Substituted 4-hydroxy-1,2,3-triazoles: synthesis, characterization and first drug design applications through bioisosteric modulation and scaffold hopping approaches

Article abstract of DOI:10.1039/c5md00182j

Bioisosterism and scaffold hopping are two widely used approaches in medicinal chemistry for the purpose of lead optimization. The study highlights the physicochemical properties of the 4-hydroxy-1,2,3-triazole scaffold, a less investigated heterocyclic system. Synthetic strategies to obtain different N-substituted 4-hydroxy-1,2,3-triazole isomers are presented, and their role as possible isosteres of the carboxylic acid is discussed. The aim is to use this system to modulate the acidic moieties present in lead compounds and, at the same time, to regiodirect substituents in set directions, through targeted substitution on the three nitrogen atoms of the triazole ring. Through this approach, compounds having enhanced binding affinity, will be sought. Two examples of bioisosteric applications of this moiety are presented. In the first example, a classical bioisosteric approach mimicking the distal (S)-glutamic acid carboxyl group using the 4-hydroxy-1,2,3-triazole moiety is applied, to obtain two promising glutamate analogs. In the second example, a scaffold hopping approach is applied, replacing the phenolic moiety present in MDG-1-33A, a potent inhibitor of Onchocerca volvulus chitinase, with the 4-hydroxy-1,2,3-triazole scaffold. The 4-hydroxy-1,2,3-triazole system appears to be useful and versatile in drug design.

Full text of DOI:10.1039/c5md00182j

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CONCISE ARTICLE
Substituted 4-hydroxy-1,2,3-triazoles: synthesis, characterization
and first drug design applications through bioisosteric modulation
and scaffold hopping approaches†
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Agnese C. Pippione,*a Franco Dosio,a Alex Ducime,a Antonella Federico,a Katia Martina,a Stefano
Sainas,a Bente Frølund,b Major Gooyit,c Kim D. Janda,c Donatella Boschia and Marco L. Lolli.a
Bioisosterism and scaffold hopping are two widely used approaches in medicinal chemistry for the purpose of lead
optimization. The study highlights the physicochemical properties of the 4-hydroxy-1,2,3-triazole scaffold,
a less
investigated heterocyclic system. Synthetic strategies to obtain different N-substituted 4-hydroxy-1,2,3-triazole isomers
are presented, and their role as possible isosteres of the carboxylic acid is discussed. The aim is to use this system to
modulate the acidic moieties present in lead compounds and, at the same time, to regiodirect substituents in set
directions, through targeted substitution on the three nitrogen atoms of the triazole ring. Through this approach,
compounds having enhanced binding affinity, will be sought. Two examples of bioisosteric applications of this moiety are
presented. In the first example, a classical bioisosteric approach mimicking the distal (S)-glutamic acid carboxyl group using
the 4-hydroxy-1,2,3-triazole moiety is applied, to obtain two promising glutamate analogs. In the second example, a
scaffold hopping approach is applied, replacing the phenolic moiety present in MDG-1-33A, a potent inhibitor of
Onchocerca volvulus chitinase, with the 4-hydroxy-1,2,3-triazole scaffold. The 4-hydroxy-1,2,3-triazole system appears to
be useful and versatile in drug design.
profile, they are known as bioisosteres.2 Whereas chemistry
dictates the isosteric similarity of groups, only the biological
target can determine their bioisosteric similarity. Scaffold
Introduction
Bioisosteric replacement and scaffold hopping are two
techniques widely used in drug design to enhance potency,
selectivity, and other properties of a lead compound, and to
find new chemical entities with improved features. These
techniques may be defined as replacing part of a bioactive
molecule with a substructure that is similar in size and that
exhibits similar properties.1 Bioisosterism is an extension of the
concept of isosterism in which two functional groups, known
as isosteres, that present similar physicochemical properties,
are related. When isosteres also possess a common biological
hopping is a subset of the bioisosteric replacement process in
which the core structure of a small molecule is replaced. The
core may be of direct functional importance in interacting with
the biological target, or it may provide the necessary
scaffolding that allows substitution with functional groups in
the appropriate geometric configuration.1 For the appropriate
application of these powerful strategies to drug design,
continuous efforts must be made to develop innovative
isosteres that meet specific target requirements. An
application of bioisosteric substitution as a tool for generating
new antidiabetic lead structures using a family of acidic
hydroxyazoles was made in 1995 by a team at Wyeth.3 In that
paper the Structure-Activity Relationship studies determined
that the 3-hydroxypirazols were the most promising new class
of potential antidiabetic agent while the 4-hydroxy-1,2,3-
triazole ring system was not effective. Although it must be
emphasized that the study was in vivo and some unknown
mechanism could interfere with the activity of the studied
compounds. To our knowledge no other similar application
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was subsequently reported on the 4-hydroxy-1,2,3-triazole pentatomic heterocyclics (i.e. furazan and thiadiazole).
system. Moreover, the carbon substituent offers the potential to
This study focuses on the bioisosteric application of the modulate the pKa of the hydroxyl moiety. This approach might
pentatomic 4-hydroxy-1,2,3-triazole system (hereafter the be used to explore additional binding areas during bioisosteric
hydroxytriazole system, structure
A, Fig. 1). The attractive applications. Because of its versatility, this system can also be
feature of this system is the possibility it offers to regiodirect employed in scaffold hopping, for example it is well suited to
substituents in set directions, by substituting the three replace a generic hydroxylated aromatic ring (Fig. 1).
nitrogen atoms of the triazole ring; this is absent in other
Fig. 1 General structure of the hydroxytriazole system (A). The system may be used both to (bio)isosterically modulate a carboxylic acid (left), mainly thanks to the comparatively
low pKa values of hydroxyl group, and to substitute a generic hydroxylated (hetero)aromatic ring (right), exploiting a scaffold hopping approach.
This article presents and rationalizes the synthetic strategy to potent and specific Onchocerca volvulus chitinase (OvCHT1)
obtain different N-regio-substituted 4-hydroxy-1,2,3-triazoles. inhibitor.5 OvCHT1 is hypothesized to play a key role in the life
Structural characterization and physicochemical properties cycle of the worm parasite responsible for Onchocerciasis, a
(pKa and ClogP) of some N-substituted isomeric neglected tropical disease claimed to be the world's second
hydroxytriazoles are also presented. This preparatory study leading infectious cause of blindness.
lays the foundation for future exploitation of hydroxytriazoles
as bioisosteres of acidic functions. We also present two
successful bioisosteric applications using this moiety. The first
Result and Discussion
is an example of a classical bioisosteric application, in which
the hydroxytriazole system was successfully used to replace
Chemistry
the distal carboxyl group of (S)-glutamic acid (Glu), the
A straightforward and robust chemical strategy to obtain a
excitatory
neurotransmitter
responsible
for
major
series of hydroxytriazole isomers (type
The isomers bearing different substituents on the three
nitrogen atoms are shown as formulas and (Fig. 2).
A, Fig. 2) is presented.
neurodegenerative diseases.4 The second example is of
scaffold hopping, in which the hydroxytriazole system was
used to replace the phenol ring present in MDG-1-33A, a
1,
2
3
Fig. 2 Hydroxytriazole system (structure A) and its related hydroxytriazole isomers 1, 2 and 3 together with the benzyl protected forms (general structure B), and related triazole
isomers 4, 5 and 6. Substitutions on the nitrogen atoms are arbitrarily named N(a), N(b) and N(c) for easy reference.
The carboxylate substituent offers additional opportunities for
synthetic applications, as it can easily be transformed into
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other functional groups to link the triazole scaffold to obtain hydroxytriazoles 1, 2 and 3 is shown in Scheme 1.
additional molecule portions. The general synthetic scheme to
Scheme 1 Synthesis of differently substituted hydroxytriazoles 1, 2 and 3 and their protected forms 4, 5 and 6; (a) diethyl malonate, EtO-Na+, EtOH, reflux; (b) BnBr, K2CO3, DMF, rt;
(c) CAN, CH3CN/H2O (9/1), rt; (d) R-X, K2CO3, CH3CN, rt; (e) H2/Pd, THF, rt; (f) MeNH2; (g) 0.5 N HCl.
4-Methoxybenzylazide
reaction with diethyl malonate and sodium ethoxide in ethanol three isomeric benzyl derivatives (4b
described by Buckle et al,6 to obtain compound
(yield 71%). assigned by heteronuclear 2D-NMR (HSQC and HMBC). The
Subsequent treatment of with benzyl bromide in DMF structure elucidation of these isomeric series will be
afforded the corresponding O-benzyl protected in 55% yield extensively discussed in a forthcoming full article. The data
The structure of was unequivocally assigned using indicated alkylation reactivity of 10 at both the N(b) and N(c)
7
first underwent regioselective halides afforded comparable results.8 The structures of the
5b and 6b) were
,
8
8
9
.
9
heteronuclear 2D-NMR (HSQC and HMBC) techniques. The ring positions, but not at N(a); this is in line with findings
results of these studies confirmed the reported regioselectivity reported for other ethyl triazole-4-carboxylates.8 In all cases,
of the cycloaddition reaction used to obtain 8 6,7
.
The 4- compounds with structure
methoxybenzyl moiety was selectively removed using cerium (47% and 45% for compounds 5a and 5b, respectively)
ammonium nitrate (CAN) to afford triazole 10 in 35% yield compared to compounds with structure (30% for both 6a
The reactivity of 10 towards alkylating agents was then and 6b). Hydroxytriazoles and were obtained from the
investigated. Reaction of 10 with different alkyl and benzyl corresponding O-benzylated derivatives and by catalytic
5 were obtained in higher yields
.
6
2
3
5
6
halides in acetonitrile at room temperature afforded a mixture hydrogenation at atmospheric pressure pressure in 90% yield
of two isomers, which was separable by flash chromatography. in both cases.
Two exemplifying compounds (structures
5 and 6) are To avoid the risk due to the explosive behavior of azides with
presented in Scheme 1; other examples using different alkyl low C/N ratios, two different synthetic strategies were
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designed for 1a and 1b. For 1a, diethyl diazomalonate 129 was
treated with methylamine to yield the desired triazole as the
primary ammonium salt 13 (yield 79%). After acidification and
extraction with ethyl acetate, the free hydroxytriazole 1a was
isolated without any further purification (quantitative yield).
Conversely, for 1b, benzylazide was treated with diethyl
malonate and sodium ethoxide in ethanol, similar to the
Table 1. Measured pKa and CLogP values for compunds 1a-3a, 1b-3b, 14-16.
a
Compound
Structure
pKa
CLogPb
0.46
1a
6.21±0.02
5.14 ±0.02
5.54 ±0.02
preparation of 8 (70% yield). Benzylation of compounds 1a and
1b with benzylbromide in dimethylformamide afforded the
corresponding O-benzyl protected compounds 4a and 4b (93%
and 83% yield, respectively).
2a
3a
0.95
0.46
Physicochemical profile and isosteric evaluation.
In order to correctly modulate a functional group through a
(bio)isosteric approach, a wide range of isosteric replacements
must be evaluated. The selection should be initially guided by
using some important physicochemical parameters such as pKa
or other.10 Usually being the first to be taken into account in
handling acidic isosteres of the carboxyl group, the pKa value
1b
6.22±0.02
2.23
of selected hydroxytriazoles (compounds 1a-b, 2a-b and 3a-b
)
were measured by potentiometric titration, in order to find a
correlation between the hydroxytriazole ring substitution and
the pKa value (Table 1). For comparison purposes, the pKa
values of three other hydroxylated pentatomic heterocyclic
systems (hydroxypyrazole 14
,
hydroxythiadiazole 15 and
2b
5.14±0.02
2.72
hydroxyfurazan 16) are also given.
Together with dissociation constants, lipophilicity is an
important physicochemical parameter that should be taken
into account in seeking bioisosterism. To evaluate this effect,
the calculated LogP (CLogP) values are included in Table 1.
In the hydroxytriazole system, the pKa value appears to be
influenced by substitution on the three endocyclic nitrogens
N(a), N(b) and N(c). The N(a)-substituted isomers 1a and 1b
present higher pKa values (6.21 and 6.22, respectively), similar
to that of the hydroxypyrazole (14, pKa = 6.71). N(b)- and N(c)-
3b
14
5.38 ±0.01
6.71 ±0.01
2.23
1.19
substituted compounds (2a-b, 3a-b) are slightly more acidic,
with pKa values in the 5.14 - 5.54 range. Although less acidic
compared to either hydroxythiadiazole 15 (pKa = 4.21) or
hydroxyfurazan 16 (pKa = 3.24), compounds 2a and 2b may still
be fully deprotonated at physiological pH. Comparison of
CLogP values showed smaller values for the N-methyl
substituted hydroxytriazoles 1a, 2a and 3a compared to those
15
16
4.21 ±0.01
3.24 ±0.05
2.09
1.61
of compounds 14, 15 and 16. Hydroxytriazoles 1a-b, 2a-b and
3a-b may thus be considered potential bioisosteres of a
carboxylic acid, since they exist predominantly in their
deprotonated states at physiological pH.
a
Determined by potentiometry (see supplementary); each dataset
b
represents mean ± SD of five independent experiments. Calculated by
Bioloom for Windows v. 1.5, Biobite Corp., Claremont, CA, USA.
Examples of successful bioisosteric application of the
hydroxytriazole moiety
The first application here presented is an example of a classical
bioisosteric modulation, in which the carboxylic acid of a lead
compound is replaced with a series of isosteric moieties. The
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biological activities of the resulting compounds are evaluated presented
(R,S)-2-amino-3-(4-hydroxy-1,2,5-oxadiazol-3-
to determine the best bioisostere. The compound selected for yl)propionic acid (HFPA, Table 2) as Glu analogue obtained
modulation is a natural compound, (S)-Glu, for which specific using the hydroxy-1,2,5-oxadiazole scaffold.13
agonists and antagonists are necessary in order to characterize In the attempt to enhance selectivity for AMPA GluRs, the
the physiological and pathological roles of the different types hydroxytriazole scaffold was used to bioisosterically replace
of glutamate receptors (GluRs).11, 12
the distal group of Glu. From a preliminary comparison
In the second application, the potent OvCHT1 inhibitor MDG-1- between the pKa values of the ethylcarboxylate 4-hydroxy-
33A was modulated.5 The lack of any crystallographic data triazole derivatives 2a and 3a and the Glu biosiosteres 17 and
makes this a challenging task, as such data would be useful to 18, we observed that replacement of the electronwithdrawing
guide the rational design of inhibitors. By modulating the carboxyl substituent entails an increase of pKa (see Table 1 and
triazole scaffold substitution, an advantageous positioning of 2). In both isomeric methyl analogues, 2-Me-TRPA 17 and 3-
the lipophilic substituent was achieved.
MeTRPA 18, the pKa values of the hydroxytriazole moiety arise
6.11 and 6.42 respectively and an increase of 0.9 was
measured versus 2a and 3a respectively. When the
1) Targeting ionotropic AMPA GluRs using the hydroxyfurazan series was studied, we observed an increase of
hydroxytriazole moiety as a bioisosteric replacement: (S)-Glu pKa of 0.24, as depicted in Table 1 and 2 (derivatives 16 and
(Table 2) plays a major role in the transmission of excitatory (+)-HFPA). The extent of this effect depends on the hydroxyl
signals in the central nervous system, and is implicated in e.g. substituted heterocycle and, being the furazan a strongly
epilepsy, pain, memory, excitotoxicity, and brain electron-acceptor group14, a weaker effect on the acidic
development.4 (S)-Glu exerts its effects through two types of properties was observed. On the basis of these results the
receptors: ligand-gated ionotropic receptors (iGluRs) and G- derivatives 17 and 18 more closely mimic the distal carboxylic
protein-coupled metabotropic receptors (mGluRs).13 iGluRs are acid of Glu, maintaining the correct degree of deprotonation of
classified, depending on their selective agonists, into NMDA the hydroxyl group. Moreover, the hydroxytriazole scaffold
(N-methyl-D-aspartate), AMPA ((RS)-2-amino-3-(3-hydroxy-5- also provides the opportunity of placing a small lipophilic
methylisoxazol-4-yl)propionic acid, Table 2) and KA (kainic portion oriented in two different directions, being useful for
acid) receptors. The second class, mGluRs, are divided into increasing the potency and/or selectivity towards a receptor
three groups (I, II, III), each containing at least two subtypes. subtype. It is known that AMPA GluRs present lipophilic
The vast majority of both iGluR and mGluR subtypes are cavities close to the distal carboxyl group in contrast to other
considered to be interesting therapeutic targets for the Glu receptor subtypes. The N-methyl groups in both 17 and 18
treatment of a number of neurologic and psychiatric diseases. are directed in well-defined areas within the Glu receptor
Specific agonists and antagonists are necessary to characterize binding site, unlike other examples, such as TDPA15 or HFPA,
the physiological roles of the individual receptors.12 Since the where unsubstituted ring systems have been used to mimic
late 1970s, the bioisosteric approach has been the strategy of the distal carboxyl group.
election to design selective agonists and antagonists for The synthetic preparation of compounds 17 and 18 is shown in
AMPA/KA/NMDA receptors. In this connection, a number of Scheme 2.
bioisosteric replacements of the distal Glu carboxylic group
have been reported. Among them, the authors recently
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Scheme 2 Synthesis of AMPA GluRs agonists 17 and 18; (a) NaBH4, absolute ethanol, rt; (b) NBS, P(Ph)3, dry CH2Cl2, 0°C; (c) NaH, diethyl acetamidomalonate, dry THF,
rt; (d) H2/Pd, dry THF, rt; (e) 6 N HCl, reflux.
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hydroxytriazoles 22a (95% yield) and 22b (85% yield). Final
deprotection of 22a and 22b in 6 HCl at reflux gave
Table 2 Receptor binding affinities at native rat iGluRs a.
N
hydrochloride salts of desired compounds 2-Me-TRPA (17) and
3-Me-TRPA (18) in quantitative yields.
IC50 (μM)
The preliminary pharmacological results concerning iGluR
receptor subtypes are shown in Table 2. Both 17 and 18, tested
as racemic mixtures, present good selectivity within their
receptor subtypes. In particular, both compounds presented a
pharmacological profile quite similar to that of AMPA, being
neither compound able to show any activity on KA or NMDA
receptors. The binding affinity of 18 toward the AMPA receptor
was expected, as the AMPA receptor is capable of
accommodating bulky groups in the area adjacent to the
aminoacidic function. At the same time, if compared both with
AMPA, HFPA and TDPA, the low pKa value of the
hydroxyltriazoles should be behind the low activity showed by
these compounds. To the opposite, the N(b) substituted 2-Me-
TRPA (17) analogue presented in prospective a quite interesting
activity profile. In recent years, it was investigated the possibility
to replace the 3-hydroxyisoxazole in AMPA with other isosters
being able to extrasubstitute the ring and by effect to poses
lipophilic bulkiness in a topographic receptor space. Recently, it
Distal
carboxylate
or its mimic
pKa
KA
Ki (μM)
AMPA
Compound
Structure
receptor NMDA
receptors
s
receptors
(S)-Glu16
0.34
0.38
0.20
4.217
AMPA18
0.039
>100
>100
4.819
(+)-HFPA13
0.11
18
13
3.48
was the case of 3-hydroxypyrazole AMPA analogue20
(cpd6 in
the reference) able to weakly but selectively inhibit AMPA
receptor with IC50 of 8.9 μM. This quite poor result was
explained by the author with the increased pKa value of the 3-
hydroxypyrazole compared to the 3-hydroxyisoxazole in AMPA.
- not
(S)-TDPA15
0.065
>100
>100 b
determined
2-Me-TRPA
hydroxypyrazole cpd6 20
AMPA receptor with IC50 of 3.1 μM, being almost three time
more active than cpd6 20
This preliminary quite interesting
(17
)
analogues, showing a lower pKa value of
,
was found able to selectively inhibit
.
results will open the possibility to design subtype selective
ligands by insertion of substituents on the N(b) triazole position
that may reach non-conserved regions of the receptor.
3.1
17
[5.54 ± > 100
0.09] c
> 100 6.11 ± 0-02d
(2-Me-TRPA)
2) Targeting OvCHT1 using the hydroxytriazole moiety within
a scaffold hopping approach: Onchocerciasis, also known as
river blindness, is a neglected tropical disease caused by the
parasitic worm Onchocerca volvulus (O. volvulus), and is the
world's second leading infectious cause of blindness. An
estimated 37 million people are infected with river blindness,
mostly in developing countries, in sub-Saharan Africa and
Central and South America.21 Additionally, the possible
emergence of Ivermectin-resistant O. volvulus accelerates the
need to develop new innovative therapeutic strategies in this
field, since the broad-spectrum antiparasitic drug Ivermectin is
the product currently most widely used to treat the disease.22
OvCHT1 is a larval-stage-specific chitinase that was recently
identified as a potential biological target affecting nematode
development.23 As a result of a blind screening program,
Closantel was discovered to be a potent and selective inhibitor
of OvCHT1. Starting from this lead, a series of Closantel
analogues was prepared to afford chitinase inhibitors with IC50
values in the low nanomolar range.5 Of these, compound MDG-
1-33A (Table 3) was found to inhibit OvCHT1 with an IC50 value
1.4
18
[5.87 ± > 100
0.08] c
> 100 6.42 ± 0.01d
(3-Me-TRPA)
AMPA, KA, and NMDA receptors were studied using [3H]AMPA, [3H]KA, and
[3H]CGP 39653 as radioligands, respectively.b [3H]CPP binding. c Mean pIC50 ± SEM
(shown in brackets) and the corresponding IC50 values were calculated from full
a
d
concentration-response curves of three individual experiments. Determined by
potentiometry (see supplementary).
Compounds 5a or 6a were first reduced to their corresponding
alcohols 19a-b (64% and 81% yield, respectively) and
subsequently reacted with N-bromosuccinimide (NBS) to yield
20a (90% yield) and 20b (93% yield), respectively. Reaction with
diethyl [(tert-butoxycarbonyl)amino]malonate in the presence of
NaH (70% yield in both cases) followed by catalytic
hydrogenation
at
atmospheric
pressure
furnished
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of 1.98 µM.5 Using
a scaffold hopping approach, the
Table 3 IC50 of OvCHT1 inhibition
.
diiodophenolic core, found to be essential for chitinase
inhibition, in MDG-1-33A was replaced by the hydroxytriazole
Structure
R=
Compound
MDG-1-33A
IC50 (μM)a
scaffold to give derivatives 23
-
27. The synthetic routes for the
27 are outlined in Scheme 3.
preparation of compounds 23
-
Alkylation of compound 10 produced two isomers, which were
separable by flash chromatography. As previously discussed,
compounds with alkyl substituent on the N(b) atom were
obtained in higher amounts (range: 45-50% yield) than
compounds with alkyl substituent on the N(c) atom (range 15-
30% yield). Basic hydrolysis yielded the carboxylic acid (around
90% yield in all cases), which was subsequently converted into
the corresponding acyl chloride. Reaction with 4-phenoxyaniline
1.984
23
24
4.69 ± 0.29
yielded compounds 25
- 28 (40-65% range of yield). Catalytic
hydrogenation of 28 and 25 afforded hydroxytriazoles 23 and 24
respectively, in quantitative yields.
During the subsequent fine tuning modulation of the
hydroxytriazole scaffold (data not shown), the role of four
possible substitutions on the triazole ring was determined
considering all the N(a), N(b) and N(c) positions. Because the fact
that the phenolic group in MDG-1-33A analogues can be
successfully alkylated without affecting chitinase inhibition, the
O-alkylated triazole analogues were also considered. From these
studies, it was found that the presence of either N(b) or N(c)
alkyl substituents is beneficial to the potency that follow the size
of the alkyl substituent. The combination of this facts together
the presence of a benzyl substituent of the hydroxyl group
afforded submicromolar range compounds. IC50 values of MDG-
5.11 ± 0.06
25
26
27
0.52 ± 0.03
0.41 ± 0.01
0.41 ± 0.04
1-33A triazole derivatives 23
results of these scaffold hopping procedures afforded three
promising MDG 1-33A derivatives, compounds 25 26 and 27,
- 27 are shown in Table 3. The
-
,
that inhibit OvCHT1 chitinase in the nM range.
.
a in vitro OvCHT1 inhibition activity was measured by fluorescence-based
assay (see supplementary for details).23 Each dataset represents mean ± SD of
three independent assays.
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Scheme 3. Synthesis of OvCHT1 chitinase inhibitors 23 - 27; (a) R-X, K2CO3, CH3CN, rt; (b) 2 N LiOH, THF, rt; (c) 2 N HCl, H2O; (d) (COCl)2, DMF, dry THF; (e) 4-phenoxyaniline, dry
pyridine, dry THF, rt; (f) H2/Pd, dry THF, rt.
Conclusions
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