s, 2H).HRMS (ESI-
TOF) m/z: C17H14FN7O2 experimental 368.1261 [MþH]þ, theoretical
368.1266 [MþH]þ,
D
¼ 0.0005. Purity HPLC (Method B): 97.1%.
N5-(3-methylbenzyl)-2-(3,5-dihydroxyphenyl)-[1,2,4]-triazolo-
[1,5-a][1,3,5]-triazine-5,7-diamine (54): preparative TLC eluent:
DCM-MeOH 94:6. Yield 7% (11.4 mg from 0.43 mmol of 44); white
solid; mp > 300 ꢀC (EtOEt-light petroleum). 1H NMR (400 MHz,
First, enzyme activity percentage was determined at 40 mM for
each inhibitor with respect to DMSO; subsequently, for the most
active compounds the IC50 values were determined. Data were
analyzed using Excel and GraphPad Prism software (version
Methanol-d4)
d 7.23e7.12 (m, 3H), 7.09e7.01 (m, 3H), 6.38 (t,
J ¼ 2.3 Hz, 1H), 4.58 (s, 2H), 2.32 (s, 3H). HRMS (ESI-TOF) m/z:
C
18H17N7O2 experimental 364.1516 [MþH]þ, theoretical 364.1522
8.0).Assay with full length CK1d. Compounds were evaluated to-
[MþH]þ,
D
¼ 0.0006; experimental 386.1336 [MþH]þ, theoretical
wards CK1 (full length, ThermoFisher) with the KinaseGlo®
d
386.1338 [MþH]þ,
D
¼ 0.0002. Purity HPLC (Method B): 98.5%.
luminescence assay (Promega). In detail, luminescent assays were
performed in black 96-well plates, using the following buffer:
50 mM HEPES (pH 7.5),1 mM EDTA, 1 mM EGTA, and 15 mM
magnesium acetate. Compound PF-670462 was used as positive
N5-cyclohexyl-2-(3,5-dihydroxyphenyl)-[1,2,4]-triazolo-[1,5-a]
[1,3,5]-triazine-5,7-diamine (55):
preparative TLC eluent: DCM-MeOH 94:6. Yield 43% (13.9 mg
from 0.088 mmol of 45); white solid; mp > 300 ꢀC (EtOEt-light
control for CK1
control. In a typical assay, 10
DMSO at 10 mM concentration and diluted in assay buffer to the
desired concentration) and 10 L (26 nM) of enzyme solution were
added to the well, followed by 20 L of assay buffer containing 0.1%
casein substrate and 4 M ATP. The final DMSO concentration in the
reaction mixture did not exceed 1e2%. After 10 min of incubation at
30 ꢀC the enzymatic reactions were stopped with 40
L of Kin-
d
while DMSO/buffer solution was used as negative
petroleum). 1H NMR (400 MHz, CD3OD)
d
7.07 (d, J ¼ 1.6 Hz, 2H),
mL of inhibitor solution (dissolved in
6.37 (s, 1H), 3.84 (s, 1H), 2.00 (s, 2H), 1.85e1.56 (m, 3H), 1.55e1.08
(m, 5H). HRMS (ESI-TOF) m/z: C16H19N7O2 experimental 342.1674
m
[MþH]þ, theoretical 342.1673 [MþH]þ,
D
¼ 0.0001; experimental
m
364.1496 [MþH]þ, theoretical 364.1492 [MþH]þ,
D
¼ 0.0004. Purity
m
HPLC (Method B): 96.9%.
N5-cyclohexyl-2-(3-hydroxyphenyl)-(5-methoxyphenyl)-[1,2,4]-
triazolo-[1,5-a][1,3,5]-triazine-5,7-diamine (56): preparative TLC
eluent: DCM-MeOH 94:6. Yield 32% (10.5 mg from 0.088 mmol of
45); white solid; mp > 300 ꢀC (EtOEt-light petroleum). 1H NMR
m
aseGlo ® reagent (Promega). Luminescence signal (relative light
unit, RLU) was recorded after 10 min at 30 ꢀC using Tecan Infinite
M100. For IC50 determination, ten different inhibitor concentra-
(400 MHz, CD3OD)
d
7.29e7.07 (m, 2H), 6.46 (t, J ¼ 2.2 Hz, 1H),
tions ranging from 100 to 0.026
ported as means standard errors of three independent
experiments. Data were analyzed using GraphPad Prism software
(version 8.0).
mM were used. IC50 values are re-
3.99e3.71 (m, 4H), 2.00 (s, 2H), 1.78 (m, 2H), 1.66 (m, 1H), 1.56e1.21
(m, 5H). HRMS (ESI-TOF) m/z: C17H21N7O2 experimental 356.1832
[MþH]þ, theoretical 356.1829 [MþH]þ,
D
¼ 0.0003. Purity HPLC
23

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
7.2. Competition binding assays on other kinases
permeability coefficient (Pe) of each drug, in centimeter per second,
was calculated applying the following formula [64]:
KINOMEscan™ platform by DiscoverX has been applied to 6
Vd Vr
100:Vd
human protein kinase CK1 isoforms: CK1a1, CK1a1L, CK1g1-3,
Pe
ðVd VrÞ: S: t 100:Vd ꢁ %TðVd VrÞ
Vr : Ar
CK1 and CK1ε, by testing compound 51 at a concentration of 10 mM
d
following a protocol reported in literature [60].
%T ¼
100
Ad : Vd
7.3. Cytotoxicity in non-tumoral cells
where Vd and Vr are the volumes of the donor and the receptor
solutions (0.18 cm3), S is the membrane area (0.266 cm2), t is the
time of incubation (2.5 h ¼ 9000 s), Ar is the absorbance of the
receptor plate after the experiment and Ad0 is the absorbance in
the donor compartment before incubation. Results are given as the
mean and the average of the two runs standard deviation (SD) is
reported.
Obtained results for quality control drugs were then correlated
to permeability data found in the literature. The linear correlation
between experimental and literature permeability values was used
for the classification of compounds in those able to cross the BBB by
passive permeation (CNS þ which correlate with a bibliographic
Pe > 4) and those not (CNS- which correlate with a bibliographic
Pe < 2).
Peripheral blood lymphocytes (PBL) from healthy donors were
obtained from human peripheral blood (leucocyte rich plasma-
buffy coats) from healthy volunteers using the Lymphoprep (Fre-
senius KABI Norge AS) gradient density centrifugation.
Buffy coats were obtained from the Blood Transfusion Service,
Azienda Ospedaliera of Padova and provided at this institution for
research purposes. Therefore, no informed consent was further
needed. In addition, buffy coats were provided without identifiers.
The experimental procedures were carried out in strict accordance
with approved guidelines.
After extensive washing, cells were resuspended (1.0 ꢂ 106 cells/
mL) in RPMI-1640 with 10% FBS and incubated overnight. For
cytotoxicity evaluations in proliferating PBL cultures, non-adherent
cells were resuspended at 5 ꢂ 105 cells/mL in growth medium,
Compounds correlating with reported Pe values from 2 to 4
10ꢁ6 cm sꢁ1 are classified as CNSþ/-. Complete data were reported
in supporting information.
containing 2.5 mg/mL Phytohematoagglutinin (PHA, Irvine Scienti-
fic). For cytotoxicity evaluations in resting PBL cultures, non-
adherent cells were resuspended (5 ꢂ 105 cells/mL) and treated
for 72 h with the test compounds, as described above.
7.5. Molecular modeling
Normal human adult fibroblast were purchased by ATCC (PCS-
201-012) and were cultivated in DMEM additioned of 10% fetal
Bovine serum. 2 ꢂ 104 cells were plated in 96 well plate and after
24 h, different concentrations of the test compounds were added,
and viability was determined 72 h later. For both cell lines viability
was evaluated by resazurin-based cell, measuring the relative
fluorescence intensity at 590 nm with a Spark (Tecan) multiwell
plate reader during a 4 h timespan.
Most of the in-silico operations described in the following par-
agraphs were performed on a Desktop Linux workstation (Intel®
Core™ CPU i9-9820 ꢂ 3.30 GHz). The collection of MD trajectories
instead was made possible using a Linux Server equipped with a
Titan V GPU.
7.6. Three-dimensional structures of CK1
d
7.4. CNS permeation prediction: PAMPA-BBB assay
Among the 31 crystallographic structures available for the hu-
man protein kinase CK1 , two were selected for this computational
d
Prediction of the brain penetration of active compounds was
evaluated using a parallel artificial membrane permeability assay
(PAMPA) [62]. Ten drugs of known BBB permeability (2e6 mg of
Caffeine, Enoxacine, Hydrocortisone, Desipramine, Ofloxacine, Pir-
oxicam, Testosterone, 12e15 mg of Promazine and Verapamil and
study. For molecular docking calculations, as well as for SuMD
simulations, the holo-form of the protein kinase (PDB ID: 4HNF)
was chosen, while for the investigation of the hydrodynamic
behavior of the solvent within the catalytic binding pocket, the apo-
form (PDB ID: 5IH4) was instead selected. The two structures were
retrieved from the RCSB Protein Data Bank database and were
processed with the protein preparation tool as implemented in the
Molecular Operating Environment (MOE) suite 2019.01 version
[65,66]. In detail, missing hydrogen atoms were added, establishing
the more probable ionization states for all titratable residues at pH
7.4 using the Protonate-3D tool [67]. Missing atoms in protein side
chains were built according to AMBER14 force field topology and
non-natural N-terminal and C-terminal were capped to mimic the
previous residue. Crystallographic co-solvents or ligand were sub-
sequently removed.
23 mg of Atenolol dissolved in 1000 mL of ethanol) were included in
each experiment to validate the analysis set. Compounds were
dissolved in a 70/30 PBS pH ¼ 7.4 buffer/ethanol solution in a
concentration that ensures adequate absorbance values in the UV-
VIS light spectrum. 5 mL of these solutions were filtered with PDVF
membrane filters (diameter 30 mm, pore size 0.45
acceptor indented 96-well microplate (MultiScreen 96-well Culture
Tray clear, Merck Millipore) was filled with 180 L of PBS/ethanol
(70/30). The donor 96-well filtrate plate (Multiscreen® IP Sterile
Plate PDVF membrane, pore size is 0.45 M, Merck Millipore) was
coated with 4 L of porcine brain lipid (Spectra 2000) in dodecane
(20 mg/mL) and after 5 min, 180 L of each compound solution
mm). The
m
m
m
m
were added. Then the donor plate was carefully put on the acceptor
plate to form a “sandwich”, which was left undisturbed for 2.5 h at
25 ꢀC. During this time the compounds diffused from the donor
plate through the brain lipid membrane into the acceptor plate.
After incubation, the donor plate was removed. The concentrations
of compounds and commercial drugs were determined by
measuring the absorbance in the donor (before the incubation) and
the acceptor wells (after the incubation) with UV plate reader Tecan
Infinite M1000. Every sample was analyzed at two to five wave-
lengths in 3 replicates and in two independent experiments. The
7.7. Molecular docking
Three-dimensional structures of the ligands were built by the
MOE-builder tool. Ionization states were predicted using the MOE-
protonate 3D tool and structures were minimized by the MMFF94x
until the root mean square (RMS) gradient fell below
0.1 kcal molꢁ1Åꢁ1. PLANTS docking protocol was selected as a
conformational search program and ChemPLP as a scoring function
[68,69]. For each compound investigated, 20 docking simulation
runs were performed, searching on a sphere of 10 Å radius, build
24

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
around the coordinates of ligand 16W center of mass (PDB ID:
4HNF).
7.11. Supervised molecular dynamics (SuMD) setup
SuMD is a computational protocol written in python program-
ming language allowing the exploration of the entire ligand-
receptor binding mechanism, from the unbound to the bound
state by collecting short unbiased MD simulations (hereinafter
defined SuMD steps) at the end of which variations in the ligand-
receptor distances is checked [80,81]. The supervision, more spe-
cifically, acts accepting all those SuMD steps in which a shortening
in ligand-receptor distances has been sampled while rejecting and
simulating again from the previous set of coordinates the other.
When the ligand-protein center of mass distance falls below a
defined cut-off (here 5 Å), the supervision algorithm is switched off
and classical MD is exploited to relax the predicted complex. The
merging of all the collected SuMD steps hence results in a SuMD
trajectory describing a putative binding mechanism. SuMD code
has several input parameters user-editable, such as the MD time-
step (here, 2 fs), the number of MD steps within a SuMD step (here,
300000e600ps), and the residues were chosen to dynamically
define the binding site location (here I23, A36 and I148). The SuMD
trajectory discussed was analyzed exploiting an in-house tool
written in tcl and python languages, deeply described in the orig-
inal publication [82].
7.8. Docking results analysis
For each derivate herein investigated, a single conformation was
chosen as representative of the bound state, based on the docking
score and a visual inspection of each complex, in such a way to
optimize the ligand-protein interaction network. Electrostatic and
hydrophobic interactions, respectively IEele and IEhyd
, were
computed between each selected ligand pose and each protein
residue involved in binding (residues within 10 Å with respect to
the binding site center of mass). Both these contributions were
computed using MOE software and, in particular, IEele were calcu-
lated as non-bonded electrostatic interactions energy term of the
force field (expressed in kcal/mol). Instead, IEhyd were computed as
contact hydrophobic surfaces and are associated with an adimen-
sional score (the higher the better). The data obtained by this
analysis were reported in a graphic, representing residues (x-axis)
in the form of equally high rectangles rendered according to a
colorimetric scale. As regards IEele, colors from blue to red represent
energy values ranging from negative to positive values; for IEhyd
colors from white to dark green depict scores going from 0 to
positive values.
,
Declaration of competing interest
None.
7.9. Molecular dynamics (MD)simulations setup
The tleap software, as part of the AmberTools20 suite, was
exploited to explicitly solvate each molecular system by a cubic
water box with cell borders placed at least 15 Å away from any
protein or ligand atoms [70]. The protein force field parameters
were assigned using the AMBER ff14SB and the TIP3P water model
was used [71,72]. In the case also small organic molecules were
present (e.g. SuMD simulations), parameters were assigned by
means of parmchk tools, as implementer on AmberTools20 pack-
age, using the GAFF. The compound partial charges were instead
calculated using the semiempirical method AM1-BCC [70,73,74]. To
neutralize the total charge of each system, Naþ/Clꢁ counterions
were added to a final salt concentration of 0.154 M. The energy of
each system was minimized by performing 500 steps with the
conjugate-gradient method, then sampling 500000 MD steps (1 ns)
in the canonical ensemble (NVT) followed by 500000 steps (1 ns) in
the isothermal-isobaric ensemble (NPT), in both of the cases using 2
fs as a time step. Harmonic positional constraints have been applied
during the system equilibration phases on protein-heavy atoms,
with a force constant of 1 kcal molꢁ1 Åꢁ2, gradually reduced with a
scaling factor of 0.1. The temperature was maintained at 273 K by a
Langevin thermostat and the pressure at 1 atm by a Monte Carlo
barostat [75,76]. All MD simulations were performed exploiting
ACEMD3 platform, which is based on the OpenMM 7.4.2 engine
[77,78].
Acknowledgments
MMS lab is very grateful to Chemical Computing Group, Open-
Eye, and Acellera for the scientific and technical partnership. MMS
lab gratefully acknowledges the support of NVIDIA Corporation
with the donation of the Titan V GPU used for this research. This
scientific work has been financially supported by MIUR (PRIN
2010e2011, n.20103W4779_006; PRIN2017, n. 2017MT3993).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113331.
Supplementary material
additional experimental data, video 1 description and all spectra
of final compounds were reported in supplementary material.
References
[1] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein
kinase complement of the human genome, Science 298 (80-) (2002)
1912e1934, https://doi.org/10.1126/science.1075762.
[2] K. Yasojima, J. Kuret, A.J. Demaggio, E. McGeer, P.L. McGeer, Casein kinase 1
delta mRNA is upregulated in Alzheimer disease brain, Brain Res. 865 (2000)
116e120, https://doi.org/10.1016/S0006-8993(00)02200-9.
[3] G. Cozza, L.A. Pinna, Casein kinases as potential therapeutic targets, Expert
Opin. Ther. Targets 8222 (2015) 1e22, https://doi.org/10.1517/
14728222.2016.1091883.
7.10. AquaMMapS analysis
To characterize the hydrodynamic behavior of water molecules
[4] Y. Xu, Q.S. Padiath, R.E. Shapiro, C.R. Jones, S.C. Wu, N. Saigoh, K. Saigoh,
within the CK1
sampled in the NVT ensemble with the temperature set at 273 K.
The trajectory has then been superposed and aligned on the
d binding site,100 ns of classical MD simulation were
ꢁꢂ
L.J. Ptacek, Y.-H. Fu, Acknowledgements We Thank M. Rees for Comments on
Statistical Analyses Functional Consequences of a CKId Mutation Causing Fa-
milial Advanced Sleep Phase Syndrome, 2005. www.nature.com/nature.
[5] K.C. Brennan, E.A. Bates, R.E. Shapiro, J. Zyuzin, W.C. Hallows, Y. Huang,
ꢁꢂ
a
-
carbons atoms of the protein at the first frames and wrapped into
an image of the system simulated under periodic boundary con-
ditions (PBC). AquaMMapS algorithm was then exploited to analyze
this trajectory and only the hydration spots characterized by a
water occupancy (% ORMSF) higher than 15% were retained and used
in this study [79].
H.Y. Lee, C.R. Jones, Y.H. Fu, A.C. Charles, L.J. Ptacek, Casein kinase Id mutations
in familial migraine and advanced sleep phase, Sci. Transl. Med. 5 (2013),
https://doi.org/10.1126/scitranslmed.3005784, 183ra56-183ra56.
[6] F. Vena, S. Bayle, A. Nieto, V. Quereda, M. Aceti, S.M. Frydman, S.S. Sansil,
W. Grant, A. Monastyrskyi, P. McDonald, W.R. Roush, M. Teng, D. Duckett,
Targeting casein kinase 1 delta sensitizes pancreatic and bladder cancer cells
to gemcitabine treatment by upregulating deoxycytidine kinase, Mol. Canc.
25

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
Therapeut. 19 (2020) 1623e1635, https://doi.org/10.1158/1535-7163.MCT-
19-0997.
H. Sun, Small molecule modulators targeting protein kinase CK1 and CK2, Eur.
J. Med. Chem. 181 (2019), https://doi.org/10.1016/j.ejmech.2019.111581.
[28] T. Chijiwa, M. Hagiwara, H. Hidaka, A newly synthesized selective casein ki-
nase I inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, and
affinity purification of casein kinase I from bovine testis, J. Biol. Chem. 264
(1989) 4924e4927, https://doi.org/10.1016/s0021-9258(18)83679-1.
[7] I. Bar, A. Merhi, L. Larbanoix, M. Constant, S. Haussy, S. Laurent, J.-L. Canon,
ꢁ
P. Delree, Silencing of Casein Kinase 1 Delta Reduces Migration and Metastasis
of Triple Negative Breast Cancer Cells, 2018. www.oncotarget.com.
ꢀ
[8] T. Liu, D. Vergara, E. Laura Mazzoldi, S. Pavan, A. Pasto, E. Ceppelli, G. Pilotto,
V. Barbieri, A. Amadori, Casein kinase 1 delta regulates cell proliferation,
response to chemotherapy and migration in human ovarian cancer cells,
Front. Oncol. | Www.Frontiersin.Org. 9 (2019) 1211, https://doi.org/10.3389/
fonc.2019.01211.
[29] G. Rena, J. Bain, M. Elliott, P. Cohen, D4476, A cell-permeant inhibitor of CK1,
suppresses the site-specific phosphorylation and nuclear exclusion of
FOXO1a,
EMBO Rep.
5
(2004) 60e65, https://doi.org/10.1038/
sj.embor.7400048.
ꢀ
[9] Y.C. Lin, M.C. Chen, T.H. Hsieh, J.P. Liou, C.H. Chen, CK1
d
as a potential ther-
[30] L. Behrend, D.M. Milne, M. Stoe Ter, W. Deppert, L.E. Campbell, D.W. Meek,
U. Knippschild, IC261, a speci®c inhibitor of the protein kinases casein kinase
1-delta and-epsilon, triggers the mitotic checkpoint and induces p53-
dependent postmitotic eVects. www.nature.com/onc.
apeutic target to treat bladder cancer, Aging 12 (2020) 5764e5780, https://
doi.org/10.18632/aging.102966.
[10] U. Knippschild, M. Krüger, J. Richter, P. Xu, Balbina García-Reyes, C. Peifer,
J. Halekotte, V. Bakulev, J. Bischof, The CK1 family: contribution to cellular
stress response and its role in carcinogenesis, Front. Oncol. 4 (2014) 1e33,
https://doi.org/10.3389/fonc.2014.00096. MAY.
[11] M.B. Batty, R.B. Schittenhelm, D. Dorin-Semblat, C. Doerig, J.F. Garcia-Bustos,
Interaction of Plasmodium falciparum casein kinase 1 with components of
host cell protein trafficking machinery, IUBMB Life 72 (2020) 1243e1249,
https://doi.org/10.1002/iub.2294.
[31] S.D. Gross, R.A. Anderson, Casein kinase I: spatial organization and positioning
of a multifunctional protein kinase family, Cell. Signal. 10 (1998) 699e711.
[32] U. Knippschild, A. Gocht, S. Wolff, N. Huber, M. St, The casein kinase 1 family :
participation in multiple cellular processes in eukaryotes 17 (2005) 675e689,
https://doi.org/10.1016/j.cellsig.2004.12.011.
[33] L. Badura, T. Swanson, W. Adamowicz, J. Adams, J. Cianfrogna, K. Fisher,
J. Holland, R. Kleiman, F. Nelson, L. Reynolds, K.S. Germain, E. Schaeffer,
B. Tate, J. Sprouse, An inhibitor of casein kinase I induces phase delays in
circadian rhythms under, Free-Running and Entrained Conditions 322 (2007)
730e738, https://doi.org/10.1124/jpet.107.122846.loops.
[12] E. Durieu, E. Prina, O. Leclercq, N. Oumata, N. Gaboriaud-Kolar,
K. Vougogiannopoulou, N. Aulner, A. Defontaine, J.H. No, S. Ruchaud, A.-
€
L. Skaltsounis, H. Galons, G.F. Spath, L. Meijer, N. Rachidi, From Drug Screening
to Target Deconvolution:
Leishmania Casein Kinase 1 Isoform 2 to Identify Compounds with Anti-
leishmanial Activity, 2016, https://doi.org/10.1128/AAC.00021-16.
a
Target-Based Drug Discovery Pipeline Using
[34] T.T. Wager, R.Y. Chandrasekaran, J. Bradley, D. Rubitski, H. Berke, S. Mente,
T. Butler, A. Doran, C. Chang, K. Fisher, J. Knafels, S. Liu, J. Ohren, M. Marconi,
G. DeMarco, B. Sneed, K. Walton, D. Horton, A. Rosado, A. Mead, Casein kinase
[13] P. Gami-Patel, I. Van Dijken, Lieke H. Meeter, S. Melhem, H.J. Tjado, Morrema,
Wiep Scheper, J.C. Van Swieten, J.M. Annemieke, Rozemuller, A.A. Dijkstra,
J.M. Jeroen, Hoozemans, J.J.M. Hoozemans, Unfolded protein response acti-
vation in C9orf72 frontotemporal dementia is associated with dipeptide pa-
thology and granulovacuolar degeneration in granule cells, Brain Pathol. 31
(2021) 163e173, https://doi.org/10.1111/bpa.12894.
1d/ε inhibitor PF-5006739 attenuates opioid drug-seeking behavior, ACS
Chem. Neurosci. 5 (2014) 1253e1265, https://doi.org/10.1021/cn500201x.
[35] N. Oumata, K. Bettayeb, Y. Ferandin, L. Demange, A. Lopez-Giral, M.L. Goddard,
V. Myrianthopoulos, E. Mikros, M. Flajolet, P. Greengard, L. Meijer, H. Galons,
Roscovitine-derived, dual-specificity inhibitors of cyclin-dependent kinases
and casein kinases 1, J. Med. Chem. 51 (2008) 5229e5242, https://doi.org/
10.1021/jm800109e.
[14] C. Schwab, A.J. Demaggio, N. Ghoshal, L.I. Binder, J. Kuret, P.L. McGeer, Casein
kinase 1 delta is associated with pathological accumulation of tau in several
neurodegenerative diseases, Neurobiol. Aging 21 (2000) 503e510, https://
doi.org/10.1016/S0197-4580(00)00110-X.
[36] M. Bibian, R.J. Rahaim, J.Y. Choi, Y. Noguchi, S. Schürer, W. Chen, S. Nakanishi,
K. Licht, L.H. Rosenberg, L. Li, Y. Feng, M.D. Cameron, D.R. Duckett,
J.L. Cleveland, W.R. Roush, Development of highly selective casein kinase 1d/
€
[15] P. Xu, C. Ianes, F. Gartner, C. Liu, T. Burster, V. Bakulev, N. Rachidi,
1ε (CK1
Chem.
d
/ε) inhibitors with potent antiproliferative properties, Bioorg, Med.
Lett. 23 (2013) 4374e4380, https://doi.org/10.1016/
U. Knippschild, J. Bischof, Structure, regulation, and (patho-)physiological
functions of the stress-induced protein kinase CK1 delta (CSNK1D), Gene 715
(2019), https://doi.org/10.1016/j.gene.2019.144005, 144005.
j.bmcl.2013.05.075.
[37] C.R. Keenan, S.Y. Langenbach, F. Jativa, T. Harris, M. Li, Q. Chen, Y. Xia, B. Gao,
M.J. Schuliga, J. Jaffar, D. Prodanovic, Y. Tu, A. Berhan, P.V.S. Lee, G.P. Westall,
[16] P. Adler, J. Mayne, K. Walker, Z. Ning, D. Figeys, Therapeutic targeting of casein
^
kinase 1I/ε in an alzheimer’s disease mouse model, J. Proteome Res. 18 (2019)
A.G. Stewart, Casein kinase 1
effects of transforming growth factor-
d
/ε inhibitor, PF670462 attenuates the fibrogenic
in pulmonary fibrosis, Front. Phar-
3383e3393, https://doi.org/10.1021/acs.jproteome.9b00312.
[17] D.P. Hanger, H.L. Byers, S. Wray, K.Y. Leung, M.J. Saxton, A. Seereeram,
C.H. Reynolds, M.A. Ward, B.H. Anderton, Novel phosphorylation sites in Tau
from Alzheimer brain support a role for casein kinase 1 in disease patho-
genesis, J. Biol. Chem. 282 (2007) 23645e23654, https://doi.org/10.1074/
jbc.M703269200.
b
macol. 9 (2018) 738, https://doi.org/10.3389/fphar.2018.00738.
[38] H. Zuo, M. Trombetta-Lima, I.H. Heijink, C.H.T.J. Van Der Veen, L. Hesse, K.N.
Faber, W.J. Poppinga, H. Maarsingh, V.O. Nikolaev, M. Schmidt, A-kinase
anchoring proteins diminish TGF-b 1/cigarette smoke-induced epithelial-to-
mesenchymal transition, 9 (n.d) 356. doi:10.3390/cells9020356.
[18] G. Li, H. Yin, J. Kuret, Casein kinase 1 delta phosphorylates tau and disrupts its
binding to microtubules*, J. Biol. Chem. 279 (2004) 15938e15945, https://
doi.org/10.1074/jbc.M314116200.
[39] S. Sundaram, S. Nagaraj, H. Mahoney, A. Portugues, W. Li, K. Millsaps,
J. Faulkner, A. Yunus, C. Burns, C. Bloom, M. Said, L. Pinto, S. Azam, M. Flores,
A. Henriksen, J. Gamsby, D. Gulick, Inhibition of Casein Kinase 1d/ε Improves
[19] K. Yasojima, J. Kuret, A.J. Demaggio, E. Mcgeer, P.L. Mcgeer, Casein kinase 1
delta mRNA is upregulated in Alzheimer disease brain. www.elsevier.com/
locate/bres, 2000.
Cognitive-Affective Behavior and Reduces Amyloid Load in the APP-PS1
Mouse Model of Alzheimer’s Disease Open, 2019, https://doi.org/10.1038/
s41598-019-50197-x.
[20] I.G. Salado, M. Redondo, M.L. Bello, C. Perez, N.F. Liachko, B.C. Kraemer,
L. Miguel, M. Lecourtois, C. Gil, A. Martinez, D.I. Perez, Protein kinase CK-1
inhibitors as new potential drugs for amyotrophic lateral sclerosis, J. Med.
Chem. 57 (2014), https://doi.org/10.1021/jm500065f.
[40] P. Janovska, J. Verner, J. Kohoutek, L. Bryjova, M. Gregorova, M. Dzimkova,
H. Skabrahova, T. Radaszkiewicz, P. Ovesna, O.V. Blanarova, T. Nemcova,
Z. Hoferova, K. Vasickova, L. Smyckova, A. Egle, S. Pavlova, L. Poppova,
K. Plevova, S. Pospisilova, V. Bryja, Casein Kinase 1 Is a Therapeutic Target in
Chronic Lymphocytic Leukemia, 2018. http://ashpublications.org/blood/
article-pdf/131/11/1206/1405389/blood786947.pdf.
[41] D.W. Kim, C. Chang, X. Chen, A.C. Doran, F. Gaudreault, T. Wager, G.J. DeMarco,
J.K. Kim, Systems approach reveals photosensitivity and <scp>PER</scp> 2
level as determinants of clock-modulator efficacy, Mol. Syst. Biol. 15 (2019),
https://doi.org/10.15252/msb.20198838 e8838.
[42] T. Ihara, Y. Nakamura, T. Mitsui, S. Tsuchiya, M. Kanda, S. Kira, H. Nakagomi,
N. Sawada, M. Kamiyama, E. Shigetomi, Y. Shinozaki, M. Yoshiyama, A. Nakao,
S. Koizumi, M. Takeda, Intermittent Restraint Stress Induces Circadian
Misalignment in the Mouse Bladder, Leading to Nocturia, Sci. Rep (9), 2019,
10069, https://doi.org/10.1038/s41598-019-46517-w.
ꢁ
ꢁ
[21] C. Alquezar, I.G. Salado, A. De La Encarnacion, D.I. Perez, F. Moreno, C. Gil,
ꢁ
ꢁ
A. Lopez De Munain, A. Martínez, A. Martín-Requero, Targeting TDP-43
Phosphorylation by Casein Kinase-1 Inhibitors: a Novel Strategy for the
d
Treatment of Frontotemporal Dementia, 2016, https://doi.org/10.1186/
s13024-016-0102-7.
ꢁ
ꢁ
[22] L. Martínez-Gonzalez, C. Rodríguez-Cueto, D. Cabezudo, F. Bartolome,
ꢁ
ꢁ
ꢁ
P. Andres-Benito, I. Ferrer, C. Gil, A. Martín-Requero, J. Fernandez-Ruiz,
A. Martínez, E. de Lago, Motor Neuron Preservation and Decrease of in Vivo
TDP-43 Phosphorylation by Protein CK-1
d Kinase Inhibitor Treatment, Sci
.Rep. 10, 2020, 4449, https://doi.org/10.1038/s41598-020-61265-y.
[23] M. Bissaro, S. Federico, V. Salmaso, M. Sturlese, G. Spalluto, S. Moro, Targeting
protein kinase CK1
d
with Riluzole: could it be one of the possible missing
[43] A. Alli, L. Yu, M. Holzworth, J. Richards, K.-Y. Cheng, I. Jeanette Lynch,
C.S. Wingo, M.L. Gumz, Direct and indirect inhibition of the circadian clock
protein Per1: effects on ENaC and blood pressure, Am. J. Physiol. Ren. Physiol.
316 (2019) 807e813, https://doi.org/10.1152/ajprenal.00408.2018.-Circadian.
[44] K. Bettayeb, N. Oumata, A. Echalier, Y. Ferandin, J.A. Endicott, H. Galons,
L. Meijer, CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-
dependent kinases, Oncogene 27 (2008) 5797.
bricks to interpret its effect in the treatment of ALS from a molecular point of
view? ChemMedChem (2018) https://doi.org/10.1002/cmdc.201800632.
[24] J. Kosten, A. Binolfi, M. Stuiver, S. Verzini, F.-X. Theillet, B. Bekei, M. Van
Rossum, P. Selenko, Efficient modification of alpha-synuclein serine 129 by
protein kinase CK1 requires phosphorylation of tyrosine 125 as a priming
event, ACS Chem. Neurosci. 5 (2014), https://doi.org/10.1021/cn5002254.
[25] M. Okochi, J. Walter, A. Koyama, S. Nakajo, M. Baba, T. Iwatsubo, L. Meijer,
P.J. Kahle, C. Haass, Constitutive phosphorylation of the Parkinson’s disease
associated a-synuclein, J. Biol. Chem. 275 (2000) 390e397, https://doi.org/
10.1074/jbc.275.1.390.
[45] A. Monastyrskyi, N. Nilchan, V. Quereda, Y. Noguchi, C. Ruiz, W. Grant,
M. Cameron, D. Duckett, W. Roush, Development of dual casein kinase 1
(CK1 /ε) inhibitors for treatment of breast cancer, Bioorg. Med. Chem. 26
(2018) 590e602, https://doi.org/10.1016/j.bmc.2017.12.020.
d/1ε
d
[26] T. De Wit, & V Baekelandt, E. Lobbestael, Inhibition of LRRK2 or Casein Kinase
1 Results in LRRK2 Protein Destabilization, (2035). doi:10.1007/s12035-018-
1449-2.
[46] L.H. Rosenberg, M. Lafitte, V. Quereda, W. Grant, W. Chen, M. Bibian,
Y. Noguchi, M. Fallahi, C. Yang, J.C. Chang, W.R. Roush, J.L. Cleveland,
D.R. Duckett, Therapeutic targeting of casein kinase 1
d in breast cancer, Sci.
[27] Y. Qiao, T. Chen, H. Yang, Y. Chen, H. Lin, W. Qu, F. Feng, W. Liu, Q. Guo, Z. Liu,
Transl. Med. 7 (2015) 1e13, https://doi.org/10.1126/scitranslmed.aac8773.
26

I. Grieco, M. Bissaro, D.B. Tiz et al.
European Journal of Medicinal Chemistry 216 (2021) 113331
[47] S. Federico, A. Ciancetta, N. Porta, S. Redenti, G. Pastorin, B. Cacciari, K.N. Klotz,
S. Moro, G. Spalluto, Scaffold decoration at positions 5 and 8 of 1,2,4- triazolo
[1,5-c]Pyrimidines to explore the antagonist profiling on adenosine receptors:
a preliminary StructureꢁActivity relationship study, J. Med. Chem. 57 (2014)
6210e6225.
G. Bottegoni, P. Bisignano, M. Bissaro, S. Moro, A. Martinez, P. Storici,
G. Spalluto, A. Cavalli, S. Federico, A triazolotriazine-based dual GSK-3b/CK-1d
ligand as a potential neuroprotective agent presenting two different mecha-
nisms of enzymatic inhibition, ChemMedChem 14 (2019) 310e314, https://
doi.org/10.1002/cmdc.201800778.
[48] S. Federico, E. Margiotta, V. Salmaso, G. Pastorin, S. Kachler, K.N. Klotz, S. Moro,
G. Spalluto, [1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antago-
nists: modifications at the 8 position to reach selectivity towards A3adenosine
receptor subtype, Eur. J. Med. Chem. 157 (2018) 837e851, https://doi.org/
10.1016/j.ejmech.2018.08.042.
[64] A. Avdeef, Absorption and Drug Development, John Wiley
& Sons, Inc.,
Hoboken, NJ, USA, 2012, https://doi.org/10.1002/9781118286067.
[65] R. Wang, X. Fang, Y. Lu, C.Y. Yang, S. Wang, The PDBbind database: method-
ologies and updates, J. Med. Chem. 48 (2005) 4111e4119, https://doi.org/
10.1021/jm048957q.
[49] S. Federico, S. Paoletta, S.L. Cheong, G. Pastorin, B. Cacciari, S. Stragliotto,
K.N. Klotz, J. Siegel, Z.-G. Gao, K.A. Jacobson, S. Moro, G. Spalluto, Synthesis and
biological evaluation of a new series of 1,2,4-triazolo[1,5-a]-1,3,5-triazines as
human A(2A) adenosine receptor antagonists with improved water solubility,
J. Med. Chem. 54 (2011) 877e889, https://doi.org/10.1021/jm101349u.
[50] G. Deganutti, A. Zhukov, F. Deflorian, S. Federico, G. Spalluto, R.M. Cooke,
S. Moro, J.S. Mason, A. Bortolato, Impact of proteineligand solvation and
desolvation on transition state thermodynamic properties of adenosine A2A
ligand binding kinetics, Silico Pharmacol 5 (2017) 16, https://doi.org/10.1007/
s40203-017-0037-x.
[51] A. Dalpiaz, B. Cacciari, C.B. Vicentini, F. Bortolotti, G. Spalluto, S. Federico,
B. Pavan, F. Vincenzi, P.A. Borea, K. Varani, A novel conjugated agent between
dopamine and an A 2A adenosine receptor antagonist as a potential anti-
Parkinson multitarget approach, Mol. Pharm. 9 (2012) 591e604, https://
doi.org/10.1021/mp200489d.
[66] Molecular Operating Environment (MOE), (2019).
[67] P. Labute, Protonate 3D: Assignment of Macromolecular Protonation State and
Geometry, Chem. Comput. Gr. Inc., 2007.
[68] O. Korb, T. Stützle, T.E. Exner, An ant colony optimization approach to flexible
proteineligand docking, Swarm Intell (2007), https://doi.org/10.1007/s11721-
007-0006-9.
[69] O. Korb, T. Stützle, T.E. Exner, Empirical scoring functions for advanced
Protein-Ligand docking with PLANTS, J. Chem. Inf. Model. (2009), https://
doi.org/10.1021/ci800298z.
[70] D.M. Y. and P.A.K. D.A. Case, K. Belfon, I.Y. Ben-Shalom, S.R. Brozell, D.S. Cer-
utti, T.E. Cheatham, III, V.W.D. Cruzeiro, T.A. Darden, R.E. Duke, G. Giambasu,
M.K. Gilson, H. Gohlke, A.W. Goetz, R. Harris, S. Izadi, S.A. Izmailov, K. Kasa-
vajhala, A. Kovalenko, R. Krasny, T, AmberTools20, (2020).
[71] J.A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K.E. Hauser,
C. Simmerling, ff14SB: improving the accuracy of protein side chain and
backbone parameters from ff99SB, J. Chem. Theor. Comput. 11 (2015)
3696e3713, https://doi.org/10.1021/acs.jctc.5b00255.
[52] S. Federico, A. Ciancetta, N. Porta, S. Redenti, G. Pastorin, B. Cacciari, K.N. Klotz,
S. Moro, G. Spalluto, 5,7-Disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazines as
pharmacological tools to explore the antagonist selectivity profiles toward
adenosine receptors, Eur. J. Med. Chem. 108 (2016), https://doi.org/10.1016/
j.ejmech.2015.12.019.
[72] W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein, Com-
parison of simple potential functions for simulating liquid water, J. Chem.
Phys. (1983), https://doi.org/10.1063/1.445869.
[53] H. Tsumoki, J. Shimada, H. Imma, A. Nakamura, H. Nonaka, S. Shiozaki,
S. Ichikawa, T. Kanda, Y. Kuwana, M. Ichimura, F. Suzuki, [1,2,4]Triazolo[1,5-c]
Pyrimidine Derivatives, EP0976753A1, 1998.
[54] J. Hutton, Chemical Process, US 5 (1994). https://patents.google.com/patent/
US5326869/pt.
[55] C. Slobutsey, L. Audrieth, R. Campbell, Acid catalysis in liquid ammonia in
ammonolysis of diethylmalonate, Proc. Natl. Acad. Sci. U.S.A. 23 (1937)
611e615.
[73] K.G. Sprenger, V.W. Jaeger, J. Pfaendtner, The general AMBER force field
(GAFF) can accurately predict thermodynamic and transport properties of
many ionic liquids, J. Phys. Chem. B 119 (2015) 5882e5895, https://doi.org/
10.1021/acs.jpcb.5b00689.
[74] A. Jakalian, B.L. Bush, D.B. Jack, C.I. Bayly, Fast, efficient generation of high-
quality atomic charges. AM1-BCC model: I. Method, J. Comput. Chem. 21
(2000) 132e146, https://doi.org/10.1002/(SICI)1096-987X(20000130)21:
2<132::AID-JCC5>3.0. CO;2-P.
[56] J. Matasi, J. Caldwell, D. Tulshian, L. Silverman, B. Neustadt, Adenosine A2a
Receptor Antagonists, EP1453835, 2006.
[75] R.J. Loncharich, B.R. Brooks, R.W. Pastor, Langevin dynamics of peptides: the
frictional dependence of isomerization rates ofN-acetylalanyl-N?-methyl-
amide, Biopolymers 32 (1992) 523e535, https://doi.org/10.1002/
bip.360320508.
[76] W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein, Com-
parison of simple potential functions for simulating liquid water, J. Chem.
Phys. 79 (1983) 926e935, https://doi.org/10.1063/1.445869.
[77] M.J. Harvey, G. Giupponi, G. De Fabritiis, ACEMD: accelerating biomolecular
dynamics in the microsecond time scale, J. Chem. Theor. Comput. 5 (2009)
1632e1639.
[78] P. Eastman, J. Swails, J.D. Chodera, R.T. McGibbon, Y. Zhao, K.A. Beauchamp, L.-
P. Wang, A.C. Simmonett, M.P. Harrigan, C.D. Stern, R.P. Wiewiora, B.R. Brooks,
V.S. Pande, OpenMM 7: rapid development of high performance algorithms
for molecular dynamics, PLoS Comput. Biol. 13 (2017), e1005659, https://
doi.org/10.1371/journal.pcbi.1005659.
[79] A. Cuzzolin, G. Deganutti, V. Salmaso, M. Sturlese, S. Moro, AquaMMapS: an
alternative tool to monitor the role of water molecules during protein-ligand
association, ChemMedChem 13 (2018) 522e531, https://doi.org/10.1002/
cmdc.201700564.
[80] D. Sabbadin, S. Moro, Supervised molecular dynamics (SuMD) as a helpful tool
to depict GPCReligand recognition pathway in a nanosecond time scale,
J. Chem. Inf. Model. 54 (2014) 372e376.
[81] A. Cuzzolin, M. Sturlese, G. Deganutti, V. Salmaso, D. Sabbadin, A. Ciancetta,
S. Moro, Deciphering the complexity of ligandeprotein recognition pathways
using supervised molecular dynamics (SuMD) simulations, J. Chem. Inf. Model.
56 (2016) 687e705, https://doi.org/10.1021/acs.jcim.5b00702.
[82] V. Salmaso, M. Sturlese, A. Cuzzolin, S. Moro, Exploring protein-peptide
ꢂ
ꢁ
[57] O. Písa, S. Radl, Synthesis of 4-quinolones: N , O -Bis(trimethylsilyl)acetamide-
Mediated cyclization with cleavage of aromatic C-O bond, Eur. J. Org Chem.
2016 (2016) 2336e2350, https://doi.org/10.1002/ejoc.201600178.
[58] P.W. Caulkett, G. Jones, M. McPartlin, N. Renshaw, S. Stewart, B. Wright,
Adenine isosteres with bridgehead nitrogen. Part 1. Two independent syn-
theses of the [1,2,4]triazolo[1,5-a]triazine ring system leading to a range of
substituents in the 2,5 and 7 positions, J. Chem. Soc. Perkin Trans. 1 (1995)
801e808.
ꢁ
[59] S. Redenti, I. Marcovich, T. De Vita, C. Perez, R. De Zorzi, N. Demitri, D.I. Perez,
G. Bottegoni, P. Bisignano, M. Bissaro, S. Moro, A. Martinez, P. Storici,
G. Spalluto, A. Cavalli, S. Federico, A triazolotriazine-based dual GSK-3b/CK-1d
ligand as a potential neuroprotective agent presenting two different mecha-
nisms of enzymatic inhibition, ChemMedChem 14 (2019) 310e314, https://
doi.org/10.1002/cmdc.201800778.
[60] M.A. Fabian, W.H. Biggs, D.K. Treiber, C.E. Atteridge, M.D. Azimioara,
M.G. Benedetti, T.A. Carter, P. Ciceri, P.T. Edeen, M. Floyd, J.M. Ford, M. Galvin,
J.L. Gerlach, R.M. Grotzfeld, S. Herrgard, D.E. Insko, M.A. Insko, A.G. Lai, J.-
ꢁ
M. Lelias, S.A. Mehta, Z.V. Milanov, A.M. Velasco, L.M. Wodicka, H.K. Patel,
P.P. Zarrinkar, D.J. Lockhart, A small moleculeekinase interaction map for
clinical kinase inhibitors, Nat. Biotechnol. 23 (2005) 329e336, https://doi.org/
10.1038/nbt1068.
[61] J.T. Henderson, M. Piquette-Miller, Blood-brain barrier: an impediment to
neuropharmaceuticals, Clin. Pharmacol. Ther. 97 (2015) 308e313, https://
doi.org/10.1002/cpt.77.
[62] L. Di, E.H. Kerns, K. Fan, O.J. McConnell, G.T. Carter, High throughput artificial
membrane permeability assay for blood-brain barrier, Eur. J. Med. Chem. 38
(2003) 223e232, https://doi.org/10.1016/S0223-5234(03)00012-6.
recognition pathways using
a supervised molecular dynamics approach,
Structure 25 (2017) 655e662.e2, https://doi.org/10.1016/j.str.2017.02.009.
ꢁ
[63] S. Redenti, I. Marcovich, T. De Vita, C. Perez, R. De Zorzi, N. Demitri, D.I. Perez,
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