Synthesis and MAO inhibitory activity of novel thiazole-hydrazones

Article abstract of DOI:10.3906/kim-1612-78

A series of new thiazole-hydrazones (3a-3n) were synthesized, characterized, and screened for their hMAO-A and hMAO-B inhibitory activity by an in vitro uorometric method. Selectivity indexes (SIs) were expressed as IC₅₀ (MAO-A) / IC₅₀

Full text of DOI:10.3906/kim-1612-78

Turk J Chem
2017) 41: 685 – 699
Turkish Journal of Chemistry
(
http://journals.tubitak.gov.tr/chem/
¨
˙
⃝
c TUBITAK
Research Article
doi:10.3906/kim-1612-78
Synthesis and MAO inhibitory activity of novel thiazole-hydrazones
1
2,∗
¨
¨
Ozlem ATLI , Yusuf OZKAY
1
2
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eski ¸s ehir, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eski ¸s ehir, Turkey
Received: 28.12.2016
•
Accepted/Published Online: 30.03.2017
•
Final Version: 10.11.2017
Abstract: A series of new thiazole-hydrazones (3a–3n) were synthesized, characterized, and screened for their hMAO-
A and hMAO-B inhibitory activity by an in vitro fluorometric method. Selectivity indexes (SIs) were expressed
as IC50 (MAO-A) / IC50 (MAO-B). Compound 3f showed promising hMAO-A inhibition with an IC50 value of
1
.20 µM and displayed a very significant SI of 0.04 towards hMAO-A. The mechanism of hMAO-A inhibition was
investigated by enzyme kinetics using Lineweaver–Burk graphics. Compound 3f was further screened for its cytotoxicity
by using a healthy NIH/3T3 mouse embryonic fibroblast cell line (ATCC CRL1658) and was evaluated as nontoxic at its
effective concentration against hMAO-A. The ADME prediction of the compounds revealed that they may have good
pharmacokinetic profiles, which is necessary for drug candidates.
Key words: Thiazole, hydrazone, hMAO enzymes, enzyme inhibition
1. Introduction
Monoamine oxidase (MAO) is the key enzyme of brain function, responsible for the metabolism of neurotrans-
mitters by regulating the oxidative deamination of amines in neuronal, glial, and other cells in the brain as well
as peripheral tissues.1 Two main forms of this enzyme are present as MAO-A and MAO-B. MAO-A is mainly
present in catecholaminergic neurons of cortex, and MAO-B is found in the serotonergic neurons in the brain.3,5
The common substrates for these enzymes are dopamine, tyramine, and tryptamine, whereas serotonin and no-
radrenaline are particularly metabolized by MAO-A. MAO-B metabolizes small amines like benzylamine and
−4
phenethylamine.3
,5−7
Endogenous amine metabolism results in the formation of toxic reactive oxygen species
responsible for oxidative damage and neurodegeneration.4,5 Therefore, MAO inhibitors are used for the treat-
ment of neurodegenerative and neurological disorders.3,4,8 As the two forms have different substrate selectivities
and levels in different regions of brain, their activities are involved in distinct clinical cases.4 MAO-B inhibitors
are used in multiple therapies for the movement disorders of Parkinson disease and Alzheimer disease,4,7,9
whereas MAO-A inhibitors are potent antidepressants and anxiolytic agents.3,4,7,8 MAO-A activity has been
also found to be associated with neuropsychiatric disorders, including autism, major depressive disorder, and
attention deficit hyperactivity disorder.4,6 The first-generation irreversible MAO inhibitors were used as an-
tidepressants, but most of these were later withdrawn from the market due to their severe adverse effects. They
were reported to cause tachycardia, photophobia, palpitation, nausea, hepatotoxicity, and drug and food inter-
actions. In particular, “tyramine reaction” is a serious health problem from the interaction between the MAO
∗
Correspondence: yozkay@anadolu.edu.tr
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inhibitor and certain foods, and it causes death by manifesting hypertensive crisis.7,10 The potentiation effects
of MAO-A (not MAO-B) inhibitors on indirectly acting sympathomimetic amines and their minimal tyramine
potentiation pointed out the importance of MAO-A inhibitors and led to their reemergence for clinical use in
the treatment of depression.11 Since MAO-A is the major enzyme for endogenous amine metabolism, it is the
main target of neurological and neurodegenerative disorders commonly caused by this oxidative metabolism.6
Thus, design and development of potent and selective MAO-A inhibitors are required because of their features
as explained above and the tight involvement of dysregulation of monoamines in neurodegenerative and mental
disease development. Reversible and selective MAO-A inhibitory drug development efforts will also lead to the
discovery of useful therapeutic agents, which are devoid of unwanted life-threating adverse effects.3
,5−7,10
The development of MAO inhibitors arises from hydrazine derivatives, since the first one developed was
the drug iproniazid. Subsequently, heterocyclic hydrazines, hydrazides, and hydrazones were synthesized as
potential MAO inhibitors.5
,12−16
The common structural feature of inhibitors in these studies was an amino
or imino group, which seems to play an essential role in orientation and complex formation at the active site of
the enzyme1 .
Thiazole was reported as another important heterocyclic moiety for MAO inhibition. From molecular
modeling studies, it was found that C4 of the thiazole ring was responsible for the interactions between the
inhibitor and the FAD cofactor, which controls the oxidative activity of the MAO enzyme.10 It was concluded
that even a small modification of the substituent’s dimension at the C4 position could attenuate the activity of
the compound.10 MAO inhibition potencies of hydrazone and thiazole have directed researchers to synthesize
combination products of these two moieties for the development of new MAO inhibitors.5,14,17,18 Prompted by
this knowledge, in the present study, we synthesized new thiazole-hydrazone compounds to screen their hMAO
inhibition potency.
2
2
. Results and discussion
.1. Chemistry
Compounds 3a–3n were synthesized as outlined in the Scheme. 4-(4- Methoxyphenoxy)benzaldehyde (1a)
and 4-(4-methoxyphenylsulfanyl)benzaldehyde (1b) were prepared under microwave irradiation by reacting 4-
fluorobenzaldehyde with 4-methoxyphenol or 4-methoxythiophenol. Compounds 1a and 1b were reacted with
thiosemicarbazide to gain 4-(4-methoxyphenoxy)benzaldehyde thiosemicarbazone (2a) and 4-(4-methoxyphenyl-
sulfanyl)benzaldehyde (2b). Hantzsch reaction of compounds 2a and 2b with an appropriate α-bromoacetophe-
none under microwave conditions afforded 4-(4-methoxyphenoxy)benzaldehyde [4-(4-substituted phenyl)-1,3-
thiazol-2-yl] hydrazones (3a–3g) and 4-(4-methoxyphenylsulfanyl)benzaldehyde [4-(4-substituted phenyl)-1,3-
thiazol-2-yl] hydrazones (3h–3n) in high yields (88%–96%) and short reaction times (5 min).
Structural elucidation of the synthesized compounds (3a–3n) was performed by spectral analyses. The
−
1
FT-IR spectra of the final products showed characteristic absorption bands at 3155–3346 cm
for NH and at
−
1
for the azomethine group (CH = N). The 1 H NMR spectra of the compounds showed signals
1489–1566 cm
at δ 7.94–8.03 and δ 12.07–12.24 corresponding to the azomethine (CH = N) proton and hydrazide (CONH)
proton, respectively. The C5-H proton of the thiazole was observed as a singlet at δ 7.20–7.38. The appearance
of a pair of doublets and/or multiplets at δ 6.90–7.90 was due to the aromatic protons of phenyl rings. In the
1
3
C NMR spectra, methoxy carbons (OCH3) were observed at 55.80–55.91 ppm. The carbon of azomethine
CH = N) was assigned at 140.89–141.69 ppm, respectively. The C2 carbon of the thiazole ring appeared at
(
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ATLI and OZKAY/Turk J Chem
K CO / DMF
2
3
H CO
XH
F
3
CHO
o
H CO
X
CHO
MW, 110 C, 5 Bar
3
1
a, 1b
S
S
CH COOH / C H OH
3
2
5
H CO
X
CH NNH C NH2
NH NH C NH2
2
3
Reflux
R1
2
a, 2b
R2
O
N
C H OH
2
5
R1
Br
H CO
X
CH NNH
o
3
MW, 100 C, 10 Bar
S
3
a - 3n
R2
3
a
3b
X : -O- -O- -O- -O- -O- -O- -O- -S- -S-
R : -H -CH3 -Br -Cl -F -Cl -F -H -CH3 -Br -Cl -F -Cl -F
3c 3d 3e 3f
3h 3i
3k 3l 3m 3n
3g
3j
-S- -S- -S- -S- -S-
1
R : -H -H
-H -H -H -Cl -F
-H -H -H -Cl -F -H -H
2
Scheme. Synthesis pathway for compounds 3a–3n.
166.75–168.85 ppm. The aromatic carbons were recorded in the region of 103.02–162.05 ppm. Due to the
presence of a fluoro substituent in compounds 3e, 3g, 3l, and 3n, splittings relating to neighboring atoms were
detected in the spectra. In the HR-MS spectra of the final compounds (3a–3n), the M + 1 peak was observed
in accordance with their molecular formulas. The M + 1 peaks were determined at an accuracy of 2–17 ppm.
2.2. Enzyme inhibition
The synthesized 4-(4-methoxyphenoxy)benzaldehyde [4-(4-substituted phenyl)-1,3-thiazol-2-yl] hydrazones (3a–
3
3
g) and 4-(4-methoxyphenylsulfanyl)benzaldehyde [4-(4-substituted phenyl)-1,3-thiazol-2-yl] hydrazones (3h–
n) were screened for their hMAO-A and hMAO-B inhibitory activity by an in vitro fluorometric method.19,20
The assay is based on the fluorometric detection of H2 O2 , one of the products generated during the oxidative
deamination of the MAO substrate (tyramine), using the OxiRed probe reagent, a highly sensitive and stable
probe for H2 O2 . Clorgiline and selegiline were used as reference drugs. The inhibitory activity results are listed
in Table 1.
Against hMAO-A, the most active compound 3f displayed an IC50 of 1.20 µM, whereas reference drug
clorgiline had an IC50 of 0.0071 µM. On the other hand, compound 3f showed an IC50 of 33.47 µM against
hMAO-B, while reference drug selegiline (IC50 = 0.040 µM) also displayed a significant inhibition against
hMAO-B. Selectivity indexes (SIs) were expressed as IC5
/IC
. Selectivity towards MAO-
0(MAO−A)
50(MAO−B)
A increased as the corresponding SI decreased, while selectivity towards the MAO-B isoform increased as the
corresponding SI increased. It was observed that the synthesized compounds have selective inhibition potency
against hMAO-A. Compound 3f displayed a very significant SI of 0.04.
In the design of the target compounds (3a–3n), the substitution pattern was performed on two regions
to discuss the substitution effect on biological activity. The first region shows the difference in terms of 4-(4-
methoxyphenoxy) or 4-(4-methoxyphenylsulfanyl) substructures. There is a substituent difference in the second
region, which includes the 4-(4-substituted phenyl)-1,3-thiazol substructure. It was observed that compounds
6
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Table 1. IC50 (µM) and selectivity of compounds 3a–3n against hMAO isoforms.
Compound IC50 (µM) hMAO-A IC50 (µM) hMAO-B SI
Selectivity
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
8.54 ꢀ 0.39
12.79 ꢀ 0.58
10.58 ꢀ 0.45
7.72 ꢀ 0.37
9.32 ꢀ 0.51
1.20 ꢀ 0.08
17.62 ꢀ 0.86
26.78 ꢀ 1.73
21.05 ꢀ 1.69
23.06 ꢀ 1.74
21.01 ꢀ 1.91
24.24 ꢀ 1.88
20.71 ꢀ 1.76
26.74 ꢀ 1.53
0.0071 ꢀ 0.0004
-
50.43 ꢀ 2.48
68.15 ꢀ 3.61
58.40 ꢀ 2.96
39.75 ꢀ 2.73
53.60 ꢀ 3.09
33.47 ꢀ 1.98
58.93 ꢀ 2.54
91.13 ꢀ 4.59
164.17 ꢀ 9.21
95.99 ꢀ 5.47
338.30 ꢀ 14.89
92.21 ꢀ 4.29
69.28 ꢀ 3.75
279.70 ꢀ 13.27
-
0.17 MAO-A
0.19 MAO-A
0.18 MAO-A
0.19 MAO-A
0.17 MAO-A
0.04 MAO-A
0.30 MAO-A
0.29 MAO-A
0.13 MAO-A
0.24 MAO-A
0.06 MAO-A
0.26 MAO-A
0.30 MAO-A
0.10 MAO-A
g
h
i
j
k
l
m
n
Clorgiline
Selegiline
-
-
MAO-A
MAO-B
0.044 ꢀ 0.003
3a–3g have more potency to inhibit the MAO enzymes when compared to compounds 3h–3n. This result
suggests that the first structural region has an impact on enzyme inhibition, and it may be declared that the
presence of oxygen instead of sulfur enhances the enzyme inhibitory activity. The substituent change in the
second structural region also has an effect on enzyme inhibitory activity. Among compounds 3a–3g, compound
3f has a higher potency to inhibit the MAO enzymes, which may be the result of a more lipophilic character
due to dichloro substitution.
2.3. Kinetic studies of enzyme inhibition
The mechanism of hMAO-A inhibition was investigated by enzyme kinetics, following a procedure similar to
the MAO inhibition assay. The linear Lineweaver–Burk graphics were used to estimate the type of inhibition.21
The enzyme was analyzed by recording substrate-velocity curves in the absence and presence of the most potent
compound, 3f, which was prepared at concentrations of IC50 / 4 (0.30 µM), IC50 / 2 (0.60 µM), IC50 (1.20
µM), 2 × IC50 (2.40 µM), and 4 × IC50 (4.80 µM) (Figures 1A and 1B). In each case, the initial velocity
measurements were gained at different substrate (tyramine) concentrations ranging from 20 µM to 0.625 µM.
Replots of the slopes of the Lineweaver–Burk plots versus inhibitor concentration are presented in Figure 1. The
Lineweaver–Burk plot introduces the inhibition type as mixed type, competitive or noncompetitive. In mixed-
typed inhibition, the lines cross neither the x- nor the y-axis at the same point. Noncompetitive inhibition
has plots with the same intercept on the x-axis, but there are different slopes and intercepts on the y-axis.
Competitive inhibitors possess the same intercept on the y-axis, but there are diverse slopes and intercepts on
the x-axis between the two datasets, as seen in Figure 1. Therefore, this pattern indicates that the mechanism
of hMAO-A inhibition of 3f is competitive, explaining that the inhibitor can bind to the enzyme in competition
with the substrate. The Ki value for compound 3f was calculated as 1.453 µM for the inhibition ofhMAO-A.
688

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ATLI and OZKAY/Turk J Chem
(A)
2
1
0.00
6.00
Control
0
0
1
2
4
.30 µM
.60 µM
.20 µM
.40 µM
.80 µM
(B)
12.00
1
2
0
8
6
4
2
0
2
y = 1.8278x + 2.6756
R² = 0.99
1
8
4
0
.00
.00
.00
-
3
-2
-1
0
1
2
3
-2
-1
0
1
2
3
4
5
-
1
/[S] µM-1
Compound 3f (µM)
-
4.00
Figure. A) Lineweaver–Burk plots for the inhibition of hMAO-A by compound 3f. [S], substrate concentration [µM];
−
1
−1
V, reaction velocity [nmol min
mg ]. Inhibitor concentrations are shown on the left. Vmax value for competitive
values from 4.80 µM to control: 7.411, 4.242, 3.280, 2.611, 2.181,
.463 µM. B) Replots of the slopes of the Lineweaver–Burk plots versus inhibitor concentration. K was calculated as
.453 µM.
inhibition was calculated as 0.6410. Respective K
m
1
1
i
2.4. Cytotoxicity test
The toxicity of compound 3f was investigated by MTT assay, which is based on the reduction of yellow MTT
dye by metabolically active eukaryotic and prokaryotic cells to form the purple formazan product. This assay is
mainly preferred to establish an understanding about cell viability and to observe the growth of cell cultures.22,23
MTT assay was carried out using the healthy NIH/3T3 mouse embryonic fibroblast cell line (ATCC CRL1658),
which is recommended for cytotoxicity screening by ISO 10993-5.24 As seen in Table 2, the IC50 values of the
reference drugs and 3f against NIH/3T3 cells were found to be ≥1000 µM, which is significantly higher than
their IC50 values against hMAO enzymes. Thus, it can be stated that compound 3f is nontoxic at its effective
concentration against hMAO-A.
Table 2. IC50 (µM) values of the reference drugs and 3f against the NIH/3T3 cell line.
Compound Clorgiline Selegiline 3f
IC50 (µM)
≥ 1000
≥ 1000
≥ 1000
2.5. Theoretical prediction of ADME properties and BBB permeability
Essential pharmacological activity and low toxicological effects are not adequate for a compound to become a
drug candidate. A good pharmacokinetics profile is also very important for new drug candidates and should be
assessed earlier in the process of drug development. In recent years, noteworthy developments in combinatorial
chemistry have made the estimation of absorption, distribution, metabolism, and excretion (ADME) relatively
easy.25 The theoretical calculation of the ADME properties (molecular weight, log P, tPSA, number of hydrogen
donors and acceptors, volume) of compounds (3a–3n) was carried out and this is presented in Table 3 along
with violations of Lipinski’s rule.26 This rule suggests that an orally active drug should not have more than
one violation. According to the findings in Table 3, all compounds obey Lipinski’s rule. On the other hand, all
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ATLI and OZKAY/Turk J Chem
calculated physicochemical parameters for compound 3f are compatible with Lipinski’s rule except the log P
value. Although the log P (7.05) of compound 3f exceeds Lipinski’s limit, it shows that the related compound
has a lipophilic character, which is suitable to cross the central nervous system (CNS). Furthermore, tPSA,
described to be a predictive indicator of membrane penetration, is positive (55.75), and as MAO inhibitors have
to pass different membranes and reach the CNS, this supports the potential of compound 3f.27
Table 3. Some physicochemical parameters of the compounds 3a–3n and reference drugs used in the prediction of
ADME profiles.
Comp.
MW
logP tPSA nON nOHNH MV
Vio BBB (+ / -)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
g
h
i
j
k
l
m
n
401.49 5.77 55.75
415.52 6.22 55.75
480.39 6.58 55.75
435.94 6.45 55.75
419.48 5.93 55.75
470.38 7.05 55.75
437.47 6.02 55.75
417.56 5.98 46.52
431.59 6.43 46.52
496.45 6.79 46.52
452.00 6.66 46.52
435.55 6.14 46.52
486.45 7.26 46.52
453.54 6.24 46.52
5
5
5
5
5
5
5
5
5
5
5
5
5
5
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
355.02
371.58
372.90
368.55
359.95
382.09
364.88
364.16
380.72
382.05
377.70
369.09
391.23
374.02
202.64
238.91
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Selegiline 187.29 2.64 3.24
Clorgiline 272.18 3.74 12.47
MW: Molecular weight; log P: log octanol/water partition coefficient; tPSA: total polar surface area; nON: number
of hydrogen acceptors; nOHNH: number of hydrogen donors; MV: molecular volume; Vio: violations of Lipinski’s rule
were calculated using the Molinspiration Calculation of Molecular Properties toolkit. BBB (+ / -): Blood–brain barrier
permeability was calculated with the CBLigand BBB prediction server.
Drugs that specifically target the CNS must first pass the blood–brain barrier (BBB). Although the
BBB is protective in nature, the inability of drug molecules to permeate the BBB is a significant impediment
for CNS drug candidates and should be addressed early in the drug discovery process. Thus, the task of
predicting the BBB permeability of new compounds is of great importance.28 From this point of view, BBB
permeability of the synthesized compounds (3a–3n) was calculated by the CBLigand BBB prediction server
(
http://www.cbligand.org/BBB/index.php). This predictor uses two different algorithms, AdaBoost and sup-
port vector machine (SVM), combined with four different fingerprints to predict if a compound can pass (+) or
cannot pass (-) the BBB. In each case, the predictor generates scores higher than 0 if the compound can pass
the BBB. As presented in Table 3, all calculations for the synthesized compounds resulted in BBB (+), which
is necessary for MAO inhibitors.
2.6. Conclusions
In summary, preliminary evaluation of new 4-(4-methoxyphenoxy)benzaldehyde [4-(4-substituted phenyl)-1,3-
thiazol-2-yl] hydrazones (3a–3g) and 4-(4-methoxyphenylsulfanyl)benzaldehyde [4-(4-substituted phenyl)-1,3-
thiazol-2-yl] hydrazones (3h–3n) as hMAO inhibitory agents resulted in promising findings. Compound 3f
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displayed good hMAO-A inhibition. Furthermore, this compound did not show any cytotoxicity. Consequently,
the findings of the present study will not only direct our research group to further studies but may also encourage
medicinal chemists to synthesize more effective and safer compounds bearing chemical structures similar to those
of compound 3f.
3
3
. Experimental
.1. Chemistry
All chemicals were purchased from Sigma Aldrich Chemicals (Sigma Aldrich Corp., St. Louis, MO, USA)
and Merck Chemicals (Merck KGaA, Darmstadt, Germany). All melting points (mp) were determined with
an MP90 digital melting point apparatus (Mettler Toledo, Columbus, OH, USA) and were uncorrected. All
reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 F254 TLC plates (Merck
KGaA, Darmstadt, Germany). Spectroscopic data were recorded with the following instruments: IR, Shimadzu
Affinity 1S spectrophotometer (Shimadzu, Tokyo, Japan); NMR, Bruker DPX 300 NMR spectrometer (Bruker
Bioscience, Billerica, MA, USA), in DMSO-d6 , using TMS as internal standard; M + 1 peaks, Shimadzu LC/MS
IT-TOF system (Shimadzu).
3.2. Synthesis of 4-(4-methoxyphenoxy)benzaldehyde (1a) and 4-(4-methoxyphenylsulfanyl)ben-
zaldehyde (1b)
A mixture of 4-fluorobenzaldehyde (40 mmol, 4.28 mL), 4-methoxyphenol (40 mmol, 4.96 g), or 4-methoxythiop-
henol (40 mmol, 4.92 mL) and dimethylformamide (DMF) (10 mL) were added into a vial (30 mL) of microwave
synthesis reactor (Anton-Paar, Monowave 300, Austria). The reaction mixture was heated under conditions of
◦
1
10 C and 5 bar for 15 min. After cooling, the mixture was poured into ice water and the precipitated product
was washed with water, dried, and recrystallized from ethanol to give 4-(4-methoxyphenoxy)benzaldehyde (1a)
at yield: 92%; mp: 61 C; ref. mp: 60 C29 and 4-(4-methoxyphenylsulfanyl)benzaldehyde (1b) at yield: 94%;
◦
◦
mp: 46 C; ref. mp: 46–46.5 C30
◦
◦
.
3
.3. Synthesis of 4-(4-methoxyphenoxy)benzaldehyde thiosemicarbazone (2a) and 4-(4-methoxyp-
henylsulfanyl)benzaldehyde (2b)
A mixture of thiosemicarbazide (30 mmol, 2.7 g) and compound 1a (30 mmol, 6.84 g) or 1b (30 mmol,
.32 g) in ethanol (20 mL) was refluxed for 2 h. The progress of the reaction was monitored by TLC. The
resulting mixture was cooled, poured into ice water, filtered, and then recrystallized from ethanol to afford
7
-(4-methoxyphenoxy)benzaldehyde thiosemicarbazone (2a) at yield: 86%; mp: 175 C; ref. mp: 174 C31
◦
◦
4
◦
and 4-(4-methoxyphenylsulfanyl)benzaldehyde thiosemicarbazone (2b) at yield: 88%, mp: 165.3 C, FTIR
ATR, cm 1): 3414 (N - H), 3282 (N - H), 3149 (N - H), 1487 (C = N), 1274 (C - N), 815, 804. H NMR
−
1
(
(
300 MHz, DMSO-d6): δ = 3.79 (3H, s, OCH3), 7.03 (2H, d, J =8.80 Hz, methoxyphenyl H3,3′ ), 7.07 (2H,
d, J =8.40 Hz, benzylidene H2,2′ ), 7.45 (2H, d, J = 8.80 Hz, methoxyphenyl H2,2′ ), 7.70 (2H, d, J = 8.40 Hz,
benzylidene H3,3′ ), 7.95 (1H, br, NH2), 7.97 (1H, s, azomethine CH), 8.18 (1H, br, NH2), 11.41 (1H, s, NH).
1
3
C NMR (75 MHz, DMSO-d6): δ = 55.83 (OCH3), 115.99 (methoxyphenyl C3,3′ ), 122.28 (methoxyphenyl
C1), 127.37 (methoxyphenyl C2,2′ ), 128.42 (benzylidene C2,2′ ), 132.19 (benzylidene C1), 136.37 (benzylidene
6
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ATLI and OZKAY/Turk J Chem
C3,3′ ), 140.92 (benzylidene C4), 141.94 (C = N), 160.50 (methoxyphenyl C4), 178.33 (C = S). HRMS (m / z):
[
M + H]+ calcd for C15 H15 N3 OS2 : 318.0729; found: 318.0721.
3.4. Synthesis of 4-(4-methoxyphenoxy)benzaldehyde [4-(4-substituted phenyl)-1,3-thiazol-2-yl]
hydrazones (3a–3g) and 4-(4-methoxyphenylsulfanyl)benzaldehyde [4-(4-substituted phenyl)-
1,3-thiazol-2-yl] hydrazones (3h–3n)
A mixture of appropriate α-bromoacetophenone (2 mmol) and compound 2a (2 mmol, 0.600 g) or 2b (2 mmol,
0
3
.632 g) in ethanol (10 mL) was added to a vial (30 mL) of microwave synthesis reactor (Anton-Paar, Monowave
◦
00). The reaction mixture was heated under conditions of 100 C and 10 bar for 5 min. After cooling, the
mixture was poured into ice water and the precipitated product was washed with water, dried, and recrystallized
from ethanol to give 4-(4-methoxyphenoxy)benzaldehyde [4-(4-substituted phenyl)-1,3-thiazol-2-yl] hydrazones
(
(
3a–3g) and 4-(4-methoxyphenylsulfanyl)benzaldehyde [4-(4-substituted phenyl)-1,3-thiazol-2-yl] hydrazones
3h–3n).
3.4.1. 4-(4-Methoxyphenoxy)benzaldehyde (4-phenyl-1,3-thiazol-2-yl) hydrazone (3a)
Yield: 89%, mp = 203.3 C, FTIR (ATR, cm ): 3169 (N - H), 1494 (C = N), 1222 (C - N), 827, 707. 1 H NMR
◦
−1
(
(
300 MHz, DMSO-d6): δ = 3.76 (3H, s, OCH3), 6.96 (2H, d, J =8.58 Hz, methoxyphenyl H3,3′ ), 7.00–7.07
4H, m, methoxyphenyl H2,2′ , benzylidene H2,2′ ), 7.29–7.31 (2H, m, monosubstituted phenyl H4 , thiazole H),
7
.40 (2H, t, J =7.32 Hz, monosubstituted phenyl H3,3′ ), 7.63 (2H, d, J =8.73 Hz, benzylidene H2,2′ ), 7.85
2H, d, J =7.32 Hz, monosubstituted phenyl H2,2′ ), 8.00 (1H, s, azomethine CH), 12.09 (1H, s, NH). 13
NMR (75 MHz, DMSO-d6): δ = 55.88 (OCH3), 103.97 (thiazole C5), 115.64 (methoxyphenyl C3,3′ ), 117.77
benzylidene C1), 121.55 (benzylidene C3,3′ ), 125.96 (monosubstituted phenyl C4), 127.97 (methoxyphenyl
(
C
(
C2,2′ ), 128.45 (monosubstituted phenyl C3,3′ ), 129.06 (monosubstituted phenyl C2,2′ ), 129.29 (benzylidene
C2,2′ ), 135.17 (monosubstituted phenyl C1), 141.22 (C = N), 149.22 (methoxyphenyl C1), 151.16 (thiazole
C4), 156.41 (benzylidene C4), 159.52 (methoxyphenyl C4), 168.70 (thiazole C2). HRMS (m / z): [M + H]+
calcd for C23 H19 N3 O2 S: 402.1271; found: 402.1265.
3.4.2. 4-(4-Methoxyphenoxy)benzaldehyde [4-(4-methylphenyl)-1,3-thiazol-2-yl] hydrazone (3b)
◦
−1
1
Yield: 92%, mp = 131.3 C, FTIR (ATR, cm ): 3302 (N - H), 1496 (C = N), 1228 (C - N), 839, 727.
H
NMR (300 MHz, DMSO-d6): δ = 2.32 (3H, s, CH3), 3.77 (3H, s, OCH3), 6.95–7.03 (4H, m, benzylidene H2,2′
methoxyphenyl H3,3′ ), 7.06 (2H, d, J =9.21 Hz, methoxyphenyl H2,2′ ), 7.20–7.25 (3H, m, methylphenyl H3,3′
,
,
thiazole H), 7.63 (2H, d, J =8.85 Hz, benzylidene H3,3′ ), 7.74 (2H, d, J =8.13 Hz, methylphenyl H2,2′ ), 8.00
1H, s, azomethine CH), 12.07 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 21.26 (CH3), 55.89 (OCH3),
(
1
03.02 (thiazole C5), 115.62 (methoxyphenyl C3,3′ ), 117.76 (benzylidene C1), 121.54 (benzylidene C3,3′ ),
1
25.91 (methylphenyl C4), 128.43 (methoxyphenyl C2,2′ ), 129.31 (benzylidene C2,2′ ), 129.62 (methylphenyl
C4), 132.50 (methylphenyl C2,2′ ), 137.23 (methylphenyl C1), 141.19 (C = N), 149.21 (methoxyphenyl C1),
1
/
50.97 (thiazole C4), 156.40 (benzylidene C4), 159.50 (methoxyphenyl C4), 168.61 (thiazole C2). HRMS (m
z) : [M + H]+ calcd for C24 H21 N3 O2 S : 416.1427; found: 416.1417.
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3.4.3. 4-(4-Methoxyphenoxy)benzaldehyde [4-(4-bromophenyl)-1,3-thiazol-2-yl] hydrazone (3c)
Yield: 89%, mp = 208.5 C, FTIR (ATR, cm ): 3305 (N - H), 1496 (C = N), 1230 (C - N), 1051, 839, 729. 1
◦
−1
H
NMR (300 MHz, DMSO-d6): δ = 3.76 (3H, s, OCH3), 6.94–6.99 (4H, m, benzylidene H2,2′ , methoxyphenyl
H3,3′ ), 7.06 (2H, d, J =9,18 Hz, methoxyphenyl H2,2′ ), 7.38 (1H, s, thiazole H), 7.59 (2H, d, J =8.58 Hz,
bromophenyl H2,2′ ), 7.63 (2H, d, J =8.82 Hz, benzylidene H3,3′ ), 7.80 (2H, d, J =8.58 Hz, bromophenyl
H3,3′ ), 8.00 (1H, s, azomethine CH), 12.10 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.88 (OCH3),
1
04.87 (thiazole C5), 115.63 (methoxyphenyl C3,3′ ), 117.76 (benzylidene C1), 120.95 (bromophenyl C1), 121.55
benzylidene C3,3′ ), 127.99 (methoxyphenyl C2,2′ ), 128.49 (bromophenyl C2,2′ ), 129.22 (benzylidene C2,2′ ),
31.99 (bromophenyl C3,3′ ), 134.37 (bromophenyl C4), 141.45 (C = N), 149.20 (methoxyphenyl C1), 149.81
thiazole C4), 156.41 (benzylidene C4), 159.56 (methoxyphenyl C4), 168.85 (thiazole C2). HRMS (m / z): [M
H]+ calcd for C23 H18 BrN3 O2 S: 480.0376; found: 480.0360.
(
1
(
+
3.4.4. 4-(4-Methoxyphenoxy)benzaldehyde [4-(4-chlorophenyl)-1,3-thiazol-2-yl] hydrazone (3d)
◦
−1
Yield: 93%, mp = 192.9 C, FTIR (ATR, cm ): 3305 (N - H), 1504 (C = N), 1244 (C - N), 839, 731.
1
H NMR (300 MHz, DMSO-d6): δ = 3.76 (3H, s, OCH3), 6.96 (2H, d, J =8.61 Hz, methoxyphenyl H3,3′ ),
6
.99–7.07 (4H, m, benzylidene H2,2′ , methoxyphenyl H2,2′ ), 7.37 (1H, s, thiazole H), 7.46 (2H, d, J =8.61 Hz,
chlorophenyl H2,2′ ), 7.63 (2H, d, J =8.79 Hz, benzylidene H3,3′ ), 7.86 (2H, d, J =8.58 Hz, chlorophenyl H3,3′ ),
.00 (1H, s, azomethine CH), 12.09 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.91 (OCH3), 104.79
8
(
(
thiazole C5), 115.64 (methoxyphenyl C3,3′ ), 117.76 (benzylidene C1), 121.55 (benzylidene C3,3′ ), 127.67
methoxyphenyl C2,2′ ), 128.48 (chlorophenyl C1), 129.08 (benzylidene C2,2′ ), 129.23 (chlorophenyl C2,2′ ),
1
32.35 (chlorophenyl C3,3′ ), 134.03 (chlorophenyl C4), 141.42 (C = N), 149.20 (methoxyphenyl C1), 149.79
thiazole C4), 156.41 (benzylidene C4), 159.56 (methoxyphenyl C4), 168.84 (thiazole C2). HRMS (m / z): [M
H]+ calcd for C23 H18 ClN3 O2 S: 436.0881; found: 436.0874.
(
+
3.4.5. 4-(4-Methoxyphenoxy)benzaldehyde [4-(4-fluorophenyl)-1,3-thiazol-2-yl] hydrazone (3e)
◦
−1
1
Yield: 91%, mp = 224.9 C, FTIR (ATR, cm ): 3305 (N - H), 1496 (C = N), 1226 (C - N), 842, 827.
H
NMR (300 MHz, DMSO-d6): δ = 3.75 (3H, s, OCH3), 6.90–7.06 (6H, m, benzylidene H2,2′ , methoxyphenyl
H2,3,2′,3′ ), 7.21 (3H, m, fluorophenyl H2,2′ , thiazole), 7.62 (2H, d, J =8.79 Hz, benzylidene H3,3′ ), 7.88 (2H, d,
J =8.82 Hz, fluorophenyl H3,3′ ), 8.00 (1H, s, azomethine CH), 12.09 (1H, s, NH). 13 C NMR (75 MHz, DMSO-
d6): δ = 55.87 (OCH3), 103.69 (thiazole C5), 115.74 (methoxyphenyl C3,3′ ), 117.75 (benzylidene C1), 121.54
(
(
(
benzylidene C3,3′ ), 127.93 (JCF − 3 =8.05 Hz, fluorophenyl C2,2′ ), 128.46 (methoxyphenyl C2,2′ ), 128.99
fluorophenyl C1), 129.08 (benzylidene C2,2′ ), 129.45 (JCF − 2 =27.14 Hz, fluorophenyl C3,3′ ), 131.78, 141.34
C = N), 146.41 (thiazole C4), 149.20 (methoxyphenyl C1), 156.40 (benzylidene C4), 159.43 (methoxyphenyl
+
C4), 162.05 (JCF − 1 = 242.71 Hz, fluorophenyl C4), 168.82 (thiazole C2). HRMS (m / z): [M + H] calcd
for C23 H18 FN3 O2 S: 420.1170; found: 419.1182.
3.4.6. 4-(4-Methoxyphenoxy)benzaldehyde [4-(2,4-dichlorophenyl)-1,3-thiazol-2-yl] hydrazone (3f)
Yield: 89%, mp = 190.1 C, FTIR (ATR, cm ): 3157 (N - H), 1494 (C = N), 1230 (C - N), 827, 738. 1 H NMR
◦
−1
(
(
300 MHz, DMSO-d6): δ = 3.76 (3H, s, OCH3), 6.94–6.97 (2H, d, J =8.82 Hz, benzylidene H2,2′ ), 6.97–7.06
4H, m, methoxyphenyl H2,3,2′,3′ ), 7.38 (1H, s, thiazole H), 7.49 (1H, dd, J =2.22–8.52 Hz, dichlorophenyl
6
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ATLI and OZKAY/Turk J Chem
H5), 7.63 (2H, d, J =8.82 Hz, benzylidene H3,3′ ), 7.68 (1H, d, J =2.16 Hz, dichlorophenyl H3), 7.89 (1H, d,
J =8.52 Hz, dichlorophenyl H6), 8.01 (1H, s, azomethine CH), 12.11 (1H, s, NH). 13 C NMR (75 MHz, DMSO-
d6): δ = 55.90 (OCH3), 109.53 (thiazole C5), 115.63 (methoxyphenyl C3,3′ ), 117.77 (benzylidene C1), 121.54
(
benzylidene C3,3′ ), 127.95 (methoxyphenyl C2,2′ ), 128.50 (benzylidene C2,2′ ), 129.21 (dichlorophenyl C3),
30.22 (dichlorophenyl C5), 132.00 (dichlorophenyl C1), 132.63 (dichlorophenyl C6), 132.66 (dichlorophenyl
C4), 132.91 (dichlorophenyl C2), 141.52 (C = N), 146.38 (thiazole C4), 149.20 (methoxyphenyl C1), 156.41
benzylidene C4), 159.58 (methoxyphenyl C4), 167.94 (thiazole C2). HRMS (m / z): [M + H]+ calcd for
C23 H17 Cl2 N3 O2 S: 470.0491; found: 470.0474.
1
(
3
.4.7. 4-(4-Methoxyphenoxy)benzaldehyde [4-(2,4-difluorophenyl)-1,3-thiazol-2-yl] hydrazone (3g)
−1
◦
Yield: 95%, mp = 146.1 C, FTIR (ATR, cm ): 3346 (N - H), 1496 (C = N), 1222 (C - N), 831, 742.
1
H NMR (300 MHz, DMSO-d6): δ = 3.76 (3H, s, OCH3), 6.96 (2H, d, J =8.34 Hz, methoxyphenyl H3,3′ ),
6
.99–7.07 (4H, m, benzylidene H2,2′ , methoxyphenyl H2,2′ ), 7.14–7.20 (2H, m, difluorophenyl H5 , thiazole
H), 7.30–7.37 (1H, m, difluorophenyl H3), 7.63 (2H, d, J =8.73 Hz, benzylidene H3,3′ ), 8.00–8.03 (2H, m,
azomethine CH, difluorophenyl H6), 12.15 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.90 (OCH3),
104.70 (t, J =26.3 Hz, difluorophenyl C3), 108.11 (d, J =13.8 Hz, thiazole C4), 112.30 (dd, J =2.8–21.0
Hz, difluorophenyl C5), 115.63 (methoxyphenyl C3,3′ ), 117.75 (benzylidene C1), 119.51 (dd, J =3.9–11.2
Hz, difluorophenyl C1), 121.56 (benzylidene C3,3′ ), 128.52 (methoxyphenyl C2,2′ ), 129.18 (benzylidene C2,2′ ),
1
30.87 (dd, J =4.2–10.2 Hz, difluorophenyl C6), 141.69 (C = N), 143.63 (thiazole C4), 149.18 (methoxyphenyl
C1), 156.41 (benzylidene C4), 159.60 (methoxyphenyl C4), 160.07 (dd, J =12.0–250.3 Hz, difluorophenyl C4),
61.75 (dd, J =12.6–245.7 Hz, difluorophenyl C2), 168.19 (thiazole C2). HRMS (m / z): [M + H]+ calcd for
C23 H17 F2 N3 O2 S: 438.1082; found: 438.1078
1
3
.4.8. 4-(4-Methoxyphenylsulfanyl)benzaldehyde (4-phenyl-1,3-thiazol-2-yl) hydrazone (3h)
◦
−1
Yield: 96%, mp = 162.9 C, FTIR (ATR, cm ): 3313 (N - H), 1566 (C = N), 1242 (C - N), 823, 711.
1
H NMR (300 MHz, DMSO-d6): δ = 3.80 (3H, s, OCH3), 7.04 (2H, d, J =8.58 Hz, methoxyphenyl H3,3′ ),
7
.00–7.07 (4H, m, benzylidene H3,3′ , methoxyphenyl H2,2′ ), 7.38–7.40 (2H, m, monosubstituted phenyl H4 ,
thiazole H), 7.46 (2H, d, J =8.85 Hz, monosubstituted phenyl H3,3′ ), 7.63 (2H, d, J =8.49 Hz, benzylidene
H2,2′ ), 7.83–7.86 (2H, m, monosubstituted phenyl H2,2′ ), 7.96 (1H, s, azomethine CH), 12.17 (1H, s, NH). 13
NMR (75 MHz, DMSO-d6): δ = 55.81 (OCH3), 104.15 (thiazole C5), 115.99 (methoxyphenyl C3,3′ ), 125.96
methoxyphenyl C1), 127.41 (monosubstituted phenyl C4), 127.85 (methoxyphenyl C2,2′ ), 128.26 (benzylidene
C
(
C2,2′ ), 128.72 (monosubstituted phenyl C3,3′ ), 129.07 (monosubstituted phenyl C2,2′ ), 132.47 (benzylidene
C1), 135.97 (monosubstituted phenyl C1), 136.16 (benzylidene C3,3′ ), 140.03 (benzylidene C4), 141.00 (C =
N), 148.46 (thiazole C4), 160.15 (methoxyphenyl C4), 168.21 (thiazole C2). HRMS (m / z): [M + H]+ calcd
for C23 H19 N3 OS2 : 418.1042; found: 418.1038.
3
.4.9. 4-(4-Methoxyphenylsulfanyl)benzaldehyde [4-(4-methylphenyl)-1,3-thiazol-2-yl] hydrazone
3i)
Yield: 93 %, mp = 179.7 C, FTIR (ATR, cm ): 3155 (N - H), 1489 (C = N), 1246 (C - N), 815, 727. 1 H NMR
300 MHz, DMSO-d6): δ = 2.31 (3H, s, CH3), 3.80 (3H, s, OCH3), 7.04 (2H, d, J =8.82 Hz, methoxyphenyl
(
◦
−1
(
H3,3′ ), 7.13 (2H, d, J =8.40 Hz, benzylidene H2,2′ ), 7.19 (2H, d, J =8.13 Hz, methylphenyl H3,3′ ), 7.23 (1H, s,
thiazole H), 7.46 (2H, d, J =8.79 Hz, methoxyphenyl H2,2′ ), 7.56 (2H, d, J =8.46 Hz, benzylidene H3,3′ ), 7.73
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2H, d, J =8.13 Hz, methylphenyl H2,2′ ), 7.95 (1H, s, azomethine CH), 12.13 (1H, s, NH). 13 C NMR (75 MHz,
(
DMSO-d6): δ = 21.28 (CH3), 55.82 (OCH3), 103.23 (thiazole C5), 115.98 (methoxyphenyl C3,3′ ), 122.53
methoxyphenyl C1), 125.91 (methylphenyl C3,3′ ), 127.39 (methylphenyl C4), 127.86 (methoxyphenyl C2,2′ ),
(
1
29.63 (benzylidene C2,2′ ), 132.50 (benzylidene C1), 136.21 (methylphenyl C2,2′ ), 137.25 (methylphenyl C1),
139.99 (benzylidene C4), 140.89 (C = N), 148.29 (thiazole C4), 160.43 (methoxyphenyl C4), 168.47 (thiazole
C2). HRMS (m / z): [M + H]+ calcd for C24 H21 N3 OS2 : 432.1199; found: 432,1191.
3
.4.10. 4-(4-Methoxyphenylsulfanyl)benzaldehyde [4-(4-bromophenyl)-1,3-thiazol-2-yl] hydrazone
3j)
Yield: 88%, mp = 205.7 C, FTIR (ATR, cm ): 3159 (N - H), 1489 (C = N), 1247 (C - N), 819, 725.
NMR (300 MHz, DMSO-d6): δ = 3.79 (3H, s, OCH3), 7.03 (2H, d, J =8.79 Hz, methoxyphenyl H3,3′ ), 7.13
2H, d, J =8.40 Hz, benzylidene H2,2′ ), 7.38 (1H, s, thiazole H), 7.45 (2H, d, J =8.76 Hz, methoxyphenyl
(
◦
−1
1
H
(
H2,2′ ), 7.54–7.60 (4H, m, benzylidene H3,3′ , bromophenyl H2,2′ ), 7.79 (2H, d, J =8.55 Hz, bromophenyl
H3,3′ ), 7.96 (1H, s, azomethine CH), 12.17 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.82 (OCH3),
1
1
05.06 (thiazole C5), 115.97 (methoxyphenyl C3,3′ ), 120.98 (bromophenyl C1), 122.50 (methoxyphenyl C1),
27.43 (bromophenyl C2,2′ ), 127.82 (methoxyphenyl C2,2′ ), 127.98 (benzylidene C2,2′ ), 131.99 (bromophenyl
C3,3′ ), 132.40 (benzylidene C1), 134.33 (bromophenyl C4), 136.23 (benzylidene C3,3′ ), 140.13 (benzylidene
C4), 141.18 (C = N), 149.82 (thiazole C4), 160.44 (methoxyphenyl C4), 168.73 (thiazole C2). HRMS (m / z):
[
M + H]+ calcd for C23 H18 BrN3 OS2 : 496.0147; found: 496.0131.
3
.4.11. 4-(4-Methoxyphenylsulfanyl)benzaldehyde [4-(4-chlorophenyl)-1,3-thiazol-2-yl] hydrazone
3k)
Yield: 90%, mp = 196.4 C, FTIR (ATR, cm ): 3205 (N - H), 1489 (C = N), 1247 (C - N), 821, 725.
NMR (300 MHz, DMSO-d6): δ = 3.79 (3H, s, OCH3), 7.03 (2H, d, J =8.76 Hz, methoxyphenyl H3,3′ ), 7.13
2H, d, J =8.37 Hz, benzylidene H2,2′ ), 7.37 (1H, s, thiazole H), 7.44–7.46 (4H, m, methoxyphenyl H3,3′
(
◦
−1
1
H
(
,
chlorophenyl H2,2′ ), 7.56 (2H, d, J =8.43 Hz, benzylidene H3,3′ ), 7.86 (2H, d, J =8.55 Hz, chlorophenyl
H3,3′ ), 7.96 (1H, s, azomethine CH), 12.17 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.82 (OCH3),
1
1
04.96 (thiazole C5), 115.97 (methoxyphenyl C3,3′ ), 122.50 (methoxyphenyl C1), 127.42 (chlorophenyl C1),
27.67 (chlorophenyl C2,2′ ), 127.82 (methoxyphenyl C2,2′ ), 129.08 (benzylidene C2,2′ ), 131.22 (chlorophenyl
C3,3′ ), 132.41 (benzylidene C1), 133.98 (chlorophenyl C4), 136.22 (benzylidene C3,3′ ), 140.12 (benzylidene
C4), 141.17 (C = N), 149.79 (thiazole C4), 160.44 (methoxyphenyl C4), 168.72 (thiazole C2). HRMS (m / z):
[
M + H]+ calcd for C23 H18 ClN3 OS2 : 452.0653; found: 451.0642.
3
.4.12. 4-(4-Methoxyphenylsulfanyl)benzaldehyde [4-(4-fluorophenyl)-1,3-thiazol-2-yl] hydrazone
3l)
Yield: 92%, mp = 182.1 C, FTIR (ATR, cm ): 3284 (N - H), 1489 (C = N), 1247 (C - N), 821, 732.
NMR (300 MHz, DMSO-d6): δ = 3.80 (3H, s, OCH3), 6.98–7.07 (2H, m, methoxyphenyl H3,3′ ), 7.09–7.14
2H, m, benzylidene H2,2′ ), 7.20–7.30 (3H, m, fluorophenyl H2,2′ , thiazole H), 7.42–7.47 (2H, m, methoxyphenyl
H2,2′ ), 7.56 (2H, d, J =8.49 Hz, benzylidene H3,3′ ), 7.85–7.90 (2H, m, fluorophenyl H3,3′ ), 7.94–7.96 (1H, m,
(
◦
−1
1
H
(
6
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ATLI and OZKAY/Turk J Chem
azomethine CH), 12.16 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.80 (OCH3), 103.91 (thiazole
C5), 115.98 (methoxyphenyl C3,3′ ), 122.65 (methoxyphenyl C1), 127.41 (JCF − 2 = 11.0 Hz, fluorophenyl C2,2′ ),
1
27.83 (methoxyphenyl C2,2′ ), 127.88 (benzylidene C2,2′ ), 128.97 (fluorophenyl C1), 130.26 (JCF − 2 = 21.2
Hz, fluorophenyl C3,3′ ), 132.44 (benzylidene C1), 136.22 (benzylidene C3,3′ ), 140.07 (benzylidene C4), 141.07
C = N), 149.86 (thiazole C4), 160.44 (methoxyphenyl C4), 162.04 (JCF − 1 = 243.8 Hz, fluorophenyl C4),
68.67 (thiazole C2). HRMS (m / z): [M + H]+ calcd for C23 H18 FN3 OS2 : 436.0948; found: 436.0946.
(
1
3.4.13. 4-(4-Methoxyphenylsulfanyl)benzaldehyde [4-(2,4-dichlorophenyl)-1,3-thiazol-2-yl] hydra-
zone (3m)
◦
−1
Yield: 92%, mp = 103.7 C, FTIR (ATR, cm ): 3168 (N - H), 1490 (C = N), 1249 (C - N), 1051, 821,
1
8
10. H NMR (300 MHz, DMSO-d6): δ = 3.80 (3H, s, OCH3), 7.04 (2H, d, J =8.85 Hz, methoxyphenyl
H3,3′ ), 7.13 (2H, d, J =8.43 Hz, benzylidene H2,2′ ), 7.39 (1H, s, thiazole H), 7.45 (2H, d, J =8.85 Hz,
methoxyphenyl H2,2′ ), 7.49 (1H, dd, J =2.22–8.49 Hz, dichlorophenyl H5), 7.56 (2H, d, J =8.52 Hz, ben-
zylidene H3,3′ ), 7.68 (1H, d, J =2.16 Hz, dichlorophenyl H3), 7.88 (1H, d, J =8.49 Hz, dichlorophenyl
H6), 7.98 (1H, s, azomethine CH), 12.24 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.80 (OCH3),
1
1
09.73 (thiazole C5), 115.98 (methoxyphenyl C3,3′ ), 122.48 (methoxyphenyl C1), 127.46 (dichlorophenyl C3),
27.82 (methoxyphenyl C2,2′ ), 127.96 (benzylidene C2,2′ ), 130.21 (dichlorophenyl C5), 132.04 (dichlorophenyl
C1), 132.36 (dichlorophenyl C6), 132.50 (benzylidene C1), 132.68 (dichlorophenyl C4), 132.99 (dichlorophenyl
C2), 136.22 (benzylidene C3,3′ ), 140.19 (benzylidene C4), 141.41 (C = N), 146.23 (thiazole C4), 160.44
(
methoxyphenyl C4), 167.81 (thiazole C2). HRMS (m / z): [M + H]+ calcd for C23 H17 Cl2 N3 OS2 : 486.0263;
found: 486.0261.
3.4.14. 4-(4-Methoxyphenylsulfanyl)benzaldehyde [4-(2,4-difluorophenyl)-1,3-thiazol-2-yl] hydra-
zone (3n)
◦
−1
Yield: 88%, mp = 201.4 C, FTIR (ATR, cm ): 3170 (N - H), 1489 (C = N), 1249 (C - N), 850, 821.
1
H NMR (300 MHz, DMSO-d6): δ = 3.79 (3H, s, OCH3), 6.99 (1H, m, difluorophenyl H5), 7.03–7.09
(
5H, m, methoxyphenyl H3,3′ , difluorophenyl H3 , benzylidene H2,2′ ), 7.21 (1H, s, thiazole), 7.40–7.47 (2H,
m, methoxyphenyl H2,2′ ), 7.50–7.57 (2H, m, benzylidene H3,3′ ), 7.56 (1H, d, J =8.50 Hz, difluorophenyl
H6), 7.98 (1H, s, azomethine CH), 12.18 (1H, s, NH). 13 C NMR (75 MHz, DMSO-d6): δ = 55.81 (OCH3),
1
04.75 (t, J =26.3 Hz, difluorophenyl C3), 108.15 (d, J =13.8 Hz, thiazole C5), 112.34 (dd, J =2.8–21.0
Hz, difluorophenyl C5), 115.68 (methoxyphenyl C3,3′ ), 119.52 (dd, J =3.9–11.2 Hz, difluorophenyl C1),
21.58 (methoxyphenyl C1), 128.58 (methoxyphenyl C2,2′ ), 129.26 (benzylidene C2,2′ ), 130.83 (t, J =10.2 Hz,
difluorophenyl C6), 137.70 (benzylidene C3,3′ ), 141.62 (benzylidene C4), 143.71 (C = N), 149.20 (thiazole C4),
1
156.43, 159.66 (methoxyphenyl C4), 160.02 (dd, J =12.0–250.3 Hz, difluorophenyl C4), 161.76 (dd, J =12.6–
2
45.7 Hz, difluorophenyl C2), 166.75 (thiazole C2). HRMS (m / z): [M + H]+ calcd for C23 H17 F2 N3 OS2 :
454.0854; found: 454.0849.
3.5. MAO-A and MAO-B inhibition assay
hMAO inhibitory activity of the compounds (3a–3n) was determined by a ?uorometric method using the Bio-
Vision MAO A/B (MAO-A/B) Inhibitor Screening Kit (USA) according to the manufacturer’s instructions.19,20
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All pipetting processes were performed using a BioTek Precision XS robotic system (USA). Measurements were
carried out with a BioTek Synergy H1 microplate reader (USA) based on the fluorescence generated (excitation,
535 nm; emission, 587 nm) over a 20-min period, in which the fluorescence increased linearly. Clorgiline and
selegiline were used as the specific inhibitors of hMAO-A and hMAO-B, respectively.
−3
−9
M concentrations using
Standard drugs and synthesized compounds were prepared at 10
to 10
2% DMSO. Recombinant enzymes and developer were diluted in the reaction buffer. Substrate was diluted
in bidistilled H2 O. In order to prepare the MAO working solution, 37 µL of assay buffer, 1 µL of developer
solution, 1 µL of substrate solution, and 1 µL of OxiRed probe were mixed for each well.
The solutions of inhibitors and standard drugs (10 µL/well) and recombinant enzyme solution (50
◦
µL/well) were added to a black flat-bottomed 96-well microplate and incubated for 10 min at 25 C and
◦
37
C for the MAO-A and MAO-B assay, respectively. After this incubation period, the reaction was started
by adding a working solution (40 µL/well). The mixture was incubated for 30 min at a proper temperature.
Fluorescence was measured using excitation at 535 nm and emission at 587 nm at 5-min intervals. Control
experiments were carried out simultaneously by replacing the inhibitor solution with 2% DMSO (10 µL). To
check the probable inhibitory effect of the inhibitors on the developer, a parallel reading was performed by
replacing enzyme solutions with 10 mM H2 O2 solution (50 µL/well). In addition, the possible capacity of
the inhibitors to modify the fluorescence generated in the reaction mixture due to nonenzymatic inhibition was
determined by mixing the inhibitor and working solutions.
The specific fluorescence values (used to obtain the final results) were calculated after subtraction of
the background activity, which was determined from wells containing all components except for the hMAO
isoforms, which were replaced by phosphate buffer (50 µL/well). The blank, control, and all concentrations
of inhibitors were analyzed in quadruplicate and inhibition percentage was calculated by using the following
equation:
(
FCt2 − FCt1) − (FIt2 − FIt1)
FCt2 − FCt1
%
Inhibition =
× 100
FCt2 = Fluorescence of a control well measured at time t2 ;
FCt1 = fluorescence of a control well measured at time t1 ;
FIt2 = fluorescence of an inhibitor well measured at time t2 ;
FIt1 = fluorescence of an inhibitor well measured at time t1 .
The IC50 values were calculated from a dose–response curve obtained by plotting the percentage inhibition
versus the log concentration with the use of Microsoft Office Excel 2013. The results were displayed as mean
ꢀ
standard deviation. The SI was calculated as IC50 (hMAO-A) / IC50 (hMAO-B).
3.6. Enzyme kinetic studies
The same materials were used in the MAO inhibition assay. The most active compound against the hMAO-A
enzyme, 3f, was tested at five different concentrations (IC50 / 4, IC50 / 2, IC50 , 2 × IC50 , and 4 × IC50).
The inhibitor (10 µL/well) and MAO-A enzyme solution (50 µL/well) were added to the black flat-bottomed
◦
9
6-well microplate and incubated at 37 C for 10 min. After the incubation period, the working solution,
including the reaction buffer, developer solution, OxiRed, and various concentrations (20, 10, 5, 2.5, 1.25, and
.625 µM) of tyramine (40 µL/well), was added. The increase of the fluorescence (Ex/Em = 535 / 587 nm)
was recorded for 30 min. A parallel experiment was carried out without inhibitor. All processes were assayed
0
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in quadruplicate. The results were analyzed as Lineweaver–Burk plots21 using Microsoft Office Excel 2013.
Km and Vmax values belonging to the inhibitor and control were calculated using junction points on the x-
and y-axis. The slopes of the Lineweaver–Burk plots were replotted versus the inhibitor concentration and the
inhibitory constant (Ki) was calculated.32
3.7. Cytotoxicity assay
Cytotoxicity of compound 3f was tested using the NIH/3T3 mouse embryonic fibroblast cell line (ATCC
CRL1658, London, UK). NIH/3T3 cells were incubated according to the supplier’s recommendations. NIH/3T3
4
cells were seeded at 1 × 10 cells into each well of 96-well plates. The MTT assay was performed as previously
3
3,34
described.
The compounds were tested between 0.0316 and 1000 µM concentrations. The IC50 values were
determined by plotting a dose–response curve of inhibition % versus compound concentrations tested.35
3.8. Prediction of ADME parameters and BBB permeability
Physicochemical parameters of the compounds (3a–3n) were analyzed by the online Molinspiration property
calculation program (http://www.molinspiration.com/services/properties.html). BBB permeability of the com-
pounds was assigned by the online BBB Predictor (http://www.cbligand.org/BBB/index.php).
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