Synthesis and anti-Candida activity of novel 2-hydrazino-1,3-thiazole derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.01.039

Eighteen new hydrazino-1,3-thiazole derivatives were evaluated against 8 strains of multi-resistant Candida spp. Introduction of an indolyl moiety linked to the hydrazone function enhanced the in vitro anti-Candida activity, with an activity spectrum towards Candida albicans strains. Introduction of a (S)-2-aminoethyl chain on the thiazole nucleus largely enhanced the in vitro antifungal activity, with a selectivity oriented towards non-C. albicans species.

Full text of DOI:10.1016/j.bmcl.2013.01.039

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1803–1807
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-Candida activity of novel 2-hydrazino-1,3-thiazole
derivatives
b
a
d
c
Ludovic T. Maillard a, , Sébastien Bertout , Ophélie Quinonéro , Gülßsen Akalin , Gülhan Turan-Zitouni ,
⇑
Pierre Fulcrand a, Fatih Demirci e, Jean Martinez a, Nicolas Masurier a
a
´
´
Institut des Biomolecules Max Mousseron, UMR 5247, CNRS, Universites Montpellier I et II, UFR des Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault,
34093 Montpellier Cedex 5, France
b UMI 233 IRD-UM1-UY1-UCAD ‘TransVIHMI’ Equipe Infections parasitaires et fongiques liées au VIH UFR Sciences Pharmaceutiques et Biologiques, 15 Avenue Charles Flahault, BP
14491, 34093 Montpellier Cedex 5, France
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskißsehir, Turkey
d Department of Biochemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskißsehir, Turkey
e Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470 Eskißsehir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Eighteen new hydrazino-1,3-thiazole derivatives were evaluated against 8 strains of multi-resistant Can-
dida spp. Introduction of an indolyl moiety linked to the hydrazone function enhanced the in vitro anti-
Candida activity, with an activity spectrum towards Candida albicans strains. Introduction of a (S)-2-ami-
noethyl chain on the thiazole nucleus largely enhanced the in vitro antifungal activity, with a selectivity
oriented towards non-C. albicans species.
Received 30 October 2012
Revised 8 January 2013
Accepted 12 January 2013
Available online 23 January 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Thiazole
Hydrazone
Candida spp.
Antifungal activity
Candida albicans is a diploid opportunistic fungal pathogen pres-
ent in the gastrointestinal and genitourinary flora of most healthy
humans and other mammals. Among opportunistic fungal patho-
gens, C. albicans is the main causative agent of mucosal and deep-
seated mycoses. However, over the last two decades it was gradually
replaced by other Candida species in mucosal and systemic infec-
tions.1 One of the principal problems in the treatment of Candida
infections is the spread of antifungal drug resistance mainly in pa-
tients chronically subjected to antimycotic therapy and more
especially in severely immuno-compromised subjects, cancer,
transplants as well as surgery patients. The frequency of invasive
fungal infectionsas well as the antifungal resistance dramatically in-
crease despite the introduction of new drugs, like new azole or ech-
inocandin derivatives.2,3 In particular, a significant number of
Candida species (i.e., Candida glabrata and Candida krusei) that exhi-
bit high minimum inhibitory resistance (MIC) to azole-based agents
can acquire echinocandin resistance.4,5 As consequences, new drugs
are urgently needed to enhance the therapeutic arsenal to overcome
the rapid development of multi-resistant strains.
set of multi-resistant Candida strains (data not shown). Among the
tested compounds, the substituted 2-hydrazino-1,3-thiazole 1a
has shown an inhibitory activity (MIC of 16 lg/mL) against one
resistant strain of Candida krusei (Fig. 1 and Table 1). Few other
2-hydrazino-1,3-thiazole analogues that exhibit significant antimi-
crobial activity have been recently reported in the literature.6–9
Those bearing a bicyclic or heterocyclic ring on the hydrazone
function and a phenyl at C-4 position of the thiazole nucleus are
particularly active and present micromolar or submicromolar
MIC against several Candida spp. strains.6–8 Thus, such template
could be seen as a starting point for further optimization of novel
antifungal agents.
In order to develop new potent anti-fungal compounds, a
new set of 2-hydrazino-1,3-thiazoles based on compound 1a was
synthesized and their anti-Candida activity were studied. Two series
of 2-hydrazino-1,3-thiazoles were designed. In the first series (series
A, Fig. 1), the disubstituted phenyl ring was preserved and the in-
dane moiety on the hydrazino-part was replaced by unsaturated
and saturated carbocycles (cyclohexyl and naphthyl), oxo-
heterocycles (furyl and benzofuryl) and aza-heterocycles (pyrrolyl,
imidazolyl, indolyl, imidazo[1,2-a]pyridinyl and imidazo[2,1-b]
thiazolyl). The choice of such aza-heterocycles was due to our
particular interest in this field.12 In series B, substituted
In ongoing work to identify new antifungal derivatives, we have
preliminary screened a library of heterocyclic compounds toward a
⇑
Corresponding author. Tel.: +33 4 11 75 96 04; fax: +33 4 11 75 96 41.
E-mail address: ludovic.maillard@univ-montp1.fr (L.T. Maillard).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.039

1804
L. T. Maillard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1803–1807
H
S
N
N
N
HO
OCH3
1a
Compound
Modulation of the
indane moity
Replacement of the phenol
moiety by substituted amino-
methyl groups
R1
H
N
H
S
S
N
R2
N
N
N
N
H
N
R4
HO
OCH3
R3
Serie A, componds 1b-k
Serie B, compounds 2a-h
R1, R2 = H, carbocyles or heterocycles
R
3 = lateral chains of natural amino-acids
R4 = H or Z
Figure 1. Structures of lead compound 1a and new synthesized compounds.
aminomethyl groups were introduced instead of the phenol part to
increase the solubility of indanyl-thiazolylhydrazones.
diazoketones 7a–d were obtained by carboxylic acid activation
with isobutyl chloroformate (IBCF) in the presence of triethyl-
amine at 0 °C followed by subsequent diazomethane treatment.
The 2-hydrazino-1,3-thiazole scaffold was easily accessed by
Hantzsch cyclization between appropriate thiosemicarbazones
Compounds 7a–d conversion into the corresponding
a-chlorom-
and
a
-chloromethylketones.6–8,10,11 To synthesize series A, appro-
ethylketones 8a–d was performed by treatment with HCl in
diethyl ether. Thereafter, 8a–d were successively condensed with
thiosemicarbazone 4a to access 2-hydrazino-1,3-thiazoles 2a–d
in 53–81% yields. Final deprotection of the benzyloxycarbonyl
group was initially envisioned by hydrogenolysis with activated
palladium on barium sulfate. However, under these conditions,
deprotection failed. Finally, treatment of compounds 2a–d with
33% HBr in acetic acid and subsequent treatment by HCl in
diethyl ether offered the corresponding amino derivatives 2e–h
as hydrochloride salts. Compounds 2e, f, h were isolated in good
yields (67–93%) while 2g was obtained in only 20% yield. For this
reason, this compound was not evaluated for its antifungal
activity. As expected the introduction of a basic nitrogen on
the scaffold enhanced the water solubility of compounds 2e–h
as compared to 1a (Table 1).
priate carbonyl compounds 3a–k were reacted with thiosemicar-
bazide in refluxing ethanol. Compounds 3a–j were commercially
available, while compound 3k was prepared by Vilsmeier–Hack
formylation of 3-methyl-6-phenylimidazo[2,1-b][1,3]thiazole 6,
according to the method of Andreani (Scheme 1).13 The selectivity
of the formylation was attested by 1H NMR analysis: disappearance
of the 7.57 ppm singlet signal of H-5 of compound 6 and presence
of a singlet at 6.50 ppm of H-2 of the thiazole nucleus. Finally, the
thiosemicarbazones 4a–k were subsequently condensed with the
2-chloro-1-(2-hydroxy-5-methoxyphenyl)-ethanone 514 to offer
thiazolylhydrazones 1a–k, which were isolated in 37–92% yields
(Scheme 1).
For series B,
a-chloromethylketones 8a–d were synthesized
from natural N-benzyloxycarbonyl (Z) protected amino-acids,
that were, alanine, phenylalanine, tryptophan or proline
(Scheme 2), according to literature procedures.15–17 Briefly,
All synthesized compounds were fully characterized by analyt-
ical and spectral data. Hydrazones 1a–k and 2a–h could exist as
O
OH
Cl
HO
S
S
S
R1
R1
R2
R1
R2
NH2
5
Ethanol
Δ
NH2
OMe
N
N
HN
+
O
N
NH
H2N NH
Ethanol, Δ R2
OMe
3a-k
4a-k
1a-k
: R1 = Me, R2 = Indanyl
a
b: R1 = H, R2 = 1,3-Benzodioxol-5-yl
c: R1, R2 = Cyclohexyl
d: R1 = H, R2 = Napht-2-yl
S
S
N
1
: R = H, R2 = Indol-3-yl
e
POCl3
DMF, CHCl3
N
1
2
N
f
: R = H, R = Imidazo[1,2-a]pyridin-3-yl
N
Me
g: R1 = H, R2 = Benzofuran-2-yl
h: R1 = H, R2 = Pyrrol-3-yl
i: R1 = H, R2 = Fur-2-yl
Me
Ph
Ph
6
3k
CHO
1
2
j
k
: R = H, R = Imidazol-5-yl
1
2
: R = H, R = 3-Methyl-6-phenylimidazo-
[2,1-b]thiazol-5-yl
Scheme 1. Synthesis of compounds 1a–k (series A).

L. T. Maillard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1803–1807
1805
O
O
O
1. NEt3, IBCF
2. CH2N2
N2
NHZ
NHZ
HCl
Cl
NHZ
HO
Et2O
R
R
R
8a-d
7a-d
H
N
NH2
H
N
N
S
R
S
a, e : R = Me
b f
4a
N
,
: R = Bn
N
c g
,
: R = CH2-indole
EtOH, Δ
NHX
2a-c
2e-g
: X = Z
HBr, AcOH
DCM, RT
: X = H
1. NEt3, IBCF
H
N
O
2. CH2N2
S
X
N
N
3. HCl, Et2O
HO
N
4a
4. , EtOH, Δ
NX
2d
: X = Z
HBr, AcOH
DCM, RT
2h
: X = H
Scheme 2. Synthesis of compounds 2a–h (series B).
couples of diastereoisomers E/Z. However, in each case only one
isomer was detected by HPLC, 1H and 13C NMR analysis. Configura-
tion of 1a–k and 2a–h could not be unambiguously assigned by
NMR studies. However analytical studies of the E/Z hydrazine
interconversion reported in the literature indicates that the (E)-
form dominates on (Z)-form.18
Table 1
Minimal inhibitory concentration (MIC) of compounds 1a–k, 2a–f and 2h, amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin against eight strains of Candida
spp.
aCalculated logD (pH 7.4) of compounds 1 and 2 (data obtained from ACD/Labs, ACD/cLogD, Advanced Chemistry Development, Inc., Toronto, Canada, 2010).
bDetermined in PBS buffer at pH 7.0.
cClinical fluconazole resistant C. albicans isolate.
dClinical azoles resistant C. albicans isolate.
eC. albicans caspofungin resistant laboratory strain.
fClinical voriconazole resistant C. glabrata isolate.
gClinical voriconazole resistant isolate.
hC. glabrata fluconazole resistant reference strain.
iC. glabrata laboratory strain of C. parapsilosis, anidulafungin resistant.
jReference strain of C. parapsilosis sensible to reference drugs.

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L. T. Maillard et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1803–1807
Table 2
hydrophobicity balance was already proved to be important in
such series to obtain anti-Candida activity.8 Finally, some activity
was recovered for the substituted imidazo[2,1-b]thiazolyl deriva-
tive (compound 1k). This compound showed a relative high clogD
(5.19), which suggested the partial influence of this parameter to
obtain activity in this series.
In the second series (series B, compounds 2a–h, Scheme 2, Ta-
ble 2), we replaced the phenol moiety at C-4 of the thiazole ring
in 1a by different aminoalkyl substituents. When the amino group
was protected with a Z group, no significant anti-Candida activity
was detected (compounds 2a–d). Among the unprotected deriva-
tives (compounds 2e–f and 2h), compound 2e showed an interest-
ing activity against several Candida spp., with a selectivity oriented
towards non-Candida albicans species. In particular, a high activity
was detected against the C. krusei 6258 strain, which presents a
multi-resistant profile against several reference drugs. To deter-
mine the selectivity of 2-hydrazino-1,3-thiazoles towards fungi,
the most active compounds 1d, 1e, 1k and 2e were finally evalu-
ated for their cytotoxic activity against mouse fibroblast cells.
Cytotoxicity of selected compounds to mouse fibroblast (NIH/3T3) cell line
Concentration (
l
g/mL)a
0.5
1
31.3
62.5
1d
1e
1k
2e
83.7 1.3
91.9 10.7
94.6 6.3
93.6 4.0
69.2 0.2
80.6 8.2
90.0 0.1
92.6 1.8
45.3 2.4
70.6 3.2
64.1 2.1
36.2 4.1
52.7 3.9
50.2 4.6
59.5 2.9
32.4 1.2
a
Values represent mean standard deviation of triplicate determinations.
Compounds 1a–k, 2a–f and 2h were evaluated for their anti-
fungal activity against 8 Candida spp. strains (C. albicans, C. glabra-
ta, C. krusei and Candida parapsilosis) and compared with five
reference drugs, that were, amphotericin B, itraconazole, fluconaz-
ole, voriconazole and caspofungin (Table 1). Several resistant
strains to one or several reference drugs (see Table 1) were chosen
to evaluate the potential activity of synthesized compounds
against multi-resistant Candida spp. Prior testing, each Candida
spp. isolate was subcultured on a qualified medium to ensure pur-
ity and optimal growth. The susceptibility assays were determined
by the microbroth dilution method performed in sterile flat-bot-
tom 96-well microplates (Difco Laboratories, Detroit, USA) as de-
scribed in CLSI guidelines, M27-A3 document.19 Compounds 1d,
1e, 1k and 2e that achieved the strongest anti-Candida activity
were also analyzed to estimate their cytotoxic effects (Table 2).
Cytotoxicity was evaluated using the MTT cell proliferation/viabil-
ity assay on mouse 3T3/NIH fibroblast cells, which were originally
obtained from the American Type Culture Collection (ATCC, USA).
The level of cellular MTT reduction was quantified as previously
described in literature with small modifications.20–22
The cytotoxic profile (Table 2) showed a weak toxicity at 0.5 lg/
mL for all tested compounds, with more than 83% of cell viability.
In conclusion, we have synthesized a series of hydrazino-1,3-
thiazoles based on compound 1a. Their in vitro anti-Candida activ-
ity was evaluated against 8 strains of multi-resistant Candida spp.
Preliminary structure activity relationships have shown that sub-
stitution of the hydrazine by a hydrogen bond donating heterocy-
cle enhanced the anti-Candida activity. In particular, introduction
of an indolyl substituent instead of the indanyl led to interesting
compounds, especially active against C. albicans strains. Moreover,
replacement of the phenol moiety in compound 1a by a (S)-2-ami-
noethyl chain (compound 2e) largely enhanced the antifungal
activity, with a selectivity oriented towards non Candida albicans
species. Based on these results, derivatives 1e and 2e represent
good starting points for the development of novel anti-Candida
spp. agents. Synthesis of next generation compounds combining
modifications on both sides of the scaffold will be reported in
due course.
In an initial screening program to determine potential anti-
Candida activities of 2-hydrazino-1,3-thiazoles, compound 1a was
chosen as the first hit for further derivatization (MIC of 16
lg/mL
against fluconazole/voriconazole resistant C. krusei strain).
a
Replacement of the indanyl nucleus by various aliphatic or aro-
matic carbocycles or heterocycles was initially studied (series A
compounds). Surprisingly when the indanyl nucleus was replaced
by the structurally related 1,3-benzodioxol-5-yl moiety (com-
pound 1b, Scheme 1, Table 1), the anti-Candida activity was lost.
Introduction of a cyclohexyl or a naphtyl ring was previously used
to design 2-hydrazino-1,3-thiazoles with good anti-Candida activi-
ties.7 In our case, insertion of a cyclohexyl (compounds 1c) or a
naphthyl ring (compound 1d) led to only a weak activity enhance-
ment. These disappointing results led us to switch to other modu-
lations consisting in the introduction of various aza-heterocycles.
The best activities were obtained when the indanyl substituent
was replaced by an indole ring: compound 1e presented a good
anti-Candida activity, with a selectivity oriented towards C. albi-
cans. In particular, this compound showed similar or better activity
than reference drugs for several Candida strains. We assumed that
the biological activity was enhanced by the presence of a NH
hydrogen bond donor in the carbocyclic part. This hypothesis
was confirmed when we introduced an imidazo-[1,2-a]pyridinyl
substituent (compound 1f), which is usually viewed as an aza-
indolyl isostere.23,24 Imidazo[1,2-a]pyridine does not have any
hydrogen bond donating group making 1f inactive toward all Can-
dida strains. That was also the case when indole was replaced by
benzofurane (compound 1g). Similar results were obtained with
series containing a five-membered heteroaromatic substituent:
the pyrrole derivative 1h was more active than its furyl counter-
part 1i. Surprisingly, although imidazole had a hydrogen bond
donor, compound 1j was totally inactive whatever the strain con-
sidered. This might be due to a higher hydrophilicity of the mole-
cule (clogD = 1.53) compared to compound 1h (clogD = 2.03). The
Acknowledgments
The authors are grateful to G. Tambutet (Université Montpellier
2, France), C. Lamoureux and M. Aussedat (Faculté de Pharmacie de
Montpellier, France) for technical assistance.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.039.
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lg/mL for FCZ and 1600 lL/mL
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l
l
CAS. After 24 h and 48 h of incubation at 35 °C, MIC (minimum inhibitory
concentration) were determined visually by comparing its turbidity with the
drug-free growth control well. The MIC values were defined as lower drug