Facile synthesis and biological assays of novel 2,4-disubstituted hydrazinyl-thiazoles analogs

Article abstract of DOI:10.1007/s11030-015-9654-7

A convenient, one-pot multi-component synthesis of new 2,4-disubstituted hydrazinyl-thiazoles was accomplished using different aldehydes/ketones, thiosemicarbazide, and 4-methoxy phenacyl bromide in the presence of a catalytic amount of AcOH in EtOH. Prod

Full text of DOI:10.1007/s11030-015-9654-7

Mol Divers
DOI 10.1007/s11030-015-9654-7
ORIGINAL ARTICLE
Facile synthesis and biological assays of novel 2,4-disubstituted
hydrazinyl-thiazoles analogs
Fateme Ghanbari Pirbasti1 · Nosrat O. Mahmoodi1
Received: 23 July 2015 / Accepted: 29 December 2015
© Springer International Publishing Switzerland 2016
Abstract A convenient, one-pot multi-component syn-
thesis of new 2,4-disubstituted hydrazinyl-thiazoles was
accomplished using different aldehydes/ketones, thiosemi-
carbazide, and 4-methoxy phenacyl bromide in the presence
of a catalytic amount of AcOH in EtOH. Products were
obtained in reasonable yields and high purity. The in vitro
antioxidant activity of hydrazinyl-thiazoles was evaluated by
DPPH radical scavenging activity in comparison to ascorbic
acid. Synthesized thiazoles 14c and 14g possessed the low-
est IC50 values. Also, hydrazinyl-thiazoles were screened for
their in vitro antibacterial activity against six strains of bac-
teria including S. aureus, M. luteus, E. coli, Ps. aeruginosa,
B. subtilis, and A. hydrophila where some products showed
good antibacterial activity. Moreover, compound 14a showed
anticancer activity against melanoma cancerous cell lines
A375 with LC50 = 0.55 mg/mL, slightly selective versus
normal cell lines (Hu-2) with LC50 = 1.19 mg/mL.
is one of the B-complex vitamins that contains a thiazole
ring, which is vitally important to keep a living organism
operating properly. Commercial drugs, such as sulfathia-
zole 2 (antimicrobial drug), ritonavir 3 (antiretroviral drug),
and abafungin 4 (antifungal drug) contain a thiazole moiety
(Fig. 1).
Synthesized compounds containing a thiazole moiety
have been used for the treatment of allergies [1], hyper-
tension [2], inflammation [3], human immunodeficiency
virus (HIV) infections [4], and schizophrenia [5]. Thiazoles
also exhibit other biological activities, such as antibac-
terial [6,7], antitumor [8], analgesic [9], anticonvulsant
[10], antimelanogenesis [11], antipsychotic [12], and antiox-
idant [13]. Also, thiazoles can act as cyclin-dependent
kinase (CDK) inhibitors [14] and β-glucuronidase inhibitors
[15].
Analogs such as 2-substituted-6-fluorobenzo[d]thiazoles
[16], (thiazol-2-yl)hydrazine derivatives [17]. 3-Allyl-2-
(substituted imino)-4-phenyl-3H-thiazoles, and 2,2ꢀ-(1,3-
phenylene)bis(3-substituted-2-imino-4-phenyl-3H-thiazole)
derivatives [18] showed anti-candida, antibacterial, and
acetylcholinesterase activities. Moreover, thiazoles have
been used as semiconducting materials [19], thiazole-iridium
complexes as phosphorescent organic light-emitting diodes
[20], and naphthol-substituted thiazoles as ratiometric fluo-
rescent chemosensors [21].
Keywords One-pot three-component reaction · Dis-
ubstituted hydrazinyl-thiazoles · Antioxidant · DPPH ·
Antibacterial · Anticancer · MCR
Introduction
Thiazoles have attracted a great deal of attention because of
their diverse biological activities. Thiamine (vitamin B1, 1)
Recently, our research group reported the one-pot syn-
thesis of new thiazolyl-pyrazoline derivatives 5–6 [22], bis-
thiazoles 7–8 [23], and thiazolylpyridazinones 9 [24] exhibit-
ing antibacterial activity, and new 1,4-dihydropyridines 10
bearing a thiazole moiety exhibiting high antioxidant activity
[25] (Fig. 2). In continuation of our previous investiga-
tion [26], we report herein the one-pot MCR synthesis of
new hydrazinyl-thiazole analogs and their biological activi-
ties.
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-015-9654-7) contains supplementary
material, which is available to authorized users.
Nosrat O. Mahmoodi
nosmahmoodi@gmail.com; mahmoodi@guilan.ac.ir
B
1
Department of Chemistry, Faculty of Science,
University of Guilan, P.O. Box 41335-1914, Rasht, Iran
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Fig. 1 Vitamin B1 and drugs containing a thiazole moiety
Fig. 2 Thiazoles accessed via one-pot synthesis
Results and discussion
Chemistry
is a toxic solvent, EtOH was chosen as it is an eco-friendly
and green solvent.
Our results show that the products are obtained in high
yields and are easily purified using non-chromatographic
methods. The advantages of this method are simple set-up
and product isolation, reproducibility, using cheap and eco-
friendly solvent.
New 2,4-disubstituted hydrazinyl-thiazoles 14a–g were syn-
thesized via the one-pot reaction of different aldehydes/ket-
ones 11a–g, thiosemicarbazide 12, and 4-methoxy phenacyl
bromide 13 in the presence of AcOH in EtOH under
reflux conditions (Scheme 1). A literature survey indi-
cated that hydrazone-thiazoles are generally synthesized in
a two-step procedure. To the best of our knowledge, this
is the first one-pot synthesis report for 14 analogs [27–
30].
At the onset of our research, we explored the one-pot,
multi-component preparation of 14a in EtOH at room tem-
perature in the absence of a catalyst (entry 1) which after 6h
afforded the desired product in 56 % yield. In the presence of
AcOH, the yield of 14a increased from 56 to 60 % (entry 2).
Then, the scope of the reaction was investigated under reflux
conditions. As shown in Table 1, 14a was obtained after 2h
in EtOH or DMF in high yield (entries 4 & 8). Since DMF
In most cases, upon addition of 13 to a solution of 12 in
EtOH after 2 h under reflux condition with aldehydes/ketones
11a–g in the presence of few drop of AcOH, the desired prod-
uct precipitated. This is a good sign for the start of the reac-
tion. Hydrazinyl-thiazoles 14a, 14b, and 14c were obtained
as pure solids without the need for further purification.
The structures of hydrazinyl-thiazoles 14a–g were deter-
1
mined by IR, H NMR, and 13C NMR spectra. In the IR
spectra, the disappearance of the carbonyl and thiosemi-
carbazone N–H stretching vibration bands indicates the
participation of thioamide moiety in the cyclization reac-
tion formation of desired products. In 1H NMR spectra, the
presence of a singlet at 7.09–6.06 ppm confirms the thi-
azole ring closure. Also, the OCH3 signal appeared as a
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Table 1 Optimization of 14a
Entry
Catalyst
Solvent
Temp
Time (h)
Yield %a
1
2
3
4
5
6
7
8
a
–
EtOH
r.t.
6
4
2
2
4
3
3
2
56
60
75
80
65
70
70
80
AcOH (20 mol%)
AcOH (20 mol%)
AcOH (20 mol%)
AcOH (20 mol%)
AcOH (20 mol%)
AcOH (20 mol%)
AcOH (20 mol%)
EtOH
r.t.
EtOH
Reflux (60 ◦C)
Reflux (80 ◦C)
Reflux
EtOH
H2O
EtOH:H2O (1:1)
MeOH
DMF
Reflux
Reflux
Reflux
After purification
Scheme 1 Synthesis of new
thiazoles 14a–g
singlet at 3.87–3.67 ppm. Other aromatic protons appeared
in the expected region around 8.94–6.68 ppm. The N–H
signal appeared as a broad singlet at 14.50–11.45 ppm.
In addition, the N–H signal can be endo- or exocyclic.
However, according to literature [31], the signal at 14.50–
11.45 ppm is related to an exocyclic N-H. The 13C NMR
spectra of 14a–g provided the expected number and types
of carbons. The C–H of the thiazole ring appeared at
100.4–98.7 ppm. The signal at 55.9–55.3 is attributed to
OCH3 and other aromatic carbons appeared at 112.6–171.5
ppm.
The 1H NMR spectra did not show a single product;
rather, it showed a mixture of two sets of stereoisomer 16A/B
and C/D. The protons of the adamantane moiety appeared
at 2.20–1.25 ppm, OCH3 of isomers 16C/D appeared at
expected region at 3.96–3.72 ppm, and the proton of the thi-
azole moiety as well as other aromatic protons appeared at
7.80–6.71 ppm. Several signals at 3.25–3.01 and 2.74–2.58
ppm corresponded to CH–N=N isomers C and D.
Biology
In order to apply this procedure to carbonyl compounds
withsterichindrance, weinvestigatedthe3MCRsofcamphor
and 2-adamantanone 15 with thiosemicarbazide 12, and 4-
methoxy phenacyl bromide 13. The reaction of camphor was
unsuccessful and produced several by-products. However,
15 led to mixture of products 16A–16D in moderate yield
(Scheme 2).
Antioxidant assay
Using DPPH (diphenylpicrylhydrazyl) is one of the sim-
plest methods to evaluate antioxidant activity. DPPH is a
stable, free radical of violet color. When a compound donates
a radical hydrogen atom to a DPPH molecule, it reduces
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Scheme 2 Synthesis of thiazoles
16A–D from adamantanone and
proposed structures
Fig. 3 DPPH radical
scavenging activity of
hydrazinyl-thiazoles 14a–g
120
100
80
60
40
20
0
14a
14b
14c
14d
14e
14f
14g
Ascorbic acid
4000
2000
1000
500
250
125
62.5
Concentration in µg/mL
DPPH and so the absorbance of DPPH decreases. In brief,
the higher the antioxidant activity, the lighter the violet hue.
The antioxidant activity of hydrazinyl-thiazoles 14a–g was
evaluated by the DPPH radical scavenging activity method.
In this method, a decrease in absorption band at 517 nm
indicates that the test compound possesses antioxidant activ-
ity. The radical scavenging activity of 14a–g was screened
at a concentration of 4000–62.5 μg/mL and monitored at
517 nm. Also, IC50 values (the concentration of 14a–g to
scavenge 50 % of DPPH radical concentration) were cal-
culated from line equation which can be obtained from
Fig.3. Ascorbic acid was used as standard. All hydrazinyl-
thiazoles 14a–g showed dose-dependent antioxidant activity
(Fig. 3).
The IC50 values of 14a–g are in the range 2.90–0.23 μM
(Table 2). It is worth noting that hydrazinyl-thiazole 14c has
the lowest IC50 value (0.23 μM) while compound 14a has the
highest IC50 value (2.90 μM) compared to the IC50 value of
ascorbicacid(antioxidantcompound) (1.30 μM). Thehigher
antioxidant activity is reflected in a lower IC50. Compounds
14a–g were determined to exhibit potent radical scavenging
activity in a DPPH assay with IC50 values in the following
order14c, 14g, 14b, 14e, ascorbic acid, 14f, 14d, and 14a
respectively.
According to the literature [32] and our recent report [25],
the high antioxidant activity of 14a–g is attributed to the
presence of the thiazole ring. The N–H at the C2 position
of the thiazole ring can readily donate a hydrogen radical
to DPPH radical and form a radical of the test compound.
Therefore, the radical is highly resonance-stabilized through
the thiazole ring and =C–N–NH–C moiety (Scheme 3).
In addition to the presence of the thiazole moiety, the
high antioxidant activity of 14g can be due to the pres-
ence of benzylic hydrogens. These benzylic hydrogens are
easily converted to the stable benzyl radical by DPPH. Fur-
thermore, the antioxidant activity of 14b and 14c can be
due to the presence of aliphatic hydrogens which can form
tertiary, secondary, and primary carbon radicals. As it is men-
tioned in Table 2, these compounds exhibit better activity
than ascorbic acid. Therefore, the presence of benzylic and
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Scheme 3 Proposed mechanism of radical scavenging activity
Table 2 IC50 values for DPPH radical scavenging activity of
hydrazinyl-thiazoles 14a–g
limeter. According to the results, only 14d was active against
all six bacterial strains, and 14f had the highest antibac-
terial activity against S. aureus and M. luteus. Compound
14d was more active against E. coli and also showed sig-
nificant inhibition activity against B. subtilis. In addition,
14e showed significant inhibition activity against Ps. aerug-
inosa. Only 14b showed good antibacterial activity against
A. hydrophila.
Some reports showed moderate antibacterial activity of
hydrazinyl-thiazoles. For example, Bharti and co-workers
[33] studied the antibacterial activity of several hydrazinyl-
thiazole derivatives, finding that their thiazoles showed no
antibacterial activity against E. coli and only a few were
active against Ps. aeruginosa. However, in our study, 14c–
e had moderate antibacterial activity against E. coli, while
14e good antibacterial activity against Ps. aeruginosa. More-
over, Lee et al. [34] studied the antibacterial activity of
hydrazinyl-thiazoles on several bacterial strains. According
to their report, none of their synthesized compounds had
antibacterial activity against Ps. aeruginosa, and only a few
of them had good activity against S. aureus, B. subtilis, and E.
coli. Also, the hydrazinyl-thiazoles showed no antibacterial
activity against A. hydrophila.
Compound
DPPH assay IC50(μM)
14a
2.90
0.87
0.23
2.10
0.88
1.69
0.28
1.30
14b
14c
14d
14e
14f
14g
Ascorbic acid
aliphatic radicals as well thiazole and =C–N–NH–C moi-
eties possibly play an effective role in antioxidant activity
(Scheme 3).
Antibacterial assay
Products 14a–g were screened for their in vitro antibacterial
activity against Gram-positive and Gram-negative bacte-
ria strains including Staphylococcus aureus (S. aureus),
Micrococcus luteus (M. luteus), Escherichia coli (E. coli),
Pseudomonas aeruginosa (Ps. aeruginosa), Bacillus subtilis
(B. subtilis), Aeromonas hydrophila (A. hydrophila) using a
well-diffusion method. DMSO was used as negative control
and showed no activity against the above bacterial strains.
Penicillin G and Cefixime were used as positive controls
(Table 3).
Anticancer assay
Melanoma is a cancer that develops in melanocytes (the pig-
ment cells present in the skin). It is known that melanoma
can spread to other parts of the body causing serious ill-
ness and death. Melanoma can be more serious than two
other forms of skin cancers, e.g., basal-cell cancer (BCC)
and squamous-cell cancer (SCC). The primary treatment
for melanoma is surgery. There are a few drugs used in
chemotherapy of melanoma cancer; however, none of them
Hydrazinyl-thiazoles 14a–g were evaluated for their
antibacterial activity at a concentration of 4000 μg/mL in
DMSO. The experiments were performed in triplicate. The
results are presented as mean standard deviation in mil-
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Table 3 Antibacterial activity of hydrazinyl-thiazoles 14a–g (4000 μg/mL) as zone of inhibition in millimeter
Antibacterial activity (mean SD) (mm)
Compound
S. aureus
M. luteus
E. coli
Ps. aeruginosa
B. subtilis
A. hydrophila
14a
–
8.33 0.57
–
7.00 0.73
7.33 1.52
9.66 0.57
9.33 0.57
17.33 0.57
7.66 0.57
–
6.66 0.57
7.66 0.57
11.00 1.0
21.33 0.57
12.33 1.15
–
–
14b
7.33 1.15
6.66 0.57
7.66 0.57
8.66 0.57
11.33 1.52
7.66 1.15
–
–
9.66 1.15
11.00 1.0
14c
7.66 0.57
11.00 1.0
–
14d
8.66 0.57
13.00 1.0
7.66 0.57
14e
–
11.33 0.57
–
14f
10.33 0.57
–
–
14g
–
–
7.66 0.57
–
6.66 0.57
DMSOa
Penicillin Gb
Cefiximeb
–
–
–
–
23
55
35
45
39
24
33
48
41
38
30
32
a
Negative control
Positive control
b
100
90
80
70
60
50
40
30
20
10
0
Table 4 The LC50 values of cytotoxic effects of 14a on melanoma
cancer cell lines (A375) (mean SD)
Hu-02 24h
A375 24h
Hu-02 48 h
A375 48h
Entry LC50 (mg/mL)
Hu-02 (24 h) A375 (24 h) Hu-02 (48 h) A375 (48 h)
14a
1.19 0.12
0.55 0.01
0.81 0.06
0.47 0.02
at 48 h time of exposure at a concentration of 0.5 mg/mL
(P ≤ 0.05).
0.037 0.075 0.125
0.25
0.5
The LC50 values of 14a are shown in Table 4. The LC50
values are between 1.19 and 0.55 mg/mL. As it is shown,
the LC50 values on melanoma cancer cells (A375) are lower
than on skin cells (Hu-02). However, increasing the time of
exposure from 24 h to 48 h did not have a significant effect
on the LC50 values.
Concentration mg/mL
Fig. 4 In vitro cytotoxic activity of 14a on human melanoma can-
cer cell lines A375. Results are presented as the percentage of cell
viability SD using the MTT protocol
contain hydrazinyl-thiazole moiety, e.g., dacarbazine and
temozolomide contain imidazole and carboxamide moi-
eties, respectively. An in vitro cytotoxicity study of 14a
was carried out on human melanoma cancer cell lines
A375 and Hu-02 (normal human skin cell lines) obtained
from the National Centre for Cell Science, Tehran, Iran.
Cell viability of 14a was evaluated using the colorimetric
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay protocol at concentrations of 0.5, 0.25, 0.125,
0.075, 0.037 mg/mL in DMSO. DMSO was used as neg-
ative control. The MTT method is based on the reduction
of soluble MTT by mitochondrial reductase of the viable
cells to insoluble purple crystals of formazan ((E, Z)-5-
(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan). The time
of exposure was 24 h and 48 h (Fig. 4). Results are reported
as the percent of cell viability standard deviation. Statis-
tical analysis demonstrated that 14a had cytotoxic activity
on Hu-02 (69.4 %) and A375 (58.8 %) cell lines at 24 h
time of exposure and on Hu-02 (64.3 %) and A375 (51.2 %)
Conclusions
Several new hydrazinyl-thiazoles derivatives 14a–g were
synthesized via one-pot reaction of aldehydes/ketones 11a–
g, thiosemicarbazide 12, and 4-methoxy phenacyl bromide
13 through a reliable procedure for high yields and high
purity. Hydrazinyl-thiazoles 14a, 14b, and 14c were obtained
as pure solids without further purification. All products
showed high antioxidant activity in comparison to ascorbic
acid. Compounds 14b and 14e had the highest antioxidant
activity; their IC50 was less than that of ascorbic acid. Com-
pounds 14a–g showed low to moderate antibacterial activity.
Compound 14d possessed the highest antibacterial activity
against B. subtilis. Compound 14a had cytotoxic activity at
a concentration of 0.5 mg/mL upon 24 h and 48 h time of
exposure on melanoma cancer cell lines.
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Experimental section
Materials and instruments
Anal. calcd. for C21H21N3O2S: C, 66.49, H, 5.56, N, 11.10.
Found: C, 66.51; H, 5.59; N, 11.06 %.
4-(4-Methoxyphenyl)-2-(2-(4-phenylcyclohexylidene)
Starting materials, reagents, biological cultures, and solvents
were obtained from commercial suppliers Fluka, Merck, Sd-
fine, Quelab and were used without further purification. All
reactions were monitored by silica gel-coated TLC plates
(60 F254 Merck). IR spectra were recorded on a Shimadzu
IR-470 spectrophotometer in anhydrous KBr. 1H NMR and
13C NMR spectra were recorded on a 400 MHz and 500
MHz Bruker spectrometers using DMSO-d6 and CDCl3 as
solvents. Chemical shifts are expressed relative to TMS as
singlet (s), doublet (d), triplet (t), multiple (m), doublet of
doublet (dd), doublet of triplet (dt), triplet of triplet (tt), dou-
blet of doublet of doublet (ddd), and quintet (quin). Coupling
constants are expressed in Hertz (Hz). Elemental analyses
were carried out on a Carlo–Erba EA1110 CNNO-S analyzer.
Melting points were determined with a Mettler Fp5 appara-
tus, and were uncorrected. Absorbance in the antioxidant
assay was recorded on an Unico 2100 spectrophotometer.
hydrazinyl)thiazole (14b)
Orange solid (0.3 g, 85 %); mp 186–189 ◦C (from EtOH);
ν
IR (KBr)
3210 (stretch N–H), 3020 (stretch C–H aro-
max
matic), 2920 (stretch C–H aliphatic), 1610 (stretch C=N),
1560, 1540, 1510 (stretch C=C), 1250, 1040 (stretch C–O),
820, 740, 690 (OOP. C–H) cm−1; R f (80 %, hexane: EtOAc
6:3); 1H NMR (400 MHz, CDCl ) 12.45 (s, 1H, NH), 7.67
δ
3
(d, J = 8.8 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 7.26–7.22 (m,
3H), 7.01 (d, J = 8.8 Hz, 2H), 6.56 (s, 1H), 3.86 (s, 3H), 3.32
(d, J = 14.8 Hz, 1H), 2.88 (tt, J = 12, 3.3 Hz, 1H), 2.72
(d, J = 14.4 Hz, 1H), 2.47 (dt, J = 14, 4.9 Hz, 1H), 2.32
(dt, J = 14.1, 5.3 Hz, 1H), 2.27–2.17 (m, 2H), 1.79 (ddd,
J = 25.5, 13.1, 4.3 Hz, 2H) ppm; 13C NMR (125 MHz,
CDCl ) 170.1, 164.2, 161.5, 145.1, 140.8, 129.0, 127.5,
δ
3
127.1, 127.0, 120.3, 115.4, 98.8, 55.8, 43.5, 35.2, 34.6, 33.3,
29.4 ppm; Anal. calcd. for C22H23N3OS: C, 70.03; H, 6.11;
N, 11.09. Found: C, 69.98; H, 6.15; N, 11.12 %.
General procedure for the synthesis of
hydrazinyl-thiazoles 14a–g
2-(2-(Heptan-4-ylidene)hydrazinyl)-4-(4-methoxyphenyl)
thiazole (14c)
To a solution of thiosemicarbazide 12 (1 mmol) in EtOH,
aldehydes/ketones 11a–g (1 mmol) and a few drops of AcOH
were added and this mixture was refluxed and stirred. After
a few minutes (1–15 min), 4-methoxy phenacyl bromide
13 (1 mmol) was added and the reaction was refluxed and
stirred until completion (1.5–2 h). Thin layer chromatogra-
phy (TLC) was used to monitor the progress of the reaction
(EtOAc:n-hexane 3:6). After completion of the reaction, the
reaction mixture was cooled down to room temperature. The
resulting solid was filtered and washed with or recrystallized
in EtOH.
Orange solid (0.26 g, 83 %); mp 127–131 ◦C (from EtOH);
ν
IR (KBr)
3210 (stretch N–H), 3100 (stretch C–H aro-
max
matic), 2950, 2850 (stretch C–H aliphatic), 1610 (stretch
C=N), 1560, 1540, 1505 (stretch C=C), 1250, 1020 (stretch
C–O), 830 (OOP. C–H) cm−1; R f (82 %, hexane: EtOAc
6:3); 1H NMR (400 MHz, CDCl ) 12.39 (s, 1H, NH), 7.66
δ
3
(d, J = 8.2 Hz, 2H), 6.99 (d, J = 7.8 Hz, 2H), 6.53 (s,
1H), 3.85 (s, 3H), 2.49 (t, J = 9 Hz, J = 8 Hz, 2H), 2.34 (t,
J = 7.6 Hz, 2H), 1.70–1.61 (m, 4H), 1.11 (t, J = 7.2 Hz,
3H), 0.98 (t, J = 7.4 Hz, 3H) ppm; 13C NMR (125 MHz,
DMSO − d6) 170.2, 166.1, 161.5, 140.7, 127.5, 120.4,
δ
2-(2-(6-Methoxy-3,4-dihydronaphthalen-1(2H)-
ylidene)hydrazinyl)-4-(4-methoxyphenyl)thiazole (14a):
115.3, 98.7, 55.8, 38.9, 33.8, 19.8, 19.7, 14.6, 14.1 ppm;
Anal. calcd. for C17H23N3OS: C, 64.35; H, 7.28; N, 13.21.
Found: C, 64.30; H, 7.31; N, 13.17 %.
Cream solid (0.3 g, 80 %); mp 233–236 ◦C (from EtOH);
ν
IR (KBr)
3210 (stretch N–H), 3100 (stretch C–H aro-
2-(2-((4-Chlorophenyl)(phenyl)methylene)hydrazinyl)-4-
max
matic), 2920, 2830 (stretch C–H aliphatic), 1610 (stretch
C=N), 1570, 1500 (stretch C=C), 1250, 1240, 1045 (stretch
(4-methoxyphenyl)thiazole (14d)
C–O), 825, 760, 700 (OOP. C–H) cm−1; R f (83 %, hexane:
Orange crystal (0.32 g, 78 %); mp 239–242 ◦C (from EtOH);
1
ν
max
EtOAc 6:3); H NMR (400 MHz, CDCl ) 12.46 (s, 1H,
IR (KBr)
3120 (stretch C–H aromatic), 1610 (stretch
δ
3
NH), 8.02 (d, J = 8.8Hz, 1H), 7.67 (d, J = 8.0 Hz, 2H),
6.99 (d, J = 8.8 Hz, 2H), 6.82 (dd, J = 8.8, 2.8 Hz, 1H),
6.68 (d, J = 2.4 Hz, 1H), 6.06 (s, 1H), 3.84 (s, 3H), 3.83 (s,
3H), 2.90 (t, J = 6.4 Hz, 2H), 2.79 (t, J = 6.0 Hz, 2H), 2.02
(quin, J = 6.3 Hz, 2H) ppm; 13C NMR (100 MHz, CDCl )
C=N), 1580, 1560, 1540 (stretch C=C), 1250, 1030 (stretch
C–O), 830, 740, 690 (OOP. C–H) cm−1; R f (73 %, hexane:
1
EtOAc 6:3); H NMR (400 MHz, CDCl ) 12.07 (s, 1H,
δ
3
NH), 7.69 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H),
7.60 (dd, J = 7.8, 1.3 Hz, 2H), 7.49 (tt, J = 6.6, 1.8 Hz,
1H), 7.42 (tt, J = 8.8, 1.2, 2H), 7.35 (d, J = 8.8 Hz, 2H),
6.85 (d, J = 8.8 Hz, 2H), 6.63 (s, 1H) 3.85 (s, 3H) ppm; 13C
δ
3
169.3, 161.2, 160.7, 154.0, 142.5, 127.1, 126.9, 123.7, 121.5,
114.7, 113.5, 112.6, 99.1, 55.4, 55.3, 29.7, 27.2, 21.6 ppm;
123

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NMR (125 MHz, CDCl /DMSO−d ) 170.0, 161.3, 137.2,
126.8, 120.6, 115.0, 100.2, 55.7, 22.2, 21.5 ppm. Anal. calcd.
for C20H21N3OS: C, 68.37; H, 6.05; N, 11.94. Found: C,
68.32; H, 5.98; N, 11.98 %.
δ
6
3
135.7, 131.3, 130.7, 130.6, 130.4, 129.7, 129.6, 128.9, 128.5,
128.3, 127.6, 115.1, 114.6, 100.4, 55.7 ppm; Anal. calcd. for
C23H18ClN3OS: C, 65.81; H, 4.29; N, 9.98. Found: C, 65.85;
H, 4.33; N, 10.03 %.
2-(Adamantan-2-ylidenehydraono)-4-(4-methoxyphenyl)-
2,3-dihydrothiaole (16)
4-(4-Methoxyphenyl)-2-(phenyl(pyridin-2-yl)methylene)
hydrazinyl)thiazole (14e)
Orange solid (0.17g, 50 %); mp 140–148 ◦C (from EtOH); IR
ν
(KBr) max 2917, 2849 (stretch C-H aliphatic), 1605 (stretch
Red solid (0.27 g, 71 %); mp 215–220 ◦C (from EtOH); IR
C=N), 1549, 1503 (stretch C=C) cm−1; R f (35 %, hexane:
EtOAc 6:3); 1H NMR (500 MHz, CDCl ) 7.80–6.71 (m, Ar,
ν
max
(KBr)
1605 (stretch C=N), 1550, 1510, 1480 (stretch
δ
3
C=C), 1240, 1050 (stretch C–O), 830, 760, 730, 700 (OOP.
C–H) cm−1; R f (74 %, hexane: EtOAc 6:3); 1H NMR (400
MHz, CDCl ) 14.51 (s, 1H, NH), 8.93 (d, J = 3.6 Hz,
NH, H-thiazole), 3.96–3.72(OCH3), 3.01–2.55(CH–C=Nof
C and D), 2.20–1.25 (CH and CH2 adamantan) ppm.
δ
3
1H), 7.83–7.77 (m, 3H), 7.64–7.60 (m, 2H), 7.52–7.39 (m,
5H), 6.97 (d, J = 8.8 Hz, 2H), 6.78 (s, 1H), 3.87 (s, 3H)
ppm; 13C NMR (125 MHz, CDCl ) 171.5, 160.6, 150.2,
Biological methods
δ
3
DPPH radical scavenging assay
148.6, 138.7, 136.0, 129.9, 129.6, 129.3, 128.7, 128.6, 127.6,
127.3, 126.0, 124.7, 114.6, 100.3, 55.3 ppm; Anal. calcd. for
C22H18N4OS: C, 68.41; H, 4.72; N, 14.53. Found: C, 68.36;
H, 4.67; N, 14.48 %.
The DPPH radical scavenging activity of 14a–d was eval-
uated according to the literature [25]. A 2,2-diphenyl-2-
picrylhydrazyl (DPPH) solution was prepared by dissolving
an appropriate amount of DPPH in MeOH to give a con-
centration of 6.25 × 10−5 M. Compounds 14a–g and DPPH
with different concentrations (4000, 2000, 1000, 500, 250,
125, 62.5 μg/mL) in MeOH were prepared. Then, 0.1 mL
of each hydrazinyl-thiazole solution was added to 3.9 mL
of DPPH solution and was shaken vigorously. Samples were
kept in darkness for 30 min and then their absorbance was
measured at 517 nm. MeOH was used as blank. Radical scav-
enging activity was calculated as follows:
2-Methoxy-4-((2-(4-(4-methoxyphenyl)thiazol-2-yl)
hydrazono)methyl)-6-nitrophenol (14f)
Red solid (0.3 g, 75 %); mp 207–209 ◦C (from EtOH); IR
νmax
(KBr)
3250 (stretch N–H), 3100 (stretch C–H aro-
matic), 2980, 2830 (stretch C–H aliphatic), 1610 (stretch
C=N), 1570, 1480 (stretch C=C), 1540, 1340 (stretch NO2),
1250, 1050 (stretch C–O), 840, 760, 740, 690 (OOP. C–H)
cm−1; R f (60 %, hexane: EtOAc 6:3); 1H NMR (500 MHz,
DMSO − d ) ∼ 11.45 (s, 1H, NH), 7.95 (s, 1H), 7.74
ꢀ
ꢁ
Acontrol − Asample
δ
6
Radical scavenging activity %=
×100
(d, J = 8.76 Hz, 2H), 7.67 (dd, J = 1.52 Hz, 1H), 7.49
(dd, J = 1.48 Hz, 1H), 7.09 (s, 1H), 6.92 (d, J = 8.7 Hz,
2H), 3.90 (s, 3H), 3.74 (s, 3H) ppm; 13C NMR (125 MHz,
Acontrol
where Acontrol is the absorbance of the negative control
(containing all reagents except test compounds), Asample the
absorbance of the test compounds. IC50 values of the test
compounds were determined by plotting the radical scav-
enging activity percentage against concentration of the test
compound.
DMSO − d6) 168.8, 159.6, 151.1, 150.6, 144.2, 140.2,
δ
138.1, 128.3, 127.6, 126.2, 115.1, 114.8, 113.1, 102.3, 57.4,
55.9 ppm; Anal. calcd. for C18H16N4O5S: C, 54.03; H, 4.06;
N, 13.97. Found: C, 53.97; H, 4.02; N, 14.04 %.
4-(4-Methoxyphenyl)-2-(2-(2,4,6-trimethylbenzylidene)
hydrozinyl)thiazole (14g)
Antibacterial assay
Cream solid (0.26 g, 75 %); mp 229–233 ◦C (from EtOH);
The antibacterial activity of hydrazinyl-thiazoles was eval-
uated biologically using a well-diffusion method. First,
nutrient agar and nutrient broth cultures were prepared
according to manufacturer’s instructions and were incubated
at 37 ◦C. After incubation for the appropriate time, a suspen-
sion of 30 μL of each bacterium was added to the nutrient
agar plates. Cups (5 mm in diameter) were cut in the agar
using sterilized glass tube. Each well received 30 μL of the
test compounds at concentration of 4000 μg/ml in DMSO.
Then, plates were incubated at 37 ◦C for 24 h, after this
ν
IR (KBr)
3400 (stretch N–H), 3100 (stretch C–H aro-
max
matic), 2950, 2820 (stretch C–H aliphatic), 1610 (stretch
C=N), 1510 (stretch C=C), 1250, 1020 (stretch C–O), 850,
750, 740, (OOP. C–H) cm−1; R f (75 %, hexane: EtOAc 6:3);
1H NMR (500 MHz, DMSO − d ) ∼ 13.00 (s, 1H, NH),
δ
6
8.65 (s, 1H), 7.52 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 6.9 Hz,
2H), 6.75 (s, 2H), 6.56 (s, 1H), 3.67 (s, 3H), 2.31 (s, 6H), 2.13
(s, 3H)ppm;13CNMR(125MHz, DMS0−d ) DMSO−d )
δ
6
6
∼ 170, 161.3, 151.2, 141.1, 140.9, 139.1, 130.4, 127.9,
δ
123

Mol Divers
time the zone of inhibition was measured. The values are
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Cefixime as standards. DMSO was used as negative control.
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Mean Absorbance of sample
% cell viability =
× 100
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The LC50 values (lethal concentration of the test compound
required to kill 50 % of sample population) were determined
by plotting cell viability percentage against concentration of
the sample.
Acknowledgments The authors are greatly thankful to Dr. Zohreh
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and helpful comments of Professor Dr. Guillermo A. Morales, Editor-
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