Thermal behaviour of some biologically active species based on complexes with a triazolopyrimidine pharmacophore

Article abstract of DOI:10.1007/s10973-016-5425-7

A series of complexes of type M(pmtp)(CH₃COO)₂·nH₂O ((1) M: Co, n?=?4.5; (2) M: Ni, n?=?3.5; (3) M: Cu, n?=?0.5; (4) M: Zn, n?=?0; pmtp: 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine) were synthesised by one-pot condensation. The complexes were characterized by elemental analysis, ESI–MS, IR, UV–Vis-NIR, EPR spectra, magnetic susceptibility at room temperature as well as thermogravimetric analysis. The modifications in the IR spectra of complexes are in accordance with the condensation process and indicate the triazolopyrimidine coordination as unidentate. The electronic and EPR spectra together with magnetic moments indicate an octahedral stereochemistry for paramagnetic ions, except for Cu(II) with a square pyramidal geometry. Processes such as water elimination, acetate into carbonate transformation, as well as oxidative degradation of the triazolopyrimidine derivative were evidenced during thermal decomposition. The complexes exhibited an improved antimicrobial activity in comparison with the ligand, on both planktonic and biofilm-embedded microbial cells. The best antimicrobial activity in both susceptible and resistant strains was obtained for complex (3) followed by complex (1), which can be related to both oxidation state and stereochemical versatility. At high concentrations, the Cu(II) complex exhibits both cytotoxicity on HT 29 tumour cell line and anti-inflammatory activity.

Full text of DOI:10.1007/s10973-016-5425-7

J Therm Anal Calorim
DOI 10.1007/s10973-016-5425-7
Thermal behaviour of some biologically active species based
on complexes with a triazolopyrimidine pharmacophore
Rodica Olar1 Larisa Calu1,2 Mihaela Badea1 Mariana Carmen Chifiriuc3,4
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Coralia Bleotu5 Bruno Velescu6 Oana Stoica1 Gabriela Ionita7
Nicolae Stanica7 Luigi Silvestro8 Constant¸a Dulea9 Valentina Uivarosi6
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Received: 30 October 2015 / Accepted: 24 March 2016
´
´
Ó Akademiai Kiado, Budapest, Hungary 2016
Abstract A series of complexes of type M(pmtp)(CH3
COO)2ÁnH2O ((1) M: Co, n = 4.5; (2) M: Ni, n = 3.5; (3) M:
Cu, n = 0.5; (4) M: Zn, n = 0; pmtp: 5-phenyl-7-methyl-
1,2,4-triazolo[1,5-a]pyrimidine) were synthesised by one-pot
condensation. The complexes were characterized by ele-
mental analysis, ESI–MS, IR, UV–Vis-NIR, EPR spectra,
magnetic susceptibility at room temperature as well as ther-
mogravimetricanalysis. Themodificationsinthe IRspectraof
complexes are in accordance with the condensation process
and indicate the triazolopyrimidine coordination as uniden-
tate. The electronic and EPR spectra together with magnetic
moments indicate an octahedral stereochemistry for param-
agnetic ions, except for Cu(II) with a square pyramidal
geometry. Processes such as water elimination, acetate into
carbonate transformation, as well as oxidative degradation of
the triazolopyrimidine derivative were evidenced during
thermal decomposition. The complexes exhibited an
improved antimicrobial activity in comparison with the
ligand, on both planktonic and biofilm-embedded microbial
cells. The best antimicrobial activity in both susceptible and
resistant strains was obtained for complex (3) followed by
complex (1), which can be related to both oxidation state and
stereochemical versatility. At high concentrations, the Cu(II)
complex exhibits both cytotoxicity on HT 29 tumour cell line
and anti-inflammatory activity.
& Valentina Uivarosi
valentina_uivarosi@yahoo.com
1
Department of Inorganic Chemistry, Faculty of Chemistry,
University of Bucharest, 90-92 Panduri Str., Sector 5,
050663 Bucharest, Romania
2
Cellular Dynamics and Flow Cytometry Department,
National Institute R&D for Biological Sciences, 296 Splaiul
Independentei, 060031 Bucharest, Romania
Keywords Complex Á Cytotoxicity Á Biofilm Á
5-Phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine Á
Thermal behaviour
3
Department of Microbiology, Faculty of Biology, University
of Bucharest, 1-3 Aleea Portocalelor Str., 60101 Bucharest,
Romania
4
Research Institute of the University of Bucharest, Spl.
Independentei 91-95, Bucharest, Romania
Introduction
5
Stefan S Nicolau Institute of Virology, 285 Mihai Bravu
Ave., Bucharest, Romania
During the last decades, fused heterocycles bearing 1,2,4-
triazolo[1,5-a]pyrimidine scaffold proved to be of phar-
maceutical interest due to the diverse biological activities
of these derivatives [1–5]. For example, a derivative
functionalized with N-substituted piperidine proved a very
good antiviral activity against the wild-type HIV-1 strain,
being more potent than the currently used drugs [3].
Polycondensed species with steroid nucleus showed potent
inhibitory activity against PC-3 (human prostatic carci-
noma), MCF-7 (human breast carcinoma) and EC9706
(human oesophageal carcinoma) cell lines [4].
6
Department of Inorganic Chemistry, Carol Davila University
of Medicine and Pharmacy, 6 Traian Vuia Str., Bucharest,
Romania
7
‘‘Ilie Murgulescu’’ Physical Chemistry Institute, Romanian
Academy, 202 Splaiul Independentei, 77208 Bucharest,
Romania
8
3S-Pharmacological Consultation and Research GmbH, 1
Koenigsbergerstrasse, 27243 Harpstedt, Germany
9
Pharma Serv. International SRL, 52 Sabinelor Str., Sector 5,
050853 Bucharest, Romania
123

R. Olar et al.
The interest aroused by this type of compounds comes
not only from their resemblance with naturally occurring
purine nucleobases [6], but also from their ability to act as
suitable building blocks for supramolecular complexes
[6–9]. As a result, considering the remarkable importance
of these heterocycles from the pharmacological point of
view, great efforts were made to improve their activity by
coordination. These studies revealed that triazolopyrim-
idines have several nitrogen atoms that can bind to a
metallic ion. However, binding through the endocyclic N3
represents the main coordinative site for the former species,
as well as for their derivatives substituted with non-coor-
dinative groups [6].
1,2,4-triazolo[1,5-a]pyrimidine (dbtp) and 5,7-diphenyl-
1,2,4-triazolo[1,5-a]pyrimidine (dptp) have been active
against staphylococcal strains, susceptible or resistant to
methicillin [35].
The thermal behaviour of a Pd(II) complex with thiourea
and uncoordinated dmtp was reported [36]. After dehy-
dration, the pyrolitic decomposition starts at 300 °C.
In this work we report the synthesis, physico-chemical
and biological characterization of some metal(II) com-
plexes with mixed ligand (acetate and 5-phenyl-7-methyl-
1,2,4-triazolo[1,5-a]pyrimidine (pmtp)), in order to design
novel metal based biologically active candidates for the
antimicrobial therapy. The thermal behaviour of these
derivatives was investigated in synthetic air by thermo-
gravimetric analysis (TG, DTG, DTA) in order to evidence
the modifications of complexes at heating. The bioevalu-
ation of antimicrobial, anti-inflammatory and cytotoxic
activity of the obtained compounds was also performed.
So far, the study concerning these derivatives has been
mainly focused on unsubstituted 1,2,4-triazolo-[1,5-
a]pyrimidine (tp) [10–15], its monomethylated derivative
in position 5 or 6 (mtp) [14] and dimethylated (dmtp)
[7, 12, 19–32] or other dialkylated species in positions 5
and 7 with bulky substituents [6, 12, 13, 23, 32, 34, 35].
Concerning biological properties, Cu(II) compounds
with mixed ligands (tp/dmtp and 2,20-bipyrimidine) exhibit
a low cytotoxicity and in vitro antiparasitic activity against
the aetiological agents of leishmaniasis (Leishmania
infantum, Leishmania braziliensis) and Chagas disease
(Trypanosoma cruzi) [17]. Complexes of Co(II), Ni(II) and
Cu(II) with dmtp exhibited also a strong inhibitory poten-
tial against these parasites [33].
Experimental
Materials
The high purity reagents were purchased from Sigma-
Aldrich (Co(CH3COO)2Á4H2O, Ni(CH3COO)2Á4H2O,
Cu(CH3COO)2ÁH2O, Zn(CH3COO)2Á2H2O), Merck (ben-
zoylacetone) and Fluka (3-amino-4H-1,2,4-triazole) and
used as received without further purification.
The antitumour activity was studied mostly for Pt(II)
and Pt(IV) complexes with a cisplatin like structure.
In vitro antiproliferative activity against acute promyelo-
cytic leukaemia, rectal adenocarcinoma and bladder cancer
has been reported for platinum(IV) complex with ditert-
butyl substituted triazolopyrimidine [31] and one superior
to cisplatin on B16 melanoma cell lines for Pt(II) with
dmtp and hexafluoroglutarate [32]. Platinum(II) complexes
with mixed ligands (malonate and 7-isobutyl-5-methyl-
1,2,4-triazolo[1,5-a]pyrimidine (ibmtp)/5,7-ditertbutyl-1,
2,4-triazolo[1,5-a]pyrimidine (dbtp)) exhibited substantial
growth inhibitory properties against T47D (cisplatin-re-
sistant human ductal breast epithelial tumour line) cells
[32]. The bulky substituent presence might be an important
factor in the modulation of antitumour activity [31].
Also, NAMI(-A)-like compounds of Ru(III) with dmtp
inhibit significantly the metastatic growth in mice with
advanced mammary carcinoma tumours [30], and a high
cytotoxicity was evidenced as well for complexes with
bulky triazolopyrimidine ligands [34].
Instruments
Chemical analysis of carbon, nitrogen and hydrogen has
been performed using a Perkin Elmer PE 2400 analyzer.
The molar conductance was determined for 10-3
M
solutions of complexes in DMSO with a multiparameter
analyser CONSORT C861.
IR spectra were recorded in KBr pellets with a Bruker
Tensor 37 spectrometer in the range 400–4000 cm-1
.
1H NMR spectra were recorded on a Bruker DPX200
spectrometer (working frequency 200 MHz) at 25 °C.
Chemical shifts were measured in parts per million from
the internal standard TMS.
Mass spectra were recorded with API 6500 ABSCIEX-
Canada mass spectrometer with ESI source (Turbo spray
model ABSCIEX) operating in a positive mode. Samples
were dissolved in DMF at 1 mg mL-1, and then, the
solution was diluted with methanol up to a final concen-
tration of 1 lg mL-1. Molecular ions scanning range (m/z)
was 0–1250.
Some dmtp complexes with Co(II) are good in vitro
inhibitors of both Gram positive and Gram negative bac-
terial strains [21], while diorganotin(IV) species were
found to have promising antibacterial activity, against both
planktonic and antibiofilm-embedded strains [25]. More-
over, new diorganotin(IV) derivatives of 5,7-ditertbutyl-
Electronic spectra by diffuse reflectance technique, with
spectralon as standard, were recorded in the range
200–1500 nm, on a Jasco V670 spectrophotometer.
123

Thermal behaviour of some biologically active species based on complexes…
Anti-inflammatory assays
Magnetic measurements were done by Faraday’s
method, at room temperature, using Hg[Co(NCS)4] as
standard. The molar magnetic susceptibilities were calcu-
lated and corrected for the atomic diamagnetism.
Male Wistar rats weighing (202 20 g) from the Can-
tacuzino Institute, Bucharest, Romania animal facility
(rodent farm), were used. Inflammation was evaluated in
two acute inflammation experimental models by plethys-
mometry as reported [38]. The anti-inflammatory effect
was compared with a control group (untreated rats), a
reference substance (diclofenac) and a group treated with
the ligand. For both inflammatory agents (dextran and
kaolin), the control rats were treated by gavage with dis-
tilled water, 1 mL/100 g body weight (b.w.); the reference
rats were treated by gavage with 1 % solution sodium
diclofenac, 100 mg/kg b.w., and the experimental groups
were treated by gavage with 100 mg/kg b.w. of 1 %
aqueous suspension of ligand or complexes. After the
administration of substances, all animals were anaes-
thetized intraperitoneally with urethane, and the initial paw
volume was measured. The inflammation was induced by
intraplantar administration of 0.2 mL inflammatory agent
(0.6 % solution of dextran and 10 % aqueous suspension of
kaolin) into the rat’s inferior right paw. The paw oedema
was further evaluated at 1, 2, 3, 4, 5 and 24 h, respectively.
The X-band EPR measurements were recorded at 293 K
for solid samples and at 100 K for DMSO solutions, on a
JEOL FA100 spectrometer. The general settings used were
as follows: sweep field 1000 G, frequency 100 kHz, gain in
the range 100–200, sweep time 1800 s, time constant 1 s,
modulation width 2 G, microwave power 1 mW. The
magnetic field calibration was performed with a DPPH
(diphenylpicrylhydrazyl) standard marker, exhibiting a
narrow EPR line at g = 2.0036.
The heating curves (TG, DTG and DTA) were recorded
using a Labsys 1200 SETARAM instrument with a sample
mass of 8–11 mg over the temperature range of
293–1173 K, using a heating rate of 10 K min-1. The
measurements were carried out in synthetic air atmosphere
(flow rate 16.7 cm3 min-1) by using alumina crucibles.
The X-ray powder diffraction patterns were collected on
a DRON-3 diffractometer with a nickel-filtered Cu Ka
˚
radiation (k = 1.5418 A) in 2h 5°–70° range a step width
of 0.05° and an acquisition time of 2 s per step.
Synthesis, analytical and spectral data for ligand
and complexes
Antimicrobial assays
The antimicrobial activity of the obtained compounds was
assayed on a large spectrum of microbial strains, including
the following: Gram negative (Escherichia coli ATCC
25922, E. coli 832, Klebsiella pneumoniae ATCC 134202,
K. pneumoniae 806, Pseudomonas aeruginosa ATCC
27853, P. aeruginosa 392) and Gram positive (Staphylo-
coccus aureus MRSA 1263, S. aureus ATCC 25923,
Bacillus subtilis ATCC 6633) bacterial strains, as well as
the Candida albicans 22 fungal strain. The qualitative
screening was performed by an adapted disc diffusion
method, as previously reported [37]. The assessment of
influence of compounds on the microbial ability to colonize
an inert substratum was performed by the microtiter
method, following the previously described protocols [37].
All biological experiments were performed in triplicates.
To a solution containing 1 mmol metal (II) acetate
hydrated (M: Co, Ni, Cu and Zn) in 15 mL ethanol,
1 mmol 3-amino-4H-1,2,4-triazole dissolved in 5 mL
ethanol and 1 mmol benzoylacetone dissolved in 5 mL
ethanol were added. The reaction mixture was magneti-
cally stirred at 50 °C for 7 h, until a sparingly soluble
species was formed. The precipitate was filtered off,
washed several times with cold ethanol and air-dried.
[Co(pmtp)(CH3COO)2(OH2)2]Á2.5H2O (1): Yield 66 %;
ESI–MS (CH3OH:DMF) m/z: [M?H?CH3OH–4.5H2O]?,
420; [M?9H–CH3COO–4.5H2O]?, 338; [pmtp?H]?, 211.
[Ni(pmtp)(CH3COO)2(OH2)2]Á1.5H2O (2): Yield 59 %;
ESI–MS (CH3OH:DMF) m/z: [M?9H?CH3OH–3.5H2O–
CH3]?, 413; [M?9H–CH3COO–3.5H2O]?, 328; [pmtp?
H]?, 211.
[Cu(pmtp)(CH3COO)2]Á0.5H2O (3): Yield 56 %; ESI–
MS (CH3OH:DMF) m/z: [M?H?CH3–0.5H2O]?, 408;
[M?H–CH3–0.5H2O]?, 329; [pmtp?H]?, 211.
Cytotoxicity assay
[Zn(pmtp)(CH3COO)2] (4): Yield 62 %; ESI–MS
(CH3OH:DMF) m/z: [M?H?CH3OH]?, 428; [M?3H–
CH3COO]?, 338; [pmtp?H]?, 211.
The cytotoxicity of compounds was evaluated on human
tumour cell line HT 29 (human colon adenocarcinoma) by
using the CellTiter method [37]. Cell cycle was determined
after incubation with RNase A and propidium iodide. After
staining of cells, the acquisition was done using Epics
Beckman Coulter flow cytometer. Data were analysed
using FlowJo software and expressed as fractions of cells in
the different cell cycle phases [37].
The organic derivative 5-phenyl-7-methyl-1,2,4-tria-
zolo[1,5-a]pyrimidine was synthesized by [1 ? 1] con-
densation of benzoylacetone with 3-amino-4H-1,2,4-
triazole and identified through chemical analysis (Table 1),
1
IR (Table 2), H NMR and 13C NMR spectra and single-
123

R. Olar et al.
Table 1 Analytical data of complexes
Compound
Chemical formula
Mw/gmol-1
% Found (calcd.)
C
KM/X-1cm2 mol-1
H
N
pmtp
(1)
C12H10N4
210.24
936.65
900.14
801.76
787.47
68.67 (68.56)
41.11 (41.03)
42,78 (42,70)
48.02 (47.94)
48.87 (48.81)
4.65 (4.79)
5.29 (5.38)
5,01 (5.15)
4.16 (4.27)
3,98 (4.10)
26.72 (26.65)
11.98 (11.96)
12.53 (12.45)
14.12 (13.97)
14.32 (14.23)
–
Co2C32H50N8O17
Ni2C32H46N8O15
Cu2C32H34N8O9
Zn2C32H32N8O8
9.5
(2)
13.0
11.8
15.0
(3)
(4)
Table 2 IR absorption bands (cm-1) for 5-phenyl-7-methyl-1,2,4-
triazolo[1,5-a]pyrimidine (pmtp) and complexes (1)–(4)
3-amino-4H-1,2,4-triazole and benzoylacetone. The com-
plexes were characterized based on data provided by ESI MS,
IR, UV–Vis-NIR, and EPR spectroscopy, and magnetic and
thermalanalyses. Allcomplexesaresparinglysolubleinwater
and the common organic solvents, except for DMSO and
DMF.
pmtp
(1)
(2)
(3)
(4)
Assignment
–
3425m
3093m
2926w
2828w
1640m
1594m
1578vs
1562vs
1420s
1397s
181
3437m
3094m
2939w
2818w
1643m
covered
1598vs
1548vs
1424s
1400s
198
3481m
3095m
2954w
2879w
1621s
–
m(OH2)
m(CH)
3001m
2932w
2833w
1613s
1540vs
–
3099m
2912w
2824w
1622s
covered
1594vs
1526s
1406m
1399s
195
mas(CH3)
ms(CH3)
m(C=N) mtp
mpy
ESI–MS and NMR spectra
1594m
1518vs
The m/z values identified in the MS spectra confirm the
structures proposed in Scheme 1 for complexes. In the posi-
tive mode the pseudomolecular ion was found as
[Co(pmtp)(CH3COO)2?CH3OH?H]? (m/z: 420), [Ni(pmtp)
(CH3COO)2?9H?CH3OH–CH3]? (m/z: 413), [Cu(pmtp)
(CH3COO)2?H?CH3]? (m/z: 408) and [Zn(pmtp)(CH3
COO)2?2H?CH3OH]? (m/z: 428), respectively that repre-
sent the proof for a mononuclear structure. For complexes (1),
(2) and (4), these pseudomolecular ions represent the adducts
with methanol formed after elimination of all water molecules
and ligand protonation. In the case of Ni(II) complex, the
elimination of methyl radical together with four double bonds
reduction in pmtp structure was observed. For complex (3)
the addition of one methyl radical after ligand protonation
leads to the pseudomolecular ion. Moreover, other fragments
without one acetate anion or that of the free ligand (m/z: 211)
were identified in all complexes’ spectra.
mas(COO)
–
–
1378s
140
ms(COO)
DCOO
142
124
120
840w
843w
842w
849w
690m
547w
491w
843w
684m
580w
489w
c(CH)
–
–
–
692 m
548w
687 m
587w
d(COO)
m(M–O)
m(M–N)
485w
479w
vs
very
strong,
s
strong,
m
medium,
w
weak,
DCOO = mas(COO) - ms(COO)
crystal X-ray analysis, data reported elsewhere. ESI–MS
(CH3OH:DMF) m/z: [M?H]?, 211.
1
In the H NMR spectrum of ligand the peak for proton
of triazolo group appears as a singlet at 8.62 ppm, while
the singlet at 7.68 ppm is characteristic for pyrimidine ring
[40, 41]. The low solubility of compound (4) in all
deuterated solvents does not allow 1H NMR spectrum
registration.
Results and discussion
The condensation of 3-amino-4H-1,2,4-triazole and benzoy-
lacetone in the presence of cobalt(II), nickel(II), copper(II) or
zinc(II) acetate produced the species [Co(pmtp)(CH3
COO)2(OH2)2]Á2.5H2O (1), [Ni(pmtp)(CH3COO)2(OH2)2]Á
1.5H2O (2) [Cu(pmtp)(CH3COO)2]Á0.5H2O (3), [Zn(pmtp)
(CH3COO)2] (4) (pmtp: 5-phenyl-7-methyl-1,2,4-triazolo
[1,5-a]pyrimidine) as chemical analyses indicated (Table 1).
This formulation is sustained also by non-electrolyte beha-
viour of these compounds, since the molar conductivities in
DMSO range between 9.5 and 15.0 X-1cm2 mol-1 (Table 1)
[39]. The ligand 5-phenyl-7-methyl-1,2,4-triazolo[1,5-
a]pyrimidine(pmtp)was obtainedby[1 ? 1]condensationof
Infrared spectra
The most relevant IR bands and assignments for the
complexes are summarized in Table 2. In the IR spectrum
of pmtp two strong bands appear, one at 1613 cm-1
assigned to stretching vibration of triazolopyrimidine
moiety (mtp) and a second one at 1540 cm-1 assigned to the
stretching vibration for pyrimidine ring (mpy) [7, 9, 11–36].
123

Thermal behaviour of some biologically active species based on complexes…
The band corresponding to mtp stretching vibration is
The Ni(II) complex exhibits also an octahedral geometry
shifted towards higher wavenumbers by 8–30 cm-1 in
complexes’ spectra, as an indicative of ligand coordination
through the triazolo nitrogen N3 [7, 9, 11–36].
indicated by the three low intensity bands that can be
assigned to spin-allowed transitions [45]. These bands are
broad as result of distortion. The splitting parameter value
of 9305 cm-1 is according to an [Ni(II)NO5] chromophore
having a high number of oxygen donor atoms that generate
a low field, while the nephelauxetic parameter of 0.85
indicates also a lower degree of covalency. This stereo-
chemistry is further sustained by the room temperature
magnetic moment of 3.11 B.M., which is close to the spin-
only magnetic moments (3.3 B.M.) for two unpaired
electrons, as usually observed for complexes with an
A ground term [47].
On the other hand, the acetate function in complexes is
very well documented by comparison of IR data with single-
crystal X-ray diffraction studies. It was observed for a wide
range of acetate complexes with a known structure, that the
difference (D) between mas(COO) and ms(COO) vibrations,
provides useful information concerning the acetate presence
as free ion or as ligand, respectively [42]. Taking into con-
sideration these data, D values in the range 120–142 cm-1
can be considered an indicative of the acetate coordination as
bidentate for all complexes. A second criterion used to dif-
ferentiate the chelate for bridge coordination takes into
consideration the shifting of the band assigned to mas(COO)
in comparison with CH3COONa, for which the two bands
corresponding to mas(COO) and ms(COO) appear at 1578 and
1414 cm-1, respectively [42]. For all complexes, the shifting
of one of the bands assigned to mas(COO) to lower
wavenumbers suggests the acetate presence rather as chelate
[42, 43]. Instead, for complexes (1), (2) and (4) in the char-
acteristic ranges for these vibrations four strong bands can be
noticed. The difference between symmetrical and asym-
metrical vibration modes in the range 180–198 cm-1 could
indicate the acetate coordination as unidentate as well [42].
In the IR spectra of complexes, a broadband assigned to
m(OH) appears in the 3405–3481 cm-1 range, except for
complex (4) [44].
The electronic spectrum of the Cu(II) complex shows
the characteristic broad band with a maximum at
14,600 cm-1 tentatively assigned to dxz, dyz ? dðx2Ày2Þ
transition in a square pyramidal stereochemistry, since the
maximum absorption is located at higher energy [45]. The
value of 1.60 B.M. for the magnetic moment indicates a
weak interaction between paramagnetic ions with S = 1/2
at room temperature that may arise from an antiferro-
magnetic interaction [47].
Powder EPR spectrum of Cu(II) complexes usually
reveals information concerning both local metal ion sur-
rounding and monomeric or dimeric structure. The X-band
EPR spectra, in both powder and DMSO frozen solution, of
complex (3) exhibit at room temperature a broad and low-
intensity signal with gav = 2.1119, characteristic to a local
symmetry of copper ion lower than an octahedral, such as
the square pyramidal one [48]. This aspect of the spectrum
can be related to a dimeric structure for which the ion pairs
can be antiferromagnetically coupled along the z axis, the
dimeric units being preserved in DMSO.
Moreover, the new bands in the ranges 540–580 and
479–491 cm-1 assigned to m(M–O) and m(M–N) support
the formation of coordinative bonds with nitrogen and
oxygen donor atoms, respectively [44].
Thermal behaviour of complexes
Electronic, EPR spectra and magnetic moments
Thermal analysis provides useful information concerning
the composition and stability of complexes. Moreover, the
thermal behaviour understanding in the case of biologically
active species represents an important step in order to
develop new metallodrugs.
Three bands can be noticed in the UV region of the pmtp
spectrum that is assigned to p ? p* intraligand transitions
(Table 3). In the complexes’ spectra, these bands are
shifted either to higher or to lower wavenumbers as result
of coordination.
In order to obtain such information, the thermal beha-
viour of complexes was investigated by simultaneous TG/
DTA analysis, while the final residue was examined by
powder X-ray diffraction. Some intermediates were iso-
lated after the water elimination and characterized in order
to establish its presence as ligand or as hydration species.
The TG/DTG/DTA curves registered for complex (1)
are shown in Fig. 1 and indicate that this species undergoes
a thermal decomposition that follows three steps. The data
collected from these curves are summarized in Table 4.
The water is eliminated in a single step up to 204 °C
indicating that some water molecules are coordinated while
The electronic spectrum of the cobalt(II) complex
exhibits a pattern characteristic for octahedral species with
the three bands assigned to spin-allowed transitions [45].
The splitting parameter value of 10,430 cm-1 is close to an
average value for a [Co(II)NO5] chromophore, while the
nephelauxetic parameter of 0.90 is an indicative of a lower
degree of covalency. The crystal field parameters (10Dq, B
¨
and b) have been calculated with Konig’s formulas [46].
The Co(II) complex exhibits a magnetic moment of
5.10 B.M. at room temperature, value in the range accepted
for octahedral species (4.3–5.2 B.M.) with three unpaired
electrons and an orbital contribution [47].
123

R. Olar et al.
Table 3 Absorption maxima, assignments and magnetic moments for ligand and complexes
Compound
pmtp
Absorption maxima/cm-1
Assignment
Magnetic moment/B.M.
–
38,460
29,850
23,255
39,215
32,255
18,870
14,600
8440
p ? p*
[Co2(pmtp)2(CH3COO)4(OH2)4]Á5H2O (1)
p ? p*
5.10
3.11
4
4T1g ? T1g
4
4T1g ? A2g
4
4T1g ? T2g
[Ni2(pmtp)2(CH3COO)4(OH2)4]Á3H2O (2)
38,465
29,855
22,990
16,390
9305
p ? p*
3
3A2g ? T1g (P)
3
3A2g ? T1g (F)
3
3A2g ? T2g
[Cu2(pmtp)2(CH3COO)4]ÁH2O (3)
[Zn2(pmtp)2(CH3COO)4] (4)
38,465
29,855
14,600
11,240
44,440
29,415
p ? p*
1.60
dxz, dyz ? dðx2Ày2Þ
dz2 ! dðx2Ày2Þ
p ? p*
–
[43, 49, 50]. The next step is not a single one being an
overlapping of several exothermic processes, according to
profile of both DTG and DTA curves. The oxidative
degradation of the both phenyl and triazolopyrimidine core
up to triazole ring occurs in the third step as mass loss
indicates. The degradation proceeds with the rest of organic
residue, process accompanied by carbonate decomposition
and cobalt oxidation as mass loss indicates. All these
transformations lead to Co3O4 as final product (ASTM
78-1970) (found/calcd. overall mass loss: 83.1/82.9 %).
For complex (2), the decomposition proceeds in three
steps (Fig. 2). In the first endothermic step, the hydration
water is eliminated up to 130 °C [51], while the coordi-
nation water molecules are eliminated up to 230 °C [52].
The oxidative degradation of both ligands (acetate and
triazolopyrimidine) occurs in the last step, that is not a
single one but an overlapping of several processes,
according to profile of both DTG and DTA curves. NiO is
formed as powder X-ray diffraction indicates (ASTM
78-0429) (found/calcd. overall mass loss: 83.4/83.4 %).
This product is in the nonstoichiometric form and contin-
uously loss oxygen from the network according to TG
curve.
0.8
0.6
0.4
0.2
TG
0
60
40
20
0
–20
–40
–60
–80
DTG
0.0
–0.2
–0.4
–0.6
–0.8
–1.0
–1.2
–1.4
–1.6
DTA
–20
200
400
600
Temperature/°C
800
1000
0
Fig. 1 TG, DTG and DTA curves for [Co(pmtp)(CH3COO)2(OH2)2]-
2.5H2O (1)
the hydration ones are trapped in the network through
multiple hydrogen bonds. In the next step, the conversion
of acetate into carbonate occurs together with methyl group
eliminations from triazolopyrimidine derivative. The small
exothermic peaks observed on DTA curve in the
204–300 °C temperature range are probably due to both
endothermic and exothermic reactions that occur simulta-
neously, such as cleavage and rearrangement of the bonds,
as well as some moieties oxidative degradation.
The thermal decomposition for complex (3) proceeds in
three steps, one endothermic and two exothermic (Fig. 3).
In the range 59–128 °C hydration water molecules elimi-
nation occurs [51]. In the second step takes place the
transformation of acetate into carbonate and degradation of
Otherwise, such behaviour was observed for other
complexes with mixed ligand for which the coordinated
acetate decomposes before other organic derivatives
123

Thermal behaviour of some biologically active species based on complexes…
Table 4 Thermal behaviour data (in synthetic air atmosphere) for complexes
Compound
Step
Thermal effect
Temperature range/°C
Dmexp/%
Dmcalc/%
[Co(pmtp)(CH3COO)2(OH2)2]Á2.5H2O (1)
1
2
3
4
Endothermic
Exothermic
Exothermic
Exothermic
59–200
200–300
300–440
440–862
16.7
16.0
27.5
22.9
16.9
5.6
17.3
15.6
27.1
22.9
17.1
6.0
(Residue Co3O4)
[Ni(pmtp)(CH3COO)2(OH2)2]Á1.5H2O (2)
[Cu(pmtp)(CH3COO)2]Á0.5H2O (3)
[Zn(pmtp)(CH3COO)2] (4)
1
2
3
Endothermic
59–130
130–230
230–783
Endothermic
Exothermic
8.2
8.0
69.6
16.6
2.4
69.4
16.6
2.2
(Residue NiO)
1
2
3
Endothermic
59–128
128–270
270–900
Exothermic
Exothermic
34.3
43.4
19.9
18.8
60.8
20.7
33.7
44.3
19.8
18.6
60.7
20.7
(Residue CuO)
1
2
Exothermic
Exothermic
240–370
370–707
(Residue ZnO)
50
3.0
20
10
0.4
TG
0
TG
2.5
0
–20
–40
–60
–80
40
0.2
2.0
DTG
–20
30
0.0
1.5
20
10
0
–0.2
–0.4
–0.6
–0.8
–1.0
–1.2
DTA
DTG
1.0
0
–40
0.5
0.0
DTA
–60
–80
–10
–20
–0.5
–1.0
–1.5
–2.0
–10
–20
–100
0
200
400
600
800
1000
200
400
Temperature/°C
800
1000
600
0
Temperature/°C
Fig. 2 TG, DTG and DTA curves for [Ni(pmtp)(CH3COO)2(OH2)2]Á
1.5H2O (2)
Fig. 3 TG, DTG and DTA curves for [Cu(pmtp)(CH3COO)2]Á0.5H2
O (3)
38 % from triazolopyrimidine ligand that corresponds with
phenyl moiety elimination. In the final step, the rest of
organic part oxidative degradation together with carbonate
decomposition occurs and several exothermic processes
end with CuO formation at 900 °C (ASTM 5-661)
(found/calcd. overall mass loss: 80.1/80.2 %).
processes, as it indicates the DTA curve profile, that cor-
respond to the organic residue pyrolysis and carbonate
decomposition with zinc (II) oxide generation (ASTM
036-1451) (found/calcd. overall mass loss: 79.6/79.6 %).
Having in view all these data a mononuclear structure
was proposed for complexes in order to correlate all phy-
sico-chemical data, as shown in Fig. 5.
Complex (4) is an anhydrous species, and as a result,
their thermal degradation starts at 250 °C (Fig. 4). The first
step corresponds to acetate into carbonate transformation
and elimination of about 12 % from the ligand, mass loss
that corresponds with methyl group elimination from tria-
zolopyrimidine derivative. The oxidative degradation of
organic components occurs as a main process in this step,
considering the strong exothermic effect noticed on DTA
curve. The final step comprises several exothermic
Antimicrobial activity assay
The antimicrobial activity of compounds was assayed
using a wide range of bacterial and fungal strains, both
reference and of clinical origin, belonging to the major
pathogenic genera that are produced which are difficult to
123

R. Olar et al.
Table 5 MIC values (lg mL-1) for complex combinations (1)–(4)
0.4
TG
0.3
40
30
20
10
0
0
–20
–40
–60
–80
Strain
(1)
(2)
(3)
(4)
0.2
0.1
E. coli ATCC 25922
E. coli 832
125
250 62.50 62.50
[1000
DTG
0.0
[1000 [1000 250
–0.1
–0.2
–0.3
–0.4
–0.5
–0.6
K. pneumoniae ATCC 134202 62.50 [1000 62.50 [1000
K. pneumoniae 806
P. aeruginosa ATCC 27853
P. aeruginosa 392
250
500 250
250 62.50 [1000
[1000
62.50 [1000 62.50 125
[1000
[1000
DTA
[1000 [1000 250
S. aureus MRSA 1263
S. aureus ATCC 25923
B. subtilis ATCC 6633
C. albicans 22
0
200
400
600
800
1000
125
[1000 62.50 [1000
125 62.50 125
Temperature/°C
125
[1000
125 125
125
Fig. 4 TG, DTG and DTA curves for [Zn(pmtp)(CH3COO)2] (4)
ATCC 25923, B. subtilis ATCC 6633 and C. albicans 22
fungal strain with a minimal biofilm eradication concen-
tration (MBEC) of 31.25 and 62.50 lg mL-1. Complexes
(1) exhibited the most evident inhibitory effect upon the
adherence ability of E. coli ATCC 25922, K. pneumoniae
ATCC 134202, K. pneumoniae 806, S. aureus MRSA
1263, S. aureus ATCC 25923 and B. subtilis ATCC 6633
with MBEC of 15.62 or 62.50 lg mL-1. In a similar range
of MBEC, complex (4) inhibited the adherence of E. coli
ATCC 25922, S. aureus MRSA 1263, B. subtilis ATCC
6633 and C. albicans 22. Complex (2) exhibited the nar-
rowest antibiofilm effect against B. subtilis ATCC 6633
treat human infections. It is to be pointed that the E. coli
832 and K. pneumoniae 806 strains were isolated from
urine culture and wound secretion, respectively and
exhibited a multidrug resistance phenotype. The assays
revealed a good antimicrobial activity for complex (3) with
a MIC value of 62.50 lg mL-1 in the case of E. coli ATCC
25922, K. pneumoniae ATCC 134202, P. aeruginosa
ATCC 27853, S. aureus MRSA 1263, S. aureus ATCC
25923 and B. subtilis ATCC 6633. Complex (1) exhibited a
good activity with an MIC of 62.50 lg mL-1 against K.
pneumoniae ATCC 134202 and S. aureus MRSA 1263. It
is also worth to mention the good activity exhibited by
complex (4) with a MIC value of 62.50 lg mL-1 in the
case of E. coli ATCC 25922. Compounds (2), (3) and (4)
inhibited the C. albicans 22 fungal growth up to an MIC of
125 lg mL-1 (Table 5).
Complexes were investigated also concerning their
efficiency against the adherent cells grown in biofilms
developed in plastic wells. From this point of view, com-
plex (3) proved to be the most active, exhibiting an
important inhibitory effect upon the adherence ability of
the E. coli ATCC 25922, K. pneumoniae ATCC 134202, P.
aeruginosa ATCC 27853, S. aureus MRSA 1263, S. aureus
and C. albicans 22 (MBEC: 31.25–62.50 lg mL-1
)
(Table 6).
The complexes (1)–(4) could have a lipophilic character
having in view their non-electrolytic behaviour as well as
the presence of hydrophobic periphery. As a result, after
diffusion through the microbial membrane these species
may interfere with the cytoplasmatic components, affecting
thus both the microbial cell viability and pathogenicity.
The good activity of complexes (1) and (3) can be related
to oxidation state and stereochemical versatility,
respectively.
N
N
N
N
N
N
OH2
N
N
O
O
N
N
M
N
O
N
OH2
O
O
O
O
Zn
(4)
O
Cu
(3)
OH2
O
O
M: Co (1)
M: Ni (2)
Fig. 5 Coordination proposed for complexes
123

Thermal behaviour of some biologically active species based on complexes…
Table 6 Influence of complexes on biofilm formation (the threshold concentration (lg mL-1) value corresponding to MBEC)
Strain
(1)
(2)
(3)
(4)
E. coli ATCC 25922
Inhibition
62.50
–
Inhibition
Inhibition
31.25
Inhibition
15.62
–
125
–
E. coli 832
Inhibition
62.50
K. pneumoniae ATCC 134202
K. pneumoniae 806
P. aeruginosa ATCC 27853
P. aeruginosa 392
Inhibition
15.62
–
Inhibition
31.25
–
–
–
–
62.50
Inhibition
125
Inhibition
125
–
–
Inhibition
125
Inhibition
31.25
–
Inhibition
125
S. aureus MRSA 1263
S. aureus ATCC 25923
B. subtilis ATCC 6633
C. albicans 22
Inhibition
31.25
–
–
Inhibition
31.25
Inhibition
62.50
–
Inhibition
31.25
Inhibition
31.25
Inhibition
62.50
Inhibition
62.50
Inhibition
31.25
Inhibition
62.50
–
Inhibition
31.25
Inhibition
31.25
Inhibition
62.50
Cytotoxicity assay
Table 7 Complex (3) effect upon the HT 29 cell cycle progression
Species
% G0/G1
% S
% G2/M
The tumour cells HT 29 were treated with 25, 50 and 100 s¸i
200 lg mL-1 solution of compounds. Complexes (1), (2)
and (4) proved to be non-cytotoxic on the tested cell line.
In case of the complex (3) a dose-dependent effect was
obtained, i.e. at concentrations of 200, 100 and
50 lg mL-1, this inhibits the cellular proliferation since
the viability decreased up to 16.96 %. Instead, at
25 lg mL-1, the cytotoxicity was very low, as revealed by
the good viability per cent of 82.50 %. Taking into account
these results, the cell cycle assay by flow cytometry was
performed and evidenced that complex (3) induces a
decrease in DNA content of the cells found in G0/G1 phase
(Table 7). This effect is due to either metallic ion coordi-
nation or aromatic moieties intercalation. Moreover, the
Cu(II) complexes can induce DNA strands split through
redox processes and by ROS generation [53].
HT 29
63.26
48.33
26.36
25.02
10.21
20.52
(3)
80
60
40
20
0
(3)
(4)
pmtp
Control
Refence
Anti-inflammatory activity assay
Group
Complexes of Cu(II) and Zn(II) have been studied also as
anti-inflammatory agents. The ligand itself did not exert
anti-inflammatory activity; the paw oedema evolution was
similar for the pmtp treated rats to the control group (one
way ANOVA, p [ 0.05), in both studied models. As a
result, the anti-inflammatory effect was evaluated for
complexes (3) and (4) in comparison with diclofenac,
Fig. 6 Paw oedema evolution at 4 h after the inflammation induction
through paw oedema evolution for the rats with dextran
and kaolin induced inflammation (Table 7).
For the dextran induced inflammation model it was
observed that the group treated with complexes (3) and (4)
123

R. Olar et al.
2. Salem MA, Thabet HKH, Helal MH, Abdelaal AS, Ammar YA.
Synthesis and pharmacological evaluation of some pyrazoles,
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exhibits an anti-inflammatory effect compared to the con-
trol group, but the effect was smaller in comparison with
the diclofenac treated animals.
For the kaolin-induced inflammation model, complex
(3) showed a maximum anti-inflammatory effect at 4 h
after the inflammation induction (Fig. 6). The paw oedema
evolution at 4 h (35.2 %) was significantly different in
comparison with the control group and similar with the
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The series of cobalt(II), nickel(II), copper(II) and zinc(II)
complexes with 5-phenyl-7-methyl-1,2,4-triazolo[1,5-
a]pyrimidine have been synthesised by the one-pot
condensation.
´
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´
´
7. Caballero AB, Rodriguez-Dieguez A, Quiros M, Lezama L, Salas
JM. New copper(II), nickel(II) and zinc(II) complexes with 1,2,4-
triazolo[1,5-a]pyrimidines and the chelating ligand 1,3-propane-
diamine: an unexpected coordination behavior for the 7-amine-
derivative. Inorg Chim Acta. 2011;378:194–201.
The ligand nature and stereochemistry were character-
ized through IR and UV–Vis-NIR spectroscopy. The
modifications in the IR spectra of complexes are in
accordance with the condensation process and coordination
of 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine as
unidentate through nitrogen and of acetate as unidentate/
chelate ligand. The thermal degradation occurs in several
steps, involving water elimination, acetate into carbonate
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Compounds revealed a large spectrum of antimicrobial
activity, towards bacterial and fungal strains, either sus-
ceptible or resistant to antibiotics. The best antimicrobial
activity against both planktonic and adherent microbial
strains was obtained for complex (3) followed by complex
(1), which can be related, to both oxidation state and
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At high concentrations (50–200 lg mL-1), complex (3)
inhibited the HT 29 tumour cells proliferation, the viability
decreasing up to 16.96 % and also induced an arrest of
tumour cells in the G0/G1 phase, suggesting its anti-pro-
liferative potential. Copper complex was found also to
present anti-inflammatory effect with a maximum value at
4 h after the inflammation induction in the kaolin model.
´
´
8. Caballero AB, Rodrıguez-Dieguez A, Lezama L, Salas JM.
Toward a new type of multifunctional metal-organic systems based
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´
9. Caballero AB, Maclaren JK, Rodrıguez-Dieguez A, Vidal I,
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azolopyrimidine complex: synthesis and structural characteriza-
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Acknowledgements This work was partially supported by Univer-
sity of Bucharest.
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