Structural investigation of anti-trypanosoma cruzi 2-iminothiazolidin-4- ones allows the identification of agents with efficacy in infected mice

Article abstract of DOI:10.1021/jm301518v

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

Full text of DOI:10.1021/jm301518v

Article
pubs.acs.org/jmc
Structural Investigation of Anti-Trypanosoma cruzi
2‑Iminothiazolidin-4-ones Allows the Identification
of Agents with Efficacy in Infected Mice
Diogo Rodrigo Magalhaes Moreira,*,†,‡ Salvana Priscylla Manso Costa,‡ Marcelo Zaldini Hernandes,‡
Marcelo Montenegro Rabello,‡ Gevanio Bezerra de Oliveira Filho,‡ Cristiane Moutinho Lagos de Melo,§
Lucas Ferreira da Rocha,§ Carlos Alberto de Simone,∥ Rafaela Salgado Ferreira,+
Jordana Rodrigues Barbosa Fradico,+ Cassio Santana Meira,⊥ Elisalva Teixeira Guimaraes,⊥,#
́
̃
Rajendra Mohan Srivastava,† Valeria Rego Alves Pereira,§ Milena Botelho Pereira Soares,⊥,⧫
́
̂
and Ana Cristina Lima Leite‡
†Departamento de Química Fundamental, Centro de Cien
50670-901, Recife, PE, Brazil
‡Departamento de Cien
cias Farmaceu
Recife, PE, Brazil
§Centro de Pesquisas Aggeu Magalhaes, Fundaca
̂
cias Exatas and da Natureza, Universidade Federal de Pernambuco,
̂
̂
ticas, Centro de Cien
̂
cias da Saude, Universidade Federal de Pernambuco, 50740-520,
́
̧
o Oswaldo Cruz, CEP, 50670-420, Salvador-PE, Brazil
̃
∥Departamento de Física and Informat
+Departamento de Bioquímica and Imunologia, Universidade Federal de Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil
#Departamento de Cien
cias da Vida, Universidade Estadual da Bahia, CEP 41150-000, Salvador, BA, Brazil
⊥Centro de Pesquisas Gonca
́
ica, Instituto de Física, Universidade de Sao Paulo, CEP 13560-970, Sao Carlos, SP, Brazil
̃ ̃
̂
̧
lo Moniz, Fundaca̧ o Oswaldo Cruz, CEP 40296-710, Salvador, BA, Brazil
̃
⧫Centro de Biotecnologia and Terapia Celular, Hospital Sao Rafael, CEP 41253-190, Salvador, BA, Brazil
̃
S
* Supporting Information
ABSTRACT: We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6−24. Compounds with a
phenyl at position N3, 15−19, 22−24, exhibited better inhibitory properties for cruzain and against the parasite than
2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which
inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for
splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine
if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26−45
and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired
when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.
although a clinical trial (BENEFIT) aiming to investigate this
is expected to be concluded in 2013.4,5 However, many patients
experience drug intolerance and adverse effects for Bdz.6 Thus,
there is a need to look for other drugs to treat Chagas disease.
A useful strategy for anti-T. cruzi drug design is to inhibit the
cysteine protease cruzain. Most of the functional properties of
INTRODUCTION
■
The World Health Organization estimates that 5−10% of Latin
America’s population is afflicted by Chagas disease, which is
caused by the protozoan Trypanosoma cruzi.1 Current treatment is
solely based on benznidazole (Bdz), a nitroimidazole compound.
Bdz is cidal for bloodstream trypomastigotes and thus efficient
in eliminating parasite in circulation during the initial stages of
infection (acute and asymptomatic chronic).2,3 The efficacy of Bdz
for patients with Chagas-related cardiomyopathy is still unknown,
Received: July 25, 2012
© XXXX American Chemical Society
A
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Figure 1. Structural design of new 2-iminothiazolin-4-ones as trypanocidal agents.
cruzain have been revealed in the recent years.7−12 For example,
cruzain prevents macrophage activation, probably by degrading
macrophage proteins such as the nuclear factor NF-κB. Cruzain-
deficient T. cruzi is viable but unable to invade host cells.9 On
the other hand, cruzain protects infected cardiomyocytes from
apoptosis, which is essential for a pathogenic outcome.10 Cruzain
is also responsible for the proteolytic cleavage of kinins that,
once released, trigger bradykinin receptors. These receptors in
cooperation with endothelin receptors are involved in vascular
permeability, which is beneficial for parasite invasion and its
persistence in the tissue.11,12 Given these functional properties,
cruzain is a valid T. cruzi drug target.
Since the discovery that thiosemicarbazones are cruzain
inhibitors,13−16 many attempts have been made to explore the
structure−activity relationships (SAR) with a view to designing
compounds with improved trypanocidal effects.17−21 Recently,
potent triazole-based inhibitors that covalently entrap cruzain
have been identified. The triazole family of inhibitors bind to the
amino acid CYS25 and establish polar interactions with amino
acids GLY66 and GLN19 in this binding pocket to stabilize
the cruzain−ligand complex.22,23 Similar interactions have been
observed for other cruzain inhibitors.24 Some variants of this
family of triazole inhibitors are cidal for the parasite. One good
example is the aryloxymethyl ketone triazole 54, which eradicates
blood parasitemia when administered to T. cruzi-infected mice
intraperitoneally at 20 mg/kg.23 The results support the feasibi-
lity of employing multiple tactics to inhibit cruzain, which is
essential for T. cruzi infection.
equipotent to Bdz but less potent than their thiosemicarbazone
analogues for cruzain and parasite inhibition assays. However, we
also observed that 2-iminothiazolidin-4-ones had low toxicity for
mammalian cells (mouse splenocytes), a feature not generally
observed in thiosemicarbazones.25−27
For the design of new anti-T. cruzi 2-iminothiazolidin-4-ones,
we combined some chemical features of cruzain inhibitor 54 and
of our previously tested 2-iminothiazolidin-4-ones (compounds
1 and 2, Figure 1). All these newly designed 2-iminothiazolidin-
4-ones share an aryloxypropylimine group. This was introduced
because of the similarity, in terms of geometry and flexibility, to
the aryloxymethyl ketone group of the cruzain inhibitor 54. Our
previous model for the docking of 2-iminothiazolidin-4-ones
with cruzain suggests that the thiazolidinic ring establishes
both polar and hydrophobic interactions with cruzain. As a way
of locating the structural determinants of cruzain affinity, we
derived a series of 2-iminothiazolidin-4-ones 5−24 by varying
substituents around the thiazolidinic ring at positions N3,
C4, and C5. Bearing in mind the bioisosteric relationship
between 2-imino-1,3-thiazoles and 2-iminothiazolidin-4-ones,28
we prepared the 2-imino-1,3-thiazoles 26−45. Therefore, we
synthesized 2-iminothiazolidin-4-ones 5−19, their analogues
2-iminothiazolidin-4-thiones 20 and 21, and 2-imino-1,3-
thiazoles 26−45 and evaluated their activity against cruzain
and the parasite.
RESULTS
■
Chemistry. The thiosemicarbazones 4a−c were prepared
by reacting commercially available phenoxy-2-propanone 1 with
the appropriate thiosemicarbazide in an ultrasound bath in the
presence of catalytic AcOH. 2-Iminothiazolidin-4-ones 5−19
We selected 2-iminothiazolidin-4-ones for development as
trypanocidal compounds because of their structural similarity
to cruzain-inhibiting thiosemicarbazones. The most potent
2-iminothiazolidin-4-ones we had previously identified were
B
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a
Scheme 1. Synthetic Procedures for 2-Iminothiazolidin-4-ones 5-19 and 2-Iminothiazolidin-4-thiones 20, 21
a
Reagents and conditions: (A) 2-chloroacetone, K2CO3, butanone, ultrasound bath, rt, 2 h, yield of 68% (B) thiosemicarbazides, EtOH, AcOH,
ultrasound bath, rt, 30 min, yields of 65−82%; (C) ethyl 2-bromoacetate or ethyl 2-substituted-2-bromoacetates; NaOAc, EtOH, reflux, overnight,
yields of 38−84%; (D) Lawesson’s reagent, 1,4-dioxane/toluene, reflux, 20 h, yields of 54% (20) and 60% (21).
a
Scheme 2. General Synthetic Procedures for 2-Imino-1,3-thiazoles 26−45
a
Reagents and conditions: (A) substituted 2-bromoacetophenones, 2-propanol, ultrasound irradiation, rt, 30−60 min, yields of 48−85%.
were prepared by reacting the respective thiosemicarbazones
4a−c with commercially available ethyl 2-bromoacetate or
preparing the desired 2-substituted-2-bromoacetates. These
reactions were carried out in the presence of an excess of
anhydrous NaOAc under reflux, affording compounds 5−19
in acceptable yields (38−84%). For the conversion of
2-iminothiazolidin-4-ones 7 and 19 into 2-iminothiazolidin-4-
thiones 20 and 21, we used Lawesson’s reagent as the thionation
reagent (yields of 54% and 60%, respectively) (Scheme 1).
2-Imino-1,3-thiazoles 26−45 were prepared via a Hantzsch
synthesis between thiosemicarbazones 4a−c and substituted
2-bromoacetophenones (Scheme 2). These reactions proceed
well upon refluxing with ethanol (1−3 h), but we adapted them
under ultrasound conditions29 using propanol as a solvent30 and
observed excellent yields in general (48−85%) and shorter times
(30 min in most cases).
We attempted to define the relative configuration of the iminic
bond. Chemical shifts in the methyl and methylene of the ary-
loxypropylimine group were diagnostically useful for identifying
an E-isomer (for details, see the Experimental Section).
Moreover, we succeeded in crystallizing the 2-iminothiazolidin-
4-one 12 and the thiosemicarbazones 4a and 4c suitable for
X-ray analysis. High-performance chromatography revealed a
mixture of isomers (9.5/0.5) for thiosemicarbazones 4a and 4c,
and the major isomer has an E-geometry. On the other hand,
chromatographic analysis in practice revealed only one major
isomer for compounds 5−24 and 26−45. 2-Iminothiazolidin-4-
one 12 has an E-geometry (Figure 2). It is therefore highly
probable that all 2-iminothiazolidin-4-ones 5−24 as well as
2-imino-1,3-thiazoles 26−45 have the same E-geometry. For com-
pounds 5−9, an important structural assignment is the location
of an iminic bond in C2. X-ray analysis and quantum chemistry
calculations for other 2-iminothiazolidin-4-ones structurally
related to compounds 5−9 had suggested that the iminic bond
is exocyclic in relation to heterocyclic ring (Figure S1, Supporting
Information). It would, therefore, seem fair to suggest that the
structures proposed for 5−9 are the correct ones.
Structure−Activity Relationships (SAR). We first assayed
the viability of BALB/c mouse splenocytes treated with
2-iminothiazolidin-4-ones 5−19 and 22−24, 2-iminothiazoli-
din-4-thiones 20 and 21, or 2-imino-1,3-thiazoles 26−45. Once
the toxicity to mammalian cells had been determined, we focused
on evaluating their effects in T. cruzi in vitro assays. We tested
against epimastigotes (axenic culture) and bloodstream
trypomastigotes of the Y strain (Tables 1, 2, and 4). Compounds
that showed IC50 values comparable to benznidazole (Bdz) or
C
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Figure 2. Structures of compounds 4a and 12 determined by X-ray analysis. Anisotropic thermal ellipsoids are drawn at a 50% level. For details, see the
Experimental Section and Supporting Information.
Table 1. Anti-T. cruzi Activities of 2-Iminothiazolidin-4-ones 5-19 and 2-Iminothiazolidin-4-thiones 20, 21
a
b
Determined 24 h after incubation of trypomastigotes with the compounds. Determined 11 days after incubation of epimastigotes with the
c
compounds. IC50 was calculated from at least five concentrations using concentrations in triplicate (SD 10%). Highest nontoxic concentration for
mouse splenocytes after 24 h of incubation in the presence of the compounds. Bdz: benznidazole; Nfx: nifurtimox; Sap: saponin.
D
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most active compounds. We also evaluated the compounds against
the mammalian proteases cathepsins L and S (Tables 3 and 4).
Of all the tested compounds, 2-iminothiazolidin-4-one 18
possessed the highest overall trypanocidal potency, with IC50
values of 6.1 and 4.9 μM for bloodstream trypomastigotes
and axenic epimastigotes of the Y strain, respectively (Table 1).
It also had trypanocidal properties comparable to those of Bdz.
However, 2-iminothiazolidin-4-one 18 does not affect the cell
viability of mouse splenocytes even at concentrations of up to
100 μg/mL (262 μM).
Table 2. Modification of the 2-Iminothiazolidin-4-one 17
toward Compounds 22−24
a
The SAR study which led to the identification of
2-iminothiazolidin-4-one 18 began with two kinds of modifica-
tions of the thiazolidinic ring: (a) variation of the substituents
at C5; (b) replacement of NH with N-methyl and N-phenyl.
These modifications yielded 2-iminothiazolidin-4-ones 5−19.
For cruzain inhibition, we observed that modifications at N3
affect cruzain inhibition more than those at C5. For example, most
compounds with a free NH (5−9) or an N-methyl (10−14) did
not inhibit cruzain, while compounds with an N-phenyl (15−19)
did (Table 3). For T. cruzi, we observed somewhat similar
behavior, except for compounds 5, 8, and 13 which displayed
good trypanocidal properties. In general, N-phenyl compounds
15−19 were potent trypanocidal agents.
a
See footnote in Table 1. ND, not determined.
We observed interesting SAR when different substituents were
attached at C5 in 2-iminothiazolidin-4-ones 15−19. Inserting
a methyl (16), ethyl (17), or isopropyl (18) at position C5 did
not increase the affinity for cruzain when compared to the
nonsubstituted 2-iminothiazolidin-4-one 15; all of them had
nifurtimox (Nfx) were further selected for assays of in vitro
infection of host cells. The inhibitory activity against cruzain was
measured based on the cleavage of the substrate Z-FR-AMC.31
Compounds were first tested at 20 or 100 μM, and a dose−
response curve was determined and the IC50 calculated for the
Table 3. Protease Inhibition
a
T. cruzi cruzain
IC50 μM (SD)
b
compd
% inhibition at 20 μM
IC50, μM
cathepsin L (% inhibition at 100 μM)
cathepsin L
>100
cathepsin S
5
0
2
1
1.5
>100
NT
6
3
5
0
5
7
ND
ND
NT
8
60.8
63
18.9 0.9
11
2
d
9
2
4
ND
NT
NT
NT
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
0
0
NT
NT
50
81
31.9 1.0
8.0 0.1
>100
>100
>100
>100
NT
0
2
d
27
ND
NT
0
66
50
10.2 0.5
10.8 1.0
9.5 0.07
6.6 0.05
>100
>100
NT
0
c
c
66 (64)
78 (81)
4
1
>100
>100
>100
>100
NT
10
15
89
81
4
d
43
2
ND
2
5
6
c
72 5.0 (79)
7.0 0.4
18.7 2.0
11.8 0.5
8.7 0.1
10.8 0.7
13.8 2.0
NT
48 1.0
65
NT
NT
NT
71
NT
NT
NT
10
2
NT
a
b
After 10 min of preincubation with the inhibitor before adding the fluorescent cruzain substrate Z-FR-AMC. After 45 min of incubation before
adding the fluorescent cathepsin substrate Z-FR-pNA. Percentage of inhibition is the average of triplicate runs determined in one experiment. IC50
c
d
was calculated from at least seven concentrations. In parentheses, experiments using 0.01% of Triton X-100 in the reaction buffer. Not determined
because of the low solubility in 0.1 M sodium acetate pH 5.5. NT is not tested.
E
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Table 4. Cruzain and Anti-T. cruzi Activities of 2-Imino-1,3-thiazoles 26−45
Y strain T. cruzi, IC50 (μM)
a
b
c
d
compd
Ar/R
% of cruzain inhibition at 20 μM
trypomastigotes
19.6
epimastigotes
ND
toxicity splenocytes μg/mL (μM)
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Ph
no inhib
no inhib
no inhib
50 (154)
2-Py
34.8
12.3
3.9
11.8
20.5
4.0
100 (308)
100 (308)
100 (308)
100 (296)
100 (283)
>100 (293)
100 (280)
100 (249)
100 (271)
100 (271)
25 (63.9)
<1 (2.8)
3-Py
e
4-Py
13.3 3.8
4-CH3Ph
4-CH3OPh
4-FPh
no inhib
no inhib
no inhib
49.7
17.8
11.5
67.0
14.9
41.3
57.1
49.8
1.9
4-ClPh
13
25
6
1
ND
ND
5.7
4-BrPh
4-NO2Ph
3-NO2Ph
3,4-diClPh
2,4-diClPh
2,3,4-triClPh
4-tBuPh
βNaph
no inhib
no inhib
3.8
17.0
17.0
4.0
20
40
6
6
19.0
42.2
no inhib
no inhib
no inhib
ND
ND
36.4
ND
ND
83.0
>1 (2.3)
50 (131)
>100 (268)
>100 (319)
50 (128)
e
2-furfuryl
2-thiophene
CH3
4.7 3.2
17.7
37.6
26.6
16.6
25.6
2.6
e
30
6
no inhib
no inhib
174.7
61.1
10 (29.5)
>100 (250)
Ph
a
b
After 10 min of preincubation with the inhibitor before adding the fluorescent protease substrate Z-FR-AMC. Determined 24 h after incubation of
c
trypomastigotes with the compounds. Determined 11 days after incubation of epimastigotes with the compounds. IC50 was calculated from at least
five concentrations using concentrations in triplicate (SD 10%). Highest nontoxic concentration for mouse splenocytes after 24 h of incubation in
the presence of the compounds. Compounds tested at 100 μM. no inhib mean no cruzain inhibition was observed.
d
e
the same range of IC50s. However, varying substituents at C5
resulted in different trypanocidal compounds. For example, ethyl
(17), and isopropyl (18) compounds were 4-times more potent
cidal agents than compound 15. 2-Iminothiazolidin-4-ones 17
and 18 have an IC50 of 5.8 and 6.1 μM for trypomastigotes, which
is equal in potency to Bdz (IC50 of 6.1 μM) and roughly half
as potent as Nfx (IC50 of 2.7 μM). Of the five N-phenyl
2-iminothiazolidin-4-ones 15−19, only the compound which has
a phenyl at C5 (19) showed low inhibitory properties for cruzain
but exhibited trypanocidal effects.
Given these promising results, we next wanted to determine
whether the conversion of 2-iminothiazolidin-4-ones into
2-iminothiazolidin-4-thiones leads to enhanced potency. We
selected 2-iminothiazolidin-4-ones 12 (inactive as trypanocidal
and cruzain inhibitor) and 19 (a potent trypanocidal and weak
inhibitor of cruzain) to convert into their respective thione
variants (Tables 1 and 3). The data revealed that attaching a
thionyl at C4 improves cruzain affinity. 2-Iminothiazolidin-4-one
12 did not inhibit cruzain at 20 μM, while 2-iminothiazolidin-4-
thione 21 had an IC50 of 18.7 2.0 μM. This was even more
pronounced when we compared 2-iminothiazolidin-4-thione 20
with 2-iminothiazolidin-4-one 19. Compound 20 exhibited an
IC50 of 7.0 0.4 μM for cruzain, while compound 19 had an
IC50 > 20 μM. In relation to T. cruzi, 21 showed low trypanocidal
activity while its counterpart 12 was inactive and compounds
19 and 20 were almost equipotent. In general, replacing a
carbonyl with thiocarbonyl enhances the inhibitory properties
of 2-iminothiazolidin-4-ones against cruzain, but it does not
necessarily lead to a significant enhancement of potency against
the parasite.
Of the compounds described in Table 1, the phenyl group in
N3 was identified as the best substituent for conferring anti-
T. cruzi and cruzain inhibition properties, along with an ethyl
or isopropyl attached to C5. We aimed to enhance potency
̀
by preparing 17 analogues. To this end, we attached other sub-
stituents to N3. We succeeded in preparing 2-iminothiazolidin-4-
ones 22, 23, and 24 (Table 2). Replacing an N-phenyl (17)
with N-cyclohexyl (24) was deleterious for cruzain affinity and
for anti-T. cruzi activity, while replacing it with N-tolyl (22) or
N-(4-methoxyphenyl) (23) retains cruzain affinity. Similar to
N-phenyl 17, variants 22 and 23 were toxic for trypomastigotes.
The inhibitory property of 2-iminothiazolidin-4-ones 5−24
for cruzain is centered on the key role of the thiazolidinic ring.
We decided to explore the thiazolidine−thiazole isosterism.
To this end, a congener series of 2-imino-1,3-thiazoles 26−45
was prepared, covering a variety of substitutions at position C4.
However, none of the thiazoles inhibited cruzain activity
(Table 4). 2-Iminothiazolidin-4-ones and 2-imino-1,3-thiazoles
can be viewed as potential bioisosteric heterocycles,28 but this
did not prove to be the case, at least not in terms of cruzain
inhibition. We first thought the lack of cruzain inhibition for
thiazoles 26−45 was merely due to the presence of an aromatic
ring at C4. However 2-iminothiazolidin-4-ones 9 and 19 bear
a phenyl group at C5, and they still have some activity against
cruzain (Figure 3). It seems that the cruzain binding site requires
stereo- and electronic-specificity. Nonetheless, 2-imino-1,3-
thiazoles 26−45 have a broad range of trypanocidal properties.
F
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Figure 3. Structure−activity relationships of 2-iminothiazolidin-4-ones versus 2-imino-1,3-thiazoles. Biochemical data for cruzain.
Of all the tested compounds, 2-imino-1,3-thiazoles 29 and 36
possessed the highest overall trypanocidal potency. Compounds
29 and 36 had trypanocidal activity comparable to that of Bdz,
with IC50 values of 3.9 and 3.8 μM for bloodstream Y strain
trypomastigotes, respectively. 2-Imino-1,3-thiazole 29 was identi-
fied when the phenyl ring at C4 was replaced with a pyridine ring.
Replacing phenyl (26) with a pyridine ring (compounds 27−29)
changes the magnitude of trypanocidal properties for Y strain
trypomastigotes. A 2-pyridine ring (27) was less potent than a
phenyl (26), but a 3-pyridine (27) and a 4-pyridine (29) increased
the trypanocidal property without affecting mouse splenocytes.
While appending a pyridine ring at C4 is beneficial, this is not the
case when a 2-furfuryl (42) or a 2-thiophene (43) ring is attached.
We also analyzed the effect of para substitution on the phenyl
ring. A 4-tolyl (30) is two times less cidal for trypomastigotes
than phenyl (26), while a 4-methoxyphenyl (31) has the same
potency as phenyl (26). When halogens were attached to the
phenyl ring, a pronounced substituent effect was observed.32
4-Fluorphenyl (32) is twice as toxic for trypomastigotes as
phenyl thiazole (26) but still less potent than Bdz or Nfx.
However, the same trypanocidal property was not observed
when other halogen atoms were attached, such as 4-chloro (33),
4-bromo (34), or dichloro (37−39), all of them having poor
trypanocidal properties. Dichloro (compounds 37 and 38) and
trichloro (39) thiazoles were also toxic to mouse splenocytes.
However, inserting a nitro group at the para position (35) and
ortho position (36) produced the most potent anti-T. cruzi
thiazoles. For example, 4-nitrophenyl thiazole 35 inhibited the
proliferation of epimastigotes (IC50 of 1.9 μM) and is toxic for
trypomastigotes (IC50 of 5.7 μM) without affecting the cell viability
of mouse splenocytes, i.e., nontoxic at 100 μg/mL (271 μM).
Finally, we prepared and tested 2-imino-1,3-thiazoles contain-
ing a methyl (44) or phenyl (45) located at N3. In comparison
20 and 21 inhibited cathepsins. As we can infer from Table 3,
some of the N-phenyl 2-iminothiazolidin-4-ones, such as 17 and
18, were at least 10 times more selective for cruzain than for
cathepsins.
A referee suggested that the weak to moderate potency of
2-iminothiazolidin-4-ones in inhibiting cruzain may be caused
by a nonspecific reactivity toward enzymatic nucleophiles or
compound aggregation involving nonspecific attachment to
the enzyme surface. The N-phenyl 2-iminothiazolidin-4-ones
15−19, 22−24 do not possess general inhibitory properties for
cysteine proteases; they only display inhibitory properties for
cruzain. Therefore, a nonspecific reactivity toward enzymatic
nucleophiles is not the case for these compounds. The same
cannot be said for 2-iminothiazolidin-4-thiones 20 and 21, which
inhibit both cruzain and cathepsin without selectivity. But to
ascertain that N-phenyl 2-iminothiazolidin-4-ones 15−19 and
22−24 do inhibit cruzain without compound aggregation, we
needed to repeat some reactions by adding 0.01% of detergent.31
As shown in Table 3, cruzain inhibition by compounds 17, 18, or
20 was not altered once the detergent (Triton X-100) was added,
reinforcing the notion that cruzain inhibition by 2-iminothiazo-
lidin-4-ones is specific.
We also wanted to gain information about the cruzain
inhibition in the parasite cells. However, there is no cruzain gene
knockout T. cruzi cell culture, and the existing cruzain-deficient
cell line is also resistant to cruzain inhibitors. To overcome this
limitation, we decided to treat the parasite with the N-phenyl
2-iminothiazolidin-4-one 18 and examine the ultrastructural
alterations as well as the protease activity.
Bloodstream trypomastigotes as well infected macrophages
were treated with 3.9 μM 18 and examined with transmission
electron microscopy (TEM) and scanning electron microscopy
(SEM). This compound caused severe ultrastructural alterations
in the parasite (for complete details, see the Figures S2 and S3
in Supporting Information). In particular, we saw an atypical
morphology in the Golgi complex, such as dilation of cisternae
and atypical vacuoles, which are characteristic alterations caused
by cruzain inhibitors.33
Besides the evidence collected with electron microscopy, we
also gained information by performing zymography assays with
epimastigotes. Epimastigotes were treated with the 2-iminothia-
zolidin-4-ones 18 and incubated for 48 h. The SDS-page gel
containing 0.1% gelatin was loaded per slot with 20 μg of parasite
protein. As seen in Figure S4 (Supporting Information), the
nontreated epimastigote extract displayed proteolytic activity
for gelatin, while the cells treated with the cruzain inhibitor
E-64 or a protease inhibitor cocktail did not. The treatment of
epimastigotes with the 2-iminothiazolidin-4-ones 18 reduces the
proteolytic activity in a concentration-dependent manner.
Putative Binding Model. The cruzain inhibition of com-
pounds 5−24 indicates that a hydrophobic substituent at C5,
such as ethyl (17) and isopropyl (8, 13, 18), renders excellent
cruzain inhibitors, better than if a phenyl is attached at
to nonsubstituted 2-imino-1,3-thiazole 26, which has IC50
=
19.6 μM for trypomastigotes, thiazoles 44 and 45 exhibited an
IC50 of 174.7 and 61.1 μM, respectively. This suggests that a NH is
important for maintaining trypanocidal properties.
To sum up the SAR for 2-imino-1,3-thiazoles 26−45, optimal
trypanocidal thiazoles resulted when electron-withdrawing
groups such as 4-fluoro, 3-nitro, and 4-nitro were attached to
the phenyl ring at C4. Chloro, bromo, and dichloro proved to be
detrimental. Electron-releasing groups such as methyl, methoxy,
and tert-butyl are of little or no benefit when compared to non-
substituted thiazole 26. Exchanging phenyl (26) with a naphthyl
group (41) is not good, 41 being half as potent as 26.
Once we determined that N-phenyl 2-iminothiazolidin-4-ones
15−19, 22−24 as well as 2-iminothiazolidin-4-thiones 20, 21
inhibited the catalytic activity of cruzain to some degree, we
wondered whether this inhibitory property was only for cruzain
and not for other cysteine proteases. To this end, we assayed
these compounds for inhibition of cathepsins L and S. At 100 μM,
none the N-phenyl 2-iminothiazolidin-4-ones 15−19 and 22−24
inhibited cathepsins, while the 2-iminothiazolidin-4-thiones
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C5 (9, 19). These findings are consistent with another report
showing that the attachment of a hydrophilic substituent at
C5 in 2-iminothiazolidin-4-ones, such as N-methylacetamide
and esters, does not enhance the inhibitory properties of
2-iminothiazolidin-4-ones with regard to cruzain.34 In the case
of position N3, a phenyl group is well tolerated, but a methyl
(10, 11) and cyclohexyl (24) are not so well tolerated. To clarify
these relationships, these compounds were investigated using
molecular docking with cruzain.
The binding mode for these ligands was determined as the
highest (most positive) score among the possible solutions for
each ligand, generated according to the Goldscore Fitness
Function. Figure 4 shows the superimposition of the best docking
for pIC50, are usually those with the higher docking scores, demon-
strating that the molecules with more stable or positive docking
scores (i.e., greater in silico affinity for cruzain) are also the most
potent cruzain inhibitors (i.e., greater in vitro pIC50 values).
To identify the molecular reasons for the two extremes of
potency (highest and lowest in vitro results, 18S and 12R,
respectively), we performed a detailed analysis of the inter-
molecular interactions. The enantiomer with higher in silico
affinity was selected in each case. The differences between these
two molecules are as follows: (i) the presence of a phenyl ring
linked to the N3 nitrogen atom of the thiazolidinic ring in
molecule 18S, instead of a methyl group in molecule 12R; (ii) an
isopropyl group at position C5 of the thiazolidinic ring for
molecule 18S, rather than an ethyl group for 12R. It seems that
the additional hydrophobicity of the phenyl and isopropyl groups
in 2-iminothiazolidin-4-one 18S provides a greater contact
surface for interactions with cruzain hydrophobic residues, as shown
in Figures 6 and 7.
Figure 4. Superimposition of the docking solutions (blue lines) for
compounds that have IC50 for cruzain: 8R, 8S, 12R, 12S, 13R, 13S, 15,
16R, 16S, 17R, 17S, 18R, 18S, 20R, 20S, 21R, 21S, 22R, 22S, 23R, 23S,
and the crystallographic structure of the “KB2” (54) cocrystallized
ligand (red-stick). Figure generated with Pymol.49
Figure 6. Superimposition of the docking solutions for compounds 12R
(blue stick) and 18S (orange stick) and the crystallographic structure of
the “KB2” (54) cocrystallized ligand (gray line).
solutions for compounds that have IC50 values experimentally
determined for cruzain, which are: 8R, 8S, 12R, 12S, 13R, 13S,
15, 16R, 16S, 17R, 17S, 18R, 18S, 20R, 20S, 21R, 21S, 22R, 22S,
23R, 23S, and the crystallographic structure of the “KB2” (triazole
54) cocrystallized ligand. To compare in silico versus in vitro
cruzain data, IC50 values were first converted into pIC50 values
(equals −log IC50 for cruzain inhibition, at molar concentration).
Figure 5 shows the association observed between the in silico
docking scores and the pIC50 data, which indicates that the most
potent compounds, or the compounds with the highest values
The difference between the binding modes of these molecules
is shown in detail in Figure 7 and Table 5. The hydrophobic
residues are highlighted in green, those that participate in the
hydrogen bond are highlighted in cyan, and π−π stacking
interactions are highlighted in orange. The benzene ring linked to
the N3 nitrogen atom is capable of forming a π−π interaction
with the HIS162 amino acid residue, which may be a key inter-
action for positioning the phenoxyl group close to SER61,
SER64, GLY65, GLY66, and LEU67 residues, providing greater
stability for the complex by way of hydrophobic contact and one
hydrogen bond, highlighted in Figure 7B. These differences
ensured that the docking solution for molecule 18S was the only
one in the series of compounds that came closer to the crystal
position of the “KB2” ligand, as shown in Figures 4 and 6.
In Vitro and in Vivo Infection. Given the selectivity of these
compounds against extracellular forms of T. cruzi, we next
examined the activity for the intracellular parasite. To this end,
we assayed an in vitro model of parasite development using
mouse macrophages infected with Y strain trypomastigotes. Four
days postinfection, untreated control had 35−40% of macro-
phages infected and a high mean number of intracellular
amastigotes. In this assay, 10 μM Bdz reduces roughly half of
infected cells as well the number of intracellular amastigotes,
indicating a severe impairment of parasite development. Once
we had this assay validated, we first tested the three most potent
Figure 5. Trend observed between the in vitro (pIC50 for cruzain
inhibition) and in silico (docking score) results. The S and the R
symbols stand for the respective enantiomer.
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Figure 7. Detailed view of the docking solutions for (A) compound 12R and (B) compound 18S. Hydrophobic interactions (green), hydrogen bonds
(cyan), and the π−π interactions (orange). The highlighted residues (blue circle) interacting with molecule 18S are involved with the additional
intermolecular interactions responsible for the greater affinity of this inhibitor, as shown in Table 5.
(IC50 of 13.9 0.39 μM). 2-Iminothiazolidin-4-one 5, which
had an IC50 of 19.0 μM for bloodstream parasites but did not
inhibit cruzain, showed an IC50 of 19.5 1.1 μM to reduce the
percentage of infected macrophages.
Table 5. Molecular Interaction of Cruzain with Molecules 12R
and 18S
a
molecules
cruzain residues
12R
18S
Table 1 shows that in concentrations up to 100 μg/mL
(262 μM), the 2-iminothiazolidin-4-one 18 does not affect cell
viability of mouse splenocytes. We next wanted to determine the
IC50 value. Strikingly, compound 18 does not affect either cell
viability or proliferation (under mitogen challenge) of splenocytes
(IC50 values >262 μM), in contrast to an IC50 of 10.1 0.09 μM
for intracellular T. cruzi (Table 6). The selectivity index (SI) value
can be estimated to be over 25. For comparison, compound 5, our
simplest 2-iminothiazolidin-4-one variant, has an SI of 10.
On the one hand, we have potency enhancement from
2-iminothiazolidin-4-one 5 to 18 (Table 6). On the other hand,
we have observed that even at a concentration of 50 μM,
2-iminothiazolidin-4-one 18 does not eradicate the infection in a
culture of macrophages. The same is observed under treatment
with Bdz. As seen in Figure 8C, we only approached eradication
of the in vitro intracellular parasite under the simultaneous
treatment of Bdz and 18.
Chemically inhibiting parasite invasion is a desirable functional
property for any Chagas disease drug candidate. For that reason,
we assayed an invasion experiment with Y strain trypomastigotes
in mouse macrophages.35 After 60 min of parasite exposure,
about 35% of macrophages are infected (untreated control).
Bdz did not impair parasite viability in the invasion assay, even
at 50 μM; so we decided to use amphotericin B (AmpB)
as a reference drug. At 50 μM, AmpB was quite efficient in
inhibiting parasite invasion into macrophages. Our best cruzain
and T. cruzi inhibitor, N-phenyl 2-iminothiazolidin-4-one 18,
also decreased parasite invasion (P < 0.01) albeit less than AmpB
did. 2-Iminothiazolidin-4-one 5 only attenuated the invasion
(P < 0.05), and thiazole 29 did not have any significant effect in
this assay (Figure 8D).
GLN19
GLY23
CYS25
TRP26
SER61
3.0
HC
2.5
HC
HC
HC
HC
HC
HC
2.9
SER64
GLY65
GLY66
LEU67
ALA138
LEU160
ASP161
HIS162
GLY163
TRP184
HC
HC
HC
HC
PI
HC
HC
HC
HC
HC
HC
a
HC means hydrophobic contacts, PI means π−π stacking, and the
numbers are the hydrogen bond distances in angstroms.
cruzain inhibitors, 2-iminothiazolidin-4-ones 16, 17, and 18 as
well as the 2-imino-1,3-thiazoles 29 and 35.
At a concentration of 10 μM, we observed that 2-iminothia-
zolidin-4-one 18 was able to reduce the number of infected cells
with efficacy more pronounced than that seen with Bdz-treated
cells. 2-Iminothiazolidin-4-ones 16 and 17 were less potent than
Bdz. 2-Imino-1,3-thiazole 29, which was a trypanocidal
compound for bloodstream parasites, had no significant activity
in reducing the infection in macrophages. Yet 4-nitro derivative
35 inhibited the in vitro infection similarly to Bdz (Table S1,
Supporting Information).
We next examined the 2-iminothiazolidin-4-ones 5 and 18 at a
concentration of 50 μM. As seen in Figure 8A and B, compound
18 as well as Bdz substantially reduces the intracellular parasites
in most of the microscopy fields (P < 0.001). We determined the
IC50 values of these compounds to reduce the percentage of infected
macrophages (Table 6), and we found that 2-iminothiazolidin-4-
one 18 has an IC50 of 10.1 0.09 μM, almost identical to that of Bdz
Finally, we evaluated the in vivo efficacy of N-phenyl
2-iminothiazolidin-4-one 18 to reduce blood parasitemia (acute
phase). Starting on day 5 postinfection (dpi), compound 18 was
orally administered once a day at 250 μmol/kg for 5 consecutive
days in BALB/c mice infected with 104 Y strain trypomastigotes.
Controls included untreated and Bdz-treated infected mice.
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Figure 8. 2-Iminothiazolidin-4-one 18 severely impairs intracellular parasite development. The infection ratio (A) and the mean number of intracellular
amastigotes per host cell (B) are higher in untreated controls than in cultures treated with test inhibitors 5, 18, or Bdz. Macrophages were infected with Y
strain trypomastigotes for 2 h, and test inhibitors were then added at 50 μM. Cell cultures were incubated for 4 days. Significance: ***, P < 0.001; *,
P < 0.01. (C) Combined treatment of 2-iminothiazolidin-4-one 18 (40 μM) and Bdz (40 μM) approaches an eradication of in vitro infection.
Macrophages were infected with Y strain trypomastigotes for 2 h, and the combination of drugs was simutaneously added. Cell cultures were incubated
for 4 days. Significance: ***, P < 0.001. (D) 2-Iminothiazolidin-4-one 18 attenuates parasite invasion in host cells, while Bdz does not. Y strain
trypomastigotes and each test inhibitor (at 50 μM) were simultaneously added to the cell culture of macrophages and incubated for 2 h. ***, P < 0.001;
**, P < 0.01; *, P < 0.05. All test inhibitors were tested in triplicate concentrations, and two independent experiments were carried out. Standard
deviations are shown as error bars. Bdz is benznidazole; AmpB is amphotericin B.
Table 6. Comparison of the Pharmacological Properties between the 2-Iminothiazolidin-4-ones 5 and 18
IC50 ( SD), μM
a
b
c
d
e
compd
intracellular parasite
toxicity for splenocytes
antiproliferative for spleenocytes
index (SI)
% parasitemia reduction in mice
5
19.5 1.1
>200
>262
98 10
>262
10
25
−
−
89.6
18
10.1 0.09
controls
13.9 0.39 (benznidazole)
1.7 0.002 (saponin)
0.01 μg/mL (cyclosporine A)
>99 (benznidazole)
a
Determined 4 days after incubation of infected macrophages. IC50 values calculated in regard to the percent of treated infected cells in comparison
b
to untreated infected cells. Determined 24 h after incubation of mouse splenocytes. Cell viability measured using the incorporation of tritiated
thymidine. Splenocyte proliferation was induced by concanavalin A. The effect on the proliferation was determined 72 h after incubation of mouse
splenocytes using the AlamarBlue. SD is the standard deviation. Cellular selective index was estimated using the ratio (IC50 for splenocytes)/
(IC50 for amastigotes). Calculated as ([average vehicle group − average treated group)/average vehicle group] × 100%). Values of blood
parasitemia were taken from day 5 of treatment.
c
d
e
The course of infection was monitored by counting blood
parasites in a hemocytometer, and animal survival was followed
for one month.36 As seen in Figure 9 and Table 6, compound 18
was efficient in reducing 89.6% of blood parasitemia (P < 0.001 of
significance) when compared to untreated control. Bdz was even
more effective than 18, nearly eradicating blood parasites (>99%
of parasitemia reduction). No signs of toxicity or body weight loss
were observed in mice of the treatment groups.
synthetic redesign. We found that 2-iminothiazolidin-4-ones are
structural analogues of thiosemicarbazones and endowed with in
vitro trypanocidal properties. In previous studies, we discovered
that 2-iminothiazolidin-4-ones were less potent trypanocidal agents
than thiosemicarbazones, but at least 2-iminothiazolidin-4-ones
were more selective for parasite than for mammalian cells.25−27
Here, our principal aim was to improve the cidal potency by
molecular modification. We knew from past studies that the
thiazolinidic ring is more important for trypanocidal activity than
the hydrazone and aryl groups. We chose the aryloxypropylimine
group as a common core for designing compounds 5−45 because
of the remarkable functional property of the drug candidate
aryloxymethyl ketone triazole 54.23 We next wanted to identify the
main structural determinants for trypanocidal and cruzain inhibition
in this class of 2-iminothiazolidin-4-ones. Here, we have identified
most of them after screening the 2-iminothiazolidin-4-ones 5−24
and comparing them with 2-imino-1,3-thiazoles 26−45.
DISCUSSION
■
Thiosemicarbazones are known for their powerful in vitro
trypanocidal and cruzain inhibition effects.13−17 However, cur-
rently, very few thiosemicarbazones that are effective in reducing
in vivo T. cruzi infection have been described.37 One method
for increasing the efficacy of anti-T. cruzi thiosemicarbazones is
through complexation with transition metals.38−40 Another pos-
sibility is the chemical modification of thiosemicarbazones by
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Our findings corroborate a very recent work describing that
2-imino-1,3-thiazoles are trypanocidal agents by altering the
ergosterol biosynthesis instead of inhibiting the catalytic activity
of cruzain.41 In agreement with this, we also found 2-imino-1,3-
thiazoles are trypanocidal agents, and a few of them were
equipotent to Bdz, such as thiazoles 29, 35, and 36. There was a
clear trend toward trypanocidal potency as polar substituents on
the phenyl at C4 are attached to 2-imino-1,3-thiazoles. This led
to some of the most potent thiazole variants examined, such as
compounds 29 (4-pyridine), 32 (4-fluor), 35 (4-nitro), and 36
(3-nitro). This contrasts with the incorporation of an alkyl
(30, 31, 40) or naphthyl (41) group, which, in the same position,
decreased the cidal effects of the thiazole.
The selectivity of 2-iminothiazolidin-4-ones seems to be more
consistent than that of 2-imino-1,3-thiazoles, because some
thiazole variants were toxic to mouse splenocytes. Moreover, the
mechanismthrough which thiazoles arrest parasite growth remains
unknown. In view of these findings, we decided to search for more
information on the trypanocidal effects of 2-iminothiazolidin-4-
ones. We selected 2-iminothiazolidin-4-one 18, the most potent
cruzain inhibitor among the examined compounds. With regard to
the functional activity of 18, it inhibits the catalytic activity of
cruzain but not that of cathepsins L and S, clearly arrests parasite
growth (epimastigotes) and motility (trypomastigotes), impairs
intracellular trypomastigote development into amastigotes, and
attenuates trypomastigote invasion of macrophages. Using
electron microscopy, we observed that 2-iminothiazolidin-4-one
18 alters the parasite morphology, in particular, altering the
structure of the Golgi complex in a way very similar to that for
other cruzain inhibitors. Moreover, 2-iminothiazolidin-4-one 18
reduces the protease activity in epimastigotes; both experiments
suggest that cruzain is involved with the trypanocidal activity
of 2-iminothiazolidin-4-one 18. Finally, the antiparasitic effect of
compound 18 was reinforced by its efficacy in reducing blood
parasitemia in T. cruzi-infected mice.
Figure 9. 2-Iminothiazolidin-4-one 18 substantially reduces the
parasitemia in mice. Female BALB/c mice were infected with 104 Y
strain trypomastigotes. Five days postinfection (dpi) mice were orally
treated once a day with compound 18 or benznidazole (Bdz) at a dose of
250 μmol/kg during five consecutive days. Parasitemia was monitored
by counting the number of trypomastigotes in fresh blood samples.
Values represent the mean
SEM of six mice per group. Two
independent experiments; data are representative from one experiment.
Significance (***, P < 0.0001) compared to untreated infected group
(vehicle).
We first observed that, with few exceptions, most of the
nonsubstituted 2-iminothiazolidin-4-ones 5−9 or those contain-
ing a methyl at N3 10−14 are inactive or poor cruzain inhibitors,
and there was no reliable relationship between trypanocidal
effects and chemical structure. In contrast, 2-iminothiazolidin-4-
ones containing a phenyl group at N3 (15−19, 22−24) inhibit
cruzain and share trypanocidal properties. They are effective
against the parasite but with the notable advantage that they do
not affect either the viability or the proliferation of host cells.
With regard to the mechanism of action, we cannot confirm
whether the anti-T. cruzi property is entirely due to the inhibi-
tion of cruzain alone. We argue that the trypanocidal properties
of compounds 15−24 are, to some degree, a result of cruzain
inhibition. This statement is based on the ability of 2-iminothia-
zolidin-4-ones to inhibit the catalytic activity of recombinant
cruzain (competition experiments), to alter the morphology of
the Golgi complex (electron microscopy), and to impair the
proteolytic activity in the whole parasite (zymography assays
with epimastigotes).
One potential downside to 18 is that it does not eradicate
parasitemia in mice (acute infection), a desirable property
already demonstrated for other Chagas disease drug candi-
dates.23,42−44 However, the design of anti-T. cruzi 2-iminothia-
zolidin-4-one 18 is a remarkable achievement for our program,
especially because it worked orally. Synthetic redesign of the
aryloxypropylimine group is one possible way to optimize the
efficacy of compound 18.
The model of docking for 2-iminothiazolidin-4-one 18 with
cruzain shows that the phenyl ring linked to the N3 nitrogen is
capable of forming a π−π interaction with the HIS162 amino acid.
This interaction would seem to be the reason for the phenoxy
group being located at a position which allows important
hydrophobic contacts and hydrogen bond interactions, giving
greater stability to the complex. The same is not predicted for
2-iminothiazolidin-4-one containing a methyl at N3, such as
compound 12. This model is consistent with recombinant cruzain
inhibition and also provides an explanation for why the
attachment of the phenyl on the heterocyclic ring is important
for cruzain inhibition, in addition to explaining the importance of
an alkyl group, such as ethyl and isopropyl, at position C5.
Despite the structural analogy, the functional properties of
2-iminothiazolidin-4-ones 5−24 and 2-imino-1,3-thiazoles 26−
45 are distinguishable. 2-Iminothiazolidin-4-ones inhibited the
catalytic activity of cruzain while none of the tested thiazoles did.
Therefore, they do not behave as antiparasitic bioisosteres.
CONCLUSIONS
■
A detailed SAR study of anti-T. cruzi 2-iminothiazolidin-4-ones
was conducted. It included molecular modifications on the
thiazolidinic ring, as well isosteric exchanges of the carbonyl
carbon and of the heterocycle itself. We observed a remarkable
improvement in trypanocidal properties and cruzain inhibi-
tion only when positions N3 and C5 in the thiazolidinic ring
were simultaneously substituted. This led to the identification
of potent N-phenyl 2-iminothiazolidin-4-ones 15−19, 22−24.
Replacement of the carbonyl with thiocarbonyl (20, 21) did not
produce a clear enhancement in potency, and exchanging the
thiazolidinic for a thiazole ring 26−45 was deleterious for cruzain
affinity, although some thiazoles did exhibit trypanocidal pro-
perties. Of compounds 15−24, we found consistent trypanocidal
effects for 18, thereby corroborating the notion that this class
of heterocycles can lead to useful cidal agents for Chagas disease
treatment.
K
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(t, J 7.2 Hz, 3H, CH3), 1.73−1.81 (m, 1H, J 7.2 and 4.4 Hz, CH2), 1.92−
1.98 (m, 1H, J 7.2 and 4.4 Hz, CH2), 2.02 (s, 3H, CH3), 4.12−4.15 (q,
1H, J 7.6 and 4.4 Hz, CH asymmetric), 4.67 (s, 2H, CH2), 6.92−7.00 (m,
3H, Ar), 7.28 (t, 2H, Ar), 11.94 (broad s, 1H, NH). 13C NMR (100
MHz, DMSO-d6): 10.1 (CH3), 14.5 (CH3), 25.4 (CH2), 48.8 (CH),
70.9 (CH2), 114.6 (CH, Ar), 120.8 (CH, Ar), 129.3 (CH, Ar), 158.1
(CN), 161.9 (C−O, Ar), 162.1 (S−CN), 175.6 (CO). Anal.
Calcd for C14H17N3SO2: C, 57.71; H, 5.88; N, 14.42; S, 11.00; found: C,
57.64; H, 5.69; N, 14.25; S, 10.89.
EXPERIMENTAL SECTION
■
Equipment and Reagents. All reagents were used as purchased
from commercial sources (Sigma-Aldrich, Acros Organics, Vetec, or
Fluka). Moisture-sensitive reactions were carried out under a nitrogen
atmosphere. Progress of the reactions was followed by thin-layer
chromatography (silica gel 60 F254 in aluminum foil). Purity of the target
compounds was confirmed by combustion analysis (for C, H, N, S)
performed by a Carlo-Erba instrument (model EA 1110). Chemical
identity was confirmed with NMR and IR spectroscopy and accurate
mass. IR was determined in KBr pellets. For NMR, we used a Varian
2-((1-Phenoxypropan-2-ylidene)hydrazono)-5-isopropylthiazoli-
din-4-one (8). Crystallization from cyclohexane afforded colorless
crystals, yield = 0.14 g, 38%. Mp (°C): 139. IR (KBr): 2967 (C−H),
UnityPlus 400 MHz (400 MHz for H and 100 MHz for 13C) and
1
Bruker AMX-300 MHz (300 MHz for H and 75.5 MHz for 13C)
1
1
1725 (CO), 1640 and 1580 (CN) cm−1. H NMR (400 MHz,
instruments. DMSO-d6, acetone-d6, CDCl3, and D2O were purchased
from CIL or Sigma-Aldrich. Chemical shifts are reported in ppm, and
multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), and dd (double doublet), and coupling constants (J) in
hertz. NH signals were localized in each spectrum after the addition of a
few drops of D2O. Structural assignments were corroborated by DEPT,
HMBC and HSBC experiments. Mass spectrometry experiments were
performed on a Q-TOF spectrometer (nanoUPLC-Xevo G2 Tof,
Waters) or LC-IT-TOF (Shimadzu). When otherwise specified, ESI was
carried out in the positive ion mode. Typical conditions were capillary
voltage of 3 kV, cone voltage of 30 V, and a peak scan between 50 and
1000 m/z. To determine the molecular structure of thiosemicarbazones
4a and 4c as well as 2-iminothiazolidin-4-one 12, a crystal of the
corresponding compound was mounted on a Enraf-Nonius Kappa
diffractometer equipped with a CCD detector (95 mm camera on
κ-goniostat), and reflections were measured using monochromatic Mo
Kα radiation (0.71073 Å) at room temperature.
General Procedure for the Synthesis of 2-Iminothiazolidin-4-ones
5−19 and 22−24. Example for compound 5: Thiosemicarbazone 4a
(1.12 g, 5 mmol), anhydrous sodium acetate (1.2 g, 15 mmol), and
100 mL of ethanol were added to a 250 mL round-bottom flask under
magnetic stirring and slightly warmed for 10−15 min. Then ethyl
2-bromoacetate (1.6 g, 10 mmol) was added, and the colorless reaction
was refluxed for 18 h. After cooling back to rt, the precipitate was filtered
off and the solvent was evaporated to half of its volume and then cooled
DMSO-d6): δ 0.86 (d, 3H, J 4.0 Hz, CH3), 0.96 (d, 3H, J 4.0 Hz, CH3),
1.97 and 2.01 (two s, 3H, CH3), 2.35 (broad s, 1H, CH), 4.25 (s, 1H,
CH), 4.61 and 4.67 (two s, 2H, CH2), 6.98 (t, 3H, Ar), 7.28 (d, 2H, Ar),
11.86 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): 14.7 (CH3),
16.2 (CH3), 20.2 (CH3), 29.8 (CH), 54.4 (CH), 70.9 (CH2), 114.7
(CH, Ar), 120.9 (CH, Ar), 129.4 (Ar), 158.1 (CN), 162.1 (C), 175.2
(CO). HRMS (ESI): 306.1265 [M + H]+. Anal. Calcd for
C15H19N3SO2: C, 58.99; H, 6.27; N, 13.76; S, 10.50; found: C, 59.53;
H, 6.21; S, 8.69; N, 13.38.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-5-phenylthiazolidin-
4-one (9). Crystallization from toluene afforded colorless crystals,
yield = 0.14 g, 41%. Mp (°C): 150. IR (KBr): 3391 (N−H), 1709 (C
O), 1640 and 1599 (CN) cm−1. 1H NMR (400 MHz, acetone-d6): δ
1.92 and 2.06 (s, 3H, CH3), 3.58 (br s, NH+HDO), 4.67 and 5.01 (s, 2H,
CH2), 5.34 (s, 1H, CH), 6.90−7.01 (m, 3H, Ar), 7.27 (br s, 7H, Ar). 13
C
NMR and DEPT (100 MHz, acetone-d6): 14.8 and 26.3 (CH3), 51.4
(CH), 65.1 and 71.0 (CH2), 114.2 (CH, Ar), 114.2 (CH, Ar), 120.9
(CH, Ar), 128.3 (CH, Ar), 128.7 (CH, Ar), 129.4 (CH, Ar), 130.4 (CH,
Ar), 137.4 (C−N, Ar), 157.0 and 157.1 (CN), 161.2 and 162.8
(C−O, Ar), 164.4 (S−CN), 176.1 (CO). HRMS (ESI): 340.1129
[M + H]+. Anal. Calcd for C18H17SO2N3: C, 63.70; H, 5.05; N, 12.38; S,
9.45; found: C, 64.01; N, 12.60; H, 5.60; S, 8.73.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-methylthiazolidin-
4-one (10). Crystallization from cyclohexane afforded colorless crystals,
yield = 0.15 g, 54%. Mp (°C): 112. IR (KBr): 1721 (CO), 1636 and
to 0 °C. A white solid was obtained, filtered in a Buchner funnel with a
̈
1
1580 (CN) cm−1. H NMR (400 MHz, DMSO-d6): δ 2.06 (s, 3H,
sintered disc filter, washed with cold water, and then dried over SiO2.
Products were purified by column chromatography or recrystallization
using the solvent system detailed below for each compound. Purity of
compounds were >95% as determined by combustion analysis.
2-((1-Phenoxypropan-2-ylidene)hydrazono)thiazolidin-4-one (5).
After elution with hexane/toluene (1:1), colorless crystals were
obtained, yield = 0.7 g, 53%. Mp (°C): 130−131. IR (KBr): 3196
(N−H), 3006 (C−H), 1734 (CO), 1649 and 1595 (CN) cm−1. 1H
NMR (300 MHz, DMSO-d6): δ 1.98 (s, 3H, CH3), 3.83 (s, 2H, S−CH 2),
4.66 (s, 2H, CH2), 6.92−6.99 (m, 3H, Ar), 7.28 (t, 2H, Ar), 11.90 (s, 1H,
NH). 13C NMR (75.5 MHz, DMSO-d6): δ 14.8 (CH3), 32.8 (CH2,
heterocycle), 70.9 (CH2), 114.5 (CH, Ar), 120.9 (CH, Ar), 129.5
(CH, Ar), 158.1 (CN), 161.9 (C−O, Ar), 164.1 (S−CN), 173.9
(CO). HRMS (ESI): 286.0599 [M + Na]+. Anal. Calcd for
C12H13N3O2S: C, 54.74; H, 4.98; N, 15.96; S, 12.18. Found: C, 54.42;
H, 5.02; S, 11.91; N, 15.78.
CH3), 3.13 (s, 3H, N−CH3), 3.87 (s, 2H, CH2), 4.70 and 5.02 (two s,
2H, CH2−O), 6.94−7.00 (m, 3H, Ar), 7.27−7.30 (m, 2H, Ar). 13C
NMR (100 MHz, DMSO-d6): 14.4 and 19.4 (CH3), 29.2 (N−CH3),
31.9 (CH2), 64.9 and 70.8 (CH2), 114.2 (CH, Ar), 114.7 (CH, Ar),
120.9 (CH, Ar), 129.4 (CH, Ar), 158.0 (CN), 163.1 (C), 172.0 (C
O). HRMS (ESI): 278.0995 [M + H]+. Anal. Calcd for C13H15N3SO2:
C, C, 56.30; H, 5.45; N, 15.15; S, 11.56; found: C, 56.54; H, 5.40; S, 9.89;
N, 14.91.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3,5-dimethylthiazo-
lidin-4-one (11). Crystallization from toluene afforded colorless crystals,
yield = 0.18 g, 60%. Mp (°C): 67. IR (KBr): 1720 (CO), 1632 (C
1
N), 1581 (CN) cm−1. H NMR (300 MHz, DMSO-d6): δ 1.48 (d,
3H, J 7.2 Hz, CH3), 2.06 (s, 3H, CH3), 3.14 (s, 3H, N−CH3), 4.16−4.23
(q, 1H, J 7.2 Hz, CH), 4.69 (s, 2H, CH2), 6.92−7.00 (m, 3H, Ar), 7.26−
7.31 (m, 2H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 14.6 (CH3), 18.7
(CH3), 29.4 (N−CH3), 41.2 (CH), 70.8 (CH2), 114.7 (CH, Ar), 120.9
(CH, Ar), 129.4 (CH, Ar), 158.1 (CN), 161.9 (C−O, Ar), 163.3
(S−CN), 175.1 (CO). HRMS (ESI): 292.1134 [M + H]+. Anal.
Calcd for C14H17N3SO2: C, 57.71; H, 5.88; N, 14.42; S, 11.00; found: C,
57.70; H, 5.91; N, 14.38; S, 11.10.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-5-methylthiazolidin-
4-one (6). After elution with hexane/ethyl acetate (3:1), yellowish
crystals were obtained, yield = 0.11 g, 33%. Mp (°C): 117−120. IR
(KBr): 3195 (N−H), 1710 (CO), 1640 and 1602 (CN) cm−1. 1H
NMR (300 MHz, DMSO-d6): δ 1.46 (d, 3H, J 7.8 Hz, CH3), 2.02 (s, 3H,
CH3), 4.11 (t, 1H, J 7.8 Hz, CH), 4.66 (s, 2H, CH2), 6.93−6.99 (m, 3H,
Ar), 7.28 (t, 2H, Ar), 11.79 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6): δ 14.5 (CH3), 18.6 (CH3), 41.7 (CH), 70.9 (CH2), 114.6 (CH, Ar),
120.8 (CH, Ar), 129.3 (CH, Ar), 158.0 (CN), 161.8 (C−O, Ar),
161.9 (S−CN), 176.5 (CO). HRMS (ESI): 278.0995 [M + H]+.
Anal. Calcd for C13H15N3SO2: C, 56.30; H, 5.45; N, 15.15; S, 11.56.
Found: C, 56.16; H, 5.38; S, 11.21; N, 15.09.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-methyl-5-ethyl-
thiazolidin-4-one (12). Crude was washed with ethyl ether, affording
colorless crystals, yield = 0.26 g, 76%. Mp (°C): 129. IR (KBr): 1718
(CO), 1636 and 1582 (CN) cm −1. 1H NMR (300 MHz, DMSO-
d6): δ 0.92 (t, 3H, J 7.5 Hz, CH3), 1.74−1.82 (m, 1H, CH2), 1.95−1.98
(m, 1H, CH2), 2.06 (s, 3H, CH3), 3.18 (s, 3H, N−CH 3), 4.21−4.25 (q,
1H, J 4.2 and 7.8 Hz, CH), 4.70 (s, 2H, CH2), 6.93−7.01 (m, 3H, Ar),
7.26−7.32 (m, 2H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 10.3 (CH3),
14.6 (CH3), 25.5 (CH2), 29.3 (N−CH 3), 48.2 (CH), 70.8 (CH2), 114.7
(CH, Ar), 120.9 (CH, Ar), 129.5 (CH, Ar), 158.0 (CN), 161.9
(C−O, Ar), 163.4 (S−CN), 174.2 (CO). HRMS (ESI): 306.1283
2-((1-Phenoxypropan-2-ylidene)hydrazono)-5-ethylthiazolidin-4-
one (7). After elution with hexane/toluene (2:1), yellowish crystals were
obtained, yield = 0.15 g, 51%. Mp (°C): 133. IR (KBr): 1715 (CO),
1614 and 1596 (CN) cm −1. 1H NMR (400 MHz, DMSO-d6): δ 0.94
L
dx.doi.org/10.1021/jm301518v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
[M + H]+. Anal. Calcd for C15H19N3O2S: C, 58.99; N, 13.76; H, 6.27; S,
10.50; found: C, 59.15; H, 6.09; N, 13.32; S, 9.43.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-phenyl-5-isopro-
pylthiazolidin-4-one (18). Crude was washed with ethyl ether, affording
colorless crystals, yield = 0.3 g, 78%. Mp (°C): 128−130. IR (KBr): 1724
(CO), 1632 and 1579 (CN) cm−1. 1H NMR (400 MHz, DMSO-
d6): 1.01 (d, 3H, J 4.0 Hz, CH3), 1.08 (d, 3H, J 4.0 Hz, CH3), 1.86 (s, 3H,
CH3), 2.51 (t, 1H, J 4.0 and 3.4 Hz, CH), 4.52 (m, 1H, J 3.4 Hz, CH),
4.72 (s, 2H, CH2), 6.97−7.03 (m, 3H, Ar), 7.31−7.38 (m, 4H, Ar),
7.46−7.56 (m, 3H, Ar). 13C NMR (100 MHz, DMSO-d6): 14.6 (CH3),
16.4 (CH3), 20.1 (CH3), 30.4 (CH), 53.6 (CH), 70.6 (CH2), 114.7
(CH, Ar), 120.9 (CH, Ar), 127.8 (CH, Ar), 128.5 (CH, Ar), 128.9 (CH,
Ar), 129.4 (CH, Ar), 134.8 (C−N, Ar), 158.0 (CN), 161.4 (C−O,
Ar), 163.8 (S−CN), 173.4 (CO). HRMS (ESI): 382.1580
[M + H]+. Anal. Calcd for C21H23SO2N3: C, 66.12; H, 6.08; N, 11.01;
S, 8.41; found: C, 66.48; H, 5.70; S, 6.73; N, 10.81.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3,5-diphenylthiazo-
lidin-4-one (19). Crystallization from cyclohexane afforded colorless
crystals, yield = 0.32 g, 74%. Mp (°C): 159. IR (KBr): 1715 (CO),
1628 and 1590 (CN) cm−1. 1H NMR (400 MHz, acetone-d6): 1.01
(d, 3H, J 4.0 Hz, CH3), 1.08 (d, 3H, J 4.0 Hz, CH3), 1.86 (s, 3H, CH3),
2.51 (t, 1H, J 4.0 and 3.4 Hz, CH), 4.52 (m, 1H, J 3.4 Hz, CH), 4.72 (s,
2H, CH2), 6.97−7.03 (m, 3H, Ar), 7.31−7.38 (m, 4H, Ar), 7.46−7.56
(m, 3H, Ar). 13C NMR (100 MHz, acetone-d6): 14.6 (CH3), 16.4
(CH3), 20.1 (CH3), 30.4 (CH), 53.6 (CH), 70.6 (CH2), 114.7 (CH,
Ar), 120.9 (CH, Ar), 127.8 (CH, Ar), 128.5 (CH, Ar), 128.9 (CH, Ar),
129.4 (CH, Ar), 134.8 (C−N, Ar), 158.0 (CN), 161.4 (C−O, Ar),
163.8 (S−CN), 173.4 (CO). HRMS (ESI): 416.1433 [M + H]+.
Anal. Calcd for C24H21SO2N3: C, 69.37; H, 5.09; N, 10.11; S, 7.72;
found: C, 69.35; H, 5.00; S, 8.01; N, 10.21.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-methyl-5-isopro-
pylthiazolidin-4-one (13). Crystallization from cyclohexane afforded
colorless crystals, yield = 0.25 g, 70%. Mp (°C): 138. IR (KBr): 1717
(CO), 1634 and 1581 (CN) cm−1. 1H NMR (300 MHz, DMSO-
d6): δ 0.80 (d, 3H, J 6.6 Hz, CH3), 0.97 (d, 3H, J 6.9 Hz, CH3), 2.07 (s,
3H, CH2), 2.39−2.42 (m, 1H, CH), 3.151 and 3.34 (s, 3H, N−CH3),
4.31 (d, 1H, J 3.9 Hz, CH), 4.70 (s, 2H, CH2), 6.92−7.00 (m, 3H, Ar),
7.26−7.31 (m, 3H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 14.5 (CH3),
16.4 (CH3), 20.2 (CH3), 30.4 (CH), 31.3 (CH3−N), 53.7 (CH),
70.8 (CH2), 114.7 (CH, Ar), 120.9 (CH, Ar), 129.4 (CH, Ar), 158.1
(CN), 161.9 (C−O, Ar), 163.5 (S−CN), 173.7 (CO). HRMS
(ESI): 320.0915 [M + H]+. Anal. Calcd for C16H21N3SO2: C, 60.16; H,
6.63; N, 13.16; S, 10.04; found: C, 60.40; H, 6.14; N, 13.11; S, 9.66.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-methyl-5-phenyl-
thiazolidin-4-one (14). Crystallization from toluene afforded colorless
crystals, yield = 0.25 g, 70%. Mp (°C): 157. IR (KBr): 1716 (CO),
1628 and 1589 (CN) cm−1. 1H NMR (300 MHz, DMSO-d6): 1.98
and 2.12 (s, 3H, CH3), 3.23 and 3.36 (s, 3H, N−CH3), 4.71 (s, 2H,
CH2), 5.49 (s, 1H, CH), 6.94−7.00 (m, 3H, Ar), 7.25−7.30 (m, 3H, Ar),
7.37 (br s, 4H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 14.7 (CH3), 26.3
and 29.8 (N−CH3), 50.0 (CH), 70.7 (CH2), 114.7 (CH, Ar), 120.9
(CH, Ar), 128.3 (CH, Ar), 128.5 (CH, Ar), 128.8 (CH, Ar), 129.4
(CH, Ar), 136.5 (C−N, Ar), 158.0 (CN), 161.5 (C−O, Ar), 163.8
(S−CN), 172.9 (CO). HRMS (ESI): 354.0728 [M + H]+. Anal.
Calcd for C19H19SO2N3: C, 64.57; H, 5.42; N, 11.89; S, 9.07; found: C,
64.41; N, 12.01; H, 5.39; S, 9.13.
Synthesis of 2-Iminothiazolidine-4-thiones 20 and 21. Example
for Compound 21: A solution of 2-iminothiazolidin-4-one 9 (0.34 g,
1.5 mmol) in anhydrous 1,4-dioxane/toluene (1:1, 50 mL) was added to
a 100 mL round-bottom flask under magnetic stirring in N2 and slightly
warmed. Then Lawesson’s reagent (1.0 g, 2.6 mmol) was added in
portions, and the brownish mixture was kept under stirring and heating
overnight. After cooling back to rt, the solvent was evaporated to half
volume. Water was added and extracted with ethyl ether and then brine.
The organic layers were then combined and dried with MgSO4, and
solvent was removed under reduced pressure and then dried over SiO2.
The compound was isolated after column chromatography.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3,5-diphenylthiazo-
lidin-4-thione (20). After elution with hexane/ethyl acetate (7:3) to a
short silica gel column, yellowish crystals were obtained, yield = 0.2 g,
54%. Mp (°C): 131. IR (KBr): 1620 and 1596 (CN) cm−1. 1H NMR
(400 MHz, acetone-d6): 1.90 (s, 3H, CH3), 4.69 (s, 2H, CH2), 5.40 (s,
1H, CH), 6.91−7.03 (m, 3H, Ar), 7.24−7.30 (m, 2H, Ar), 7.35−7.40
(m, 3H, Ar), 7.42−7.7.56 (m, 6H, Ar). 13C NMR (100 MHz, acetone-
d6): 27.5 (CH3), 56.1 (CH), 72.0 (CH2), 115.2 (CH, Ar), 115.7
(CH, Ar), 121.9 (CH, Ar), 129.01 (CH, Ar), 129.32 (CH, Ar), 129.38
(CH, Ar), 129.72 (CH, Ar), 129.84 (CH, Ar), 130.4 (CH, Ar), 136.6
(C−N, Ar), 138.0 (C−N, Ar), 159.6 (CN), 162.4 (C−O, Ar), 168.0
(S−CN), 189.1 (CS). Anal. Calcd for C24H21S2ON3: C, 66.79; H,
4.90; N, 9.74; S, 14.86; found: C, 66.81; H, 5.00; S, 15.11; N, 14.01.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-5-ethylthiazolidin-4-
thione (21). After elution with hexane/ethyl acetate (7:3), yellowish
crystals were obtained, yield = 0.28 g, 60%. Mp (°C): 111. IR (KBr):
1620 and 1590 (CN) cm−1. 1H NMR (300 MHz, DMSO-d6): 0.92
(t, 3H, J 7.5 Hz, CH3), 1.74−1.82 (m, 1H, CH2), 1.95−1.98 (m, 1H,
CH2), 2.06 (s, 3H, CH3), 4.20−4.24 (q, 1H, J 4.2 and 7.8 Hz, CH), 4.70
(s, 2H, CH2), 6.93−7.00 (m, 3H, Ar), 7.26−7.32 (m, 2H, Ar). 13C NMR
(75.5 MHz, DMSO-d6): 10.3 (CH3), 14.6 (CH3), 25.5 (CH2), 49.0
(CH), 70.8 (CH2), 114.7 (CH, Ar), 121.0 (CH, Ar), 129.5 (CH, Ar),
158.0 (CN), 161.9 (C−O, Ar), 163.4 (S−CN), 188.9 (CS).
Anal. Calcd for C14H17S2ON3: C, 54.69; H, 5.57; N, 13.67; S, 20.86;
found: C, 54.71; H, 5.60; S, 19.99; N, 14.05.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-phenylthiazolidin-
4-one (15). Crystallization from toluene afforded colorless crystals,
yield = 0.25 g, 70%. Mp (°C): 139. IR (KBr): 1716 (CO), 1628 and
1589 (CN) cm−1. 1H NMR (300 MHz, DMSO-d6): 1.98 and 2.12 (s,
3H, CH3), 3.23 and 3.36 (s, 3H, N−CH3), 4.71 (s, 2H, CH2), 5.49 (s,
1H, CH), 6.94−7.00 (m, 3H, Ar), 7.25−7.30 (m, 3H, Ar), 7.37 (broad s,
4H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 14.7 (CH3), 26.3 and 29.8
(N−CH3), 50.0 (CH), 70.7 (CH2), 114.7 (CH, Ar), 120.9 (CH, Ar),
128.3 (CH, Ar), 128.5 (CH, Ar), 128.8 (CH, Ar), 129.4 (CH, Ar), 136.5
(C−N, Ar), 158.0 (CN), 161.5 (C−O, Ar), 163.8 (S−CN), 172.9
(CO). HRMS (ESI): 354.0728 [M + H]+. Anal. Calcd for
C18H17SO2N3: C, 63.70; H, 5.05; N, 12.38; S, 9.45; found: C, 63.65;
N, 12.28; H, 5.13; S, 10.31.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-phenyl-5-methyl-
thiazolidin-4-one (16). Crystallization from toluene afforded yellowish
crystals, yield = 0.3 g, 84%. Mp (°C): 138. IR (KBr): 1722 (CO),
1631 and 1577 (CN) cm−1. 1H NMR (400 MHz, DMSO-d6): 1.61 (d,
3H, J 7.2 Hz, CH3), 1.80 and 1.97 (s, 3H, CH3), 4.40 (q, 1H, J 7.2 Hz,
CH), 4.67 (s, 2H, CH2), 6.73 (d, 2H, J 8.0 Hz, Ar), 6.93 (m, 1H, Ar),
7.23 (t, 2H, J 7.9 Hz, Ar), 7.38−7.51 (m, 5H, Ar). 13C NMR (100 MHz,
DMSO-d6): 18.8 and 19.4 (CH3), 26.2 (CH3), 41.2 (CH), 64.5 (CH2),
114.0 (CH, Ar), 121.0 (CH, Ar), 127.9 (CH, Ar), 128.4 (CH, Ar), 128.7
(CH, Ar), 129.6 (CH, Ar), 135.0 (C−N, Ar); 157.6 (CN), 160.9
(C−O, Ar), 164.4 (S−CN), 174.6 (CO). HRMS (ESI): 354.1264
[M + H]+. Anal. Calcd for C19H19SO2N3: C, 64.57; H, 5.42; N, 11.89; S,
9.07; found: C, 64.50; H, 5.39; S, 10.89; N, 11.80.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-phenyl-5-ethyl-
thiazolidin-4-one (17). Crystallization from cyclohexane/toluene (1:1)
afforded colorless crystals, yield = 0.26 g, 70%. Mp (°C): 118. IR (KBr):
1
1722 (CO), 1634 and 1576 (CN) cm−1. H NMR (400 MHz,
DMSO-d6): 1.01 (t, J 7.2 Hz, 3H, CH3), 1.81 (s, 3H, CH3), 1.89−1.98
(m, 1H, CH2), 2.03−2.09 (m, 1H, CH2), 4.38−4.41 (q, 1H, J 7.2 and 4.4
Hz, CH), 4.66 (s, 2H, CH2), 6.92−6.98 (m, 3H, Ar), 7.26−7.30 (m, 2H,
Ar), 7.34 and 7.36 (two s, 2H, Ar), 7.41−7.44 (m, 1H, Ar), 7.48−7.51
(m, 2H, Ar). 13C NMR (100 MHz, DMSO-d6): 10.1 (CH3), 14.6
(CH3), 25.7 (CH2), 48.1 (CH), 70.6 (CH2), 114.7 (CH, Ar), 120.9
(CH, Ar), 127.9 (CH, Ar), 128.5 (CH, Ar), 128.8 (CH, Ar), 129.4
(CH, Ar), 134.9 (C−N, Ar), 158.0 (CN), 161.5 (C−O, Ar), 163.6
(S−CN), 173.8 (CO). HRMS (ESI): 368.1422 [M + H]+. Anal.
Calcd for C20H21SO2N3: C, 65.37; H, 5.76; N, 11.44; S, 8.73; found: C,
65.31; H, 5.64; S, 8.07; N, 11.27.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-(4-tolyl)-5-ethyl-
thiazolidin-4-one (22). Crystallization from cyclohexane/toluene (1:1)
afforded colorless crystals, yield = 0.26 g, 68%. Mp (°C): 140. IR (KBr):
1
1722 (CO), 1634 and 1575 (CN) cm−1. H NMR (400 MHz,
DMSO-d6): 1.01 (t, J 7.2 Hz, 3H, CH3), 1.81 (s, 3H, CH3), 1.89−1.98
(m, 1H, CH2), 2.03−2.09 (m, 1H, CH2), 2.67 (s, 3H, CH3) 4.38−4.40
M
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(q, 1H, J 7.2 and 4.5 Hz, CH), 4.66 (s, 2H, CH2), 6.92−6.98 (m, 3H,
Ar), 7.20−7.27 (m, 2H, Ar), 7.29 (d, 2H, Ar), 7.60 (d, 2H, Ar). 13C
NMR (100 MHz, DMSO-d6): 10.1 (CH3), 14.6 (CH3), 23.5 (CH3),
25.7 (CH2), 48.1 (CH), 70.6 (CH2), 114.7 (CH, Ar), 120.9 (CH, Ar),
127.9 (CH, Ar), 128.5 (CH, Ar), 129.4 (CH, Ar), 134.9 (C−N, Ar),
158.0 (CN), 161.5 (C−O, Ar), 163.6 (S−CN), 174.1 (CO).
Anal. Calcd for C21H23SO2N3: C, 66.12; H, 6.08; N, 11.01; S, 8.41;
found: C, 66.10; H, 6.01; S, 9.10; N, 11.11.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(3-pyridinyl)-1,3-
thiazole (28). Crystallization from methanol afforded greenish crystals,
yield = 0.2 g, 61%. Mp (°C): 136. IR (KBr): 1621 and 1586 (CN)
cm−1. 1H NMR (400 MHz, DMSO-d6): 2.05 (s, 3H, CH3), 4.64 (s, 2H,
CH2), 5.5−6.5 (broad s, 1H, NH), 6.92−7.02 (m, 3H, Ar), 7.27−7.30
(m, 2H, Ar), 7.82 (t, 1H, J 10 Hz, Ar), 8.14 (s, 1H, CH of thiazole), 8.40
(d, 2H, J 10 Hz, Ar), 8.46 (t, 1H, J 10 Hz, Ar), 8.81 (s, 1H, Ar). 13C NMR
(100 MHz, DMSO-d6): 14.1 (CH3), 69.71 and 71.4 (CH2), 114.4
(CH, thiazole), 114.7 (CH, Ar), 120.9 (CH, Ar), 125.5 (CH, Ar), 129.1
(CH, Ar), 129.4 (CH, Ar), 131.2 (Ar), 137.0 (Ar), 148.2, 149.1 (CN),
158.0 (C−O, Ar), 169.4 (S−CN).
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-(4-anisyl)-5-ethyl-
thiazolidin-4-one (23). Crystallization from toluene afforded colorless
crystals, yield = 0.28 g, 70%. Mp (°C): 162. IR (KBr): 1722 (CO),
1630 and 1576 (CN) cm−1. 1H NMR (400 MHz, DMSO-d6): 1.00
(t, J 7.2 Hz, 3H, CH3), 1.81 (s, 3H, CH3), 1.89−1.98 (m, 1H, CH2),
2.02−2.08 (m, 1H, CH2), 3.82 (s, 3H, OCH3) 4.38−4.40 (q, 1H, J 7.2
and 5 Hz, CH), 4.66 (s, 2H, CH2), 6.92−6.98 (m, 3H, Ar), 7.20−7.27
(m, 2H, Ar), 7.29 (d, 2H, J 9.1 Hz, Ar), 7.69 (d, 2H, J 9.1 Hz, Ar). 13C
NMR (100 MHz, DMSO-d6): 11.0 (CH3), 14.6 (CH3), 25.7 (CH2),
48.1 (CH), 54.0 (CH3), 63.5 and 70.6 (CH2), 114.7 (CH, Ar), 120.9
(CH, Ar), 127.9 (CH, Ar), 128.5 (CH, Ar), 129.4 (CH, Ar), 134.9
(C−N, Ar), 155 (C−O, Ar) 157.0 (CN), 161.5 (C−O, Ar), 163.6
(S−CN), 174.1 (CO). Anal. Calcd for C21H23SO3N3: C, 63.45; H,
5.83; N, 10.57; S, 8.07; found: C, 63.44; H, 5.91; S, 8.99; N, 10.42.
2-((1-Phenoxypropan-2-ylidene)hydrazono)-3-(cyclohexyl)-5-eth-
ylthiazolidin-4-one (24). Crystallization from cyclohexane afforded
colorless crystals, yield = 0.26 g, 71%. Mp (°C): 139−141. IR (KBr):
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-pyridinyl)-1,3-
thiazole (29). Crystallization from methanol afforded greenish crystals,
yield = 0.23 g, 70%. Mp (°C): 140. IR (KBr): 3339 (N−H), 1617 and
1
1567 (CN) cm−1. H NMR (400 MHz, DMSO-d6): 2.03 (s, 3H,
CH3), 4.63 (s, 2H, CH2), 6.94−7.00 (m, 3H, Ar), 7.28 (broad s, 2H, Ar),
7.28 (d, J 9 Hz, 2H, Ar), 7.70 (d, J 9 Hz, 2H, Ar), 11.28 (broad s, 1H,
NH). 13C NMR and DEPT (100 MHz, DMSO-d6): 14.3 (CH3), 71.2
(CH2), 114.4 (CH, thiazole), 114.7 (CH, Ar), 120.9 (CH, Ar), 125.5
(CH, Ar), 129.1 (CH, Ar), 129.4 (CH, Ar), 131.2 (Ar), 137.0 (Ar),
148.2, 149.1 (CN), 158.0 (C−O, Ar), 169.4 (S−CN).
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-tolyl)-1,3-thia-
zole (30). Crystallization from methanol afforded yellowish crystals,
1
yield = 0.2 g, 60%. Mp (°C): 160. IR (KBr): 1618 (CN) cm−1. H
NMR (400 MHz, DMSO-d6): 2.03 (s, 3H, CH3), 2.32 (s, 3H, CH3),
4.63 (s, 2H, CH2), 6.17 (broad s, 1H, N−H), 6.92−7.00 (m, 3H, Ar),
7.20−7.30 (m, 5H, 4H of Ar and 1H of thiazole), 7.71−7.74 (m, 2H,
Ar). 13C NMR (100 MHz, DMSO-d6): 14.8 (CH3), 21.2 (CH3), 71.6
(CH2), 103.6 (CH, thiazole), 115.2 (CH, Ar), 121.4 (CH, Ar), 126.0
(CH, Ar), 128.0 (CH, Ar), 128.2 (CH, Ar), 129.3 (CH, Ar), 129.6
(CH, Ar), 129.9 (CH, Ar), 130.1 (Ar), 137.5 (Ar), 158.5 (C−O, Ar),
169.9 (S−CN). HRMS (ESI): 338.1327 [M + H]+. Anal. Calcd for
C19H19SON3: C, 67.63; H, 5.68; N, 12.45; S, 9.50; found: C, 67.60; H,
5.64; N, 12.23; S, 9.40.
1
1728 (CO), 1630 and 1576 (CN) cm−1. H NMR (400 MHz,
DMSO-d6): 1.00 (t, J 7.2 Hz, 3H, CH3), 1.81 (s, 3H, CH3), 1.89−1.98
(m, 1H, CH2), 2.02−2.08 (m, 1H, CH2), 3.82 (s, 3H, OCH3) 4.38−4.40
(q, 1H, J 7.2 and 5 Hz, CH), 4.66 (s, 2H, CH2), 6.92−6.98 (m, 3H, Ar),
7.20−7.27 (m, 2H, Ar), 7.29 (d, 2H, J 9.1 Hz, Ar), 7.69 (d, 2H, J 9.1 Hz,
Ar). HRMS (ESI): 374.1919 [M + H]+. Anal. Calcd for C20H27SO2N3:
C, 64.31; H, 7.29; N, 11.25; S, 8.58.; found: C, 64.31; H, 7.21; N, 11.39;
S, 9.00.
General Procedure for the Synthesis of 2-Imino-1,3-thiazoles 26−
45. Example for Compound 26: Thiosemicarbazone 4a (0.24 g,
1 mmol) dissolved in 2-propanol (5 mL) was transferred to a boiling
tube and placed in an ultrasound bath (40 MHz, 180 V). Then
2-bromoacetophenone (0.21 g, 1.1 mmol) was added to the mixture
and allowed to react until the consumption of the starting materials
(30−45 min). The reaction was cooled, and the colorful precipitate
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-anisyl)-1,3-thia-
zole (31). Crystallization from ethanol afforded pale greenish crystals,
yield = 0.25 g, 70%. Mp (°C): 168. IR (KBr): 1613 and 1601 (CN)
1
cm−1. H NMR (300 MHz, DMSO-d6): 1.99 and 2.04 (s, 3H, CH3),
3.76 (s, 3H, OCH3), 4.64 and 4.90 (s, 2H, CH2), 6.91−7.01 (m, 5H, Ar),
7.17 (s, 1H, CH of thiazole), 7.24−7.32 (d, 2H, J 2.5 Hz, Ar), 7.72−7.81
(d, 2H, J 2.5 Hz, Ar), 10.55 (broad s, 1H, N−H). 13C NMR and DEPT
(75.5 MHz, DMSO-d6): 14.6 (CH3), 55.2 (OCH3), 71.1 (CH2), 102.3
(CH, thiazole), 114.1 (CH, Ar), 113.7 (CH, Ar), 114.8 (CH, Ar), 121.0
(CH, Ar), 125.8 (CH, Ar), 127.1 (CH, Ar), 129.3 (CH, Ar), 129.6
(CH, Ar), 146.8 (CHC), 149.6 (CN), 158.1 (C−O, Ar), 159.2
(C−O, Ar), 169.5 (S−CN). HRMS (ESI): 354.1282 [M + H]+. Anal.
Calcd for C19H19SO2N 3: C, 64.57; H, 5.42; N, 11.89; S, 9.07; found: C,
64.60; H, 5.35; N, 11.80; S, 8.91.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-fluorophenyl)-
1,3-thiazole (32). Off-white crystals, yield = 0.29 g, 85%. Mp (°C): 157.
IR (KBr): 1619 and 1608 (CN) cm−1. 1H NMR (300 MHz, DMSO-
d6): 1.98 and 2.03 (s, 3H, CH3), 4.59 and 4.62 (s, 2H, CH2), 6.89−6.97
(m, 3H, Ar), 7.20−7.30 (m, 5H, 4H of Ar and 1CH of thiazole), 7.82−
7.87 (m, 2H, Ar), 10.53 (broad s, 1H, N−H). After addition of D2O, the
signal at δ 10.53 ppm decreased. 13C NMR (75.5 MHz, DMSO-d6): 14.7
(CH3), 71.2 (CH2), 104.3 (CH, thiazole), 114.5 (CH, Ar), 114.8 (CH,
Ar), 115.2 (CH, Ar), 121.1 (CH, Ar), 127.8 (CH, Ar), 127.9 (CH, Ar),
129.6 (CH, Ar), 129.8 (C−F), 130.0 (C−F), 146.7 (Ar), 146.9
(CHC), 148.5 (CN), 158.1 (C−O), 169.4 and 169.7 (S−CN).
HRMS (ESI): 342.1099 [M + H]+. Anal. Calcd for C18H16SOFN3: C,
63.32; H, 4.72; N, 12.31; S, 9.39; found: C, 63.40; H, 4.69; N, 12.30; S,
9.22.
was separated in a Buchner funnel with a sintered disc filter, washed
̈
with cold propanol followed by cold water, and then dried over SiO2
in a glass desiccator under vacuum. Pure products were obtained after
recrystallization using the solvent system detailed below for each
compound.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-phenyl-1,3-thia-
zole (26). Crystallization from methanol afforded pinkish crystals,
yield = 0.25 g, 77%. Mp (°C): 157. IR (KBr): 1618 (CN), cm−1. 1H
NMR (400 MHz, DMSO-d6): 2.03 (s, 3H, CH3), 4.63 (s, 2H, CH2),
6.07 (broad s, 1H, N−H), 6.93−7.01 (m, 3H, 2H Ar and 1H thiazole),
7.27−7.42 (m, 6H, Ar), 7.83−7.86 (m, 2H, Ar). 13C NMR and DEPT
(100 MHz, DMSO-d 6): 14.7 (CH3), 71.8 (CH2), 104.5 (CH, thiazole),
115.2 (CH, Ar), 121.4 (CH, Ar), 126.0 (CH, Ar), 128.0 (CH, Ar), 128.2
(CH, Ar), 128.7 (CH, Ar), 129.4 (CH, Ar), 129.9 (Ar), 134.7 (Ar),
148.1 (CCH, thiazole), 158.1 (C−O), 169.9 (S−CN). HRMS
(ESI): 324.1171 [M + H]+. Anal. Calcd for C18H17SON3: C, 66.85; H,
5.30; N, 12.99; S, 9.91; found: C, 66.52; H, 5.40; N, 13.00; S, 9.71.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(2-pyridinyl)-1,3-
thiazole (27). Crystallization from methanol afforded greenish crystals,
yield = 0.24 g, 74%. Mp (°C): 136. IR (KBr): 3364 (N−H), 1620 and
1
1582 (CN) cm−1. H NMR (400 MHz, DMSO-d6): 2.05 (s, 3H,
CH3), 4.65 (s, 2H, CH2), 5.5−6.5 (broad s, 1H, NH), 6.92−7.02 (m,
3H, Ar), 7.27−7.31 (m, 2H, Ar), 7.82 (t, 1H, J 8.0 and 9.6 Hz, Ar), 8.14
(s, 1H, CH thiazole), 8.35 (d, 1H, J 10 Hz, Ar), 8.46 (t, 1H, J 10 Hz, Ar),
8.75 (d, 1H, J 8 and 9.6 Hz, Ar). 13C NMR and DEPT (100 MHz,
DMSO-d6): 13.0 and 14.6 (CH3), 69.71 and 71.4 (CH2), 114.4 (CH,
thiazole), 114.8 (CH, Ar), 121.0 (CH, Ar), 123.1 (CH, Ar), 124.7 (CH,
Ar), 129.5 (CH, Ar), 143.3 (CH, Ar), 143.6 (CH, Ar), 144.8 (CH, Ar),
146.4 (Ar), 149.2 (CCH, thiazole), 158.0 (CN), 158.7 (C−O, Ar),
170.0 and 170.6 (S−CN). HRMS (ESI): 325.1123 [M + H]+.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-chlorophenyl)-
1,3-thiazole (33). Crystallization from ethanol afforded reddish crystals,
yield = 0.26 g, 72%. Mp (°C): 164−165. IR (KBr): 1620 and 1586
1
(CN) cm−1. H NMR (300 MHz, DMSO-d6): 2.09 (s, 3H, CH3),
4.63 and 4.90 (s, 2H, CH2), 6.90−7.00 (m, 3H, Ar), 7.25−7.46 (m, 5H,
4H of Ar and 1CH of thiazole), 7.80−7.85 (d, 2H, J 7.8 Hz, Ar), 10.61
(broad s, 1H, N−H). 13C NMR (75.5 MHz, DMSO-d6): 14.6 (CH3),
71.2 (CH2), 104.4 (CH, thiazole), 114.8 (CH, Ar), 121.0 (CH, Ar),
N
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125.7 (CH, Ar), 127.4 (CH, Ar), 127.9 (CH, Ar), 128.0 (CH, Ar), 128.4
(CH, Ar), 128.6 (CH, Ar), 129.6 (CH, Ar), 133.3 (Ar), 148.3 (CHC),
149.4 (CN), 158.1 (C−O, Ar), 169.6 (S−CN). HRMS (ESI):
358.0909 [M + H]+. Anal. Calcd for C18H16SOClN3: C, 60.41; H, 4.51;
N, 11.74; S, 8.96; found: C, 61.01; H, 4.62; N, 12.03; S, 8.81.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-bromophenyl)-
1,3-thiazole (34). Brownish crystals, yield = 0.8 g, 66%. Mp (°C): 171.
IR (KBr): 3317 (N−H), 1615 and 1572 (CN) cm−1. 1H NMR (400
MHz, DMSO-d6): 2.02 (s, 3H, CH3), 4.62 (s, 2H, CH2), 6.92−7.01 (m,
3H, Ar), 7.26−7.35 (m, 2H, Ar), 7.35 (s, 1H, CH thiazole), 7.58 (d, 2H,
J 7.5 Hz, Ar), 7.80 (d, 2H, J 7.5 Hz, Ar), 11.10 (s, 1H, N−H). 13C NMR
(100 MHz, DMSO-d6): 14.2 (CH3), 71.3 (CH2), 104.8, 114.7 (CH, Ar),
120.4 (CH, Ar), 120.9 (CH, Ar), 127.4 (CH, Ar), 129.4 (CH, Ar), 131.4
(CH, Ar), 133.9 (Ar), 147.1 (CC, thiazole), 149.3 (CN), 158.1
(C−O, Ar), 169.6 (S−CN). HRMS (ESI): 402.0272 [M + H]+. Anal.
Calcd for C18H16SOBrN3: C, 53.74; H, 4.01; N, 10.44; S, 7.97; found: C,
53.72; H, 4.02; N, 10.55; S, 7.57.
7.81 (d, 1H, J 8.4 Hz, Ar), 11.17 (broad s, 1H, N−H). 13C NMR (75.5
MHz, DMSO-d6): 14.2 (CH3), 71.3 (CH2), 110.2 (CH, thiazole), 114.8
(CH, Ar), 120.9 (CH, Ar), 128.7 (CH, Ar), 129.4 (CH, Ar), 129.9 (CH,
Ar), 130.7 (Ar), 131.1 (Ar), 131.7 (Ar), 134.3 (Ar), 145.9 (CHC),
147.3 (Ar), 158.1 (C−O), 168.7 (S−CN). HRMS (ESI): 426.7810
[M + H]+.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(tert-butylphenyl)-
1,3-thiazole (40). Crystallization from toluene afforded yellowish
crystals, yield = 0.3 g, 79%. Mp (°C): 180−182. IR (KBr): 1610 and
1
1586 (CN) cm−1. H NMR (300 MHz, DMSO-d6): 1.23 (broad s,
9H, 3xCH3), 2.00 (s, 3H, CH3), 4.89 (s, 2H, CH2), 6.92−7.00 (m, 3H,
Ar), 7.16−7.20 (d, 2H, J 7.1 Hz, Ar), 7.37−7.42 (m, 3H, 2H Ar, J 7.1 Hz,
and 1H of thiazole), 7.48−7.52 (m, 2H, Ar), 10.91 (broad s, 1H, N−H).
13C NMR (75.5 MHz, DMSO-d6): 14.2 (CH3), 31.1 (CH3), 32.8
(CCH ), 71.6 (CH2), 103.6 (CH, thiazole), 115.2 (CH, Ar), 121.4
3
(CH, Ar), 126.0 (CH, Ar), 128.1 (CH, Ar), 128.2 (CH, Ar), 129.1 (CH,
Ar), 129.2 (CH, Ar), 129.9 (Ar), 130.1 (Ar), 135.5 (Ar), 149.1 (CN),
158.5 (C−O, Ar), 169.9 (S−CN).
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(4-nitrophenyl)-
1,3-thiazole (35). Crystallization from 2-propanol afforded yellowish
crystals, yield = 0.3 g, 80%. Mp (°C): 171−173. IR (KBr): 3337 (N−H),
1598 and 1565 (CN) cm−1. 1H NMR (300 MHz, DMSO-d6): 2.03 (s,
3H, CH3), 4.63 (s, 2H, CH2), 6.92−7.01 (m, 3H, Ar), 7.29 (t, 2H, Ar),
7.70 (s, 1H, CH of thiazole), 8.07 (d, 2H, J 8.7 Hz, Ar), 8.29 (d, 2H, J 9.0
Hz, Ar), 11.26 (s, 1H, N−H). 13C NMR (75.5 MHz, DMSO-d6): 14.3
(CH3), 71.3 (CH2), 108.9 (CH, thiazole), 114.8 (CH, Ar), 121.0 (CH,
Ar), 124.1 (CH, Ar), 126.3 (CH, Ar), 129.5 (CH, Ar), 140.7 (CH, Ar),
146.1 (Ar), 147.6 (CHC), 148.5 (CN), 158.1 (C−O), 169.9
(S−CN). HRMS (ESI): 369.1072 [M + H]+.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(2-naphthalenyl)-
1,3-thiazole (41). Crystallization from toluene afforded brownish
crystals, yield = 0.26 g, 70%. Mp (°C): 180−182 (decomp). IR (KBr):
1614 and 1490 (CN) cm −1. 1H NMR (400 MHz, DMSO-d6): 2.05
(s, 3H, CH3), 4.64 (s, 2H, CH2), 6.95−7.02 (m, 3H, Ar), 7.30 (s, 2H, 1H
of Ar and 1H of thiazole), 7.47 (d, 3H, Ar), 7.91−8.01 (m, 4H, Ar), 8.39
(s, 1H, Ar), 11.14 (broad s, 1H, N−H). 13C NMR and DEPT (100 MHz,
DMSO-d6): 14.2 (CH3), 71.4 (CH2), 104.7 (CH, thiazole), 114.7 (CH,
Ar), 120.9 (CH, Ar), 123.9 (CH, Ar), 124.0 (CH, Ar), 125.9 (CH, Ar),
126.3 (CH, Ar), 127.5 (CH, Ar), 128.0 (CH, Ar), 128.0 (CH, Ar), 129.4
(CH, Ar), 132.1 (CH, Ar), 132.3 (CH, Ar), 138.1 (Ar), 147.1 (CHC),
158.1 (C−O, Ar), 169.5 (S−CN). HRMS (ESI): 374.1270 [M + H]+.
Anal. Calcd for C22H19SON3: C, 70.75; H, 5.13; N, 11.25; S, 8.59; found:
C, 70.68; H, 5.10; N, 11.27; S, 8.62.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(3-nitrophenyl)-
1,3-thiazole (36). Crystallization from toluene afforded yellowish
crystals, yield = 0.18 g, 48%. Mp (°C): 159−160. IR (KBr): 3337 (N−
1
H), 1598 and 1565 (CN) cm−1. H NMR (400 MHz, DMSO-d6):
2.64 (s, 3H, CH3), 5.15 (s, 2H, CH2), 7.40 (t, 1H, Ar), 7.49 (d, 2H, Ar),
7.75 (t, 2H, Ar), 7.89 (s, 1H, CH of thiazol), 8.10 (t, 1H, J 8.0 Hz, Ar),
8.56 (d, 1H, J 8.0 Hz, Ar), 8.70 (d, 1H, J 8.0 Hz Ar), 10.58 (broad s, 1H,
N−H). 13C NMR and DEPT (100 MHz, DMSO-d6): 23.5 (CH3), 82.0
(CH2), 116.7 (CH, thiazole), 125.4 (CH, Ar), 130.8 (CH, Ar), 131.5
(CH, Ar), 132.3 (CH, Ar), 139.9 (CH, Ar), 140.3 (CH, Ar), 141.8 (Ar),
147.3 (Ar) 157.8 (CHC), 159.3 (CN), 169.2 (C−O, Ar), 180.1
(S−CN). HRMS (ESI): 369.1042 [M + H]+.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(2-furanyl)-1,3-
thiazole (42). Crystallization from methanol afforded brownish crystals,
yield = 0.15 g, 50%. Mp (°C): 210. IR (KBr): 1600 and 1576 (CN)
cm−1. 1H NMR (400 MHz, DMSO-d6): 2.02 (s, 3H, CH3), 4.61 (s, 2H,
CH2), 6.58 (two s, 2H, Ar and CH thiazole), 7.00 (broad s, 4H Ar), 7.25
(d, 2H, Ar), 7.67 (s, 1H, Ar), 11.21 (s, 1H, N−H). 13C NMR and DEPT
(100 MHz, DMSO-d6): 14.2 (CH3), 71.3 (CH2), 102.9 (CH, thiazole),
106.0 (CH, Ar), 111.6 (CH, Ar), 114.7 (CH, Ar), 120.9 (CH, Ar), 129.5
(CH, Ar), 142.4 (CH, Ar), 147.2 (C−O, Ar), 150.3 (CN), 158.1
(C−O, Ar), 170.0 (S−CN).
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(3,4-dichlorophen-
yl)-1,3-thiazole (37). Crystallization from methanol afforded reddish
crystals, yield = 0.26 g, 66%. Mp (°C): 160. IR (KBr): 3338 (N−H),
1615 and 1565 (CN) cm−1. 1H NMR (400 MHz, DMSO-d6): 2.01 (s,
3H, CH3), 4.61 (s, 2H, CH2), 6.92−7.00 (m, 3H, Ar), 7.26−7.30 (m,
3H, Ar), 7.50 (s, 1H, 1H thiazole), 7.64 (d, 1H, J 8 Hz, Ar), 7.82 (d, 1H,
J 8 Hz, Hz, Ar), 8.07 (s, 1H, Ar), 11.10 (broad s, 1H, N−H). 13C NMR
and DEPT (100 MHz, DMSO-d6): 14.2 (CH3), 71.3 (CH2), 106.2 (CH,
thiazole), 114.7 (CH, Ar), 120.9 (CH, Ar), 125.5 (CH, Ar), 127.1
(CH, Ar), 129.4 (CH, Ar), 129.6 (CH, Ar), 130.8 (CH, Ar), 131.4 (Ar),
135.2 (Ar), 147.5 (CHC), 147.7 (CN), 158.1 (C−O, Ar), 169.7
(S−CN). HRMS (ESI): 392.0378 [M + H]+.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(2-thiophenyl)-1,3-
thiazole (43). Crystallization from methanol afforded brownish crystals,
yield = 0.15 g, 53%. Mp (°C): 190−191. IR (KBr): 1620 and 1590
1
(CN) cm−1. H NMR (400 MHz, DMSO-d6): 2.02 (s, 3H, CH3),
4.61 (s, 2H, CH2), 6.58 (two s, 2H, Ar and CH thiazole), 7.00 (broad s,
4H Ar), 7.25 (d, 2H, Ar), 7.67 (s, 1H, Ar), 10.53 (s, 1H, N−H). 13C
NMR (100 MHz, DMSO-d6): 14.2 (CH3), 71.3 (CH2), 104.0 (CH,
thiazole), 106.0 (CH, Ar), 111.6 (CH, Ar), 114.7 (CH, Ar), 120.9 (CH,
Ar), 129.5 (CH, Ar), 141.3 (CH, Ar), 142.4 (CH, Ar), 147.2 (C−O, Ar),
150.3 (CN), 158.1 (C−O, Ar), 170.0 (S−CN).
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(2,4-dichlorophen-
yl)-1,3-thiazole (38). Crystallization from methanol afforded reddish
crystals, yield = 0.17 g, 55%. Mp (°C): 200. IR (KBr): 1568 (CN)
cm−1. 1H NMR (400 MHz, DMSO-d6): 2.01 (s, 3H, CH3), 4.63 (s, 2H,
CH2), 6.92−7.00 (m, 3H, Ar), 7.26−7.30 (m, 2H, Ar), 7.36 (s, 1H, CH
of thiazole), 7.47−7.49 (d, 1H, J 8.4 Hz, Ar), 7.66 (s, 1H, Ar), 7.89 (d,
1H, J 8.4 Hz, Ar), 11.25 (broad s, 1H, N−H). 13C NMR (100 MHz,
DMSO-d6): 14.2 (CH3), 71.3 (CH2), 109.4 (CH, thiazole), 114.8 (CH,
Ar), 120.9 (CH, Ar), 127.4 (CH, Ar), 129.4 (CH, Ar), 129.6 (CH, Ar),
131.5 (CH, Ar), 132.1 (CH, Ar), 132.2 (CH, Ar), 132.3 (Ar), 135.2 (Ar),
145.8 (CHC), 147.2 (CN), 158.1 (C−O, Ar), 168.6 (S−CN).
HRMS (ESI): 392.0378 [M + H]+.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-3-methyl-4-phenyl-
1,3-thiazole (44). Prepared from thiosemicarbazone 4b, crystallization
from toluene afforded yellowish crystals, yield = 0.25 g, 73%. Mp (°C):
135. IR (KBr): 1591 (CN) cm−1. 1H NMR (300 MHz, DMSO-d6):
2.03 and 2.16 (s, 3H, CH3), 3.47 (s, 3H, N−CH3), 4.76 and 5.07 (s, 2H,
CH2), 6.91−6.99 (m, 3H, Ar), 7.02 (s, 1H, S−CH), 7.25−7.31 (m, 2H,
Ar), 7.52 (m, 6H, Ar). 13C NMR (75.5 MHz, DMSO-d6): 15.4 (CH3),
35.4 (N−CH3), 70.6 (CH2), 114.3, 114.8, 121.0 (Ar), 128.9 (Ar), 129.1,
129.2, 129.5, 129.7, 129.8, 141.8 (CHC), 158.0 (CN), 158.7
(C−O, Ar), 169.6 (S−CN). HRMS (ESI): 338.1326 [M + H]+. Anal.
Calcd for C19H19SON3: C, 67.63; H, 5.68; N, 12.45; S, 9.50; found: C,
67.70; H, 5.80; N, 12.53; S, 9.31.
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-4-(2,3,4-trichloro-
phenyl)-1,3-thiazole (39). Crystallization from toluene afforded reddish
crystals, yield = 0.24 g, 50%. Mp (°C): 200 (decomp). IR (KBr): 3321
2-((1-Phenoxypropan-2-ylidene)hydrazinyl)-3,4-diphenyl-1,3-
thiazole (45). Prepared from thiosemicarbazone 4c, isolated as yellowish
crystals, yield = 0.3 g, 75%. Mp (°C): 136. IR (KBr): 1622 and 1591
1
1
(N−H), 1615 and 1565 (CN) cm−1. H NMR (300 MHz, DMSO-
(CN) cm−1. H NMR (400 MHz, DMSO-d6): 1.84 (s, 3H, CH3),
d6): 2.01 (s, 3H, CH3), 4.63 (s, 2H, CH2), 6.92−7.01 (m, 3H, Ar),
7.29 (t, 2H, Ar), 7.40 (s, 1H, CH of thiazole), 7.72 (d, 1H, J 8.5 Hz, Ar),
4.63 (s, 2H, CH2), 6.58 (s, 1H, CH thiazole), 6.92−7.00 (m, 3H,
Ar), 7.15 (d, 2H, Ar), 7.22−7.35 (m, 10H, Ar). 13C NMR (100 MHz,
O
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DMSO-d6): 14.6 (CH3), 71.2 (CH2), 101.3 (CH, thiazole), 114.7 (CH,
Ar), 120.7 (CH, Ar), 127.5 (CH, Ar) 128.0 (CH, Ar), 128.20 (CH, Ar),
128.28 (CH, Ar), 128.3 (CH, Ar), 128.6 (CH, Ar), 129.4 (CH, Ar), 130.8
(Ar), 137.6 (Ar), 139.3 (CCH), 156.9 (CN), 158.2 (C−O, Ar),
168.6 (S−CN). Anal. Calcd for C24H21SON3: C, 72.15; H, 5.30; N,
10.52; S, 8.03; found: C, 72.14; H, 5.31; N, 10.42; S, 7.93.
and maintained in RPMI-1640 medium (Sigma Co.) supplemented with
10% FBS and 50 μg/mL gentamycin at 37 °C and 5% CO2.
Toxicity to Mouse Splenocytes. Splenocytes from BALB/c mice
were placed into 96-well plates at a cell density of 5 × 106 cells/well in
RPMI-1640 medium supplemented with 10% of FBS and 50 μg mL−1 of
gentamycin. Each test inhibitor was used in at least three concentrations
(1.0, 10, and 100 μg mL−1) in triplicate. To each well, an aliquot of test
inhibitor suspended in DMSO was added. Negative (untreated) and
positive (saponin) controls were used with every plate. The plate was
incubated for 24 h at 37 °C and 5% CO2. After incubation, 1.0 μCi of
3H-thymidine (Perkin-Elmer, Waltham, MA) was added to each well,
and the plate was returned to the incubator. The plate was then
transferred to a β-radiation counter (Multilabel Reader, Finland), and the
Molecular Modeling. The optimized structures of all compounds
were obtained by using the RM145 method, available as part of the
SPARTAN '08 program,46 using internal default settings for
convergence criteria. Some of these new molecules were synthesized
as racemic mixtures, so the molecular modeling treated the two isomers
(R and S) independently, when appropriate, and the docking procedure
used both isomers for each compound. Docking calculations and
analysis were carried using the T. cruzi cruzain (PDB ID code: 3IUT) as
the target,23 in which there was a cocrystallized complex with inhibitor
54 (referred to as “KB2”). The active site was defined as all atoms within
a radius of 6.0 Å from the cocrystallized ligand. The residues GLN19,
CYS25, SER61, LEU67, MET68, ASN70, ASP161, HIS162, TRP184,
and GLU208 were treated as flexible, using the conformation library for
each one. The GOLD 5.1 program47 was used for docking calculations,
followed by the Binana program,48 which was used to analyze the mole-
cular interactions present in the best docking solutions, using the default
setting, except for hydrogen bond distance, which was changed to a
maximum of 3.5 Å. Figures were generated with Pymol.49
Cruzain Inhibition. Recombinant cruzain was dissolved in acetate
buffer (0.1 M; pH 5.5) to a concentration of 0.1 μM. Ten microliters of
cruzain in DTT (5 mM) was distributed into a 96-well plate, and 150 μL
of the test inhibitor (previously dissolved in DMSO) in phosphate buffer
(in the presence of 0.001% EDTA, 100 mM NaCl, and DTT) was added
to the respective wells. The plate was incubated at room temperature
for 10 min. After this time, 340 μL of a solution containing the protease
substrate Cbz-Phe-Arg-7-aminomethylcoumarin (Z-FR-AMC, Sigma-
Aldrich Co., St. Louis, MO) was added to each well and the plate
incubated. The plate was read 1, 5, and 10 min after the addition of the
substrate (concentration of protein and substrate in the reaction was
0.5 nM and 2 μM, respectively). The percentage of cruzain inhibition
was calculated using the following equation: 100 − (A1/A × 100), where
A1 means the RFU of the cruzain in the presence of the test inhibitor,
and A means the RFU for the control well (cruzain plus substrate; no
test inhibitor). Each test inhibitor concentration was measured in
duplicate, and the IC50 values were estimated employing nonlinear
regression from at least nine different concentrations.
3
percent of H-thymidine was determined. Cell viability was measured
as the percent of 3H-thymidine incorporation for treated cells in
comparison to untreated cells. The highest nontoxic concentration was
estimated.
Antiproliferative Activity for Splenocytes. BALB/c mouse
splenocytes were placed into 96-well plates at a cell density of 5 × 106
cells/well in RPMI-1640 medium supplemented with 10% of FBS and
100 μg mL−1 of gentamycin. Then 2.0 μg/mL of concanavalin A (con A)
suspended in HEPES buffer and test inhibitors 5 and 18 (suspended in
DMSO) were added to each well. Controls include untreated plus con A,
without con A, and con A plus 0.01 μg/mL of cyclosporin A. Each
concentration was assayed in triplicate. The plate was incubated for 72 h
at 37 °C and 5% CO2. According to manufacturer’s recommendations,
AlamarBlue was added and the plate was incubated for 1 h until
AlamarBlue reduction by the living cells to the red fluorescent dye
resorufin. After excitation at 530 nm and fluorescence at 590 nm, the
signal was measured using a fluorescence microplate reader. Counts of
untreated plus con A wells were subtracted from counts of untreated
without con A wells. Inhibition (%) of cell proliferation was calculated in
comparison to untreated cells. The concentration that results in 50%
inhibition of cell proliferation (IC50) was calculated from six different
concentrations using nonlinear regression (Origin software).
Antiproliferative Activity for Epimastigotes. Epimastigotes
were counted in a hemocytometer and then dispensed into 96-well
plates at a cell density of 106 cells/well. Test inhibitors, dissolved in
DMSO, were diluted to five different concentrations (1.23, 3.70, 11.11,
33.33, and 100 μg/mL) and added to respective wells in triplicate.
The plate was incubated for 11 days at 26 °C, aliquots of each well
were collected, and the number of viable parasites were counted in a
Neubauer chamber and compared to untreated parasite culture. IC50s
were calculated using nonlinear regression on Prism 4.0 GraphPad
software. Two independent experiments were carried out; benznidazole
(LAFEPE, Brazil) and nifurtimox (Lampit, Roche) were used as the
reference inhibitors.
Toxicity for Y Strain Trypomastigotes. Trypomastigotes col-
lected from the supernatant of LLC-MK2 cells were dispensed into
96-well plates at a cell density of 4 × 105 cells/well. Test inhibitors,
dissolved in DMSO, were diluted to five different concentrations and
added into their respective wells, and the plate was incubated for 24 h
at 37 °C and 5% of CO2. Aliquots of each well were collected, and the
number of viable parasites, based on parasite motility, was assessed in
a Neubauer chamber. The percentage of inhibition was calculated in
relation to untreated cultures. IC50 calculation was also carried out using
nonlinear regression with Prism 4.0 GraphPad software. Benznidazole
and nifurtimox were used as the reference drugs.
Intracellular Parasite Development. Peritoneal exudate macro-
phages were seeded at a cell density of 2 × 105 cells/well in a 24-well
plate with rounded coverslips on the bottom in RPMI supplemented
with 10% FBS and incubated for 24 h. Cells were then infected with Y
strain trypomastigotes at a ratio of 10 parasites per macrophage for 2 h.
Free trypomastigotes were removed by successive washes using saline
solution. Each test inhibitor was dissolved in DMSO at 10 μM and
incubated for 6 h. The medium was replaced by a fresh medium, and
the plate was incubated for 4 days. Cells were fixed in methanol, and the
percentage of infected macrophages and the mean number of
amastigotes/infected macrophages were determined by manual counting
after Giemsa staining under an optical microscope (Olympus, Tokyo, Japan).
Cathepsins L and S Inhibition. Recombinant cathepsin L was
dissolved in acetate buffer (0.1 M; pH 5.5) and activated with 1 mM of
DTT. Test inhibitors were dissolved in DMSO and then diluted with
phosphate buffer containing BSA (0.1%) and EDTA (1 mM) in 0.001%
Tween. Protease substrate (Cbz-Phe-Arg-p-nitroanilide hydrochloride;
denoted ZFR-pNA) in DMSO was suspended in phosphate buffer.
Reactions were performed in 24-well plates. First, 90 μL of test inhibitor
was distributed into the respective well, followed by the addition of
10 μL of cathepsin L. The plate was incubated for 45 min at room
temperature. Then 100 μL of protease substrate was added, and the
reaction was incubated for 5 min. The concentration of the protein in
the reaction was 30 nM, and the concentration of the substrate was
5.0 μM. IC50 values were calculated as described for cruzain. The same
experiment was carried out for cathepsin S.
Animals. Female BALB/c mice (6−8 weeks old) were supplied by
the animal breeding facility at Centro de Pesquisas Gonca̧ lo Moniz
(Fundaca
̧
o Oswaldo Cruz, Bahia, Brazil) and Centro de Pesquisas
̃
Aggeu Magalhaes (Fundaca̧ o Oswaldo Cruz, Pernambuco, Brazil) and
̃
maintained in sterilized cages under a controlled environment, receiving
a balanced diet for rodents and water ad libitum. All experiments were
carried out in accordance with the recommendations from Ethical Issues
Guidelines and were approved by the local Animal Ethics Committee.
Parasites. Epimastigotes of T. cruzi (Y strain) were maintained at
26 °C in LIT medium (liver infusion tryptose) supplemented with 10%
fetal bovine serum (FBS) (Cultilab, Brazil), 1% hemin (Sigma Co., St.
Louis, MO), 1% R9 medium (Sigma Co), and 50 μg/mL gentamycin
(Novafarma, Brazil). Bloodstream trypomastigote forms of T. cruzi were
obtained from the supernatant of LLC-MK2 cells previously infected
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The percentage of infected macrophages and the number of amastigotes
per macrophage were determined by counting 100 cells per slide. To
estimate the IC50 values, compounds were tested in triplicate at 1.0, 10,
25, and 50 μM.
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Trypomastigote Invasion. Peritoneal macrophages (105 cells)
were plated onto 13-mm glass coverslips in a 24-well plate and allowed
to stand for 24 h. The plate was washed with saline solution, and then
trypomastigotes were added at a cell density of 1.25 × 107 along with the
addition of test inhibitor (at 50 μM). Amphotericin B (50 μM) was used
as reference inhibitor. The plate was incubated for 2 h at 37 °C and 5%
CO2, followed by five washes with saline solution to remove extracellular
trypomastigotes. Plates were maintained in RPMI medium supple-
mented with 10% FBS at 37 °C for 2 h. The number of infected cells was
counted with optical microscopy using a standard Giemsa staining.
Infection in Mice. Female BALB/c mice (6−8 weeks old) were
infected with bloodstream trypomastigotes by intraperitoneal injection
of 104 parasites in 100 μL of saline solution. Mice were then randomly
divided into groups (six mice per group). After day 5 of postinfection,
treatment with 250 μmol/kg weight of drug 18 was given orally by
gavage once a day for five consecutive days. For the control group,
benznidazole was also given orally at a dose of 250 μmol/kg weight.
Infection was monitored daily by counting the number of motile
parasites in 5 μL of fresh blood sample drawn from the lateral tail veins as
recommended by standard protocols.36
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Immune Evasion. Plos Pathog. 2011, 7, e1002139.
Statistical Analyses. To determine the statistical significance of
each group in the in vitro/in vivo experiments of infection, the one-way
ANOVA test and the Bonferroni for multiple comparisons were used.
A P value <0.05 was considered significant. The data are representative
of at least two experiments.
ASSOCIATED CONTENT
* Supporting Information
■
S
(10) Aoki, M. P.; Cano, R. C.; Pellegrini, A. V.; Tanos, T.; Guinazu, N.
́
̃
L.; Coso, O. A.; Gea, S. Different Signaling Pathways Are Involved in
Cardiomyocyte Survival Induced by a Trypanosoma cruzi Glycoprotein.
Microbes Infect. 2006, 8, 1723−1731.
NMR, IR, and HRMS spectral data for intermediate compounds,
and details for the electron microscopy studies and pharmaco-
logical tests. This material is available free of charge via the
Internet at http://pubs.acs.org. Crystallographic data for
compounds 2, 4, and 12 can be obtained free of charge from
the Cambridge Crystallographic Data Centre (deposition
numbers 874915, 874916, and 897962, respectively, www.ccdc.
cam.ac.uk/data_request/cif).
(11) Monteiro, A. C.; Schmitz, V.; Svensjo, E.; Gazzinelli, R. T.;
Almeida, I. C.; Todorov, A.; de Arruda, L. B.; Torrecilhas, A. C.;
Pesquero, J. B.; Morrot, A.; Bouskela, E.; Bonomo, A.; Lima, A. P.;
Muller-Esterl, W.; Scharfstein, J. Cooperative Activation of TLR2 and
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Bradykinin B2 Receptor is Required for Induction of Type 1 Immunity
in a Mouse Model of Subcutaneous Infection by Trypanosoma cruzi. J.
Immunol. 2006, 177, 6325−6735.
AUTHOR INFORMATION
Corresponding Author
*Phone: +55 81-21268511, fax: +55 81-21268510; e-mail:
diogollucio@gmail.com.
(12) Andrade, D.; Serra, R.; Svensjo, E.; Lima, A. P.; Ramos-Jr, E. S.;
̈
■
Fortes, F. S.; Morandini, A. C.; Morandi, V.; Soeiro, M. N.; Tanowitz, H.
B.; Scharfstein, J. Trypanosoma cruzi Invades Host Cells Through the
Activation of Endothelin and Bradykinin Receptors: a Converging
Pathway Leading to Chagasic Vasculopathy. Br. J. Pharmacol. 2012, 165,
1333−1347.
Notes
(13) Du, X.; Guo, C.; Hansell, E.; Doyle, P. S.; Caffrey, C. R.; Holler, T.
P.; McKerrow, J. H.; Cohen, F. E. Synthesis and Structure−Activity
Relationship Study of Potent Trypanocidal Thio Semicarbazone
Inhibitors of the Trypanosomal Cysteine Protease Cruzain. J. Med.
Chem. 2002, 45, 2695−2707.
(14) Chiyanzu, I.; Hansell, E.; Gut, J.; Rosenthal, P. J.; McKerrow, J. H.;
Chibale, K. Synthesis and Evaluation of Isatins and Thiosemicarbazone
Derivatives Against Cruzain, Falcipain-2 and Rhodesain. Bioorg. Med.
Chem. Lett. 2003, 13, 3527−3530.
(15) Greenbaum, D. C.; Mackey, Z.; Hansell, E.; Doyle, P.; Gut, J.;
Caffrey, C. R.; Lehrman, J.; Rosenthal, P. J.; McKerrow, J. H.; Chibale, K.
Synthesis and Structure−Activity Relationships of Parasiticidal
Thiosemicarbazone Cysteine Protease Inhibitors against Plasmodium
falciparum, Trypanosoma brucei, and Trypanosoma cruzi. J. Med. Chem.
2004, 47, 3212−3219.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work received support from CNPq (grant 471461/2011-3
to A.C.L.L.), CAPES (grant 23038.003155/2011-37 to
V.R.A.P.), FAPESB (PRONEX grant to M.B.P.S.), and European
Union ChemBioFight (grant reference 269301 to M.B.P.S.).
D.R.M.M. received a CNPq doctoral scholarship and a CAPES-
Fulbright Foundation exchange doctoral sponsorship. A.C.L.L.,
C.A.S., and M.B.P.S. hold a CNPq senior fellowship. The authors
acknowledge Prof. James McKerrow for providing recombinant
́
cruzain and plasmid. C.A.S. also thanks the Instituto de Fisica de
Sao Carlos (University of Sao Paulo, Brazil) for allowing the use
̃
of a Kappa CCD diffractometer.
(16) Fujii, N.; Mallari, J. P.; Hansell, E. J.; Mackey, Z.; Doyle, P.; Zhou,
Y. M.; Gut, J.; Rosenthal, P. J.; McKerrow, J. H.; Guy, R. K. Discovery of
Potent Thiosemicarbazone Inhibitors of Rhodesain and Cruzain. Bioorg.
Med. Chem. Lett. 2005, 15, 121−123.
(17) Siles, R.; Chen, S. E.; Zhou, M.; Pinney, K. G.; Trawick, M. L.
Design, Synthesis, and Biochemical Evaluation of Novel Cruzain
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