Synthesis and antibacterial evaluation of several novel tripod pyrazoline with triazine core (TPTC) compounds

Article abstract of DOI:10.1007/s11164-016-2786-2

A series of new thiocarbamoyl tris-pyrazoline (tripod pyrazoline with triazine core) compounds was obtained by tris-cyclization of chalcone with thiosemicarbazide under basic condition in dry dimethylformamide (DMF) as solvent. Tris-chalcones were prepare

Full text of DOI:10.1007/s11164-016-2786-2

Res Chem Intermed
DOI 10.1007/s11164-016-2786-2
Synthesis and antibacterial evaluation of several novel
tripod pyrazoline with triazine core (TPTC) compounds
Nosrat O. Mahmoodi1 Masoud Mohammadi Zeydi2
•
•
Manouchehr Mamaghani1 Naser Montazeri3
•
Received: 9 June 2016 / Accepted: 14 October 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A series of new thiocarbamoyl tris-pyrazoline (tripod pyrazoline with
triazine core) compounds was obtained by tris-cyclization of chalcone with
thiosemicarbazide under basic condition in dry dimethylformamide (DMF) as sol-
vent. Tris-chalcones were prepared by condensation of tris-acetophenone and
appropriate aldehydes in basic medium. The newly synthesized compounds were
completely identified. The antibacterial activity of the tris-pyrazolines and tris-
chalcones was evaluated against Staphylococcus aureus, Micrococcus luteus,
Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis. Biological data
indicated that these compounds are effective against these bacteria.
Keywords Tris-pyrazoles Á Tris-chalcones Á Thiosemicarbazide Á Tripod Á
Antibacterial
Introduction
In recent decades, nitrogen-containing heterocyclic compounds have represented
one of the most important areas of research in organic chemistry. N-Containing
heteroaromatics have always received great attention due to their numerous
pharmaceutical applications [1, 2]. Because of this great interest in heterocycles,
synthesis of such compounds has always been among the most important research
fields in synthetic chemistry. The importance of nitrogen-containing heterocyclic
&
Nosrat O. Mahmoodi
mahmoodi@guilan.ac.ir
1
Department of Organic Chemistry, Faculty of Sciences, University of Guilan,
PO Box 41335-1914, Rasht, Iran
2
3
Department of Organic Chemistry, University Campus 2, University of Guilan, Rasht, Iran
Department of Chemistry, Faculty of Sciences, Islamic Azad University, Tonekabon Branch,
Tonekabon 46819-89711, Iran
123

N. O. Mahmoodi et al.
compounds for biomedical and material science applications has led to an increase
in the number of synthetic approaches available for preparation of this type of
heterocyclic compound [3]. Five-membered heterocycles constitute a central
component of a diverse range of biologically active natural compounds and
pharmaceuticals [4, 5]. Generally, such ring systems can be constructed by three
distinct approaches: annulation, ring expansion, and intramolecular cyclization
[6–9]. To combat infections, a wide variety of antibacterial and antifungal agents
have been synthesized.
Pyrazoles are nitrogen-containing heterocyclic compounds that exhibit important
pharmacological activities and are therefore useful materials in drug studies. These
compounds synthesized from chalcone intermediates possess high medicinal
activities and are present in various biologically active compounds such as
amidopyrene, phenazone, azolid/tandearil, methampyrone, anturane, indoxacarb,
etc. Considerable interest has been focused on the pyrazole moiety.
The pyrazole nucleus and its derivatives have been found to exhibit potential
activities such as anticonvulsant [10], analgesic [11], antihypotensive [12],
psychoanaleptic [13], muscle relaxant [14], and antidepressant actions [15]. Such
derivatives have also been found to exhibit cytotoxic, herbicidal [16], and platelet
aggregation inhibitory activity [17], and to act as cannabinoid CB1-receptor
modulators [18]. Pyrazoline derivatives are used as anticancer [19], antifungal [20],
antibacterial [21], antitumor [22], and antitubercular [23] agents. Several groups of
pyrazoles have been reported to possess antidiabetic [24], antimalarial [25],
antiinflammatory [26], and anti-human immunodeficiency virus (HIV) properties
[27]. They are also used as dyestuffs, analytical reagents, and agrochemicals [28].
Several methods for synthesis of pyrazoles have been described, the commonest
employing condensation of a,b-unsaturated carbonyl compounds with hydrazine or
phenyl hydrazine [29–32]. Recently, such compounds were also prepared by using
microwave or ultrasonic irradiation [33, 34]. However, according to our survey of
literature, there are no synthetic reports related to preparation of tris-pyrazole. In
continuation of our previous interest in new preparation routes for synthesis of
chalcones, bis-chalcones, pyrazoles, bis-pyrazoles, and other related heterocyclic
compounds [35–42], we report herein synthesis of several novel tris-pyrazole
derivatives (P1–P6). Products P1–P6 were prepared via reaction of tris-chalcones
(S1–S6) followed by regioselective cyclization with three equivalents of thiosemi-
carbazide through quadruple cascade multicomponent reaction (QCMCR) in good
yield. Furthermore, the antibacterial activity of the tris-pyrazolines and tris-
chalcones was evaluated against a series of Gram-positive and Gram-negative
bacteria.
Experimental
General
Compounds were obtained from Merck and used without further purification.
Melting points were obtained on an Electrothermal capillary melting point apparatus
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Synthesis and antibacterial evaluation of several novel…
and are uncorrected. Thin-layer chromatography (TLC) was performed using HF254
fluorescent silica gel plates (Merck), examined under ultraviolet (UV) light at 254
and 365 nm. Infrared spectra (m/cm-1) were recorded on a Shimadzu IR-470, using
KBr disks. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on
a DRX-400 MHz spectrometer at 293 K in CDCl3 and dimethyl sulfoxide (DMSO)-
d6. Spectra were internally referenced to tetramethylsilane (TMS). Elemental
analyses were carried out using a Carlo-Erba EA1110 CNNO-S analyzer and agreed
with calculated values. High-resolution mass spectra (HRMS) were recorded on an
Agilent 6520 (QTOF) ESI-HRMS instrument.
Preparation of 1,10,100-(((1,3,5-triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-
diyl))tris(ethan-1-one) (T)
A mixture of acetophenone (3 mmol, 0.360 g), NaOH (3 mmol, 0.120 g), and
20 mL distilled water was added to a suspension of cyanuric chloride (1 mmol,
0.180 g) in 25 mL dry acetone at 0–5 °C. The mixture was stirred at this
temperature for 4 h, monitoring the reaction by TLC (CHCl3:MeOH 3:1). After
reaction completion, the solid product was removed by filtration and washed twice
with distilled H2O. All synthesized compounds were recrystallized from EtOAc
(Scheme 1).
General procedure for preparation of tris-chalcone derivatives S1–S6
Tris-acetophenone (1 mmol) and substituted benzaldehyde (3 mmol) were dis-
solved in 5 mL MeOH. To this mixture was added NaOH (3.5 mmol), followed by
refluxing for 8 h. Reaction progress was monitored by TLC (CHCl3:MeOH 5:2).
After reaction completion, the reaction mixture was poured into crushed ice and
neutralized with dilute 2 N HCl. Finally, the product was filtered, washed with
water, dried, and recrystallized from EtOH 96 % to recover the products in good
yield with high purity. All of the synthesized tris-chalcones S1–S6 were charac-
1
terized by IR, H and 13C NMR spectroscopy, and elemental analysis (Scheme 2).
Scheme 1 Preparation of tris-acetophenone (T) from cyanuric chloride
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N. O. Mahmoodi et al.
Scheme 2 Preparation of tris-chalcones S1–S6 from tris-acetophenone
General procedure for preparation of tris-pyrazole derivatives P1–P6
In a round-bottomed flask, substituted tris-chalcone S1–S6 (0.01 mol) was dissolved in
10 mL dry DMF, to which was added thiosemicarbazide (0.03 mol). To the resulting
mixture of solution, NaOH (0.03 mol) was added as catalyst, then the reaction mixture
was stirred for 5 days at reflux conditions. Reaction progress was monitored by TLC
(CHCl3:MeOH 5:3). After reaction completion, the reaction mixture was poured into
crushed ice and neutralized with 2 N HCl. Finally, the product was filtered, dried, and
purified by recrystallization from EtOH 96 % (Scheme 3).
Characterization data of compounds
1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(ethan-1-one) (T)
White powder; yield 98 %; m.p. 175–177 °C; IR (tmax/cm-1) 3065 (C–Harom), 1680
1
(C=O), 1572, 1520 (C=N, C=C), 1210 (C–O); H NMR (400 MHz, CDCl3) d 2.62
Thiosemicarbazide
Scheme 3 Preparation of tris-pyrazoles P1–P6 from tris-chalcones S1–S6
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Synthesis and antibacterial evaluation of several novel…
(s, 3H), 7.25 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz,
CDCl3) d 196.6, 173.3, 154.9, 135.1, 130.0, 121.6, 26.6; HRMS ((?)-ESI): m/
z = 483.1433 (calcd. 483.1430 for C27H21N3O6): Anal. Calcd. for: C, 67.08; H,
4.38; N, 8.69. Found: C, 67.05; H, 4.41; N, 8.66 %.
(2E,20E,200E)-1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(3-
phenylprop-2-en-1-one) (S1)
Yellow crystal; yield 89 %; m.p. 132–135 °C; IR (tmax/cm-1) 3138 (C–Harom),
1643 (C=O), 1586, 1565, 1444 (C=N, C=C), 1070 (C–O); H NMR (400 MHz,
1
CHCl3) d 6.90 (d, J = 8.8, 1H, CH=CH–CO), 6.97 (d, J = 8.4 Hz, 2H), 7.31–7.35
(m, 2H), 7.71 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 8.4 Hz,
2H), 8.12 (d, J = 15.6 Hz, 1H, CH=CH–CO); 13C NMR (100 MHz, DMSO-d6) d
187.0, 162.6, 136.2, 135.5, 135.5, 131.8, 130.3, 129.7, 129.3, 128.6, 125.2, 115.7;
HRMS ((?)-ESI): m/z = 747.2366 (calcd. 747.2369 for C48H33N3O6): Anal. Calcd.
for: C, 77.10; H, 4.45; N, 5.62. Found: C, 77.13; H, 4.47; N, 5.64 %.
(2E,20E,200E)-1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(3-
(p-tolyl)prop-2-en-1-one) (S2)
Yellow crystal; yield 94 %; m.p. 193–194 °C; IR (tmax/cm-1) 3111 (C–Harom),
1643 (C=O), 1600 (C=N), 1570, 1510 (C=C), 1220 (C–O); H NMR (400 MHz,
1
DMSO-d6) d 2.36 (s, 3H), 6.97 (d, J = 15.6 Hz, 1H,CH=CH–CO), 8.08 (d,
J = 8.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d 187.5, 162.5, 143.2, 140.8,
132.6, 131.6, 129.9, 129.6, 129.2, 121.4, 115.8, 21.5; HRMS ((?)-ESI): m/
z = 789.2836 (calcd. 789.2839 for C51H39N3O6): Anal. Calcd. for: C, 77.55; H,
4.98; N, 5.32. Found: C, 77.51; H, 4.96; N, 5.35 %.
(2E,20E,200E)-1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(3-
(3-nitrophenyl)prop-2-en-1-one) (S3)
Yellow crystal; yield 87 %; m.p. 232–234 °C; IR (tmax/cm-1) 3127 (C–Harom),
1643 (C=O), 1602 (C=N), 1584, 1540 (C=C), 1520, 1340 (NO2), 1223 (C–O); H
1
NMR (400 MHz, CDCl3) d 6.93 (d, J = 8.8 Hz, 2H), 7.72–7.81 (m, 2H, Ar–H,
CH = CH–CO), 8.13–8.17 (m, 3H, Ar–H, CH=CH–CO), 8.25 (m, 1H), 8.34 (d,
J = 6.0 Hz, 1H), 8.76 (s, 1H); 13C NMR (100 MHz, CDCl3) d 187.4, 162.8, 148.8,
140.6, 137.3, 135.4, 131.9, 130.7, 129.3, 125.3, 124.8, 123.3, 115.8; HRMS ((?)-
ESI): m/z = 882.1925 (calcd. 882.1922 for C48H30N6O12): Anal. Calcd. for: C,
65.31; H, 3.43; N, 9.52. Found: C, 65.30; H, 3.46; N, 9.48 %.
1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(3-(4-
nitrophenyl)prop-2-en-1-one) (S4)
Yellow crystal; yield 95 %; m.p. 244–245 °C; IR (tmax/cm-1) 3105 (C–Harom),
1645 (C=O), 1621 (C=N), 1583, 1562 (C=C), 1517, 1338 (NO2), 1220 (C–O); H
1
NMR (400 MHz, DMSO-d6) d 6.93 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 15.6 Hz, 1H,
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N. O. Mahmoodi et al.
CH=CH–CO), 8.11-8.18 (m, 5H, Ar–H, CH=CH–CO), 8.29 (d, J = 8.8 Hz, 2H);
13C NMR (100 MHz, DMSO-d6) d 187.2, 162.9, 148.3, 141.8, 140.3, 131.9, 130.1,
129.2, 126.6, 124.3, 115.9; HRMS ((?)-ESI): m/z = 882.1919 (calcd. 882.1922 for
C48H30N6O12): Anal. Calcd. for: C, 65.31; H, 3.43; N, 9.52. Found: C, 65.34; H,
3.45; N, 9.48 %.
1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(3-(4-chlorop
henyl)prop-2-en-1-one) (S5)
Yellow crystal; yield 90 %; m.p. 184–186 °C; IR (tmax/cm-1) 3115 (C–Harom),
1640 (C=O), 1597 (C=N), 1541, 1505 (C=C), 1220 (C–O); H NMR (400 MHz,
1
DMSO-d6) d 6.91 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.06 (d,
J = 15.6 Hz, 1H, CH=CH–CO), 7.92 (d, J = 8.8 Hz, 2H), 7.95 (d, J = 16.0 Hz,
1H, CH=CH–CO), 8.10 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d
168.4, 162.7, 141.7, 135.2, 134.3, 131.7, 130.8, 129.5, 129.3, 123.3, 115.8; HRMS
((?)-ESI): m/z = 849.1205 (calcd. 849.1200 for C48H30Cl3N3O6): Anal. Calcd. for:
C, 67.74; H, 3.55; N, 4.94. Found: C, 67.71; H, 3.57; N, 4.92 %.
1,10,100-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(3-(2,4-dichloro
phenyl)prop-2-en-1-one) (S6)
Yellow crystal; yield 99 %; m.p. 158–159 °C; IR (tmax/cm-1) 3100 (C–Harom),
1640 (C=O), 1595 (C=N), 1580, 1483 (C=C), 1222 (C–O); H NMR (400 MHz,
1
CDCl3) d 6.29 (d, J = 8.8 Hz, 2H), 7.55 (m, 1H), 7.74 (s, 1H), 7.92 (d,
J = 15.6 Hz, 1H, CH=CH–CO), 8.02 (d, J = 15.6 Hz, 1H, CH=CH–CO), 8.11 (d,
J = 8.8 Hz, 2H), 8.25 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 187.1,
162.9, 136.5, 135.7, 135.4, 132.0, 131.8, 130.1, 129.8, 129.2, 128.3, 125.8, 115.9;
HRMS ((?)-ESI): m/z = 953.0006 (calcd. 953.0002 for C48H27Cl6N3O6): Anal.
Calcd. for: C, 60.40; H, 2.85; N, 4.40. Found: C, 60.43; H, 2.83; N, 4.45 %.
3,30,300-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(5-phenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide) (P1)
Pale-brown crystals; yield 86 %; m.p. 152–155 °C; IR (tmax/cm-1) 3429, 3271 (N–
H), 3090 (C–Harom), 1594, 1512 (C=N), 1361 (C=S), 1278 (C–O); 1H NMR
(400 MHz, DMSO-d6) d 3.04 (dd, J = 13.6, 3.2 Hz, 2H, CHa), 3.82 (dd, J = 18.4,
11.2 Hz, 1H, CHb), 5.86 (dd, J = 11.2, 3.2 Hz, 1H, CHx), 6.85 (d, J = 6.4 Hz, 2H),
7.02 (d, J = 8.4 Hz, 2H), 7.09-7.16 (3H, m, 3CH), 7.02 (d, J = 6.4 Hz, 2H), 10.04
(s, 2H, NH2); 13C NMR (100 MHz, DMSO-d6) d 175.8, 157.8, 155.1, 151.5, 140.0,
130.4, 128.1, 127.9, 122.7, 121.2, 115.8, 60.7, 41.7; HRMS ((?)-ESI): m/
z = 966.2661 (calcd. 966.2665 for C51H42N12O3S3): Anal. Calcd. for: C, 63.34;
H, 4.38; N, 17.38. Found: 63.37; H, 4.36; N, 17.35 %.
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Synthesis and antibacterial evaluation of several novel…
3,30,300-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(5-(p-tolyl)-
4,5-dihydro-1H-pyrazole-1-carbothioamide) (P2)
Brown crystals; yield 88 %; m.p. 136–137 °C; IR (tmax/cm-1) 3460, 3330 (N–H),
3061 (C–Harom), 1580, 1484 (C=N), 1344 (C=S), 1165 (C–O); 1H NMR (400 MHz,
DMSO-d6) d 2.26 (s, 3H), 3.05 (dd, J = 18.0, 3.2 Hz, 2H, CHa), 3.84 (dd, J = 17.6,
11.2 Hz, 1H, CHb), 5.86 (dd, J = 10.8, 3.2 Hz, CHx), 6.82 (d, J = 8.0 Hz, 2H),
7.02 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 10.04
(s, 2H, NH2); 13C NMR (100 MHz, DMSO-d6) d 175.9, 160.3, 156.5, 140.6, 136.3,
130.9, 129.4, 127.4, 125.7, 122.2, 116.1, 62.8, 43.0, 21.3; HRMS ((?)-ESI): m/
z = 1008.3129 (calcd. 1008.3134 for C54H48N12O3S3): Anal. Calcd. for: C, 64.27;
H, 4.79; N, 16.65. Found: C, 64.28; H, 4.76; N, 16.67 %.
3,30,300-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(5-(3-nitroph
enyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (P3)
Brown crystals; yield 79 %; m.p. 150–152 °C; IR (tmax/cm-1) 3460, 3267 (N–H),
3061 (C–Harom), 1600, 1481 (C=N), 1355 (C=S), 1167 (C–O); 1H NMR (400 MHz,
DMSO-d6) d 2.90-2.99 (m, 2H, CHa), 3.86 (dd, J = 17.6, 12.0 Hz, 1H, CHb), 5.97
(dd, J = 11.2, 3.2 Hz, 1H, CHx), 6.84 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.8 Hz,
1H), 7.56 (t, J = 8.4 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 8.12 (d, J = 8.4 Hz, 1H),
8.18 (s, 1H, NH2), 10.06 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d 180.7, 159.6,
155.4, 151.5, 140.0, 136.0, 133.8, 132.0, 129.0, 128.1, 127.1, 122.7, 121.2, 60.7,
41.5; HRMS ((?)-ESI): m/z = 1101.2213 (calcd. 1101.2217 for C51H39N15O9S3):
Anal. Calcd. for: C, 55.58; H, 3.57; N, 19.06. Found: C, 55.61; H, 3.53; N, 19.02 %.
3,30,300-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(5-(4-nitroph
enyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (P4)
Brown crystals; yield 75 %; m.p. 141–144 °C; IR (tmax/cm-1) 3355, 3213 (N–H),
3052 (C–Harom), 1598, 1501 (C=N), 1539, 1338 (NO2), 1338 (C=S), 1167 (C–O);
1H NMR (400 MHz, DMSO-d6) d 3.10-3.15 (m, 2H, CHa), 3.82 (dd, J = 17.6,
11.2 Hz, 2H, CHb), 5.68 (m, 1H, CHx), 6.82 (d, J = 8.0 Hz, 2H), 7.47 (d,
J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H), 10.00 (s, 2H,
NH2); 13C NMR (100 MHz, DMSO-d6) d 177.6, 163.0, 160.1, 157.4, 151.9, 132.4,
129.2, 128.3, 126.8, 126.5, 115.8, 62.2, 42.4; HRMS ((?)-ESI): m/z = 1101.2214
(calcd. 1101.2217 for C51H39N15O9S3): Anal. Calcd. for: C, 55.58; H, 3.57; N,
19.06 Found: C, 55.61; H, 3.60; N, 19.03 %.
3,30,300-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-3,1-diyl))tris(5-(4-chloroph
enyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (P5)
Brown crystals; yield 84 %; m.p. 159–161 °C; IR (tmax/cm-1) 3417, 3321 (N–H),
3120 (C–Harom), 1630, 1543 (C=N), 1355 (C=S), 1139 (C–O); 1H NMR (400 MHz,
DMSO-d6): d 3.09 (dd, J = 18.0, 3.2 Hz, 2H, CHa), 3.84 (dd, J = 18.0, 11.2 Hz,
1H, CHb), 5.88 (dd, J = 12.8, 2.8 Hz, 1H, CHx), 6.82 (d, J = 8.8 Hz, 2H), 7.15 (d,
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N. O. Mahmoodi et al.
J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 10.08 (s, 1H,
NH2); 13C NMR (100 MHz, DMSO-d6) d 175.9, 160.9, 155.7, 142.5, 131.8, 129.5,
129.3, 128.9, 127.8, 121.6, 116.1, 62.5, 42.7; HRMS ((?)-ESI): m/z = 1068.1492
(calcd. 1068.1496 for C51H39Cl3N12O3S3): Anal. Calcd. for: C, 57.22; H, 3.67; Cl,
9.93; N, 15.70 Found: C, 57.25; H, 3.64; Cl, 9.95; N, 15.68 %.
3,30,300-(((1,3,5-Triazine-2,4,6-triyl)tris(oxy))tris(benzene-4,1-diyl))tris(5-(2,4-dichlor
ophenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (P6)
Brown crystals; yield 81 %; m.p. 146–148 °C; IR (tmax/cm-1) 3410, 2275 (N–H),
3145 (C–Harom), 1597, 1467 (C=N), 1334 (C=S), 1166 (C–O); 1H NMR (400 MHz,
DMSO-d6) d 3.03-3.11 (m, 2H, CHa), 3.86 (dd, J = 17.6, 12.0 Hz, 1H, CHb), 5.59
(dd, J = 11.6, 3.2 Hz, 1H, CHX), 6.81 (d, J = 8.2 Hz, 2H), 7.10 (d, J = 8.4 Hz,
1H), 7.41 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H); 7.87 (s, 1H), 10.06 (1H, s,
NH2); 13C NMR (100 MHz, DMSO-d6) d 176.3, 157.8, 155.1, 151.5, 140.0, 136.0,
134.8, 133.8, 132.0, 130.4, 127.1, 126.7, 121.2, 116.1, 60.7, 41.7; HRMS ((?)-ESI):
m/z = 1170.0324 (calcd. 1170.0327 for C51H36Cl6N12O3S3): Anal. Calcd. for: C,
52.19; H, 3.09; N, 14.32 Found: C, 52.22; H, 3.12; N, 14.30 %.
Determination of antimicrobial activity
The antibacterial activity of the compounds was measured biologically using the
agar well-diffusion method. Mu¨ller–Hinton agar (Merck) plates were readied
similar to manufacturer instructions to compare the antibacterial activity of the
compounds. Sterile Mu¨ller–Hinton agar plates were inoculated with bacteria. Test
samples (0.001 g) were dissolved in 1 mL dimethyl sulfoxide to obtain stock
solution. For each sample, 0.1 mL was dropped into each labeled well aseptically.
The inculcated plates were then incubated for 24 h at 37 °C. Penicillin was used as
positive control and DMSO as negative control. After incubation time, the
antimicrobial activity was evaluated by measuring the zone of inhibition against the
test organisms and compared with that of the standard. The results of the tests are
presented as inhibition zones in millimeters (mm). The experiment was repeated,
and the average inhibition zone estimated.
Results and discussion
Synthesis of pyrazoles P1–P6 was performed as shown in Scheme 1. Initially, tris-
acetophenone (T) was synthesized by reaction of cyanuric chloride (1) and
4-hydroxybenzaldehyde (2) in presence of NaOH in acetone/water at 0–5 °C.
Subsequently, premade tris-acetophenone (T) was further converted to correspond-
ing tris-chalcones S1–S6 via reaction of diverse substituted benzaldehydes 4a–f in
MeOH. Initially, the color of the reaction mixture was pale yellow, but at the end of
reaction with addition of thiosemicarbazone, the color of the reaction mixture
changed to dark yellow. Detailed studies of this process confirmed that the first
aldol-type condensation reaction completed within 30 min in a variety of solvents.
123

Synthesis and antibacterial evaluation of several novel…
In contrast, formation of second and third chalcones was more time consuming and
more sluggish. This procedure gave high yields of the desired tris-chalcones S1–S6
with no undesirable side reactions. Products S1–S6 precipitated from the reaction
mixture in relatively pure state and were easily recrystallized.
Reaction of 1 equiv. tris-chalcones S1–S6 with 3 equiv. thiosemicarbazide while
refluxing in DMF/NaOH solution directly produced cyclization with thiosemicar-
bazide to afford tris-pyrazoles P1–P6 3,30,300-((1,3,5-triazine-2,4,6-triyl)tris(oxy))-
tris(5-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide). It is significant that,
when this reaction was performed under various other solvents, e.g., MeOH, EtOH,
and DMSO, the yields were unsatisfactory.
Transformation of tris-chalcones S1–S6 to corresponding tris-pyrazolines P1–P6
was accomplished by reaction of 1 mmol tris-chalcones S1–S6 with 3 mmol
thiosemicarbazide over 5 days. The first pyrazoline reaction was completed within
*6 h in a variety of solvents and was attended by loss of water followed by
regioselective, endo-fashion, intramolecular cyclization. Formation of second and
third pyrazolines happened at a much slower rate than the first one; this can
probably be attributed to increased steric hindrance around the second and
especially third chalcone moiety relative to the first and second one, or decreased
electrophilicity and/or lipophilicity of this intermediate. The mechanism of this
reaction involves formation of hydrazones followed by subsequent addition of N–H
to the olefinic bond, leading to formation of the target tris-pyrazolines (Scheme 3).
The purity of the compounds was determined by TLC and melting point
measurements. The chemical structures of the synthesized compounds were
1
established by spectroscopic data (FT-IR, H NMR, and 13C NMR), HRMS, and
elemental analyses. The FT-IR spectrum of tris-chalcones S1–S6 exhibited the
characteristic band at 1650–1700 cm-1 indicating presence of a,b-unsaturated C=O
group. The a-H and b-H of tris-chalcones resonate at d 7.06–7.92 and 7.79–8.12 as
two doublets (J = 15.6 Hz) in the 1H NMR spectra. The IR spectra of tris-
pyrazolines P1–P6 revealed presence of thiocarboxamide amino stretching vibration
bands at 3250–3400 cm-1, absorption bands in the region 1509–1610 cm-1
corresponding to C=N stretching bands because of ring closure, and peaks in the
regions 1355–1365 and 1055–1096 cm-1, indicating presence of C=S and C–N
groups. The structure of P1–P6 was confirmed by elemental analysis, FT-IR, and 1H
1
and 13C NMR. The H NMR spectrum of the tris-pyrazoles in DMSO-d6 showed
three multiplet signals characteristic of CH and CH2 pyrazole ring, near 3.10, 3.86,
and 8.88 ppm, as well as singlet for NH2 at approximately 10.10 ppm. The 13C
NMR spectrum showed signals in agreement with the proposed structure, and data
were found to be in good agreement with the proposed structure.
The in vitro antibacterial activity of tris-chalcones S1–S6 and tris-pyrazoles P1–
P6 was assessed against three representative Gram-positive bacteria, viz. Staphy-
lococcus aureus (MTCC 96), Bacillus subtilis (MTCC 441), and Micrococcus luteus
(ATCC 9341), and two Gram-negative bacteria, viz. Pseudomonas aeruginosa
(MTCC 741) and Escherichia coli (E. coli) ATCC 25922. Penicillin was used as
positive control and DMSO as negative control.
According to Table 1, almost all the tris-chalcones and tris-pyrazoles were
inactive against Gram-negative bacteria. Compounds P1, P2, P3, P5, P6 showed
123

N. O. Mahmoodi et al.
Table 1 Antimicrobial activity of compounds S1–S6 and P1–P6
Entry
Compound
Antimicrobial activity (zone of inhibition in mm)
Gram-positive
Gram-negative
S. aureus
B. subtilis
M. luteus
E. coli
P. aeruginosa
1
S1
–
7
7
6
–
–
6
–
–
–
–
–
–
–
–
–
21
–
–
–
7
–
–
–
–
–
–
–
–
–
47
2
S2
7
10
7
11
5
3
S3
–
4
S4
–
7
7
5
S5
–
7
8
6
S6
10
13
11
10
–
7
7
7
P1
11
9
15
20
16
–
8
P2
9
P3
13
10
10
8
10
11
12
13
14
P4
P5
11
7
18
10
–
P6
DMSO
Penicillin
–
–
48
33
49
antibacterial activity against Micrococcus luteus, and compounds P2, P5 had good
antibacterial activity. Compounds P1–P6, S2 had good antibacterial activity against
Bacillus subtilis, and compound P3 had remarkable activity against Bacillus subtilis.
Compounds P1–P3, P5, P6, S6 showed good antibacterial activity against Staphy-
lococcus aureus.
Conclusions
We describe an efficient protocol for synthesis of tris-pyrazoles P1–P6 in good yield
starting with tris-chalcones S1–S6 and thiosemicarbazide in DMF/NaOH. Tris-
chalcones S1–S6 were synthesized from reaction of tris-acetophenone (T) with
substituted benzaldehydes 4a–f in MeOH. Thus, the present method constitutes a
novel synthesis of tris-pyrazoles P1–P6 with good yield. The analysis results
confirmed the correct structures for synthesized products. The tris-pyrazolines
showed better antibacterial activity than the tris-chalcones.
Acknowledgments Partial support of this research by the Research Committee of the University of
Guilan is gratefully acknowledged.
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