Synthesis, characterization and antimicrobial evaluation of some new schiff, mannich and acetylenic mannich bases incorporating a 1,2,4-triazole nucleus

Article abstract of DOI:10.3390/molecules191118897

A series of Schiff and Mannich bases derived from 4-amino-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione were synthesized. The alkylation of 4-phenyl-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with propargyl bromide afforded the corresponding thiopropargylated derivative which upon treatment with the appropriate secondary amines in the presence of CuCl2 furnished the desired acetylenic Mannich bases. The synthesized compounds were characterized on the basis of their spectral (IR, 1H- and 13C-NMR) data and evaluated for their biological activities. Some of the compounds were found to exhibit significant antimicrobial activity.

Full text of DOI:10.3390/molecules191118897

Molecules 2014, 19, 18897-18910; doi:10.3390/molecules191118897
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Characterization and Antimicrobial Evaluation of
Some New Schiff, Mannich and Acetylenic Mannich Bases
Incorporating a 1,2,4-Triazole Nucleus
Mohamed R. Aouad 1,2
1
Department of Chemistry, Faculty of Sciences, Taibah University, Al-Madinah Al-Munawarah 30002,
Saudi Arabia; E-Mail: mr_aouad@yahoo.fr; Tel.: +966-540-953-573
2
Laboratoire de Chimie & Electrochimie des Complexes Métalliques (LCECM) USTO-MB,
Department of Chemistry, Faculty of Sciences, University of Sciences and Technology Mohamed
Boudiaf, B.p. 1505 El M`nouar, Oran 31000, Algeria
External Editor: Derek J. McPhee
Received: 31 August 2014; in revised form: 29 October 2014 / Accepted: 3 November 2014 /
Published: 18 November 2014
Abstract: A series of Schiff and Mannich bases derived from 4-amino-5-(3-fluoro-
phenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione were synthesized. The alkylation of
4-phenyl-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with propargyl bromide
afforded the corresponding thiopropargylated derivative which upon treatment with the
appropriate secondary amines in the presence of CuCl2 furnished the desired acetylenic
Mannich bases. The synthesized compounds were characterized on the basis of their
13
spectral (IR, 1H- and C-NMR) data and evaluated for their biological activities. Some of
the compounds were found to exhibit significant antimicrobial activity.
Keywords: Schiff bases; Mannich bases; acetylenic Mannich bases; 1,2,4-triazole;
antimicrobial activity
1. Introduction
The 1,2,4-triazole core is considered a privileged fragment in modern heterocyclic chemistry
principally due to its incorporation into a wide variety of drugs such as fluconazole, itraconazole,
ribavirine, alprazolam and rizatriptan [1–4]. Moreover, some fluorinated 1,2,4-triazoles including
flusilazole, fluotrimazole, epoxiconazole and flutriafol are reported to be effective fungicides [5,6].

Molecules 2014, 19
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The presence of fluorine in organic molecules often results in unexpected biological activity, which is
rationalized as being due to their higher lipholicity which enhances the rate of penetration and
transport of the drug to an active site [7,8]. Furthermore, the incorporation of a Schiff base moiety
within the 1,2,4-triazole ring gave compounds with enhanced biological activities [9,10]. On the other
hand, Mannich bases of 1,2,4-triazoles have gained importance due to their biological properties such
as anticancer, antifungal, anti-inflammatory and antimalarial activities [11–15]. Among these, some
1,2,4-triazole Mannich bases incorporating N-methylpiperazine or morpholine moieties were
recently found to possess antimicrobial activity [16–18]. In view of these facts and in an attempt to
design and synthesize some novel fluorine-containing 1,2,4-triazoles with improved biological activity,
a new series of Schiff, Mannich and acetylenic Mannich bases bearing fluorophenyl-1,2,4-triazole
moieties were synthesized with a view to explore their potential as better antibacterial and
antifungal agents.
2. Results and Discussion
2.1. Chemistry
The reaction sequences employed for the synthesis of title compounds are shown in Schemes 1–3.
The key intermediate 4-amino-5-(3-fluorophenyl)-2H-1,2,4-triazole-3-thione (1) was synthesized
in 85% yield by the fusion of 3-fluorobenzoic acid with thiocarbohydrazide for 20–25 min
(Scheme 1). The resulting triazole 1 was identical to that previously obtained via multi-steps
synthesis [19].
Scheme 1. Synthesis of 4-amino-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1).
H
N N
N N
F
COOH
F
F
Fusion
SH
S
NH2NHCSNHNH2
N
N
+
NH2
NH2
1
Scheme 2. Synthesis of Schiff Bases 2a–2b.
H
H
N N
N N
F
F
EtOH/HCl
S
ArCHO
S
+
N
N
N
H
NH2
Ar
1
2a Ar = 4F-C6H4
2b Ar = 3,4-diF-C6H3

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1
13
The structure of the triazole 1 was confirmed by IR, H-NMR, C-NMR, and elemental analysis.
In the IR spectrum of compound 1, the NH group of the triazole ring was observed at 3227–3373 cm−1,
thus confirming the formation of the aminotriazole. The absorption band observed at 1284 cm−1 could
be attributed to the C=S group. The 1H-NMR spectrum of compound 1 showed two singlets at δH 5.82
and 13.80 ppm and a multiplet at δH 7.32–7.93 ppm corresponding to NH2 and NH protons, as well as
the phenyl protons, respectively. The condensation of the aminotriazole 1 with 4-fluorobenzaldehyde
and/or 3,4-difluorobenzaldehyde in the presence of a catalytic amount of hydrochloric acid gave Schiff
bases 2a and 2b in good yields (Scheme 2).
All Schiff bases displayed IR, 1H- and 13C-NMR absorptions and elemental analyses consistent with
the assigned structures. In the IR spectra of compounds 2a and 2b, the most characteristic absorptions
were observed at 3288–3315 cm−1 (N–H), 1604–1614 cm−1 (C=N) and 1280–1298 cm−1 (C=S). Lack
of resonances attributable to NH2 protons and the appearance of a sharp H-C=N group singlet at δH
9.79–9.89 in their 1H-NMR spectra agreed with the formation of Schiff bases. The 13C-NMR signals at
δc 160.55–161.86 ppm were due to the azomethine-carbon. Moreover, the C=S group resonated at δC
164.34–165.91 ppm, thus confirming the presence of Schiff bases 2a and 2b in the thione form.
Scheme 3. Synthesis of Mannich Bases 3a–3f, 4a–4b, and 5a–5b.
N
Y
N
N
F
H
N
Y
S
N
N
H
Ar
3a Ar = 4-F-C6H4, Y = O
3b Ar = 3,4-diF-C6H3, Y = O
3c Ar = 4-F-C6H4, Y = CH2
3d Ar = 3,4-diF-C6H3, Y = CH2
3e Ar = 4-F-C6H4, Y = NCH3
3f Ar = 3,4-diF-C6H3, NCH3
H
N
N
EtOH, HCHO
F
S
N
N
H
Ar
N
N
N
N
N N
H
N
N H
2a Ar = 4-F-C6H4
2b Ar = 3,4-diF-C6H3
F
F
S
S
N
N
N
N
H
H
Ar
Ar
4a Ar = 4-F-C6H4
4b Ar = 3,4-diF-C6H3
H3C
H3C
N
N
N
N
N N
H
N
N H
F
F
S
S
N
N
N
N
H
H
Ar
Ar
5a Ar = 4-F-C6H4
5b Ar = 3,4-diF-C6H3
Mannich reactions on 3,4,5-trisubstituted-1,2,4-triazole which exist as thiol-thione tautomers
gave the new Mannich bases 3a–3f, 4a–4b, and 5a–5b via aminomethylation of the endocyclic
nitrogen (N-2) of the triazole ring with formaldehyde and the appropriate secondary amine in ethanol
(Scheme 3).The structural assignments of Mannich bases 3a–3f, 4a–4b and 5a–5b were based on their

Molecules 2014, 19
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1
1
elemental analysis and spectral (IR, H-NMR and 13C-NMR) data. In the H-NMR spectra of
compounds 3a–3f, the N-CH2-N protons resonated as singlet at δH 5.14–5.30 ppm integrating
for two protons. The -CH2-O-CH2 protons corresponding to the morpholine ring resonated as a triplet
at δH 3.59 ppm (J = 4.4 Hz) in 3a and at 3.65 ppm (J = 4.6 Hz) in 3b, while the -CH2-N-CH2-
protons of the morpholine ring resonated as a triplet at δH 2.77 ppm (J = 4.4 Hz) and 2.82 ppm
(J = 4.6 Hz), respectively. The methyl protons of 3e and 3f appeared as singlets at δH 2.12 and
2.16 ppm, respectively.
1
In addition, the H-NMR spectrum of compound 4a showed a characteristic singlet integrating for
four protons at δH 5.19 ppm attributed to two N-CH2-N groups (Figure 1) which appeared as a
multiplet at δH 5.11–5.30 in the 1H-NMR spectrum of compound 5b (Figure 2).
Figure 1. 1H-NMR spectrum of compound 4a.
Figure 2. 1H-NMR spectrum of compound 5b.

Molecules 2014, 19
18901
The 13C-NMR spectra of Mannich bases 3a–3f, 4a–4b, and 5a–5b showed signals at
δc 160.53–166.15 and 164.89–166.64 ppm characteristic for the heterocyclic C(3) and C(5) carbons
and at δC 68.60–70.21 ppm due to N-CH2-N.
The commercially available 5-(3-fluorophenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8)
[CAS Registry Number: 330646-49-6] was prepared starting from 3-fluorobenzoic acid hydrazide (6)
as outlined in Scheme 4.
Scheme 4. Synthesis of 5-(3-fluorophenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8).
H
N N
F
F
CONHNH2
F
CONHNHCSNHPh
EtOH/ PhNCS
10% NaOH
S
N
Ph
6
7
8
The reaction sequences employed for the synthesis of the acetylenic Mannich bases are shown in
Scheme 5. Thus, the alkylation of compound 8 with propargyl bromide in the presence of potassium
carbonate as base gave 5-(3-fluorophenyl)-4-phenyl-1,2,4-triazole-3-thio(prop-2-yne) (9) in good yield.
Scheme 5. Synthesis of Acetylenic Mannich Bases 10a–10c.
H
N
N
N N
F
F
BrCH2
DMF, K2CO3
C
CH
SCH2
S
C CH
N
N
Dioxane, HCHO
CuSO4
Ph
Ph
8
9
H
N
Y
N
N
F
SCH2
C
C
N
N
Ph
Y
10a Y = O
10b Y = CH2
10c Y = NCH3
The disappearance of the NH stretch at 3345 cm−1 in the IR spectrum of compound 9 and
appearance of the characteristic C≡C and ≡C-H bands at 2145 and 3290 cm−1, respectively, confirmed
1
the formation of thiopropargylated triazole 9. In the H-NMR spectrum of compound 9, a triplet
corresponding to the ≡C-H group was observed at δH 2.26 ppm and a doublet at δH 4.01 ppm
13
integrating for two protons of SCH2 group and a peak at δC 79.85 ppm due to C≡C in the C-NMR
spectrum confirmed the formation of compound 9. The IR, 1H-NMR, 13C-NMR and elemental analysis
data of compound 9 was in agreement with the assigned structure. The acetylenic Mannich bases
10a–10c were synthesized in one pot multi-component Mannich reaction involving the
thiopropargylated triazole 9, formaldehyde, CuSO4 and the appropriate secondary amine in refluxing
dioxane. The structures of the newly synthesized acetylenic Mannich bases 10a–10c have been
1
13
established on the basis of their elemental analysis, IR, H-NMR, and C-NMR data. The IR spectra
of compounds 10a–10c showed characteristic C≡C group bands at 2148–2152 cm−1. In addition, their

Molecules 2014, 19
18902
1H-NMR spectra showed two singlets at δH 3.43–3.46 and 4.01–4.05 ppm characteristic for C-CH2-N
1
and SCH2 groups, respectively. The H-NMR spectrum of compound 10a gave signals at δH 2.81 and
3.62 ppm characteristic for NCH2 and OCH2 morpholine protons, respectively.
1
Moreover, the H-NMR spectrum of compound 10c gave a characteristic singlet at δH 2.24 ppm
integrating for three protons of NCH3 group which resonated at 42.35 ppm in its 13C-NMR spectrum.
The 13C-NMR spectra of all acetylenic Mannich bases 10a–10c showed characteristic signals at
22.60–22.97, 60.29–61.11 and 77.87–78.96 ppm due to SCH2, C-CH2-N and C≡C groups,
respectively.
2.2. Antibacterial and Antifungal Activity
Both antimicrobial studies were assessed by minimum inhibitory concentration (MIC) assays
carried out by the broth dilution method [20–22]. MIC is the highest dilution of a compound which
shows clear fluid with no development of turbidity.
The antibacterial and antifungal screening revealed that some of the tested compounds showed good
to excellent activity at 4–62.5 μg/mL in DMSO. 4-(4-Fluorobenzylideneamino)-5-(3-fluorophenyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (2a) and 4-(3,4-difluorobenzylideneamino)-5-(3-fluorophenyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (2b) showed comparatively good activity against Gram positive
bacterial strains at MIC 16–31.25 μg/mL and excellent activity towards fungal strains at MIC 4–8
μg/mL. The Mannich bases 3a and 3b bearing a morpholine moiety showed excellent antibacterial
activity against all bacterial strains and good activity against fungal species at 16–31.25 μg/mL. On the
contrary, compounds 3c and 3d possessing a piperidine exhibited good to moderate antibacterial
activity but lost the activity against the tested fungal species.
On the other hand, Mannich bases 3a–3f, 4a–4b, and 5a–5b incorporating piperazine moieties
showed excellent and greater antibacterial activity at MIC 4–16 μg/mL than antifungal action.
Furthermore, 5-(3-fluorophenyl)-4-phenyl-1,2,4-triazole-3-thio(prop-2-ynyl) (9) exhibited moderate
activity with a MIC value of 16–62.5 μg/mL. Evaluation of the antibacterial activity of the acetylenic
Mannich bases 10a–10c, revealed these compounds to be more effective against Gram positive
bacteria at MIC 8 to 31.25 μg/mL. Particularly, acetylenic Mannich bases carrying a morpholine
10a and/or a piperazine 10c moiety exhibited excellent inhibition at MIC 8–16 μg/mL against
Gram positive bacteria. Antifungal screening of all acetylenic Mannich bases revealed that
compounds 10a and 10c showed excellent antifungal activity against all tested fungal strains at MIC
8–31.5 μg/mL.
The remaining compounds were found to be active at higher concentrations, e.g., 62.5 and 125 mg/mL.
It was therefore concluded that the presence of a morpholine and/or piperazine moiety, in addition to
3-fluorophenyl groups, was essential for high antibacterial and antifungal activities in these
compounds. The results of antibacterial and antifungal screening of the newly prepared Schiff,
Mannich and acetylenic Mannich bases, expressed as MIC values, are summarized in Table 1.

Molecules 2014, 19
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Table 1. Antimicrobial activity expressed as MIC (μg/mL).
Gram-Positive
Gram-Negative
Organisms b
Ec
Fungi c
Organisms a
Compounds
Sp
16
16
8
Bs
31.25
16
Sa
Pa
125
31.25
8
Kp
Af
8
Ca
4
2a
16
31.25
62.5
8
62.5
31.25
4
2b
16
4
4
3a
4
8
16
31.25
16
3b
8
4
4
8
4
4
16
3c
31.25
62.5
4
16
31.25
8
31.25
62.5
16
31.25
16
8
16
125
125
31.25
16
250
250
16
3d
16
16
3e
4
8
4
3f
4
8
8
8
4
8
16
4a
8
8
4
8
4
16
31.25
31.25
62.5
31.25
125
8
16
4b
16
8
4
4
8
4
8
31.25
16
5a
16
16
62.5
8
4
16
4
8
5b
4
4
31.25
8
8
4
16
16
9
10a
16
16
16
8
16
16
62.5
125
62.5
≤1
-
31.25
31.25
62.5
31.25
≤1
250
16
62.5
31.25
31.25
≤5
-
10b
31.25
16
≤1
-
31.25
8
31.25
8
62.5
31.25
-
10c
Ciprofloxacin
≤5
-
≤5
-
Fluconazole
-
-
≤1
≤1
a
Notes: : Gram-positive bacteria: Streptococcus pneumonia (RCMB 010010, Sp), Bacillus subtilis (RCMB
010067, Bs), Staphylococcus aureus (RCMB 010025, Sa); b: Gram-negative bacteria: Pseudomonas
aeuroginosa (RCMB 010043, Pa), Escherichia coli (RCMB 010052, Ec), Klebsiella pneumonia (RCMB
010058, Kp); c: yeasts: Aspergillus fumigatus (RCMB 02568, Af), Candida albicans (RCMB 05036, Ca).
3. Experimental Section
3.1. General Information
1
Melting points were determined on a Melt-temp apparatus and are uncorrected. The H- and
13C-NMR spectra were recorded on a Bruker AC-400 NMR spectrometer operating at 400 MHz for
13
1H-NMR, 100 MHz for C-NMR. Compounds were dissolved in DMSO-d6 and chemical shifts were
13
referenced to TMS (1H- and C-NMR). The IR spectra were measured as potassium bromide pellets
using a Perkin-Elmer 1430 series FT-IR spectrometer. The elemental analyses were performed by the
microanalysis unit at the Faculty of Science, Cairo University.
3.2. Synthesis of 4-Amino-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1)
A mixture of 3-fluorobenzoic acid (1, 0.01 mol) and thiocarbohydrazide (0.015 mol) was heated on
a mantle in a round-bottomed flask until the contents melted. The mixture was maintained at this
temperature for 15–20 min. The product obtained on cooling was treated with sodium bicarbonate
solution to neutralize the unreacted carboxylic acid if any. The solid mass was then washed with water
and collected by filtration. The product was recrystallized from a mixture of dimethylformamide and

Molecules 2014, 19
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ethanol yielded 81% of the desired compound as white crystals: m.p. 221–222 °C, Lit m.p. 220 °C [19].
IR (υ, cm−1): 3227–3373 (NH, NH2), 3070 (Ar-H), 1617 (C=N), 1284 (C=S). 1H-NMR: δ 5.82 (s, 2H,
NH2), 7.32–7.93 (m, 4H, ArH), 13.80 (s, 1H, NH triazole). 13C-NMR: δ 114.64, 114.88, 117.23,
117.44, 124.04, 127.64, 130.71, 148.23, 160.51, 162.93, 167.17 (ArC, C=N). Anal. Calcd. for
C8H7FN4S: C 45.70, H 3.36, N 26.65. Found: C 45.92, H 3.13, N 26.40.
3.3. General Procedure for the Synthesis of Schiff Bases 2a–2b
A mixture of compound 1 (10 mmol) and the appropriate benzaldehyde derivative (10 mmol) was
refluxed in ethanol (30 mL) containing HCl (1 mL) for 6 h. The solution was cooled and a yellow solid
appeared. The obtained precipitate was filtered and recrystallized from ethanol to afford the
desired product.
4-(4-Fluorobenzylideneamino)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2a). This
compound was obtained as colorless crystals, Yield 89%; m.p. 167–168 °C. IR (υ, cm−1): 3,288 (NH),
1
3,085 (Ar-H), 1,604 (C=N), 1,298 (C=S). H-NMR: δ 7.33–7.84 (m, 8H, ArH), 9.89 (s, 1H, H-C=N),
14.39 (s, 1H, NH triazole). 13C-NMR: δ 116.30, 116.52, 124.89, 127.41, 128.50, 128.53, 129.70,
131.00, 131.09, 132.65, 132.80, 133.21, 147.54, 161.86, 163.41, 163.93, 165.91 (ArC, C=N). Anal.
Calcd. for C15H10F2N4S: C 56.95, H 3.19, N 17.71. Found: C 56.78, H 3.36, N 17.43.
4-(3,4-Difluorobenzylideneamino)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(2b).
This compound was obtained as colorless crystals, Yield 87%; m.p. 195–196 °C. IR (υ, cm−1): 3,315
1
(NH), 3,064 (Ar-H), 1,614 (C=N), 1,280 (C=S). H-NMR: δ 7.42–8.01 (m, 7H, ArH), 9.79 (s, 1H,
H-C=N), 14.40 (s, 1H, NH triazole). 13C-NMR: δ 114.93, 115.18, 116.97, 117.15, 117.60, 117.81,
118.58, 118.75, 124.48, 124.50, 126.68, 127.24, 127.32, 129.54, 130.97, 131.05, 147.43, 151.20,
160.55, 162.45, 162.97, 164.34 (ArC, C=N). Anal. Calcd. for C15H9F3N4S: C 53.89, H 2.71, N 16.76.
Found: C 54,13, H 2.50, N 16.93.
3.4. General Procedure for the Synthesis of Mannich Bases
A solution of Schiff base 2a and/or 2b (10 mmol), formaldehyde (40%, 1.5 mL) and the appropriate
secondary amine (10 mmol) in ethanol (25 mL) was stirred for 2 h and left overnight at room
temperature. The solid mass thus separated was collected by filtration, dried and recrystallized from
ethanol/DMF.
4-(4-Fluorobenzylideneamino)-2-(morpholin-4-ylmethyl)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3a). This compound was obtained as colorless crystals, Yield: 87%; m.p.
230–231 °C. IR (υ, cm−1): 3,046 (Ar-H), 2,826–2,971 (CH str.), 1,608 (C=N), 1,284 (C=S). 1H-NMR:
δ 2.77 (t, 4H, J = 4.4 Hz, N-CH2), 3.59 (t, 4H, J = 4.4 Hz, OCH2), 5.21 (s, 2H, N-CH2-N),
7.32–7.86 (m, 8H, ArH), 9.81 (s, 1H, H-C=N). 13C-NMR: δ 50.32 (NCH2), 66.05 (OCH2),
69.06 (N-CH2-N), 116.33, 116.55, 124.48, 127.45, 128.36, 128.39, 129.75, 131.14, 131.23, 132.63,
132.97, 133.23, 146.16, 162.75, 163.52, 165.09, 166.02 (ArC, C=N). Anal. Calcd. for C20H19F2N5OS:
C 57.82, H 4.61, N 16.86. Found: C 57.62, H 4.45, N 16.77.

Molecules 2014, 19
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4-(3,4-Difluorobenzylideneamino)-2-(morpholin-4-ylmethyl)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3b). This compound was obtained as colorless crystals, Yield: 86%; m.p.
1
256–257 °C. IR (υ, cm−1): 3063 (Ar-H), 2842–2961 (CH str.), 1612 (C=N), 1292 (C=S). H-NMR:
δ 2.82 (t, 4H, J = 4.6 Hz, N-CH2), 3.65 (t, 4H, J = 4.6 Hz, OCH2), 5.30 (s, 2H, N-CH2-N), 7.45–8.03
(m, 7H, ArH), 9.73 (s, 1H, H-C=N). 13C-NMR: δ 50.96 (NCH2), 68.30 (OCH2), 69.82 (N-CH2-N),
115.49, 117.23, 117.67, 117.89, 118.11, 118.48, 118.84, 124.67, 126.79, 126.90, 127.09, 127.40,
129.80, 131.22, 131.71, 145.77, 148.52, 149.50, 151.12, 151.30, 151.93, 153.67, 153.98, 160.74,
163.18, 163.69, 165.25 (ArC, C=N). Anal. Calcd. for C20H18F3N5OS: C 55.42, H 4.19, N 16.16.
Found: C 58.67, H 4.34, N 16.37.
4-(4-Fluorobenzylideneamino)-2-(piperidin-1-ylmethyl)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3c). This compound was obtained as colorless crystals solid, Yield: 85%; m.p.
1
204–205 °C. IR (υ, cm−1): 3082 (Ar-H), 2833–2967 (CH str.), 1600 (C=N), 1280 (C=S). H-NMR: δ
1.31–1.34 (m, 2H, CH2CH2CH2), 1.48 (t, 4H, J = 4.4 Hz, NCH2CH2), 2.74 (t, 4H, J = 4.4 Hz, NCH2),
5.18 (s, 2H, N-CH2-N), 7.31–7.85 (m, 8H, ArH), 9.83 (s, 1H, H-C=N). 13C-NMR: δ 23.43
(CH2CH2CH2), 25.48 (NCH2CH2), 51.20 (NCH2), 69.99 (N-CH2-N), 116.29, 116.51, 124.58, 127.42,
128.41, 128.43, 129.74, 131.10, 131.19, 132.59, 132.90, 133.25, 146.03, 162.58, 163.49, 164.78, 165.99
(ArC, C=N). Anal. Calcd. for C21H21F2N5S: C 61.00, H 5.12, N 16.94. Found: C 61.23, H 5.01, N 16.65.
4-(3,4-Difluorobenzylideneamino)-2-(piperidin-1-ylmethyl)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-
triazole-3-thione (3d). This compound was obtained as white solid, Yield: 85%; m.p. 237–238 °C.
1
IR (υ, cm−1): 3,095 (Ar-H), 2,847–2,983 (CH str.), 1,622 (C=N), 1,299 (C=S). H-NMR: δ 1.29–1.33
(m, 2H, CH2CH2CH2), 1.49 (t, 4H, J = 4.5 Hz, NCH2CH2), 2.73 (t, 4H, J = 4.5 Hz, NCH2), 5.14 (s, 2H,
13
N-CH2-N), 7.41–8.01 (m, 7H, ArH), 9.76 (s, 1H, H-C=N). C-NMR: δ 23.33 (CH2CH2CH2), 25.47
(NCH2CH2), 51.13 (NCH2), 70.21 (N-CH2-N), 115.27, 116.98, 117.17, 117.72, 117.93, 118.52,
118.70, 124.59, 126.69, 126.74, 126.86, 126.95, 129.44, 130.97, 131.05, 145.98, 148.60, 148.73,
150.96, 151.07, 151.20, 153.49, 153.61, 160.54, 162.96, 163.19, 164.89 (ArC, C=N). Anal. Calcd. for
C21H20F3N5S: C 58.46, H 4.67, N 16.23. Found: C 58.17, H 4.81, N 16.41.
4-(4-Fluorobenzylideneamino)-5-(3-fluorophenyl)-2-[(4-methylpiperazin-1-yl)methyl]-2,4-dihydro-
3H-1,2,4-triazole-3-thione (3e). This compound was obtained as white solid, Yield: 84%; m.p.
1
191–192 °C. IR (υ, cm−1): 3,061 (Ar-H), 2838–2978 (CH str.), 1604 (C=N), 1290 (C=S). H-NMR: δ
2.12 (s, 3H, NCH3), 2.78 (t, 4H, J = 4.6 Hz, NCH2), 3.31 (t, 4H, J = 4.6 Hz, NCH2), 5.18 (s, 2H,
N-CH2-N), 7.40–8.02 (m, 8H, ArH), 9.68 (s, 1H, H-C=N). 13C-NMR: δ 45.72 (NCH3), 49.69 (NCH2),
54.51 (NCH2), 69.14 (N-CH2-N), 115.03, 115.27, 116.41, 116.63, 117.74, 117.95, 124.56, 124.59,
126.94, 127.03, 128.35, 128.37, 131.00, 131.08, 131.37, 131.46, 146.01, 146.04, 160.53, 162.96,
163.33, 163.64, 166.15, 166.64 (ArC, C=N). Anal. Calcd. for C21H22F2N6S: C 58.86, H 5.17, N 19.61.
Found: C 59.10, H 5.32, N 19.46.
4-(3,4-Difluorobenzylideneamino)-5-(3-fluorophenyl)-2-[(4-methylpiperazin-1-yl)methyl]-2,4-dihydro-
3H-1,2,4-triazole-3-thione (3f). This compound was obtained as white solid, Yield: 82%; m.p. 224–225 °C.
1
IR (υ, cm−1): 3042 (Ar-H), 2830–2989 (CH str.), 1612 (C=N), 1288 (C=S). H-NMR: δ 2.16 (s, 3H,
NCH3), 2.36 (t, 4H, J = 4.6 Hz, NCH2), 2.79 (t, 4H, J = 4.6 Hz, NCH2), 5.19 (s, 2H, N-CH2-N),

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7.39–8.05 (m, 7H, ArH), 9.74 (s, 1H, H-C=N). 13C-NMR: δ 45.61 (NCH3), 49.59 (NCH2), 54.44
(NCH2), 69.11 (N-CH2-N), 114.81, 115.05, 115.11, 115.36, 116.89, 117.07, 117.37, 117.58, 117.83,
118.04, 118.55, 118.60, 118.72, 118.78, 124.37, 124.68, 126.83, 126.92, 127.50, 130.91, 131.06,
131.14, 146.15, 147.44, 160.56, 162.55, 162.97, 163.24, 163.73, 165.35 (ArC, C=N). Anal. Calcd. for
C21H21F3N6S: C 56.49, H 4.74, N 18.82. Found: C 56.71, H 4.89, N 18.59.
1,4-Bis{[4-(4-fluorobenzylideneamino)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione]-2-
methyl}piperazine (4a). This compound was obtained as colorless crystals, Yield: 81%; m.p. 263–264 °C.
1
IR (υ, cm−1): 3088 (Ar-H), 2827–2965 (CH str.), 1611 (C=N), 1296 (C=S). H-NMR: δ 2.73 (t, 2H,
J = 4.8 Hz, NCH2), 2.76 (bs, 2H, NCH2), 2.81 (t, 3H, J = 4.8 Hz, NCH2), 2.89 (t, 1H, J = 4.8 Hz,
NCH2), 5.19 (s, 4H, N-CH2-N), 7.28–7.85 (m, 16H, ArH), 9.79 (s, 2H, 2 × H-C=N). 13C-NMR: δ
51.57 (NCH2), 69.42 (N-CH2-N), 115.22, 115.51, 116.33, 116.94, 117.41, 118.72, 124.79, 125.68,
126.79, 127.90, 128.43, 131.46, 131.64, 131.81, 132.19, 146.20, 146.33, 163.56, 164.02, 165.49,
166.36 (ArC, C=N). Anal. Calcd. for C36H30F4N10S2: C 58.21, H 4.07, N 18.86. Found: C 58.40, H
4.26, N 18.61.
1,4-Bis{[4-(3,4-difluorobenzylideneamino)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione]-2-methyl}piperazine (4b). This compound was obtained as colorless crystals, Yield: 80%; m.p.
1
277–278 °C. IR (υ, cm−1): 3067 (Ar-H), 2840–2969 (CH str.), 1603 (C=N), 1291 (C=S). H-NMR: δ
2.73 (t, 2H, J = 4.4 Hz, NCH2), 2.78 (bs, 2H, NCH2), 2.82 (t, 3H, J = 4.4 Hz, NCH2), 2.89 (t, 1H,
J = 4.4 Hz, NCH2), 5.17 (s, 4H, N-CH2-N), 7.42–8.00 (m, 14H, ArH), 9.71 (s, 2H, 2 × H-C=N);
13C-NMR: δ 51.78 (NCH2), 69.90 (N-CH2-N), 114.65, 115.19, 115.35, 115.90, 117.08, 117.15, 117.69,
117.89, 118.22, 118.70, 118.97, 119.26, 125.80, 126.74, 126.95, 127.17, 127.83, 130.57, 131.43,
131.60, 146.42, 147.92, 161.34, 162.42, 162.84, 163.50, 164.88, 165.04 (ArC, C=N). Anal. Calcd. for
C36H28F6N10S2: C 55.52; H 3.62, N 17.99. Found: C 55.78, H 3.49, N 17.73.
1,4-Bis{[4-(4-fluorobenzylideneamino)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione]-2-
methyl}-2-methylpiperazine (5a). This compound was obtained as colorless crystals, Yield: 78%; m.p.
257–258 °C. IR (υ, cm−1): 3,084 (Ar-H), 2,841–2,973 (CH str.), 1,606 (C=N), 1,293 (C=S). 1H-NMR:
δ 1.29 (d, 3H, J = 4.0 Hz, CH3), 2,13 (t, 1H, J = 4.0 Hz, NCH2), 2.66–2.89 (m, 3H, CHCH3, NCH2),
3.02 (d, 2H, J = 4.0 Hz, NCH2), 3.09 (d, 1H, J = 4.0 Hz, NCH2), 5.15–5.31 (m, 4H, N-CH2-N),
7.42–78.00 (m, 16H, ArH), 9.63, 9.66 (2s, 2H, 2 × H-C=N). 13C-NMR: δ 28.85 (NCH3), 49.26, 51.72,
51.96 (NCH2), 69.17, 69.73 (N-CH2-N), 115.12, 115.48, 115.70, 116.42, 117.43, 117.63, 118.05,
118.30, 118.96, 119.22, 119.54, 124.82, 125.74, 126.39, 127.18, 127.79, 129.14, 130.45, 130.78,
131.33, 132.50, 145.42, 146.36, 148.70, 161.45, 162.00, 162.52, 163.70, 164.47, 165.55 (ArC, C=N).
Anal. Calcd. for C37H32F4N10S2: C 58.72, H 4.26, N 18.51. Found: C 58.48, H 4.06, N 18.76.
1,4-Bis{[4-(3,4-difluorobenzylideneamino)-5-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione]-2-methyl}-2-methylpiperazine (5b). This compound was obtained as white solid, Yield: 76%;
m.p. 284–285 °C. IR (υ, cm−1): 3,070 (Ar-H), 2,842–2,982 (CH str.), 1,602 (C=N), 1,295 (C=S).
1H-NMR: δ 1.28 (d, 3H, J = 4.0 Hz, CH3), 2,32 (t, 1H, J = 4.0 Hz, NCH2), 2.63–2.85 (m, 3H, CHCH3,
NCH2), 3.00 (d, 2H, J = 4 Hz, NCH2), 3.11 (d, 1H, J = 4 Hz, NCH2), 5.11–5.30 (m, 4H, N-CH2-N),
7.40–7.95 (m, 14H, ArH), 9.71, 9.73 (2s, 2H, H-C=N). 13C-NMR: δ 28.66 (NCH3), 49.43, 50.98, 51.48

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(NCH2), 68.60, 69.04 (N-CH2-N), 113.88, 114.79, 115.48, 116.11, 117.27, 117.34, 117.84, 118.15,
118.50, 118.86, 119.24, 119.58, 119.90, 125.96, 126.56, 126.98, 127.30, 128.23, 129.66, 130.83,
131.57, 131.88, 132.77, 145.65, 146.55, 148.30, 161.61, 162.12, 162.76, 162.89, 163.62, 164.62,
165.32, 166.58 (ArC, C=N). Anal. Calcd. for C37H30F6N10S2: C 56.05, H 3.81, N 17.67. Found:
C 55.87, H 3.67, N 17.91.
3.5. Synthesis of 5-(3-Fluorophenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8)
A mixture of compound 7 (10 mmol) and 10% sodium hydroxide solution (100 mL) was refluxed
for 4 h. The mixture was then cooled to room temperature and filtered. The filtrate was acidified by
the addition of hydrochloric acid. The resulting solid was collected by filtration, dried and
recrystallized from ethanol yielded 83% of 8 as white solid; m.p. 175–176 °C. IR (υ, cm−1): 3,345
1
(N-H), 3,095 (Ar-H), 1,619 (C=N), 1,289 (C=S). H-NMR: δ 7.03–7.56 (m, 9H, Ar-H), 14.22 (s, 1H,
NH). 13C-NMR: δ 115.00, 115.24, 117.22, 117.43, 124.49, 127.80, 127.89, 128.70, 129.35, 129.53,
130.75, 130.83, 134.28, 149.34, 160.27, 162.70, 168.74 (ArC, C=N). Anal. Calcd. for C14H10FN3S: C
61.98, H 3.72, N 15.49. Found: C 61.70, H 3.51, N 15.77.
3.6. Synthesis of 5-(3-Fluorophenyl)-4-phenyl-1,2,4-triazole-3-thio(prop-2-yne) (9)
To a stirred solution of compound 8 (10 mmol) and triethylamine (10 mmol) in ethanol (25 mL),
was added propargyl bromide (10 mmol) dropwise. The mixture was refluxed for one hour. Excess
ethanol was removed in vacuo. The product was collected and crystallized from ethanol yielded 90%
of 9 as white solid; m.p. 148–149 °C. IR (υ, cm−1): 3290 (≡C-H), 3058 (Ar-H), 2854–2984 (CH str.),
2145 (C≡C), 1,623 (C=N). 1H-NMR: δ 2.26 (t, 1H, J = 4.5 Hz, ≡CH), 4.01 (d, 2H, J = 4.5 Hz, SCH2),
7.15–7.60 (m, 9H, Ar-H). 13C-NMR: δ 21.30 (SCH2), 75.15 (≡CH), 79.85 (C≡C), 115.08, 115.24,
117.21, 117.35, 124.56, 124.58, 128.19, 129.07, 129.13, 130.52, 130.80, 131.29, 131.35, 133.93,
151.32, 153.95, 161.30, 162.91 (ArC, C=N). Anal. Calcd. for C17H12FN3S; C 66.00, H 3.91, N 13.58.
Found: C 66.17, H 3.72, N 13.74.
3.7. General Procedure for Preparation of Acetylenic Mannich Bases 10a–10c
To a stirring solution of compound 9 (5 mmol) in dioxane (25 mL) was added cuprous chloride
(0.0025 g) and the mixture was heated for a few min, then paraformaldehyde (5 mmol) and the
appropriate secondary amine (5 mmol) were added. The mixture was heated at 90 °C for four h. After
cooling, the mixture was filtered then poured onto ice water (100 mL). The residue was extracted with
chloroform (3 × 25 mL) and purified on a column of silica gel using ethylacetate-hexane (1:3).
1-{4-[5-(3-Fluorophenyl)-4-phenyl-1,2,4-triazol-3-ylthio]but-2-ynyl}-morpholine (10a). This compound
was obtained as white solid, Yield: 81%; m.p. 190–191 °C. IR (υ, cm−1): 3,046 (Ar-H), 2829–2980 (CH
str.), 2148 (C≡C), 1607 (C=N). 1H-NMR: δ 2.81 (t, 4H, J = 4.6 Hz, N-CH2), 3.44 (s, 2H, ≡C-CH2-N), 3.62
(t, 4H, J = 4.6 Hz, OCH2), 4.05 (s, 2H, SCH2), 7.10–7.53 (m, 9H, ArH). 13C-NMR: δ 22.78 (SCH2), 53.46
(NCH2), 60.29 (≡C-CH2-N), 78.96 (C≡C), 115.17, 115.41, 117.45, 117.66, 124.69, 124.72, 127.37, 127.46,
128.73, 129.39, 129.67, 130.82, 130.90, 134.75, 147.94, 160.24, 162.67, 169.71 (ArC, C=N). Anal. Calcd.
for C22H21FN4OS: C 64.69, H 5.18, N 13.72. Found: C 64.93, H 5.01, N 13.54.

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1-{4-[5-(3-Fluorophenyl)-4-phenyl-1,2,4-triazol-3-ylthio]but-2-ynyl}-piperidine (10b). This compound
was obtained as white solid, Yield: 80%; m.p. 221–222 °C. IR (υ, cm−1): 3037 (Ar-H), 2828–2980
(CH str.), 2152 (C≡C), 1613 (C=N). 1H-NMR: δ 1.38–1.42 (m, 2H, CH2CH2CH2), 1.60 (t, 4H, J = 5.0 Hz,
NCH2CH2), 2.68 (t, 4H, J = 5.0 Hz, NCH2), 3.46 (s, 2H, ≡C-CH2-N), 4.02 (s, 2H, SCH2), 7.08–7.53 (m,
13
9H, ArH). C-NMR: δ 22.60 (SCH2), 23.37 (CH2CH2CH2), 25.48 (NCH2CH2), 51.13 (NCH2), 60.76
(≡C-CH2-N), 77.87 (C≡C), 115.09, 115.33, 117.42, 117.63, 124.62, 124.65, 127.39, 127.48, 128.71,
129.40, 129.65, 130.84, 130.92, 134.77, 147.74, 160.26, 162.69, 169.57 (ArC, C=N). Anal. Calcd. for
C23H23FN4S: C 67.95, H 5.70, N 13.78. Found: C 67.67, H 5.98, N 13.62.
1-{4-[5-(3-Fluorophenyl)-4-phenyl-1,2,4-triazol-3-ylthio]but-2-ynyl}-4-methylpiperazine (10c). This
compound was obtained as white solid, Yield: 78%; m.p. 173–174 °C. IR (υ, cm−1): 3,061 (Ar-H),
1
2836–2960 (CH str.), 2149 (C≡C), 1,603 (C=N). H-NMR: δ (s, 3H, NCH3), 2.44 (t, 4H, J = 5.0 Hz,
NCH2), 2.63 (t, 4H, J = 5.0 Hz, NCH2), 3.43 (s, 2H, ≡C-CH2-N), 4.01 (s, 2H, SCH2), 7.10–7.56 (m,
13
9H, ArH). C-NMR: δ 22.97 (SCH2), 42.35 (NCH3), 51.32 (NCH2), 56.62 (NCH2), 61.11 (≡C-CH2-
N), 78.16 (C≡C), 115.29, 115.54, 117.80, 118.27, 124.46, 124.95, 127.90, 128.26, 128.54, 129.16,
129.80, 131.36, 131.52, 134.63, 147.64, 160.42, 162.80, 169.98 (ArC, C=N). Anal. Calcd. for
C23H24FN5S: C 65.53, H 5.74, N 16.61. Found: C 65.42, H 5.48, N 16.85.
3.8. Biological Assays
3.8.1. Cells
The newly synthesized compounds 2a–2b, 3a–3f, 4a–4b, 5a–5b, 9 and 10a–10c were tested for
their in vitro growth inhibitory activity against the standard pathogenic strains of the Regional Center
for Mycology and Biotechnology (RCMB) namely; Streptococcus pneumonia RCMB 010010,
Bacillus subtilis RCMB 010067, Staphylococcus aureus RCMB 010025 (Gram-positive bacteria),
Pseudomonas aeuroginosa RCMB 010043, Escherichia coli RCMB 010052, Klebsiella pneumonia
RCMB 010058 (Gram-negative bacteria), and the yeast-like pathogenic fungus Aspergillus fumigatus
RCMB 02568 and Candida albicans RCMB 05036.
3.8.2. Antibacterial and Antifungal Assays
Preliminary antimicrobial activities of the newly synthesized compounds 2a–2b, 3a–3f, 4a–4b,
5a–5b, 9 and 10a-were tested by broth microdilution method [20–22]. The MIC determination of
the synthesized compounds was carried out in side-by-side comparison with ciprofloxacin against
Gram-positive bacteria (S. pneumonia, B. subtilis, S. aureus) and Gram-negative (P. aeruginosa,
E. coli, K. penumonia). The antifungal activity was assayed against yeasts (A. fumigatus, C. albicans).
The minimum inhibitory concentrations of the compounds were recorded as the lowest concentration
of each chemical compounds in the tubes with no turbidity (i.e., no growth) of inoculated
bacteria/fungi. Test compounds (10 mg) were dissolved in dimethylsulfoxide (DMSO, 1 mL) then
diluted in culture medium (Mueller-Hinton Broth for bacteria and Sabouraud Liquid Medium for
fungi), further progressive dilutions to obtain final concentrations of 1, 2, 4, 8, 16, 31.25, 62.5, 125,
250 and 500 mg/mL. DMSO never exceeded 1% v/v. The tubes were inoculated with 105 cfu·mL−1

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(colony forming unit/mL) and incubated at 37 °C for 24 h. The growth control consisting of media and
media with DMSO at the same dilutions as used in the experiments was employed.
4. Conclusions
New Schiff, Mannich and acetylenic Mannich bases containing 1,2,4-triazole and fluorophenyl
moieties were successfully synthesized. Antimicrobial activity screening revealed that some of the
tested compounds exhibited good antibacterial and antifungal activities. The combination of three
biologically potent units, namely Schiff base, morpholine/piperazine and 1,2,4-triazole in one
framework is essential for significant antimicrobial activity.
Acknowledgments
The author is very grateful to Linda Aouad, Faculty of Medicine, University of Djilali Liabes,
Algeria, for her valuable help in the interpretation of the antimicrobial results.
Author Contributions
The work presented in this manuscript was done by Mohamed Reda Aouad. The author read and
approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Not available.
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