Synthesis of novel Schiff bases and azol-β-lactam derivatives starting from morpholine and thiomorpholine and investigation of their antitubercular, antiurease activity, acethylcolinesterase inhibition effect and antioxidant capacity

Article abstract of DOI:10.1016/j.bioorg.2019.102928

In this study, new Schiff bases and β-lactam derivatives containing morpholine and thio morpholine nuclei were synthesized. Antimicrobial, antioxidant, antimicrobial and antioxidant properties of all synthesized compounds were investigated and highly effe

Full text of DOI:10.1016/j.bioorg.2019.102928

Accepted Manuscript
Synthesis of novel schiff bases and azol-β-lactam derivatives starting from mor-
pholine and thiomorpholine and investigation of their antitubercular, antiurease
activity, acethylcolinesterase inhibition effect and antioxidant capacity
Yıldız Uygun Cebeci, Hacer Bayrak, Yakup Şirin
PII:
S0045-2068(19)30328-1
https://doi.org/10.1016/j.bioorg.2019.102928
102928
DOI:
Article Number:
Reference:
YBIOO 102928
Bioorganic Chemistry
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Revised Date:
Accepted Date:
28 February 2019
10 April 2019
15 April 2019
Please cite this article as: Y.U. Cebeci, H. Bayrak, Y. Şirin, Synthesis of novel schiff bases and azol-β-lactam
derivatives starting from morpholine and thiomorpholine and investigation of their antitubercular, antiurease
activity, acethylcolinesterase inhibition effect and antioxidant capacity, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.102928
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Synthesis of Novel Schiff Bases and Azol-β-Lactam Derivatives starting
from Morpholine and Thiomorpholine and Investigation of their
Antitubercular, Antiurease activity, Acethylcolinesterase Inhibition Effect
and Antioxidant Capacity
Yıldız Uygun Cebeci1, Hacer Bayrak2*, Yakup Şirin1
1Department of Chemistry, Karadeniz Technical University, 61080, Trabzon, Turkey
2Department of Chemistry and Chemical Processing Technology, Karadeniz Technical
University, 61080, Trabzon, Turkey
*Address correspondence to: Hacer Bayrak, Karadeniz Technical University, Maçka
Vocational School, Department of Chemistry and Chemical Processing Technology, 61750
Trabzon, Turkey, Phone: +90 462 3777639, Fax: +90 4625123552, e-mail:
h.bayrak@ktu.edu.tr
Abstract: In this study, new Schiff bases and β-lactam derivatives containing morpholine and
thio morpholine nuclei were synthesized. Antimicrobial, antioxidant, antimicrobial and
antioxidant properties of all synthesized compounds were investigated and highly effective
products were obtained. In this context, new effective structures were introduced to the
literature.
Keywords: Schiff Base, β-Lactam, Morpholine, Thiomorpholine, Azole, Antitubercular
Activity, Antiurease Activity, Acethylcolinesterase, Antioxidant
1. Introduction
The azetidine-2-one ring also known as β-lactam is a four-membered ring that is a class of
important bioactive compound. Penicillin, Klavam, Penem, Carbapenem, Cephalosporin,
Oxasefem, Izosefem and Izooxasefem are known drugs and contain a β-lactam ring in their
molecular structure and are also known β-lactam antibiotics [1]. Recent clinical studies on
bacterial infections and the discovery of β-lactam chemistry and biology have led to important
advances [2-7].
H
N
H
N
H
H
N
R
O
R
O
X
H
N
H
N
R
O
X
X
R
O
R
O
S
O
R
SR
R
R
COOH
COOH
COOH
COOH
Penicillin
COOH
Klavam
X: S - Cephalosporin
X: O - Oxasefem
X: S - Izosefem
X: O - Izooxasefem
X: S - Penem
X: CH2 - Carbapenem

Tuberculosis (TB) is a devastating disease caused by poverty, and according to World Health
Organization (WHO) 2016 data, 95% of 1.4 million people infected with TB in developing
countries are killed and 10 million children are orphaned as a result [8]. Moreover, the
emergence of a drug-resistant microorganism with the lethal combination of TB, particularly
multidrug-resistant TB and HIV-1 infection, makes this disease one of the greatest global
health problems of our time [9]. Therefore, new antitubercular drugs and drug targets are
urgently needed. The need to identify, detect and synthesize agents that will be effective
against rapid action and intracellular Mycobacterium tuberculosis by reducing treatment time
is of great importance nowadays.
Azole rings are heterocyclic compounds containing nitrogen atoms and constitute a class of
compounds with a broad biological effect. Some of these effects are anti-bacterial, anti-
malarial, anti-fungal, anti-HIV, anti-inflammatory and anti-TB [8, 10]. The antibacterial
resistance of pathogenic microorganisms against the drugs used clinically is developing very
rapidly all over the world and this threatens public health. As a result of the low number of
antimicrobial compounds used and the rapid development of bacterial resistance, the synthesis
of new antimicrobial compounds has become an attractive subject for organic medicinal
chemists. In last decades in order to obtain antitumor drugs, many compounds with different
structure have been synthesized. These include complex molecules containing an azole ring as
well as structurally simple azole derivatives [8, 11-14]. Some examples of azole drugs are
Trazodone, Ribavirin, Rizatriptan, Vorazol, Alprazolam and Nefazonon that contain a 1,2,4-
triazole ring in their molecular structure [15-19]. Cefazedone and Sevazolin, which are
cephalosporin-derived antibiotics, are important drugs containing 1,3,4-thiadiazole ring [20].
Acetozolamide and methazolamide, which are used in the treatment of glaucoma and epilepsy
diseases, are another important molecule in the structure of thiadiazole ring [21, 22]. Also,
Raltegravir (HIV-infections), Furamizol (antibacterial), Zibotentan (anticancer candidate
drug) and Tiodazosin (antihypertensive agent) are an important drug or drug candidate
containing oxadiazole nucleus [23, 24].
In the point of this information, in this study, the effects of antimicrobial, antioxidant
properties, antiureas and acetylcholinesterase effects were investigated by the synthesis of
new hybrid compounds by combining different active groups in a single molecule.
2. Results and Discussion
2.1. Chemistry

In this study there are two starting material thiomorpholine and morpholine. Separately
thio/morpholine was reacted with ethyl bromoacetate and hydrazinhydrate to afford the
corresponding asetohydrazide compounds 2a and 2b and then they were converted to 1,3,4-
oxadiazole ring (3a/3b) separately with carbondisulfide in basic media. The carbonyl group of
compounds 2a/2b disappeared and the C=S bond was occurred at 1295 cm-1 in IR spectrum.
And in 1H NMR spectrum the 1,3,4-oxadiazole –NH was resonated at 8.67 ppm of compound
3a where in compound 3b –NH was not observed. Compound 4a and 4b were synthesized by
the reaction of compound 3 with acrylonitrile in basic media, separately. At the N-3 position
of 1,3,4-oxadiazole the nitrile moiety was linked by addition reaction. In the 13C NMR spectra
the –CN group was resonated at 118.30-118.34 ppm of the target compounds 4a and 4b.
1,3,4-Thiadiazole ring were introduced to compound 4 by cycloaddition reaction with
thiosemicarbazide in triflouroacetic acid to afford the corresponding compound 5a and 5b
separately. Compared with the starting material –NH2 protons were resonated at 7.02-7.20
ppm in 1H NMR, and in 13C NMR the carbon atoms of thiadiazole were resonated at 153.88-
155.10 ppm as thiadiazole C-3 and 158.30-159.36 ppm as thiadiazole C-5 of compounds 5a
and 5b. To afford the Schiff bases shown at Figure 1, compound 5 was firstly reacted with
hydrazine hydrate to convert 1,3,4-oxadiazole ring to 1,2,4-triazole-4-amino (6a, 6b), then,
compound 6 was reacted with different aldehydes to synthesized the corresponding Schiff
bases with the value of 8.60-10.89 ppm of –N=CH- (imine) peak of compounds 7a-7f and 8a-
8f. The beta lactam ring of compounds 9a-9f and 10a-10f were occurred by the reaction of
Schiff bases with chloroacetyl chloride in dioxane solvent and in IR spectrum the carbonyl
13
group was seen between 1710-1730 cm-1 and in C NMR the signal was resonated between
167.87-170.23 ppm respectively.

O
O
N NH
O
ii
i
iii
(O)S
N
(O)S
N
(O)S
NH
(O)S
N
NHNH2
OCH2CH3
S
2a, 2b
3a, 3b
1a, 1b
iv
N
N
N
N
CN
NH2
S
NH2
N
N
S
N
N
N
O
N
(O)S
N
vi
v
(O)S
N
S
6a, 6b
(O)S
N
N
S
O
S
NH2
5a, 5b
4a, 4b
vii
N
N
N
N
NH2
S
N
N
NH2
S
N
N
(O)S
N
S
N
N
viii
(O)S
N
S
N
N
O
R
R
Cl
7a-7f and 8a-8f
9a-9f and 10-10f
Cl
N
R=
R=
R=
OCH3
R=
N
F
R=
CH3 R=
7b, 8b, 9b, 10b
7a, 8a, 9a, 10a
7d, 8d, 9d, 10d
7f, 8f, 9f, 10f
7c, 8c, 9c, 10c
Cl
7e, 8e, 9e, 10e
Figure 1. i. BrCH2COOEt, EtOH; ii. NH2NH2, EtOH; iii. CS2, triethylamine, EtOH; iv. CH2CHCN,
EtOH; v. Thiosemicarbazide, TFA, NH3; vi. NH2NH2, EtOH; vii. Aldehyde, MW; viii. ClCH2COOH,
dioxane, triethylamine.
2.2. Biological Activity
2.2.1. Antituberculosis Activity
When the antitubercular effect of thiomorpholine and morpholine serie compounds were
examined, it was seen that the thiomorpholine derivative 7d and Schiff base 7e exhibited very
good activity against streptomycin which is the standard drug with the value of 7.81 µg / mL.
The compounds of formula 2b and 3b, which have the acetohydrazide and oxadiazole ring in
the morpholine derivative structure, were equally effective against streptomycin.
Table 1. Antitubercular Activity of newly synthesized compounds
Microorganism and Minimal Inhibition Concentrations
(MIC) (µg/mL)
Comp.
No
No Gram / Ms
Comp.
No
No Gram / Ms
15.2
7.8
7.8
31.2
31.2
31.2
1b
2b
3b
8c
8d
8e

31.2
15.6
7.8
7.8
15.6
4
31.2
31.2
15.6
15.6
31.2
4
7a
7c
9a
9b
7d
7e
9d
9e
7f
9f
Strep.
Strep.
Ms: Mycobacterium smegmatis ATCC607, Strep.: Streptomycin
Compounds 7c, 7f which are Schiff bases of thiomorpholine serie and 9d, 9e which are beta-
lactam derivatives of thiomorpholine serie exhibit moderate-good activity compared with the
standart drug of steptomycin of Mycobacterium smegmatis.
The 4-methoxybenzaldehyde group of thiomorpholine serie, compound 7a and 9a showed
significant antitubercular activity with the value of 31.2 9 µg / mL. β-lactam derivative
compounds 9b containing 4-methylphenyl group and 9f containing 4-fluorophenyl group, and
Schiff base derivatives of compounds 8c (2-pyridinylcarbaldehyde), 8d (4-
pyridinylcarbaldeyde) and 8e (2,6-dichlorophenyl) also showed moderate antituberculosis
activity.
2.2.2. Anti-Urease Activity
Nearly all the newly synthesized compounds were found to have highly effective urease
inhibition activity.
Table 2. Anti-Urease Activity results of newly Synthesized compounds.
Comp.
No
Anti Urease
IC50 (mg/mL) + SD
Comp.
No
Anti Urease
IC50 (mg/mL) + SD
4.75 ± 0.01
5.57 ± 0.06
2.23 ± 0.02
6.57 ± 0.09
1.48 ± 0.03
1.18 ± 0.01
1.26 ± 0.02
2.28 ± 0.02
4.19 ± 0.09
0.85 ± 0.04
5.96 ± 0.02
4.23 ± 0.01
0.45 ± 0.07
1.01 ± 0.02
1.25 ± 0.05
2.83 ± 0.06
1.38 ± 0.05
1.57 ± 0.09
0.48 ± 0.00
0.96 ± 0.00
1.45 ± 0.02
1.19 ± 0.06
0.85 ± 0.05
1.33 ± 0.03
2.14 ± 0.09
2a
3a
3b
4a
6a
6b
7b
7c
7d
7e
7f
8c
8d
8e
9a
9b
9c
9e
10b
10c
10d
10f
10e
8a
8b
1.56 ± 0.03
1.56 ± 0.03
FP-T58
FP-T58
12.02 ± 0.06
12.02 ± 0.06
thiourea
thiourea
2.2.3. Acetylcholinesterase Inhibition Effects

Compounds that exhibit moderate inhibition compared to Donepezil reference drug which is
an acetylcholinesterase inhibitor, are 6a, 7b, 7e, 9b, 9c, 10e and 10f respectively.
Table 3. Acetylcholinesterase Inhibition results of newly synthesized compounds.
Comp.
No
EeAChE Activity
IC50 (mg/mL) + SD
Comp.
No
EeAChE Activity
IC50 (mg/mL) + SD
2.32 ± 0.03
0.95 ± 0.00
2.45 ± 0.04
0.46 ± 0.02
1.28 ± 0.03
4.19 ± 0.06
0.75 ± 0.04
6.21 ± 0.01
1.73 ± 0.02
2.12 ± 0.03
2.86 ± 0.03
0.03 ± 0.00
1.13 ± 0.05
5.18 ± 0.02
0.87 ± 0.02
0.97 ± 0.02
1.19 ± 0.01
2.01 ± 0.00
1.25 ± 0.01
1.13 ± 0.01
0.95 ± 0.01
0.88 ± 0.01
3b
6a
8e
9a
6b
7b
7c
7d
7e
8a
8b
8c
8d
9b
9c
9e
10b
10c
10d
10e
10f
0.03 ± 0.00
Donepezil
Donepezil
2.2.4. Antioxidant Capacity
According to the CUPRAC method, compounds with excellent antioxidant activity results
were found to have 8b, 8c and 8d Schiff derivatives containing 4-methylphenyl, 3-pyridine
and 4-pyridine ring respectively. The good-moderate characteristics were found to be the β-
lactam derivative compounds with 10b-10f (Table 4). The compounds carrying the phenyl
group together with the β-lactam ring of compound 9a, 9b and 9f have antioxidant properties
because they contain the phenyl ring in the structures of Schiff base 7b, and have the potential
to reduce the Cu (II) complexes.
The compounds exhibiting potent antioxidant properties by DPPH method were 2b, 8b, 8a,
8d, 8e, 10b-10e, 7b, 7c, 7e and 9a-9c respectively, and DPPH radical was found to be among
the classes that could perform cleansing function. Among all of these compounds, compounds
3b, 7c and 10e that are closest to the trolox standard (0.04 ± 0.00) mg / mL have excellent
antioxidant capacity with values of 0.06-0.09 mg / mL
Table 4. Comparing Antioxidant capacity of newly synthesized compounds with CUPRAC /
DPPH and FRAP Methods.
Comp.
No
CUPRAC
(µmol TE/g)
DPPH
(mg/mL) SC50
FRAP
(µmol TE/g)
65.29 ± 2.00
4578.11 ± 29.45
3048.61 ± 9.48
--
0.473 ± 0.004
4.228 ± 0.014
4.649 ± 0.021
1b
2b
3b
0.13 ± 0.00
0.06 ± 0.00

106.18 ± 5.64
65.74 ± 5.00
--
--
0.044 ± 0.003
0.096 ± 0.008
2.908 ± 0.012
3.187 ± 0.001
1.329 ± 0.016
4.878 ± 0.030
3.830 ± 0.080
0.386 ± 0.020
2.478 ± 0.040
4.076 ± 0.008
2.648 ± 0.046
3.368 ± 0.060
3.044 ± 0.002
4.911 ± 0.007
2.977 ± 2.638
4b
5b
6b
8b
8a
8c
8d
8f
8e
10b
10a
10c
10d
10e
10f
2484.80 ± 17.00
7178.35 ± 4.00
1978.35 ± 14.00
7265.29 ± 12.00
8048.11 ± 16.00
45.12 ± 1.00
1.11 ± 0.01
0.48 ± 0.01
4.29 ± 0.01
0.17 ± 0.01
0.26 ± 0.01
--
0.23 ± 0.01
0.32 ± 0.01
0.27 ± 0.01
0.52 ± 0.03
0.33 ± 0.02
0.09 ± 0.00
0.37 ± 0.01
678.35 ± 4.00
4765.74 ± 5.00
6048.68 ± 36.42
7024.24 ± 22.10
5106.18 ± 5.64
6879.54 ± 23.00
6004.45 ± 10.16
7986.12 ± 23.42
0.29 ± 0.03
0.04 ± 0.00
4.119 ± 0.030
FP-T58
TROLOX
The FRAP method is based on the reduction of Fe (III) ion in Fe (III) complex in the presence
of an antioxidant. Fe (III) ions form the (Fe (III)-TPTZ-2,4,6-tris(2-pyridyl)-S-triazine
complex with the ligand called TPTZ. Evaluation was made by calculating trolox equivalent
antioxidant capacity in µmol TE / g. Compounds 2b, 8c, 8d, 10b, 10e, 7c, 7f, 7e, 9a and 9e
were found to exhibit trolox equivalent antioxidant properties (Table 4).
3. Conclusion
In summary we report here the synthesis of some new kind of Schiff base and of β-lactam
derivatives starting from morpholine and thiomorpholine. And all the newly synthesized
compounds were screened for their antituberculosis and anti-Urease activity,
acetylcholinesterase inhibition effects and antioxidant capacity. Among them compounds 3b,
7d and 7e showed very good antituberculosis activity, compounds 6a, 7b, 7e, 9b, 9c, 10e and
10f showed Acetylcholinesterase Inhibition effect, compounds 3b, 8b, 8c, 8d, 10a, 10c, 10d,
10e and 10f showed antioxidant capacity against standard drugs mentioned at Table 1, Table 2
and Table 4. And also all synthesized compounds showed excellent anti-ureas activity against
thiourea.
4. Experimental
4.1. Chemistry

All the chemicals were purchased from FlukaChemie AG Buchs (Switzerland) and used
without further purification. Melting points of the synthesized compounds were determined in
open capillaries on a Büchi B-540 melting point apparatus and are uncorrected. Reactions
were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminium sheets.
The mobile phase was ethyl acetate:diethyl ether (1:2), and detection was made using UV
1
light. FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR spectrometer. H
13
NMR and C NMR spectra were registered in DMSO-d6 on a BRUKER AVENE II 400 MHz
1
NMR Spectrometer (400.13 MHz for H and 100.62 MHz for 13C). The chemical shifts are
given in ppm, J values are given in Hz. The Mass spectra were obtained on a Quattro LC-MS
(70 eV) Instrument.
4.2. General Synthesis Method for Compounds 3a and 3b
Compound 2a/2b (10 mmol) and CS2 (20 mmol) were added to a solution of KOH (10 mmol)
in 20 mL H2O and 20 mL ethanol and the reaction mixture was refluxed for 13 h. Then, the
reaction content was acidified with conc. HCl to pH 6. The precipitate formed was filtered
off, washed with H2O and recrystallized from ethanol to afford the desired compound.
4.2.1. 5-(Morpholin-4-ylmethyl)-1,3,4-oxadiazol-2(3H)-thione (3a)
1
Yield 73 %, m.p: 147-149 °C. FT-IR (υmax, cm-1): 1587 (C=N), 1295 (C=S). H NMR
13
(DMSO-d6, δ ppm): 3.30 (6H, s, 3CH2+H2O), 4.48 (4H, s, CH2), 8.67 (1H, s, NH). C NMR
(DMSO-d6, δ ppm): 55.78 (CH2), 58.36 (2CH2), 63.25 (2CH2), 160.20 (oxadiazole C-2),
168.85 (oxadiazole C-5). EI MS m/z (%): 202.25 ([M+1]+, 100), 170.21 (85), 161.35 (63).
Elemental analysis for C7H11N3O2S calculated (%), C, 41.78; H, 5.51; N, 20.88; O, 15.90,
found (%), C, 41.72; H, 5.49; N, 20.83; O, 15.86.
4.2.2. 5-(Thiomorpholin-4-ylmethyl)-1,3,4-oxadiazol-2(3H)-thione (3b)
Yield 76 %, m.p: 133-135 °C. FT-IR (υmax, cm-1): 3318 (NH), 1570 (C=N), 1458 (C=S).
1H NMR (DMSO-d6, δ ppm): 2.57-2.60 (4H, m, 2CH2), 2.69 (4H, d, J= 4.0 Hz, 2CH2), 3.65
(2H, s, CH2). NH was not observed. 13C NMR (DMSO-d6, δ ppm): 27.52 (CH2), 52.55
(2CH2), 54.18 (2CH2), 161.09 (oxadiazole C-2), 178.60 (oxadiazole C-5). Elemental analysis
for C7H11N3OS2 calculated (%), C, 38.69; H, 5.10; N, 19.34; O, 7.36, found (%), C, 38.72; H,
5.15; N, 19.37; O, 7.41.

4.3. General Synthesis Method for Compounds 4a and 4b
To a solution of the compound 3a/3b (10 mmol) in ethanol was added acrylonitrile (50 mmol)
in the presence of triethylamine (50 mmol) and reaction mixture was refluxed for 6h. After
evaporating the solvent under reduced pressure, a solid obtained. The crude product was
recrystallized from ethanol to afford the desired product.
4.3.1. 3-[5-(Morpholin-4-ylmethyl)-2-thioxo-1,3,4-oxadizol-3(2H)-yl]propan nitrile (4a)
1
Yield 78 %, m.p: 115-117 °C. FT-IR (υmax, cm-1): 2250 (C=N), 1252 (C=S). H NMR
(DMSO-d6, δ ppm): 1.98 (4H, s, 2CH2), 3.57-3.60 (6H, m, 3CH2), 3.68 (4H, s, 2CH2). 13C
NMR (DMSO-d6, δ ppm): 16.12 (CH2), 44.58 (CH2), 51.58 (CH2), 52.81 (2CH2), 66.44
(2CH2), 118.34 (C≡N), 159.76 (oxadiazole C-2), 176.61 (oxadiazole C-5). EI MS m/z (%):
277.13 (89), 255.17 ([M+1]+, 40), 148.99 (71), 136.04 (22), 102.13 (100). Elemental analysis
for C10H14N4O2S calculated (%), C, 47.23; H, 5.55; N, 22.03; O, 12.58, found (%), C, 47.17;
H, 5.47; N, 21.98; O, 12.51.
4.3.2. 3-[5-(Thiomorpholin-4-ylmethyl)-2-thioxo-1,3,4-oxadizol-3(2H)-yl]propan nitrile
(4b)
1
Yield 71 %, m.p: 114-116 °C. FT-IR (υmax, cm-1): 2251 (C=N), 1462 (C=S). H NMR
(DMSO-d6, δ ppm): 2.60 (4H, d, J= 4.0 Hz, 2CH2), 2.73 (4H, d, J=4.0 Hz, 2CH2), 3.03-3.06
(2H, m, CH2), 3.74 (2H, s, CH2), 4.26-4.29 (2H, m, CH2). 13C NMR (DMSO-d6, δ ppm):
16.17 (CH2), 27.46 (CH2), 44.61 (CH2), 52.43 (2CH2), 54.96 (2CH2), 118.30 (C≡N), 159.89
(oxadiazole C-2), 176.73 (oxadiazole C-5). EI MS m/z (%): 271.01 ([M+1]+, 154.23 (100).
Elemental analysis for C10H14N4OS2 calculated (%), C, 44.42; H, 5.22; N, 20.72; O, 5.92,
found (%), C, 44.47; H, 5.25; N, 20.74; O, 5.94.
4.4. General Synthesis Method for Compounds 5a and 5b
A mixture of the compound 4a/4b (10 mmol) and thiosemicarbazide (10 mmol) were
combined in ethanol in the presence of trifluoroacetic acid (5 mL) in an oil bath and then was
refluxed for 9 h. Then the reaction content was neutralized with diluted NH3 to pH 6. Water
was decanted from the mixture and the remaining fatty fraction was treated with ether to
solidify the crude product. The resulting solid was filtered and purified by crystallization from
acetone.

4.4.1.
3-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(morpholin-4-ylmethyl)-1,3,4-
oxadiazol-2(3H)-thione (5a)
Yield 53 %, m.p: 121-123 °C. FT-IR (υmax, cm-1): 3280 (NH2), 3092 (NH), 1527(C=N),
1
1298 (C=S). H NMR (DMSO-d6, δ ppm): 2.42 (2H, s, CH2), 2.48 (2H, s, CH2), 3.27-3.30
(2H, m, CH2), 3.55 (4H, s, H2O+2CH2), 3.63 (2H, s, CH2), 4.27-4.30 (2H, s, CH2), 7.06 (2H,
s, NH2). 13C NMR (DMSO-d6, δ ppm): 27.57 (CH2), 47.92 (CH2), 51.81 (CH2), 52.82 (2CH2),
66.43 (2CH2), 153.88 (thiadiazole C-3), 159.36 (thiadiazole C-5), 169.28 (oxadiazole C-2),
176.54 (oxadiazole C-5). EI MS m/z (%): 329.25 ([M+1]+, 100), 245.22 (22), 171.13 (31),
128.05 (42). Elemental analysis for C11H16N6O2S2 calculated (%), C, 40.23; H, 4.91; N,
25.59; O, 9.74, found (%), C, 40.29; H, 4.95; N, 25.57; O, 9.78.
4.4.2.
3-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(thiomorpholin-4-ylmethyl)-1,3,4-
oxadiazol-2(3H)-thione (5b)
1
Yield 62 %, m.p: 146-148 °C. FT-IR (υmax, cm-1): 3368 (NH2), 1465 (C=S). H NMR
(DMSO-d6, δ ppm): 2.02 (2H, s, CH2), 2.78 (4H, s, 2CH2), 3.00 (4H, s, 2CH2), 3.18 (2H, s,
13
CH2), 3.78 (2H, s, CH2), 7.20 (2H, brs, NH2). C NMR (DMSO-d6, δ ppm): 28.12 (CH2),
48.12 (2CH2), 50.12 (2CH2), 53.96 (CH2), 55.30 (CH2), 155.10 (thiadiazole C-3), 158.30
(thiadiazole C-5), 168.52 (oxadiazole C-2), 177.18 (oxadiazole C-5). EI MS m/z (%): 345.20
([M+1]+, 100), 278.12 (85), 260.10 (76), 178.96 (55). Elemental analysis for C11H16N6OS3
calculated (%), C, 38.36; H, 4.68; N, 24.40; O, 4.64, found (%), C, 38.39; H, 4.73; N, 24.34;
O, 4.69.
4.5. Genaral Synthesis Method for Compounds 6a and 6b
To the solution of compound 5a/5b (10 mmol) in absolute ethanol hydrazinehydrate (25
mmol) was added and the reaction was refluxed for 10 hours. Evaporation of the solvent
under reduced pressure gave the oily substance solidified by treatment with n-butylacetate /
diethylether. After standing overnight in cold, the precipitated solid was filtered off and
purified by crystallization from ethanol.
4.5.1. 4-Amino-2-[2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(morpholin-4-ylmethyl)-2,4-
dihydro-3H-1,2,4-triazol-3-thione (6a)

Yield 68 %, m.p: 168-170 °C. FT-IR (υmax, cm-1): 3239 ve 3133 (NH2), 1571 (C=N),
1326 (C=S). 1H NMR (DMSO-d6, δ ppm): 2.40 (4H, s, 2CH2), 3.27-3.35 (4H, m, 2CH2), 3.54
(4H, d, J= 12.0 Hz, H2O+2CH2), 4.34-4.37 (2H, m, CH2), 5.63 (2H, s, NH2), 7.02 (2H, s,
13
NH2). C NMR (DMSO-d6, δ ppm): 28.31 (CH2), 48.29 (CH2), 50.98 (CH2), 53.16 (2CH2),
66.46 (2CH2), 147.92 (thiadiazole C-3), 154.47 (thiadiazole C-5), 165.82 (oxadiazole C-2),
169.18 (oxadiazole C-5). EI MS m/z (%): 343.20 ([M+1]+, 100), 216.06 (32), 200.11 (82),
100.13 (65). Elemental analysis for C11H18N8OS2 calculated (%), C, 38.58; H, 5.30; N, 32.72;
O, 4.67, found (%), C, 38.52; H, 5.22; N, 32.69; O, 4.69.
4.5.2. 4-Amino-2-[2-(5-amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(thiomorpholin-4-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazol-3-thione (6b)
Yield 67 %, m.p: 127-129 °C. FT-IR (υmax, cm-1): 3302 (NH2), 3109 (NH2), 1494 (C=S).
1H NMR (DMSO-d6, δ ppm): 2.10 (2H, s, CH2), 2.65 (4H, s, 2CH2), 2.78 (4H, s, 2CH2), 3.10
(2H, s, CH2), 3.28 (2H, s, CH2), 6.12 (2H, s, NH2), 7.35 (2H, s, NH2). 13C NMR (DMSO-d6, δ
ppm): 29.10 (CH2), 49.63 (CH2), 51.34 (CH2), 57.30 (2CH2), 65.40 (2CH2), 146.89
(thiadiazole C-3), 153.20 (thiadiazole C-5), 161.20 (oxadiazole C-2), 168.83 (oxadiazole C-
5). EI MS m/z (%): 359.60 ([M+1]+, 100), 200.69 (85), 218.54 (62), 107.67 (40). Elemental
analysis for C11H18N8S3 calculated (%), C, 36.85; H, 5.06; N, 31.26, found (%), C, 36.89; H,
5.12; N, 31.32.
4.6. Genaral Synthesis Method for Compounds 7a-7f and 8a-8f
To a solution of compound 6a (for compounds 7a-7f) or 6b (for compounds 8a-8f) (10 mmol)
and corresponding aromatic aldehyde (20 mmol) was refluxed for 2 h at 125 ºC in an oil bath.
After completion of the reaction, pure water was added to the flask and exracted with
dichloromethane (1:3). Organic phase was dried with Na2SO4. After filtration off the solvent
was evaporated under reduced pressure, a solid obtained. The crude product was
recrystallized from an appropriate solvent (Table 1) to afford the desired product.
4.6.1. 2-[(5-Amino-1,3,4-thiadiazol-2-yl)methyl]-4-{(4-metoxyphenyl)methyleneamino}-
5-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (7a)
Yield 77 %. FT-IR (υmax, cm-1): 3221 (NH2), 3058 (aromatic CH), 1583 (C=N), 1245
1
(C=S). H NMR (DMSO-d6, δ ppm): 3.08 (4H, s, 2CH2), 3.31 (2H, d, J= 8.0 Hz, CH2), 3.41
(2H, d, J= 4.0 Hz, CH2), 3.78 (3H, d, J= 12.0 Hz, CH3), 4.30-4.41 (6H, m, 3CH2), 7.03 (2H,

13
d, J= 8.0 Hz, arH), 7.45 (2H, brs, NH2), 7.79 (2H, d, J= 8.0 Hz, arH), 8.60 (1H, s, CH). C
NMR (DMSO-d6, δ ppm): 28.30 (CH2), 48.48 (CH2), 48.76 (CH2), 52.22 (CH2), 55.84 (CH3),
56.49 (CH2), 64.30 (2CH2), 114.84 (CH), arC: [126.96 (C), 130.44 (2CH), 144.12 (C), 160.95
(2CH)], 154.81 (thiadiazole C-3), 162.11 (thiadiazole C-5), 166.72 (triazole C-3), 169.48
(triazole C-5). ). EI MS m/z (%): 460.60 ([M]+, 95), 169.12 (100), 103.05 (25). Elemental
analysis for C19H24N8O2S2 calculated (%), C, 49.55; H, 5.25; N, 24.33; O, 6.95, found (%), C,
49.51; H, 5.23; N, 24.29; O, 6.92.
4.6.2. 2-[(5-Amino-1,3,4-thiadiazol-2-yl)methyl]-4-{(4-methylphenyl)methylenamino}-5
(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (7b)
Yield 68 %. FT-IR (υmax, cm-1): 3217 (NH2), 3053 (aromatic CH), 1582 (C=N), 1284
1
(C=S). H NMR (DMSO-d6, δ ppm): 2.36 (3H, d, J= 12.0 Hz, CH3), 2.94 (2H, s, CH2), 3.07
(2H, s, CH2), 3.70 (2H, s, CH2), 3.78 (2H, s, CH2), 4.20 (2H, s, CH2), 4.28 (2H, s, CH2), 4.46
(2H, d, J= 4.0 Hz, CH2), 7.36-7.42 (4H, m, arH), 7.83 (2H, d, J= 8.0 Hz, NH2), 9.87 (1H, s,
13
CH). C NMR (DMSO-d6, δ ppm): 21.76 (CH3), 28.18 (CH2), 47.98 (2CH2), 52.51 (2CH2),
65.14 (2CH2), 129.36 (CH), arC: [129.62 (2CH), 130.24 (2CH), 131.12 (C), 131.98 (C)],
143.85 (thiadiazole C-3), 157.23 (thiadiazole C-5), 161.53 (triazole C-3), 169.39 (triazole C-
5). EI MS m/z (%): 470.02 ([M+Na]+, 35), 447.56 ([M+2]+, 84), 315.12 (100). Elemental
analysis for C19H24N8OS2 calculated (%), C, 51.33; H, 5.44; N, 25.20; O, 3.60, found (%), C,
51.32; H, 5.46; N, 25.22; O, 3.63.
4.6.3. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(morpholin-4-ylmethyl)-4-{[pyridin-3-
ylmethylene]amino}-2,4-dihydro-3H-1,2,4-triazol-3-thione (7c)
1
Yield 71 %. FT-IR (υmax, cm-1): 3317 (NH2), 3060 (aromatic CH), 1578 (C=N). H
NMR (DMSO-d6, δ ppm): 1.17- 1.21 (8H, m, 4CH2), 3.05-3.08 (6H, m, 3CH2), 6.92 (2H, d,
J= 8.0 Hz, NH2), 7.20-7.25 (2H, m, arH), 7.43 (2H, s, arH), 9.69 (1H, s, CH). 13C NMR
(DMSO-d6, δ ppm): 28.61 (CH2), 45.99 (CH2), 47.40 (CH2), 47.83 (CH2), 48.20 (CH2), 51.45
(CH2), 61.83 (CH2), 114.90 (CH), arC: [116.20 (CH), 116.44 (CH), 123.26 (C), 124.22 (CH),
124.96 (CH)], 141.95 (thiadiazole C-3), 143.16 (triazole C-3), 161.99 (thiadiazole C-5),
167.09 (triazole C-5). EI MS m/z (%): 432.67 ([M+1]+, 65), 414.32 ([M-H2O]+, 77), 309.00
(100). Elemental analysis for C17H21N9OS2 calculated (%), C, 47.32; H, 4.91; N, 29.21; O,
3.71, found (%), C, 47.27; H, 4.88; N, 29.22; O, 3.67.

4.6.4. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(morpholin-4-ylmethyl)-4-{[pyridin-4-
ylmethylene]amino}-2,4-dihydro-3H-1,2,4-triazol-3-thione (7d)
1
Yield 78 %. FT-IR (υmax, cm-1): 3331 (NH2), 3058 (aromatic CH), 1587 (C=N). H
NMR (DMSO-d6, δ ppm): 1.89-1.93 (8H, m, 4CH2), 2.78-2.85 (6H, m, 3CH2), 6.07 (2H, s,
13
NH2), 7.25-7.30 (2H, m, arH), 7.45-7.52 (2H, m, arH), 8.90 (1H, s, CH). C NMR (DMSO-
d6, δ ppm): 41.95 (CH2), 45.97 (CH2), 47.34 (CH2), 47.81 (CH2), 48.20 (CH2), 51.30 (CH2),
61.88 (CH2), 114.88 (CH), arC: [116.03 (CH), 116.24 (CH), 124.02 (CH), 124.90 (CH),
129.28 (C)], 151.47 (thiadiazole C-3), 152.63 (triazole C-3), 154.73 (thiadiazole C-5), 155.49
(triazole C-5). EI MS m/z (%): 433.53 ([M+2]+, 70), 415.72 ([M-H2O]+, 68), 310.02 (100).
Elemental analysis for C17H21N9OS2 calculated (%), C, 47.32; H, 4.91; N, 29.21; O, 3.71,
found (%), C, 47.28; H, 4.88; N, 29.23; O, 3.68.
4.6.5.
2-[(5-Amino-1,3,4-thiadiazol-2-yl)methyl]-4-{(2,6-dichlorophenyl)methylene
amino}-5-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (7e)
Yield 72 %. FT-IR (υmax, cm-1): 3178 (NH2), 3065 (aromatic CH), 1578 (C=N), 1239
(C=S). 1H NMR (DMSO-d6, δ ppm): 3.09 (2H, s, CH2), 3.19 (2H, s, CH2), 3.72-3.79 (4H, m,
2CH2), 4.35-4.50 (6H, m, 3CH2), 5.92 (2H, s, NH2), 7.41-7.67 (3H, m, arH), 10.89 (1H, s,
CH). 13C NMR (DMSO-d6, δ ppm): 48.43 (CH2), 52.13 (CH2), 52.46 (CH2), 56.47 (CH2),
61.71 (CH2), 64.01 (CH2), 64.53 (CH2), 130.30 (CH), arC: [128.47 (C), 133.73 (2CH), 135.25
(C), 143.63 (C), 156.96 (C)], 154.92 (thiadiazole C-3), 161.14 (thiadiazole C-5), 166.93
(triazole C-3), 169.61 (triazole C-5). EI MS m/z (%): 517.25 ([M+H2O]+, 100), 497.23 ([M-
2]+, 67). Elemental analysis for C18H20N8OS2Cl2 calculated (%), C, 43.29; H, 4.04; N, 22.44;
O, 3.20, found (%), C, 43.33; H, 4.00; N, 22.39; O, 3.17.
4.6.6. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-4-{[(4-fluorophenyl)methylene]amino}-
5-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (7f)
1
Yield 65 %. FT-IR (υmax, cm-1): 3299 (NH2), 3188 (aromatic CH), 1510 (C=N). H
NMR (DMSO-d6, δ ppm): 1.42 (2H, s, CH2), 2.14 (2H, s, CH2), 2.22 (2H, s, CH2), 3.24 (4H,
s, 2CH2), 3.74-3.82 (4H, m, 2CH2), 7.28-7.30 (3H, m, arH), 7.50-7.53 (1H, m, arH), 7.78 (2H,
13
s, NH2), 8.74 (1H, s, CH). C NMR (DMSO-d6, δ ppm): 28.51 (CH2), 45.21 (CH2), 48.35
(CH2), 51.77 (CH2), 52.15 (CH2), 56.49 (CH2), 61.75 (CH2), 127.50 (CH), arC: [116.18 ve
116.39 (2CH, d, J= 21.0 Hz), 130.88 ve 130.97 (2CH, d, J= 9.0 Hz), 143.49 ve 153.30 (CF, d,
J= 981.0 Hz), 161.70 (C)], 164.21 (triazole C-3), 167.21 (triazole C-5), 169.91 (thiadiazole C-

3), 180.17 (thiadiazole C-5). EI MS m/z (%): 429.50 [M-F]+, 23), 312.70 (100), 223.12 (67).
Elemental analysis for C18H21N8OS2F calculated (%), C, 48.20; H, 4.72; N, 24.98; O, 3.57,
found (%), C, 48.25; H, 4.74; N, 24.94; O, 3.53.
4.6.7.
2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-4-{[(4-methoxyphenyl)methylene]
amino}-5-(thiomorpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (8a)
1
Yield 71 %. FT-IR (υmax, cm-1): 3298 (NH2), 3065 (aromatic CH), 1577 (C=N). H
NMR (DMSO-d6, δ ppm): 1.04-1.07 (2H, m, CH2), 2.82 (4H, s, 2CH2), 3.18 (4H, s, 2CH2),
3.44 (3H, s, CH3), 4.20 (2H, s, CH2), 4.43 (2H, s, CH2), 7.05 (2H, d, J= 8.0 Hz, arH), 7.81
(2H, d, J= 8.0 Hz, arH), 7.98 (2H, brs, NH2), 8.63 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm):
25.71 (CH2), 29.33 (CH2), 48.49 (CH2), 49.75 (CH2), 54.16 (CH2), 55.86 (CH3), 56.75 (CH2),
114.86 (CH), arC: [124.73 (C), 127.03 (C), 130.45 (CH), 131.40 (CH), 142.99 (CH), 146.02
(CH)], 154.08 (triazole C-3), 161.62 (triazole C-5), 162.14 (thiadiazole C-3), 163.59
(thiadiazole C-5). EI MS m/z (%): 503.15 (55), 477.02 ([M]+, 63), 402.45 (100). Elemental
analysis for C19H24N8OS3 calculated (%), C, 47.88; H, 5.08; N, 23.51; O, 3.36, found (%), C,
47.92; H, 5.13; N, 23.52; O, 3.37.
4.6.8. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-4-{[(4-methylphenyl)methylene amino}-
5-(thiomorpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (8b)
1
Yield 80 %. FT-IR (υmax, cm-1): 3189 (NH2), 3063 (aromatic CH), 1580 (C=N). H
NMR (DMSO-d6, δ ppm): 1.04-1.08 (3H, m, CH3), 2.28 (6H, s, 3CH2), 2.36 (4H, s, 2CH2),
3.45 (4H, s, 2CH2), 7.14 (2H, d, J= 8.0 Hz, NH2), 7.28-7.32 (2H, m, arH), 7.38-7.78 (2H, m,
arH), 8.65 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 19.01 (CH3), 27.30 (CH2), 28.52 (CH2),
48.31 (CH2), 54.33 (CH2), 56.50 (CH2), 58.29 (CH2), 61.73 (CH2), 125.71 (CH), arC: [128.79
(CH), 129.56 (CH), 129.99 (CH), 131.66 (C), 133.98 (C), 139.97 (CH)], 133.37 (triazole C-
3), 141.81 (thiadiazole C-3), 161.68 (triazole C-5), 167.78 (thiadiazole C-5). EI MS m/z (%):
462.71 ([M+2]+, 82), 442.60 (([M-H2O-1]+, 54), 412.20 (100). Elemental analysis for
C19H24N8S3 calculated (%), C, 49.54; H, 5.25; N, 24.33, found (%), C, 49.50; H, 5.25; N,
24.32.
4.6.9. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-4-{[pyridin-3-ylmethylene]amino}-5-
(thiomorpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (8c)

1
Yield 73 %. FT-IR (υmax, cm-1): 3210 (NH2), 3051 (aromatic CH), 1583 (C=N). H
NMR (DMSO-d6, δ ppm): 2.63 (2H, d, J= 4.0 Hz, CH2), 2.79 (2H, s, CH2), 3.42-3.47 (4H, m,
2CH2), 3.74-3.80 (4H, m, 2CH2), 4.41 (2H, d, J= 8.0 Hz, CH2), 7.35-7.38 (2H, m, NH2), 7.52-
7.55 (1H, m, arH), 7.73 (1H, s, arH), 7.89 (1H, d, J= 8.0 Hz, arH), 8.26 (1H, d, J= 8.0 Hz,
arH), 9.03 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 28.33 (CH2), 48.35 (CH2), 51.13 (CH2),
54.39 (CH2), 56.52 (CH2), 61.58 (CH2), 61.84 (CH2), arC: [124.33 (CH), 124.58 (CH), 129.10
(C), 132.53 (CH), 134.89 (CH), 135.38 (CH)], 146.66 (thiadiazole C-3), 147.96 (triazole C-3),
150.37 (thiadiazole C-5), 152.44 (triazole C-5). EI MS m/z (%): 449.70 ([M+2]+, 51), 425.32
([M-Na+1]+, 34), 400.01 (100). Elemental analysis for C17H21N9S3 calculated (%), C, 45.62;
H, 4.73; N, 28.16, found (%), C, 45.59; H, 4.75; N, 28.09.
4.6.10. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-5-(morpholin-4-ylmethyl)-4-{[pyridin-
4-ylmethylene]amino}-2,4-dihydro-3H-1,2,4-triazol-3-thione (8d)
1
Yield 81 %. FT-IR (υmax, cm-1): 3310 (NH2), 3067 (aromatic CH), 1582 (C=N). H
NMR (DMSO-d6, δ ppm): 1.76 (4H, s, 2CH2), 2.15 (4H, s, 2CH2), 3.39 (4H, s, 2CH2), 3.75
(2H, d, J= 8.0 Hz, CH2), 5.46 (2H, s, NH2), 7.63-7.65 (2H, m, arH), 7.97-7.99 (2H, m, arH),
10.90 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 19.06 (CH2), 24.48 (CH2), 30.63 (CH2),
40.63 (CH2), 42.75 (CH2), 63.05 (CH2), 65.37 (CH2), 111.93 (CH), arC: [110.75 (C), 118.94
(CH), 119.04 (CH), 121.57 (CH), 123.80 (CH)], 145.41 (thiadiazole C-3), 153.56 (triazole C-
3), 159.64 (thiadiazole C-5), 178.54 (triazole C-5). EI MS m/z (%): 449.53 ([M+2]+, 49),
423.20 ([M-Na+3]+, 42), 405.22 (100). Elemental analysis for C17H21N9S3 calculated (%), C,
45.62; H, 4.73; N, 28.16, found (%), C, 45.60; H, 4.76; N, 28.12.
4.6.11.
2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-4-{[(2,6-dichlorophenyl)methylene]
amino}-5-(thiomorpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (8e)
Yield 79 %, m.p: 152-154 °C. FT-IR (υmax, cm-1): 3215 (NH2), 3057 (aromatic CH),
1
1581 (C=N). H NMR (DMSO-d6, δ ppm): 1.10-1.14 (2H, m, CH2), 2.59-2.61 (2H, m, CH2),
2.70 (2H, d, J= 8.0 Hz, CH2), 3.32 (2H, s, CH2), 3.63 (2H, s, CH2), 3.77 (2H, d, J= 8.0 Hz,
CH2), 4.38-4.41 (2H, m, CH2), 5.66 (2H, s, NH2), 7.23-7.45 (3H, m, arH), 8.84 (1H, s, CH).
13C NMR (DMSO-d6, δ ppm): 48.31 (CH2), 51.43 (CH2), 54.46 (CH2), 58.72 (CH2), 61.74
(CH2), 63.68 (CH2), 66.82 (CH2), arC: [129.36 (CH), 129.42 (CH), 131.61 (C), 132.29 (C),
132.33 (2CH), 133.61 (C)], 147.98 (thiadiazole C-3), 154.49 (triazole C-3), 165.84
(thiadiazole C-5), 169.21 (triazole C-5). EI MS m/z (%): 533.53 ([M+H2O]+, 77), 500.11

(100). Elemental analysis for C18H20N8S3Cl2 calculated (%), C, 41.94; H, 3.91; N, 21.74,
found (%), C, 41.96; H, 3.95; N, 21.71.
4.6.12. 2-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-4-{[(4-fluorophenyl)methylene]amino}-
5-(thiomorpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazol-3-thione (8f)
Yield 85 %, m.p: 145-147 °C. FT-IR (υmax, cm-1): 3278 (NH2), 3065 (aromatic CH),
1578 (C=N). 1H NMR (DMSO-d6, δ ppm): 2.72 (6H, s, 3CH2), 3.65 (4H, s, 2CH2), 3.72 (4H,
s, 2CH2), 5.64 (2H, s, NH2), 7.02 (2H, s, arH), 7.39-7.44 (1H, m, arH), 8.00-8.03 (1H, m,
arH), 9.82 (1H, s, CH). 13C NMR (DMSO-d6, δ ppm): 27.61 (CH2), 28.34 (CH2), 47.96 (CH2),
48.31 (CH2), 51.29 (CH2), 51.74 (CH2), 54.15 (CH2), arC: [116.42 ve 116.64 (CH, d, J= 22.0
Hz), 116.63 ve 117.85 (CH, d, J= 122.0 Hz), 129.11 ve 129.14 (C, d, J= 3.0 Hz), 131.11 ve
131.20 (CH, d, J= 9.0 Hz), 131.72 ve 131.81 (CH, d, J= 9.0 Hz), 154.39 ve 154.48 (C, d, J=
9.0 Hz), 164.24 (CH)], 146.70 (thiadiazole C-3), 147.92 (triazole C-3), 161.49 (thiadiazole C-
5), 163.99 (triazole C-5). EI MS m/z (%): 467.52 ([M+3]+, 100). Elemental analysis for
C18H21N8S3F calculated (%), C, 46.53; H, 4.56; N, 24.12, found (%), C, 46.56; H, 4.51; N,
24.15.
4.7. General Synthesis Method for Compounds 9a-9f and 10a-10f
A mixture of compound 7 (for compound 9a-9f) or 8 (for compound 10a-10f) (10 mmol),
triethylamine (5 mmol) and chloroacetyl chloride (5 mmol) were combined in dioxane and
stirred in an ice bath (0-5 °C) for 6 h. The reaction mixture was poured into ice-water, the
resulting solid was filtered, dried and crystallized from an appropriate solvent.
4.7.1. 1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(morpholin-4-ylmethyl)-5-thioxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(4-methoxyphenyl) azetidin-2-one (9a)
1
Yield 75 %. FT-IR (υmax, cm-1): 3317 (NH2), 1715 (C=O), 1572 (C=N). H NMR
(DMSO-d6, δ ppm): 1.19-1.23 (8H, m, 4CH2), 3.03-3.09 (3H, m, 3CH2), 3.41 (3H, s, -OCH3),
3.83 (1H, s, CH), 4.27 (1H, s, CH), 7.06 (2H, s, NH2), 7.22-7.83 (4H, m, arH). 13C NMR
(DMSO-d6, δ ppm): 47.25 (CH2), 48.96 (CH2), 49.10 (CH2), 50.85 (CH2), 52.63 (CH2), 51.25
(CH3), 53.45 (CH2), 55.21 (CH2), 68.52 (CH), 69.71 (CH), arC: [125.85 (CH), 127.63 (CH),
128.96 (CH), 130.53 (CH), 139.63 (C), 140.27 (C)], 148.63 (thiadiazole C-3), 150.41 (triazole
C-3), 155.32 (thiadiazole C-5), 158.10 (triazole C-5), 169.17 (C=O). EI MS m/z (%): 536.00
([M-1]+, 66), 517.28 ([M-H2O]+, 36), 322.10 (100). Elemental analysis for C21H25N8O3S2Cl

calculated (%), C, 46.97; H, 4.69; N, 20.86; O, 8.94, found (%), C, 47.01; H, 4.71; N, 20.83;
O, 8.95.
4.7.2. 1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(morpholin-4-ylmethyl)-5-thioxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(4-methylphenyl)azetidin-2-one (9b)
1
Yield 70 %. FT-IR (υmax, cm-1): 3210 (NH2), 1710 (C=O), 1576 (C=N). H NMR
(DMSO-d6, δ ppm): 1.41 (3H, d, J= 8.0 Hz, CH3), 2.15 (2H, s, CH2), 2.49-2.51 (2H, m, CH2),
2.73 (2H, s, CH2), 2.89 (4H, s, 2CH2), 3.35 (6H, s, 3CH2), 4.59 (1H, s, CH), 5.16 (1H, s, CH),
13
6.16 (2H, s, NH2), 7.57-7.59 (4H, m, arH). C NMR (DMSO-d6, δ ppm): 14.45 (CH3), 30.35
(CH2), 33.74 (CH2), 45.99 (CH2), 50.80 (CH2), 51.95 (CH2), 61.27 (CH3), 66.83 (CH2),
101.28 (CH), 108.80 (CH), arC: [117.37 (CH), 121.15 (CH), 129.47 (CH), 129.78 (CH),
141.67 (C), 145.65 (C)], 156.15 (thiadiazole C-3), 158.73 (triazole C-3), 160.42 (thiadiazole
C-5), 168.82 (triazole C-5), 169.63 (C=O). EI MS m/z (%): 521.00 ([M]+, 98), 489.15 (100).
Elemental analysis for C21H25N8O2S2Cl calculated (%), C, 48.41; H, 4.84; N, 21.51; O, 6.14,
found (%), C, 48.41; H, 4.86; N, 21.48; O, 6.17.
4.7.3. 1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(morpholin-4-ylmethyl)-5-thioxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-pyridin-3-ylazetidin-2-one (9c)
1
Yield 84 %. FT-IR (υmax, cm-1): 3218 (NH2), 1726 (C=O), 1585 (C=N). H NMR
(DMSO-d6, δ ppm): 1.19-1.22 (8H, m, 4CH2), 3.02-3.08 (6H, m, 3CH2), 4.27 (1H, s, CH),
4.68 (1H, s, CH), 6.00 (2H, s, NH2), 7.81-7.84 (1H, m, arH), 8.46 (1H, d, J= 8.0 Hz, arH),
8.94-8.96 (2H, m, arH). 13C NMR (DMSO-d6, δ ppm): 41.96 (CH2), 45.79 (CH2), 56.47
(CH2), 63.52 (2CH2), 66.81 (2CH2), 87.23 (2CH), arC: [127.97 (CH), 127.41 (CH), 133.54
(C), 139.22 (CH), 144.24 (CH)], 148.77 (thiadiazole C-3), 149.47 (triazole C-3), 152.16
(thiadiazole C-5), 168.99 (triazole C-5), 192.05 (C=O). EI MS m/z (%): 508.00 ([M]+, 74),
432.15 (100). Elemental analysis for C19H22N9O2S2Cl calculated (%), C, 44.92; H, 4.36; N,
24.81; O, 6.30, found (%), C, 44.86; H, 4.31; N, 24.83; O, 6.33.
4.7.4. 1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(morpholin-4-ylmethyl)-5-thioxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-pyridin-4-ylazetidin-2-one (9d)

1
Yield 71 %. FT-IR (υmax, cm-1): 3320 (NH2), 1710 (C=O), 1585 (C=N). H NMR
(DMSO-d6, δ ppm): 1.19-1.22 (8H, m, 4CH2), 3.03-3.08 (6H, m, 3CH2), 3.95 (1H, s, CH),
13
4.27 (1H, s, CH), 6.07 (2H, s, NH2), 7.97-8.95 (4H, m, arH). C NMR (DMSO-d6, δ ppm):
47.20 (CH2), 48.62 (CH2), 49.41 (CH2), 50.78 (CH2), 52.63 (CH2), 53.85 (CH2), 55.67 (CH2),
68.21 (CH), 70.14 (CH), arC: [127.32 (CH), 129.21 (CH), 130.12 (CH), 131.87 (CH), 139.21
(C)], 149.21 (thiadiazole C-3), 150.17 (triazole C-3), 155.24 (thiadiazole C-5), 157.31
(triazole C-5), 169.21 (C=O). EI MS m/z (%): 508.02 ([M]+, 74), 432.07 (100). Elemental
analysis for C19H22N9O2S2Cl calculated (%), C, 44.92; H, 4.36; N, 24.81; O, 6.30, found (%),
C, 44.84; H, 4.32; N, 24.82; O, 6.35.
4.7.5.
1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(thiomorpholin-4-ylmethyl)-5-
thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(2,6-dichlorophenyl)azetidin-2-one
(9e)
1
Yield 79 %. FT-IR (υmax, cm-1): 3178 (NH2), 1723 (C=O), 1586 (C=N). H NMR
(DMSO-d6, δ ppm): 1.03-1.07 (4H, m, 2CH2), 1.18-1.21 (4H, m, 2CH2), 3.04-3.07 (4H, m,
2CH2), 3.43 (2H, d, J= 4.0 Hz, CH2), 4.15 (1H, s, CH), 4.58 (1H, s, CH), 7.55 (2H, d, J= 4.0
13
Hz, NH2), 8.31-8.34 (3H, m, arH). C NMR (DMSO-d6, δ ppm): 18.93 (CH2), 41.24 (CH2),
41.92 (CH2), 45.94 (2CH2), 56.49 (2CH2), 61.83 (CH), 66.82 (CH), arC: [129.85 (CH),
131.87 (CH), 134.39 (C), 139.68 (CH), 142.31 (2C)], 143.93 (triazole C-3), 165.19
(thiadiazole C-3), 167.20 (triazole C-5), 168.13 (thiadiazole C-5). EI MS m/z (%): 594.82
([M+K]+, 33), 576.14 ([M+1]+, 73), 521.21 (100). Elemental analysis for C20H21N8O2S2Cl3
calculated (%), C, 41.71; H, 3.68; N, 19.46; O, 5.56, found (%), C, 41.72; H, 3.71; N, 19.41;
O, 5.61.
4.7.6. 1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(morpholin-4-ylmethyl)-5-thioxo-
1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(4-fluorophenyl)azetidin-2-one (9f)
1
Yield 72 %. FT-IR (υmax, cm-1): 3218 (NH2), 1723 (C=O), 1583 (C=N). H NMR
(DMSO-d6, δ ppm): 1.18-1.22 (8H, m, 4CH2), 3.02-3.09 (6H, m, 3CH2), 3.56 (1H, s, CH),
4.27 (1H, s, CH), 6.08 (2H, brs, NH2), 7.20-7.45 (4H, m, arH). 13C NMR (DMSO-d6, δ ppm):
41.95 (CH2), 45.89 (CH2), 51.74 (CH2), 59.95 (CH2), 61.77 (CH2), 63.51 (CH2), 66.81 (CH2),
72.14 (CH), 74.28 (CH), arC: [116.38 ve 116.60 (CH, d, J= 22.0 Hz), 125.98 (C), 127.65
(CH), 131.15 ve 131.31 (CH, d, J= 16.0 Hz), 140.78 (C), 142.08 (C)], 146.12 (thiadiazole C-
3), 147.20 (triazole C-3), 150.30 (thiadiazole C-5), 159.79 (triazole C-5), 169.01 (C=O). EI

MS m/z (%): 523.00 ([M-2]+, 87), 499.01 (100). Elemental analysis for C20H22N8O2S2ClF
calculated (%), C, 45.75; H, 4.22; N, 21.34; O, 6.09, found (%), C, 45.72; H, 4.26; N, 21.39;
O, 6.15.
4.7.7.
1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(thiomorpholin-4-ylmethyl)-5-
thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(4-methoxyphenyl)azetidin-2-one
(10a)
1
Yield 75 %. FT-IR (υmax, cm-1): 3310 (NH2), 1712 (C=O), 1582 (C=N). H NMR
(DMSO-d6, δ ppm): 1.15 (2H, s, CH2), 1.31 (2H, s, CH2), 1.77 (2H, s, CH2), 2.67 (2H, s,
CH2), 2.73 (2H, s, CH2), 2.82 (2H, s, CH2), 2.88 (2H, s, CH2), 2.93 (3H, s, CH3), 4.90 (1H, s,
13
CH), 5.21 (1H, s, CH), 5.48 (2H, s, NH2), 7.05-7.48 (4H, m, arH). C NMR (DMSO-d6, δ
ppm): 15.54 (CH3), 43.18 (CH2), 46.97 (CH2), 48.15 (CH2), 51.13 (CH2), 51.48 (CH2), 51.95
(CH2), 6.29 (CH2), 67.79 (CH), 78.21 (CH), arC: [101.33 (CH), 108.71 (CH), 114.04 (CH),
120.87 (CH), 132.41 (C), 133.08 (C)], 135.83 (triazole C-3), 140.28 (thiadiazole C-3), 151.06
(triazole C-5), 154.04 (thiadiazole C-5), 157.87 (C=O). EI MS m/z (%): 553.01 ([M]+, 42),
500.11 (100). Elemental analysis for C21H25N8O2S3Cl calculated (%), C, 45.60; H, 4.56; N,
20.26; O, 5.79, found (%), C, 45.62; H, 4.61; N, 20.21; O, 5.84.
4.7.8. 1-[1-[2-(5-Amino-1,3,4-tiyadiazol-2-il)etil]-3-(tiyomorfolin-4-ilmetil)-5-tiyokso-1,5-
dihidro-4H-1,2,4-triazol-4-il]-3-kloro-4-(4-metilfenil)azetidin-2-on (10b)
1
Yield 78 %. FT-IR (υmax, cm-1): 3278 (NH2), 1710 (C=O), 1578 (C=N). H NMR
(DMSO-d6, δ ppm): 1.02-1.06 (3H, m, CH3), 1.17 (4H, s, 2CH2), 2.34 (2H, s, CH2), 3.05 (4H,
s, 2CH2), 3.43 (2H, d, J= 8.0 Hz, CH2), 3.58 (2H, s, CH2), 4.23 (1H, s, CH), 4.27 (1H, s, CH),
13
6.00 (1H, s, NH2), 7.22-7.35 (3H, m, arH), 7.73-7.75 (1H, m, arH). C NMR (DMSO-d6, δ
ppm): 18.81 (CH3), 41.87 (CH2), 46.05 (CH2), 46.66 (CH2), 56.54 (CH2), 62.14 (CH2), 63.18
(CH2), 66.79 (CH2), 126.98 (CH), 128.38 (CH), arC: [128.78 (C), 129.03 (CH), 129.22 (CH),
129.56 (CH), 129.75 (CH), 129.97 (C)], 131.50 (triazole C-3), 133.92 (thiadiazole C-3),
141.92 (triazole C-5), 161.70 (thiadiazole C-5), 169.02 (C=O). EI MS m/z (%): 557.10 ([M]+,
65), 538.65 ([M-1-H2O]+, 54), 498.14 (100). Elemental analysis for C21H25N8OS3Cl
calculated (%), C, 46.96; H, 4.69; N, 20.86; O, 2.98, found (%), C, 46.89; H, 4.74; N, 20.84;
O, 3.04.

4.7.9.
1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(thiomorpholin-4-ylmethyl)-5-
thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-pyridin-3yl-azetidin-2-one (10c)
1
Yield 77 %. FT-IR (υmax, cm-1): 3314 (NH2), 1725 (C=O), 1574 (C=N). H NMR
(DMSO-d6, δ ppm): 1.18-1.22 (8H, m, 4CH2), 3.04-3.07 (6H, m, 3CH2), 3.44 (1H, d, J= 8.0
13
Hz), 3.57 (1H, s, CH), 6.05 (2H, brs, NH2), 7.23-7.53 (4H, m, arH). C NMR (DMSO-d6, δ
ppm): 45.83 (CH2), 56.47 (CH2), 57.20 (CH2), 58.13 (CH2), 59.32 (CH2), 61.71 (CH2), 66.82
(CH2), 68.90 (CH), 69.37 (CH), arC: [123.87 (CH), 125.63 (CH), 126.98 (CH), 127.37 (CH),
130.12 (C)], 147.85 (triazole C-3), 150.39 (thiadiazole C-3), 163.85 (triazole C-5), 167.89
(thiadiazole C-5), 169.52 (C=O). EI MS m/z (%): 547.10 ([M+Na]+, 70), 523.00 ([M-1]+, 48),
490.35 (100). Elemental analysis for C19H22N9OS3Cl calculated (%), C, 43.54; H, 4.23; N,
24.05; O, 3.05, found (%), C, 43.50; H, 4.28; N, 24.06; O, 3.09.
4.7.10.
1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(thiomorpholin-4-ylmethyl)-5-
thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-pyridin-2yl-azetidin-2-one (10d)
1
Yield 70 %. FT-IR (υmax, cm-1): 3314 (NH2), 1730 (C=O), 1571 (C=N). H NMR
(DMSO-d6, δ ppm): 1.17-1.20 (8H, m, 4CH2), 3.02-3.06 (6H, m, 3CH2), 3.42 (1H, d, J= 8.0
13
Hz, CH), 3.54 (1H, s, CH), 6.26 (2H, brs, NH2), 7.26-8.00 (4H, m, arH). C NMR (DMSO-
d6, δ ppm): 41.23 (CH2), 41.59 (CH2), 41.93 (CH2), 45.98 (CH2), 53.65 (CH2), 56.53 (CH2),
59.88 (CH2), 62.17 (CH), 66.78 (CH), arC: [123.38 (CH), 125.05 (CH), 126.07 (CH), 128.33
(CH), 138.70 (C)], 146.51 (triazole C-3), 149.89 (thiadiazole C-3), 165.44 (triazole C-5),
169.06 (thiadiazole C-5), 170.01 (C=O). EI MS m/z (%): 547.13 ([M+Na]+, 67), 523.05 ([M-
1]+, 42), 490.12 (100). Elemental analysis for C19H22N9OS3Cl calculated (%), C, 43.54; H,
4.23; N, 24.05; O, 3.05, found (%), C, 43.51; H, 4.25; N, 24.02; O, 3.09.
4.7.11.
1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(thiomorpholin-4-ylmethyl)-5-
thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(2,6-dichlorophenyl)azetidin-2-one
(10e)
1
Yield 79 %. FT-IR (υmax, cm-1): 3178 (NH2), 1723 (C=O), 1586 (C=N). H NMR
(DMSO-d6, δ ppm): 1.03-1.07 (4H, m, 2CH2), 1.18-1.21 (4H, m, 2CH2), 3.04-3.07 (4H, m,
2CH2), 3.43 (2H, d, J= 4.0 Hz, CH2), 4.15 (1H, s, CH), 4.58 (1H, s, CH), 7.55 (2H, d, J= 4.0
13
Hz, NH2), 8.31-8.34 (3H, m, arH). C NMR (DMSO-d6, δ ppm): 18.93 (CH2), 41.24 (CH2),
41.92 (CH2), 45.94 (2CH2), 56.49 (2CH2), 61.83 (CH), 66.82 (CH), arC: [129.85 (CH),

131.87 (CH), 134.39 (C), 139.68 (CH), 142.31 (2C)], 143.93 (triazole C-3), 165.19
(thiadiazole C-3), 167.20 (triazole C-5), 168.13 (thiadiazole C-5). EI MS m/z (%): 592.00
([M]+, 100). Elemental analysis for C20H21N8OS3Cl3 calculated (%), C, 40.58; H, 3.58; N,
18.93; O, 2.70, found (%), C, 40.55; H, 3.61; N, 18.97; O, 3.65.
4.7.12.
1-[1-[2-(5-Amino-1,3,4-thiadiazol-2-yl)ethyl]-3-(thiomorpholin-4-ylmethyl)-5-
thipxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-chloro-4-(4-fluorophenyl)azetidin-2-one (10f)
1
Yield 72 %. FT-IR (υmax, cm-1): 3312 (NH2), 1693 (C=O), 1582 (C=N). H NMR
(DMSO-d6, δ ppm): .1.16-1.20 (8H, m, 4CH2), 3.03-3.06 (6H, m, 3CH2), 4.25 (1H, s, CH),
4.58 (1H, s, CH), 6.16 (2H, s, NH2), 7.26 (1H, s, arH), 7.38 (1H, s, arH), 7.51 (1H, s, arH),
13
10.08 (2H, s, 2OH). C NMR (DMSO-d6, δ ppm): 41.93 (CH2), 45.94 (CH2), 59.99 (CH2),
60.02 (CH2), 61.20 (CH2), 61.78 (CH2), 62.32 (CH2), 67.10 (CH), 68.63 (CH), arC: [122.10
(CH), 123.75 (CH), 126.85 (CH), 139.02 (C), 140.85 (C)], 147.85 (triazole C-3), 150.10
(thiadiazole C-3), 163.78 (triazole C-5), 168.52 (thiadiazole C-5), 170.23 (C=O). EI MS m/z
(%): 560.11 ([M+K]+, 35), 543.22 ([M+2]+, 77), 525.12 ([M+2-H2O]+, 42), 512.98 (100).
Elemental analysis for C20H22N8OS3ClF calculated (%), C, 44.40; H, 4.10; N, 20.71; O, 2.96,
found (%), C, 44.38; H, 4.04; N, 20.72; O, 2.96.
4.8. Antimicrobial Activity / Sensitivity Studies
Antimicrobial activity or susceptibility tests were performed to determine the in-vitro activity
of the antimicrobial agent against a particular bacterial species. Two techniques are used to
measure susceptibility testing of microorganisms, including "diffusion" and "dilution". Disc
diffusion technique is a frequently used technique and the sensitivity of the antibiotic
absorbed into paper discs (the solution to be measured by antibacterial activity) is based on
the diffusion of the organism to the medium in which the organism is inoculated. After the
impregnated paper discs are placed on solid media in which the microorganism is inoculated,
the discs dissolve and diffuse into the agar, while the inoculated microorganism begins to
multiply. There is no growth in the vicinity of the disc where the inhibitor concentrations of
the drug are achieved after a certain incubation period. The more sensitive the microorganism
is to the drug, the larger the zone of inhibition around the disc. The diameter of the zone of
inhibition is measured in mm and evaluations are made according to standard zone tables and
the susceptibility status of the microorganism to the antimicrobial agents used is determined.

Minimal Inhibition Test measurements are tested by applying the microdilution technique to
determine the dose value of the effectiveness of the substances determined to be effective in
the agar well. The quantity of the material is diluted with serial dilutions to the lowest doses
and the same amount of microorganism is added to each diluent. With this test, the efficacy
dose of the lowest amount of substance is determined.
4.8.1. Minimal Inhibition Concentration (MIC) Method
In order to determine the minimum amount of substance that shows antimicrobial activity, in
other words, to determine the quantitative effect value, the micro-dilution liquid method is
applied in the liquid medium and the minimal inhibition concentration (MIC) is determined as
microgram/milliliter (μg/mL) [25]. For the determination of antimicrobial activity, liquid
media were used for determining the antifungal activity of Mueller-Hinton liquid (MHB,
pH.7.3) (Difco, Detroit, MI) and yeast extract liquid medium (YEG, pH 7.0) (Difco, Detroit,
MI). ELISA plates were used for micro-dilution tests and serial dilutions were made with 0.1
mL of dissolved chemicals. McFarland 0.5 turbidity (1x108 cfu/mL) from overnight cultures
of inoculated microorganisms was prepared for reconstitution and diluted 1:10 and 0.005 mL
of microorganism (final assay concentration 5x104 cfu/well) was added to each well. Plates
were incubated at 35 °C for 16-24 hour under aerobic conditions. The MIC value was
completely inhibited by the growth of the microorganism in the micro-dilution wells and was
determined as the lowest antimicrobial concentration that could be determined by the naked
eye. Streptomycin (4 μg) and standard solvent control were used as standard control drugs.
4.9. Urease inhibition assay [26]
Urease activity was defined by using Weatherburn process. Reaction mixtures including 25
µL of Jack Bean urease, 55 µL of buffer (0.01 mol/L K2HPO4, 1 mmol/L EDTA and 0.01
mol/L LiCl, pH 8.2) and 10 mmol/L urea were incubated with 5 µL of the test compounds at
room temperature for 15 min in microtiter plates. The production of ammonia was measured
following the indophenol method and was used to determine the urease inhibitory activity.
The phenol reagent (45 µL, 1% w/v phenol and 0.005% w/v sodium nitroprusside) and alkali
reagent (70 µL, 0.5% w/v sodium hydroxide and 0.1% v/v NaOCl) were added to each well.
This mixture was incubated for 15 minutes more at 35 °C and optical density was measured at
625 nm against a blank solution including distilled water instead of enzyme. For the
determination of the IC50 value of the extracts, activity assays were conducted at five different

extract concentration and dose response curve was generated. Thiourea was used as standard
inhibitor.
4.10. Determination of Acetylcholinesterase Activity
The AChE inhibitory activity of the compounds was determined using the ELHMAN method
[27] using AChE from E. electricus (Sigma) and acetylthiocololine iodide (0.35 mM) as a
substrate. The reaction was carried out in the final volume of 3 mL of phosphate buffered
solution containing 0.35 mM 5,50-dithiobis-2-nitrobenzoic acid (DTNB), produced from 5-
thio-2-nitrobenzoic acid, 0.035 U / mL EeAChE and a yellow anion. Inhibition curves were
made by incubating with different compounds for 15 minutes; A sample without any
compound was always used to determine 100% of the enzymatic activity. After a 15 min
incubation period, color production as an indicator of enzymatic activity was assessed by
measuring the absorbance at 412 nm in a spectrophotometer plate reader.
4.11. Antioxidant activity
DPPH (2.2-diphenyl-1-picrylhydrazyl) radical scavenging activity was performed the
synthesized compounds with different chemicals was determined using the free radical DPPH
(2.2-diphenyl-1-picrylhydrazyl), as described by Blois [28]. A 100-μL: chemical solution was
mixed with 1 mL of freshly prepared methanolic DPPH solution. The reaction mixture was
incubated for 30 min at room temperature in the dark and was then measured at 520 nm. The
activity was expressed as μmol Trolox equivalent. FRAP (the ferric reducing ability of
plasma) was measured using the method described by Benzie & Strain [29] with some
modification. To 100 μL of each sample was added 2900 μL freshly prepared FRAP reagent
containing 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ (2,4,6-tripyridyle-s-
triazine) and 20 mmol/L FeCl.6H2O in proportions of 10:1:1 (v/v/v). The mixture was
incubated for 30 min at 37 ºC and measured at 593 nm. The values were given as μmol of
Trolox/g. CUPRAC (cupric ion reducing antioxidant capacity) was measured following the
procedure described by Apak et al. [30] with some modification. Briefly, 100 μL of each
chemical solution was mixed with 900 μL bidistilled water, 1 mL acetate buffer solution (1
mmol/L, pH: 7.0), 1 mL CuCl2 (10 mmol/L) and 1 mL 7.5 mmol/L neocuproine to a final
volume of 4 mL. The reaction mixture was then incubated in the dark for 30 min at room
temperature and the absorbance of the reaction mixture was measured at 450 nm against a

water blank. Trolox was used as the standard calibration curves and the results were expressed
as μmol Trolox equivalent per g.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of the paper.
Acknowledgments
This work was supported by the Scientific and Technological Research Council of Turkey
(TUBITAK) [grant numbers 115Z770].
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Graphical abstract
Synthesis of Novel Schiff Bases and Azol-β-Lactam Derivatives starting from Morpholine and Thiomorpholine and
Investigation of their Antituberculosis, Antiurease activity, Acethylcolinesterase Inhibition Effect
and Antioxidant Capacity
Yıldız Uygun Cebeci1, Hacer Bayrak2*, Yakup Şirin1
N
N
N
N
N
N
NH2
NH2
S
S
NH2
S
N
N
N
N
N
N
(O)S
NH
(O)S
N
(O)S
N
(O)S
N
S
S
N
N
N
N
S
O
5a, 5b
O
R
R
Cl