New hydrazones bearing thiazole scaffold: Synthesis, characterization, antimicrobial, and antioxidant investigation

Article abstract of DOI:10.3390/molecules200917325

New series of hydrazones 5-18 were synthesized, in good yields, by reacting 4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide with differently substituted benzaldehyde. The resulting compounds were characterized via elemental analysis, physico-chemical and spectral data. An antimicrobial screening was done, using Gram (+), Gram (-) bacteria and one fungal strain. Tested molecules displayed moderate-to-good growth inhibition activity. 2,2-Diphenyl-1-picrylhydrazide assay was used to test the antioxidant properties of the compounds. Monohydroxy (14-16), para-fluorine (13) and 2,4-dichlorine (17) derivatives exhibited better free-radical scavenging ability than the other investigated molecules.

Full text of DOI:10.3390/molecules200917325

Molecules 2015, 20, 17325-17338; doi:10.3390/molecules200917325
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Hydrazones Bearing Thiazole Scaffold: Synthesis,
Characterization, Antimicrobial, and Antioxidant Investigation
Cristina Nastasă 1,*, Brîndușa Tiperciuc 1, Mihaela Duma 2, Daniela Benedec 3
and Ovidiu Oniga 1
1
Department of Pharmaceutical Chemistry, “Iuliu Hațieganu” University of Medicine and Pharmacy
41 Victor Babeș Street, RO-400012 Cluj-Napoca, Romania; E-Mails: brandu32@yahoo.com (B.T.);
onigao65@yahoo.com (O.O.)
2
State Veterinary Laboratory for Animal Health and Safety, 1 Piața Mărăști Street, 400609 Cluj-Napoca,
Romania; E-Mail: duma.mihaelacj@yahoo.com
3
Department of Pharmacognosy, “Iuliu Hațieganu” University of Medicine and Pharmacy,
12 Ion Creangă Street, RO-400010 Cluj-Napoca, Romania; E-Mail: dbenedec@umfcluj.ro
* Author to whom correspondence should be addressed; E-Mail: cmoldovan@umfcluj.ro;
Tel.: +40-264-450529.
Academic Editor: Jean Jacques Vanden Eynde
Received: 26 July 2015 / Accepted: 9 September 2015 / Published: 18 September 2015
Abstract: New series of hydrazones 5–18 were synthesized, in good yields, by reacting
4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide with differently substituted
benzaldehyde. The resulting compounds were characterized via elemental analysis,
physico-chemical and spectral data. An antimicrobial screening was done, using Gram (+),
Gram (−) bacteria and one fungal strain. Tested molecules displayed moderate-to-good growth
inhibition activity. 2,2-Diphenyl-1-picrylhydrazide assay was used to test the antioxidant
properties of the compounds. Monohydroxy (14–16), para-fluorine (13) and 2,4-dichlorine (17)
derivatives exhibited better free-radical scavenging ability than the other investigated molecules.
Keywords: thiazole; hydrazide; acyl-hydrazone; antibacterial; antifungal; antioxidant
1. Introduction
Microbial resistance has been, for more than a few decades, a threat to the effectiveness of common
therapy. In the case of patients at risk, this can involve the extension of the disease or even death [1–4].

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Pathogens have developed several types of resistance (natural/intrinsic and acquired) and different
installation mechanisms. The most alarming resistance to conventional treatment is cited for nine
bacteria: Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae,
Salmonella and Shigella species, Neisseria gonorrhoeae. Candida species infections are a public health
problem worldwide, the most common form of fungal infection. Invasive forms have high rate of
morbidity and mortality, especially in patients with cancer, immunocompromise, new-borns, or those in
the intensive care unit. There is reported increased resistance to azoles (fluconazole) and the emergence
of strains resistant to echinocandins, the newest class of antifungal agents [5].
Globally, efforts are being made for the control of microbial resistance phenomenon, of its spread,
effects, treatment costs, and towards finding new therapeutic protocols. In this context of the alarming
development of the microbial resistance phenomenon, the discovery of new effective substances is
required urgently.
Reactive oxygen species are produced in physiological and metabolic processes of the oxidative
reactions that threaten living organisms. Normally, these radical species are removed by enzymatic and
non-enzymatic antioxidant mechanisms. However, in certain circumstances, the increase of proportion of
oxidants and decrease of antioxidants cannot be prevented. In this case, we can speak of oxidative stress,
a phenomenon involved in the onset and progression of over 100 diseases (cancer, cardiovascular diseases,
neurodegenerative disorders, rheumatoid arthritis, etc.). Antioxidants are molecules, natural or synthetic,
capable of interacting with free radicals and stopping their chain reactions before essential vital molecules
are damaged [6–8].
Scientific literature is more and more focused on Schiff bases containing heterocyclic systems,
due to their large variety of biological properties. New series of hydrazones have been synthesized and
investigated for their antitumor, antioxidant [9], and antimicrobial activities [10–13].
From pentatomic heterocycles, thiazole represents a very important scaffold in medicinal
chemistry [14,15]. There are many compounds bearing this fragment which are used in therapy, for
treating inflammation [16], oxidative stress [17,18], bacterial infections [19–21], hyperglycemia,
hyperlipidemia [22], cancer [23–25], schizophrenia, hypertension, HIV infection, hypnotics, allergy, etc.
In new drug development studies, a combination of different pharmacophores in the same molecule
may lead to new compounds having higher biological activity. Therefore the combination of thiazole- and
hydrazone-type compounds might provide new effective drugs for the treatment of multidrug resistant
microbial infections and for diminishing oxidative processes. Prompted by all these relevant data, we
propose here the synthesis of new hybrid molecules, which gather the two pharmacophores, and the
investigation of their antimicrobial and antioxidant potential.
2. Results and Discussion
2.1. Chemistry
The reaction sequences used for the synthesis of target compounds are shown in Scheme 1.
In order to obtain thiazole heterocycle, in good yield (80%), Hantzsch thiazole synthesis was applied,
using 4-(trifluoromethyl)benzothioamide 1 and ethyl 2-chloroacetoacetate 2. The key intermediate
2-(4-trifluoromethyl-phenyl)-4-methyl-thiazole-5-carbohydrazide 4 was prepared (yield: 80%) by the

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reaction of ethyl-2-(4-trifluoromethyl-phenyl)-4-methyl-thiazole-5-carboxylate 3 with hydrazine hydrate
in absolute ethanol, according to the literature [26]. The next step was the condensation of compound 4
with differently substituted benzaldehydes, in order to obtain, in good yields (70%–99%), target
acyl-hydrazones 5–18.
N
NH2
S
O
O
ii
Cl
i
F3C
F3C
+
S
O
O
1
2
O
3
N
N
O
O
iii
R
F3C
F3C
S
S
HN
HN
R1
N
NH2
4
5-18
Cp
5
R
H
H
H
H
H
H
H
R1
Cp
12
13
14
15
16
17
18
R
H
H
H
H
H
R1
2-OCH3
3-OCH3
4-OCH3
3-Cl
4-Br
4-F
6
7
2-OH
3-OH
4-OH
4-Cl
8
9
4-Cl
10
11
2-Br
2-Cl
2-Cl
3-Br
6-Cl
Scheme 1. Synthesis of new acyl-hydrazones bearing thiazole scaffold. i: ethanol/reflux;
ii: NH2NH2·H2O/ethanol/reflux; iii: substituted benzaldehyde/H2SO4/ethanol/reflux.
All newly synthesized molecules were purified by recrystallization. Purity was verified by TLC, under
1
UV light exposure. Mass spectrometry confirmed molecular mass of the compounds. H-NMR spectra
showed three characteristic signals for the two methyl and one methylene group, demonstrating the synthesis
of ethyl-2-(4-trifluoromethyl-phenyl)-4-methyl-thiazole-5-carboxylate 3. For thiazole-carbohydrazide 4, the
signals due to NH and NH2 protons confirmed the formation of this compound. In the 1H-NMR of 5–18,
the NH proton signal shifted from 9.65 ppm in carbohydrazide to 11.84–12.21 ppm region. Also, signals
of NH2 protons disappeared. The spectra showed characteristic signals for N=CH protons and also for
new phenyl protons, which confirmed that condensation with benzaldehyde derivatives took place.
2.2. Antimicrobial Screening
Newly synthesized compounds were tested for their antimicrobial activity, using agar diffusion
technique. Stock solution of each substance (1 mg/mL) was prepared in dimethylsulfoxide. The screening
was done against two gram-negative bacterial strains: Salmonella enteritidis ATCC 13,076 and
Escherichia coli ATCC 25,922, two gram-positive bacterial strains: Listeria monocytogenes ATCC 13,932
and Staphylococcus aureus ATCC 6538P, and one fungal strain: Candida albicans ATCC 10,231. The
assessment of the antimicrobial potency was realized by measuring the diameter of the growth inhibition
zone. There were two drugs used as reference: gentamicin for the antibacterial activity and fluconazole

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for the antifungal. DMSO was used as control and it did not present a growth inhibitory effect. The
results of the screening are summarized in Table 1.
Table 1. Antimicrobial activity of the new synthesized compounds.
Microbial Strain/Diameter of the Growth Inhibition Zone (mm)
Cp
Gram-Negative Bacteria
S. enteritidis E. coli
ATCC 13,076 ATCC 25,922
Gram-Positive Bacteria
L. monocytogenes S. aureus
Fungus
C. albicans
(1 mg/mL)
ATCC 13,932
ATCC 6538P ATCC 10,231
3
20
20
10
10
18
10
18
18
18
22
20
12
18
18
12
18
18
NT
18
18
16
18
18
18
18
20
20
20
18
16
18
20
20
20
22
NT
16
24
-
12
25
12
12
16
14
14
14
14
14
16
16
12
12
12
12
19
NT
18
20
25
20
14
20
18
22
20
35
14
18
18
20
18
20
NT
25
4
5
6
-
7
14
-
8
9
14
-
10
11
-
12
14
14
16
-
13
14
15
16
17
-
-
18
-
Gentamicin
Fluconazole
18
NT
NT = not tested.
Investigated compounds exerted moderate-to-good activity against microbial strains used. The growth
inhibition was more pronounced in case of gram-negative bacteria. The strain of S. enteritidis ATCC 13076
was sensitive to all new substances. Seven of the 16 compounds presented the same activity as gentamicin
used as reference, while compounds 3, 4, 12, and 13 showed to be more potent than the antibiotic. Analyzing
the inhibition of E. coli ATCC 25922, six (10–12, 16–18) of the new thiazole derivatives showed promising
potential, the growth inhibition diameters being close to that of gentamicin.
L. monocytogenes ATCC 13932 was the less inhibited bacterial strain. It is worth mentioning the
activity displayed by aryl-thiazolyl-carbohydrazide 4 was superior to the reference drug. The same
compound exerted best inhibition also against S. aureus ATCC 6538P, superior to gentamicin.
The antifungal screening revealed that all the investigated derivatives displayed modest-to-good
activity. As it can be seen in Table 1, new synthesized molecule 5 showed an inhibitory effect similar to
that of fluconazole, used as reference. A very promising candidate is acyl-hydrazone 12, obtained from
condensation with 4-bromo-benzaldehyde, which proved to be the most active, being a more powerful
growth inhibitor than the reference.
The good antimicrobial results can be explained by the capacity of acyl-hydrazones to form hydrogen
bonds. The presence of aryl ring with various substitutes in different positions attached to the double

Molecules 2015, 20
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bond of azomethine group displayed good antifungal activity, according to literature data [27,28]. Also,
the most active compounds against fungi species possess electron-withdrawing F, Cl, Br, and CF3 at the
phenyl ring [29,30]. In our series, 4-Br-phenyl derivative displayed the most powerful growth inhibitory
effect against C. albicans strain. Para-substitution of phenyl ring was endowed with better antimicrobial
properties, this aspect being in agreement with literature [31,32]. These results indicate that introduction
of different electron-withdrawing atoms or groups in different positions of the aryl ring [33] plays an
important role in the antimicrobial activity of the hydrazones tested here.
2.3. DPPH Based Free Radical Scavenging Activity
2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging is considered a good in vitro model,
being widely used to conveniently assess antioxidant efficacy, as a rapid, easy, and cost-effective
spectrophotometric method [34,35]. DPPH radicals are stable free radicals, which are neutralized in the
presence of molecules capable of donating H atoms or electrons. The color of DPPH changes from purple
(free radical) to yellow (2,2-diphenyl-1-picrylhydrazine, the non-radicalic, reduced form). This is taken
as an indicator of the hydrogen donating ability of the tested compounds. The degree of discoloration
indicates the scavenging potential of the antioxidant compounds or samples in terms of hydrogen donating
ability. The absorbance is registered at 517 nm.
The ability of Schiff bases and their derivatives to scavenge free radicals is an important property. These
chemical structures may act as metal chelators of ions involved in lipid oxidation or may react with oxygen
bearing systems. The reducing abilities of the examined compounds were determined by their interaction
with the free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) at 500 μg/mL concentration, for 30 min.
The results of the antioxidant screening are depicted in Figure 1. Ascorbic acid was used as reference.
Figure 1. DPPH radical scavenging activity of the new synthesized molecules.
By comparing structural information and antioxidant activity, it was observed that DPPH radical
scavenging antioxidant activity depends on two parameters: the functional groups on the aromatic ring
and the position of functional groups on the ring. Antioxidants donate hydrogen atoms to become stable
free radicals. The degree of stability and antioxidant potential are in direct relationship with the range of

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delocalization. All investigated compounds demonstrated an anti-radical activity. For most of the new
acyl-hydrazones, the effect was weaker than that of the reference ascorbic acid, but it is worth mentioning
the results obtained for compound 4, 2-(4-trifluoromethyl-phenyl)-4-methyl-thiazol-5-carbohydrazide.
In this case, the percentage of inhibition was superior to ascorbic acid. Generally, organic molecules
incorporating an electron donating groups, such as amine, hydroxyl, or methoxy, are inquired as promising
antioxidant molecules.
Compounds 14–16 bearing a hydroxyl group at orto, meta, or para position of the phenyl ring,
showed better DPPH activity than the others tested. This result is consistent with the concept that the
hydroxyl group enhances antioxidant ability [36,37]. The activities of monohydroxy compounds mainly
depend upon the position of the hydroxy group. Para-substitution has more stabilizing potential then
meta-substitution. This can explain why compound 16, with OH group in position 4 of phenyl ring,
displayed better activity than meta- and orto- derivatives. Lower capacity of compound 14, with OH group
in position 2, may be due to the intramolecular hydrogen bonding.
Derivatives 5–7 bearing methoxy group didn’t display a good antioxidant effect. From the results
obtained, new substances containing fluorine (13), in position para, or chlorine (17) exhibited better
activity than the others. Also, the addition of another chlorine atom on the structure of 9, in compound 17,
increased the scavenging effect.
The inhibition displayed on the DPPH radical by the test samples shows that the new synthesized
hydrazones are capable of neutralizing free radicals and thus, could represent promising candidates for
the treatment of pathological diseases and conditions caused as a result of excessive radicals or stress.
3. Experimental Section
3.1. Reagents and Solvents
Reagents and solvents were obtained from commercial sources: 4-(trifluoromethyl)-benzothioamide
from Maybridge, Cornwall, England, UK; ethyl 2-chloroacetoacetate, hydrazine hydrate from Sigma
Aldrich, Buchs, Switzerland; substituted benzaldehydes, HPLC grade methanol from Merck, Germany;
DPPH (2,2-diphenyl-1-picrylhydrazyl) from Alfa-Aesar (Karlruhe, Germany); DMSO (AppliChem-An
ITW Company, Darmstadt, Germany) and used as received. The intermediates were synthesized in our
research laboratory.
3.2. Analytical Methods
Analytical thin layer chromatography (TLC) was carried out on Merck precoated Silica Gel 60F254
sheets (Merck, Darmstadt, Germany), using UV absorption for visualization. Melting points were
determined on open glass capillaries with the help of MPM-H1 Schorpp melting point meter (Schorpp
Gerätetechnik, Überlingen, Germany), and are uncorrected. The 1H-NMR spectra were recorded at room
temperature on a Bruker Avance NMR spectrometer (Bruker, Karlsruhe, Germany) operating at 500 MHz
and were in accord with the assigned structures. Chemical shift values were reported relative to
tetramethylsilane (TMS) as internal standard and are expressed in ppm. The samples were prepared by
dissolving the synthesized powder of the compounds in DMSO-d6 (δH = 2.51 ppm) as solvent. Mass
spectra were recorded by Agilent 1100, type SL spectrometer (positive ionization, Agilent, Santa Clara,

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CA, USA) and with a Varian MAT CH-5 spectrometer (70 eV, Varian MAT, Bremen, Germany).
Elemental analysis was registered with a Vario El CHNS instrument (Elementar Analysensysteme
GmbH, Hanau, Germany).
3.3. Chemical Synthesis
3.3.1. General Procedure for the Synthesis of Ethyl-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-
carboxylate (3)
To a solution of 4-(trifluoromethyl)benzothioamide 1 (10 mmol/2.05 g) in the minimum required
quantity of absolute ethanol (10 mL), ethyl 2-chloroacetoacetate 2 (15 mmol/2.46 g) was added and the
resulting mixture was heated on the water bath for 5 h. The solution was cooled down and neutralized with
a solution of potassium carbonate. The resulting solid was filtered, washed with water, recrystallized from
ethanol to give the target compound.
Ethyl-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carboxylate (3): light brown powder, yield: 80%,
mp: 90 °C. 1H-NMR (DMSO-d6, 500 MHz, ppm): δ 1.30 (t, 3H, -CH3); 2.68 (s, 3H, -CH3); 4.30 (q, 2H,
-CH2-); 7.83 (d, 2H, phenyl-H); 8.15 (d, 2H, phenyl-H). Anal. Calcd. (%) for C14H12F3NO2S (315.31):
C, 53.33; H, 3.84; N, 4.44; S, 10.17. Found: C, 53.32; H, 3.84; N, 4.43; S, 10.18. MS (EI, 70 eV): m/z (%)
316 [M+], 288 (100%).
3.3.2. General Procedure for the Synthesis of 4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-
carbohydrazide (4)
To a solution of compound 3 (10 mmol/3.15 g) in absolute ethanol (20 mL), hydrazine hydrate
(15 mmol/0.75 g) was added and the resulting mixture was heated on water bath, for 5 h. The mixture
was filtered while still hot and the obtained solution was cooled down. A yellow precipitate was formed,
washed with water and recrystallized from ethanol, in order to obtain carbohydrazide 4.
4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (4): white-light green powder, yield:
80%, mp: 177 °C. 1H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.63 (s, 3H, -CH3); 4.60 (br, 2H, -NH2); 7.87
(d, 2H, phenyl-H); 8.13 (d, 2H, phenyl-H); 9.65 (br, 1H, -NH-). Anal. Calcd. (%) for C12H10F3N3OS
(301.29): C, 47.84; H, 3.35; N, 13.95; S, 10.64. Found: C, 47.83; H, 3.35; N, 13.94; S, 10.65. MS (EI,
70 eV): m/z (%) 302 [M+], 285 (25%), 244 (100%).
3.3.3. General Procedure for the Synthesis of Acyl-hydrazones 5–18
To a solution of thiazole-carbohydrazide 4 (1 mmol/0.301 g) in absolute ethanol (10 mL), 1 mmol of
different substituted benzaldehyde and two drops of concentrated sulphuric acid were added. The
mixture was heated for 4 h. The residue was filtered and recrystallized from ethanol in order to give
compounds 5–18, as different color powders.
N′-(2-Methoxybenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (5): yellow
1
powder, yield: 98%, mp: 289 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.80 (s, 3H, -CH3); 3.87
(s, 3H, -CH3); 7.10–7.15 (m, 2H, phenyl-H); 7.46 (t, 1H, phenyl-H); 7.92 (d, 1H, phenyl-H); 7.95 (d,

Molecules 2015, 20
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2H, phenyl-H); 8.23 (d, 2H, phenyl-H); 8.49 (s, 1H, =CH-); 11.94 (s, 1H, -NH-). Anal. Calcd. (%) for
C20H16F3N3O2S (419.42): C, 57.27; H, 3.85; N, 10.02; S, 7.65. Found: C, 57.26; H, 3.84; N, 10.01; S,
7.66. MS (EI, 70 eV): m/z (%) 420 [M+], 270 (25%), 244 (100%).
N′-(3-Methoxybenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (6): light
yellow powder, yield: 90%, mp: 249 °C. 1H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.81 (s, 3H, -CH3);
3.85 (s, 3H, -CH3); 7.03–7.05 (dd, 1H, phenyl-H); 7.33 (d, 1H, phenyl-H); 7.41 (d, 1H, phenyl-H);
7.44 (s, 1H, phenyl-H); 7.92 (d, 2H, phenyl-H); 8.12 (s, 1H, =CH-); 8.22 (d, 2H, phenyl-H); 12.02
(s, 1H, -NH-). Anal. Calcd. (%) for C20H16F3N3O2S (419.42): C, 57.27; H, 3.85; N, 10.02; S, 7.65. Found:
C, 57.26; H, 3.85; N, 10.01; S, 7.66. MS (EI, 70 eV): m/z (%) 420 [M+], 270 (7%), 244 (100%).
N′-(4-Methoxybenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (7): yellow
powder, yield: 87%, mp: 232 °C. 1H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.80 (s, 3H, -CH3); 3.82 (s,
3H, -CH3); 7.08 (d, 2H, phenyl-H); 7.74 (d, 2H, phenyl-H); 7.92 (d, 2H, phenyl-H); 8.09 (s, 1H, =CH-);
8.25 (d, 2H, phenyl-H); 11.84 (s, 1H, -NH-). Anal. Calcd. (%) for C20H16F3N3O2S (419.42): C, 57.27;
H, 3.85; N, 10.02; S, 7.65. Found: C, 57.26; H, 3.85; N, 10.02; S, 7.66. MS (EI, 70 eV): m/z (%) 420
[M+], 244 (100%).
N′-(3-Chlorobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (8): light
1
yellow powder, yield: 99%, mp: 263 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.81 (s, 3H, -CH3);
7.03–7.04 (dd, 1H, phenyl-H); 7.31 (d, 1H, phenyl-H); 7.43 (d, 1H, phenyl-H); 7.45 (s, 1H, phenyl-H);
7.92 (d, 2H, phenyl-H); 8.10 (s, 1H, =CH-); 8.21 (d, 2H, phenyl-H); 12.00 (s, 1H, -NH-). Anal. Calcd.
(%) for C19H13ClF3N3OS (423.84): C, 53.84; H, 3.09; N, 9.91; S, 7.57. Found: C, 53.85; H, 3.08; N,
9.92; S, 7.56. MS (EI, 70 eV): m/z (%) 424 [M+], 244 (100%).
N′-(4-Chlorobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (9): yellow
1
powder, yield: 86%, mp: 290 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.78 (s, 3H, -CH3); 7.08
(d, 2H, phenyl-H); 7.72 (d, 2H, phenyl-H); 7.92 (d, 2H, phenyl-H); 8.07 (s, 1H, =CH-); 8.20 (d, 2H,
phenyl-H); 11.87 (s, 1H, -NH-). Anal. Calcd. (%) for C19H13ClF3N3OS (423.84): C, 53.84; H, 3.09; N,
9.91; S, 7.57. Found: C, 53.84; H, 3.08; N, 9.92; S, 7.56. MS (EI, 70 eV): m/z (%) 424 [M+], 244 (100%).
N′-(2-Bromobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (10): light
1
yellow powder, yield: 98%, mp: 289 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.80 (s, 3H, -CH3);
7.10–7.14 (m, 2H, phenyl-H); 7.41 (t, 1H, phenyl-H); 7.94 (d, 1H, phenyl-H); 7.96 (d, 2H, phenyl-H);
8.20 (d, 2H, phenyl-H); 8.41 (s, 1H, =CH-); 11.89 (s, 1H, -NH-). Anal. Calcd. (%) for C19H13BrF3N3OS
(468.29): C, 48.73; H, 2.80; N, 8.97; S, 6.85. Found: C, 48.74; H, 2.80; N, 8.98; S, 6.86. MS (EI, 70 eV):
m/z (%) 468 [M+], 451 (6%), 244 (100%).
N′-(3-Bromobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (11): light
1
yellow powder, yield: 89%, mp: 279 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.78 (s, 3H, -CH3);
7.03–7.06 (dd, 1H, phenyl-H); 7.37 (d, 1H, phenyl-H); 7.45 (d, 1H, phenyl-H); 7.51 (s, 1H, phenyl-H);
7.90 (d, 2H, phenyl-H); 8.09 (s, 1H, =CH-); 8.23 (d, 2H, phenyl-H); 11.97 (s, 1H, -NH-). Anal. Calcd.

Molecules 2015, 20
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(%) for C19H13BrF3N3OS (468.29): C, 48.73; H, 2.80; N, 8.97; S, 6.85. Found: C, 48.74; H, 2.80; N,
8.97; S, 6.86. MS (EI, 70 eV): m/z (%) 468 [M+], 451 (6%), 244 (100%).
N′-(4-Bromobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (12): yellow
1
powder, yield: 91%, mp: 296 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.74 (s, 3H, -CH3); 7.06
(d, 2H, phenyl-H); 7.77 (d, 2H, phenyl-H); 7.91 (d, 2H, phenyl-H); 8.09 (s, 1H, =CH-); 8.23 (d, 2H,
phenyl-H); 11.91 (s, 1H, -NH-). Anal. Calcd. (%) for C19H13BrF3N3OS (468.29): C, 48.73; H, 2.80; N,
8.97; S, 6.85. Found: C, 48.73; H, 2.80; N, 8.96; S, 6.86. MS (EI, 70 eV): m/z (%) 468 [M+], 451 (6%),
244 (100%).
N′-(4-Fluorobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (13): yellow
1
powder, yield: 71%, mp: 304 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.79 (s, 3H, -CH3); 7.07
(d, 2H, phenyl-H); 7.74 (d, 2H, phenyl-H); 7.94 (d, 2H, phenyl-H); 8.10 (s, 1H, =CH-); 8.25 (d, 2H,
phenyl-H); 11.98 (s, 1H, -NH-). Anal. Calcd. (%) for C19H13F4N3OS (407.38): C, 56.02; H, 3.22; N, 10.31;
S, 7.87. Found: C, 56.01; H, 3.22; N, 10.30; S, 7.88. MS (EI, 70 eV): m/z (%) 407 [M+], 244 (100%).
N′-(2-Hydroxybenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (14): yellow
powder, yield: 70%, mp: 217 °C. 1H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.79 (s, 3H, -CH3); 6.92–7.00
(m, 2H, phenyl-H); 7.28–7.33 (m, 2H, phenyl-H); 7.41 (t, 1H, phenyl-H); 7.92 (d, 1H, phenyl-H); 8.22
(d, 2H, phenyl-H); 8.47 (s, 1H, =CH-); 9.01 (s, 1H, phenyl-OH); 11.90 (s, 1H, -NH-). Anal. Calcd. (%)
for C19H14F3N3O2S (405.39): C, 56.29; H, 3.48; N, 10.37; S, 7.91. Found: C, 56.28; H, 3.48; N, 10.36;
S, 7.92. MS (EI, 70 eV): m/z (%) 406 [M+], 270 (24%), 244 (100%).
N′-(3-Hydroxybenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (15): light
1
yellow powder, yield: 72%, mp: 270 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.76 (s, 3H, -CH3);
7.03–7.05 (dd, 1H, phenyl-H); 7.36 (d, 1H, phenyl-H); 7.46 (d, 1H, phenyl-H); 7.53 (s, 1H, phenyl-H);
7.92 (d, 2H, phenyl-H); 8.10 (s, 1H, =CH-); 8.25 (d, 2H, phenyl-H); 9.00 (s, 1H, phenyl-OH); 12.00 (s,
1H, -NH-). Anal. Calcd. (%) for C19H14F3N3O2S (405.39): C, 56.29; H, 3.48; N, 10.37; S, 7.91. Found:
C, 56.28; H, 3.48; N, 10.36; S, 7.92. MS (EI, 70 eV): m/z (%) 406 [M+], 244 (100%).
N′-(4-Hydroxybenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (16): light
1
yellow powder, yield: 95%, mp: 284 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.78 (s, 3H, -CH3);
7.04 (d, 2H, phenyl-H); 7.78 (d, 2H, phenyl-H); 7.91 (d, 2H, phenyl-H); 8.09 (s, 1H, =CH-); 8.23 (d,
2H, phenyl-H); 9.04 (s, 1H, phenyl-OH); 11.96 (s, 1H, -NH-). Anal. Calcd. (%) for C19H14F3N3O2S
(405.39): C, 56.29; H, 3.48; N, 10.37; S, 7.91. Found: C, 56.28; H, 3.48; N, 10.36; S, 7.92. MS (EI, 70 eV):
m/z (%) 406 [M+], 244 (100%).
N′-(2,4-Dichlorobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (17): light
1
yellow powder, yield: 99%, mp: 298 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.80 (s, 3H, -CH3);
7.77 (d, 1H, phenyl-H); 7.91 (d, 2H, phenyl-H); 8.09 (d, 1H, phenyl-H); 8.14 (s, 1H, phenyl-H); 8.25
(d, 2H, phenyl-H); 8.50 (s, 1H, =CH-); 12.21 (br, 1H, -NH-). Anal. Calcd. (%) for C19H12Cl2F3N3OS
(458.28): C, 49.80; H, 2.64; N, 9.17; S, 7.00. Found: C, 49.80; H, 2.64; N, 9.16; S, 7.01. MS (EI, 70 eV):
m/z (%) 459 [M+], 287 (9%), 244 (100%).

Molecules 2015, 20
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N′-(2,6-Dichlorobenzylidene)-4-methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-carbohydrazide (18): light
1
yellow powder, yield: 94%, mp: 268 °C. H-NMR (DMSO-d6, 500 MHz, ppm): δ 2.77 (s, 3H, -CH3);
7.48 (t, 1H, phenyl-H); 7.60 (d, 2H, phenyl-H); 7.89 (d, 2H, phenyl-H); 8.12 (s, 1H, phenyl-H); 8.35
(d, 1H, phenyl-H); 8.50 (s, 1H, =CH-); 12.20 (br, 1H, -NH-). Anal. Calcd. (%) for C19H12Cl2F3N3OS
(458.28): C, 49.80; H, 2.64; N, 9.17; S, 7.00. Found: C, 49.79; H, 2.64; N, 9.16; S, 7.01. MS (EI, 70 eV):
m/z (%) 459 [M+], 287 (9%), 244 (100%).
3.4. Antimicrobial Screening
The screening of antimicrobial activity was done in the State Veterinary Laboratory for Animal Health
and Safety, Cluj-Napoca, Romania, according to the guidelines of National Committee for Clinical
Laboratory Standards (NCCLS, 1997), [38], using the agar diffusion method. Gentamicin and fluconazole
were purchased from the drug market and used as reference for antibacterial and antifungal activity,
respectively. Petri plates containing 20 mL of Mueller Hinton Agar were used for all the bacteria tested
and Mueller-Hinton medium supplemented with 2% glucose (providing adequate growth of yeasts) and
0.5 g/L methylene blue (providing a better definition of the inhibition zone diameter) was used for
antifungal testing.
After 18 h, the bacterial strains were put on a saline solution of NaCl (0.9%), so that the turbidity
would be that of MacFarland (106 UFC/mL). The inoculum was spread on the surface of the solidified
media. Solutions of the tested compounds were prepared in DMSO and concentration of 1 mg/mL was used.
Six-millimeter diameter wells were cut from the agar using a sterile cork-borer. A sterile swab was
soaked in suspension and then the Mueller-Hinton agar plates were inoculated by streaking the entire
surface. After drying for 10–15 min, the six millimeter diameter wells were inoculated with 50 μL from
each solution. Gentamicin (10 μg/well) and fluconazole (25 μg/well) were used as antibacterial and
antifungal reference, respectively. Plates inoculated with bacteria were incubated for 24 h and those with
fungus 48 h, at 37 °C.
The effects of the new compounds were assessed by measuring the diameter of the growth inhibition
zone. Zone diameters were measured to the nearest whole millimeter at a point in which there will be no
visible growth after 24–48 h.
All the tests were performed in duplicate and the average was taken as final reading. All
microorganism products were distributed by MicroBioLogics®: Salmonella enteritidis ATCC 13076
(Gram−), Escherichia coli ATCC 25922 (Gram−), Listeria monocytogenes ATCC 13932 (Gram+),
Staphylococcus aureus ATCC 6538P (Gram+), and Candida albicans ATCC 10231 (fungus).
3.5. DPPH Based Free Radical Scavenging Activity
The direct antioxidant activity of the tested compounds was evaluated through a free radical scavenging
assay, namely the stable DPPH radical method. Samples were prepared by treating a series of methanolic
0.1 g/L DPPH solutions with an equal volume of the tested thiazole hydrazones solution, of 1 mg/mL
concentration. Simultaneously, a control sample was prepared by diluting 1:1 the initial DPPH solution
with methanol. The mixtures where incubated for 30 min at 40 °C and then the absorbance was measured
at 517 nm. A decrease in absorbance is associated with the reduction of the DPPH radical and thus
directly proportional to the radical scavenging activity of the tested compounds. The DPPH scavenging

Molecules 2015, 20
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ability was expressed as a percentage of absorbance reduction: DPPH scavenging ability % = [(Acontrol
−
Asample)/Acontrol] × 100, where Abscontrol is the absorbance of DPPH radical + methanol (containing all
reagents except the sample) and Abssample is the absorbance of DPPH radical + compound sample. As a
positive control we used the well-known antioxidant, ascorbic acid.
The investigation of the antioxidant potential was completed in the research laboratory of Pharmacognosy
Department of Faculty of Pharmacy, Cluj-Napoca, Romania.
4. Conclusions
We have successfully synthesized new hydrazones, derivatives of 2-(4-trifluoromethyl-phenyl)-4-
methyl-thiazol-5-carbohydrazide. The compounds were isolated in solid state, in high yields
1
(70%–99%), characterized with the help of combined physico-chemical and spectral (MS, H-NMR)
data. The antimicrobial effect of the new molecules was investigated using the diffusion technique.
5-Thiazole-carbohydrazide 4 showed a strong growth inhibitory activity, higher than that of gentamicin,
the reference antibiotic used, against Gram-positive strains and against the strain of S. enteritidis.
Compound 12, a 4-bromo-phenyl derivative, displayed a good inhibitory effect against S. enteritidis,
too. This new molecule represents a very promising candidate for the antifungal activity, the effect being
superior to that of fluconazole, the antimycotic reference, on C. albicans. All compounds manifested an
anti-radical potential. The best DPPH scavenging activity was demonstrated by carbohydrazide 4, better
than ascorbic acid. The obtained results suggest that the new hydrazones bearing thiazole scaffold may be
considered for further investigation and optimization, in designing antimicrobial and antioxidant drugs.
Acknowledgments
The research was (partially) funded by POSDRU grant no. 159/1.5/S/136893 grant with title:
“Parteneriat strategic pentru creșterea calității cercetării științifice din universitățile medicale prin
acordarea de burse doctorale și postdoctorale—DocMed.Net_2.0” and within the research contract
No. 1494/5/28.01.2014 financed by “Iuliu Hațieganu” University of Medicine and Pharmacy
Cluj-Napoca, Romania.
Author Contributions
Cristina Nastasă, Brîndușa Tiperciuc and Ovidiu Oniga designed the research, performed the chemical
synthesis and analyzed the data. Mihaela Duma was responsible for the antimicrobial screening and
Daniela Benedec for the antioxidant evaluation. All authors contributed to the elaboration of the article
and approved the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Jomha, M.Y.; Yusef, H.; Holail, H. Antimicrobial and biocide resistance of bacteria in a Lebanese
tertiary care hospital. J. Glob. Antimicrob. Resist. 2014, 2, 299–305.

Molecules 2015, 20
17336
2. Tang, S.S.; Apisarnthanarak, A.; Hsu, L.Y. Mechanisms of β-lactam antimicrobial resistance and
epidemiology of major community and healthcare-associated multidrug-resistant bacteria. Adv. Drug
Deliv. Rev. 2014, 78, 3–13.
3. Ogbolu, D.O.; Webber, M.A. High-level and novel mechanisms of carbapenem resistance in
Gramnegative bacteria from tertiary hospitals in Nigeria. Int. J. Antimicrob. Agents 2014, 43, 412–417.
4. Nayak, N.; Lenka, R.K.; Padhy, R.N. Surveillance of multidrug resistant suppurative infection causing
bacteria in hospitalized patients in an Indian tertiary care hospital. J. Acute Dis. 2014, 148–156.
5. WHO Library Cataloguing-in-Publication Data. Antimicrobial Resistance: Global Report on
Surveillance; WHO Press, World Health Organization: Geneva, Switzerland, 2015.
6. Popoola, O.K.; Marnewick, J.L.; Rautenbach, F.; Ameer, F.; Iwuoha, E.I.; Hussein, A.A. Inhibition
of oxidative stress and skin aging-related enzymes by prenylated chalcones and other flavonoids
from Helichrysum teretifolium. Molecules 2015, 20, 7143–7155.
7. Ni, N.; Liu, Q.; Ren, H.; Wu, D.; Luo, C.; Li, P.; Wan, J.B.; Su, H. Ginsenoside Rb1 protects rat
neural progenitor cells against oxidative injury. Molecules 2014, 19, 3012–3024.
8. Shakir, R.M.; Ariffin, A.; Abdulla, M.A. Synthesis of new 2,5-di-substituted 1,3,4-oxadiazoles
bearing 2,6-di-tert-butylphenol moieties and evaluation of their antioxidant activity. Molecules
2014, 19, 3436–3449.
9. Gwaram, N.S.; Ali, H.M.; Abdulla, M.A.; Buckle, M.J.C.; Sukumaran, S.D.; Chung, L.Y.;
Othman, R.; Alhadi, A.A.; Yehye, W.A.; Hadi, A.H.A.; et al. Synthesis, characterization, X-ray
crystallography, Acetyl cholinesterase inhibition and antioxidant activities of some novel ketone
derivatives of gallic hydrazide-derived Schiff bases. Molecules 2012, 17, 2408–2427.
10. Kashinath, K.; Durg, V.; Baburao, K.; Angadi, S.D. Synthesis, characterization and biological
activities of Schiff bases derived from hydrazide derivative and their Cu(II), Ni(II), Co(II), Zn(II),
Cd(II) and Hg(II), complexes. Int. J. Res. Pharm. Chem. 2014, 4, 557–563.
11. Husain, A.; Varshney, M.M.; Parcha, V.; Ahmad, A.; Khan, S.A. Synthesis and biological evaluation
of new hydrazide-Schiff bases. Bangladesh J. Pharmacol. 2015, 10, 555–561.
12. Zayed, E.M.; Zayed, M.A. Synthesis of novel Schiff’s bases of highly potential biological activities
and their structure investigation. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2015, 143, 81–90.
13. Hearn, M.J.; Cynamon, M.H.; Chen, M.F.; Coppins, R.; Davis, J.; Kang, H.J.-O.; Noble, A.;
Tu-Sekine, B.; Terrot, M.S.; Trombino, D.; et al. Preparation and antitubercular activities in vitro
and in vivo of novel Schiff bases of isoniazid. Eur. J. Med. Chem. 2009, 44, 4169–4178.
14. Hassan, F.A. Synthesis, characterization, anti-inflammatory, and antioxidant activities of some new
thiazole derivatives. Int. J. Appl. Sci. Technol. 2012, 2, 180–187.
15. Siddiqui, N.; Arshad, M.F.; Ahsan, W.; Alam, M.S. Thiazoles: A valuable insight into the recent
advances and biological activities. Int. J. Pharm. Sci. Drug Res. 2009, 1, 136–143.
16. Moldovan, C.M.; Oniga, O.; Pârvu, A.; Tiperciuc, B.; Verite, P.; Pîrnău, A.; Crişan, O.; Bojiţă, M.;
Pop, R. Synthesis and anti-inflammatory evaluation of some new acyl-hydrazones bearing
2-aryl-thiazole. Eur. J. Med. Chem. 2011, 46, 526–534.
17. Andreania, A.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M.; Cervellati, R.; Greco, E.;
Kondratyuk, T.P.; Park, E.-J.; Huang, K.; et al. Chemopreventive and antioxidant activity of 6
substituted imidazo[2,1-b]thiazoles. Eur. J. Med. Chem. 2013, 68, 412–421.

Molecules 2015, 20
17337
18. Jaishree, V.; Ramdas, N.; Sachin, J.; Ramesh, B. In vitro antioxidant properties of new thiazole
derivatives. J. Saudi Chem. Soc. 2012, 16, 371–376.
19. Abdel-Wahab, B.F.; Awad, G.E.A.; Badria, F.A. Synthesis, antimicrobial, antioxidant, anti-hemolytic
and cytotoxic evaluation of new imidazole-based heterocycles. Eur. J. Med. Chem. 2011, 46,
1505–1511.
20. Mohammad, H.; Reddy, P.V.N.; Monteleone, D.; Mayhoub, A.S.; Cushman, M.; Seleem, M.N.
Synthesis and antibacterial evaluation of a novel series of synthetic phenylthiazole compounds
against methicillin-resistant Staphylococcus aureus (MRSA). Eur. J. Med. Chem. 2015, 94, 306–316.
21. El-Wahab, H.A.; El-Fattah, M.A.; El-Khalik, N.A.; Nassar, H.S.; Abdelall, M.M. Synthesis and
characterization of coumarin thiazole derivative 2-(2-amino-1,3-thiazol-4-yl)-3H-benzo[f]chromen-
3-one with anti-microbial activity and its potential application in antimicrobial polyurethane coating.
Prog. Org. Coat. 2014, 77, 1506–1511.
22. Yang, S.J.; Lee, W.J.; Kim, E.-A.; Namd, K.D.; Hahn, H.-G.; Choi, S.Y.; Cho, S.-W. Effects of
N-adamantyl-4-methylthiazol-2-amine on hyperglycemia, hyperlipidemia and oxidative stress in
streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 2014, 736, 26–34.
23. Zhang, B.L.; Song, L.X.; Li, Y.F.; Li, Y.L.; Guo, Y.Z.; Zhang, E.; Liu, H.M. Synthesis and biological
evaluation of dehydroepiandrosterone-fused thiazole, imidazo[2,1-b]thiazole, pyridine steroidal
analogues. Steroids 2014, 80, 92–101.
24. Prashanth, T.; Thirusangu, P.; Avin, B.R.V.; Ranganatha, V.L.; Prabhakar, B.T.; Khanum, S.A.
Synthesis and evaluation of novel benzophenone-thiazole derivatives as potent VEGF-A inhibitors.
Eur. J. Med. Chem. 2014, 87, 274–283.
25. Popsavin, M.; Koji, V.; Spai, S.; Svircev, M.; Bogdanovic, G.; Jakimov, D.; Aleksic, L.; Popsavin, V.
2-Substituted thiazole-4-carboxamide derivatives as tiazofurin mimics: Synthesis and in vitro
antitumour activity. Tetrahedron 2014, 70, 2343–2350.
26. Tiperciuc, B.; Zaharia, V.; Colosi, I.; Moldovan, C.; Crisan, O.; Pirnau, A.; Vlase, L.;
Duma, M.; Oniga, O. Synthesis and Evaluation of antimicrobial activity of some new hetaryl-azoles
derivatives obtained from 2-aryl-4-methylthiazol-5-carbohydrazides and isonicotinic acid hydrazide.
J. Heterocycl. Chem. 2012, 49, 1407–1414.
27. Bharti, S.K.; Nath, G.; Tilak, R.; Singh, S.K. Synthesis, anti-bacterial and anti-fungal activities of
some novel Schiff bases containing 2,4-disubstituted thiazole ring. Eur. J. Med. Chem. 2010, 45,
651–660.
28. Chimenti, F.; Bizzarri, B.; Bolasco, A.; Secci, D.; Chimenti, P.; Granese, A.; Carradori, S.;
D’Ascenzio, M.; Lilli, D.; Rivanera, D. Synthesis and biological evaluation of novel 2,4-disubstituted-
1,3-thiazoles as anti-Candida spp. agents. Eur. J. Med. Chem. 2011, 46, 378–382.
29. Gaikwad, N.D.; Patil, S.V.; Bobade, V.D. Hybrids of ravuconazole: Synthesis and biological
evaluation. Eur. J. Med. Chem. 2012, 54, 295–302.
30. Gaikwad, N.D.; Patil, S.V.; Bobade, V.D. Synthesis and biological evaluation of some novel
thiazole substituted benzotriazole derivatives. Bioorg. Med. Chem. Lett. 2012, 22, 3449–3454.
31. Alam, M.S.; Liu, L.; Lee, Y.E.; Lee, D.W. Synthesis, antibacterial activity and quantum-chemical
studies of novel 2-arylidenehydrazinyl-4-arylthiazole analogues. Chem. Pharm. Bull. 2011, 59,
568–573.

Molecules 2015, 20
17338
32. Carradori, S.; Secci, D.; Bolasco, A.; Rivanera, D.; Mari, E.; Zicari, A.; Lotti, L.V.; Bizzarri, B.
Synthesis and cytotoxicity of novel (thiazol-2-yl)hydrazine derivatives as promising anti-Candida
agents. Eur. J. Med. Chem. 2013, 65, 102–111.
33. Secci, D.; Bizzarri, B.; Bolasco, A.; Carradori, S.; D’Ascenzio, M.; Rivanera, D.; Mari, E.; Polletta, L.;
Zicari, A. Synthesis, anti-Candida activity, and cytotoxicity of new (4-(4-iodophenyl)thiazol-2-
yl)hydrazine derivatives. Eur. J. Med. Chem. 2012, 53, 246–253.
34. Benedec, D.; Vlase, L.; Oniga, I.; Mot, A.C.; Damian, G.; Hanganu, D.; Duma, M.;
Silaghi-Dumitrescu, R. Polyphenolic Composition, antioxidant and antibacterial activities for two
romanian subspecies of Achillea distans Waldst. et Kit. ex Willd. Molecules 2013, 18, 8725–8739.
35. Lima, E.S.; Pinto, A.C.S.; Nogueira, K.L.; Rocha e Silva, L.F.; de Almeida, P.D.O.;
de Vasconcellos, M.C.; Chaves, F.C.M.; Tadei, W.P.; Pohlit, A.M. Stability and antioxidant activity
of semi-synthetic derivatives of 4-nerolidylcatechol. Molecules 2013, 18, 178–189.
36. Simić, A.; Manojlović, D.; Šegan, D.; Todorović, M. Electrochemical behavior and antioxidant and
prooxidant activity of natural phenolics. Molecules 2007, 12, 2327–2340.
37. Moalin, M.; Strijdonck, G.P.F.; Beckers, M.; Hagemen, G.J.; Borm, P.J.; Bast, A.; Haenen, G.R.
A planar conformation and the hydroxyl groups in the B and C rings play a pivotal role in the
antioxidant capacity of quercetin and quercetin derivatives. Molecules 2011, 16, 9636–9650.
38. National Committe for Clinical Laboratory Standards. NCCLS Approved Standard M27-PA; Clinical
and Laboratory Standards Institute: Wayne, PA, USA, 1997.
Sample Availability: Samples of the compounds 3–18 are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).