1,3,4-oxadiazole n-mannich bases: Synthesis, antimicrobial, and anti-proliferative activities

Article abstract of DOI:10.3390/molecules26082110

The reaction of 5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 3 with formalde-hyde solution and primary aromatic amines or 1-substituted piperazines, in ethanol at room temper-ature yielded the corresponding N-Mannich bases 3-arylaminomethyl-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 4a–l or 3-[(4-substituted piperazin-1-yl)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 5a–d, respectively. The in vitro inhibitory activity of compounds 4a–l and 5a–d was assessed against pathogenic Gram-positive, Gram-negative bacteria, and the yeast-like pathogenic fungus Candida albicans. The piperazinomethyl derivatives 5c and 5d displayed broad-spectrum antibacterial activities the minimal inhibitory concentration (MIC) 0.5–8 μg/mL) and compounds 4j, 4l, 5a, and 5b showed potent activity against the tested Gram-positive bacteria. In addition, the anti-proliferative activity of the compounds was evaluated against prostate cancer (PC3), human colorectal cancer (HCT-116), human hepatocellular carcinoma (HePG-2), human epithe-lioid carcinoma (HeLa), and human breast cancer (MCF7) cell lines. The optimum anti-proliferative activity was attained by compounds 4l, 5a, 5c, and 5d.

Full text of DOI:10.3390/molecules26082110

molecules
Article
1,3,4-Oxadiazole N-Mannich Bases: Synthesis, Antimicrobial,
and Anti-Proliferative Activities
Lamya H. Al-Wahaibi 1, Ahmed A. B. Mohamed 2, Samar S. Tawfik 3, Hanan M. Hassan 4 and Ali A. El-Emam 2,
*
1
Department of Chemistry, College of Sciences, Princess Nourah Bint Abdulrahman University,
Riyadh 11671, Saudi Arabia; lhalwahaibi@pnu.edu.sa
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt;
ahmed_smt@yahoo.com
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt; samarsamir1984@gmail.com
Department of Pharmacology and Biochemistry, Faculty of Pharmacy, Delta University for Science and
Technology, International Costal Road, Gamasa City, Mansoura 11152, Egypt; hananhafila@hotmail.com
Correspondence: elemam@mans.edu.eg; Tel.: +20-50-2258087
2
3
4
*
Abstract: The reaction of 5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 3 with formalde-
hyde solution and primary aromatic amines or 1-substituted piperazines, in ethanol at room temper-
ature yielded the corresponding N-Mannich bases 3-arylaminomethyl-5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazole-2(3H)-thiones 4a–l or 3-[(4-substituted piperazin-1-yl)methyl]-5-(3,4-dimethoxyphenyl)-
1,3,4-oxadiazole-2(3H)-thiones 5a
4a and 5a was assessed against pathogenic Gram-positive, Gram-negative bacteria, and the
yeast-like pathogenic fungus Candida albicans. The piperazinomethyl derivatives 5c and 5d displayed
broad-spectrum antibacterial activities the minimal inhibitory concentration (MIC) 0.5–8 g/mL)
and compounds 4j 4l 5a, and 5b showed potent activity against the tested Gram-positive bacteria.
–d, respectively. The in vitro inhibitory activity of compounds
–
l
–d
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
µ
Citation: Al-Wahaibi, L.H.;
,
,
Mohamed, A.A.B.; Tawfik, S.S.;
Hassan, H.M.; El-Emam, A.A.
1,3,4-Oxadiazole N-Mannich Bases:
Synthesis, Antimicrobial, and
Anti-Proliferative Activities.
In addition, the anti-proliferative activity of the compounds was evaluated against prostate cancer
(PC3), human colorectal cancer (HCT-116), human hepatocellular carcinoma (HePG-2), human epithe-
lioid carcinoma (HeLa), and human breast cancer (MCF7) cell lines. The optimum anti-proliferative
activity was attained by compounds 4l, 5a, 5c, and 5d.
Molecules 2021, 26, 2110. https://
doi.org/10.3390/molecules26082110
Keywords: 1,3,4-oxadiazoles; N-Mannich bases; antimicrobial activity; anti-proliferative activity
Academic Editor: Athina Geronikaki
1. Introduction
Received: 5 March 2021
Accepted: 31 March 2021
Published: 7 April 2021
The 1,3,4-Oxadiazoles are an important class of heterocyclic compounds with diverse
pharmacological properties [
building unit in several drugs including the broad spectrum antibacterial drug furami-
zole [ ], the antiretroviral drug raltegravir [ ], the anticancer agent zibotentan [ ], the anti-
], and the antihypertensive drugs tiodazosin [ ],
1–3]. The 1,3,4-Oxadiazole nucleus represents an essential
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
4
5
6
obesity/antidiabetic agent AZD 3988 [
7
8
and nesadipil [9].
In addition, there is a growing interest in the chemotherapeutic activities of 2,5-
disubstituted-1,3,4-oxadiazoles as antibacterial [10 12], antifungal [13 15], antitubercu-
lar [16 17], and antiviral [18 21] agents. Moreover, 1,3,4-Oxadiazole-2(3H)-thiones, their
thioether derivatives and 3-aminomethyl analogues (N-Mannich bases) are the most in-
teresting for their anticancer activities [21 22]. The 1,3,4-Oxadiazole derivatives exert
–
–
,
–
,
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
their anticancer activities via different mechanisms, such as targeting epidermal growth
factor receptors (EGFR) [23], vascular endothelial growth factor receptors (VEGF) [24],
focal-adhesion kinase (FAK) [25
,
26], histone deacetylases (HDAC) [27
,28], methionine
aminopeptidase (MetAP) [29], NF-
κ
B (nuclear factor B) [30], poly(ADP-ribose) poly-
κ
merase (PARP-1) [31], thymidine phosphorylase (TP) [32], telomerase [33], thymidylate
synthase (TS) [34], zinc-finger protein 143 (ZNF143) [35], and tubulin polymerase [36].
Molecules 2021, 26, 2110. https://doi.org/10.3390/molecules26082110
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2110
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Furthermore, 1,3,4-oxadiazoles were proved to exhibit potent anti-inflammatory [37–39]
antioxidant [40], antidiabetic [41 42], and monoamine oxidase (MAO) inhibitory activi-
ties [43 44]. Besides, 1,3,4-oxadiazole derivatives are highly attractive compounds in the
,
,
,
development of organic light-emitting diodes (OLEDs) [45,46].
Furthermore, 3,4-dimethoxyphenyl moiety represents an essential motif in various
chemotherapeutic agents with anticancer [47
activities.
–52], antibacterial [53,54], and antiviral [55]
Motivated by the above-mentioned findings and following an ongoing interest in the
pharmacological [56] and structural properties [57 59] of 1,3,4-oxadiazole-2(3H)-thione
–
N-Mannich bases, we report herein the synthesis, characterization, antibacterial, anti-
fungal, and anti-proliferative activities of related series of 5-(3,4-dimethoxyphenyl)-1,3,4-
oxadiazole-2(3H)-thione N-Mannich bases.
2. Results and Discussion
2.1. Chemical Synthesis
The 3,4-Dimethoxybenzohydrazide
methyl 3,4-dimethoxybenzoate via treatment with hydrazine in ethanol [60]. 1,3,4-
Oxadiazole-2(3H)-thione was obtained via reaction of the carbohydrazide with carbon
2 was prepared from the commercially-available
1
3
2
disulfide in ethanolic potassium hydroxide as previously described [61]. Furthermore, 1,3,4-
Oxadiazole-2(3H)-thiones were reported to undergo aminomethylation through reaction
with primary aromatic amines and formaldehyde to yield the corresponding N-Mannich
bases [62]. Consequently, treatment of
aromatic amines or 1-substituted piperazines, in ethanol at room temperature yielded
their corresponding 3-arylaminomethyl 4a or 3-piperazinomethyl 5a N-Mannich bases,
3 with formaldehyde solution and various primary
–
l
–d
respectively, in good yields (Scheme 1, Table 1).
Scheme 1. Synthesis of compounds 4a–l and 5a–d.

Molecules 2021, 26, 2110
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Table 1. Crystallization solvents, melting points (M.P.), yield percentages, molecular (Mol.) formulae, and molecular
weights (Wt.) of compounds 4a–l and 5a–d.
◦
Compound No.
X/R
Crystallization Solvents
M.P. ( C)
Yield (%)
Mol. Formula (Mol. Wt.)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
H
4-F
3-Cl
EtOH/H2O
EtOH/H2O
EtOH
147–149
135–137
143–145
166–168
211–213
181–183
222–224
220–222
206–208
169–171
212–214
227–229
151–153
118–120
121–123
141–143
82
84
77
79
88
85
92
90
86
92
90
94
85
78
75
89
C17H17N3O3S (343.40)
C17H16FN3O3S (361.39)
C17H16ClN3O3S (377.85)
C17H16ClN3O3S (377.85)
C17H16N4O5S (388.40)
C17H16N4O5S (388.40)
C17H16N4O5S (388.40)
C18H16F3N3O3S (411.40)
C18H16F3N3O3S (411.40)
C17H15F2N3O3S (379.38)
C17H15F2N3O3S (379.38)
C17H15Cl2N3O3S (412.29)
C21H24N4O3S (412.51)
C21H23FN4O3S (430.50)
C22H26N4O3S (426.53)
C23H25F3N4O3S (494.53)
4-Cl
EtOH
2-NO2
3-NO2
4-NO2
2-CF3
3-CF3
2,4-F2
2,5-F2
2,4-Cl2
C6H5
EtOH/CHCl3
EtOH/CHCl3
EtOH/CHCl3
EtOH/H2O
EtOH/H2O
EtOH
EtOH
EtOH
EtOH
EtOH
4-FC6H4
C6H5CH2
2-CF3C6H4CH2
EtOH/H2O
EtOH
The structures of compounds 4a
1H NMR and 13C NMR spectral data.
–l and 5a–d were confirmed by elemental analyses,
2.2. In Vitro Antibacterial and Antifungal Activities
The in vitro growth inhibitory activity of compounds 4a
–l and 5a–d was evaluated
towards the standard Gram-positive bacterial strains Staphylococcus aureus American type
culture collection (ATCC) 6571, Bacillus subtilis ATCC 5256 and Micrococcus luteus ATCC
27141, Gram-negative bacterial strains Escherichia coli ATCC 8726, and Pseudomonas aerugi-
nosa ATCC 27853, and the yeast-like pathogenic fungus Candida albicans MTCC 227. The
initial screening was performed by the semi-quantitative agar-disc diffusion method using
Müller-Hinton agar medium [63]. The results of the preliminary screening of compounds
4a–l and 5a–d (200 µg/disc); the antibacterial antibiotics Gentamicin sulfate, Ampicillin
trihydrate, and the antifungal drug Clotrimazole (100 µg/disc); and the calculated log P
values (Clog P) are depicted in Table 2.
The results revealed that potent antibacterial activity was displayed by the compounds
4l 5a 5b 5c, and 5d, which displayed growth inhibition zones 18 mm against one
or more of the tested microorganisms. In addition, compounds 4a 4b 4c 4d 4h 4i, and
4k showed medium activity (growth inhibition zones 14–17 mm) and compounds 4e 4f,
4j,
,
,
,
≥
,
,
,
,
,
,
and 4g showed poor antibacterial activity (growth inhibition zones 10–13 mm) against the
tested microorganisms. In general, the activity against the tested Gram-positive bacteria is
higher than the activity against the tested Gram-negative bacteria. The optimal antibacterial
activity was attained by compounds 5c and 5d, which showed potent and broad-spectrum
antibacterial activity against all the tested bacterial strains. The inhibitory activity of the
compounds against Candida albicans was generally lower than their antibacterial activity,
compounds 4g displayed medium activity, and all other compounds were either poorly
active or inactive compared with Clotrimazole.
The minimal inhibitory concentrations (MICs) of the most active compounds 4j
, 4l, 5a,
5b, 5c, and 5d, and the antibacterial antibiotics Gentamicin sulfate, Ampicillin trihydrate,
and the antifungal drug Clotrimazole were determined by the microdilution susceptibility
method in Müller–Hinton broth and Sabouraud liquid medium [64]. The MIC values were
highly consistent with their growth inhibition zones.

Molecules 2021, 26, 2110
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Table 2. In vitro activity of compounds 4a
–l
and 5a
–d
(200
µg/8 mm disc); the broad-spectrum
antibacterial drugs Gentamicin sulfate, Ampicillin trihydrate, and the antifungal drug Clotrimazole
(100 g/8 mm disc) against Staphylococcus aureus American type culture collection (ATCC) 6571 (SA),
µ
Bacillus subtilis ATCC 5256 (BS), Micrococcus luteus ATCC 27,141 (ML), Escherichia coli ATCC 8726
(EC), Pseudomonas aeruginosa ATCC 27,853 (PA), and the yeast-like pathogenic fungus Candida albicans
MTCC 227 (CA).
Diameter of Growth Inhibition Zone (mm) a
Comp.
No.
C log Pc
SA
BS
ML
EC
PA
CA
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
3.701
4.146
4.716
4.716
4.242
4.092
4.092
5.113
5.113
4.395
4.395
5.535
3.789
4.103
4.712
5.595
14
15
14
15
12
11
13
15
16
13
16
17
13
11
12
12
12
12
15
17
13
14
10
-
11
-
-
-
-
-
-
-
-
-
-
-
12
14
15
14
-
-
-
-
-
-
-
-
-
13
-
12
13
17
10
12
-
-
13
12
16
-
-
11
12
11
-
-
-
-
19 (4) b
17
12
14
16
15
19 (4) b
23 (1) b
20 (8) b
26 (1) b
28 (1) b
27 (1) b
22 (2) b
NT
21 (2) b
26 (1) b
23 (1) b
30 (0.5) b
29 (1) b
26 (2) b
23 (1) b
NT
18 (16) b
21 (1) b
20 (1) b
22 (1) b
26 (1) b
20 (2) b
20 (2) b
NT
16
19 (4) b
22 (1) b
22 (0.5) b
16 (8) b
NT
18 (4) b
20 (2) b
21 (0.5) b
16 (8) b
NT
Gentamicin sulfate
Ampicillin trihydrate
Clotrimazole
NT
NT
21 (4) b
a
b
(-): inactive (inhibition zone < 10 mm), Figures shown in parentheses represent the minimal inhibitory
concentration (MIC) values (µ
g/mL), c Calculated using the CS ChemOffice Ultra version 8.0, CambridgeSoft,
Cambridge, MA, USA), NT: not tested. SA, Staphylococcus aureus; BS, Bacillus subtilis; ML, Micrococcus luteus; EC,
Escherichia coli; PA, Pseudomonas aeruginosa; CA, Candida albicans. High activity (>18 mm) values have been bolded
for emphasis.
Based on the results of the antibacterial activity, it could be concluded that the activ-
ity of the piperazinomethyl N-Mannich bases 5a
analogues 4a . Considering the anilinomethyl analogue 4a as the basic structure of the
arylaminomethyl analogues 4a , the antibacterial activity of the monohalo derivatives
4b was slightly improved against the tested Gram-positive bacteria. Meanwhile, the
antibacterial activity of the nitro derivatives 4e was greatly declined with general im-
–d is superior to their arylaminomethyl
–
l
–
l
–
d
–
g
provement of the antifungal activity. Despite the high lipophilicity of the trifluoromethyl
derivatives 4h and 4i, the compounds only retained moderate activity against Staphylococ-
cus aureus and lacked activity against the tested Gram-positive bacteria and Candida albicans.
Introduction a difluoro- or dichlorophenyl moieties (compounds 4j–l) greatly enhanced the
Gram-positive antibacterial activity and these derivatives endowed moderate or marginal
activity against the tested Gram-negative bacteria and Candida albicans.
The replacement of the arylaminomethyl moiety with a piperazinomethyl moiety
greatly enhanced the antibacterial activity and the piperazinomethyl derivatives 5a
ited higher potency and broader antibacterial spectrum compared to their arylaminomethyl
analogues 4a , with no antifungal activity. In addition, the antibacterial activity of the
–d exhib-
–
l
piperazinomethyl derivatives seems correlated to their lipophilicity as the optimum an-
tibacterial activity against the tested Gram-negative bacteria was displayed by compounds
5c and 5d.
2.3. In Vitro Anti-proliferative Activity
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric
assay [65,66] was adopted to evaluate the in vitro anti-proliferative activity of compounds

Molecules 2021, 26, 2110
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4a–
l
and 5a
–d
against five human cancer cell lines namely; prostate cancer (PC3), human
colorectal cancer (HCT-116), human hepatocellular carcinoma (HePG-2), human epithelioid
carcinoma (HeLa) and human breast cancer (MCF7) cell lines. The results of the anti-
proliferative activity of compounds 4a–l, 5a–d, and the anticancer drug Doxorubicin [67]
are displayed in Table 3.
Table 3. In vitro anti-proliferative activity of the tested compounds 4a–l, 5a–d, and Doxorubicin
expressed as IC50 values against prostate cancer (PC3), human colorectal cancer (HCT-116), human
hepatocellular carcinoma (HePG-2), human epithelioid carcinoma (HeLa), and MCF7 human cancer
cell line.
IC50 (µM) a
Comp.
No.
PC3
HCT-116
HePG-2
HeLa
MCF7
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
63.94 ± 3.8
71.80 ± 4.0
84.52 ± 4.8
78.35 ± 4.5
>100
53.17 ± 3.3
77.52 ± 4.1
39.44 ± 2.6
48.30 ± 3.0
92.11 ± 4.9
69.38 ± 3.8
92.11 ± 4.9
35.01 ± 2.7
35.01 ± 2.7
64.07 ± 3.5
61.07 ± 3.4
19.95 ± 1.8
27.49 ± 2.3
89.26 ± 4.6
14.69 ± 1.2
32.81 ± 2.6
5.23 ± 0.3
47.23 ± 3.1
56.34 ± 3.3
78.86 ± 4.1
73.80 ± 3.9
>100
28.31 ± 2.0
49.47 ± 3.0
44.69 ± 2.8
54.02 ± 3.2
75.61 ± 3.9
57.26 ± 2.5
75.61 ± 3.9
31.72 ± 2.2
31.72 ± 2.2
61.98 ± 3.5
49.55 ± 3.2
10.96 ± 1.1
24.09 ± 1.8
67.53 ± 3.7
9.50 ± 0.8
18.37 ± 1.4
5.57 ± 0.4
55.34 ± 2.8
68.26 ± 3.4
33.86 ± 2.3
40.52 ± 2.5
88.33 ± 3.9
65.35 ± 3.2
88.33 ± 3.9
29.10 ± 2.1
29.10 ± 2.1
59.87 ± 2.7
29.56 ± 3.6
12.97 ± 1.0
17.80 ± 1.3
79.16 ± 3.6
6.49 ± 0.4
24.33 ± 1.9
4.17 ± 0.2
74.67 ± 4.4
58.41 ± 3.5
>100
>100
59.48 ± 3.5
59.48 ± 3.5
95.61 ± 5.1
75.22 ± 4.1
34.60 ± 2.3
52.53 ± 3.3
>100
23.92 ± 1.9
38.02 ± 2.5
8.87 ± 0.6
42.74 ± 2.9
42.74 ± 2.9
86.45 ± 4.5
77.40 ± 3.6
17.42 ± 1.4
36.08 ± 2.5
91.78 ± 4.9
11.93 ± 1.0
22.91 ± 1.6
4.50 ± 0.2
Doxorubicin
IC50 values presented as the mean
been bolded for emphasis.
a
±
SD of three separate determinations. Significant values (<25 µm) have
According to the anti-proliferative activity results, the tested compounds exhibited
variable degrees of activity against the tested cancer cell lines. In general, the activity
against MCF-7 and HeLa seems higher than PC-3, HCT-116, and HePG-2. In addition, the
activity of the piperazinomethyl analogues is higher than their arylaminomethyl analogues.
The optimal activity was attained by compounds 4l, 5a, 5c, and 5d with IC50 < 25 µM
against the tested cell lines.
Within the arylaminomethyl analogues 4a
PC3 cell lines (IC50 > 100 M) or weakly active (IC50 = 51–100
4l, which retained moderate activity (IC50 34.60 M). The anti-proliferative activity of
the arylaminomethyl analogues 4a seems dependent on the aryl substituents, the 2,4-
dichlorophenyl is the optimal substituents while the nitrophenyl substitution at 2- or 4- posi-
tion (compounds 4e ) greatly deteriorated the anti-proliferative activity. The anilinomethyl
analogue 4a, monohalophenylaminomethyl analogues 4b , the trifluoromethylphenyl
aminomethyl analogues 4h,i and the difluorophenylaminomethyl analogues 4j,k ana-
–
l
, the compounds were inactive against
µ
µM) except compounds
µ
–
l
,
g
–
d
logues only retained moderate anti-proliferative activity (IC50 = 26–50 µM) or poor activity
(IC50 = 51–100 µM).
Concerning the anti-proliferative activity of the piperazinomethyl derivatives 5a–d,
the 4-phenylpiperazinomethyl derivative 5a exhibited potent activity against HeLa and
MFC7 cell lines, moderate activity against HCT-116 cell lines and weak activity against PC3
cell lines. Substitution of the phenyl group of compound 5a with 4-fluorophenyl group
(compound 5b) dramatically deteriorated the anti-proliferative activity. The optimum
antiproliferative activity was attained by the 4-benzylpiperazinomethyl derivative 5c
,
which showed potent activity against all the tested cell lines. Replacement of the benzyl

Molecules 2021, 26, 2110
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group of compound 5c with 2-trifluoromethylbenzyl group (compound 5d) reduced the
anti-proliferative activity against the tested cancer cell lines.
3. Materials and Methods
3.1. General Information
Melting points (◦C, uncorrected) were determined in open glass capillaries using
a Barnstead 9100 electro-thermal melting point apparatus. Nuclear magnetic resonance
(NMR) spectra were determined in DMSO-d6 on a JEOL ECA 500 III at 500.16 MHz for
1H and 125.77 MHz for 13C. Elemental analyses (C, H, N, and S) were in agreement with
the proposed structures within
±
0.4% of the theoretical values (Table S1). Monitoring
of the reactions and checking of the purity of the final products were carried out by thin
layer chromatography (TLC) using silica gel pre-coated aluminum sheets (60 F254; Merck)
and visualization with ultraviolet light (UV) at 365 and 254 nm and/or stained with an
anisaldehyde solution and a phosphomolybdic acid solution. All chemicals and solvents
were purchased from Alfa Aesar (Germany), and used without additional purification. The
reference drugs Gentamicin sulfate (CAS 1405-41-0), Ampicillin trihydrate (CAS 7177-48-2),
Clotrimazole (CAS 23593-75-1) and Doxorubicin (CAS 23214-92-8) were purchased from
Sigma-Aldrich Chemie GmbH (Germany). Compound
previously reported procedure [61].
3 was prepared according to the
3.2. Synthesis of 3-(Arylaminomethyl)-5-(3,4-Dimethoxyphenyl)-1,3,4-Oxadiazole-2(3H)-Thiones
4a–l and 3-[(4-Substituted Piperazin-1-yl)Methyl]-5-(3,4-Dimethoxyphenyl}-1,3,4-Oxadiazole-
2(3H)-Thiones 5a–d
The appropriate primary aromatic amine or 1-substituted piperazine (0.01 mole) and
37% formaldehyde solution (1.0 mL) were added to a hot solution of 5-(3,4-dimethoxyphenyl)-
1,3,4-oxadiazole-2(3H)-thione
3 (1.19 g, 5.0 mmole), in ethanol (10 mL), and the mixture
was stirred at room temperature for 5 h and allowed to stand overnight. Water (5 mL) was
then added drop-wisely to the reaction mixture with continuous stirring for one hour. The
separated precipitate was filtered, washed with water, dried, and crystallized.
3-Anilinomethyl-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4a. Fine colorless
1
needle crystals. H NMR:
δ 3.84–3.90 (m, 7H, OCH3 & NH), 6.01–6.09 (m, 2H, CH2), 6.69 (t,
1H, Ar-H, J = 7.0 Hz), 6.91 (d, 2H, Ar-H, J = 8.5 Hz), 7.08–7.33 (m, 5H, Ar-H). 13C NMR:
55.6, 55.8 (OCH3), 57.9 (CH2), 108.4, 112.1, 112.9, 114.0, 117.9, 120.0, 129.1, 145.3, 149.1,
δ
152.4 (Ar-C), 159.0 (Oxadiazole C5), 175.3 (C=S).
3-[(4-Fluorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4b
.
1
Fine colorless needle crystals. H NMR:
δ
3.86–3.90 (m, 7H, OCH3 & NH), 5.49 (d, 2H, CH2,
J = 7.0 Hz), 6.89–6.94 (m, 2H, Ar-H), 6.99–7.06 (m, 2H, Ar-H), 7.21–7.25 (m, 1H, Ar-H), 7.32
(d, 1H, Ar-H, J = 1.5 Hz), 7.51 (d, 1H, Ar-H, J = 1.5 Hz). 13C NMR:
δ
55.7, 55.8 (OCH3), 58.3
(CH2), 108.4, 112.1, 114.0 (d, JC-F = 5.0 Hz), 115.6 (d, JC-F = 23.0 Hz), 119.1 (d, JC-F = 7.0 Hz),
120.1, 141.9, 149.1, 152.4, 155.4 (d, JC-F = 232.5 Hz) (Ar-C), 159.0 (Oxadiazole C5), 175.4
(C=S).
3-[(3-Chlorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4c
.
1
Fine colorless needle crystals. H NMR:
δ
3.87–3.88 (m, 7H, OCH3 & NH), 5.50 (d, 2H, CH2,
J = 7.0 Hz), 6.73 (d, 1H, Ar-H, J = 1.5 Hz), 6.87 (d, 1H, Ar-H, J = 1.5 Hz), 7.02–7.05 (m, 1H,
Ar-H), 7.17–7.19 (m, 1H, Ar-H), 7.33 (d, 1H, Ar-H, J = 1.5 Hz), 7.51 (d, 1H, Ar-H, J = 1.5 Hz),
7.55 (t, 1 H, Ar-H, J = 7.0 Hz). 13C NMR:
δ 55.6, 55.8 (OCH3), 57.5 (CH2), 108.4, 111.8, 112.1,
112.5, 113.9, 117.5, 120.1, 130.7, 133.8, 147.1, 149.1, 152.4 (Ar-C), 159.1 (Oxadiazole C5), 175.4
(C=S).
3-[(4-Chlorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4d
.
1
Fine colorless needle crystals. H NMR:
δ
3.87–3.88 (s, 7H, OCH3 & NH), 5.49 (d, 2H, CH2,
J = 7.0 Hz), 6.93 (d, 2H, Ar-H, J = 9.0 Hz), 7.20–7.22 (m, 2H, Ar-H), 7.32 (d, 1 H, Ar-H,
J = 1.5 Hz), 7.42 (d, 1 H, Ar-H, J = 8.0 Hz), 7.50 (d, 1 H, Ar-H, J = 1.5, 8.0 Hz). 13C NMR:
δ
55.7, 55.8 (OCH3), 57.8 (CH2), 108.4, 112.1, 113.9, 114.5, 120.1, 121.5, 128.8, 144.4, 149.1,
152.4, 159.1 (Oxadiazole C5), 175.4 (C=S).

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3-[(2-Nitrophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4e
.
1
Pale yellow block crystals. H NMR:
δ
3.85–3.89 (m, 7H, OCH3 & NH), 5.77 (d, 2H, CH2,
J = 7.0 Hz), 6.89–6.95 (m, 1H, Ar-H), 7.16–7.21 (m, 1H, Ar-H), 7.44–7.48 (m, 1H, Ar-H),
7.49–7.54 (m, 1H, Ar-H), 7.65–7.70 (m, 1H, Ar-H), 8.16 (d, 1H, Ar-H, J = 1.5 Hz), 8.85–8.91
(m, 1H, Ar-H). 13C NMR:
δ 55.7, 55.8 (OCH3), 56.6 (CH2), 108.5, 112.1, 115.4, 117.9, 119.8,
120.0, 125.4, 126.5, 136.6, 142.2, 149.2, 152.5 (Ar-C), 159.3 (Oxadiazole C5), 175.5 (C=S).
3-[(3-Nitrophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4f.
1
Pale yellow block crystals. H NMR:
δ
3.86–3.90 (m, 7H, OCH3 & NH), 5.59 (d, 2H, CH2,
J = 7.0 Hz), 7.19 (d, 1H, Ar-H, J = 9.0 Hz), 7.32–7.36 (m, 1H, Ar-H), 7.45 (t, 1 H, Ar-H,
J = 8.0 Hz), 7.51 (d, 1H, Ar-H, J = 1.5 Hz), 7.55 (d, 1H, Ar-H, J = 1.5 Hz), 7.87–7.94 (m, 2H,
Ar-H). 13C NMR:
δ 55.6, 55.8 (OCH3), 57.4 (CH2), 106.9, 108.4, 112.0, 112.4, 113.9, 119.6,
120.1, 130.2, 146.9, 148.8, 149.1, 152.4 (Ar-C), 159.0 (Oxadiazole C5), 175.5 (C=S).
3-[(4-Nitrophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione 4g
.
1
Pale yellow block crystals. H NMR:
δ
3.86–3.90 (m, 7H, OCH3 & NH), 5.61 (d, 2H, CH2,
J = 7.0 Hz), 7.07 (d, 2H, Ar-H, J = 9.0 Hz), 7.19 (d, 1H, Ar-H, J = 8.5 Hz), 7.33 (d, 1H, Ar-H,
J = 2.0 Hz), 7.52 (d, 1H, Ar-H, J = 2.0 Hz), 8.12 (d, 2H, Ar-H, J = 9.0 Hz). 13C NMR:
δ
55.7,
55.8 (OCH3), 56.7 (CH2), 108.5, 112.1, 112.5, 113.9, 120.2, 126.0, 138.1, 149.1, 152.1, 152.5
(Ar-C), 159.2 (Oxadiazole C5), 175.5 (C=S).
3-[(2-Trifluoromethylphenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-
1
thione 4h. Colorless prism crystals. H NMR:
δ
3.86–3.90 (m, 7H, OCH3 & NH), 5.61 (d, 2H,
CH2, J = 6.5 Hz), 6.89 (t, 2H, Ar-H, J = 8.0 Hz), 7.24 (d, 2H, Ar-H, J = 9.0 Hz), 7.31–7.37 (m,
2H, Ar-H), 7.50–7.52 (m, 2H, Ar-H). 13C NMR:
δ
55.7, 55.8 (OCH3), 57.3 (CH2), 108.5, 112.0,
114.6, 115.0, 116.7, 117.8, 119.8, 120.1, 126.6, 133.0, 133.7, 149.1, 152.1 (Ar-C & CF3), 160.6
(Oxadiazole C5), 177.2 (C=S).
3-[(3-Trifluoromethylphenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-
1
thione 4i. White amorphous powder. H NMR:
δ
3.86–3.90 (m, 7H, OCH3 & NH), 5.56 (d,
2H, CH2, J = 7.5 Hz), 7.02 (d, 1H, Ar-H, J = 7.5 Hz), 7.17–7.21 (m, 1H, Ar-H), 7.31–7.34
(m, 2H, Ar-H), 7.37–7.42 (m, 1H, Ar-H), 7.51 (d, 1H, Ar-H, J = 1.5 Hz), 7.71 (t, 1 H, Ar-H,
J = 7. Hz). 13C NMR:
δ 55.6, 55.8 (OCH3), 57.4 (CH2), 108.3, 109.0, 112.0, 113.9, 115.4, 117.0,
120.1, 123.3, 125.5, 130.1, 146.2, 149.1, 152.4 (Ar-C & CF3), 159.0 (Oxadiazole C5), 175.5
(C=S).
3-[(2,4-Difluorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
4j. Colorless needle crystals. 1H NMR:
δ
3.86-3.90 (m, 7H, OCH3 & NH), 5.52 (d, 2H,
CH2, J = 7.0 Hz), 6.95–7.04 (m, 2H, Ar-H), 7.17–7.21 (m, 2H, Ar-H), 7.32 (d, 1H, Ar-H,
J = 1.5 Hz), 7.49–7.52 (m, 1H, Ar-H). 13C NMR:
55.6, 55.8 (OCH3), 57.9 (CH2), 104.0 (d,
C-F = 23.05 Hz), 108.4, 111.0 (d, JC-F = 19.0 Hz), 112.0, 113.8 (d, JC-F = 8.5 Hz), 113.9, 120.1,
δ
J
130.2 (d, JC-F = 13.0 Hz), 149.1, 152.4, 154.3 (d, JC-F = 225.5 Hz), 159.7 d, JC-F = 237.5 Hz)
(Ar-C), 159.2 (Oxadiazole C5), 175.4 (C=S).
3-[(2,5-Difluorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
1
4k. Colorless needle crystals. H NMR:
δ 3.86–3.90 (m, 7H, OCH3 & NH), 5.51 (d, 2H, CH2,
J = 6.5 Hz), 7.02–7.07 (m, 1H, Ar-H), 7.12–7.16 (m, 1H, Ar-H), 7.18–7.21 (m, 1H, Ar-H), 7.33
(d, 1H, Ar-H, J = 1.5 Hz), 7.39–7.44 (m, 1H, Ar-H), 7.49–7.53 (m, 1H, Ar-H). 13C NMR:
δ
55.7, 55.8 (OCH3), 57.1 (CH2), 100.5 (d, JC-F = 27.5 Hz), 103.2 (d, JC-F = 7.0 Hz), 108.4, 111.9,
112.0, 114.6, 115.6 (d, JC-F = 11.0 Hz), 119.8, 120.1, 149.1, 152.1, 159.0 (d, JC-F = 241.0 Hz)
(Ar-C), 159.7 (Oxadiazole C5), 175.5 (C=S).
3-[(2,4-Dichlorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thione
1
4l. Fine colorless needle crystals. H NMR:
δ
3.86–3.90 (m, 7H, OCH3 & NH), 5.58 (d, 2H,
CH2, J = 7.0 Hz), 7.04–7.08 (m, 1H, Ar-H), 7.16–7.20 (m, 1H, Ar-H), 7.28–7.33 (m, 2H, Ar-H),
7.47–7.52 (m, 2H, Ar-H). 13C NMR:
δ
55.7, 55.8 (OCH3), 57.4 (CH2), 108.5, 112.0, 113.8,
113.9, 119.1, 120.1, 121.7, 127.9, 128.8, 140.5, 149.1, 152.4 (Ar-C), 159.2 (Oxadiazole C5), 175.4
(C=S).
5-(3,4-Dimethoxyphenyl)-3-[(4-phenylpiperazin-1-yl)methyl]-1,3,4-oxadiazole-2(3H)-thione
1
5a. Fine colorless needle crystals. H NMR:
δ
2.89-2.99 (m, 4H, Piperazine-H), 3.13–3.23
(m, 4H, Piperazine-H), 3.89 (s, 6H, OCH3), 5.13 (s, 2H, CH2), 6.80 (t, 1H, Ar-H, J = 7.0 Hz),

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6.96 (d, 2H, Ar-H, J = 8.0 Hz), 7.18–7.26 (m, 3H, Ar-H), 7.36–7.39 (m, 1H, Ar-H), 7.55 (d,
1H, Ar-H, J = 8.0 Hz). 13C NMR: δ 48.3, 49.6 (Piperazine-C), 55.7, 55.8 (OCH3), 69.7 (CH2),
108.5, 112.0, 114.1, 115.7, 119.0, 120.1, 128.9, 149.1, 151.0, 152.3 (Ar-C), 158.7 (Oxadiazole
C5), 177.2 (C=S).
5-(3,4-Dimethoxyphenyl)-3-{4-[(4-fluorophenyl)piperazin-1-yl]methyl}-1,3,4-oxadiazole-2(3H)-
1
thione 5b. Colorless needle crystals. H NMR:
δ
2.92–2.95 (m, 4H, Piperazine-H), 3.09–3.16
(m, 4H, Piperazine-H), 3.89 (s, 6H, OCH3), 5.12 (s, 2H, CH2), 6.96–6.98 (m, 2H, Ar-H),
7.05-7.07 (m, 1H, Ar-H), 7.08-7.19 (m, 1H, Ar-H), 7.21 (d, 1H, Ar-H, J = 1.5 Hz), 7.37 (d, 2H,
Ar-H, J = 8.0 Hz). 13C NMR:
δ 49.1, 49.6 (Piperazine-C), 55.7, 55.8 (OCH3), 69.7 (CH2), 108.5,
112.0, 114.2, 115.3 (d, JC-F = 21.5 Hz), 117.4 (d, JC-F = 7.0 Hz), 120.1, 147.0, 149.1, 152.3, 156.1
(d, JC-F = 236.5 Hz) (Ar-C),158.6 (Oxadiazole C5), 177.2 (C=S).
5-(3,4-Dimethoxyphenyl)-3-[(4-benzylpiperazin-1-yl)methyl]-1,3,4-oxadiazole-2(3H)-thione 5c
.
1
White amorphous powder. H NMR:
δ
2.30–2.50 (m, 4H, Piperazine-H), 2.74–2.88 (m, 4H,
Piperazine-H), 3.50 (s, 2H, Benzylic CH2), 3.89 (s, 6H, OCH3), 5.05 (s, 2H, CH2), 7.19 (d, 1H,
Ar-H, J = 9.0 Hz), 7.24–7.41 (m, 6H, Ar-H), 7.53 (d, 1H, Ar-H, J = 8.0 Hz). 13C NMR:
δ
49.5,
52.3 (Piperazine-C), 55.7, 55.8 (OCH3), 61.9 (Benzylic CH2), 69.7 (CH2), 108.5, 112.0, 114.3,
120.0, 127.0, 128.1, 128.8, 137.8, 149.1, 152.2 (Ar-C), 158.6 (Oxadiazole C5), 177.2 (C=S).
5-(3,4-Dimethoxyphenyl)-3-{4-[(2-trifluorobenzyl)piperazin-1-yl]methyl}-1,3,4-oxadiazole-2(3H)-
1
thione 5d. Colorless needle crystals. H NMR:
δ
2.38–2.52 (m, 4H, Piperazine-H), 2.78–2.90
(m, 4H, Piperazine-H), 3.64 (s, 2H, Benzylic CH2), 3.89 (s, 6H, OCH3), 5.06 (s, 2H, CH2),
7.20 (d, 1H, Ar-H, J = 8.0 Hz), 7.37 (d, 1H, Ar-H, J = 1.5 Hz), 7.47 (t, 1H, Ar-H, J = 7.0 Hz),
7.55 (d, 1H, Ar-H, J = 8.0 Hz), 7.64 (t, 1H, Ar-H, J = 8.0 Hz), 7.71 (d, 1H, Ar-H, J = 8.0 Hz),
7.76 (d, 1H, Ar-H, J = 8.0 Hz). 13C NMR:
δ 49.7, 52.6 (Piperazine-C), 55.7, 55.8 (OCH3), 57.5
(Benzylic CH2), 69.8 (CH2), 108.5, 112.0, 114.2, 118.5, 120.1, 123.4, 125.7, 127.3, 130.3, 132.4,
137.2, 149.1, 152.3 (Ar-C & CF3), 158.6 (Oxadiazole C5), 177.2 (C=S).
4. Conclusions
Sixteen 1,3,4-oxadiazole-linked N-Mannich bases namely; 3-arylaminomethyl-5-(3,4-
dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 4a
yl)-methyl]-5-(3,4-dimethoxyphenyl)-1,3,4-oxadiazole-2(3H)-thiones 5a
sized and their structures were confirmed by 1H NMR, 13C NMR and elemental analysis.
The in vitro inhibitory activity of compounds 4a and 5a was assessed against a panel
–l
and 3-[(4-substituted piperazin-1-
–
d
were synthe-
–l
–d
of standard pathogenic Gram-positive bacteria, Gram-negative bacteria, and the yeast-
like pathogenic fungus Candida albicans. Compounds 5c and 5d displayed potent broad
spectrum antibacterial activities and compounds 4j, 4l, 5a, and 5b showed potent activity
against the tested Gram-positive bacteria. The anti-proliferative activity of the compounds
was evaluated against prostate cancer (PC3), human colorectal cancer (HCT-116), human
hepatocellular carcinoma (HePG-2), human epithelioid carcinoma (HeLa), and human
breast cancer (MCF7) cell lines. Compounds 4l, 5a, 5c, and 5d showed potent inhibition of
cell proliferation in almost all the tested cancer cell lines. The prepared 1,3,4-oxadiazole-
linked N-Mannich bases could be considered good antibacterial and anticancer drug
candidates. The biological testing results are considered as preliminary and further investi-
gations, including experimental and theoretical investigations for the exploration of their
targets, are required for optimization of their chemotherapeutic activities.
Supplementary Materials: The micro-analytical data (C, H, N, and S), the experimental details of
the determination of in vitro antimicrobial activity, in vitro anti-proliferative activity, and the NMR
spectra can be found online.
Author Contributions: Conceptualization, A.A.E.-E. and A.A.B.M.; methodology, L.H.A.-W., S.S.T.,
and H.M.H.; validation, H.M.H..; formal analysis, L.H.A.-W., A.A.B.M., and S.S.T.; investigation,
L.H.A.-W. and H.M.H.; data curation, H.M.H. and S.S.T.; writing—original draft preparation, A.A.E.-
E.; writing—review and editing, A.A.E.-E. and A.A.B.M.; supervision, L.H.A.-W., A.A.E.-E.; project
administration, A.A.E.-E.; funding acquisition, L.H.A.-W. All authors have read and agreed to the
published version of the manuscript.

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Funding: This research was funded by the Deanship of Scientific Research at Princess Nourah bint
Abdulrahman University through the Research Groups Program (Grant No. RGP-1442-0010-4).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 4a–l and 5a–d are available from the correspond-
ing author.
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