Efficient Dimerization Disruption of Leishmania infantum Trypanothione Reductase by Triazole-phenyl-thiazoles

Article abstract of DOI:10.1021/acs.jmedchem.1c00206

Inhibition of Leishmania infantum trypanothione disulfide reductase (LiTryR) by disruption of its homodimeric interface has proved to be an alternative and unexploited strategy in the search for novel antileishmanial agents. Proof of concept was first obtained by peptides and peptidomimetics. Building on previously reported dimerization disruptors containing an imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-triazole-based chemotype that yields noncompetitive, slow-binding inhibitors of LiTryR. Several compounds bearing (poly)aromatic substituents dramatically improve the ability to disrupt LiTryR dimerization relative to reference imidazoles. Molecular modeling studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory activity over LiTryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able to kill both extracellular and intracellular parasites in cell cultures.

Full text of DOI:10.1021/acs.jmedchem.1c00206

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Article
Efficient Dimerization Disruption of Leishmania infantum
Trypanothione Reductase by Triazole-phenyl-thiazoles
Alejandro Revuelto,∥ Héctor de Lucio,∥ Juan Carlos García-Soriano, Pedro A. Sánchez-Murcia,
Federico Gago, Antonio Jiménez-Ruiz,* María-José Camarasa, and Sonsoles Velázquez*
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ABSTRACT: Inhibition of Leishmania infantum trypanothione
disulfide reductase (LiTryR) by disruption of its homodimeric
interface has proved to be an alternative and unexploited strategy
in the search for novel antileishmanial agents. Proof of concept was
first obtained by peptides and peptidomimetics. Building on
previously reported dimerization disruptors containing an
imidazole-phenyl-thiazole scaffold, we now report a new 1,2,3-
triazole-based chemotype that yields noncompetitive, slow-binding
inhibitors of LiTryR. Several compounds bearing (poly)aromatic
substituents dramatically improve the ability to disrupt LiTryR
dimerization relative to reference imidazoles. Molecular modeling
studies identified an almost unexplored hydrophobic region at the interfacial domain as the putative binding site for these
compounds. A subsequent structure-based design led to a symmetrical triazole analogue that displayed even more potent inhibitory
activity over LiTryR and enhanced leishmanicidal activity. Remarkably, several of these novel triazole-bearing compounds were able
to kill both extracellular and intracellular parasites in cell cultures.
antioxidant defense in trypanosomatids. The Try/TryR couple
in these parasites substitutes for the glutathione/glutathione-
disulfide reductase (GR) pair characteristic of most eukaryotic
organisms. Accordingly, the absence of TryR in mammalian
cells, the significant differences between TryR and human GR
(hGR) (the active sites of the two enzymes have opposite net
charges and different volumes), and its crucial role for parasite
survival make TryR an attractive target for new chemo-
therapeutics.12−15 Since this enzyme was identified, many
potent and selective competitive polycationic TryR inhibitors
(which mimic the positively charged Try in the active site)
have been described. However, there are scarce reports of
potent TryR-inhibiting compounds with adequate antiparasitic
activity.16,17 This is so because it has been shown that survival
of the parasites is only affected when TryR activity is reduced
by more than 90%, probably as a consequence of the high
intracellular concentrations of its natural substrate (Try) in the
cell.18 This implies that, to be effective against the parasites,
competitive inhibitors must have very high binding affinities so
as to give rise to inhibition constants in the low nanomolar
INTRODUCTION
■
Leishmaniasis is an infectious disease caused by intracellular
protozoan parasites from more than 20 Leishmania species and
transmitted to humans through the bite of infected female
phlebotomine sandflies. According to the World Health
Organization (WHO), leishmaniasis is one of the major
neglected tropical diseases that affects 12 million people in 98
countries, being especially prevalent in tropical and subtropical
areas.1 Visceral leishmaniasis (VL), also known as Kala-azar, is
the most severe form of the disease with more than 60 000
human deaths annually. VL is caused by Leishmania donovani
and Leishmania infantum parasites worldwide, including
Mediterranean countries.2 No effective vaccine for humans
has so far been developed, and leishmaniasis control mainly
relies on chemotherapy.3,4 However, currently available
treatments are inadequate because of high costs, toxicity,
drug resistance, and the need for parenteral administration.5,6
Therefore, there is an urgent need to find new effective
innovative drugs against this disease.
Herein, we report a target-based approach for the discovery
of novel agents against L. infantum parasites targeting a protein
that is essential for parasite survival and exclusive for these
parasites.7,8 We have focused on trypanothione (Try) disulfide
reductase (TryR) because it is one of the few genetically and
chemically validated drug targets, and validation is one
important criterion in target assessment.9−11 Among other
highly relevant functions, this enzyme is essential for
Received: February 3, 2021
Published: May 4, 2021
https://doi.org/10.1021/acs.jmedchem.1c00206
© 2021 American Chemical Society
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Figure 1. From a peptide dimerization disruptor of LiTryR (1) and in-house imidazoles 2 and 3 to the newly designed triazole-phenyl-thiazole
scaffold I.
range. Nonetheless, one of the most potent (submicromolar)
competitive inhibitors of Trypanosoma brucei (TbTryR)
described to date is almost inactive against L. donovani.19
This limitation, together with the need for very high affinities
imposed by the high intracellular concentrations of both the
natural substrate (between 3 and 50 μM under steady-state
conditions and up to 150 μM under oxidative stress) and TryR
itself (0.5−1.3 μM), make noncompetitive and/or irreversible
inhibitors emerge as more attractive alternatives for targeting
this enzyme.20a,b,21
The crystal structure of TryR from L. infantum (LiTryR) was
first solved by Baiocco et al.22 and is very similar to that of
other trypanosomatids.13,14,16,23,24 This enzyme is a twofold
symmetrical homodimer that contains two active sites and two
reduced nicotinamide adenine dinucleotide phosphate
(NADPH)- and flavin adenine dinucleotide (FAD)-binding
domains separated by a large and well-characterized inter-
face.25 Each Try binding site is located in a large solvent-
exposed cavity at the interface between the two monomers.
Several X-ray crystal structures of TryR in complex with a
variety of competitive reversible inhibitors have shown that the
large pocket allows not only several binding modes for small-
molecule inhibitors but also the simultaneous binding of more
than one inhibitor molecule.23 Such unpredictable binding
modes, due to the large size of the active site, make the rational
design of competitive, high-affinity inhibitors of TryR a very
challenging task.
Bearing in mind the homodimeric nature of this enzyme,
back in 2013 we devised an alternative inhibition strategy that
aims to disrupt the protein−protein interactions responsible
for LiTryR homodimerization.25 Initially, by means of a
combination of molecular modeling and site-directed muta-
genesis studies, we identified and validated E436an amino
acid located at an interfacial α-helix spanning from P435 to
M447as a hotspot for dimer stabilization and assessed its
importance for the catalytic activity of the enzyme. As a “proof
of concept” of this novel approach, we designed and tested a
small library of linear peptides that represent rational variations
of this α-helix. From these studies, the 13-mer-modified
peptide sequence PKIIQSVGIS-Nle-K-Nle (1) emerged as a
potent enzyme dimerization disruptor that also behaves as a
strong inhibitor (in the submicromolar range) of the
oxidoreductase activity of LiTryR.25 Next, a variety of helix-
stabilized cyclic peptides26,27 and α,β3-peptide foldamers28
were successfully explored in our search to find peptidomi-
metics with increased proteolytic stability. However, con-
jugation with cell-penetrating peptides was always required to
facilitate the cellular uptake of these peptide- or peptidomi-
metic-based LiTryR dimerization disruptors and to kill the
parasites in cell culture.28,29 Thus, the design of small
molecules with druglike properties better than those of the
prototype peptide or the previous peptidomimetics appeared
as a desirable goal.
In this regard, we recently disclosed the results of our first
steps toward nonpeptide disruptors of LiTryR dimerization
with promising leishmanicidal activity by an α-helical mimetic
approach.30 Among the reported proteomimetic scaffolds,31−33
the pyrrolopyrimidine34 and the 5-6-5 imidazole-phenyl-
thiazole35 cores were selected to dictate the spatial orientations
of the side chains of three key residues (K2, Q5, and I9) in the
linear peptide prototype 1. Imidazole-based compounds 2 and
3 (Figure 1), bearing a naphthalene or a biphenyl polyaromatic
R3 substituent to mimic the hydrophobic isoleucine residue,
emerged as potent inhibitors of the LiTryR oxidoreductase
activity, while pyrrolopyrimidines were shown to be much less
active. Although these small molecules displayed a moderate
capacity to disrupt the LiTryR dimer, this effect was much less
pronounced than that observed for the previous peptide-based
inhibitors. Remarkably, the imidazole-based compounds were
cell-permeable and showed significant leishmanicidal activity
against both amastigote and promastigote forms of Leishmania
parasites. However, these molecules displayed a cytotoxic
activity similar to that observed for their peptidic and
peptidomimetic predecessors and exhibited low selectivity
indexes (SIs).
1,2,3-Triazole-containing compounds have been widely
investigated because they are considered privileged scaffolds
in different areas of medicinal chemistry,36−38 including several
examples in the antileishmanial field.39 Interest in this
molecular architecture has also been fueled by its expedient
synthesis through “click chemistry”.40 In this scenario, we
decided to apply the bioisosteric replacement of imidazole by
1,2,3-triazole in our recently described imidazole-phenyl-
thiazoles.30 We herein report the synthesis and structure−
activity relationship (SAR) studies of novel 1,2,3-triazole-
phenyl-thiazole compounds of general formulae I (Figure 1) to
demonstrate the potential of this novel chemotype for LiTryR
dimerization disruption and leishmanicidal activity against L.
infantum parasites. A whole set of 26 triazole-based compounds
modified at four different positions in the new scaffold (R1−R4
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a
Scheme 1. Synthesis of the First Series of 1,4-Disubstituted 1,2,3-Triazole Compounds 12a−c
a
Reagents and conditions: (a) NaN3, DMSO, molecular sieves 4 Å, 100 °C, 72 h; (b) Cp*RuCI(PPh3)2 (5 or 10 mol %), DMF, rt to 100 °C; (c)
CuSO4·5H2O, sodium ascorbate, H2O/EtOH 1:1, rt, 8 h; (d) (NH4)2S 20% aq, DMF, 80 °C, 4 h; (e) α-bromomethylketones 10a−c, iPrOH, 70
°C, 5 h; (f) H2, Pd/C 10%, TFA, THF/MeOH 1:1, rt, 2 h.
substituents) was prepared and evaluated. Five of these
molecules display activities that very closely resemble that of
the peptide prototype 1 and likewise behave as non-
competitive, slow-binding inhibitors.41 Molecular modeling
studies identified a putative binding site for these compounds
at an almost unexplored central interfacial cavity of the enzyme
and provided a structural rationale to the observed SAR results
that was supported by ensuing rational design, synthesis, and
activity of a new symmetrical triazole analogue with the desired
properties.
hydrolysis of the azide group to the undesired amine. The next
step in the synthetic route was the 1,3-dipolar cycloaddition of
the azide intermediate with terminal alkynes substituted with
the appropriate R1 group (as a Lys mimetic) to generate the
1,2,3-triazole ring. Initial attempts to use the ruthenium-
catalyzed azide−alkyne cycloaddition (RuAAC)40b from 5a
and commercially available N-Cbz-protected terminal alkyne 6
in the presence of catalytic amounts (5 or 10%) of a
cyclopentadienyl Ru catalyst [Cp*RuCl(PPh3)2] at different
temperatures in dry dimethylformamide (DMF) and an inert
atmosphere did not produce the expected 1,5-disubstituted
1,2,3-triazole 7. Only unreacted starting materials or complex
decomposition mixtures were observed under these conditions.
In contrast, the 1,4-disubstituted 1,2,3-triazole regioisomer 8
could be easily obtained in 56% yield by the 1,3-dipolar
copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC)40a
of azide 5a and the terminal alkyne 6 using the CuSO4/sodium
ascorbate catalyst system at room temperature for 8 h in H2O/
EtOH (1:1 mixture) as the solvent. Subsequent treatment of
nitrile 8 with aqueous (aq) 20% ammonium sulfide at 80 °C
for 4 h provided the thioamide intermediate 9 in 74% yield.
Next, Hantzsch thiazole synthesis from 1 equiv of 9 and 1
equiv of the commercially available α-bromomethylketone 10a
or the previously synthesized 10b and 10c30 (bearing a
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of the target triazole-phenyl-
thiazole type I compounds is exemplified by the preparation of
the first series of 1,2,3-triazole derivatives 12a−c (bearing R1−
R3 substituents similar to those present in the predecessor
imidazole analogues 2 and 3), as shown in Scheme 1. The
synthetic route started from the previously described bromide
intermediate 4a30 bearing an imidazolidinone ethyl R2 group
(used as a Gln mimetic in the previous imidazole derivatives)
by treatment with sodium azide in anhydrous dimethyl
sulfoxide (DMSO) at 100 °C for 72 h to afford the azide
intermediate 5a in 85% yield. The addition of molecular sieves
with pore openings of 4 Å was required to avoid the partial
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Table 1. Half-Maximal Inhibitory Concentration (IC50) Standard Error (SE) Values for Triazole Compounds I and the
a f
,
Truncated Analogues 13 and 14 in the TryR Oxidoreductase Activity and LiTryR Monomer Displacement Assays
b
c
act
dim
compounds
R1
n
R2
R3
R4
IC (μM)
IC (μM)
50
50
1
2
3
12a
12b
12c
1.5 0.2
5.1 0.4
8.6 1.4
14.6 1.0
5.9 1.1
10.9 1.8
7.0 0.6
d
(CH2)2naphthyl
(CH2)2biphenyl
(CH2)2Ph
(CH2)2naphthyl
(CH2)2biphenyl
26%
32%
d
+
NH3
NH3
NH3
4
4
4
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
H
H
H
38.1 1.2
e
+
+
7.1 0.6
e
4.8 0.6
Modifications at R1
12d
12e
12f
12g
12h
12i
OH
CH3
CH3
COOH
NH3
4
3
3
3
3
3
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Ph
(CH2)2Ph
(CH2)2biphenyl
(CH2)2Ph
(CH2)2Ph
H
H
H
H
H
H
>75
>75
>75
>75
20.8 2.3
19.7 3.8
>75
>75
>75
>75
>75
10.8 0.1
+
+
NH3
(CH2)2Biph
Modifications at R2
+
12j
12k
NH3
NH3
4
4
Me
Me
(CH2)2Ph
(CH2)2biphenyl
H
H
20.1 2.5
26.8 5.7
>75
16.1 0.7
+
Modifications at R3
OiPr
+
12l
NH3
NH3
NH3
NH3
NH3
NH3
NH3
NH3
4
4
4
4
4
4
4
4
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
H
H
H
H
H
H
H
H
54.5 4.4
24.1 1.4
11.2 0.2
43.6 1.1
14.6 1.7
6.9 2.1
11.9 0.6
6.6 1.9
>75
>75
+
t
12m
12n
12o
12p
12q
12r
12s
CH2 Bu
Ph
CH2Ph
(CH2)3Ph
(CH2)2THQ*
(CH2)2DHB*
(CH2)2DBF*
+
29.0 1.5
61.2 2.8
19.1 1.1
>75
10.7 0.5
8.8 0.2
+
+
+
+
+
Truncated Analogues
13
14
>75
>75
>75
>75
Modifications at R3 and R4
+
19a
19b
19c
19d
19e
NH3
NH3
NH3
NH3
4
4
4
4
4
(CH2)2Im*
Ph
Ph
Ph
Ph
Ph
4.3 1.0
7.1 1.7
11.3 0.2
9.0 0.5
5.7 0.5
6.7 1.0
+
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
(CH2)2Im*
biphenyl
PhOPh
(CH2)2biphenyl
(CH2)2PhOPh
18.4 3.9
16.9 2.7
19.8 2.3
19.3 3.7
+
+
+
NH3
Ph
a
b
Linear peptide 1 and imidazole-based compounds 2 and 3 were included as reference compounds. Enzymatic activity >75 indicates that the IC50
c
value is higher than 75 μM (maximum assayed). Results are representative of three independent experiments, each performed in triplicate. Dimer
quantitation assay (enzyme-linked immunosorbent assay (ELISA)).25 Percentage of inhibition observed at 20 μM as reliable IC50 values could not
d
e
f
be determined in this case. Percentages of inhibition observed at 20 μM were 73 and 78% for 12b and 12c, respectively. Im* = imidazolidinone,
THQ* = tetrahydroquinoline (TFA salt), DHB* = dihydrobenzofuranyl, DBF* = dibenzofuranyl.
naphthyl or a biphenylethyl hydrophobic R3 substituent)
heated in isopropanol at 70 °C for 36 h gave the expected α/β-
unsaturated derivatives 11a−c monosubstituted at the 4-
position of the thiazole ring in 87, 51, and 63% yields,
respectively (Scheme 1). Finally, treatment of 11a−c with H2,
Pd/C in tetrahydrofuran (THF)/MeOH mixture in the
presence of trifluoroacetic acid (TFA) at room temperature
allowed the simultaneous hydrogenolysis of NHCbz and
hydrogenation of the double bond to afford the deprotected
target compounds 12a−c as monotrifluoroacetate salts in 35,
24, and 35% yields, respectively. Chromatographic purification
by Biotage using reverse-phase columns was always required to
obtain the final compounds with purities higher than 95% for
their biological evaluation.
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a
Scheme 2. Synthesis of Triazoles Disubstituted at the Thiazole Ring (19a−e)
a
Reagents and conditions: (a) PPh3, dry toluene, 110 °C, 6 h; (b) KOH, dry toluene, 110 °C, 3 h; (c) RCHO, 110 °C, 3 h; (d) NBS, p-TsOH,
CH3CN, rt, 14 h; (e) thioamide 9, 17a−e, iPrOH, 70 °C, 5 h; (f) H2, Pd/C 10%, TFA, THF/MeOH 1:1, rt, 2 h. *Commercially available reagents.
As will be discussed below, the 1,2,3-triazole compounds
12a−c emerged as more potent dimerization disruptors of
LiTryR than the reference imidazoles and also behaved as
strong inhibitors of the oxidoreductase activity of the enzyme.
These results prompted us to develop a novel series of triazole
derivatives 12d−s (see structures in Table 1 and Scheme S1)
modified at the R1−R3 substituents to perform a SAR study.
Regarding modifications at R1 and R2, the protonated
aminobutyl group linked to the triazole was replaced by OH,
Me, or COOH (12d−g) or by a shorter aminopropyl
substituent (12h, 12i), while the R2 imidazolidinone (attached
to the central phenyl ring through an ethyl linker) was
removed (12j, 12k). In terms of R3 modifications, bulky alkyl
substituents (12l, 12m) or a phenyl group linked to the
thiazole ring through a polymethylene spacer of a different
length (12n−p vs 12a) were explored.
Since the presence of a biphenyl or naphthyl substituent at
R3 was highly relevant for potent enzyme inhibition and
antileishmanial activity, other polyheteroaromatic rings were
also explored at R3 (12q−s). The synthesis of the novel
triazole analogues 12d−s involved (i) a high-yielding
nucleophilic aromatic substitution (SNAr) of aryl fluorides
with the appropriate alcohols (bearing the R2 substituent),30
(ii) CuAAC reactions between azide intermediates and
terminal alkynes (containing the R1 substituent), (iii)
Hanztsch thiazole synthesis of thioamides with suitable α-
bromomethylketones (with the R3 substituent), and (iv) and a
final step of catalytic hydrogenation to yield the deprotected
final compounds using similar protocols to those described
above. It should be noted that in the catalytic hydrogenation of
the protected triazole compound intermediate 11q (see
Scheme S1) bearing a quinoline moiety as the R3 substituent,
the 1,2,3,4-tetrahydroquinoline compound 12q (see Scheme
S1) was isolated as a ditrifluoroacetate salt due to the partial
hydrogenation and salt formation of the quinoline ring
nitrogen atom. The synthetic experimental details of the target
12q−s compounds and the noncommercially available key α-
bromomethyl-ketone intermediates 10m, 10p−s are included
in the Supporting Information (see Schemes S1 and S2).
Scaffold-truncated simplified compounds (13 and 14, see
Table 1), possessing only two aromatic rings and substituents,
were also prepared for the SAR studies following similar
synthetic protocols (see also the Supporting Information for
experimental details; Scheme S3). To further investigate the
SAR of the triazole-based compounds, we next focused on the
preparation of an additional series of triazole analogues 19a−e
(Scheme 2) disubstituted at both positions 4 and 5 of the
thiazole ring. Polyaromatic groups directly attached or linked
through an ethyl spacer to the 4-position and a phenyl group at
the 5-position were selected as R3 and R4 substituents,
respectively. As shown in Scheme 2, the preparation of these
compounds required the previous synthesis of the non-
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a
Scheme 3. Synthesis of the Newly Designed Symmetrical Analogue 22
a
Reagents and conditions: (a) NBS, p-TsOH, acetonitrile, rt, 14 h; (b) thioamide 9, iPrOH, 70 °C, 5 h; (c) H2 Pd/C 10%, TFA, THF/MeOH, 1:1,
rt, 2 h.
commercially available α-bromobenzylketones 17a−d. Thus,
17a−c bearing a biphenyl or a phenyloxyphenyl group at R3
directly attached to the carbonyl were synthesized by
bromination of the commercially available benzylketones
16a−c with N-bromosuccinimide (NBS) in p-toluensulfonic
acid (p-TsOH) in good to excellent yields.
characterized further to obtain their dissociation constants.
act
The IC50 values determined in the activity (IC ) and
50
dim
50
dimerization (IC ) assays are shown in Table 1.
According to the results obtained with the first series of
triazole-based compounds 12a−c (Table 1), analogues
containing a naphthyl or a biphenyl group at R3 (12b, 12c)
On the other hand, α-bromobenzyl α,β-unsaturated ketone
17d with a polyaromatic group was obtained by regioselective
bromination of the most favorable benzylic position with NBS
in p-TsOH of the corresponding benzylketones 16d, which
was previously synthesized from 1-chloro-3-phenylpropan-2-
one by treatment with PPh3 to give the phosphonium salt 15
followed by a Wittig procedure with the corresponding
polyaromatic aldehydes in moderate yields. Next, Hantzsch
thiazole synthesis from thioamide intermediate 9 with the
commercial 17e or synthetic α-bromobenzylketones 17a−d,
followed by catalytic hydrogenolysis of the N-Cbz group and
simultaneous olefin hydrogenation of 18a−e using 10% Pd/C
in the presence of TFA, afforded the desired unprotected
disubstituted thiazole derivatives 19a−e as monotrifluoroace-
tate salts in low to moderate yields. Finally, we also designed
(see section Computational Studies below) and prepared
(Scheme 3) a new symmetrical triazole analogue (22) by
treating 2 equiv of thioamide intermediate 9 with 1 equiv of α-
bromomethylketone 20 (previously synthesized by bromina-
tion with NBS of commercially available 1,3-diacetyl benzene)
under Hantzsch reaction conditions, followed by catalytic
hydrogenolysis of the NHCbz group using similar procedures
to those described above.
Biological and Structural Studies. Enzymatic Assays. L.
infantum TryR Inhibition and Dimerization Assays. All of the
novel triazole-phenyl-thiazole modified at substituents R1−R3
(12a−s), disubstituted thiazole analogues bearing R3 and R4
substituents (19a−e), and truncated simplified compounds
(13 and 14) were evaluated for their effect on both the
oxidoreductase activity and the dimerization status of
LiTryR.25 The prototype peptide 1 and imidazole-containing
compounds 2 and 3 were used as reference molecules.
Screening was performed at different concentrations ranging
from 0.1 to 75 μM. A concentration of 1 μM TS2 was used for
this initial screening. This low substrate concentration is
sufficient for comparison of compound inhibitory activity in
the oxidoreductase assay. The best candidates were then
potently inhibited the TryR oxidoreductase activity, showing
act
50
similar IC values (in the low micromolar range) to those
obtained for the reference imidazole compounds 2 and 3.
Remarkably, 12a−c were much more potent LiTryR
dimerization disruptors than their imidazole counterparts.
Thus, in contrast to 2 and 3, for which no reliable ICd50im could
be measured and only ∼30% dimerization inhibition was
observed at a concentration of 20 μM, the ICd50im values for 12b,
12c (7.1 and 4.8 μM, respectively) were similar to (or even
slightly lower than) those obtained for the potent prototype
peptide 1. Furthermore, replacement of the naphthyl or
biphenyl groups at R3 by a phenyl (12b, 12c vs 12a) slightly
act
50
increased the IC value, while it significantly decreased (in a
5−8-fold range) the ability to disrupt the LiTryR dimer. We
took this result as a clear indication that the presence of a
polyaromatic substituent at R3 is relevant to potent LiTryR
dimer disruption.
SAR studies on the second series of triazole compounds
(12d−s) with variations at the R1−R3 substituents began with
modifications at the R1 position. As shown in Table 1, the
presence of a positively charged amino alkyl group at R1
appears as an absolute requirement for inhibition of both
activity and dimerization, as evidenced by the complete loss of
effect observed when the amino group was replaced by
hydroxyl, methyl, or carboxylic acid groups (12d−g vs 12a,
12c). As regards the length of the R1 spacer attached to the
amino group, shortening the original tetramethylene linker to a
trimethylene brought about a ∼2-fold reduction of potency in
both assays (12h, 12i vs 12a, 12c). The existence of the
imidazolidinone ring as the R2 substituent on the central
phenyl ring also appeared important for optimal activity since
its removal (12j, 12k vs 12a, 12c) resulted in compounds twice
or thrice less active in both assays. Modifications at the R3
substituent (located at the 4-position of the thiazole ring) had
a pronounced effect on enzymatic activity inhibition and, even
more so, on dimerization disruption. Thus, the presence of a
(poly)aromatic substituent at R3 was crucial for enhanced
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potency since compounds 12l, 12m, with bulky aliphatic
groups, exhibit much less inhibitory activity on the enzyme and
no destabilization effect on the LiTryR homodimer in
comparison with the (poly)aromatic analogues 12a−c. In
comparison, varying the length of the linker connecting the
phenyl group to the thiazole (12n−p vs 12a) turned out to be
not so crucial because, in general, it did not significantly affect
the inhibitory activity.
an additional phenyl as R4 (19a) are highly relevant to optimal
enzyme inhibition and dimerization disruption in this series.
To gain insight into the selectivity of the compounds for
LiTryR, representative molecules 12a−c, 12n, 12r, and 19a
were studied as inhibitors of human glutathione-disulfide
reductase (hGR), the closest related host enzyme. Remarkably,
none of these compounds showed any inhibitory activity
against this enzyme. As an example, a comparison of the
activities of LiTryR and hGR in the presence of compounds
12b and 12c at 50 μM of their respective substrates is shown in
Figure S15.
In view of these results, and even though the phenylpropyl
dim
50
derivative 12p displayed the lowest IC value, for the next
round of modifications, we decided to focus on shortening the
propylene linker at R3 to an ethylene (12a) or even removing it
altogether (12n) because of the better synthetic accessibility of
the corresponding bromoketones. On the other hand,
replacement of the naphthyl or the biphenyl group at R3 in
the most potent analogues 12b, 12c by polyheteroaromatics
such as a dihydrobenzofuranyl substituent (12r) or a larger
hydrophobic tricyclic dibenzofuranyl moiety (12s) also yielded
very potent compounds but did not further increase the
inhibitory activity of the most potent analogues (12b, 12c) in
any of the two assays. In contrast, the introduction of a
positively charged tetrahydroquinoline ring (12q) annihilated
the dimerization disruption capacity of the molecules even
though potent inhibition of the redox activity of the enzyme
was retained. In contrast, no activity in any of the assays was
observed for the simplified truncated scaffold derivatives 13
and 14, in which the appropriately substituted triazole or
thiazole rings were removed. This additional evidence further
supports the key role played by these two rings (and the R1
and R3 substituents) in this type of inhibitor.
Finally, we also evaluated a new series of triazole compounds
(19a−e) disubstituted at the 4- and 5-positions of the thiazole
ring with R3 and R4 aromatic moieties. Some of these
molecules combined excellent inhibition in the redox assay
with potent LiTryR dimer disruption ability. Interestingly, both
activities were significantly improved by a factor of 2−4 for
19a, in which a phenyl group is bonded at both R3 and R4
positions, in comparison with the R3 phenyl monosubstituted
analogue 12n. Introduction of a larger biphenyl or a
diphenylether group as the R3 substituent (maintaining the
phenyl group at R4) significantly decreased the inhibitory
activity of the highly potent diphenylsubstituted analogue, but
their dimerization disruption ability was affected only slightly
(cf. 19b, 19c vs 19a). Also, the combination of R3
polyaromatics attached to the thiazole ring through an ethylene
linker with an R4 phenyl group resulted in a significant
deleterious effect on the potency of the compounds as
inhibitors of the LiTryR oxidoreductase activity, although the
potent disrupting effect on dimerization was maintained (cf.
19d, 19e vs 19b, 19c or the corresponding monosubstituted
12c).
Mechanism of LiTryR Inactivation. As already described for
1,41 the peptidic precursor of all other compounds, a
progressive loss of LiTryR activity is observed during the
course of the reaction in the presence of 12b, 12c, 12r, 12s,
and 19a (Figure 2), selected as the most potent inhibitors in
Figure 2. Time-dependent inhibition of LiTryR. Reaction progress
◆
curves of LiTryR in the absence ( ) and presence of 25 μM
□
○
△
▲
■
mepacrine ( ), 10 μM peptide 1 ( ), 10 μM 12c ( ), 10 μM 12b
◊
●
( ), 10 μM 12r ( ), 10 μM 12s ( ), or 10 μM 19a ( ). [DTNB] =
100 μM. [NADPH] = 150 μM. Data represent the mean SD of
three independent experiments.
these series. In stark contrast, reaction rates in the presence of
the classical competitive inhibitor mepacrine are almost
constant until 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
(which allows substrate regeneration) is exhausted. The
progress curves of the reactions in the presence of the selected
compounds are typical of slow-binding inhibitors.42a,b
Despite the loss of activity observed during the reaction,
dependency of the initial velocities (vi) on 19a and TS2
concentrations could be fitted to a model in which a rapid
equilibrium is reached between the free enzyme and inhibitor
(E + I) and the enzyme/inhibitor (EI) complex (Scheme 4).
This analysis, based on vi values, was performed both in a
DTNB-coupled assay and in a direct NADPH-oxidation assay
Scheme 4. Two-Step Mechanism of Time-Dependent
a
In general, the most active compounds in the enzymatic
activity assay were also the best dimerization disruptors in the
ELISA with the exception of the highly potent tetrahydroqui-
noline inhibitor 12q, which did not show any dimerization
disruption effect, or the thiazole-disubstituted analogues 19b−
e, which behave as moderate redox inhibitors but highly potent
dimerization disruptors of the enzyme. All in all, our SAR study
showed that (i) a positively charged butylamine (R1), (ii) an
imidazolidinone (R2) together with a hydrophobic poly-
(hetero)aromatic group as R3 (12b, 12c; 12r, 12s)
substituents, or (iii) the combination of a phenyl as R3 with
Inhibition of LiTryR
a
The first step is a rapid equilibrium that generates the enzyme−
inhibitor (EI) complex. This process is governed by the association
rate (k3) and the dissociation rate (k4) constants. The second step
consists of slow and reversible inactivation of the enzyme that is
governed by the forward isomerization rate constant (k5) and the
much smaller reverse rate constant (k6).
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Figure 3. Concentration dependence of the observed rate constants of LiTryR (0.8 nM) inactivation by 19a at different TS2 concentrations. Plot of
the kobs values ( standard errors) as a function of 19a concentration at six different TS2 concentrations (3.1, 6.2, 12.5, 25, 50, and 100 μM) (kobs
determinations for every 19a concentration at the different fixed TS2 concentrations assayed are shown in the Supporting Information). Curves
were fitted using eq 2, and the results of the fits are shown in Table 2.
(Figure S1 and Table S1). Our data consistently reveal a
nonlinear noncompetitive hyperbolic inhibition mechanism.
Peptide 1 has already been described as a pseudoirreversible
time-dependent noncompetitive inhibitor that is able to
promote a two-step process of enzyme isomerization (Scheme
4).41 The kinetic data obtained for 19a, the most potent
compound, were also analyzed considering a similar mecha-
nism of enzyme inactivation.
In this inactivation model, a rapid and reversible primary
inhibition event generates an enzyme−inhibitor binary
complex (EI complex). The process of generating an inactive
form of LiTryR upon 19a binding is governed by k5 and k6
first-order rate constants, which describe an isomerization
equilibrium between the initial enzyme−inhibitor complex
(EI) and a second high-affinity complex (E*I) (Scheme 4).
This slow isomerization process gives rise to a characteristic
bending of the reaction curve that is defined by the first-order
rate constant kobs, whose value describes the conversion from
the initial reaction rate (vi) to the final steady-state velocity
(vs).
vi, and vs (k6 = kobs × vi/vs) at all of the trypanothione disulfide
(TS2) and 19a concentrations assayed, k6 varies between 3.9 ×
10−6 and 6.5 × 10−5 s−1. Taking into consideration the
extremely slow process of recovery of the enzymatic activity
after complete inactivation of LiTryR (400 nM) upon
incubation with 25 μM 19a for 16 h and subsequent 2500-
fold dilution (Figure S13), a value of 3 × 10−5 s−1 was selected
as a conservative upper value for k6. Fixing this value for k6
rendered very good estimations of Kiapp and Ki*app after the
fitting process for all of the substrate concentrations assayed
(Figure 3 and Table 2).
The relationships between the different Kiapp and Ki values in
slow-binding inhibitors are the same as those for classical linear
reversible inhibitors.42a,b By applying eq 3 to fit the Kiapp values
obtained at the six different TS2 concentrations assayed
(Figure S8A), we could estimate the values of α (0.8 0.4)
Table 2. Apparent Inhibition Constants for LiTryR Time-
a
Dependent Inhibition by 19a
[TS2] (μM)
Kiapp (μM)
Ki*app (μM)
Adjustment to eq 1 (see equations in Experimental Section)
of the progress curves of the reactions catalyzed by LiTryR
allowed us to obtain the kobs values for six 19a concentrations
(4.4, 5.9, 7.9, 10.5, 14.1, and 18.7 μM) at six different TS2
concentrations (3.1, 6.2, 12.5, 25, 50, and 100 μM) (Figures
S2−S7 and Table S2). The different kobs values obtained at
each substrate concentration can be used to determine the
apparent inhibitory constants Kiapp and Ki*app defined as the
dissociation constant for the initial EI complex and the
constant for the overall dissociation from E*I to E + I,
3.1
6.2
12.5
25
50
100
8.9 3.1
7.2 1.7
5.2 2.6
8.3 3.0
9.3 3.2
5.0 2.7
0.6 0.1
0.5 0.1
0.5 0.2
0.8 0.2
1.4 0.3
1.3 0.6
a
Kiapp and Ki*app values at different TS2 concentrations were obtained
by fitting to eq 2 the kobs values for every 19a concentration at the
different fixed TS2 concentrations assayed. Results are the estimated
values of the nonlinear regression associated standard errors.
respectively (eq 2).42a,b Based on the relationship among kobs
,
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and Ki (7.8 1.7 μM). A similar approach (eq 4 and Figure
S8B) was followed to estimate the values of α* (5.5 2.9) and
Ki* (0.5 0.1 μM) for the two-step global process of enzyme
inhibition. According to these results, formation of the initial
EI complex is not affected by the presence of substrate, a
behavior that is characteristic of a pure noncompetitive process
(α ∼ 1). The isomerization step from EI to E*I strongly
enhances the inhibitory activity of 19a, which is characterized
by an overall Ki* value of 0.5 μM. This isomerization process
is, as expected, slightly impaired by the presence of substrate
(α = 5.5) because of the generation of the enzyme−substrate−
inhibitor complex, which decreases the concentration of the EI
complex and thereafter the rate of E*I generation. Despite this
small substrate effect, an α value of 5.5 for the overall process is
still in the range of α values characteristic of noncompetitive
(mixed) inhibitors. The overall inhibitory process is depicted
in Scheme S5. Because of the ability of 19a to cause enzyme
dissociation, the characteristic isomerization of the enzyme in
this mechanism of slow-binding inhibition is expected to be
related to homodimer disruption.
Figure 4. LiTryR dimer structure. FTMap identified the central
interfacial cavity and the two adjacent sites where the two FAD
cofactors are lodged as putative ligand-binding sites (circled by yellow
lines).
Interestingly, compounds 12b and 12q have similar IC50
values in the reductase assay but only 12b interferes with
dimerization. The kinetic analyses reveal that both compounds
are time-dependent inhibitors but only inhibition caused by
12b follows a two-step process of enzyme isomerization
(Figure S11 and Table S3). Plots of kobs vs inhibitor
concentration for 12q fit to a straight line that is indicative
of a single-step binding mechanism (Figure S12, Table S4, and
Scheme S4), which agrees with its inability to disrupt the
homodimer.
Computational Studies. Despite numerous attempts to
form cocrystals with LiTryR or soak crystals of the enzyme
with moderately or highly potent triazole-based dimerization
disruptors 12a, 12b, and the heteroaromatic 12r, no
diffraction-quality crystal containing an enzyme/drug complex
could be obtained. Of note, similar problems were encountered
in previous cocrystallization efforts with the predecessor
imidazoles 2 and 3.30 Because of these so-far insurmountable
hurdles, we had to rely on molecular modeling tools to try and
shed light on the atomistic details of the binding process.
Docking the most potent triazoles 12b, 12c into the cavity of
one LiTryR monomer that lodges the 435Pro−Met447 α-helix of
the other monomer, while feasible, failed to account for the
SAR results discussed above. Indeed, the biologically essential
naphthyl or biphenyl R3 substituents could not be properly
accommodated unless the ligand folded onto itself in an
unrealistic fashion due to lack of room. Therefore, we sought
alternative binding sites in LiTryR, as found in Protein Data
Bank (PDB) entry 2JK622b using the FTMap web server.43
This procedure highlighted the existence of a large, hydro-
phobic, and putatively druggable cavity right at the center of
the dimer that is lined by residues from both monomers and
located very close to the interfacial helices that were mimicked
by the original peptides and peptidomimetics (Figure 4).
Interestingly, this central cavity at the LiTryR dimer
interface is reminiscent of that present in Plasmodium
falciparum GR44 or hGR and is shown to bind some
noncompetitive or uncompetitive inhibitors such as safranin,
menadione,45 xanthenes,46 or N-arylisoalloxazines.47 Ligand
accessibility to this site was assessed by means of the CAVER
web server,48 which identified several tunnels connecting this
intermonomer cavity to the bulk solvent (Figure 5).
Figure 5. Visualization of the potential access routes from the bulk
solvent to the interfacial cavity (2.808 Å3, 0.77 “druggability”) as
determined by the CAVER Web server using default parameters.48 In
comparison, each nicotinamide adenine dinucleotide phosphate
(NADP) binding site occupies 1.343 Å3 and its druggability is 0.63.
The tunnels are depicted as a continuous semitransparent gray
surface. The central vertical α-helices encompass the amino acid
residues originally identified as hotspots for dimerization. The FAD
prosthetic groups are displayed as stick models for reference purposes
only.
We next performed docking studies with the most potent
triazole dimerization inhibitors 12b, 12c and the disubstituted
thiazole analogue 19a centering the search within the proposed
interfacial cavity of the LiTryR homodimer (Figure 6). The
best ligand poses had in common the positioning of the
(hetero)aromatic scaffold inside the connection tunnel and the
terminal ammonium group of the R1 substituent at the
hydrogen-bonding distance from the carboxylate of Glu466′
(d(N12b···OGlu466′) = 2.2 Å, d(N12c···O Glu466′) = 3.4 Å).
The proposed binding modes also revealed the potential of
the imidazolidinone group of the R2 substituent to act as a
hydrogen bond acceptor from the hydroxyl group of either
Thr65 (d(N12b···OThr65) = 2.9 Å), for the naphthyl compound
12b, or Ser433′ (d(N12c···OSer433′) = 2.5 Å), for biphenyl
compounds 12c and 19b. The unhindered rotation of the
central benzene ring of the scaffold seems to play a
fundamental role to distinctly orient the R2 substituent in
each complex. Regarding the R3 hydrophobic substituents at
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Figure 6. Proposed ligand-binding modes within LiTryR. (a) Cartoon representation of dimeric LiTryR showing the two FAD prosthetic groups
(sticks, C atoms in gray) close to the active sites and a docked 19a molecule (C atoms in pink) filling half of the central interfacial hydrophobic
cavity and part of the tunnel connecting it to the bulk solvent. (b) Details of the best-scoring binding pose for 12b (C atoms in green). (c) Details
of the best-scoring binding pose for 12c (C atoms in cyan). (d) Details of the best-scoring binding pose for 19a (C atoms in pink).
the thiazole, the bulky naphthyl and biphenyl aromatic groups
for compounds 12b, 12c could be accommodated in one of the
hydrophobic subpockets of the interfacial cavity that is mainly
defined by Pro435′ and Phe367′, in good accord with the
previous FTMap results. In addition, for the R3 and R4
disubstituted phenyl-thiazole analogue 19a, one of the phenyl
groups attached to the thiazole ring was found to point directly
toward the second hydrophobic subpocket Leu72−Leu72′ at
the bottom of the cavity, another location pinpointed by
FTMap. This binding mode nicely accounts for the above-
mentioned dramatic improvement of inhibitory potency
relative to the monosubstituted phenyl-thiazole 12n.
Our molecular dynamics simulations and binding energy
decomposition results using 19a as a representative inhibitor
point to Glu436 as one of the major contributors to ligand
binding (see Figure S16 and Movie S1 in the Supporting
Information). We have previously shown that this residue is a
hotspot for LiTryR dimerization.25 Indeed, in all X-ray crystal
structures of TryR, this glutamate’s carboxylate from one
monomer is involved in two short hydrogen bonds with the
peptide backbone of Ser464 and Ala465 from the other
monomer. Inhibitor binding appears to promote the loss of
these interdimer hydrogen-bonding interactions (Figure S17)
due to a wedge effect brought about by hydrophobic
interactions involving the phenyl rings on the one side of the
molecule and strong electrostatic interactions on the other
side, which involve not only the free amino group but also the
triazole ring. Indeed, the negative electrostatic potential
generated by the two unsubstituted ring nitrogens faces the
positive dipole of the short 400MetGly405 α-helix. Taken
together, the theoretical calculations provide a rationale for
the observed SAR results. Furthermore, the residue in hGR
(PDB id: 1XAN) that is positionally equivalent to Leu72 in
LiTryR is Phe78, whose aromatic side chain provides a flat
platform for the stacking of ligands such as safranin or
xanthene45,46 but prevents the binding of our novel
compounds, hence their remarkable selectivity. The central
cavity putatively targeted by 19a and analogues is lined by the
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Figure 7. Rationale for the design of the C2-symmetric compound 22. (A) Best docking pose for 19a (C atoms in pink) with the tail extended into
one LiTryR monomer reproduced for the other monomer (C atoms in white). Note how the propylamino group is extended and oriented toward
the carboxylate of Glu466 in both cases. (B) Best-scoring result provided by the automated docking program49 for 22 (CPK model) inside the
central interfacial cavity and connecting tunnels. The side chains of Glu466 and Glu466′ are displayed as sticks with C atoms colored in green.
residues listed in Table S5, which also shows the positionally
equivalent residues in hGR. The overall lack of identity in
several crucial regions is in consonance with the observed
marked selectivity for TryR.
compound emerged not only as the most effective LiTryR
act
inhibitor of the entire series, with an IC value of 0.4 0.03
50
dim
50
μM, but also as a very potent dimerization disruptor (IC
=
8.0
0.2 μM). In comparison with the corresponding
To further support the feasibility of the proposed binding
mode and improve the binding affinity for the putative target
site, a new C2-symmetric compound 22 was rationally designed
with a view to exploiting the C-2 symmetry of this
homodimeric enzyme and simultaneously occupy both
entrance/exit tunnels (Figure 5). The putative LiTryR/19a
complex provided the starting point for the structure-based
design. As shown in Figure 7A, reproducing the binding mode
of 19a within one monomer on the other monomer
immediately reveals the possibility of merging both single
molecular entities into one by making use of a suitable linker
bonded to the R3 substituent. To this end, a 1,3-disubstituted
phenyl was selected as an appropriate spacer to yield the C2-
symmetric compound 22, which was also docked into the
putative binding site (Figure 7B).
monomeric triazole ligand 12n (substituted with an R3 phenyl
group), 22 was 28- and 4-fold more potent, respectively, in
both assays (see Table 1). Taken together, these experimental
results support the proposed binding mode of the monomeric
and symmetric triazole-based compounds at this interfacial site.
Inhibitory Activity on Other TryR Enzymes from the
Trypanosoma Genus. TryR is an essential enzyme not only
for Leishmania but also for Trypanosoma parasites. Given the
high degree of identity/similarity among the amino acid
sequences of TryRs from these two genera, some degree of
activity against so closely related enzymes would not be
unexpected for our compounds. To test this hypothesis, the
most active members of these series were tested as inhibitors of
the oxidoreductase activity of TryRs from T. brucei (TbTryR)
and Trypanosoma congolense (TcoTryR), the causative agents of
sleep sickness and nagana, respectively. The results shown in
Table 3 demonstrate, in fact, very similar activities for 12a−c,
12n, 12r, 19a, and 22 as inhibitors of the three enzymes.
In Vitro Antileishmanial Evaluation and Cytotoxicity.
Compound Internalization into the Parasites. Our earlier
attempts to develop LiTryR dimerization disruptors as
leishmanicidal molecules were hampered by the poor ability
of previously synthesized compounds to cross the parasite
plasma membrane. The peptidomimetics herein described,
endowed with fluorescent properties, no longer suffer from this
limitation. Figure 8 shows the intense blue fluorescence
emitted by promastigotes incubated for 1 h with compound
12a. Strong fluorescent spots can be observed in the apical
The good agreement found between the intended binding
mode for 22 and that found by the automated docking
program49 (Figure 7) is noteworthy. In addition to the
hydrophobic contacts between the aromatic rings and the walls
of the cavity, the two terminal R1 butylammonium groups of
22 are presumed to establish simultaneous salt bridges with
Glu466/Glu467 and Glu466′/Glu467′, whereas the carbonyl
groups of the two imidazolidinone moieties can establish
hydrogen bonds with the protonated nitrogens of Lys61/
Lys61′, the side-chain carboxamide of Gln68, and the
backbone NH of Ser433/Ser433′.
The activity of the new symmetric compound 22 was
evaluated in the two enzymatic assays. Encouragingly, the
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could be replaced by a phenyl resulting in an equipotent
leishmanicidal analogue (12c vs 12n), in contrast to the higher
IC50 values observed for 12n in the enzymatic assays. However,
the SI for 12n was low due to an increase in toxicity. The
introduction of polyheteroaromatic rings gave rise to a
significant reduction of the leishmanicidal activity (12q−s vs
12c) in contrast to the high potency observed for these
analogues in the enzymatic assays. Disubstituted thiazole
analogues 19a−e also showed high leishmanicidal activities
against amastigotes with EC50 values in the 8.4−22.6 μM range
but, with the exception of 19c, low SIs (SI < 2.5) due to higher
toxicities. Remarkably, the symmetrical triazole analogue 22
turned out to be one of the most potent and selective agents
against L. infantum amastigotes with an EC50a value of 4.9 μM
and a SIa of 6.2.
It should be noted that a direct correlation between in vitro
TryR inhibitory activity and in cellulo effects is not to be
expected due to differences in cellular permeability or the
possibility that this enzyme is not the only target for these
compounds. Therefore, among the newly tested triazole-based
analogues, the propylamine biphenyl derivative 12i and the C-
2 symmetric 22 displayed the best leishmanicidal activity and
the lowest in cellulo cytotoxicity with SI values that exceeded
those obtained for the imidazoles 2 and 3 by 5−6-fold. It is
interesting to point out that, despite the relatively poor IC50
value obtained for 12i in the oxidoreductase activity assay, its
behavior in the dimerization assay is similar to that of the most
potent compounds from these series. This finding suggests that
their ability to disrupt the dimer may be largely responsible for
the leishmanicidal activity of these compounds. As IC50 values
were calculated taking into consideration only the initial
velocities of the reactions and because of the time-dependent
behavior of these molecules, compounds such as 12i may show
good activities in the dimerization assay while displaying poor
IC50 values.
Table 3. IC50 SE Values (μM) for Selected Triazole
Analogues 12a−c, 12n, 12r, 19a, and 22 in the
Oxidoreductase Activity of TryR from L. infantum, T. brucei,
and T. congolense
compounds
LiTryR
TbTryR
TcoTryR
12a
12b
12c
12n
12r
19a
22
14.6 1.0
5.9 1.1
10.9 1.8
9.0 0.2
11.9 0.6
4.3 1.0
0.4 0.03
13.3 1.9
9.7 2.1
10.0 2.5
12.5 1.9
15.0 2.9
7.3 0.6
1.0 0.3
11.0 1.3
4.8 1.9
4.8 1.8
9.3 0.8
10.1 1.4
5.6 0.8
0.5 0.1
region of some parasites, just opposite the flagellar pocket that
is expected to be the main entry region for these compounds.
Activity on Extracellular Parasites and Human Cells.
Representative examples of triazole-based LiTryR enzyme
inhibitors were tested in vitro against L. infantum axenic
promastigotes and amastigotes. Cytotoxicity was assessed
employing the human liver cancer cell line HepG2.
Miltefosine, the R9 conjugate of the prototype peptide 1, and
imidazole-based compounds 2 and 3 were included as
reference drugs. According to the data shown in Table 4,
among the 18 triazole compounds tested, 9 showed half-
maximal effective concentration (EC50) values below 10 μM
against Leishmania promastigotes or amastigotes (12b, 12c,
12i−k, 12n, 19b, 19d, and 19e) that are similar to those
observed for the reference compounds.
Of all the compounds bearing a butylamine at R1, an
imidazolidinone at R2, and different R3 substituents (12a−c),
the biphenylethyl analogue 12c displayed better activity against
both promastigotes and amastigotes than those containing
naphthyl or phenylethyl substituents (12c > 12b > 12a) and
also lower cytotoxicity. This results in an improved selectivity
index (SI) of 8.8 for promastigotes (SIp) and 3.7 for
amastigotes (SIa) in comparison with the SIs of the
corresponding biphenyl imidazole analogue 3 (SIp = SIa =
2.7). On the other hand, a combination of a shorter propyl
spacer at R1 and the biphenyl group at R3 gave 12i, the most
potent and selective compound of the entire series, with EC50
values of 5.8 against promastigotes (EC50p) and 5.2 μM against
amastigotes (EC50a) and the highest SIs (SIp = 10.4 and SIa =
11.6). The imidazolidinone moiety at R2 could be eliminated
without compromising the leishmanicidal activity (12j, 12k),
but a significant increase in cytotoxicity was detected, which
suggests a positive effect of the imidazolidinone on reducing
the cytotoxicity. As regards R3, the biphenylethyl substituent
Leishmanicidal Activity in Intracellular Amastigotes. The
most potent and selective compounds were further tested for
their leishmanicidal activity in amastigote-infected THP-1 cells.
The EC50 values obtained are summarized in Table 5.
Miltefosine, edelfosine, amphotericin B, the R9-conjugate of
the prototype peptide 1, and compounds 2 and 3 from the
precursor imidazole series were included as reference
compounds. In our view, the fact that the EC50 values cover
a range from 22 to >75 μM indicates that not all of the
compounds are able to cross the plasma and phagolysosomal
membranes in the macrophages. Among the monomeric
triazole-based compounds, only the phenyl derivative 12a,
with an EC50 value of 32.4 μM, and the biphenyl derivatives
Figure 8. Fluorescence microscopy images of L. infantum promastigotes treated with 25 μM compound 12a for 1 h.
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a b
,
Table 4. In Vitro Activity on L. infantum Parasites and Cytotoxic Activity of Representative Triazole Compounds
c
c
c d
,
e
compounds
EC50 (μM) promastigotes
EC50 (μM) amastigotes
EC50 (μM) HepG2
SIp/SIa
12a
12b
12c
12h
12i
12j
12k
12m
12n
12o
12q
12r
12s
19a
19b
19c
19d
19e
16.4 0.9
8.4 0.3
4.9 0.4
12.7 0.3
5.8 0.7
8.5 0.1
13.9 0.6
15.3 1.7
11.6 0.8
31.6 5.8
5.2 0.3
7.1 0.7
4.0 0.2
25.2 0.5
11.5 0.3
>75
27.0 2.8
22.3 0.6
68.8 3.2
15.6 3.0
9.4 0.5
22.6 5.7
9.1 0.2
8.4 0.1
4.9 0.2
2.0 0.1
3.4 0.2
12.8 1.3
5.3 0.2
20.3 2.4
33.3 2.7
43.1 2.6
25.7 2.2
60.4 3.0
12.4 0.7
7.4 0.6
44.9 1.3
16.4 2.3
67.9 1.5
43.3 1.0
29.4 0.7
>75
26.5 4.1
17.3 0.1
≥ 75
12.1 0.1
6.6 0.6
30.5 4.1
>75
1.2/1.5
4.0/2.2
8.8/3.7
2.0/<1
10.4/11.6
1.8/1.7
ND/1.9
1.0/1.8
3.0/1.4
<1.0/<1.0
ND/1.6
1.2/1.3
ND/>1.1
2.5/1.7
ND/1.8
ND/≥3.3
ND/1.3
ND/<1
8.0/6.2
>1.6/>37.5
19.9/18.1
2.1/2.1
f
ND
42.6 3.9
5.4 0.08
>75
ND
23.8 0.5
ND
10.7 1.3
ND
ND
ND
ND
3.8 0.1
47.6 0.6
3.1 0.6
12.8 0.7
5.3 0.3
22
miltefosine
R9-peptide 1
2
61.7 6.1
27.1 2.4
14.2 0.9
3
2.7/2.7
a
Miltefosine, linear peptide 1 conjugated to R9, and imidazole-containing compounds 2 and 3 were included as in-house reference compounds.
b
c
Results are representative of three independent experiments, each performed in triplicate. EC50 SE values are indicated. The half-maximal
effective concentration (EC50) is defined as that causing a 50% reduction of proliferation in L. infantum promastigotes (EC50p) and amastigotes
(EC50a) or human hepatocellular carcinoma (HepG2) cells. Dead parasites were identified by their increased permeability to propidium iodide
d
e
(PI). Cytotoxicity in HepG2 cells was measured using the crystal violet method. The selectivity index (SI) is the ratio of EC50 against human cells
f
to EC50 against L. infantum promastigotes (SIp) and amastigotes (SIa). ND = not determined.
12c, 12i, with EC50 values of 46.5 and 54.5 μM, respectively,
displayed moderate effectiveness against intracellular amasti-
gotes. Interestingly, the symmetric triazole molecule 22
emerged, once again, as the most potent and selective
leishmanicidal compound of all of the triazole-based series
(EC50 = 22 μM), showing a SI of 2.2, better than that obtained
for edelfosine (SI = 1.4). Accordingly, derivatives 12a, 12c,
12i, and 22 constitute the first analogues designed against the
dimerization interface of TryR that show leishmanicidal
activity toward intracellular amastigotes. These results confirm
the interest and potential of both monomeric and symmetric
triazole-based compounds as antileishmanial agents.
dimer interface of the enzyme. In the absence of crystallo-
graphic evidence, despite extensive efforts, this putative
binding mode provided a rationale for the observed SAR and
a basis for the subsequent structure-based design of a new
symmetrical triazole analogue 22 that displays improved
LiTryR inhibitory activities over those of its parental
monomeric triazole. This rationally designed compound
shows a dramatic enhancement in leishmanicidal potency, in
particular against intracellular amastigotes.
All in all, our results provide the first evidence for ligand
binding to a so-far unexplored region of LiTryR and lay the
ground for further design of innovative TryR inhibitors that
may become valuable candidates in the process of discovering
new drugs against leishmaniasis.
CONCLUSIONS
■
The development of new and more efficient antileishmanial
drugs is a priority in the field of neglected tropical diseases.
From a small library of 26 triazole-phenyl-thiazole compounds,
we have identified the most potent small-molecule dimeriza-
tion disruptors of LiTryR described to date. Similar to
prototype peptide 1, they behave as slow-binding, non-
competitive inhibitors with overall Ki* values of 0.5 μM
(19a). These molecules are also endowed with high
antileishmanial activity against both extracellular and intra-
cellular parasites and improved selectivity indexes when
compared to those obtained for previous in-house imidazole-
based compounds.
EXPERIMENTAL SECTION
■
Chemical Procedures. Experiments dealing with air- and water-
sensitive reagents/compounds were performed in an argon atmos-
phere. Hygroscopic species were previously dried under vacuum for
24 h by P2O5 as the drying agent. Unless otherwise noted, analytical-
grade solvents and commercially available reagents were used without
further purification. Anhydrous CH2Cl2 was dried by refluxing over
CaH2. EtOH was dried with 4 Å molecular sieves previously activated
in a microwave oven. THF was dried by reflux over sodium/
benzophenone. Pressured reactions were carried out on a Sigma-
Aldrich Ace Glass flask (50 or 100 mL) supplied with a Teflon cap.
Microwave-assisted experiments were performed on a Biotage
Initiator 2.0 single-mode cavity instrument from Biotage (Uppsala).
Experiments were carried out in sealed microwave reaction vials at the
standard absorbance level (400 W maximum power). Lyophilizations
were carried out on a Telstar 6-80 lyophilizer.
Molecular modeling studies carried out for the most potent
LiTryR triazole-containing dimerization inhibitors 12b, 12c,
and 19a identified a previously unexplored but apparently
druggable binding site consisting of a large central cavity at the
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Melting points (m.p.) were measured on a Mettler Toledo M170
apparatus and are uncorrected. Infrared (IR) spectra were recorded
on a PerkinElmer Spectrum One instrument and KBr pellet as a
sample support. Mass spectrum (MS) on electrospray ionization
(ESI) mode was recorded on a Hewlett-Packard 1100SD in positive
mode. High-resolution mass spectra (HRMS) were obtained on an
Agilent 6520 Accurate-Mass Q-TOF LC-MS equipped with an HP-
1200 (Agilent) liquid chromatograph coupled to a mass spectrometer
with a Q-TOF 6520 hybrid mass analyzer. Usually, an error equal to
or lower than 5 ppm was allowed. Nuclear magnetic resonance
(NMR) spectra were recorded on a Varian INNOVA-300 operating at
300 MHz (1H) and 75 MHz (13C), a Varian INNOVA-400 operating
at 400 MHz (1H) and 100 MHz (13C), a Varian MERCURY-400
operating at 400 MHz (1H) and 100 MHz (13C), and a Varian
SYSTEM-500 operating at 500 MHz (1H) and 125 MHz (13C). The
assignments were performed by means of different standard
homonuclear and heteronuclear correlation experiments (gradient
heteronuclear single quantum coherence (gHSQC), gradient
heteronuclear multiple bond coherence (gHMBC), and nuclear
Overhauser enhancement spectroscopy (NOESY)) when required.
4-Azido-2-(2-(2-oxoimidazolidin-1-yl)ethoxy)benzo-nitrile (5a).
A solution of bromoarene 4a30 (500 mg, 1.61 mmol) and NaN3
(1.57 g, 24.2 mmol) in anhydrous DMSO (30 mL) in the presence of
4 Å molecular sieves, and under an argon atmosphere, was heated at
100 °C for 72 h. The reaction was allowed to cool to room
temperature and diluted with H2O (60 mL), and the aqueous phase
was extracted with CH2Cl2 (3 × 50 mL). The combined organic
layers were dried (Na2SO4), filtered, evaporated to dryness, and
lyophilized. The residue was purified by flash column chromatography
(CH2Cl2/MeOH, 100:3) to give 372 mg (85%) of 5a as a yellow
solid; m.p.: 158−161 °C; IR (KBr), ν (cm−1): 3230 (NH-st), 3090
(Csp−H st), 2223 (CN st), 2125 (NNN st), 1702 (CO st);
1H NMR (CDCl3, 300 MHz) δ (ppm): 7.53 (d, J = 8.3 Hz, 1H, Ar),
6.70 (dd, J = 8.3, 2.0 Hz, 1H, Ar), 6.53 (d, J = 1.8 Hz, 1H, Ar), 4.71
(bs, 1H, NHCON), 4.20 (t, J = 4.7 Hz, 2H, OCH2), 3.81−3.76 (m,
2H, CH2CH2NHCON), 3.64 (t, J = 4.9 Hz, 2H, OCH2CH2), 3.44 (t,
J = 8.1 Hz, 2H, CH2CH2NHCON); 13C NMR (CDCl3, 75 MHz) δ
Table 5. In Vitro Antileishmanial Activity for the Most
Representative Triazole-Based Compounds in Amastigote-
a
Infected THP-1 Monocyte-Derived Macrophages
EC50 (μM) intracellular
b
c
compounds
12a
12b
12c
amastigotes
EC50 (μM) THP-1
32.4 6.7
>75
46.5 1.5
45 3.1
>75
>75 (100% at
d
75 μM)
12i
54.4 15.5
>75 (100% at
d
75 μM)
d
12q
19c
22
mdelfosine
miltefosine
amphotericin B
R9-peptide 1
2
>75
>75
>75 (67% at 75 μM)
>75 (63% at 75 μM)
48.4 14.5
3.5 0.3
>25
>5
14.5 2.9
27.9 4.4
15.5 1.8
d
22.2 6.4
2.5 0.02
0.6 0.1
0.07 0.01
>75
>75
>75
3
a
Edelfosine, miltefosine, and amphotericin B were included as
reference leishmanicidal drugs. The R9-peptide 1 and the imidazole-
containing compounds 2 and 3 were included as in-house reference
b
compounds for structure−activity comparisons. The half-maximal
effective concentration (EC50) is defined as that causing a 50%
reduction in the number of L. infantum amastigotes per cell in the
culture or amastigote-infected THP-1 monocyte-derived macro-
phages. Living intracellular amastigotes were identified by their
green fluorescence due to green fluorescent protein (GFP) expression
and their lack of permeability to propidium iodide; EC50 SE values
c
are indicated. Cytotoxicity was assessed in THP-1 monocyte-derived
macrophages by measuring lactate dehydrogenase (LDH) activity.
d
Cell viability of amastigote-infected THP-1 monocyte-derived
macrophages at 75 μM (maximum concentration assayed). Results
are representative of three independent experiments.
(ppm): 162.8 (NHCON), 161.8 (OCAr), 146.8 (CAr), 134.9 (CHAr),
116.2 (CN), 111.8 (CHAr), 103.2 (CHAr), 98.3 (CAr), 69.4 (OCH2),
47. 8 (CH2 CH2 NHCON), 43. 2 (OC H2 CH2 ), 38. 6
(CH2CH2NHCON); MS (ESI, positive mode) m/z: 567.2 [2M +
Na]+, 295.0 [M + Na]+, 273.0 [M + H]+. NaN3 may be toxic and
explosive. Thus, for safety precautions, a polycarbonate safety screen
in a properly functioning fume hood was always used to perform this
reaction.
Monitoring of the reactions was performed by analytical thin-layer
chromatography (TLC) on silica gel 60 F254 (Merck) or by high-
performance liquid chromatography coupled to mass spectrometry
(HPLC−MS) using a Waters Alliance 2695 Separation Module with a
Waters Micromass ZQ Detector.
Compounds were purified by (a) flash column chromatography
(Merck) with silica gel 60 (230−400 mesh); (b) high-performance
flash column chromatography (HPFC) on a Biotage Isolera One
using cartridges of Claricep i-Series, Silica, 40 g (40−60 μm, 60 Å) for
normal phase chromatography, and KP-C18-HS 12g (21 × 55 mm2)
for the reverse phase; (c) centrifugal circular thin-layer chromatog-
raphy (CCTLC) on a Chromatotron (Kiesegel 60 PF254 gipshaltig,
Merck), with layer thicknesses of 1 and 2 mm and flow rates of 2 and
3 mL/min; or (d) semipreparative HPLC purifications on a Waters
2695 HPLC system equipped with a Photodiode Array 2998 coupled
to a 3100 Mass Detector mass spectrometer using a C18 Sunfire
column (19 mm × 150 mm).
The purity of the compounds was checked by analytical HPLC on
a Waters 600 system equipped with a C18 Sunfire column (4.6 mm ×
150 mm, 3.5 μm) and a selectable wavelength UV detector.
Alternatively, an Agilent Technologies 1120 Compact LC provided
with a reverse-phase column ACE 5 C18-300 (4.6 mm × 150 mm, 3.5
μm) and integrated diode detector PDA 996 was used. As the mobile
phase, mixtures of A (CH3CN) and B (H2O) (0.05% TFA) on
isocratic or gradient mode with a flow rate of 1 mL/min were used.
The following gradients were used: from 10% A to 100% A in 10 min
(gradient A) and from 1% A to 100% A in 10 min (gradient B). The
purity of the final compounds was also determined to be >95% by
elemental analysis with a LECO CHNS-932 apparatus. Deviations of
the elemental analysis results from the calculated value were within
0.4%.
Benzyl-4-(1-(4-cyano-3-((1-(2-oxoimidazolidin-1-yl)ethoxy)-
phenyl)-1H-1,2,3-triazol-4-yl)butyl)carbamate (8). To a solution of
azide 5a (450 mg, 1.65 mmol), commercially available benzyl-5-
hexynylcarbamate (523 mg, 2.15 mmol), and CuSO4·5H2O (42 mg,
0.17 mmol) in EtOH (25 mL) were added sodium ascorbate (131
mg, 0.66 mmol) and H2O (25 mL). The reaction mixture was stirred
at room temperature in the dark overnight. The mixture was
evaporated to dryness, and the residue dissolved in CH2Cl2 (50 mL)
was washed with H2O (3 × 50 mL), dryed (Na2SO4), filtered, and
evaporated under reduced pressure. The residue was purified by flash
column chromatography (CH2Cl2/MeOH, 100:3) to provide 8 (573
1
mg, 69%) as a colorless oil. H NMR (CDCl3, 400 MHz) δ (ppm):
7.86 (s, 1H, Ar), 7.61 (d, J = 8.3 Hz, 1H, Ar), 7.49 (s, 1H, Ar), 7.34
(d, J = 7.1 Hz, 1H, Ar), 7.32−7.17 (m, 5H, Ar), 5.05 (bs, 1H,
NHCbz), 5.02 (s, 2H, NHCOOCH2), 4.68 (bs, 1H, NHCON), 4.26
(t, J = 5.2 Hz, 2H, OCH2), 3.66 (t, J = 7.9 Hz, 2H,
CH2CH2NHCON), 3.57 (t, J = 5.2 Hz, 2H, OCH2CH2), 3.33 (t, J
= 8.3 Hz, 2H, CH2CH2NHCON), 3.18 (q, J = 6.7 Hz, 2H,
CH2NHCbz), 2.76 (t, J = 7.3 Hz, 2H, TrizCH2), 1.71 (quin, J = 7.4
Hz, 2H, TrizCH2CH2), 1.52 (quin,
J = 7.2 Hz, 2H,
CH2CH2NHCbz); 13C NMR (CDCl3, 75 MHz) δ (ppm): 162.8
(NHCON), 161.5 (OCAr), 156.6 (NHCOO), 149.2 (CAr), 141.5
(CAr), 136.6 (CHAr), 135.1 (CHAr), 128.6 (CHAr), 128.2 (CHAr),
119.1 (CHAr), 115.7 (CN), 112.1 (CHAr), 104.1 (CHAr), 101.5 (CAr),
69.0 (OCH2), 66.7 (NHCOOCH2), 47.3 (CH2CH2NHCON), 42.8
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i
(OCH2CH2), 40.8 (CH2NHCbz), 38.5 (CH2CH2NHCON), 29.4
(CH2CH2NHCbz), 26.1 (TrizCH2CH2), 25.1 (TrizCH2); HRMS
(ES, positive mode) m/z: calcd for C26H29N7O4 503.2281; found
503.2279 (−0.33 ppm).
mg, 0.97 mmol) were reacted at 70 °C for 5 h in PrOH. After the
workup, the residue was purified by flash column chromatography
(CH2Cl2/MeOH, 100:4) to give 11b (365 mg, 51%) as a yellow oil.
1H NMR (CDCl3, 400 MHz) δ (ppm): 8.61 (d, J = 8.5 Hz, 1H, Ar),
7.90−7.64 (m, 7H, Ar, ThiazCHCH), 7.55 (s, 1H, Ar), 7.45−7.36
(m, 3H, Ar), 7.30−7.14 (m, 7H, Ar, ThiazCHCH), 5.03 (s, 2H,
NHCOOCH2), 4.97 (bs, 1H, NHCON), 4.48 (bs, 1H, NHCbz), 4.39
(t, J = 5.7 Hz, 2H, OCH2), 3.73 (t, J = 5.7 Hz, 2H, OCH2CH2), 3.52
(dd, J = 9.0, 6.6 Hz, 2H, CH2CH2NHCON), 3.28 (t, J = 7.9 Hz, 2H,
CH2CH2NHCON), 3.20 (q, J = 6.7 Hz, 2H, CH2NHCbz), 2.78 (t, J
= 7.3 Hz, 2H, TrizCH2), 1.74 (quin, J = 7.5 Hz, 2H, TrizCH2CH2),
1.59−1.51 (m, 2H, CH2CH2NHCbz); 13C NMR (CDCl3, 75 MHz) δ
Benzyl-4-(1-(4-carbamothioyl-3-((1-(2-oxoimidazolidin-1-yl)-
ethoxy)phenyl)-1H-1,2,3-triazol-4-yl)butyl)carbamate (9). To a
solution of benzonitrile 8 (400 mg, 0.79 mmol) in DMF (25 mL),
20% aq (NH4)2S (3.78 mL, 55.6 mmol) was added and then heated
to 80 °C for 4 h. The reaction mixture was allowed to cool to room
temperature, and then, CH2Cl2 (50 mL) was added. The mixture was
successively washed with HCl 0.1 N (2 × 50 mL), H2O (1 × 50 mL),
and brine (1 × 50 mL). The organic layers were dried (Na2SO4),
filtered, and evaporated to dryness. The crude was purified by flash
column chromatography (CH2Cl2/MeOH, 100:5) to give 9 (354 mg,
74%) as a yellow oil. 1H NMR (CDCl3, 400 MHz) δ (ppm): 9.63 (bs,
1H, SCNH2), 9.50 (bs, 1H, SCNH2), 8.71 (d, J = 8.7 Hz, 1H, Ar),
7.78 (s, 1H, Ar), 7.45 (d, J = 2.0 Hz, 1H, Ar), 7.36−7.17 (m, 5H, Ar),
7.10 (dd, J = 8.7, 2.0 Hz, 1H, Ar), 5.96 (bs, 1H, NHCbz), 5.02 (s, 2H,
NHCOOCH2), 4.98 (bs, 1H, NHCON), 4.16 (t, J = 4.4 Hz, 2H,
OCH2), 3.64 (t, J = 4.4 Hz, 2H, OCH2CH2), 3.50 (dd, J = 9.7, 6.7
(ppm): 162.7 (NHCON), 161.0 (OCAr), 156.6 (CAr), 156.2 (CAr),
153.5 (CAr), 148.8 (CAr), 138.6 (CAr), 136.7 (CAr), 134.7 (CHAr),
133.8 (CAr), 133.3 (CAr), 131.5 (CHAr), 130.3 (CHAr), 128.7 (CHAr),
128.4 (CHAr), 128.3 (CHAr), 128.2 (CHAr), 127.8 (CHAr), 127.2
(CAr), 126.5 (CHAr), 126.1 (CHAr), 123.6 (CHAr), 121.8 (CHAr),
119.2 (CHAr), 117.1 (CHAr), 112.5 (CHAr), 104.5 (CHAr), 67.7
(OCH2), 66.8 (NHCOOCH2), 46.4 (CH2CH2NHCON), 43.0
(OCH2CH2), 40.9 (CH2NHCbz), 38.4 (CH2CH2NHCON), 29.5
(CH2CH2NHCbz), 26.4 (TrizCH2CH2), 25.2 (TrizCH2); HRMS
(ES, positive mode) m/z: calcd for C40H39N7O4S 713.2784; found
713.2779 (−0.72 ppm).
Benzyl-(E)-(4-(1-(4-(4-(2-([1,1′-biphenyl]-4-yl)vinyl) thiazol-2-yl)-
3-(2-(2-oxoimidazolidin-1-yl)ethoxy)phenyl)-1H-1,2,3-triazol-4-yl)-
butyl)carbamate (11c). Following the general Hantzsch synthesis, 9
(200 mg, 0.37 mmol) reacted with α-bromoketone 10c30 (112 mg,
0.37 mmol) for 5 h. After the workup, the residue was purified by
flash column chromatography (CH2Cl2/MeOH, 100:3) to give 174
Hz, 2H, CH2CH2NHCON), 3.40 (dd, J = 9.1, 6.2 Hz, 2H,
CH2CH2NHCON), 3.17 (q, J = 6.7 Hz, 2H, CH2NHCbz), 2.73 (t,
J = 7.5 Hz, 2H, TrizCH2), 1.70 (quin, J = 7.5 Hz, 2H, TrizCH2CH2),
1.53 (quin, J = 7.5 Hz, 2H, CH2CH2NHCbz); 13C NMR (CDCl3, 75
MHz) δ (ppm): 196.5 (SCNH2), 163.5 (NHCON), 156.3
(NHCOO), 149.1 (CAr), 140.3 (CAr), 138.4 (CHAr), 136.7 (CHAr),
128.7 (CHAr), 128.2 (CHAr), 125.1 (CAr), 119.1 (CHAr), 111.3
(CHAr), 104.0 (CHAr), 66.8 (OCH2), 66.6 (NHCOOCH2), 45.4
(CH2CH2NHCON), 43.2 (OCH2CH2), 40.9 (CH2NHCbz), 38.3
(CH2CH2NHCON), 29.8 (CH2CH2NHCbz), 26.4 (TrizCH2CH2),
25.3 (TrizCH2); MS (ESI, positive mode) m/z: 538.3 [M + H]+.
General Procedure for the Synthesis of Thiazoles 11a−c by
Hantzsch Cyclization. A solution of thioamide 9 (1 equiv) in iPrOH
(15 mL) was treated with the appropriated α-haloketone (10a−c) (1
equiv). The reaction mixture was stirred at 70 °C for 3−6 h in a
pressure flask, and then, it was allowed to cool to room temperature
and concentrated to dryness under reduced pressure. The residue was
purified by flash column chromatography or CCTLC on a
Chromatotron (eluents are specified in each case).
1
mg (63%) of 11c as a colorless oil. H NMR (CDCl3, 400 MHz) δ
(ppm): 8.57 (d, J = 8.5 Hz, 1H, Ar), 7.81 (s, 1H, Ar), 7.61−7.45 (m,
8H, Ar, ThiazCHCH), 7.40−7.31 (m, 3H, Ar), 7.31−7.16 (m, 7H,
Ar), 7.12 (d, J = 16.0 Hz, 1H, ThiazCHCH), 5.02 (s, 2H,
NHCOOCH2), 4.68 (bs, 1H, NHCON), 4.36 (t, J = 5.7 Hz, 2H,
OCH2), 3.70 (t, J = 5.8 Hz, 2H, OCH2CH2), 3.50 (dd, J = 9.0, 6.7
Hz, 2H, CH2CH2NHCON), 3.26 (t,
J = 7.9 Hz, 2H,
CH2CH2NHCON), 3.16 (q, J = 6.6 Hz, 2H, CH2NHCbz), 2.76 (t,
J = 7.3 Hz, 2H, TrizCH2), 1.75 (quin, J = 7.5 Hz, 2H, TrizCH2CH2),
1.55 (quin, J = 7.3 Hz, 2H, CH2CH2NHCbz); 13C NMR (CDCl3, 75
Benzyl-(4-(1-(3-(2-(2-oxoimidazolidin-1-yl)ethoxy)-4-(4-phene-
thylthiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)butyl)carbamate
(11a). Following the general procedure, thioamide 9 (100 mg, 0.19
mmol) and the commercially available 1-bromo-4-phenylbutan-2-one
(10a 42 mg, 0.19 mmol) in iPrOH (15 mL) reacted at 70 °C for 4 h.
After the workup, the residue was purified by CCTLC on the
Chromatotron (CH2Cl2/MeOH, 100:3) to yield 108 mg (87%) of
11a as a colorless oil. 1H NMR (CDCl3, 400 MHz) δ (ppm): 8.46 (d,
J = 8.5 Hz, 1H, Ar), 7.81 (s, 1H, Ar), 7.51 (d, J = 2.0 Hz, 1H, Ar),
7.38−7.06 (m, 11H, Ar), 6.86 (s, 1H, Ar), 5.06 (t, J = 6.0 Hz, 1H,
NHCbz), 5.01 (s, 2H, NHCOOCH2), 4.34 (t, J = 5.7 Hz, 2H,
MHz) δ (ppm): 162.7 (NHCON), 160.9 (OCAr), 156.6 (CAr), 156.2
(CAr), 153.4 (CAr), 148.8 (CAr), 140.7 (CAr), 140.6 (CAr), 138.6
(CHAr), 136.7 (CAr), 136.2 (CHAr), 130.9 (CHAr), 130.3 (CHAr),
128.9 (CHAr), 128.6 (CHAr), 128.3 (CHAr), 127.5 (CHAr), 127.0
(CHAr), 122.4 (CAr), 121.5 (CHAr), 119.1 (CHAr), 117.4 (CHAr),
112.4 (CHAr), 104.4 (CHAr), 67.7 (OCH2), 66.8 (NHCOOCH2),
46.4 (CH2CH2NHCON), 42.9 (OCH2CH2), 40.9 (CH2NHCbz),
38.4 (CH2CH2NHCON), 29.4 (CH2CH2NHCbz), 26.4
(TrizCH2CH2), 25.2 (TrizCH2); HRMS (ES, positive mode) m/z:
calcd for C42H41N7O4S 739.2941; found 739.2972 (4.21 ppm).
General Procedure for N-Cbz Deprotection. A solution of the
corresponding Cbz-protected compound (1 equiv) in a 1:1 mixture of
THF/MeOH (20 mL) containing Pd/C (10%) (20% w/w) and TFA
(0.5−1.5 mL) was hydrogenated at room temperature for 2 h under
atmospheric pressure using a balloon filled with hydrogen gas (three
cycles of vacuum + hydrogen). The Pd/C was filtered through
Whatman poly(tetrafluoroethylene) (PTFE) filter paper, and the
solvent was removed under reduced pressure and co-evaporated with
mixtures of CH2Cl2/MeOH (5 × 10 mL). The residue was purified
by HPFC on an SP1 Isolera Biotage using reverse-phase columns
(from 0% of CH3CN to 100% of CH3CN in 45 min) to afford to give
the final deprotected compounds as trifluoroacetate salts.
1-(2-(5-(4-(4-Ammoniumbutyl)-1H-1,2,3-triazol-1-yl)-2-(4-phe-
nethylthiazol-2-yl)phenoxy)ethyl)imidazolidin-2-one 2,2,2-Trifluor-
oacetate (12a). According to the general hydrogenolysis procedure, a
solution of 11a (55 mg, 0.08 mmol), in 1:1 THF/MeOH (20 mL)
containing Pd/C (10%) (20% w/w) (18 mg) and TFA (0.5 mL), was
hydrogenated. The residue was purified by HPFC on the SP1 Isolera
Biotage using reverse-phase columns (from 0% of CH3CN to 100% of
CH3CN in 45 min) to afford 12a (19 mg, 35%) as a colorless oil. 1H
NMR (CD3OD, 400 MHz) δ (ppm): 8.47 (s, 1H, Ar), 8.46 (d, J = 8.5
OCH2), 3.69 (t, J = 5.7 Hz, 2H, OCH2CH2), 3.50 (dd, J = 9.0, 6.7
Hz, 2H, CH2CH2NHCON), 3.26 (t,
J = 7.9 Hz, 2H,
CH2CH2NHCON), 3.18 (q, J = 7.4 Hz, 2H, CH2NHCbz), 3.12−
2.97 (m, 4H, CH2CH2Ph), 2.76 (t, J = 7.4 Hz, 2H, TrizCH2), 1.71
(quin, J = 7.4 Hz, 2H, TrizCH2CH2), 1.59 (quin, J = 7.1 Hz, 2H,
CH2CH2NHCbz); 13C NMR (CDCl3, 75 MHz) δ (ppm): 162.8
(NHCON), 160.3 (OCAr), 156.6 (CAr), 156.0 (CAr), 155.9 (CAr),
148.7 (CAr), 141.7 (CAr), 138.3 (CAr), 136.7 (CAr), 130.0 (CHAr),
128.6 (CHAr), 128.6 (CHAr), 128.4 (CHAr), 128.2 (CHAr), 126.1
(CHAr), 122.7 (CAr), 119.1 (CHAr), 115.1 (CHAr), 112.4 (CHAr),
104.5 (CHAr), 67.8 (OCH2), 66.7 (NHCOOCH2), 46.4
(CH2CH2NHCON), 43.0 (OCH2CH2), 40.8 (CH2NHCbz), 38.4
(CH2CH2NHCON), 35.6 (CH2CH2Ph), 33.4 (CH2CH2Ph), 29.4
(CH2CH2NHCbz), 26.4 (TrizCH2CH2), 25.2 (TrizCH2); HRMS
(ES, positive mode) m/z: calcd for C36H39N7O4S 665.2784; found
665.2791 (1.00 ppm).
Benzyl-(E)-(4-(1-(4-(4-(2-(naphthalen-2-yl)vinyl)thiazol-2-yl)-3-
(2-(2-oxoimidazolidin-1-yl)ethoxy)phenyl)-1H-1,2,3-triazol-4-yl)-
butyl)carbamate (11b). According to the general procedure,
thioamide 9 (520 mg, 0.97 mmol) and α-bromoketone 10b30 (267
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Hz, 1H, Ar), 7.70 (d, J = 2.0 Hz, 1H, Ar), 7.58 (dd, J = 8.6, 2.0 Hz,
1H, Ar), 7.33−7.17 (m, 5H, Ar), 7.15 (s, 1H, Ar), 4.48 (t, J = 5.5 Hz,
2H, OCH2), 3.74 (t, J = 5.5 Hz, 2H, OCH2CH2), 3.60 (dd, J = 9.3,
6.9 Hz, 2H, CH2CH2NHCON), 3.36 (dd, J = 9.3, 6.9 Hz, 2H,
CH2CH2NHCON), 3.15−3.03 (m, 4H, CH2CH2Ph), 3.00 (t, J = 7.4
(gradient A, Agilent): Rt = 8.4 min; HRMS (ES, positive mode) m/z:
calcd for C34H37N7O2S 607.2729; found 607.2740 (1.73 ppm); anal.
calcd for C34H37N7O2S·TFA: C, 59.91; H, 5.31; N, 13.58; S, 4.44;
found: C, 60.08; H, 5.09; N, 13.18; S, 4.20.
Phosphonium (2-Oxo-3-phenylpropyl)triphenyl Chloride (15). A
solution of 1-chloro-3-phenylpropan-2-one (250 mg, 1.48 mmol) and
triphenylphosphine (583 mg, 2.22 mmol) in anhydrous toluene (8
mL) and under an argon atmosphere was heated to 110 °C for 6 h in
a pressure tube. After cooling to room temperature, the white solid
was filtered and washed with cooled diethyl ether (3 × 10 mL) to give
+
Hz, 2H, CH2NH3 ), 2.87 (t, J = 7.2 Hz, 2H, TrizCH2), 1.89−1.81 (m,
+
2H, TrizCH2CH2), 1.81−1.70 (m, 2H, CH2CH2NH3 ); 13C NMR
(CD3OD, 100 MHz) δ (ppm): 165.1 (NHCON), 162.0 (OCAr),
157.4 (CAr), 157.1 (CAr), 149.3 (CAr), 142.8 (CAr), 139.7 (CAr), 130.8
(CHAr), 129.5 (CHAr), 129.4 (CHAr), 127.0 (CHAr), 123.8 (CAr),
121.6 (CHAr), 116.9 (CHAr), 113.6 (CHAr), 105.7 (CHAr), 68.4
(OCH2), 47.0 (CH2CH2NHCON), 43.9 (OCH2CH2), 40.4
15 (498 mg, 78%) as a white solid;50 m.p.: 200−202 °C; H NMR
1
(DMSO-d6, 400 MHz) δ (ppm): 7.93−7.64 (m, 15H, Ar), 7.31−7.20
(m, 3H, Ar), 7.12 (d, J = 6.7 Hz, 2H, Ar), 5.91 (d, J = 12.7 Hz, 2H,
Ar), 4.14 (s, 2H, CH2CO).
+
(CH2NH3 ), 39.3 (CH2CH2NHCON), 36.7 (CH2CH2Ph), 34.3
+
(CH2CH2Ph), 28.0 (CH2CH2NH3 ), 27.1 (TrizCH2CH2), 25.6
(E)-4-([1,1′-Biphenyl]-4-yl)-1-phenylbut-3-en-2-one (16c). A
stirred solution of 15 (250 mg, 0.58 mmol) and KOH (130 mg,
2.37 mmol) in dry toluene (15 mL) was heated at 110 °C in a
pressure flask for 3 h. After cooling to room temperature, [1,1′-
biphenyl]-4-carboxaldehyde (158 mg, 0.87 mmol) was added
dropwise. The reaction mixture was stirred at 110 °C for 3 additional
hours, cooled, and evaporated under reduced pressure. The residue
was dissolved in EtOAc (30 mL), washed with H2O (2 × 30 mL) and
brine (1 × 30 mL), dried (Na2SO4), filtered, and evaporated to
dryness. The residue was purified by flash column chromatography
(hexane/EtOAc, 95:5) to yield 16c (107 mg, 62%) as a white solid;
m.p.: 121−126 °C; 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.59 (d, J
= 16.1 Hz, 1H, CHCHCO), 7.56−7.48 (m, 6H, Ar), 7.37 (t, J =
7.4 Hz, 2H, Ar), 7.34−7.25 (m, 3H, Ar), 7.24−7.15 (m, 3H, Ar), 6.74
(d, J = 16.0 Hz, 1H, CHCHCO), 3.88 (s, 2H, CH2CO); 13C NMR
(CDCl3, 75 MHz) δ (ppm): 197.4 (CO), 143.5 (ArCHCH), 143.1
(CAr), 140.2 (CHAr), 134.6 (CAr), 133.5 (CAr), 129.6 (CHAr), 129.0
(CHAr), 129.0 (CHAr), 128.9 (CHAr), 128.1 (CAr), 127.7 (ArCH
CH), 127.2 (CHAr), 127.1 (CHAr), 125.1 (CHAr), 48.6 (CH2CO);
MS (ESI, positive mode) m/z: 321.0 [M + Na]+, 299.3 [M + H]+.
(E)-4-(4-Phenyloxyphenyl)-1-phenylbut-3-en-2-one (16d). Fol-
lowing a procedure similar to that described for 16c, a solution of
phosphonium salt 15 (700 mg, 1.62 mmol), KOH (728 mg, 13
mmol), and 4-phenoxybenzaldehyde (0.34 mL, 1.95 mmol) in dry
toluene (20 mL) was reacted in a pressure flask. After the workup, the
residue was purified by flash column chromatography (hexane/
EtOAc, 95:5) to give 241 mg (47%) of 16d as a white solid; m.p.:
95−97 °C; 1H NMR (CDCl3, 400 MHz) δ (ppm): 7.51 (d, J = 16.0
Hz, 1H, CHCHCO), 7.47−7.33 (m, 2H, Ar), 7.30−7.21 (m, 4H,
Ar), 7.19−7.13 (m, 3H, Ar), 7.06 (tt, J = 7.4, 1.1 Hz, 1H, Ar), 6.97−
6.90 (m, 2H, Ar), 6.89−6.83 (m, 2H, Ar), 6.60 (d, J = 16.0 Hz, 1H,
CHCHCO), 3.83 (s, 2H, CH2CO); 13C NMR (CDCl3, 75 MHz) δ
(ppm): 197.3 (CO), 159.9 (OCAr), 156.1 (OCAr), 142.8 (ArCH
CH), 134.7 (CAr), 130.2 (CHAr), 130.0 (CHAr), 129.6 (CHAr), 129.2
(CAr), 128.9 (CHAr), 127.1 (ArCHCH), 124.3 (CHAr), 124.0
(CHAr), 119.8 (CHAr), 118.5 (CHAr), 48.5 (CH2CO); MS (ESI,
positive mode) m/z: 337.0 [M + Na]+, 315.0 [M + H]+.
(TrizCH2); HPLC (gradient A, Agilent): Rt = 7.1 min; HRMS (ES,
positive mode) m/z: calcd for C28H33N7O2S 531.2416; found
531.2424 (1.35 ppm); anal. calcd for C28H33N7O2S·TFA: C, 55.80;
H, 5.31; N, 15.18; S, 4.97; found: C, 55.94; H, 5.50; N, 15.08; S, 4.51.
1-(2-(5-(4-(4-Ammoniumbutyl)-1H-1,2,3-triazol-1-yl)-2-(4-(2-
(naphthalen-2-yl)ethyl)thiazol-2-yl)phenoxy)ethyl)imidazolidin-2-
one 2,2,2-Trifluoroacetate (12b). Following the deprotection
procedure, compound 11b (218 mg, 0.31 mmol), Pd/C (10%)
(20% w/w) (44 mg), and TFA (1.3 mL) in a 1:1 mixture of THF/
MeOH (30 mL) were hydrogenated. After the workup, the residue
was purified by reverse phase on the Biotage to yield 51 mg (24%) of
12b as a colorless oil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.77
(s, 1H, Ar), 8.49 (d, J = 8.6 Hz, 1H, Ar), 7.89−7.66 (m, 8H, Ar,
+
NH3 ), 7.52−7.34 (m, 3H, Ar), 7.34 (s, 1H, Ar), 4.47 (t, J = 5.7 Hz,
2H, OCH2), 3.63 (t, J = 5.6 Hz, 2H, OCH2CH2), 3.48 (dd, J = 8.9
Hz, J = 6.7 Hz, 2H, CH2CH2NHCON), 3.33−3.04 (m, 6H,
+
CH2CH2NHCON, CH2NH3 , ThiazCH2CH2), 2.93−2.86 (m, 2H,
ThiazCH2CH2), 2.77 (t, J = 7.2 Hz, 2H, TrizCH2), 1.84−1.71 (m,
+
2H, TrizCH2CH2), 1.67−1.56 (m, 2H, CH2CH2NH3 ); 13C NMR
(DMSO-d6, 75 MHz) δ (ppm): 162.1 (NHCON), 159.4 (OCAr),
155.6 (CAr), 155.3 (CAr), 147.7 (CAr), 139.0 (CAr), 137.9 (CAr), 133.2
(CHAr), 131.6 (CAr), 129.1 (CHAr), 127.7 (CHAr), 127.4 (CHAr),
127.3 (CAr), 126.2 (CHAr), 125.9 (CHAr), 125.2 (CHAr), 121.4
(CHAr), 120.4 (CHAr), 116.1 (CHAr), 112.0 (CHAr), 104.3 (CHAr),
67.6 (OCH2), 45.3 (CH2CH2NHCON), 42.4 (OCH2CH2), 37.5
(CH2CH2NHCON), 34.9 (ThiazCH2CH2), 32.5 (ThiazCH2CH2),
+
26.5 (CH2CH2NH3 ), 25.7 (TrizCH2CH2), 24.8 (TrizCH2); HPLC
(gradient A, Agilent): Rt = 7.8 min; HRMS (ES, positive mode) m/z:
calcd for C32H35N7O2S 581.2573; found 581.2572 (−0.13 ppm); anal.
calcd for C32H35N7O2S·TFA: C, 58.69; H, 5.22; N, 14.09; S, 4.61;
found: C, 58.20; H, 5.01; N, 14.09; S, 4.12.
1-(2-(2-(4-(2-([1,1′-Biphenyl]-4-yl)ethyl)thiazol-2-yl)-5-(4-(4-am-
moniumbutyl)-1H-1,2,3-triazol-1-yl)phenoxy)ethyl)imidazolidin-2-
one 2,2,2-Trifluoroacetate (12c). Following the hydrogenolysis
procedure, compound 11c (50 mg, 0.07 mmol), Pd/C (10%) (20%
w/w) (10 mg), and TFA (0.5 mL) in a 1:1 mixture of THF/MeOH
(20 mL) were hydrogenated. After the workup, the residue was
purified by reverse phase on the Biotage to give 12c (17 mg, 35%) as
a colorless oil. 1H NMR (DMSO-d6, 500 MHz) δ (ppm): 8.77 (s, 1H,
Ar), 8.47 (d, J = 8.6 Hz, 1H, Ar), 7.75 (d, J = 2.1 Hz, 1H, Ar), 7.69
(dd, J = 8.6, 2.1 Hz, 1H, Ar), 7.66−7.62 (m, 2H, Ar), 7.60−7.56 (m,
2H, Ar), 7.48−7.39 (m, 3H, Ar), 7.38−7.29 (m, 3H, Ar), 6.41 (bs,
1H, NHCON), 4.47 (t, J = 5.7 Hz, 2H, OCH2), 3.63 (t, J = 5.7 Hz,
2H, OCH2CH2), 3.49 (dd, J = 9.0, 6.7 Hz, 2H, CH2CH2NHCON),
General Procedure for the Synthesis of α-Bromomethylketones
17a−d. To a solution of the corresponding ketone (1 equiv) in
CH3CN, p-TsOH (1.3−2.6 equiv) and NBS (1.3−2.6 equiv) were
successively added at room temperature and the mixture was stirred
for 6 h/overnight. After quenching with H2O (30 mL), the reaction
mixture was carefully concentrated under reduced pressure without
heating. The aqueous crude was extracted with EtOAc (3 × 30 mL),
and the organic layers were dried (Na2SO4), filtered, and evaporated
to dryness. The residue was purified by flash column chromatography
or CCTLC on the Chromatotron (the eluents are specified in each
case) to give the desired α-bromoketones 17a−d.
3.21 (dd, J = 9.3, 6.5 Hz, 2H, CH2CH2NHCON), 3.17−2.96 (m, 4H,
+
CH2NH3 , ThiazCH2CH2), 2.74 (t, J = 7.4 Hz, 2H, TrizCH2), 2.69
(t, J = 7.2 Hz, 2H, ThiazCH2CH2), 1.72 (quin, J = 7.6 Hz, 2H,
+
TrizCH2CH2), 1.58−1.42 (m, 2H, CH2CH2NH3 ); 13C NMR
2-([1,1′-Biphenyl]-4-yl)-1-bromo-1-phenylethan-2-one (17a).
Following the general bromination procedure, p-TsOH (173 mg,
0.92 mmol) and NBS (165 mg, 0.92 mmol) were successively added
to a solution of the commercially available 16a (50 mg, 0.18 mmol) in
CH3CN (15 mL). The reaction was stirred at room temperature
overnight. After the workup, the residue was purified by CCTLC on
the Chromatotron (hexane/EtOAc, 80:20) to give 62 mg (98%) of
(DMSO-d6, 100 MHz) δ (ppm): 162.1 (NHCON), 159.4 (OCAr),
155.6 (CAr), 155.3 (CAr), 148.1 (CAr), 140.7 (CAr), 140.0 (CAr), 138.0
(CAr), 137.8 (CAr), 129.0 (CHAr), 128.9 (CHAr), 128.9 (CHAr), 127.2
(CHAr), 126.5 (CHAr), 126.5 (CHAr), 121.3 (CAr), 120.3 (CHAr),
116.0 (CHAr), 112.0 (CHAr), 104.3 (CHAr), 67.6 (OCH2), 45.4
+
(CH2CH2NHCON), 42.4 (OCH2CH2), 37.7 (CH2NH3 ), 37.5
(CH2CH2NHCON), 34.3 (ThiazCH2CH2), 32.5 (ThiazCH2CH2),
+
1
30.1 (CH2CH2NH3 ), 25.9 (TrizCH2CH2), 24.7 (TrizCH2); HPLC
17a as a white solid; m.p.: decompose without melting; H NMR
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(CDCl3, 400 MHz) δ (ppm): 8.08 (d, J = 8.2 Hz, 2H, Ar), 7.67 (d, J
= 8.4 Hz, 2H, Ar), 7.63−7.54 (m, 4H, Ar), 7.47 (t, J = 7.5 Hz, 2H,
Ar), 7.43−7.29 (m, 4H, Ar), 6.43 (s, 1H, BrCHCO).
128.2 (CHAr), 127.9 (CAr), 122.5 (CHAr), 119.1 (CHAr), 112.4
(CHAr), 104.4 (CHAr), 67.7 (OCH2), 66.7 (NHCOOCH2), 46.4
(CH2CH2NHCON), 42.9 (OCH2CH2), 40.8 (CH2NHCbz), 38.4
(CH2CH2NHCON), 29.4 (CH2CH2NHCbz), 26.3 (TrizCH2CH2),
25.2 (TrizCH2); HRMS (ES, positive mode) m/z: calcd for
C40H39N7O4S 713.2784; found 713.2786 (0.23 ppm).
Benzyl-(4-(1-(3-(2-(2-oxoimidazolidin-1-yl)ethoxy)-4-(4-([1,1′-bi-
phenyl]-4-yl)-5-phenylthiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-yl)-
butyl)carbamate (18b). Following the general procedure for the
synthesis of thiazoles by Hantzsch cyclization, thioamide 9 (194 mg,
0.36 mmol) reacted with α-bromoketone 17a (127 mg, 0.36 mmol) in
iPrOH (20 mL). After the workup, the residue was purified by
2-(4-Phenyloxyphenyl)-1-bromo-1-phenylethan-2-one (17b).
The general bromination procedure was followed with the
commercially available 16b (150 mg, 0.52 mmol), p-TsOH (297
mg, 1.56 mmol), and NBS (278 mg, 1.56 mmol) in CH3CN (22 mL)
at room temperature overnight. The residue was purified by CCTLC
on the Chromatotron (hexane/CH2Cl2, 70:30) to afford 17b (140
mg, 73%) as a white solid; m.p.: 116-118 °C; 1H NMR (CDCl3, 400
MHz) δ (ppm): 7.97 (d, J = 8.9 Hz, 2H, Ar), 7.53 (dd, J = 8.1, 1.6 Hz,
2H, Ar), 7.44−7.29 (m, 5H, Ar), 7.22 (tt, J = 7.3, 1.0 Hz, 1H, Ar),
7.06 (dd, J = 7.9, 1.6 Hz, 2H, Ar), 6.96 (d, J = 8.9 Hz, 2H, Ar), 6.34
(s, 1H, BrCHCO); 13C NMR (CDCl3, 75 MHz) δ (ppm): 189.7
(CO), 162.7 (OCAr), 155.1 (OCAr), 136.2 (CAr), 131.7 (CHAr), 130.3
(CHAr), 129.2 (CHAr), 129.2 (CHAr), 128.5 (CAr), 125.1 (CHAr),
120.6 (CHAr), 117.4 (CHAr), 51.2 (BrCHCO); MS (ESI, positive
mode) m/z: 389 [M + Na]+ with a Br isotopic pattern.
CCTLC on the Chromatotron (CH2Cl2/MeOH, 95:5) to give 18b
1
(244 mg, 84%) as a colorless oil. H NMR (CDCl3, 400 MHz) δ
(ppm): 8.66 (d, J = 8.6 Hz, 1H, Ar), 7.60 (s, 1H, Ar), 7.71 (d, J = 8.1
Hz, 2H, Ar), 7.65−7.51 (m, 3H, Ar), 7.56 (d, J = 8.1 Hz, 2H, Ar),
7.52−7.18 (m, 14H, Ar), 5.13 (bs, 1H, NHCON), 5.10 (s, 2H,
NCOOCH2), 4.67 (bs, 1H, NHCbz), 4.47 (t, J = 5.9 Hz, 2H, OCH2),
3.78 (t, J = 5.9 Hz, 2H, OCH2CH2), 3.59 (dd, J = 9.0, 6.7 Hz, 2H,
CH2CH2NHCON), 3.37−3.22 (m, 4H, CH2CH2NHCON,
CH2NHCbz), 2.84 (t, J = 7.3 Hz, 2H, TrizCH2), 1.86−1.74 (m,
2H, TrizCH2CH2), 1.64 (quin, J = 7.2 Hz, 2H, CH2CH2NHCbz);
13C NMR (CDCl3, 75 MHz) δ (ppm): 162.7 (NHCON), 158.4
(OCAr), 156.6 (CAr), 156.0 (NHCOO), 149.0 (CAr), 148.8 (CAr),
140.8 (CAr), 140.5 (CAr), 138.5 (CAr), 136.7 (CAr), 134.4 (CAr), 134.1
(CHAr), 132.3 (CAr), 129.8 (CHAr), 129.5 (CHAr), 128.9 (CHAr),
128.9 (CHAr), 128.6 (CHAr), 128.3 (CHAr), 128.2 (CHAr), 127.5
(CAr), 127.1 (CHAr), 122.5 (CHAr), 119.2 (CAr), 112.5 (CHAr), 104.5
(CHA r ), 67.7 (NHCOOCH2 ), 66.8 (OCH2 ), 46.4
(CH2CH2NHCON), 42.9 (OCH2CH2), 40.9 (CH2NHCbz), 38.4
(CH2CH2NHCON), 29.4 (CH2CH2NHCbz), 26.4 (TrizCH2CH2),
25.2 (TrizCH2); HRMS (ES, positive mode) m/z: calcd for
C46H43N7O4S 789.3097; found 789.3063 (−4.31 ppm).
(E)-4-([1,1′-Biphenyl]-4-yl)-1-bromo-1-phenylbut-3-en-2-one
(17c). Following the general procedure, compound 16c (200 mg, 0.67
mmol) dissolved in CH3CN (25 mL) was sequentially treated with p-
TsOH (191 mg, 1.00 mmol) and NBS (179 mg, 1.00 mmol) at room
temperature for 4 h. After the workup, the residue was purified by
flash column chromatography (hexane/EtOAc, 90:10) to give 17c
(253 mg, 79%) as a white solid; m.p.: 118−120 °C; 1H NMR
(CDCl3, 400 MHz) δ (ppm): 7.71 (d, J = 15.8 Hz, 1H, CH
CHCO), 7.56−7.49 (m, 6H, Ar), 7.47−7.42 (m, 2H, Ar), 7.41−7.34
(m, 2H, Ar), 7.35−7.24 (m, 4H, Ar), 6.94 (d, J = 15.8 Hz, 1H, CH
CHCO), 5.61 (s, 1H, BrCHCO); 13C NMR (CDCl3, 75 MHz) δ
(ppm): 190.4 (CO), 145.0 (ArCHCH), 143.9 (CAr), 140.1
(CHAr), 135.4 (CAr), 133.2 (CAr), 129.3 (CHAr), 129.3 (CHAr),
129.2 (CHAr), 129.1 (CHAr), 128.1 (CAr), 127.7 (ArCHCH), 127.2
(CHAr), 127.1 (CHAr), 121.4 (CHAr), 55.5 (BrCHCO).
(E)-4-(4-Phenyloxyphenyl)-1-phenylbut-3-en-2-one (17d). Ac-
cording to the general bromination procedure, the α,β-unsaturated
compound 16d (211 mg, 0.67 mmol), p-TsOH (140 mg, 0.74 mmol),
and NBS (131 mg, 0.74 mmol) dissolved in CH3CN (25 mL) were
reacted at room temperature for 4 h. After the workup, the crude was
purified by flash column chromatography (hexane/EtOAc, 97:3) to
give 17d (150 mg, 39%) as a yellow oil. 1H NMR (CDCl3, 400 MHz)
δ (ppm): 7.72 (d, J = 15.7 Hz, 1H, CHCHCO), 7.56−7.47 (m,
4H, Ar), 7.43−7.32 (m, 5H, Ar), 7.18 (tt, J = 7.4, 1.1 Hz, 1H, Ar),
7.10−7.01 (m, 2H, Ar), 6.97 (d, J = 8.8 Hz, 2H, Ar), 6.89 (d, J = 15.7
Hz, 1H, CHCHCO), 5.66 (s, 1H, BrCHCO); 13C NMR (CDCl3,
75 MHz) δ (ppm): 190.4 (CO), 160.4 (OCAr), 156.0 (OCAr), 144.8
(ArCHCH), 135.5 (CAr), 130.6 (CHAr), 130.1 (CHAr), 129.2
(CHAr), 129.1 (CAr), 129.1 (CHAr), 128.9 (ArCHCH), 124.5
(CHAr), 120.2 (CHAr), 120.0 (CHAr), 118.5 (CHAr), 55.5
(BrCHCO); MS (ESI, positive mode) m/z: 417.0 [M + Na]+,
393.0 [M + H]+ with a Br isotopic pattern.
Benzyl-(4-(1-(3-(2-(2-oxoimidazolidin-1-yl)ethoxy)-4-(4-(4-phe-
nyloxyphenyl-1-yl)-5-phenylthiazol-2-yl)phenyl)-1H-1,2,3-triazol-4-
yl)butyl)carbamate (18c). Following the general procedure for the
synthesis of thiazoles by Hantzsch cyclization, thioamide 9 (190 mg,
0.35 mmol) and α-bromoketone 17b (130 mg, 0.35 mmol) reacted in
iPrOH (20 mL). The final residue was purified by CCTLC in the
Chromatotron (CH2Cl2/MeOH, 95:5) to yield 235 mg (81%) of 18c
a colorless oil. 1H NMR (CDCl3, 400 MHz) δ (ppm): 8.63 (d, J = 8.5
Hz, 1H, Ar), 7.90 (s, 1H, Ar), 7.65−7.55 (m, 3H, Ar), 7.47−7.24 (m,
13H, Ar), 7.11 (t, J = 7.4 Hz, 1H, Ar), 7.04 (d, J = 8.0 Hz, 2H, Ar),
6.95 (d, J = 8.3 Hz, 2H, Ar), 5.14 (bs, 1H, NHCON), 5.09 (s, 2H,
NCOOCH2), 4.73 (bs, 1H, NHCbz), 4.46 (t, J = 5.9 Hz, 2H, OCH2),
3.77 (t, J = 5.9 Hz, 2H, OCH2CH2), 3.59 (dd, J = 9.0, 6.7 Hz, 2H,
CH2CH2NHCON), 3.37−3.21 (m, 4H, CH2CH2NHCON,
CH2NHCbz), 2.84 (t, J = 7.3 Hz, 2H, TrizCH2), 1.84−1.74 (m,
2H, TrizCH2CH2), 1.63 (quin, J = 7.2 Hz, 2H, CH2CH2NHCbz);
13C NMR (CDCl3, 75 MHz) δ (ppm): 162.7 (NHCON), 158.3
(OCAr), 157.2 (CAr), 157.0 (OCAr), 156.6 (OCAr), 156.0 (NHCOO),
148.8 (CAr), 138.5 (CAr), 136.7 (CAr), 133.7 (CAr), 132.3 (CAr), 130.7
(CHAr), 130.1 (CAr), 129.9 (CHAr), 129.7 (CHAr), 129.7 (CHAr),
128.9 (CHAr), 128.6 (CHAr), 128.2 (CAr), 128.2 (CHAr), 123.6
(CHAr), 122.5 (CAr), 119.3 (CHAr), 119.1 (CAr), 118.6 (CHAr), 112.5
(CHAr), 104.5 (CHAr), 67.7 (NHCOOCH2), 66.8 (OCH2), 46.4
(CH2CH2NHCON), 42.9 (OCH2CH2), 40.9 (CH2NHCbz), 38.4
(CH2CH2NHCON), 29.4 (CH2CH2NHCbz), 26.3 (TrizCH2CH2),
25.2 (TrizCH2); HRMS (ES, positive mode) m/z: calcd for
C46H43N7O5S 805.3046; found 805.3054 (0.92 ppm).
Benzyl-(4-(1-(4-(4,5-diphenylthiazol-2-yl)-3-(2-(2-oxoimidazoli-
din-1-yl)ethoxy)phenyl)-1H-1,2,3-triazol-4-yl)butyl)carbamate
(18a). Following the general procedure of Hantzsch synthesis, the
thioamide 9 (150 mg, 0.28 mmol) and the commercially available 1-
bromo-1,2-diphenylethan-2-one 17e (77 mg, 0.28 mmol) reacted in
iPrOH (20 mL). After the workup, the final residue was purified by
CCTLC in the Chromatotron (CH2Cl2/MeOH, 97:3) to provide 191
1
mg (94%) of 18a as a colorless oil. H NMR (CDCl3, 400 MHz) δ
(ppm): 8.62 (d, J = 8.6 Hz, 1H, Ar), 7.89 (s, 1H, Ar), 7.64−7.57 (m,
3H, Ar), 7.44−7.23 (m, 14H, Ar), 5.19 (t, J = 6.0 Hz, 1H, NHCbz),
5.08 (s, 2H, NHCOOCH2), 4.87 (bs, 1H, NHCON), 4.44 (t, J = 5.9
Hz, 2H, OCH2), 3.75 (t, J = 5.8 Hz, 2H, OCH2CH2), 3.57 (dd, J =
9.0, 6.7 Hz, 2H, CH2CH2NHCON), 3.29 (t, J = 8.0 Hz, 2H,
CH2CH2NHCON), 3.25 (q, J = 6.9 Hz, 2H, CH2NHCbz), 2.82 (t, J
= 7.4 Hz, 2H, TrizCH2), 1.84−1.71 (m, 2H, TrizCH2CH2), 1.62
(quin, J = 6.2 Hz, 2H, CH2CH2NHCbz); 13C NMR (CDCl3, 75
MHz) δ (ppm): 162.2 (NHCON), 158.3 (OCAr), 156.6 (CAr), 155.9
(CAr), 149.3 (CAr), 148.7 (CAr), 138.4 (CAr), 136.7 (CAr), 135.1
(CAr), 134.3 (CAr), 132.2 (CHAr), 129.7 (CHAr), 129.6 (CHAr), 129.2
(CHAr), 129.1 (CHAr), 128.8 (CHAr), 128.6 (CHAr), 128.4 (CHAr),
Benzyl-(E)-(4-(1-(4-(4-(2-([1,1′-biphenyl]-4-yl)vinyl)-5-phenyl-
thiazol-2-yl)-3-(2-(2-oxoimidazolidin-1-yl)ethoxy) phenyl)-1H-
1,2,3-triazol-4-yl)butyl)carbamate (18d). Following the general
Hantzsch cyclization procedure, thioamide 9 (200 mg, 0.37 mmol)
i
and α-bromoketone 17c (140 mg, 0.37 mmol) reacted in PrOH (20
mL). After the workup, the residue was purified by CCTLC on the
Chromatotron (CH2Cl2/MeOH, 95:5) to give 18d (183 mg, 59%) as
1
a yellow oil. H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.72 (s, 1H,
Ar), 8.61 (d, J = 8.4 Hz, 1H, Ar), 7.80−7.71 (m, 3H, Ar, ThiazCH
CH), 7.70−7.53 (m, 10H, Ar), 7.51−7.42 (m, 3H, Ar), 7.39−7.27
6153
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J. Med. Chem. 2021, 64, 6137−6160

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(m, 3H, Ar, NHCbz), 7.24 (d, J = 15.7 Hz, 1H, ThiazCHCH), 6.42
(bs, 1H, NHCON), 5.02 (s, 2H, NHCOOCH2), 4.48 (t, J = 5.7 Hz,
2H, OCH2), 3.66 (t, J = 5.6 Hz, 2H, OCH2CH2), 3.49 (dd, J = 9.0,
6.6 Hz, 2H, CH2CH2NHCON), 3.23 (dd, J = 9.0, 6.7 Hz, 2H,
CH2CH2NHCON), 3.08 (q, J = 6.6 Hz, 2H, CH2NHCbz), 2.72 (t, J
= 7.1 Hz, 2H, TrizCH2), 1.70 (quin, J = 7.5 Hz, 2H, TrizCH2CH2),
1.52 (quin, J = 7.2 Hz, 2H, CH2CH2NHCbz); 13C NMR (DMSO-d6,
1-(2-(5-(4-(4-Ammoniobutyl)-1H-1,2,3-triazol-1-yl)-2-(4-([1,1′-bi-
phenyl]-4-yl)-5-phenylthiazol-2-yl)phenoxy) ethyl)imidazolidin-2-
one 2,2,2-Trifluoroacetate (19b). The general N-deprotection
procedure described for 12a−c was followed with 18b (100 mg,
0.13 mmol), Pd/C 10% (20 mg), and TFA (0.8 mL) to give, after
workup and purification, compound 19b (15 mg, 15%) as a colorless
oil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.79 (s, 1H, Ar), 8.57
(d, J = 8.6 Hz, 1H, Ar), 7.80 (d, J = 8.1 Hz, 1H, Ar), 7.77−7.60 (m,
75 MHz) δ (ppm): 161.8 (NHCON), 158.0 (OCAr), 155.8
(NHCOO), 147.9 (CAr), 146.6 (CAr), 139.4 (CAr), 139.3 (CAr),
138.3 (CAr), 137.1 (CAr), 135.8 (CAr), 135.0 (CAr), 131.6 (CAr), 130.9
(CHAr), 129.9 (CHAr), 128.9 (CHAr), 128.6 (CHAr), 128.0 (CAr),
128.0 (CHAr), 127.4 (CHAr), 127.3 (CAr), 127.2 (CHAr), 126.9
(CHAr), 126.7 (CAr), 126.2 (CHAr), 120.9 (CHAr), 119.9 (CHAr),
119.5 (CAr), 112.0 (CHAr), 104.3 (CHAr), 67.3 (NHCOOCH2), 64.9
(OCH2), 45.0 (CH2CH2NHCON), 42.3 (OCH2CH2), 39.9
(CH2NHCbz), 37.3 (CH2CH2NHCON), 28.7 (CH2CH2NHCbz),
25.8 (TrizCH2CH2), 24.5 (TrizCH2); HRMS (ES, positive mode) m/
z: calcd for C48H45N7O4S 815.3254; found 815.3266 (1.56 ppm).
Benzyl-(E)-(4-(1-(4-(4-(2-([4-phenyloxyphenyl]-1-yl)vinyl)-5-phe-
nylthiazol-2-yl)-3-(2-(2-oxoimidazolidin-1-yl)ethoxy)phenyl)-1H-
1,2,3-triazol-4-yl)butyl)carbamate (18e). According to the general
Hantzsch procedure, thioamide 9 (183 mg, 0.34 mmol) was reacted
+
10H, Ar, NH3 ), 7.54−7.41 (m, 7H, Ar), 7.37 (t, J = 7.5 Hz, 1H, Ar),
6.40 (bs, 1H, NHCON), 4.53 (t, J = 5.6 Hz, 2H, OCH2), 3.66 (t, J =
5.6 Hz, 2H, OCH2CH2), 3.51 (dd, J = 9.0, 6.7 Hz, 2H,
CH2CH2NHCON), 3.23 (t, J = 7.8 Hz, 2H, CH2CH2NHCON),
+
2.85 (t, J = 7.5 Hz, 2H, CH2NH3 ), 2.78 (t, J = 7.2 Hz, 2H, TrizCH2),
1.76 (quin, J = 7.2 Hz, 2H, TrizCH2CH2), 1.63 (quin, J = 7.8 Hz, 2H,
+
CH2CH2NH3 ); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 162.1
(NHCON), 157.9 (OCAr), 155.8 (CAr), 148.0 (CAr), 147.7 (CAr),
139.4 (CAr), 139.4 (CAr), 138.3 (CAr), 133.9 (CAr), 133.7 (CAr), 131.5
(CHAr), 129.3 (CHAr), 129.2 (CHAr), 129.1 (CHAr), 129.0 (CHAr),
128.8 (CHAr), 128.5 (CHAr), 127.6 (CHAr), 126.5 (CHAr), 126.5
(CHAr), 121.1 (CHAr), 120.4 (CAr), 112.1 (CHAr), 104.3 (CHAr),
67.4 (OCH2), 45.1 (CH2CH2NHCON), 42.4 (OCH2CH2), 38.6
+
+
(CH2NH3 ), 37.5 (CH2CH2NHCON), 26.5 (CH2CH2NH3 ), 25.5
(TrizCH2CH2), 24.4 (TrizCH2); HPLC (gradient A, Agilent): Rt =
9.3 min; HRMS (ES, positive mode) m/z: calcd for C38H37N7O2S
655.2729; found 655.2748 (2.87 ppm); anal. calcd for C38H38N7O2S·
TFA: C, 62.41; H, 4.98; N, 12.74; S, 4.16; found: C, 62.36; H, 5.06;
N, 12.37; S, 4.25.
i
with α-bromoketone 17d (134 mg, 0.34 mmol) in PrOH (23 mL).
After purification of the residue by CCTLC on the Chromatotron
(CH2Cl2/MeOH, 96:4), 18e (150 mg, 52%) was obtained as a
1
colorless oil. H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.74 (s, 1H,
Ar), 8.61 (d, J = 8.5 Hz, 1H, Ar), 7.85−7.67 (m, 3H, Ar, ThiazCH
CH), 7.64−7.51 (m, 5H, Ar), 7.51−7.24 (m, 9H, Ar), 7.20−7.10 (m,
2H, Ar, ThiazCHCH), 7.05 (d, J = 7.7 Hz, 2H, Ar), 7.00 (d, J = 8.6
Hz, 2H, Ar), 6.40 (bs, 1H, NHCON), 5.01 (s, 2H, NHCOOCH2),
4.49 (t, J = 5.7 Hz, 2H, OCH2), 3.66 (t, J = 5.6 Hz, 2H, OCH2CH2),
3.49 (dd, J = 9.0, 6.6 Hz, 2H, CH2CH2NHCON), 3.22 (t, J = 7.9 Hz,
2H, CH2CH2NHCON), 3.07 (q, J = 6.6 Hz, 2H, CH2NHCbz), 2.74
(t, J = 7.5 Hz, 2H, TrizCH2), 1.69 (quin, J = 7.5 Hz, 2H,
TrizCH2CH2), 1.53 (quin, J = 7.2 Hz, 2H, CH2CH2NHCbz); 13C
1-(2-(5-(4-(4-Ammoniobutyl)-1H-1,2,3-triazol-1-yl)-2-(4-([4-phe-
nyloxyphenyl]-1-yl)-5-phenylthiazol-2-yl) phenoxy)ethyl)-
imidazolidin-2-one 2,2,2-Trifluoroacetate (19c). Following the
general Cbz deprotection procedure, 18c (120 mg, 0.15 mmol),
Pd/C 10% (24 mg), and TFA (0.9 mL) were reacted. Workup and
1
purification yielded 19c (44 mg, 37%) as a colorless oil. H NMR
(DMSO-d6, 400 MHz) δ (ppm): 8.78 (s, 1H, Ar), 8.53 (d, J = 8.6 Hz,
+
1H, Ar), 7.79 (d, J = 2.1 Hz, 1H, Ar), 7.83−7.72 (m, 3H, NH3 ), 7.71
(dd, J = 8.6, 2.0 Hz, 1H, Ar), 7.56 (d, J = 8.7 Hz, 2H, Ar), 7.50−7.32
(m, 7H, Ar), 7.17 (t, J = 7.4 Hz, 1H, Ar), 7.06 (d, J = 7.9 Hz, 2H, Ar),
6.98 (d, J = 8.7 Hz, 2H, Ar), 6.39 (bs, 1H, NHCON), 4.51 (t, J = 5.6
Hz, 2H, OCH2), 3.65 (t, J = 5.6 Hz, 2H, OCH2CH2), 3.50 (dd, J =
9.0, 6.7 Hz, 2H, CH2CH2NHCON), 3.22 (t, J = 7.9 Hz, 2H,
NMR (DMSO-d6, 75 MHz) δ (ppm): 162.1 (NHCON), 158.1 (CAr),
156.6 (OCAr), 156.3 (OCAr), 156.1 (OCAr), 156.0 (NHCOO), 148.1
(CAr), 146.7 (OCAr), 138.4 (CAr), 137.3 (CAr), 134.8 (CAr), 132.0
(CAr), 131.6 (CAr), 131.1 (CAr), 130.1 (CAr), 129.4 (CHAr), 129.2
(CHAr), 128.4 (CHAr), 128.3 (CHAr), 127.7 (CHAr), 123.7 (CHAr),
120.9 (CHAr), 120.3 (CAr), 118.9 (CHAr), 118.7 (CHAr), 112.0
(CHAr), 104.3 (CHAr), 67.4 (NHCOOCH2), 65.1 (OCH2), 45.1
(CH2CH2NHCON), 42.4 (OCH2CH2), 40.0 (CH2NHCbz), 37.5
(CH2CH2NHCON), 28.9 (CH2CH2NHCbz), 26.0 (TrizCH2CH2),
24.7 (TrizCH2); HRMS (ES, positive mode) m/z: calcd for
C48H45N7O5S 831.3203; found 831.3174 (−3.45 ppm).
1-(2-(5-(4-(4-Ammoniobutyl)-1H-1,2,3-triazol-1-yl)-2-(4,5-diphe-
nylthiazol-2-yl)phenoxy)ethyl)imidazolidin-2-one 2,2,2-Trifluoroa-
cetate (19a). Following the general Cbz removal procedure, 18a (100
mg, 0.14 mmol), Pd/C 10% (20 mg) and TFA (1 mL) afforded, after
purification, compound 19a (25 mg, 26%) as a colorless oil. 1H NMR
(CD3OD, 400 MHz) δ (ppm): 8.56 (d, J = 8.6 Hz, 1H, Ar), 8.46 (s,
1H, Ar), 7.71 (d, J = 8.0 Hz, 1H, Ar), 7.59 (dd, J = 8.6, 2.0 Hz, 1H,
Ar), 7.56−7.51 (m, 2H, Ar), 7.44−7.27 (m, 8H, Ar), 4.51 (t, J = 5.5
Hz, 2H, OCH2), 3.76 (t, J = 5.5 Hz, 2H, OCH2CH2), 3.62 (dd, J =
+
CH2CH2NHCON), 2.91−2.80 (m, 2H, CH2NH3 ), 2.77 (t, J = 7.2
Hz, 2H, TrizCH2), 1.76 (quin, J = 7.2 Hz, 2H, TrizCH2CH2), 1.64
+
(quin, J = 8.1 Hz, 2H, CH2CH2NH3 ); 13C NMR (DMSO-d6, 100
MHz) δ (ppm): 162.1 (NHCON), 158.1 (CF3COO−), 157.8 (CAr),
156.5 (OCAr), 156.2 (OCAr), 155.5 (OCAr), 147.9 (CAr), 147.7 (CAr),
138.3 (CAr), 133.3 (CAr), 131.5 (CAr), 130.4 (CHAr), 130.1 (CHAr),
129.8 (CHAr), 129.2 (CHAr), 129.0 (CHAr), 128.8 (CHAr), 128.3
(CAr), 123.8 (CHAr), 121.1 (CAr), 120.4 (CHAr), 119.0 (CHAr), 118.1
(CHAr), 115.8 (CF3COO−), 112.1 (CHAr), 104.3 (CHAr), 67.3
(OCH2), 45.1 (CH2CH2NHCON), 42.4 (OCH2CH2), 38.6
+
+
(CH2NH3 ), 37.5 (CH2CH2NHCON), 26.5 (CH2CH2NH3 ), 25.5
(TrizCH2CH2), 24.4 (TrizCH2); HPLC (gradient A, Agilent): Rt =
9.8 min; HRMS (ES, positive mode) m/z: calcd for C38H37N7O3S
671.2679; found 671.2673 (−0.81 ppm); anal. calcd for
C38H38N7O3S·TFA: C, 61.14; H, 4.87; N, 12.48; S, 4.08; found: C,
61.55; H, 4.99; N, 12.12; S, 4.34.
9.3, 6.9 Hz, 2H, CH2CH2NHCON), 3.32 (dd, J = 9.3, 6.9 Hz, 2H,
+
CH2CH2NHCON), 3.00 (q, J = 6.5 Hz, 2H, CH2NH3 ), 2.86 (t, J =
1-(2-(2-(4-(2-([1,1′-Biphenyl]-4-yl)ethyl)-5-phenyl-thiazol-2-yl)-5-
(4-(4-ammoniobutyl)-1H-1,2,3-triazol-1-yl)phenoxy)ethyl)-
imidazolidin-2-one 2,2,2-Trifluoroacetate (19d). Following the
general hydrogenation procedure, a solution of 18d (91 mg, 0.11
mmol), Pd/C 10% (18 mg), and TFA (0.7 mL) was reacted to give,
after purification, 19d (14 mg, 16%) as a colorless oil. 1H NMR
(DMSO-d6, 400 MHz) δ (ppm): 8.78 (s, 1H, Ar), 8.54 (d, J = 8.6 Hz,
1H, Ar), 7.78 (d, J = 2.1 Hz, 1H, Ar), 7.72 (dd, J = 8.5, 2.0 Hz, 1H,
7.2 Hz, 2H, TrizCH2), 1.95−1.65 (m, 4H, TrizCH2CH2,
+
CH2CH2NH3 ); 13C NMR (CD3OD, 75 MHz) δ (ppm): 165.0
(NHCON), 159.9 (OCAr), 157.5 (CAr), 150.6 (CAr), 149.3 (CAr),
139.8 (CAr), 136.4 (CAr), 135.8 (CAr), 133.3 (CHAr), 130.6 (CHAr),
130.5 (CHAr), 130.3 (CHAr), 129.9 (CHAr), 129.3 (CHAr), 129.3
(CHAr), 129.0 (CHAr), 129.0 (CHAr), 123.7 (CHAr), 121.6 (CHAr),
113.6 (CHA r ), 105.6 (CHA r ), 68.2 (OCH2 ), 46.9
+
+
Ar), 7.73−7.65 (m, 3H, NH3 ), 7.62 (d, J = 7.2 Hz, 2H, Ar), 7.54 (d,
(CH2CH2NHCON), 43.9 (OCH2CH2), 40.4 (CH2NH3 ), 39.3
+
J = 8.2 Hz, 2H, Ar), 7.49−7.38 (m, 7H, Ar), 7.33 (tt, J = 7.4, 1.3 Hz,
1H, Ar), 7.26 (d, J = 8.2 Hz, 2H, Ar), 6.39 (bs, 1H, NHCON), 4.50
(t, J = 5.6 Hz, 2H, OCH2), 3.65 (t, J = 5.6 Hz, 2H, OCH2CH2), 3.49
(dd, J = 8.9, 6.7 Hz, 2H, CH2CH2NHCON), 3.21 (t, J = 7.9 Hz, 2H,
CH2CH2NHCON), 3.16−3.12 (m, 4H, ThiazCH2CH2), 2.85 (t, J =
(CH2CH2NHCON), 28.0 (CH2CH2NH3 ), 27.1 (TrizCH2CH2),
25.6 (TrizCH2); HPLC (gradient A, Agilent): Rt = 9.3 min; HRMS
(ES, positive mode) m/z: calcd for C32H33N7O2S 579.2416; found
579.242 (0.61 ppm); anal. calcd for C32H34N7O2S·TFA: C, 58.87; H,
4.94; N, 14.13; S, 4.62; found: C, 59.15; H, 5.36 N, 14.62; S, 5.03.
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J. Med. Chem. 2021, 64, 6137−6160

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Article
+
7.5 Hz, 2H, CH2NH3 ), 2.78 (t, J = 7.2 Hz, 2H, TrizCH2), 1.76
(dd, J = 9.0, 6.7 Hz, 4H, CH2CH2NHCON), 3.25 (t, J = 7.9 Hz, 4H,
CH2CH2NHCON), 3.07 (q, J = 6.5 Hz, 4H, CH2NHCbz), 2.74 (t, J
= 7.1 Hz, 4H, TrizCH2), 1.70 (quin, J = 7.6 Hz, 4H, TrizCH2CH2),
1.52 (quin, J = 7.2 Hz, 4H, CH2CH2NHCbz); 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 162.1 (NHCON), 160.2 (CAr), 156.1 (OCAr),
155.9 (NHCOO), 153.3 (CAr), 148.1 (CAr), 138.3 (CAr), 137.3
(CHAr), 134.7 (CAr), 129.3 (CHAr), 128.3 (CHAr), 127.7 (CHAr),
126.0 (CAr), 123.7 (CAr), 121.1 (CHAr), 120.3 (CHAr), 116.4 (CHAr),
112.1 (CHAr), 104.3 (CHAr), 67.6 (OCH2), 65.1 (NHCOOCH2),
45. 3 (CH2 CH2 NHCON), 42. 4 (OC H2 CH2 ), 37. 6
(CH2CH2NHCON), 28.9 (CH2CH2NHCbz), 26.0 (TrizCH2CH2),
24.7 (TrizCH2); HRMS (ES, positive mode) m/z: calcd for
C62H64N14O8S2 1196.4473; found 1196.4482 (0.72 ppm).
(quin, J = 7.6 Hz, 2H, TrizCH2CH2), 1.63 (quin, J = 7.5 Hz, 2H,
+
CH2CH2NH3 ); 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 162.1
(NHCON), 157.4 (CAr), 155.6 (OCAr), 150.1 (CAr), 147.7 (CAr),
140.7 (CAr), 140.1 (CAr), 138.1 (CAr), 137.9 (CAr), 133.4 (CAr), 131.4
(CAr), 129.0 (CHAr), 128.9 (CHAr), 128.9 (CHAr), 128.9 (CHAr),
128.8 (CHAr), 128.0 (CHAr), 127.2 (CHAr), 126.6 (CHAr), 126.5
(CHAr), 121.3 (CAr), 120.4 (CHAr), 112.1 (CHAr), 104.3 (CHAr),
67.4 (OCH2), 45.2 (CH2CH2NHCON), 42.4 (OCH2CH2), 38.7
+
(CH2NH3 ), 37.5 (CH2CH2NHCON), 34.6 (ThiazCH2CH2), 31.2
(ThiazCH2CH2), 26.5 (CH2CH2NH3 ), 25.6 (TrizCH2CH2), 24.4
+
(TrizCH2); HPLC (gradient A, Agilent): Rt = 9.8 min; HRMS (ES,
positive mode) m/z: calcd for C40H41N7O2S 683.3042; found
683.3050 (1.06 ppm); anal. calcd for C40H42N7O2S·TFA: C, 63.22;
H, 5.31; N, 12.29; S, 4.02; found: C, 63.40; H, 5.64; N, 12.50; S, 3.93.
1-(2-(2-(4-(2-([4-Phenyloxyphenyl]-1-yl)ethyl)-5-phenyl-thiazol-
2-yl)-5-(4-(4-ammoniobutyl)-1H-1,2,3-triazol-1-yl)phenoxy)ethyl)-
imidazolidin-2-one 2,2,2-Trifluoroacetate (19e). Following the
general Cbz deprotection procedure, reaction of 18e (80 mg, 0.10
mmol), Pd/C 10% (16 mg), and TFA (0.6 mL) afforded 19e (20 mg,
1,1′-((((1,3-Phenylenebis(thiazole-4,2-diyl))bis(5-(4-(4-ammonio-
butyl)-1H-1,2,3-triazol-1-yl)-2,1-phenylene)) bis(oxy))bis(ethane-
2,1-diyl))bis(imidazolidin-2-one)bis(2,2,2-trifluoroacetate) (22).
Following the general Cbz removal procedure, 21 (120 mg, 0.11
mmol), Pd/C 10% (24 mg), and TFA (1.0 mL) reacted to gave, after
workup and purification, compound 22 (22 mg, 19%) as a colorless
oil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.81 (s, 2H, Ar), 8.75
(s, 1H, Ar), 8.69 (d, J = 8.5 Hz, 2H, Ar), 8.40 (s, 2H, Ar), 8.13 (dd, J
1
26%) as a colorless oil. H NMR (DMSO-d6, 400 MHz) δ (ppm):
8.78 (s, 1H, Ar), 8.56−8.50 (m, 1H, Ar), 7.77 (d, J = 2.0 Hz, 1H, Ar),
7.81−7.64 (m, 4H, Ar, NH3 ), 7.62 (d, J = 7.2 Hz, 1H, Ar), 7.54 (d, J
+
= 7.8, 1.7 Hz, 2H, Ar), 7.94−7.67 (m, 10H, Ar, NH3 ), 7.62 (t, J = 7.7
+
Hz, 1H, Ar), 6.42 (bs, 2H, NHCON), 4.54 (t, J = 5.7 Hz, 4H,
OCH2), 3.69 (t, J = 5.6 Hz, 4H, OCH2CH2), 3.53 (dd, J = 8.9, 6.7
= 8.1 Hz, 1H, Ar), 7.50−7.31 (m, 7H, Ar), 7.26 (d, J = 7.9 Hz, 1H,
Ar), 7.16 (d, J = 8.5 Hz, 1H, Ar), 7.10 (t, J = 7.4 Hz, 1H, Ar), 6.94 (d,
J = 8.0 Hz, 1H, Ar), 6.90 (d, J = 8.5 Hz, 1H, Ar), 6.39 (bs, 1H,
NHCON), 4.49 (t, J = 5.7 Hz, 2H, OCH2), 3.64 (t, J = 5.7 Hz, 2H,
OCH2CH2), 3.48 (dd, J = 9.0, 6.6 Hz, 2H, CH2CH2NHCON), 3.21
(t, J = 7.9 Hz, 2H, CH2CH2NHCON), 3.15−3.06 (m, 4H,
Hz, 4H, CH2CH2NHCON), 3.25 (t,
J = 7.9 Hz, 4H,
+
CH2CH2NHCON), 2.90−2.80 (m, 4H, CH2NH3 ), 2.78 (t, J = 7.3
Hz, 4H, TrizCH2), 1.76 (quin, J = 7.4 Hz, 4H, TrizCH2CH2), 1.64
+
(quin, J = 7.7 Hz, 4H, CH2CH2NH3 ); 13C NMR (DMSO-d6, 100
MHz) δ (ppm): 162.1 (NHCON), 160.2 (OCAr), 158.0
(CF3COO−), 155.9 (CAr), 153.3 (CAr), 147.7 (CAr), 138.2 (CAr),
134.7 (CHAr), 129.4 (CHAr), 126.0 (CHAr), 123.1 (CAr), 121.2 (CAr),
120.5 (CHAr), 116.5 (CHAr), 112.1 (CHAr), 104.3 (CHAr), 67.6
(OCH2), 45.3 (CH2CH2NHCON), 42.4 (OCH2CH2), 38.6
+
ThiazCH2CH2), 2.85 (t, J = 7.5 Hz, 2H, CH2NH3 ), 2.78 (t, J =
7.2 Hz, 2H, TrizCH2), 1.75 (quin, J = 7.4 Hz, 2H, TrizCH2CH2),
+
1.64 (quin, J = 7.0 Hz, 2H, CH2CH2NH3 ); 13C NMR (DMSO-d6,
100 MHz) δ (ppm): 162.1 (NHCON), 157.3 (CAr), 157.1 (CAr),
155.6 (OCAr), 154.6 (CAr), 150.0 (CAr), 147.7 (CAr), 140.7 (CAr),
138.1 (CAr), 137.9 (CAr), 136.6 (CAr), 133.4 (CAr), 131.4 (CHAr),
129.9 (CHAr), 129.8 (CHAr), 129.0 (CHAr), 129.0 (CHAr), 128.9
(CHAr), 128.9 (CHAr), 128.9 (CHAr), 128.0 (CHAr), 126.6 (CHAr),
126.5 (CHAr), 123.1 (CHAr), 121.3 (CAr), 118.8 (CHAr), 118.1
(CHAr), 112.1 (CHAr), 104.3 (CHAr), 67.4 (OCH2), 45.2
(CH2CH2NHCON), 42.4 (OCH2CH2), 38.7 (CH2NH3 ), 37.5
( C H 2 C H 2 N H C O N ) , 3 4 . 6 ( C H 2 C H 2 P h O P h ) , 3 1 . 2
(CH2CH2PhOPh), 26.5 (CH2CH2NH3 ), 25.6 (TrizCH2CH2), 24.4
(TrizCH2); HPLC (gradient A, Agilent): Rt = 9.7 min; HRMS (ES,
positive mode) m/z: calcd for C40H41N7O3S 699.2992; found
699.2993 (0.17 ppm); anal. calcd for C40H42N7O3S·TFA: C, 61.98;
H, 5.20; N, 12.05; S, 3.94; found: C, 62.23; H, 5.34; N, 11.68; S, 3.50.
1-(3-(2-Bromo)acetylphenyl)-2-bromoethan-1-one (20). Follow-
ing the general procedure described for the synthesis α-bromome-
thylketones 17a−e, p-toluensulfonic acid (761 mg, 4.00 mmol) and
NBS (713 mg, 4.00 mmol) were successively added to a solution of
commercially available diacetyl benzene (250 mg, 1.54 mmol) in
CH3CN (20 mL) and stirred at room temperature overnight. After
the workup, the residue was purified by flash column chromatography
(hexane/EtOAc, 90:10) to give 20 (366 mg, 74%) as a white solid.51
m.p.: 87−88 °C; 1H NMR (CDCl3, 400 MHz) δ (ppm): 8.55 (t, J =
1.8 Hz, 1H, Ar), 7.20 (dd, J = 7.8, 1.8 Hz, 2H, Ar), 7.64 (t, J = 7.8 Hz,
1H, Ar), 4.78 (s, 4H, BrCH2CO).
+
+
(CH2NH3 ), 37.6 (CH2CH2NHCON), 26.5 (CH2CH2NH3 ), 25.6
(TrizCH2CH2), 24.4 (TrizCH2); HPLC (gradient A, Agilent): Rt =
6.4 min; HRMS (ES, positive mode) m/z: calcd for C46H52N14O4S2
928.3737; found 928.3740 (0.27 ppm); anal. calcd for
C46H52N14O4S2.2TFA: C, 51.90; H, 4.70; N, 16.95; S, 5.54; found:
C, 51.38; H, 5.16; N, 16.98; S, 5.83.
+
Biological Methods. Chemical Compounds. For LiTryR
oxidoreductase activity and dimer quantitation assays, stock solutions
of synthesized compounds were prepared in anhydrous DMSO at 5
mM. For the leishmanicidal and cytotoxicity assays, stock solutions of
synthesized compounds were prepared in anhydrous DMSO at 15
mM. All reagents for the LiTryR activity assay were obtained from
Sigma-Aldrich (St. Louis, MO) except for TS2, which was purchased
from Bachem (Bubendorf, Switzerland).
+
LiTryR Purification. Recombinant L. infantum TryR (LiTryR) HIS-
tagged and HIS-FLAG-tagged versions were used for all LiTryR
oxidoreductase activity and dimer quantitation assays. These LiTryR
versions were purified from Escherichia coli as previously described.25
Briefly, pRSETA-HIS-LiTryR construct alone or in combination with
the pET24a-FLAG-LiTryR construct was transformed into BL21
(DE3) Rossetta E. coli strain. An overnight E. coli culture grown at 37
°C in Luria−Bertani (LB) medium with the appropriate antibiotics
and vigorous shaking was diluted (1:100) in the same medium and
allowed to grow in the same conditions until the OD600 was 0.5. Then,
LiTryR expression was induced by addition of 1 mM isopropyl-β-D-1-
thiogalactopyranoside (IPTG) for 16 h at 26 °C. The cells were
centrifuged for 5 min at 9000g and 4 °C and the wet pellet
resuspended in lysis buffer (50 mM Tris, pH 7, 300 mM NaCl, 25
mM imidazole, protease inhibitor cocktail, and 1 mg/mL lysozyme).
Following a 30 min incubation on ice, the cell lysate was sonicated on
wet ice (50% pulses, potency 7) for 30 min using a Sonifier Cell
Disruptor B15 (Branson, Danbury, CT) and centrifuged for 1 h at
50 000g and 4 °C. The supernatant was sonicated again as previously
described for 10 min and loaded on a HisTrap column (GE
Healthcare, Chicago, IL) for 16 h at 4 °C using a P-1 peristaltic pump
(GE Healthcare, Chicago, IL). Once loaded, the HisTrap column was
Dibenzyl-((((1,3-phenylenebis(thiazole-4,2-diyl))bis(3-(2-(2-oxoi-
midazolidin-1-yl)ethoxy)-4,1-phenylene))bis(1H-1,2,3-triazole-1,4-
diyl))bis(butane-4,1-diyl))dicarbamate (21). Following the general
procedure of Hantzsch synthesis, 9 (100 mg, 0.19 mmol) was reacted
i
with α-bromoketone 20 (30 mg, 0.09 mmol) in PrOH (15 mL).
After the workup, the residue was purified by CCTLC on the
Chromatotron (CH2Cl2/MeOH, 96:4) to give 21 (68 mg, 30%) as a
pink oil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 8.75 (s, 2H, Ar),
8.68 (d, J = 8.5 Hz, 2H, Ar), 8.37 (s, 2H, Ar), 8.12 (dd, J = 7.6, 1.7
Hz, 2H, Ar), 7.79 (d, J = 2.0 Hz, 2H, Ar), 7.75 (dd, J = 8.7, 1.8 Hz,
2H, Ar), 7.60 (t, J = 7.7 Hz, 1H, Ar), 7.46−7.10 (m, 12H, Ar,
NHCbz), 6.42 (s, 2H, NHCON), 5.01 (s, 4H, NHCOOCH2), 4.52
(t, J = 5.6 Hz, 4H, OCH2), 3.69 (t, J = 5.6 Hz, 4H, OCH2CH2), 3.53
̈
connected to an AKTA purifier UPC 10 (GE Healthcare, Chicago,
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Article
IL) and washed using a 5−10% gradient between buffer A (50 mM
Tris, pH 7 and 300 mM NaCl) and B (50 mM Tris, pH 7, 300 mM
NaCl, and 500 mM imidazole). LiTryR was eluted using a 40%
gradient between buffers A and B. Fractions containing recombinant
LiTryR were pooled and loaded into a HiPrep 26/10 Desalting
column (GE Healthcare, Chicago, IL) previously equilibrated with
buffer A. Finally, LiTryR was concentrated to 2 mg/mL using an
Amicon Ultra-15 50K (Merck Millipore, Burlington, MA) and an
equal volume of glycerol was added before storing at −20 °C.
TbTryR and TcoTryR Purification. The DNA coding sequences for
Homo sapiens GR, T. brucei TryR, and T. congolense TryR were
polymerase chain reaction (PCR)-amplified and cloned in the
pRSETA plasmid. The DNA coding for hGR (transcript variant 1;
NCBI Reference Sequence: NM_000637.5) was purchased from
GeneScript. Genomic DNA from T. congolense parasites was kindly
provided by Dr. Stefan Magez (University of Vrije, Brussels).
with agitation in a humid atmosphere. The plates were washed five
times with TTBS buffer (2 mM Tris, pH 7.6, 138 mM NaCl, and
0.1% Tween-20) and incubated with diluted monoclonal α-HIS
horseradish peroxidase (HRP)-conjugated antibody (200 μL,
1:50 000, Abcam, Cambridge, U.K.) in fatty acid- and essentially
globulin-free bovine serum albumin (BSA) (5%) prepared in TTBS
buffer for 1 h at room temperature. The plates were washed five times,
and o-phenylenediamine dihydrochloride (OPD) substrate prepared
according to the manufacturer’s instructions was added. The
enzymatic reaction was stopped after 5 min with H2SO4 (100 μL,
0.5 μM), and the absorbances were measured at 490 nm in an
EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA). All of
the assays were conducted in triplicate in at least three independent
experiments. Data were analyzed using a nonlinear regression model
with GraFit 6 software (Erithacus, Horley, Surrey, U.K.).
Determination of 19a Inhibitory Constants. The inhibitory
constants Ki and Ki* for 19a were calculated following a slightly
modified version of the standard DTNB-coupled assay.52 TS2 was
serially diluted (sixfold dilution from 1666 to 52 μM) in a buffer
containing 40 mM HEPES (pH 7.5) and 1 mM EDTA. The different
TS2 solutions were dispensed (15 μL) in a 96-well microplate. 19a
was serially diluted in DMSO (sixfold dilution from 1875 to 445 μM).
The different aliquots of 19a and the equivalent amount of the vehicle
(DMSO) were added to a preassay mixture, yielding different
mixtures containing 40 mM HEPES (pH 7.5), 1 mM EDTA, 416.67
μM NADPH, 208.33 μM DTNB, 1.45% DMSO, and different 19a
concentrations ranging from 6.2 to 26 μM. These mixtures (180 μL)
were subsequently added to the appropriate wells previously filled
with different TS2 solutions. The assay was initiated by addition of 55
μL of buffer containing 40 mM HEPES (pH 7.5), 1 mM EDTA,
272.73 μM NADP+, 0.01% glycerol, and 3.5 nM recombinant LiTryR
using an automated dispensing system (PerkinElmer, Waltham, MA).
The order of addition was essential to avoid enzyme preincubation
with the inhibitor or the substrates. The final 250 μL assay contained
40 mM HEPES buffer (pH 7.5) 1 mM EDTA, 300 μM NADPH, 60
μM NADP+, 150 μM DTNB, 6.25−50 μM TS2, 1.04% DMSO,
0.002% glycerol, and 0.8 nM recombinant LiTryR. TryR oxidor-
eductase activity was monitored at 26 °C by an increase in absorbance
at 412 nm in an EnSpire Multimode Plate Reader (PerkinElmer,
Waltham, MA). Coupling an automated dispenser module to the
spectrophotometer enabled real-time detection of the enzymatic
reaction, which was crucial for high-quality nonlinear fits of the
experimental data and, especially, to estimate the initial velocity of the
progress curves in the presence of 19a. To guarantee linearity of the
enzyme reaction in the absence of an inhibitor, the analysis was
performed with data obtained during the first 20 000 s to avoid TS2
depletion. Under this condition, all uninhibited reactions followed
straight lines until the generation of 230 μM TNB (Figure S10). This
product concentration was never reached in the inhibited reactions
during the first 20 000 s. All of the assays were conducted in three
independent experiments.
Genomic DNA from the T. brucei S16 cell line was kindly provided
́
́
́
by Jose M. Perez-Victoria. Instituto de Parasitologia y Biomedicina
́
“LopezNeyra” CSIC. HIS-tagged recombinant T. brucei and T.
congolense TryRs (TbTryR and TcoTryR, respectively) were purified
from E. coli as already explained for LiTryR.
TryR Oxidoreductase Activity. TryR oxidoreductase activity was
determined spectrophotometrically using a modified version of the
DTNB-coupled assay described by Hamilton et al.52 Briefly, reactions
were carried out at 26 °C in 250 μL of N-(2-hydroxyethyl)piperazine-
N′-ethanesulfonic acid (HEPES) buffer (pH 7.5, 40 mM) containing
ethylenediaminetetraacetic acid (EDTA) (1 mM), NADP+ (30 μM),
DTNB (25 μM), TS2 (1 μM), NADPH (150 μM), glycerol (0.02%),
DMSO (1.75%), and recombinant TryR (7 nM). DTNB, glycerol,
and DMSO concentrations used in this assay do not have any relevant
effect on the kinetics of the enzymatic reaction. Reactions were started
by addition of a mixture of NADPH and TS2.
TryR oxidoreductase activity was monitored at 26 °C by an
increase in absorbance at 412 nm in an EnSpire Multimode Plate
Reader (PerkinElmer, Waltham, MA). 2-Nitro-5-meta mercaptoben-
zoic acid (TNB) concentration was obtained by multiplying the
absorbance values by 100 (50 μM TNB generates 0.5 arbitrary units
of absorbance at 412 nm). In this DTNB-coupled assay, one molecule
of T(SH)2 reduces one molecule of DTNB, producing two TNB
molecules. All of the assays were conducted in at least three
independent experiments. Data were analyzed using a nonlinear
regression model with GraFit 6 software (Erithacus, Horley, Surrey,
U.K.).
hGR Oxidoreductase Activity. hGR oxidoreductase activity was
determined spectrophotometrically using a DTNB-coupled assay.
Briefly, reactions were carried out at 37 °C in 250 μL of HEPES buffer
(pH 7.5, 40 mM) containing EDTA (1 mM), NADP+, (60 μM),
DTNB (150 μM), oxidized glutathione (50 μM), NADPH (300 μM),
glycerol (0.02%), DMSO (1.75%), and recombinant hGR (7 nM).
DTNB, glycerol, and DMSO concentrations used in this assay do not
have any relevant effect on the kinetics of the enzymatic reaction.
hGR oxidoreductase activity was monitored at 37 °C by an increase in
absorbance at 412 nm using an EnSpire Multimode Plate Reader
(PerkinElmer, Waltham, MA). TNB concentration was obtained by
multiplying the absorbance values by 100 (50 μM TNB generates 0.5
arbitrary units of absorbance at 412 nm). All of the assays were
conducted in at least three independent experiments. Data were
analyzed using a nonlinear regression model with GraFit 6 software
(Erithacus, Horley, Surrey, U.K.).
Dimer Quantitation Assay. The stability of the LiTryR dimeric
form in the presence of 1,2,3-triazole-phenyl-thiazole-based com-
pounds was evaluated using the novel enzyme-linked immunosorbent
assay (ELISA) developed in our laboratory.25 Briefly, HIS-FLAG-
tagged LiTryR (400 nM) was incubated in dimerization buffer (1 mL
of 300 mM NaCl, 50 mM Tris, pH 8.0) for 16 h at 37 °C with
agitation and in a humid atmosphere in the presence of the different
compounds ranging from 75 to 3.12 μM. Next, the different solutions
were centrifuged at 18 000g for 15 min at room temperature. The
supernatants (200 μL/well) were added to the α-FLAG-coated plates
(Sigma-Aldrich, St. Louis, MO) and incubated for 30 min at 37 °C
Data and Statistical Analysis. GraFit 6.0 software (Erithacus,
Horley, SRY, U.K.) was used to perform linear and nonlinear
regressions. All experiments were undertaken in triplicate to ensure
the reliability of single values.
kobs values were estimated for every 19a concentration at all TS2
concentrations by fitting the values of the TNB concentration
produced during each enzymatic reaction to eq 1.42b,53
vi − v
kobs
[P] = vt +
s [1 − exp(−kobst)] + d
s
(1)
where vi and vs are the initial and the steady-state velocities,
respectively, kobs is the apparent first-order rate constant for the
conversion of vi into the steady-state velocity (vs), and d is a
parameter that indicates the displacement of the curve on the vertical
ordinate and takes into account any nonzero value of the measured
signal at time zero caused by some of the reagents. The apparent
inhibition constants (Kiapp and Ki*app) for each TS2 concentration
were determined by fitting the kobs values obtained at different 19a
concentrations to eq 2
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Article
Ä
É
Å
Ñ
Å
Ñ
Human Cell Line Culture. THP-1 cells were grown in RPMI-1640
medium (Gibco, Leiden, The Netherlands) supplemented with 10%
heat-inactivated FCS, antibiotics, 10 mM HEPES, 2 mM glutamine,
and 1 mM sodium pyruvate, pH 7.2, at 37 °C and 5% CO2.
Liver hepatocellular carcinoma HepG2 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St.
Louis, MO) with 10% heat-inactivated FCS, antibiotics, and 10 mM
HEPES.
Determination of Cellular Toxicity. Drug treatment of liver
hepatocellular carcinoma HepG2 cells was performed at a
concentration of 105 cells/mL at 37 °C and 5% CO2 for 24 h. EC50
was evaluated by the crystal violet assay. Briefly, cells were washed
with phosphate-buffered saline (PBS) and stained with 200 μL of
crystal violet solution (0.2% crystal violet, 2% ethanol) for 10 min at
room temperature. Next, the plates were washed twice with tap water
and allowed to dry. The stained cells were solubilized with 400 μL of
1% sodium dodecyl sulfate (SDS), and the color intensity was
quantified at 570 nm using an EnSpire Multimode Plate Reader
(PerkinElmer, Waltham, MA). All of the assays were conducted in
triplicate in at least three independent experiments. Data were
analyzed using a nonlinear regression model with GraFit 6 software
(Erithacus, Horley, Surrey, U.K.).
[I]
Å
Ñ
Å
Ñ
Å1 +
Ñ
Ñ
Å
Ki*app
Å
Å
Ñ
Ñ
Å
Å
Å
Å
Ñ
Ñ
Ñ
Ñ
kobs = k
6
Å
Å
Ñ
Ñ
[I]
Å
Ñ
Å
Ñ
Å1 +
Ñ
Ñ
Ñ
Ñ
Å
Å
Å
Kiapp
(2)
Å
Ç
Ñ
Ö
where Kiapp is the apparent value of the Ki for the initial encounter
complex (EI) and Ki*app is the apparent value of the overall inhibition
constant Ki* to form a higher-affinity complex (E*I). k6 is the first-
order rate constant that defines the reverse isomerization step that
returns E*I to EI.42a,53
The Ki value for the formation of the EI complex was determined
by fitting the Kiapp values obtained at different TS2 concentrations to
eq 3. In addition, this graph was used to assess the inhibition modality
of 19a during the first equilibrium of its two-step binding mechanism.
The value obtained for α defines the mode of inhibition (competitive,
α → infinite; uncompetitive, α → 0; pure noncompetitive, α = 1;
mixed, 1 < α < 10).42a
[S] + Km
Kiapp
=
Km
Ki
[S]
αKi
+
(3)
In Vitro Infection of THP-1-Derived Macrophages with GFP-
Expressing L. infantum Axenic Amastigotes. THP-1 monocytes
were seeded in a 48-well plate at a concentration of 5 × 105 cells/mL
(250 μL/well) in RPMI complete medium supplemented with
phorbol 12-myristate 13-acetate (PMA) (100 ng/mL). Differentiation
of THP-1 monocytes to macrophages was allowed for 24 h at 37 °C
and 5% CO2. THP-1-derived macrophages were infected with GFP-
expressing axenic amastigotes at 5 × 106 parasites/mL in RPMI
complete medium for 24 h (250 μL/well). Extracellular amastigotes
were washed twice with PBS. The remaining extracellular parasites
were challenged for 24 h with THP-1 medium supplemented with
10% horse serum (Gibco) instead of FCS. Cells were washed twice
with PBS, and treatments were performed in 250 μL of fresh RPMI
medium supplemented with 10% horse serum for 72 h at 37 °C and
5% CO2. Afterward, treatments were removed by two PBS washes.
Lysis of infected cells was performed with 120 μL of SDS 0.005% (w/
v) in PBS. After a 30 min incubation at 37 °C, lysis was stopped by
adding RPMI complete medium with PI at 20 μg/mL (120 μL/well).
The lysates were transferred to a 96-well plate (200 μL/well), and the
GFP+ amastigotes extracted from the infected cells were acquired by a
Beckman Coulter FC500 flow cytometer. Each sample (100 μL) was
acquired for 20 s.
The overall inhibition constant Ki* value was determined by fitting
the Ki*app values obtained at different TS2 concentrations to eq 4. In
addition, this graph was used to assess the overall inhibition modality
of 19a during its two-step binding mechanism. As explained above,
the value obtained for α defines the mode of inhibition.42a
[S] + Km
Ki*app
=
Km
Ki
[S]
+
*
αKi
(4)
Leishmania Cell Lines and Culture. L. infantum promastigotes
(MCAN/ES/89/IPZ229/1/89) were grown in Roswell Park
Memorial Institute (RPMI)-1640 medium (Sigma-Aldrich, St.
Louis, MO) supplemented with 10% heat-inactivated fetal calf
serum (FCS), antibiotics, and 25 mM HEPES (pH 7.2) at 26 °C.
L. infantum axenic amastigotes (MCAN/ES/89/IPZ229/1/89) were
grown in M199 medium (Invitrogen, Leiden, The Netherlands)
supplemented with 10% heat-inactivated FCS, 1 g/L β-alanine, 100
mg/L L-asparagine, 200 mg/L sucrose, 50 mg/L sodium pyruvate, 320
mg/L malic acid, 40 mg/L fumaric acid, 70 mg/L succinic acid, 200
mg/L α-ketoglutaric acid, 300 mg/L citric acid, 1.1 g/L sodium
bicarbonate, 5 g/L morpholineethanesulfonic acid (MES), 0.4 mg/L
hemin, and 10 mg/L gentamicine, pH 5.4, at 37 °C.
Cytotoxicity of THP-1 infected macrophages was determined using
an in-house developed lactate dehydrogenase (LDH) activity assay.
LDH activity assays from the remaining cell lysates (100 μL/well)
were carried out at 37 °C in 250 μL of Tris/HCl buffer (pH 8.9, 50
mM) containing DL-lactate (840 mM), NAD+ (450 μM), 1-
methoxyphenazine methosulfate (45 μM), and nitro blue tetrazolium
chloride (40 μM). The color intensity of the generated formazan dye
was monitored at 570 nm using an EnSpire Multimode Plate Reader
(PerkinElmer, Waltham, MA). CC50 values were calculated using a
nonlinear regression model with GraFit 6 software (Erithacus, Horley,
Surrey, U.K.).
Axenization of L. infantum Promastigotes. Axenization was
performed by diluting 0.5 mL of a 7-day stationary phase culture of L.
infantum promastigotes (2−3 × 107 parasites/mL) in 4.5 mL of
amastigotes medium and incubating the culture at 37 °C for 2 or 3
days. L. infantum axenic amastigotes apparition was followed by
phase-contrast microscopy using an Eclipse Ti inverted microscope
(Nikon, Tokyo, Japan).
Leishmanicidal Activity. Drug treatment of L. infantum promas-
tigotes was performed during the logarithmic growth phase at a
concentration of 2 × 106 parasites/mL at 26 °C for 24 h. Drug
treatment of L. infantum axenic amastigotes was performed during the
logarithmic growth phase at a concentration of 1 × 106 parasites/mL
at 37 °C for 24 h. EC50 was evaluated by flow cytometry by the
propidium iodide (PI) exclusion method. After selection of the
parasite population based on their forward scatter (FSC) and side
scatter (SSC) values, live and dead parasite cells were identified by
their permeability to PI. All of the assays were conducted in triplicate
in at least three independent experiments. Data were analyzed using a
nonlinear regression model with GraFit 6 software (Erithacus, Horley,
Surrey, U.K.).
The relative number of GFP+ intracellular amastigotes per cell in
each sample was estimated after the division of the total number of
GFP+ amastigotes by the LDH activity value of the sample. Results
were normalized to the vehicle-treated controls. EC50 values of
intracellular amastigotes were calculated using a nonlinear regression
model with GraFit 6 software (Erithacus, Horley, Surrey, U.K.). All of
the assays were conducted in at least three independent experiments.
Screening for Pan-Assay Interference Compounds (PAINS) and
Aggregation. Screening of all tested compounds for pan-assay
interference compounds (PAINS) and aggregation via public tools
(http://zinc15.docking.org/patterns/home/ and https://www.cbli-
Fluorescence Microscopy. Logarithmic growth phase promasti-
gotes were treated with 25 μM 12a for 1 h at 26 °C. Parasites were
fixed using a solution containing EDTA (450 mM) and mounted on
polylysine slides (Thermo Scientific, Waltham, MA). Images were
captured with an Eclipse Ti inverted microscope (Nikon, Tokyo,
Japan).
gand.org/PAINS/) gave no hits. Dose−response curves for all
experiments and compounds were recorded. None of the curves
showed Hill slopes that could be a hint for PAINS (curves for
representative compounds 12b and 12c are presented in Figure S14,
6157
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J. Med. Chem. 2021, 64, 6137−6160

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Article
Supporting information). Counter-screening against related (gluta-
thione reductase; GR) and unrelated (superoxide dismutase) targets
discarded any nonspecific activity of the compounds. Reaction curves
for TryR and GR in the presence of compounds 12b and 12c are
shown in Figure S15. Nifurtimox is included as a well-characterized
inhibitor of GR. SAR, considered the most relevant criterion that
distinguishes a PAIN from a non-PAIN, is deeply discussed in the
manuscript.54
Computational Methods. Automated Ligand Docking. A
three-dimensional cubic grid consisting of 65 × 65 × 65 points
with a spacing of 0.375 Å centered midway between the two active
sites of LiTryR was defined for ligand docking. Electrostatic,
desolvation, and affinity maps for the atom types present in the
ligands of this series were calculated using AutoGrid 4.2.6. Then, the
Lamarckian genetic algorithm implemented in AutoDock449 was used
to generate up to 100 feasible binding poses of the ligands studied.
The best poses were selected on the basis of results from intra- and
intermolecular energy evaluations.
Molecular Dynamics Simulations. The leaprcff14SB AMBER
force field and the graphics processing unit (GPU)-based
implementation of the pmemd.cuda module of Amber16 in the
single-precision−fixed-precision (SPFP) mode were used, as
described before.30 The molecular dynamics trajectories were
analyzed using the cpptraj module of AmberTools18, and the binding
energy analysis was carried out with our in-house tool MM-ISMSA.55
The molecular graphics program PyMOL56 was employed for
molecular editing, visualization, and figure preparation.
Henares, Madrid, Spain; orcid.org/0000-0001-8415-
870X
́
Federico Gago − Area de Farmacología, Departamento de
Ciencias Biomédicas, Unidad Asociada al IQM-CSIC,
Universidad de Alcalá, E-28805 Alcalá de Henares, Madrid,
Spain; orcid.org/0000-0002-3071-4878
María-José Camarasa − Instituto de Química Médica (IQM-
CSIC), E-28006 Madrid, Spain; orcid.org/0000-0002-
4978-6468
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00206
Author Contributions
∥A.R. and H.d.L. contributed equally to this work.
Author Contributions
F.G., M.-J.C., A.J.-R., and S.V.: participated in research design.
A.R.: carried out the synthesis. H.d.L. and J.C.G.-S.: performed
biological evaluations. A.R., P.A.S.-M., and F.G.: conducted
computational studies. F.G., A.J.-R., and S.V.: performed data
analysis and supervised experiments. A.R., H.d.L., F.G., A.J.-R.,
M.-J.C., and S.V.: wrote or contributed to the writing of the
manuscript. All authors have given approval to the final version
of the manuscript.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00206.
■
sı
ACKNOWLEDGMENTS
■
This work has been supported by the Spanish MICINN
(Projects PID2019-104070RB-C21 and PID2019-104070RB-
C22), the Spanish Agencia Estatal Consejo Superior de
Investigaciones Científicas (CSIC, Projects CSIC-PIE-
201980E100 and CSIC-PIE-201980E028), and the Comuni-
dad de Madrid (PLATESA2-CM ref S-2018/BAA-4370). The
Spanish MEC is also acknowledged for FPU grants to A.R. and
J.C.G.-S.
Additional synthesis; biological and computational
assays; screening of representative compounds for
1
PAINS; H NMR, 13C NMR, and HRMS spectra and
HPLC chromatograms for all of the target compounds
(PDF)
Molecular formula strings of the prepared compounds
and biological data (CSV)
ABBREVIATIONS
■
Movie of LiTryR-19a complex (Movie S1) (AVI)
CCTLC, centrifugal circular thin-layer chromatography;
CuAAC, 1,3-dipolar copper(I)-catalyzed azide−alkyne cyclo-
addition; EC50a, half-maximal effective concentration in
AUTHOR INFORMATION
Corresponding Authors
■
amastigotes; EC50p, half-maximal effective concentration in
promastigotes; HepG2, liver hepatocellular carcinoma; IC50
act
Antonio Jiménez-Ruiz − Departamento de Biología de
Sistemas, Universidad de Alcalá, E-28805 Alcalá de Henares,
Madrid, Spain; Phone: (+34) 918855109;
Email: antonio.jimenez@uah.es
Sonsoles Velázquez − Instituto de Química Médica (IQM-
CSIC), E-28006 Madrid, Spain; orcid.org/0000-0003-
2209-1751; Phone: (+34) 912587689; Email: iqmsv29@
iqm.csic.es
,
half-maximum inhibitory concentration in the activity assay;
IC50dim, half-maximum inhibitory concentration in the
dimerization assay; GR, glutathione reductase; LiTryR,
Leishmania infantum trypanothione reductase; LDH, lactate
dehydrogenase; PI, propidium iodide; RuAAC, ruthenium-
catalyzed azide−alkyne cycloaddition; SIa, selectivity index in
amastigotes; SIp, selectivity index in promastigotes; TbTryR,
Trypanosoma brucei trypanothione reductase; TcoTryR,
Trypanosoma congolense trypanothione reductase; Try/TryR,
thypanothione/trypanothione reductase; TS2, trypanothione
disulfide; VL, visceral leishmaniasis
Authors
Alejandro Revuelto − Instituto de Química Médica (IQM-
CSIC), E-28006 Madrid, Spain
Héctor de Lucio − Departamento de Biología de Sistemas,
Universidad de Alcalá, E-28805 Alcalá de Henares, Madrid,
Spain; orcid.org/0000-0002-9840-0779
Juan Carlos García-Soriano − Departamento de Biología de
Sistemas, Universidad de Alcalá, E-28805 Alcalá de Henares,
Madrid, Spain
Pedro A. Sánchez-Murcia − Area de Farmacología,
Departamento de Ciencias Biomédicas, Unidad Asociada al
IQM-CSIC, Universidad de Alcalá, E-28805 Alcalá de
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