Synthesis of 1,6-hexanediyl-bis(semicarbazides) and 1,6-hexanediyl-bis(1,2, 4-triazol-5-ones) and their antiproliferative and antimicrobial activity

Article abstract of DOI:10.2298/JSC110212157P

A series of 1,6-bis(3-substituted 1,5-dihydro-5-oxo-4H-1,2,4-triazol- -4-yl)hexanes 3a-g were synthesized by the cyclization reaction of 1,6-bis{[(2- -substituted hydrazinyl)carbonyl]amino}hexanes 2a-g in alkaline medium. The new derivatives 3a-c were screened in vitro for their antiproliferative and anticancer activity in human tumor cell lines derived from breast and lung carcinoma cells. Compounds 3a (at a concentration of 0.18 mM), 3b (at concentrations of 0.12 and 0.02 mM) and 3c (at concentrations of 0.23 and 0.11 mM) were found to be the most effective against the lung cell line. Compound 3a had the greatest antiproliferative effect on the breast carcinoma cell line. Representative compounds were established and evaluated as antimicrobial agents. All the tested derivatives showed minimum inhibitory concentrations (MIC) in the range 1.87-7.5 μg mL^-1. Compound 3b was the most effective against Candida albicans (MIC 1.87 μg mL^-1). Copyright 2012 (CC) SCS.

Full text of DOI:10.2298/JSC110212157P

J. Serb. Chem. Soc. 77 (1) 1–8 (2012)
UDC 547.497.1+547.792+542.913:57–188
JSCS–4243
Original scientific paper
Synthesis of 1,6-hexanediyl-bis(semicarbazides) and
1,6-hexanediyl-bis(1,2,4-triazol-5-ones) and their
antiproliferative and antimicrobial activity
1
2
3
*
MONIKA PITUCHA , JOLANTA RZYMOWSKA , ALINA OLENDER
and LUDMIŁA GRZYBOWSKA-SZATKOWSKA4
1Department of Organic Chemistry, Medical University, 20-081 Lublin, Poland, 2Department
of Biology and Genetics, Medical University, 20-081 Lublin, Poland, 3Chair and Department
of Medical Microbiology, Medical University, 20-093 Lublin, Poland and 4Department of
Oncology, Medical University, 20-090 Lublin, Poland
(Received 12 February, revised 14 August 2011)
Abstract. A series of 1,6-bis(3-substituted 1,5-dihydro-5-oxo-4H-1,2,4-triazol-
-4-yl)hexanes 3a–g were synthesized by the cyclization reaction of 1,6-bis{[(2-
-substituted hydrazinyl)carbonyl]amino}hexanes 2a–g in alkaline medium. The
new derivatives 3a–c were screened in vitro for their antiproliferative and anti-
cancer activity in human tumor cell lines derived from breast and lung car-
cinoma cells. Compounds 3a (at a concentration of 0.18 mM), 3b (at concen-
trations of 0.12 and 0.02 mM) and 3c (at concentrations of 0.23 and 0.11 mM)
were found to be the most effective against the lung cell line. Compound 3a
had the greatest antiproliferative effect on the breast carcinoma cell line. Re-
presentative compounds were established and evaluated as antimicrobial agents.
All the tested derivatives showed minimum inhibitory concentrations (MIC) in
the range 1.87–7.5 µg mL-1. Compound 3b was the most effective against Can-
dida albicans (MIC 1.87 µg mL-1).
Keywords: synthesis; semicarbazide; 1,2,4–triazole; biological activity.
INTRODUCTION
The synthesis of compounds containing a 1,2,4–triazole ring in their struc-
ture has attracted widespread attention, mainly in connection with their wide range
of pharmacological properties. A variety of biological activities, such as antide-
1,2
3
4
5,6
pressant, anticonvulsant, antitumor and antimicrobial have been reported
for mono-substituted 1,2,4–triazole systems. A great number of these derivatives
7,8
display interesting anticancer activity. It was reported that compounds having
triazole moieties, such as vorozole, anastrozole and letrozole, appear to be very
9
effective aromatase inhibitors and are very useful for preventing breast cancer.
*Corresponding author. E-mail: monika.pitucha@umlub.pl
doi: 10.2298/JSC110212157P
1
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PITUCHA et al.
Moreover, several compounds involving the triazole moiety and having diverse
10–12
antibacterial and antifungal activities were reported.
1,2,4-Triazol-3-ones
have been prepared by different methods. One of the most common routes lead-
ing to the preparation of these compounds involves cyclodehydration of 1-acetyl-
13
semicarbazide with a variety of reagents, such as tris(formylamino)methane,
sodium hydroxide
14,15
16
and formic acid. 4,5-Disubstituted and 2,4,5-trisubsti-
tuted 1,2,4-triazol-3-ones have been obtained in the reaction of amidrazones salts
17
with isocyanate or urea performed in the melt. Some bis(3,4-disubstituted 5-
-oxo-1,2,4-triazol-5-4-yl)alkanes were synthesized using esters of ethoxyalkyl-
18
idene)hydrazinecarboxylic acids and diamines.
On the other hand, it is known that semicarbazides, the key intermediates
used in the synthesis of 1,2,4-triazol-3-one derivatives, are compounds with va-
19
20
rious pharmacological activities: antitubercular, anticonvulsant and antinoci-
21
ceptive. It was found that some compounds possess antibacterial activity against
Gram-positive bacteria, including staphylococci (coagulase-positive Staphylococ-
22
cus aureus and coagulase-negative Staphylococcus epidermidis). Semicarbazi-
des possessing a heterocyclic ring show anticancer activity against human gastric
23
carcinoma cell line.
In view of the above-mentioned findings and in continuation of our research
in the domain of heterocyclic compounds of the 1,2,4-triazole class with expec-
24,25
ted biological activity,
herein, the synthesis of some new bis-semicarbazides
and their cyclization derivatives from the bis-1,2,4-triazole class with potential
biological activity are described.
The newly obtained compounds were screened in vitro for their anticancer
and antimicrobial activity.
RESULTS AND DISCUSSION
Synthesis
The carboxylic acid hydrazides 1a–g, the key intermediates used for the
synthesis of the 1,6-bis{[(2-substituted hydrazinyl)carbonyl]amino}hexanes 2a–g,
26,27
were synthesized according to a literature method.
New semicarbazide deri-
vatives were obtained by the reaction of corresponding carboxylic acid hydrazide
1 with 1,6-hexamethylene diisocyanate (HDI). The reaction medium was diethyl
ether and the process was realized at room temperature. Next, the obtained semi-
carbazides were subjected to cyclization in a 2 % solution of sodium hydroxide
obtaining the corresponding 1,6-bis(3-substituted 1,5-dihydro-5-oxo-4H-1,2,4-
-triazol-4-yl)hexanes 3a–g. The synthetic pathway followed for the preparation
of the title compounds is presented in Scheme 1.
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SYNTHESIS AND ACTIVITY OF BIS-SEMICARBAZIDES AND BIS-1,2,4-TRIAZOLES
3
Comp.
R
a
b
c
CH3
C6H5
CH2
N
CH3
d
e
H
f
N
g
N
Scheme 1. Synthesis of the 1,6-bis[(hydrazinylcarbonyl)amino)hexanes 2a–g and of the
1,6-bis(5-oxo-1,2,4-triazol-4-yl)hexanes 3a–g.
Characterization
1
The structures of the synthesized compounds were elucidated by H-NMR,
C-NMR, MS and IR spectroscopy. Analytical and spectral data of the synthe-
13
sized compounds are given in the Supplementary material.
The reaction products may exist in two different tautomeric forms (Scheme
1
1). The IR and H-NMR data indicated that the obtained cyclization products
exist in the keto form both in the solution and in the solid. In the IR spectra of the
cyclic compounds containing the 1,2,4-triazole system, absorption bands of the
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PITUCHA et al.
–1
1
C=O group at 1683–1651 cm were observed. H-NMR spectra showed protons
signals for the –N–C(O)–NH– group in the δ range 8.00–10.46 ppm.
Preliminary anticancer screening
The bis-1,2,4-triazol-3-one derivatives 3a–c were evaluated for their anti-
proliferative and anticancer activity in two human cell lines derived from lung
and breast carcinoma cells. One normal cell line was included in the cytotoxicity
study – primary cell line of human skin fibroblasts (HSF). The results for each
tested compound are reported as the growth inhibition percentage of the tested
cells in comparison with the untreated cells. According to the data listed in Table
I, compounds 3a–c were found to be the most effective against human lung car-
cinoma cells in vitro. In the case of the human breast cancer cell line, a slight in-
hibition for compounds 3a and 3b was observed. A non-cytotoxic or stimulation
effect of compound 3a referring to normal cell line HSF and several-fold higher
effect on the two observed carcinoma cell lines were ascertained. The investi-
gated compounds exhibited dose-dependent effects. The most evident action was
–1
observed for all the examined compounds at a 50 μg mL dose.
TABLE I. In vitro inhibition of the growth of normal and cancer cells by compounds 3a–c;
the examined concentration of compounds: I – a concentration of 100 µg mL-1, which
corresponds to a concentration of 0.35 (3a), 0.25 (3b) and 0.23 mM (3c); II – a concentration of
50 µg mL-1, which corresponds to a concentration of 0.18 (3a), 0.12 (3b) and 0.11 mM (3c);
III – a concentration of 10 µg mL-1, which corresponds to a concentration of 0.04 (3a), 0.02
(3b) and 0.02 mM (3c)
Growth inhibition factor, GI / %
Compound
Time of incubation
Cell
3a
3b
Dose
3c
h
I
II III
I
II III
I
II III
Human skin fibroblast HSF
24
48
72
24
48
72
24
48
72
0
0
0
0
0
5
5
5
0
0
0
0
0
0
0
0
0
5
5
5
5
5
5
5
5
0
0
5
5
5
5
5
0
5
5
0
0
5
0
5
5
5
10
10
0
0
5
5
5
5
Human lung cancer cell line A549
Human breast cancer cell line T47D
22
25
25
5
10 10
10 10
10 10 10
20 10 25 25
20 10 25 25
0
10 10
10
0
0
5
0
5
5
0
0
Antimicrobial activity
Selected compounds, 2a–c and 3a–c, were screened for their possible anti-
microbial activities against Staphylococcus aureus ATCC 25923, Klebsiella pneu-
moniae ATCC 700603, Enterococcus faecalis ATCC 2912, Escherichia coli ATCC
25822, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC
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SYNTHESIS AND ACTIVITY OF BIS-SEMICARBAZIDES AND BIS-1,2,4-TRIAZOLES
5
90028, and clinical isolates of C. albicans, C. parapsilosis, C. kefyr, and C. tropi-
calis. The antimicrobial susceptibility to the tested compounds was determined
by MIC (minimum inhibitory concentration) values and compared to amphoteri-
cin B and meropenem as standard drugs. All the studied bacterial species were
–1 28
classified as resistant to the standard meropenem, MIC > 1.0 µg mL , and the
studied species Candida spp. as resistant to the standard amphotericin B, MIC >
–1 29
–1
> 16.0 µg mL . Compounds 3a and 3b at concentrations 3.75 and 1.87µg mL ,
respectively, were active against the tested C. albicans (clinical isolates, 3b), C.
albicans ATCC 90028 (3b) and C. tropicalis (3a, Table II). The other com-
pounds were also found to inhibit growth of Candida spp. at a concentration of 7.5
–1
µg mL . All the tested derivatives showed in vitro effectiveness against the five
reference species: S. aureus ATCC 25923, K. pneumoniae ATCC 700603, E. fae-
calis ATCC 2912, E. coli ATCC 25822 and P. aeruginosa ATCC 27853 at a
–1
concentration of 7.5 µg mL .
TABLE II. Minimum inhibitory concentration (MIC / µg mL-1) of compounds 2a–c and 3a–c;
Ca* – C. albicans ATCC 90028, Ca - C. albicans, Cp - C. parapsilosis, Ck - C. kefyr, Ct- C.
tropicalis, Sa- S. aureus ATCC 25923, Kp - K. pneumoniae ATCC 700603, Ef - E. faecalis
ATCC 2912, Ec – E. coli ATCC 25822, Pa- P. aeruginosa ATCC 27853, ST - Standard:
*amphotericin B, **meropenem
Microorganism
Compound
Ca*
7.5
7.5
7.5
7.5
Ca
7.5
7.5
7.5
7.5
Cp
7.5
7.5
7.5
7.5
7.5
7.5
Ck
7.5
7.5
7.5
7.5
7.5
7.5
Ct
7.5
7.5
7.5
3.75
7.5
7.5
Sa
7.5
7.5
7.5
7.5
7.5
7.5
Kp
7.5
7.5
7.5
7.5
7.5
7.5
Ef
Ec
7.5
7.5
7.5
7.5
7.5
7.5
Pa
7.5
7.5
7.5
7.5
7.5
7.5
2a
2b
2c
3a
3b
3c
ST
7.5
7.5
7.5
7.5
7.5
7.5
1.87 1.87
7.5 7.5
<0.5* <0.5* <0.5* <0.5* <0.5* 0.05** 0.05** 5.0** 0.5** 0.1**
EXPERIMENTAL
All chemicals were purchased from Merck or Alfa-Aesar and used without further puri-
fication. Melting points (m.p.) were determined in a Fisher-Johns block and are not corrected.
The IR spectra were recorded in KBr discs using a Specord IR-75 spectrophotometer. The 1H-
and 13C-NMR spectra were generally recorded at room temperature on a Bruker AC 200F
instrument (300 MHz) in DMSO-d6 with TMS as the internal standard. The mass spectra were
obtained using an AMD-604 mass spectrometer with a 70 eV electron beam. The purity of the
obtained compounds was checked by TLC on aluminum oxide 60 F254 plates (Merck) in
CHCl3/C2H5OH (10:1 and 10:2) solvent systems with UV or iodine visualization.
General procedure for the synthesis of 1,6-bis{[(2-substituted hydrazinyl)carbonyl]amino}-
hexanes 2a–g
A mixture of an appropriate carboxylic acid hydrazide 1 (20 mmol) and 1,6-hexame-
tylene diisocyanate (1.68 g, 10 mmol), and 10 mL of diethyl ether was kept at room tempera-
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PITUCHA et al.
ture for 24 h. Then, the formed compound was filtered off, washed with diethyl ether and
crystallized from ethanol.
General procedure for the synthesis of 1,6-bis(3-substituted 1,5-dihydro-5-oxo-4H-1,2,4-tri-
azol-4-yl)hexanes 3a–g
The appropriate semicarbazide 2 (10 mmol) was placed in a round-bottomed flask equip-
ped with reflux and 40–50 mL of a 2 % sodium hydroxide solution was added. The flask was
heated for 10 h. After cooling, the solution was neutralized with dilute hydrochloric acid. The
precipitate was filtered off and then crystallized from ethanol.
Proliferation of tumor cells assay
The synthesized compounds 3a–c were evaluated for their anticancer activity in human
tumor cell lines derived from lung and breast carcinoma cells. The studies was performed on
A549 (ECACC 86012804 human lung epithelial) cells and T47D (ECACC 85102201 human
breast epithelial). The influence of the newly synthesized triazoles on human skin fibroblast
cells (HSF) was also determined. The cell lines were incubated at 104 cells per mL density on
microtiter plates. The tested compounds were then added at three examined concentrations:
10, 50 and 100 µg mL-1, and the cultures were incubated under standard conditions (37 °C, 5 %
CO2 and 90 % humidity) for 24, 48 and 72 h. The determinations were realized by 5-bromo-
-2’-deoxyuridine (BrdU) labeling30,31 and detection kit (Roche) on an ELISA reader (BIO-
-TEC Instruments, USA). The viability of normal and carcinoma cells were evaluated spectro-
photometrically. The results of all spectrophotometric measurements were registered as per-
cent growth inhibition or growth stimulation. All experiments were repeated in triplicate.
Antifungal screening
Clinical isolates of Candida albicans, C. parapsilosis, C. kefyr and C. tropicalis and C.
albicans ATCC 90028, all susceptible to amphotericin B (as determined by ATB fungitest-
-bioMerieux), were tested for their susceptibility to the newly obtained compounds. Stock so-
lutions were prepared in dimethyl sulfoxide (DMSO), at a working concentration of 300 mg
mL-1. Final dilutions were prepared in RPMI 1640 medium (Sigma) buffered to pH 7.0, 0.165 M
4-morpholinepropanesulfonic acid (MOPS) buffer (Sigma) in 96-well plates (NUNC). The
concentration of the Candida species in the final inoculums was (1.5±1.0)×103 cells mL-1
RPMI 1640. All strains were incubated for 48 h at 35 °C.32-34 As controls, DMSO and ampho-
tericin B were used. The results were obtained spectrophotometrically (570 nm) and compared
to the control results.35
Antibacterial screening
Antibacterial susceptibility was determined on following strains: S. aureus ATCC 25923,
K. pneumoniae ATCC 700603, E. faecalis ATCC 2912, E. coli ATCC 25822 and P.
aeruginosa ATCC 27853. Dilutions of the tested compounds were prepared from the stock
solutions in DMSO. The susceptibilities of the bacterial strains 1.0 x 103 cells mL–1 to the
tested compounds were determined by the CLSI microlitre broth dilution method,22,36
performed in 96-well plates (NUNC) – 200 µL per well, at 35 °C for 24 h. As the control,
meropenem at the same dilutions in DMSO was used for bacterial strains cultured under the
same conditions. The results were obtained spectrophotometrically at 570 nm.
CONCLUSIONS
In the present study, novel series of 1,6-bis{[(2-substituted hydrazinyl)carbo-
nyl]amino}hexanes and 1,6-bis(3-substituted 1,5-dihydro-5-oxo-4H-1,2,4-triazol-
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SYNTHESIS AND ACTIVITY OF BIS-SEMICARBAZIDES AND BIS-1,2,4-TRIAZOLES
7
-4-yl)hexanes were synthesized and characterized. The new derivatives 3a–c
were screened in vitro for their antiproliferative and anticancer activity in human
tumor cell lines derived from breast and lung carcinoma cells. Compounds 3a (at
a concentration of 0.18 mM), 3b (at concentrations of 0.12 and 0.02 mM) and 3c
(at concentrations of 0.23 and 0.11 mM) were found to be the most effective
against the lung cell line. Compounds 2a–c and 3a–c were screened for their
antimicrobial activities. All the tested derivatives showed MIC values in range
–1
1.87–7.5 μg mL .
SUPPLEMENTARY MATERIAL
Аnalytical and spectral data of synthesized compounds are available electronically from
http://www.shd.org.rs/JSCS/, or from the corresponding author on request.
И З В О Д
СИНТЕЗА 1,6-ХЕКСАНДИИЛ-БИС(СЕМИКАРБАЗИДА) И 1,6-ХЕКСАНДИИЛ-БИС-
(1,2,4,-ТРИАЗОЛ-5-ОНА) И ЊИХОВА АНТИПРОЛИФЕРАТИВНА И
АНТИБАКТЕРИЈСКА АКТИВНОСТ
M. PITUCHA1, J. RZYMOWSKA2, A. OLENDER3 и L. GRZYBOWSKA-SZATKOWSKA4
1Department of Organic Chemistry, Medical University, 20-081 Lublin, Poland, 2Department of Biology and
Genetics, Medical University, 20-081 Lublin, Poland, 3Chair and Department of Microbiology, Medical
University, 20-093 Lublin, Poland и 4Department of Oncology, Medical University, 20-090 Lublin, Poland
Синтетисана је серија 1,6-бис(3-супституисаних 1,5-дихидро-5-оксо-4H-1,2,4-триазол-4-
-ил)хексана 3a–g реакцијом циклизације 1,6-бис{[(2-супституисаних хидразинил)карбо-
нил]}хексана 2a–g под базним условима. Испитана је in vitro антипролиферативна активност
нових деривата 3a–3c према ћелијским линијама хуманих тумора дојке и плућа. Утврђено је
да су једињењења 3а (при концентрацији 0,18 mM), 3b (при концентрацијама 0,12 и 0,02
mМ) и 3c (при концентрацијама 0,23 и 0,11 mМ) најактивнија према ћелијској линији рака
плућа. Једињење 3а је најактивније према ћелијској линији рака дојке. Репрезентативним
једињењима испитана је антимикробна активност. Сва испитана једињења показују MIC
вредности у опсегу 1,87–7,5 µg/mL. Једињење 3b је најактивније према C. albicans (MIC =
= 1,87 µg/mL).
(Примљено 12. фебруара, ревидирано 14. августа 2011)
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Available online at www.shd.org.rs/JSCS/
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