Efficient synthesis of novel 3-aryl-5-(4-chloro-2-morpholinothiazol-5-yl)- 4,5-dihydro-1h-pyrazoles and their antifungal activity alone and in combination with commercial antifungal agents

Article abstract of DOI:10.1002/ardp.201400084

The α,β-unsaturated carbonyl compounds 5a-f were prepared by reaction between 2-chloro-4-morpholinothiazol-5-carbaldehyde 3 and substituted acetophenones 4a-f. Treatment of compounds 5a-f with hydrazine hydrate employing mild reaction conditions led to the formation of 4,5-dihydro-1H-pyrazoles 6a-f. Then the treatment with acetic anhydride or formic acid afforded the expected 4,5-dihydro-1H-pyrazoles 7a-f and 8a-f. The antifungal activity of each series of synthesized compounds was determined against the clinically important fungi Candida albicans and Cryptococcus neoformans. In addition, the most active compounds 7e and 7f were tested in combination with the commercial antifungal agents: fluconazole, itraconazole, and amphotericin B. Compound 7e showed a synergistic effect with fluconazole against C. albicans while 7f showed synergistic activities with all tested antifungal drugs against the same yeast.

Full text of DOI:10.1002/ardp.201400084

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
1
Full Paper
Efficient Synthesis of Novel 3-Aryl-5-(4-chloro-2-
morpholinothiazol-5-yl)-4,5-dihydro-1H-pyrazoles and
Their Antifungal Activity Alone and in Combination with
Commercial Antifungal Agents
Juan Ramírez1, María Victoria Rodríguez2, Jairo Quiroga1, Rodrigo Abonia1, Maximiliano Sortino3,
Susana A. Zacchino3, and Braulio Insuasty1
1
Heterocyclic Compounds Research Group, Department of Chemistry, Universidad del Valle, Cali, Colombia
Área Biología Vegetal, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,
2
Rosario, Argentina
3
Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,
Rosario, Argentina
The a,b-unsaturated carbonyl compounds 5a–f were prepared by reaction between 2-chloro-4-
morpholinothiazol-5-carbaldehyde 3 and substituted acetophenones 4a–f. Treatment of compounds
5a–f with hydrazine hydrate employing mild reaction conditions led to the formation of 4,5-dihydro-
1H-pyrazoles 6a–f. Then the treatment with acetic anhydride or formic acid afforded the expected 4,5-
dihydro-1H-pyrazoles 7a–f and 8a–f. The antifungal activity of each series of synthesized compounds
was determined against the clinically important fungi Candida albicans and Cryptococcus neoformans. In
addition, the most active compounds 7e and 7f were tested in combination with the commercial
antifungal agents: fluconazole, itraconazole, and amphotericin B. Compound 7e showed a synergistic
effect with fluconazole against C. albicans while 7f showed synergistic activities with all tested
antifungal drugs against the same yeast.
Keywords: Antifungal activity / 2-Chloro-4-morpholinothiazol-5-carbaldehyde / Commercial antifungal agents / 4,5-
Dihydro-1H-pyrazoles / Synergistic effect
Received: March 3, 2014; Revised: April 21, 2014; Accepted: April 22, 2014
DOI 10.1002/ardp.201400084
Introduction
derivatives presented antimycobacterial and antimalarial
activity [15, 16]. In addition, some compounds containing
thiazole and thiazolidine nucleus are in current use as
therapeutic agents; abafungin, is a broad-spectrum anti-
fungal agent formulated as a topical cream for the
treatment of dermatomycoses [17], epalrestat is an aldo-
reductase inhibitor, approved for clinic use in Japan [18],
dasatinib, was approved as a first-line treatment for chronic
myelogenous leukemia by the National Cancer Institute
(NCI) [19, 20] (Fig. 1).
On the other hand, five membered nitrogen-containing
heterocyclic compounds like 2-pyrazolines have shown a wide
spectrum of biological activities such as antidepressant [21],
antifungal [22], anticancer [23–27], antimalarial [25, 28], and
antimicrobial activities [29, 30].
The use of thiazole-containing compounds in organic
synthesis has recently grown due to their synthetic
versatility [1], and their wide spectrum of biological
activities [2–10]. Regarding their antimicrobial activity,
new thiazole-cyano compounds containing morpholine in
their structures, showed antibacterial activity [11]; some
2,4-dichlorothiazole-5-carbaldehyde derivatives displayed
antifungal activity [12]; thiazole-based chalcones showed
antibacterial activity [13, 14], and hydrazinylthiazole
Correspondence: Dr. Braulio Insuasty, Faculty of Science, Heterocyclic
Compounds Research Group, Department of Chemistry, Universidad del
Valle, A.A. 25360 Cali, Colombia
E-mail: braulio.insuasty@correounivalle.edu.co
Fax: þ57 2 3392440
In the course of our ongoing research on the synthesis and
biological activities of pyrazolines added to our willingness to
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J. Ramírez et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Figure 1. Chemical structures of some thiazoles and thiazolidines used as therapeutic agents.
contribute to the research on the synthesis and biological Results and discussion
activities of new thiazole derivatives, we performed an
efficient synthesis of 12 novel 3-aryl-5-(4-chloro-2-morpholi-
nothiazole-5-yl)-4,5-dihydro-1H-pyrazoles containing both the
pyrazole and the thiazole moieties and tested them for
antifungal activities against two clinically important fungal
species: Candida albicans and Cryptococcus neoformans. The
selection of C. albicans as one of the target fungus species
was due to this fungus representing over 60% of all isolates
from clinical infections [31], and many treatment failures
were reported in the last 10 years associated to the high
appearance of resistant strains [32]. As a consequence, new
structures with anti-C. albicans activity are very welcome. In
turn, the selection of C. neoformans as the second fungal target
is due to this fungus producing cryptococcal meningitis that
has killed more than 650,000 immunocompromised patients
worldwide and whose treatment is based on medications that
were discovered nearly 50 years ago, since no new better drugs
have been recently incorporated to clinic [33]. To solve these
problems, many efforts have been undertaken into different
directions. One of them was to look for new structures that,
alone, possess anti-C. albicans and anti-C. neoformans activity
[34]. The other one consisted in finding new structures
that, acting in combination with antifungal agents [35–38],
produce an improvement of the activity. In this strategy, the
mixture of low doses of each component produce the same or
higher activity than each one alone, showing synergism or at
least additivism, avoiding the known toxicity of the antifun-
gal drugs and decreasing the occurrence of resistant strains.
Following this trend and driven by the fact that the
compounds of this series did not show much activity
when acting alone (see Results and Discussion section), we
tested the most active compounds in combination with
amphotericin B (AmB), itraconazole (ITZ), and fluconazole
(FCZ) against C. albicans and C. neoformans. These mixtures
showed that some compounds were synergistic only with
AmB, while other ones enhanced the activity of the three
antifungal agents.
Chemistry
New thiazole-based 4,5-dihydro-1H-pyrazoles 7a–f and 8a–f
were prepared from a,b-unsaturated carbonyl compounds
containing the thiazole ring 5a–f employing different path-
ways. In this sense to obtain the aldehyde 3, we carried out the
nucleophilic aromatic substitution (SNAr) with morpholine of
chlorine in aldehyde 1, according to the Kotlyar and co-
workers methodologies [39]. The treatment of equimolar
amounts of 3 with acetophenones 4a–f under Claisen–
Schmidt conditions afforded the a,b-unsaturated carbonyl
compounds 5a–f which, by treatment with hydrazine
hydrate, under mild reaction conditions gave 4,5-dihydro-
1H-pyrazoles 6a–f. As these compounds decomposed rapidly,
they were immediately isolated by filtration on vacuum and a
fraction of 0.3 mmol of each compound was treated with
acetic anhydride or formic acid at room temperature during
1 h to obtain the expected compounds 7a–f and 8a–f,
respectively, in good yields (54–86%) (Scheme 1).
Structure elucidation of pyrazole derivatives 7a–f and 8a–f
was performed based on their spectral data (FT-IR, H NMR,
13C NMR, and MS). The IR spectrum of compound 7b as
representative of the series showed absorption bands at 1672,
1595, and 1525 cmꢀ1 associated with C O, and C N, C C
functionalities, respectively. In the 1H NMR spectrum of
compound 7b, a singlet at d ¼ 2.40 ppm indicated the
presence of the acyl group and multiplets at d ¼ 3.39–
3.41 ppm and d ¼ 3.75–3.77 ppm integrating for 4H each
one assigned to methylenic protons of the morpholine ring
appeared. The two methylenic protons in C-4 and the
stereogenic proton in C-5 on dihydropyrazole unit form an
1
–
–
–
–
–
–
AMX spin system; in this sense, proton 4-HA appeared as a
2
double-doublet at d ¼ 3.24 ppm with coupling values JAM
¼
3
17.7 Hz and JAX ¼ 4.7 Hz, proton 4-HM exhibited a double-
2
doublet at d ¼ 3.70 ppm with coupling values JAM ¼ 17.7 Hz
and 3JMX ¼ 11.9 Hz and the proton 5-HX appeared as a double-
doublet at d ¼ 5.78 ppm with coupling constants 3JAX ¼ 4.7 Hz
3
and JMX ¼ 11.9 Hz. At aromatic region, two doublets at
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Thiazolylpyrazoles
3
Scheme 1. General methodology for the synthesis of thiazole-based 4,5-dihydro-1H-pyrazoles. i ¼ CH3CN, r.t., 4 h, ii ¼ substituted
acetophenones, EtOH, NaOH, r.t., 8 h.
d ¼ 7.43 and 7.69 ppm with coupling constant J ¼ 8.5 Hz
assigned to 4-chlorophenyl substituent were observed. The
13C NMR spectrum of compound 7b showed signals at d ¼ 21.8
and 53.2 ppm assigned to methyl from acyl group and C-5
of the dihydropyrazole unit, respectively. Also signals at
signal associated to carbonyl group at d ¼ 159.9 ppm, the
remaining signals appear with similar results as compound
7b (see Experimental section).
Antifungal activity studies
–
d ¼ 152.6 and 168.9 ppm assigned to C-3 and C O from N-acyl-
All synthesized compounds 7a–f and 8a–f were tested alone
against C. albicans ATCC 10231 and C. neoformans ATCC 32264
with the CLSI microplate design that is usually used for
determining the minimum inhibitory concentration [40]. The
inhibition percentages displayed by each compound at the
different concentrations (Table 1) were calculated with the aid
of a microplate reader, as explained in Experimental section.
As it can be observed in Table 1, 7e and 7f inhibited ꢁ50% of
C. albicans growth at 200 mg/mL, and six of them (7b, 7c, 8b, 8d,
8e, and 8f) inhibited 20–40% growth at the same concentra-
tion. Regarding C. neoformans, 7e, 7f, and 8d inhibited ꢁ50% of
C. neoformans growth at 200 mg/mL, while 7a–d, 8a–c, 8e, and 8f
showed inhibition percentages between 20 and 31% at the
same concentration. At lower concentrations (ꢂ100 mg/mL),
any compound overcame 30% inhibition of fungal growth.
–
dihydropyrazole functionality. With the help of DEPT-135
experiment, at d ¼ 40.6, 47.6, and 65.9 ppm appeared
the signals assigned to methylenic carbon atoms C-4,
–CH2–N–CH2–, and –CH2–O–CH2– of the dihydropyrazole
and morpholine units. Regarding to the N-formyl-4,5-dihydro-
1H-pyrazoles, the IR spectrum for compound 8b as represen-
tative of the series showed an absorption band associated to
–
the carbonyl group (C O) at 1679 and absorption bands at
–
1596 and 1529 cmꢀ1 associated with the C N and C C
–
–
–
–
functionalities. The 1H NMR spectrum of compound 8b
showed all signals with similar results in comparison with the
1H NMR spectrum of compound 8b, only appeared a new
singlet at d ¼ 8.89 ppm assigned to the proton from the formyl
group. The 13C NMR spectrum of compound 8b exhibited the
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Table 1. Antifungal activity (% inhibition) of compounds 7a–f and 8a–f against Candida albicans ATCC 10231 and Cryptococcus
neoformans ATCC 32264.
Candida albicans
Cryptococcus neoformans
Compound
200 mg/mL
100 mg/mL
50 mg/mL
200 mg/mL
100 mg/mL
50 mg/mL
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
0
0
0
0
31.14 ꢃ 1.9
15.35 ꢃ 1.2
27.79 ꢃ 0.1
13.60 ꢃ 1.7
69.97 ꢃ 3.9
57.59 ꢃ 1.2
20.34 ꢃ 1.7
22.63 ꢃ 1.6
17.96 ꢃ 2.3
65.35 ꢃ 1.1
24.07 ꢃ 1.5
24.77 ꢃ 1.8
100
30.33 ꢃ 3.3
12.04 ꢃ 0.2
25.52 ꢃ 0.3
12.07 ꢃ 2.8
16.16 ꢃ 1.7
23.59 ꢃ 1.3
3.13 ꢃ 0.4
18.70 ꢃ 1.3
13.72 ꢃ 1.6
20.76 ꢃ 2.1
18.42 ꢃ 1.1
3.25 ꢃ 1.1
100
27.59 ꢃ 1.7
11.87 ꢃ 1.5
22.32 ꢃ 0.4
10.51 ꢃ 0.6
10.87 ꢃ 1.4
15.14 ꢃ 2.3
0
9.29 ꢃ 0.3
12.30 ꢃ 1.3
19.91 ꢃ 0.1
15.83 ꢃ 1.2
0
40.09 ꢃ 2.5
29.16 ꢃ 1.8
0
5.80 ꢃ 0.2
4.70 ꢃ 1.2
0
2.28 ꢃ 0.1
0
0
65.90 ꢃ 2.6
50.85 ꢃ 2.8
13.59 ꢃ 0.4
23.93 ꢃ 0.5
16.30 ꢃ 2.0
33.42 ꢃ 1.7
36.20 ꢃ 0.6
27.51 ꢃ 0.5
100
8.28 ꢃ 0.5
17.29 ꢃ 1.1
7.44 ꢃ 0.6
13.00 ꢃ 1.3
7.20 ꢃ 0.3
21.20 ꢃ 1.1
19.19 ꢃ 1.6
15.02 ꢃ 0.2
100
3.77 ꢃ 0.7
5.18 ꢃ 0.1
11.84 ꢃ 1.3
4.26 ꢃ 1.0
12.72 ꢃ 1.4
8.96 ꢃ 0.2
9.81 ꢃ 1.1
100
8f
AmBa)
100
a)
Amphotericin B.
These results clearly showed that this series of compounds
possess moderate antifungal activity when acting alone. So,
we decided to test 7a–c, 8a–c in combination with the
antifungal drugs FCZ, AmB, or ITZ (each combination partner
at low doses) against C. albicans and C. neoformans, taking
advantage of both, the new structural type of these pyrazole
derivatives and the high activity of antifungal drugs. The aim
was to achieve high response rates with mixtures that contain
sub-inhibitory doses of antifungal drugs, thus avoiding the
known toxicity of them and the generation of resistance [41].
Regarding the method of choice to test the compounds in
combination with antifungal drugs, the high throughput
screening synergy assay (HTSS) developed by Zhang et al. [42],
was selected and applied to 7a–f, 8a–f at a first instance of
screening. This strategy consisted in testing (in holes of a
96-well microplate) a mixture formed by both the compound
and the antifungal drug, each one at a sub-optimal dose
(producing 10–20% of the maximum activity) against C.
albicans and C. neoformans by using FCZ as the drug partner
with the aim of finding mixtures which, in combination,
show an enhancement of activity, reaching ꢁ50% growth
inhibition (Fig. 2). This simplification, previously used to
screen microbial extracts [42] and plant extracts of Baccharis
genus [43], demonstrated to help in the identification of
synergistic mixtures.
quantified with the microplate reader, as explained in
Experimental section) is observed in (c) when an enhance-
ment of inhibition is observed. Only enhancements ꢁ50%
were considered of interest.
Results of the HTSS demonstrated that any compound in
combination with an antifungal drug showed enhanced
activity against C. neoformans. Instead, 7e and 7f were
identified as two hits against C. albicans. Compound 7f alone
displayed 17.29% inhibition of C. albicans growth (Table 1) at
the concentration tested (100 mg/mL) while the activity of
the mixture of this compound with sub-inhibitory doses
of FCZ (0.5 mg/mL) far surpassed the activity of the single
compound reaching 50% C. albicans growth inhibition.
In Fig. 2, bars represent the percentage of inhibition growth
of: (a) each tested compound (7a–f or 8a–f) at a concentration
that inhibits ꢂ20% of either C. albicans or C. neoformans growth;
(b) antifungal agent (FCZ) at a concentration that inhibits
ꢂ20% of fungal growth; (c) a mixture containing (a) and (b).
Above each bar (a), (b), or (c), it can be observed the wells after
24 h incubation at 30°C. The diminution of growth (that was
Figure 2. Top: Wells prepared as labeled, after incubation for
24 h in a moistured dark chamber at 30°C. Bars show the
percentage of growth inhibition measured with a microplate reader
of: (a) compound at a sub-inhibitory concentration (100 mg/mL) that
inhibits only ꢂ20% of fungal growth; (b) antifungal agent (flucon-
azole) at a sub-inhibitory concentration (0.05–0.5 mg/mL) that
inhibits ꢂ20% of fungal growth; (c) a mixture containing (a) and
(b) that produces ꢁ50% fungal growth inhibition.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Thiazolylpyrazoles
5
In turn, compound 7e, which produced an 8.28% inhibition of
C. albicans when acting alone, showed ꢁ50% growth inhibition
in the presence of FCZ.
Analysis of the combination effect
The nature of the interaction (synergism, additivism, or
antagonism) between 7e or 7f and FCZ, ITZ, or AmB, was
assessed by calculating the combination index (CI) method
[45]; the CI was calculated with the Eq. (1):
To determine if the enhancement observed for 7e and 7f
with the HTSS assay signified that the mixtures were
synergistic, antagonistic, or additive, a design used by Eid
et al. [44] was applied. For this study compounds 7e and 7f
were tested in mixtures not only with FCZ but with two other
antifungal drugs, ITZ and AmB against C. albicans. With this
new design, fungal cells were incubated with serial dilutions
of each antifungal drug (FCZ, ITZ, or AmB) in a 96-well
microplate, and in parallel, the same series of wells were
CAD;50
Ccomp;50
CI ¼
þ
ð1Þ
MIC50;AD MIC50;comp
CAD,50 is the concentration of AmB, FCZ, or ITZ in mixture
that produces 50% inhibition of fungal growth, datum
that was obtained from the dose–response curve of each
combination. MIC50,AD is the concentration of the antifungal
drug alone that produces 50% inhibition of the fungal growth.
Ccomp,50 is the concentration of compound (7e or 7f) in
the mixture, that produces 50% inhibition of fungal growth.
MIC50,comp is the concentration of the compound alone
that produces 50% inhibition of the fungal growth (see
Table 2).
prepared with the addition of
a fixed sub-inhibitory
concentration (100 mg/mL) of 7e or 7f in each well. Dose–
response curves were constructed for each antifungal drug
alone and with the added compound (Fig. 3).
In the two-drug combination assays, 7e and 7f produced
shifts of the dose–response curves of FCZ toward lower
concentrations (Fig. 3A). Instead, in the dose–response curves
of ITZ (Fig. 3B) and AmB (Fig. 3C), 7e produced a shift to
higher concentrations while 7f shifted the curves to lower
concentrations.
A CI < 1 is indicative of synergism; CI ¼ 1 indicates
additivism and CI > 1 denotes antagonism [44]. Values of
CI ¼ 1–0.3; 0.3–0.7; 0.7–0.85; and 0.85–1 indicate strong
Figure 3. Dose–response curves of fluconazole (A), itraconazole (B), and amphotericin B (C) alone and in combination with 100 mg/mL of 7e
and 7f against C. albicans. Results are expressed as mean ꢃ SD (n ¼ 3).
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Table 2. MIC50 values (mg/mL) of itraconazole (ITZ), fluconazole (FCZ), amphotericin B (AmB), 7e or 7f either alone or in two-drugs
combination against Candida albicans.
Compound
MIC50
CI
Effect
Potency
DRI of the AD
FCZ
ITZ
AmB
7e
2.730 ꢃ 0.260
1.780 ꢃ 0.30 ꢄ 10ꢀ5
0.071 ꢃ 0.01
180 ꢃ 0.61
7f
200 ꢃ 2.01
FCZ þ 7ea)
FCZ þ 7fa)
ITZ þ 7eb)
ITZ þ 7fb)
AmB þ 7ec)
AmB þ 7fc)
0.972 ꢃ 0.11
0.90
0.50
94.11
0.58
1.71
0.80
Synergistic
Synergistic
Antagonistic
Synergistic
Antagonistic
Synergistic
(þ)
(þ þ þ)
(ꢀ ꢀ ꢀ ꢀ ꢀ)
(þþþ)
2.80
151.66
ꢀ92
0.018 ꢃ 0.01
164 ꢃ 0.15 ꢄ 10ꢀ5
0.15 ꢃ 0.00 ꢄ 10ꢀ5
0.083 ꢃ 0.003
0.022 ꢃ 0.001
11.86
ꢀ1.16
3.55
(ꢀ ꢀ ꢀ)
(þ þ þ)
Data are represented as mean ꢃ SD. The combination index (CI) and the dose reduction index (DRI) of each antifungal drug (AD) upon
combination were calculated.
a)
FCZ þ 100 mg/mL of 7e or 7f.
b)
ITZ þ 100 mg/mL of 7e or 7f.
c)
AmB þ 100 mg/mL of 7e or 7f. Potency according to [44], (þ) slight synergism; (þ þ) moderate synergism; (þ þ þ) synergism; (þ þ þ þ)
strong synergism; (þ þ þ þ þ) very strong synergism; (ꢀ) slight antagonism; (ꢀ ꢀ) moderate antagonism; (ꢀ ꢀ ꢀ) antagonism;
(ꢀ ꢀ ꢀ ꢀ) strong antagonism; (ꢀ ꢀ ꢀ ꢀ ꢀ) very strong antagonism.
synergism, synergism, moderate, or slight synergism, respec-
tively. Instead, CI ¼ 1.10–1.20; 1.20–1.45; 1.45–3.3; 3.3–10; or
>10 indicate slight or moderate antagonism, antagonism,
strong or very strong antagonism, respectively [45].
In addition, the dose-reduction index (DRI) is a measure
of how many-fold the dose of each drug in a synergistic
combination may be reduced at a given effect level, compared
with the dose of each drug alone [45]. The DRI of each
antifungal drug was calculated as follows (Eq. 2):
which, along with the CI, pointed out that a very low dose of
FCZ forms synergistic antifungal mixtures against C. albicans.
Regarding the mixtures with ITZ and AmB, 7e showed
very strong antagonism (CI ¼ 94) or antagonism (CI ¼ 1.71),
respectively, while 7f showed synergism (CI ¼ 0.58) with ITZ
and moderate synergism (CI ¼ 0.80) with AmB. DRI values
were 11.86 and 3.55 for the mixtures with 7f, this compound
showing again that it should be a good candidate for future
research in the field of antifungal compounds.
In summary, these results demonstrated that the pyrazole-
derived compounds synthesized for the first time here,
could constitute alternatives for future developments in
antifungal therapy as binary mixtures with an antifungal
drug. In addition, it is worth to take into account that
both compounds (7e and 7f) that showed synergism with the
tested antifungal drugs possess at least one substituent
OCH3 on their aromatic ring. This structural feature appears
to be very important not only because it is present in both
active compounds but because the highest synergism was
shown by 7f, which possesses three OCH3 on the aromatic
ring, reinforcing the importance of this group in the
enhancement of antifungal activity of FCZ, ITZ, or AmB
against C. albicans.
MIC50 of AD alone
DRI ¼
ð2Þ
C
50 of AD in combination
The DRI is important to visualize the extent of reduction
of the dose of each partner in the mixture that would
concomitantly lead to a reduced toxicity to the host while
the therapeutic efficacy is retained or enhanced. However,
although a DRI > 1 or higher indicates dose-reduction for a
given therapeutic effect, it does not necessarily indicate
synergism which, as explained above, is determined with the
CI values. In Table 2, MIC50 values of each antifungal drug
(FCZ, ITZ, or AmB) alone or in dual combinations with 7e and
7f, along with the CI and the DRI are showed.
Regarding the mixtures of FCZ with 7e and 7f, it could
be observed in Table 2, columns 2 and 3, that MIC50 of FCZ Conclusions
decreased 151-fold (from 2.73 to 0.018 mg/mL) and 2.8-fold
(from 2.73 to 0.97 mg/mL) when combined with 7f and 7e,
respectively, being the CI values 0.50 (synergism) and 0.90
(slight synergism) (Table 2, columns 4–6). It is interesting to
note that the DRI is very high (151.66) for the mixture with 7f,
We performed the synthesis under mild reaction conditions,
short reaction times and good yields of novel N-acetyl
and N-formyl-4,5-dihydro-1H-pyrazoles 7a–f and 8a–f from
a,b-unsaturated carbonyl compounds containing the thiazole
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Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Thiazolylpyrazoles
7
þ
0
–
ring 5a–f. All compounds were tested for antifungal activity
against C. albicans and C. neoformans and, of them, 7e and 7f
showed the best activities, although moderate, when tested
alone. However, when 7e and 7f were tested in combination
with the antifungal agents FCZ, ITZ, or AmB, 7e showed a
synergistic effect with FCZ against C. albicans and 7f displayed
synergistic effects with all the tested antifungal agents against
the same yeast. These relevant findings open new avenues for
a future deepening of the antifungal properties of these novel
compounds in combination with antifungal drugs.
(C-2 ), 168.9 (C O) ppm; MS (IE, 70 eV) m/z (%): 392/390 [M ] (8/20),
–
355 (15), 313 (14), 287 (35), 252 (100), 130 (45), 83 (60). Anal. calcd.
for C18H19ClN4O2S: C, 55.31; H, 4.90; N, 14.33. Found: C, 55.36;
H, 5.01; N, 14.37.
1-Acetyl-3-(4-chlorophenyl)-5-(4-chloro-2-
morpholinothiazol-5-yl)-4,5-dihydro-1H-pyrazole (7b)
White solid; 86% yield; m.p. 179–180°C; FT-IR (KBr), n (cmꢀ1): 1672
1
–
–
–
–
–
–
(C O), 1595 and 1525 (C N and C C). H NMR (400 MHz, CDCl ):
3
d 2.40 (s, 3H, CH3), 3.24 (dd, JAX ¼ 4.7 Hz, JAM ¼ 17.7 Hz, 1H, H-
4A), 3.39–3.41 (m, 4H, CH2–N–CH2), 3.70 (dd, JMX ¼ 11.8 Hz,
JAM ¼ 17.7 Hz, 1H, H-4M), 3.75–3.77 (m, 4H, CH2–O–CH2), 5.78
(dd, JAX ¼ 4.7 Hz, JMX ¼ 11.8 Hz, 1H, H-5X), 7.43 (d, J ¼ 8.5Hz, 2H,
Hm), 7.69 (d, J ¼ 8.5 Hz, 2H, Ho) ppm; 13C NMR (100 MHz, CDCl3):
d 21.8 (CH3), 40.6 (C-4), 47.6 (CH2–N–CH2), 53.2 (C-5), 65.9
(CH2–O–CH2), 117.6 (C-50), 127.8 (Co), 129.0 (Cm), 129.6 (Ci), 132.3
Experimental
Reagents and solvents used were obtained from commercial
sources. Melting points were measured using a Stuart SMP3
melting point device and are uncorrected. IR spectra were
obtained with a Shimadzu IRAffinity-1. The 1H and 13C NMR
spectra were run on a Bruker DPX 400 spectrometer operating at
400 and 100 MHz, respectively, using CDCl3 as solvent and TMS as
internal standard. The mass spectrum was obtained on a
Shimadzu-GCMS-QP2010 spectrometer operating at 70 eV. Thin
layer chromatography (TLC) was performed on 0.2-mm pre-coated
plates of silica gel 60GF254 (Merck). The elemental analyses were
obtained using a Thermo Finnigan Flash EA1112 CHN (STIUJA)
elemental analyzer.
0
0
–
–
(C-4 ), 136.5 (C ), 152.6 (C-3), 167.5 (C-2 ), 168.9 (C O) ppm; MS (IE,
p
70 eV) m/z (%): 428/426/424 [Mþ] (2/10/16), 389 (13), 347 (20), 287
(33), 252 (100), 130 (43), 83 (97). Anal. calcd. for C18H18Cl2N4O2S: C,
50.83; H, 4.27; N, 13.17. Found: C, 50.89; H, 4.30; N, 13.28.
1-Acetyl-5-(4-chloro-2-morpholinothiazol-5-yl)-3-
(4-fluorophenyl)-4,5-dihydro-1H-pyrazole (7c)
White solid; 71% yield; m.p. 154–155°C; FT-IR (KBr), n (cmꢀ1): 1674
(C O), 1598 and 1523 (C N and C C). H NMR (400 MHz, CDCl ):
1
–
–
–
–
–
–
3
d 2.39 (s, 3H, CH3), 3.23 (dd, JAX ¼ 4.7 Hz, JAM ¼ 17.7 Hz, 1H, H-4A),
3.35–3.43 (m, 4H, CH2–N–CH2), 3.64–3.78 (m, 5H, H-4M,
CH2–O–CH2), 5.78 (dd, JAX ¼ 4.7 Hz, JMX ¼ 11.7 Hz, 1H, H-5X),
Chemistry
7.11–7.15 (m, 2H, Hm), 7.74 (dd, JHF ¼ 4.6 Hz, 3J ¼ 8.0 Hz 2H, Ho)
3
General procedure for the synthesis of compounds 7a–f
and 8a–f
ppm; 13C NMR (100 MHz, CDCl3): d 21.8 (CH3), 40.7 (C-4),
47.6 (CH2–N–CH2), 53.1 (C-5), 65.9 (CH2–O–CH2), 115.9 (d,
2JCF ¼ 22.0 Hz, Cm), 117.7 (C-50), 127.3 (d, JCF ¼ 3.5 Hz Ci), 128.5
4
A mixture of 2 mmol of aldehyde 3 (prepared previously by the
methodology reported by Kotlyar et al. [39]), 2 mmol of
substituted acetophenone 4a–f and 20% NaOH solution (3 mL)
in ethanol (20 mL) was stirred during 8 h at room temperature.
The precipitate formed was filtered and washed with ethanol to
obtain the compounds 5a–f as pure yellow solids. Subsequently, a
mixture of 1 mmol of compounds 5a–f and 3 mmol of hydrazine
hydrate in ethanol (5 mL) was stirred during 1 h at room
temperature. Reaction progress was monitored by TLC and the
precipitate formed was filtered and washed with water to obtain
the compounds 6a–f, which decomposed rapidly, then, 0.3 mmol
of compounds 6a–f was stirred during 1 h at room temperature in
the presence of Ac2O (1 mL) or HCOOH (1 mL) both as reagents and
solvents. Water was added to reaction mixture and the precipitate
formed was filtered and washed with ethanol/water 1:1 to
obtain compounds 7a–f and 8a–f as white solids, without further
purification process.
(d, 3JCF ¼ 8.5 Hz Co), 132.6 (C-40), 152.7 (C-3), 164.0 (d, 1JCF ¼ 251 Hz,
0
–
–
C ), 167.5 (C-2 ), 168.8 (C O) ppm; MS (IE, 70 eV)m/z (%): 410/408
p
[Mþ] (8/22), 373 (19), 331 (26), 287 (35), 252 (100), 189 (14), 83 (31).
Anal. calcd. for C18H19FClN4O2S: C, 52.87; H, 4.44; N, 13.70. Found:
C, 52.90; H, 4.51; N, 13.82.
1-Acetyl-5-(4-chloro-2-morpholinothiazol-5-yl)-3-
(4-methylphenyl)-4,5-dihydro-1H-pyrazole (7d)
White solid; 67% yield; m.p.191–192°C; FT-IR (KBr), n (cmꢀ1): 1672
(C O), 1596 and 1524 (C N and C C). H NMR (400 MHz, CDCl ):
1
–
–
–
–
–
–
3
d 2.41 (s, 3H, CH3), 2.43 (s, 3H, ArCH3), 3.24 (dd, JAX ¼ 4.6 Hz,
JAM ¼ 17.7 Hz, 1H, H-4A), 3.35–3.45 (m, 4H, CH2–N–CH2), 3.64–3.82
(m, 5H, H-4M, CH2–O–CH2), 5.77 (dd, JAX ¼ 4.6 Hz, JMX ¼ 11.7 Hz,
1H, H-5X), 7.26 (d, J ¼ 8.1 Hz, 2H, Ho), 7.65 (d, J ¼ 8.1 Hz, 2H, Hm)
ppm; 13C NMR (100 MHz, CDCl3): d 21.8 (CH3), 21.9 (ArCH3), 40.8
(C-4), 47.6 (CH2–N–CH2), 53.0 (C-5), 65.9 (CH2–O–CH2), 118.3 (C-50),
126.5 (Co), 128.3 (Ci), 129.5 (Cm), 132.1 (C-40), 140.9 (Cp), 153.8 (C-3),
1-Acetyl-5-(4-chloro-2-morpholinothiazol-5-yl)-3-phenyl-
þ
0
–
–
167.5 (C-2 ), 168.9 (C O) ppm; MS (IE, 70 eV) m/z (%): 406/404 [M ]
4,5-dihydro-1H-pyrazole (7a)
(3/8), 369 (8), 327 (10), 287 (12), 252 (46), 159 (56), 83 (100). Anal.
calcd. for C19H21ClN4O2S: C, 56.36; H, 5.23; N, 13.84. Found: C,
56.40; H, 5.28; N, 13.83.
White solid; 84% yield; m.p. 165–166°C; FT-IR (KBr), n (cmꢀ1): 1673
1
–
–
–
(C O), 1595 and 1525 (C N and C C). H NMR (400 MHz, CDCl ):
–
–
–
3
d 2.40 (s, 3H, CH3), 3.26 (dd, JAX ¼ 4.7 Hz, JAM ¼ 17.7 Hz, 1H, H-4A),
3.38–3.42 (m, 4H, CH2–N–CH2), 3.68–3.76 (m, 5H, H-4M,
CH2–O–CH2), 5.78 (dd, JAX ¼ 4.7 Hz, JMX ¼ 11.7 Hz, 1H, H-5X),
7.43–7.47 (m, 3H, Hm, Hp), 7.75 (dd, J ¼ 2.9 Hz, J ¼ 6.6 Hz, 2H,
Ho) ppm; 13C NMR (100 MHz, CDCl3): d 21.8 (CH3), 40.8 (C-4), 47.7
(CH2–N–CH2), 53.1 (C-5), 65.9 (CH2–O–CH2), 117.9 (C-50), 126.6 (Co),
128.8 (Cm), 130.5 (Cp), 131.1 (Ci), 132.2 (C-40), 153.8 (C-3), 167.5
1-Acetyl-5-(4-chloro-2-morpholinothiazol-5-yl)-3-
(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole (7e)
White solid; 62% yield; m.p. 181–182°C; FT-IR (KBr), n (cmꢀ1): 1671
1
–
–
–
(C O), 1595 and 1520 (C N and C C). H NMR (400 MHz, CDCl ):
–
–
–
3
d 2.39 (s, 3H, CH3), 3.22 (dd, JAX ¼ 4.6 Hz, JAM ¼ 17.6 Hz, 1H, H-4A),
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
J. Ramírez et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
3.34–3.43 (m, 4H, CH2–N–CH2), 3.68 (dd, JMX ¼ 11.6 Hz, JAM ¼ 17.6
Hz, 1H, H-4M), 3.73–3.80 (m, 4H, CH2–O–CH2), 3.87 (s, 3H, OCH3),
5.76 (dd, JAX ¼ 4.6 Hz, JMX ¼ 11.6 Hz, 1H, H-5X), 6.96 (d, J ¼ 8.7 Hz,
2H, Ho), 7.69 (d, J ¼ 8.7 Hz, 2H, Hm) ppm; 13C NMR (100 MHz,
CDCl3): d 21.8 (CH3), 40.8 (C-4), 47.6 (CH2–N–CH2), 53.0 (C-5),
55.4 (OCH3), 65.9 (CH2–O–CH2), 114.2 (Co), 118.3 (C-50), 123.7 (Ci),
128.2 (Cm), 132.1 (C-40), 153.8 (C-3), 161.5 (Cp), 167þ.5 (C-20),
–
5-(4-Chloro-2-morpholinothiazol-5-yl)-3-(4-fluorophenyl)-
1-formyl-4,5-dihydro-1H-pyrazole (8c)
White solid; 54% yield; m.p. 160–161°C; FT-IR (KBr), n (cmꢀ1): 1680
1
–
–
–
(C O), 1596 and 1528 (C N and C C). H NMR (400 MHz, CDCl ): d
–
–
–
3
3.27 (dd, JAX ¼ 4.9 Hz, JAM ¼ 17.8 Hz, 1H, H-4A), 3.37–3.44 (m, 4H,
CH2–N–CH2), 3.69–3.83 (m, 5H, H-4M, CH2–O–CH2), 5.75 (dd,
JAX ¼ 4.9 Hz, JMX ¼ 11.7 Hz, 1H, H-5X), 7.11–7.19 (m, 2H, Hm), 7.75
3
(dd, JHF ¼ 4.9 Hz, 3J ¼ 8.8 Hz 2H, Ho), 8.90 (s, 1H, CHO) ppm;
168.7 (C O) ppm; MS (IE, 70 eV) m/z (%): 422/420 [M ] (4/10),
–
13C NMR (100 MHz, CDCl3): d 41.0 (C-4), 47.6 (CH2–N–CH2), 52.2
341 (5), 256 (9), 149 (30), 137 (40), 83 (100). Anal. calcd. for
2
(C-5), 65.9 (CH2–O–CH2), 116.1 (d, JCF ¼ 22.1 Hz, Cm), 116.3 (C-50),
C19H21ClN4O3S: C, 54.22; H, 5.03; N, 13.31. Found: C, 54.27;
4
3
126.9 (d, JCF ¼ 3.5 Hz, Ci), 128.7 (d, JCF ¼ 8.5 Hz, Co), 133.0 (C-40),
H, 5.04; N, 13.32.
1
154.4 (C-3), 159.9 (C O), 164.2 (d, J ¼ 252.2 Hz, Cp), 167.5 (C-20)
–
–
CF
ppm; MS (IE, 70 eV) m/z (%): 396/394 [Mþ] (10/24), 273 (28),
1-Acetyl-5-(4-chloro-2-morpholinothiazol-5-yl)-3-
257 (20), 236 (40), 185 (40), 163 (60), 86 (100). Anal. calcd. for
(3,4,5-trimethoxylphenyl)-4,5-dihydro-1H-pyrazole (7f)
White solid; 60% yield; m.p. 182–183°C; FT-IR (KBr), n (cmꢀ1): 1673
C17H16FClN4O2S: C, 51.71; H, 4.08; N, 14.19. Found: C, 51.73;
1
H, 4.02; N, 14.30.
–
–
–
(C O), 1595 and 1525 (C N and C C). H NMR (400 MHz, CDCl ): d
–
–
–
3
2.41 (s, 3H, –CH3), 3.24 (dd, JAX ¼ 4.7 Hz, JAM ¼ 17.6 Hz, 1H, H-4A),
3.35–3.43 (m, 4H, CH2–N–CH2), 3.64–3.83 (m, 5H, H-4M,
CH2–O–CH2), 3.91 (s, 3H, pOCH3), 3.93 (s, 6H, mOCH3), 5.78 (dd,
JAX ¼ 4.6 Hz, JMX ¼ 11.7 Hz, 1H, H-5X), 6.96 (s, 2H, Ho) ppm; 13C NMR
(100 MHz, CDCl3): d 21.9 (CH3), 40.8 (C-4), 47.6 (CH2–N–CH2), 53.1
(C-5), 56.3 (mOCH3), 60.1 (pOCH3), 65.9 (CH2–O–CH2), 103.9 (Co),
117.9 (C-50), 126.4 (Ci), 132.1 (C-40), 136.4 (Cm), 140.4 (Cp), 153.5 (C-
5-(4-Chloro-2-morpholinothiazol-5-yl)-1-formyl-3-
(4-methylphenyl)-4,5-dihydro-1H-pyrazole (8d)
White solid; 60% yield; m.p. 177–178°C; FT-IR (KBr), n (cmꢀ1): 1677
1
–
–
–
(C O), 1596 and 1527 (C N and C C). H NMR (400 MHz, CDCl ): d
–
–
–
3
2.43 (s, 3H, ArCH3), 3.28 (dd, JAX ¼ 4.8 Hz, JAM ¼ 17.8 Hz, 1H, H-4A),
3.38–3.44 (m, 4H, CH2–N–CH2), 3.71–3.83 (m, 5H, H-4M,
CH2–O–CH2), 5.75 (dd, JAX ¼ 4.8 Hz, JMX ¼ 11.4 Hz, 1H, H-5X),
7.28 (d, J ¼ 8.1 Hz, 2H, Ho), 7.65 (d, J ¼ 8.1 Hz, 2H, Hm), 8.92 (s,
1H, CHO) ppm; 13C NMR (100 MHz, CDCl3): d 21.5 (ArCH3), 41.1
(C-4), 47.7 (CH2–N–CH2), 52.1 (C-5), 65.9 (CH2–O–CH2), 116.2 (C-50),
126.8 (Co), 127.8 (Ci), 129.6 (Cm), 132.9 (C-40), 141.3 (Cp), 155.6 (C-3),
0
–
3), 167.5 (C-2 ), 168.2 (C O) ppm; MS (IE, 70 eV) m/z (%): 482/480
–
[Mþ] (12/32), 445 (35), 403 (22), 287 (30), 256 (98), 173 (11), 130
(100). Anal. calcd. for C21H25ClN4O5S: C, 52.44; H, 5.24; N, 11.65.
Found: C, 52.51; H, 5.32; N, 11.70.
þ
0
–
159.92 (C O), 167.5 (C-2 ) ppm; MS (IE, 70 eV) m/z (%): 392/390 [M ]
–
5-(4-Chloro-2-morpholinothiazol-5-yl)-1-formyl-3-phenyl-
(4/10), 355 (7), 256 (8), 185 (14), 159 (17), 130 (24), 86 (100). Anal.
calcd. for C18H19ClN4O2S: C, 55.31; H, 4.90; N, 14.33. Found: C,
55.42; H, 4.99; N, 14.31.
4,5-dihydro-1H-pyrazole (8a)
White solid; 66% yield; m.p. 171–172°C; FT-IR (KBr), n (cmꢀ1): 1678
1
–
–
–
(C O), 1597 and 1528 (C N and C C). H NMR (400 MHz, CDCl ):
–
–
–
3
d 3.30 (dd, JAX ¼ 4.8 Hz, JAM ¼ 17.8 Hz, 1H, H-4A), 3.36–3.45 (m, 4H,
CH2–N–CH2), 3.69–3.86 (m, 5H, H-4M, CH2–O–CH2), 5.76 (dd,
JAX ¼ 4.8 Hz, JMX ¼ 11.6 Hz, 1H, H-5X), 7.46–7.47 (m, 3H, Hm, Hp),
7.75 (d, J ¼ 7.5 Hz, 2H, Ho), 8.91 (s, 1H, CHO) ppm; 13C NMR
(100 MHz, CDCl3): d 42.4 (C-4), 49.1 (CH2–N–CH2), 53.6 (C-5), 67.3
(CH2–O–CH2), 0117.9 (C-50), 128.2 (Co), 130.3 (Cm), 132.0 (Cp), 132.3
70 eV) m/z (%): 378/376 [Mþ] (25/63), 341 (34), 313 (23), 298 (22), 273
(40), 256 (43), 238 (51), 230 (22), 173 (51), 145 (58), 130 (65), 86
(100). Anal. calcd. for C17H17ClN4O2S: C, 54.18; H, 4.55; N, 14.87.
Found: C, 54.20; H, 4.56; N, 14.70.
5-(4-Chloro-2-morpholinothiazol-5-yl)-1-formyl-3-
(4-methoxylphenyl)-4,5-dihydro-1H-pyrazole (8e)
White solid; 59% yield; m.p. 213–214°C; FT-IR (KBr), n (cmꢀ1): 1673
1
–
–
–
(C O), 1596 and 1527 (C N and C C). H NMR (400 MHz, CDCl ): d
–
–
–
3
3.25 (dd, JAX ¼ 4.9 Hz, JAM ¼ 17.7 Hz, 1H, H-4A), 3.35–3.43 (m, 4H,
CH2–N–CH2), 3.66–3.82 (m, 5H, H-4M, CH2–O–CH2), 3.87 (s, 3H,
OCH3), 5.73 (dd, JAX ¼ 4.9 Hz, JMX ¼ 10.8 Hz, 1H, H-5X), 6.96 (d,
J ¼ 8.9 Hz, 2H, Ho), 7.68 (d, J ¼ 8.9 Hz, 2H, Hm), 8.89 (s, 1H, CHO)
ppm; 13C NMR (100 MHz, CDCl3): d 41.1 (C-4), 47.6 (CH2–N–CH2),
52.0 (C-5), 55.4 (OCH3), 65.9 (CH02–O–CH2), 114.3 (Co), 116.8 (C-50),
–
0
–
(C ), 134.4 (C-4 ), 156.9 (C-3), 161.4 (C O), 168.9 (C-2 ) ppm; MS (IE,
–
i
123.2 (C ), 128.3 (C ), 132.8 (C-4 ), 155.3 (C-3), 159.8 (C O), 161.7
–
i
m
(Cp), 167.7 (C-20) ppm; MS (IE, 70 eV) m/z (%): 408/406 [Mþ] (28/75),
371 (53), 293 (69), 265 (100), 230 (43), 175 (39), 130 (65), 86 (71).
Anal. calcd. for C18H19ClN4O3S: C, 53.13; H, 4.71; N, 13.77. Found:
C, 53.17; H, 4.77; N, 13.89.
5-(4-Chloro-2-morpholinothiazol-5-yl)-3-(4-chlorophenyl)-
1-formyl-4,5-dihydro-1H-pyrazole (8b)
White solid; 72% yield; m.p. 186–187°C; FT-IR (KBr), n (cmꢀ1):
1
–
–
–
1679 (C O), 1596 and 1529 (C N and C C). H NMR (400 MHz,
–
–
–
CDCl3): d 3.26 (dd, JAX ¼ 4.9 Hz, JAM ¼ 17.8 Hz, 1H, H-4A), 3.36–
3.43 (m, 4H, CH2–N–CH2), 3.67–3.85 (m, 5H, H-4M, CH2–O–CH2),
5.75 (dd, JAX ¼ 4.9 Hz, JMX ¼ 11.6 Hz, 1H, H-5X), 7.43 (d, J ¼ 8.6 Hz,
2H, Hm), 7.68 (d, J ¼ 8.6 Hz, 2H, Ho), 8.91 (s, 1H, CHO) ppm;
13C NMR (100 MHz, CDCl3): d 40.9 (C-4), 47.7 (CH2–N–CH2),
52.3 (C-5), 65.9 (CH2–O–CH2), 116.2 (C-50), 127.9 (Co), 129.1 (Ci),
5-(4-Chloro-2-morpholinothiazol-5-yl)-1-formyl-3-
(3,4,5-trimethoxylphenyl)-4,5-dihydro-1H-pyrazole (8f)
White solid; 56% yield; m.p. 223–224°C; FT-IR (KBr), n (cmꢀ1): 1677
1
–
–
–
(C O), 1596 and 1524 (C N and C C). H NMR (400 MHz, CDCl ): d
–
–
–
3
3.28 (dd, JAX ¼ 4.7 Hz, JAM ¼ 17.6 Hz, 1H, H-4A), 3.34–3.49 (m, 4H,
CH2–N–CH2), 3.67–3.85 (m, 5H, H-4M, CH2–O–CH2), 3.92 (s, 3H,
pOCH3), 3.93 (s, 6H, mOCH3), 5.77 (dd, JAX ¼ 4.7 Hz, JMX ¼ 11.5 Hz,
1H, H-5X), 6.97 (s, 2H, Ho), 8.91 (s, 1H, CHO) ppm; 13C NMR
(100 MHz, CDCl3): d 41.4 (C-4), 47.7 (CH2–N–CH2), 52.3 (C-5), 56.3
(mOCH3), 60.0 (pOCH3), 65.9 (CH2–O–CH2), 104.0 (Co), 116.4 (C-50),
125.9 (Ci), 133.0 (C-40), 140.6 (Cp), 153.5 (C-3), 155.2 (Cm), 159.8
0
–
129.2 (C ), 133.1 (C-4 ), 136.8 (C ), 154.4 (C-3), 159.9 (C O),
–
m
p
167.7 (C-20) ppm; MS (IE, 70 eV) m/z (%): 414/412/410 [Mþ] (9/47/
65), 375 (37), 347 (22), 332 (24), 290 (35), 273 (57), 238 (62),
230 (54), 173 (41), 130 (71), 86 (100). Anal. calcd. for
C17H16Cl2N4O2S: C, 49.64; H, 3.92; N, 13.62. Found: C, 49.71;
H, 4.01; N, 13.65.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–10
Thiazolylpyrazoles
9
þ
0
–
(C O), 167.5 (C-2 ) ppm; MS (IE, 70 eV) m/z (%):468/466 [M ] (37/89),
Two-drug combination of antifungal drugs with 7e and 7f
against C. albicans
–
431 (46), 388 (30), 346 (18), 273 (23), 130 (41), 86 (100). Anal. calcd.
for C20H23ClN4O5S (466.10): C, 51.44; H, 4.96; N, 12.00. Found: C,
51.48; H, 4.99; N, 12.24.
Non-inhibitory concentrations of 7e and 7f (100 mg/mL) were
combined with the same serial dilutions of AmB, FCZ, or ITZ
as those used for the dose-response curve of the drug alone
(see previous section) in a 96-well microplate (Greiner) to
determine whether a dual combination of AD with 7e or 7f
have additive, synergistic, or antagonistic interactions. An
inoculum of C. albicans (1 ꢄ 103 CFU/mL) was seeded in each well
and, after 24 h incubation, a new dose-response curve for each
mixture was constructed and the MIC50 value of each mixture
was determined. All assays were carried out in triplicate. All data
are expressed as mean ꢃ SD.
Biological evaluation
Antifungal activity
Fungal strains and inoculum preparation: For the antifungal
evaluation, C. albicans from the American Type Culture Collection
(ATCC) ATCC 10231 and C. neoformans ATCC 32264 were used. The
strains were grown on Sabouraud-chloramphenicol agar slants
for 48 h at 30°C, maintained on slopes of Sabouraud-dextrose
agar (SDA, Oxoid), and sub-cultured every 15-day to prevent
pleomorphic transformations. Each inoculum of fungal cells
was obtained according to reported procedures and adjusted to
1–5 ꢄ 103 cells with colony forming units (CFU)/mL [39].
This work was supported financially by Colciencias and Universidad
del Valle. S.A.Z. (Profesora Asociada), M.V.R. (Jefe de Trabajos Prácticos)
and M.S. (Profesor Adjunto) thank the Universidad Nacional de Rosario
and Agencia Nacional de Promoción Científica y Tecnológica de
Argentina (ANPCyT, PICTs 0608-2010) for their support. M.S. (assistant
researcher) greatly acknowledges CONICET.
High-throughput synergy screening assay (HTSS): Mix-
ture test wells (MTWs) were prepared in microtiter trays (Greiner
Bio-One Monroe, North Carolina) each one with a sub-inhibitory
concentration of antifungal drug (that produced ꢂ20% inhibition
of fungal growth) in DMSO (maximum concentration ꢂ 1%), and a
sub-inhibitory concentration of each of the testing compounds
7a–f and 8a–f (100 mg/mL). Inoculum suspension (100 mL) of either
C. albicans or C. neoformans was added to each well (total 72
combinations, 36 for each fungal species). The final volume in the
well was 200 mL. A growth control well (GCW) (containing
medium, inoculum, the same amount of DMSO used in MTW, but
mixture-free) and a sterility control well (SCW) (sample, medium,
and sterile water instead of inoculum) were included in the plate
for each fungus tested. Microtiter trays were incubated in a moist,
dark chamber 24 or 48 h at 30°C for C. albicans or C. neoformans,
respectively. Microplates were read in a VERSAMAX microplate
reader (Molecular Devices, Sunnyvale, CA, USA) and the % of
inhibition for each compound concentration was calculated
as follows: 100 ꢀ (OD405 MTW ꢀ OD405 SCW)/(OD405 GCW ꢀ OD405
SCW). Tests were made in triplicate.
The authors have declared no conflict of interest.
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