Thermal behaviour of some novel biologically active complexes with a triazolopyrimidine pharmacophore

Article abstract of DOI:10.1007/s10973-016-5515-6

A new derivative 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine (pmtp) was synthesised by [1?+?1] condensation of benzoylacetone and 3-amino-4H-1,2,4-triazole. Single crystal X-ray diffraction revealed that the pmtp crystallises in monoclinic system, P21/n spatial group. In order to modulate the biological activity, new species M(pmtp)Cl₂·nH₂O (M: Co, n?=?2; M: Ni, n?=?3; M: Cu, n?=?1; M: Zn, n?=?0) were synthesised by one-pot method. Chemical analysis, molar conductivities, IR, UV–Vis-NIR and EPR spectroscopy, as well as magnetic data recorded at room temperature provided useful information concerning the molecular formula, stereochemistry and ligand coordination mode. The modifications at heating and also the thermodynamic effects that accompany them were investigated by thermal analysis. The nature of the gaseous products formed in each step was evidenced by simultaneous TG/DSC/EGA measurements. Processes as water and chloride elimination, fragmentation and oxidative degradation of the triazolopyrimidine derivative were observed during the thermal studies. The final residue was the most stable metallic oxide as X-ray powder diffraction indicates. Zinc (II) and copper (II) complexes exhibited the most significant antimicrobial activity against a wide spectrum of Gram-positive and Gram-negative bacterial strains, both reference and clinical resistant ones, in planktonic and biofilm state. The minimal biofilm eradication values were two to four times lower than the minimal inhibitory concentrations demonstrating the potential of the obtained complexes to act as anti-pathogenic agents.

Full text of DOI:10.1007/s10973-016-5515-6

J Therm Anal Calorim
DOI 10.1007/s10973-016-5515-6
Thermal behaviour of some novel biologically active complexes
with a triazolopyrimidine pharmacophore
3
Larisa Calu1,2 Mihaela Badea1 Romana Cerc Korosec Peter Bukovec3
•
•
•
•
ˇ
Constantin G. Daniliuc4 Mariana Carmen Chifiriuc5,6 Luminit¸a Marut¸escu
5
•
•
•
˘
1
Camelia Ciulica Gina Serban1 Rodica Olar1
•
•
˘
Received: 30 October 2015 / Accepted: 29 April 2016
´
´
Ó Akademiai Kiado, Budapest, Hungary 2016
Abstract A new derivative 5-phenyl-7-methyl-1,2,4-tria-
zolo[1,5-a]pyrimidine (pmtp) was synthesised by [1 ? 1]
condensation of benzoylacetone and 3-amino-4H-1,2,4-
triazole. Single crystal X-ray diffraction revealed that the
pmtp crystallises in monoclinic system, P21/n spatial
group. In order to modulate the biological activity, new
species M(pmtp)Cl2ÁnH2O (M: Co, n = 2; M: Ni, n = 3;
M: Cu, n = 1; M: Zn, n = 0) were synthesised by one-pot
method. Chemical analysis, molar conductivities, IR, UV–
Vis-NIR and EPR spectroscopy, as well as magnetic data
recorded at room temperature provided useful information
concerning the molecular formula, stereochemistry and
ligand coordination mode. The modifications at heating and
also the thermodynamic effects that accompany them were
investigated by thermal analysis. The nature of the gaseous
products formed in each step was evidenced by simulta-
neous TG/DSC/EGA measurements. Processes as water
and chloride elimination, fragmentation and oxidative
degradation of the triazolopyrimidine derivative were
observed during the thermal studies. The final residue was
the most stable metallic oxide as X-ray powder diffraction
indicates. Zinc (II) and copper (II) complexes exhibited the
most significant antimicrobial activity against a wide
spectrum of Gram-positive and Gram-negative bacterial
strains, both reference and clinical resistant ones, in
planktonic and biofilm state. The minimal biofilm eradi-
cation values were two to four times lower than the mini-
mal inhibitory concentrations demonstrating the potential
of the obtained complexes to act as anti-pathogenic agents.
This work was partially supported by University of Bucharest.
Electronic supplementary material The online version of this
article (doi:10.1007/s10973-016-5515-6) contains supplementary
material, which is available to authorized users.
& Rodica Olar
rodica_m_olar@yahoo.com
Keywords Antimicrobial activity Á Complex Á 5-phenyl-7-
methyl-1,2,4-triazolo[1,5-a]pyrimidine Á One-pot
condensation Á Thermal behaviour
1
Department of Inorganic Chemistry, Faculty of Chemistry,
University of Bucharest, 90-92 Panduri Str.,
050663 Bucharest, Romania
2
Cellular Dynamics and Flow Cytometry Department,
National Institute R&D for Biological Sciences, 296 Splaiul
Independentei, 060031 Bucharest, Romania
Introduction
3
Department of Inorganic Chemistry, Faculty of Chemistry
Almost all nitrogen-based heterocycles are biologically
active, but triazolopyrimidine derivatives are particularly
interesting having in view their similarities with purinic
bases. These derivatives exhibit a large spectrum of bio-
logical activities, beginning with anti-parasitic [1], anti-
inflammatory [2, 3], analgesic [2], antiviral [4], antitumor
[5, 6], antimicrobial [7], and ending with cardiovascular
effects [8]. From these derivatives, cevipabulin and its
analogues were proved as potent anticancer agents with a
ˇ
and Chemical Technology, University of Ljubljana, Vecna
pot 113, 1000 Ljubljana, Slovenia
4
5
Organisch-Chemisches Institut, Corrensstrasse 40,
48149 Mu¨nster, Germany
Department of Microbiology, Faculty of Biology, University
of Bucharest, 1-3 Aleea Portocalilor St., 60101 Bucharest,
Romania
6
Research Institute of the University of Bucharest, Spl.
Independentei 91-95, Bucharest, Romania
123

L. Calu et al.
unique mechanism of action consisting in the induction of
tubulin polymerisation [6]. A novel triazolopyrimidine
antibiotic, essramycin was isolated from the culture broth
of the marine Streptomyces sp. strain and proved to be
active against both Gram-positive and Gram-negative
bacterial strains [9]. Furthermore, some triazolopyrimidine
derivatives were developed, as drugs such Trapidil, which
is vasodilator acting both as platelet-derived growth factor
antagonist and as phosphodiesterase inhibitor [10].
substituents, as well as with their electronic and stereo-
chemical effects. It was observed that the presence of a
bulky substituent (tert-butyl or phenyl) might be an
important factor in the modulation of antitumor activity
[24, 28]. Concerning their antimicrobial activity, some
Co(II) complexes with triazolopyrimidines exhibited good
in vitro inhibitory activity against both Gram-positive and
Gram-negative bacterial strains [30]. Moreover, diorgan-
otin(IV) complexes with [1, 2, 4] triazolo-[1,5-a]pyrim-
idine derivatives were found to have promising antifungal
activity [31], anti-Gram-positive bacteria [32, 33], partic-
ularly staphylococcal strains [34], both in planktonic and
biofilm state.
In order to modulate their biological effects, the com-
plexation ability of triazolopyrimidine compounds was
studied and allowed the generation of species with valuable
antiparasitic activity [11]. Therefore, Pt(II) and Ru(III)
triazolopyrimidine complexes were active on Leishmania,
Trypanosomas and Phytomonas genera [12], complexes
with the ligand 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-
7(4H)-one against Leishmania [13], nickel complexes with
a triazolopyrimidine derivative on Leishmania infantum
and Leishmania braziliensis [14], triazolopyrimidine com-
pounds containing first-row transition metals [15] and
lanthanide complexes containing 5-methyl-1,2,4-tria-
zolo[1,5-a]pyrimidin-7(4H)-one against Leishmania and
Trypanosoma cruzi [16] and 5-methyl-1,2,4-triazolo[1,5-
a]pyrimidin-7(4H)-one-based complexes on Trypanosoma
cruzi [17].
Only one report concerning the thermal behaviour of
some divalent metal ions (Co, Ni, Cu, Zn, Cd, Mn and Fe)
with anionic form of 4,7-dihydro-1,2,4-triazolo-[1,5-a]-
pyrimidine-7-one as ligand was found [35]. After water
(hydration or coordination) loss, all anhydrous compounds
are stable up to 350 °C, when pyrolytic decomposition
starts.
In view of the pharmacological importance of tria-
zolopyrimidines and their complexes, we report here the
synthesis and characterisation of Co(II), Ni(II), Cu(II) and
Zn(II) complexes with 5-phenyl-7-methyl-1,2,4-tria-
zolo[1,5-a]pyrimidine, species resulted in one-pot reaction
of benzoylacetone, 3-amino-4H-1,2,4-triazole and metal
chloride. The ligand and complexes have been charac-
terised by different analytical, spectral and magnetic
methods. With the aim to evidence the thermal behaviour
and the nature of evolved gases, these derivatives were
investigated by TG/DTA and simultaneous TG/DSC cou-
pled with the MS technique. The compounds were also
screened for their antimicrobial activity against a wide
range of Gram-negative and Gram-positive bacteria, as
well as fungal strains.
Also, complexes with this class of ligands, such as a
dinuclear silver compound with 5,6,7-trimethyl- [1, 2, 4]
triazolo[1,5-a]pyrimidine with a short Ag–Ag bond [18],
palladium(II) and platinum(II) organometallic complexes
with 4,7-dihydro-5-methyl-7-oxo [1, 2, 4] triazolo[1,5-
a]pyrimidine [19], platinum(IV) complexes with purine
analogues [20], mono- and dinuclear platinum(II) com-
pounds with 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine
[21], platinum(IV) coordination compounds containing
5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one
as
nonleaving ligand [22], hexafluoroglutarate platinum(II)
complex with 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine
[23], mononuclear platinum(II) complexes with 5,7-ditert-
butyl-1,2,4-triazolo[1,5-a]pyrimidine [24], ruthenium(III)
complexes with bulky triazolopyrimidine ligands [25],
dimeric ruthenium-triazolopyrimidine complex [26],
ruthenium(II) and ruthenium(III) ions with 5-methyl-1,2,4-
triazolo[1,5-a]pyrimidin-7(4H)-one [27], acetate plat-
inum(II) compound with 5,7-ditertbutyl-1,2,4-triazolo[1,5-
a]pyrimidine [28], organotin(IV) derivatives with 5,7-dis-
ubstituted-1,2,4-triazolo[1,5-a]pyrimidine [29] exhibited
antitumor activity. The in vitro antitumor activity assays of
Ag(I) [18], Pt(II) [19, 23, 24, 28], Pt(IV) [22], Pd(II) [19],
Ru(II) [27], Ru(III) [26, 27], and Sn(IV) [29] complexes
with triazolopyrimidines evidenced an antiproliferative
effect against rectal, breast and bladder cancer cells.
Cytotoxicity of these compounds was correlated with their
stereochemistry, type of ligands, nature of pyrimidine ring
Experimental
Materials
The high purity reagents were purchased from Sigma-
Aldrich (CoCl2Á6H2O, NiCl2Á6H2O, CuCl2Á2H2O, ZnCl2),
Merck (benzoylacetone) and Fluka (3-amino-4H-1,2,4-tri-
azole) and used without further purification.
Instrumentation
The content of carbon, nitrogen and hydrogen has been
obtained by using a Perkin Elmer PE 2400 analyzer.
Mass spectra were recorded by electrospray ionisation
tandem mass spectrometry (ESI–MS) technique operating
in the positive ion mode. The sample dissolved in
123

Thermal behaviour of some novel biologically active complexes with a triazolopyrimidine…
acetonitrile/water (1/1) was injected directly in the Waters
Micromass Quattro micro API triple quadrupole mass
spectrometer with an electrospray interface.
antimicrobial activity was performed by the adapted disc
diffusion, as previously reported [42], using Mueller–Hinton
Agar (MHA) medium for bacteria and Yeast Peptone Glu-
cose (YPG) in case of fungi. The compounds were solu-
bilised in dimethylsulfoxide (DMSO), and the starting stock
solution was of 1000 lg mL-1 concentration.
IR spectra were recorded by using KBr pellets with a
Bruker Tensor 37 spectrometer (400–4000 cm-1 range).
Electronic spectra were recorded by diffuse reflectance
technique on
a
Jasco V670 spectrophotometer
The quantitative assay of the antimicrobial activity was
performed by the liquid medium microdilution method, in
96 multi-well plates, in order to establish the minimal
inhibitory concentration (MIC) and the minimal biofilm
eradication concentration (MBEC) values [42]. All bio-
logical experiments were performed in triplicates.
(200–1500 nm range, spectralon as standard).
Magnetic measurements were taken at room temperature
on a Lake Shore’s fully integrated Vibrating Sample
Magnetometer (VSM) calibrated with Hg[Co(NCS)4].
The X-band EPR measurements were recorded with a
JEOL FA100 spectrometer, and the magnetic field was
calibrated with DPPH.
1H NMR and 13C NMR spectra were recorded on a
Bruker Avance spectrometer (working frequency
200 MHz) at 25 °C. Chemical shifts were measured in
parts per million from internal standard TMS.
Synthesis, analytical and spectral data for ligand
and complexes
To a solution containing 1 mmol metal(II) acetate hydrated
(M: Co, Ni, Cu and Zn) in 25 mL, ethanol was dropwise
added a solution containing 1 mmol benzoylacetone in
10 mL ethanol and then another one formed by 1 mmol
3-amino-4H-1,2,4-triazole dissolved in 10 mL ethanol. The
reaction mixture was magnetically stirred at 50 °C for 4 h,
until a sparingly soluble species was formed. This product
was filtered off, washed several times with cold ethanol and
air-dried.
The single crystal X-ray measurements were taken with a
Nonius Kappa CCD diffractometer. Programs used: data
collection, COLLECT [36]; data reduction Denzo-SMN [37];
absorption correction, Denzo [38]; structure solution
SHELXS-97 [39]; structure refinement SHELXL-97 [40] and
graphics, XP [41] (Bruker AXS, 2000), R-values are given for
observed reflections, and wR2 values are given for all reflec-
tions. CCDC reference number is 1433539 and contains the
supplementary crystallographic data for this compound.
The simultaneous TG/DTA measurements were recor-
[Co2(pmtp)2Cl4(OH2)4] (1): Analysis found: C, 38.40;
H, 3.59; N, 15.12, Co2C24H28N8O4Cl4 requires: C, 38.32;
H, 3.75; N, 14.90; Yield 78 %; ESI–MS (positive mode,
CH3CN:H2O (1:1)) m/z: [M ? 2CH3CN ? H]?, 763; [M–
Cl ? 2CH3CN]?, 726; [M ? H]?, 680; [M–CH3]?, 665;
[M ? H–CH3–Cl]?, 631; [1/2 M]?, 340; [M–2Cl]2?, 305;
ded in synthetic air atmosphere (flow rate 16.7 cm3 min-1
)
by using a Labsys 1200 SETARAM instrument, over
the temperature range of 20–900 °C, a heating rate of
10 K min-1 and a sample mass of 8–30 mg.
KM, 9.0 X-1 cm2 mol-1
.
Simultaneous thermogravimetric/dynamic scanning
calorimetry/evolved gas analysis measurements (TG/DSC/
EGA) were taken on a Mettler Toledo TGA/DSC1
Instrument, coupled to a Pfeiffer Vacuum ThermoStar
Mass Spectrometer.
[Ni2(pmtp)2Cl4(OH2)4] 2H2O (2): Analysis found: C,
36.43; H, 4.02; N, 14.38, Ni2C24H32N8O6Cl4 requires: C,
36.59; H, 4.09; N, 14.22; Yield 58 %; ESI–MS (positive
mode, CH3CN:H2O (1:1)) m/z: [M ? H ? 2CH3CN]?,
763; [M–Cl ? 2CH3CN]?, 726; [M ? H]?, 681; [M–
CH3]?, 665; [M ? H–CH3–Cl]?, 630; [1/2 M ? H]?, 341;
The X-ray powder diffraction patterns were collected on
a PANalytical X’Pert PRO diffractometer using CuKa
˚
[M–2Cl]2?, 304; KM, 11.5 X-1 cm2 mol-1
.
radiation (k = 1.5406 A) in the range of 2h from 5° to 80°.
[Cu2(pmtp)2Cl4(OH2)2] (3): Analysis found: C, 39.75;
H, 3.18; N, 15.62, Cu2C24H24N8O2Cl4 requires: C, 39.74;
H, 3.33; N, 15.45; Yield 65 %; ESI–MS (positive mode,
CH3CN:H2O (1:1)) m/z: [M ? H]?, 690; [M-CH3]?, 674;
Antimicrobial assays
The antimicrobial assays were performed on reference
(bearing the ATCC code) and recently isolated clinical
microbial strains, exhibiting resistance to current antibiotics
recommended for testing, i.e. Gram-positive (Staphylococ-
cus aureus ATCC 25923, methicillin resistant S. aureus -
MRSA 1263, Bacillus subtilis ATCC 6633) and Gram-
negative (Escherichia coli ATCC 832, E. coli ATCC 25922,
Klebsiella pneumoniae 832, K. pneumoniae ATCC 134202,
Pseudomonas aeruginosa ATCC 27853, P. aeruginosa 392)
bacterial strains. The qualitative evaluation of the
[M ? H–CH3–Cl]?, 640; [1/2 M ? H]?, 346; [M-2Cl]2?
309; KM, 8.5 X-1 cm2 mol-1
,
.
[Zn2(pmtp)2Cl4] (4): Analysis found: C, 41.64; H, 2.82;
N, 16.22, Zn2C24H20N4Cl4 requires: C, 41.59; H, 2.91; N,
16.17; Yield 62 %; ESI–MS (positive mode, CH3CN:H2O
(1:1)) m/z: [M ? H]?, 694; [M–CH3]?, 678; [M ? H–
CH3–Cl]?, 644; [1/2 M ? H]?, 348; [M–2Cl]2?, 311; KM,
13.5 X-1 cm2 mol-1
.
The synthesis of 5-phenyl-7-methyl-1,2,4-triazolo[1,5-
a]pyrimidine: To a solution containing 3.244 g (0.02 mol)
123

L. Calu et al.
benzoylacetone in 50 mL ethanol were added 1.682 g
(0.02 mol) 3-amino-4H-1,2,4-triazole and few drops of
acetic acid and the reaction mixture was refluxed for 12 h
until a yellow sparingly soluble product was formed. The
yellow product formed was filtered off, washed several
times with EtOH and recrystallised from chloroform/1-
propanol (1:2, v/v). Single crystals of 5-phenyl-7-methyl-
1,2,4-triazolo[1,5-a]pyrimidine (pmtp) suitable for X-ray
diffraction were obtained from slow evaporation of a DMF/
oriented compared to the triazolopyrimidine unit (h N4–
C5–C10–C11 = 5,9(2)°).
In the packing diagram, the molecules are linked into
pairs through pÁÁÁp staking interactions between phenyl and
˚
pyrimidine rings (shortest distance is 3.398 A). Involving
˚
additional C-HÁÁÁN contacts (C2-H2ÁÁÁN3 2.569 A), a linear
chain containing the corresponding dimmers is generated
along the ac-diagonal (Fig. 2).
1
EtOH (1:1) solution. H NMR (DMSO-d6) d (ppm): 2.64
ESI MS, NMR and IR data
(s, CH3), 7.68 (s, CH pyrimidine), 7.72–8.30 (multiplet,
Ar–H), 8.62 (s, CH triazole); 13C NMR (DMSO) d (ppm):
15.8 (C9; CH3), 110.2 (C6; pyrimidine), 127.8, 128.8,
129.4, 131.5 (C10–C15; Ar), 146.7 (C7; pyrimidine), 155.4
(C3a; triazole), 156.3 (C2; triazole), 165.8 (C5; pyrim-
idine). ESI–MS (positive mode, CH3CN) m/z: [M ? H]?,
211; [M–CH3]?, 195.
The complexes formulas were further supported by positive
mode ESI mass spectrometry, which indicated as base peak
corresponding to the pseudomolecular ion [M ? H]? for
all compounds. Moreover, other fragments that can be
related to complexes dimeric structure were identified with
or without chloride anions and acetonitrile molecules in
components.
1
In the H NMR spectrum of 5-phenyl-7-methyl-1,2,4-
Results and discussions
triazolo[1,5-a]pyrimidine, the signal at 2.64 ppm is
assigned to methyl protons and the multiplet at 8.10 ppm is
generated by phenyl ring protons. The signals of protons
belonging to triazole and pyrimidine rings appear at 7.68
and 8.62 ppm, respectively. 13C NMR spectrum shows the
signals characteristic for triazolopyrimidine unit at 110.2,
146.7 and 165.8 ppm [13]. The carbon atom of methyl
group generates a signal at 15.8 ppm and that of phenyl
group are responsible for the signals noticed in the range
127.8–131.5 ppm.
Synthesis and characterisation of 5-phenyl-7-
methyl-1,2,4-triazolo[1,5-a]pyrimidine
and complexes
The one-pot reaction in 1:1:1 molar mixture of cobalt(II),
nickel (II), copper (II) or zinc (II) chloride, benzoylacetone
and 3-amino-4H-1,2,4-triazole produced the species M2-
(pmpt)2Cl4ÁnH2O ((1) M:Co, n = 4; (1) M:Ni, n = 6; (3)
M:Cu, n = 2 and (4) M:Zn, n = 0; pmpt: 5-phenyl-7-
methyl-1,2,4-triazolo[1,5-a]pyrimidine) (Scheme 1).
The chemical analyses are in accordance with the pro-
posed formulas for complexes (see experimental part). The
complexes behave as non-electrolytes in DMSO, the molar
conductance values being very small [43].
The Zn(II) complex is not soluble in deuterated solvents
and as results the NMR data cannot be collected.
The IR spectra of complexes (Table 1) exhibit the
characteristic bands for triazolopyrimidine moiety at
1613 cm-1 assigned to triazolopyrimidine condensed ring
(mtp) and at 1540 cm-1 assigned to pyrimidine ring vibra-
tion (mpy), respectively [17–34]. The band corresponding to
mtp vibration is shifted by 5–30 cm-1 to higher
wavenumbers in comparison with the metal-free ligand
indicating the ligand coordination through triazolopyrim-
idine N3 atom [17–34].
A broad band in the range 3430–3440 cm-1 can be
assigned to m(OH) stretching vibrations for water mole-
cules, except for complex (4). Supplementary bands in the
range 650–780 cm-1 could support the water presence as
ligand in some complexes [45].
Description of 5-phenyl-7-methyl-1,2,4-triazolo[1,5-
a]pyrimidine structure
A summary of the crystallographic data and structure refine-
ment for 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine
(pmtp) is given at supplementary material, while the atom
numbering scheme and molecular structure is provided in
Fig. 1.
The compound crystallised with one independent
molecule in the asymmetric unit. Dihedral angles between
C7–N8–C3A, C3A–N8–N1 and N4–C3A–N3 from the
fused rings are 122.3(1), 110.5(1) and 128.5(1)° as result of
sp2 hybridisation of atoms belonging to the two rings.
Bonds lengths N8–C7, N8–C3A, N8–N1, N1–C2, N3–C2
Low-intensity bands noticed in the complexes spectra in
the range 450–570 cm-1 are an indicative of M–N and M–
O coordinative bonds formation.
Electronic, EPR spectra and magnetic moments
˚
range between 1.326(2) and 1.378(2) A, and values were
close to that observed for 5,7-dimethyl-1,2,4-triazolo[1,5-
In UV region of the electronic spectrum of ligand, three
a]pyrimidine [44]. The phenyl ring is almost planar
intense bands assigned to intraligand p ? p* transitions
123

Thermal behaviour of some novel biologically active complexes with a triazolopyrimidine…
N
N
O
N
N
H2N
–2 H2O
+
N
H
N
OH
N
(pmtp)
+ MCl2 (1:1:1)
N
N
N
N
N
N
Cl
M
N
N
N
OH2
OH2
Cl
H2O
H2O
N
Cl
Cl
Cl
Cl
Cl
Cu
M
Cl
Cu
N
OH2
H2O
N
N
N
N
N
(3)
M: Co (1)
M: Ni (2)
N
N
N
Cl
N
Cl
Zn
Zn
N
N
Cl
Cl
N
N
(4)
Scheme 1 Synthetic route for complexes preparation and the proposed coordination
¨
parameters (10Dq, B and b) were calculated with Konig’s
formulas [47]. The value of the magnetic moment at room
temperature is also characteristic for such a stereochem-
istry with a T ground term and an orbital contribution as a
result [48].
C9
N8
C7
C15
C6
C14
The broad bands that can be assigned to spin allowed
transitions in an octahedral distorted geometry can be
observed in electronic spectrum of Ni(II) complex as
well [46]. These bands leads to a splitting parameter of
8845 cm-1 is according with an [Ni(II)NO2Cl3] chro-
mophore having a high number of donor atoms that
generate a low field, such oxygen and chloride ones. A
value of 0.83 for the nephelauxetic parameter indicates
also a lower degree of covalency. This stereochemistry is
further sustained by the room temperature magnetic
moment of 3.12 B.M., as usually is observed for com-
plexes with an A ground term [48].
C10
N1
C5
C13
C2
C11
C3A
N4
C12
N3
Fig. 1 The atom numbering and molecular structure of 5-phenyl-7-
methyl-1,2,4-triazolo[1,5-a]pyrimidine (30 % probability thermal
ellipsoids)
can be observed. In the complexes spectra, these bands are
differently shifted as a result of coordination and interac-
tions with the other ligands or hydration water molecules.
Three low-intensity bands appear in the electronic
spectrum of compound (1) that can be assigned to spin
The electronic spectrum of the Cu(II) complex shows
the characteristic broad band with a maximum at
14,925 cm-1 tentatively assigned to dz2 ? dxy transition in
a square pyramidal stereochemistry and a C2v symmetry
[46]. The value of 2.25 B.M. for the magnetic moment is
characteristic for species with isolated paramagnetic cen-
tres [48].
4
4
4
4
allowed transitions T1g ? T2g, T1g ? A2g and
4
4T1g ? T1g, in an octahedral stereochemistry (Table 2)
[46]. The splitting parameter value of 8410 cm-1 is close
to the average value for a [Co(II)NO2Cl3] chromophore,
while the nephelauxetic parameter of 0.88 is an indicative
of a lower degree of covalency. The crystal field
Powder EPR spectrum of complex (1) exhibits a low-
intensity signal with giso = 2.119 characteristic to a local
123

L. Calu et al.
Fig. 2 Packing diagram
presenting the pÁÁÁp and C–
HÁÁÁN interactions of 5-phenyl-
7-methyl-1,2,4-triazolo[1,5-
a]pyrimidine
c
b
C3A
N3
C2
0
C15
a
Table 1 IR absorption bands (cm-1) for 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine and complexes
pmtp
(1)
(2)
(3)
(4)
Assignment
–
3441vs
3056w
2926w
2828w
1625s
1582vs
1403m
1079w
843w
3438s
3061w
2939w
2818w
1629s
1597s
1409m
1076w
842w
3433m
3065w
2954w
2879w
1635vs
1560vs
1444m
1032w
869w
–
m(OH2)
m(CH)
3101m
2932w
2833w
1613s
1540vs
1465m
1038w
810w
–
3059w
2912w
2824w
1617s
1544vs
1432m
1028w
863w
–
mas(CH3)
ms(CH3)
m(C=N)
m(C=C) ? das(CH3)
m(N–N)
c(CH)
754w
772w
768w
qr(H2O)
–
671w
673w
656w
–
qw(H2O)
m(M–N)
–
562w
530w
552w
500w
453w
–
469w
453w
478w
m(M–O)
vs very strong, s strong, m medium, w weak
symmetry of copper ion lower than octahedral [49]. The
aspect of the spectrum is preserved in frozen DMSO.
vibration of secondary amine group, while that at 3064,
1618, 1535 and 805 cm-1 is associated with aromatic ring
vibration modes. The methylene group was identified based
on vibrations located at 2914 and 2844 cm-1, respectively.
This step comprises two processes according with both
DTG and DTA curves. The next step results also from
overlapping of several exothermic processes and corre-
sponds with dibenzylhydrazine oxidative degradation.
The simultaneous TG/DTA curves registered for com-
plex (1) are shown in Fig. 4 and indicate that complex
undergoes a four steps thermal decomposition pattern. The
first step consists in an endothermic elimination of water
molecules up to 184 °C. The presence of ion peaks with m/
z 18 in the MS spectra confirms this assumption, while the
temperature range that corresponds to this transformation
indicates the coordination nature of water molecules
[50, 51]. The second step, endothermic also, corresponds to
chloride anion and methyl group elimination. These
decomposition processes gives rise to fragments as Cl,
Thermal behaviour of ligand and complexes
The thermal behaviour of these derivatives was investi-
gated in air by both TG/DTA and TG/DSC techniques. The
results were similar and in the following the TG/DTA data
will be detailed and correlated with results provided by the
evolved gases analysis.
The pmtp is stable up to 192 °C and then decomposes in
two steps (Fig. 3; Table 3). The endothermic effect noticed
on DTA curve without mass change becomes from a phase
transition such the melting is. The mass loss noticed in the
first step corresponds to 51 % oxidative degradation of
triazolopyrimidine derivative. The intermediate formed at
400 °C is dibenzylhydrazine according with chemical
analysis and IR data. In the IR spectrum of this interme-
diate, the band at 3319 cm-1 is assigned to stretching
123

Thermal behaviour of some novel biologically active complexes with a triazolopyrimidine…
Table 2 Absorption maxima, assignments and magnetic moments for ligand and complexes
Compound
pmtp
Absorption maxima (cm-1
)
Assignment
Magnetic moment (B.M.)
–
38,460
p ? p*
29,850
23,255
[Co2(pmtp)2Cl4(OH2)4] (1)
32,785
p ? p*
4.64
3.12
4
19,050
4T1g ? T1g
4
15,750
4T1g ? A2g
4
8365
4T1g ? T2g
[Ni2(pmtp)2Cl4(OH2)4]Á2H2O (2)
33,070
p ? p*
3
25,640
3A2g ? T1g (P)
3
15,150
3A2g ? T1g (F)
3
8845
3A2g ? T2g
[Cu2(pmtp)2Cl4(OH2)2] (3)
[Zn2(pmtp)2Cl4] (4)
40,000, 29,410
14,925
p ? p*
d2z ? dxy
p ? p*
2.25
–
40,000, 31,745
For compound (2), water molecules are eliminated in
two steps and the corresponding temperature ranges indi-
cate the nature of this species both as hydration and as
coordination (Fig. 5). The chloride anions are eliminated in
the second step also, considering the products with m/z 35
and 37 noticed in the gaseous products. The fragmentation
of triazolopyrimidine moiety occurs in the same step giving
rise to CH3Cl. Moreover, the oxidation of some fragments
generates products as CO2 and NO. Two endothermic
effects accompany these processes according with DTA
curve. The organic ligand fragmentation proceeds with
aromatic ring elimination since C6H5 and C6H4 fragments
were identified by EGA. This step is not a single-process
one, being an overlapping of two weak exothermic pro-
cesses accordingly to the DTA curves profile. This beha-
viour can be the result of several reactions that occur
simultaneously, such as bonds cleavage, their rearrange-
ments, as well as aromatic moiety oxidative degradation. In
the next step, the rest of organic part suffers oxidative
degradation with NO, NO2, CO2 and H2O as products. All
these transformations finally lead to nickel (II) oxide
(found/calcd. overall mass loss: 80.6/81.0 %). The nature
of final product was confirmed by powder X-ray diffraction
data (ASTM 78-0429).
TG
0
1.0
100
80
0.5
20
DTG
0.0
60
40
20
0
40
60
–0.5
–1.0
–1.5
–2.0
–2.5
80
DTA
100
–20
200
600
Temperature/°C
1000
0
800
400
Fig. 3 TG, DTG and DTA curves of 5-phenyl-7-methyl-1,2,4-
triazolo[1,5-a]pyrimidine (pmtp)
HCl, H2Cl and CH3Cl. Otherwise, such behaviour was
observed for other halide complexes during thermal
decomposition [52]. The decomposition continues with
nitrogen containing fragments eliminations, since NO was
identified along H2O in the gaseous mixture eliminated in
this step. In the next step, the rest of organic part suffers
both fragmentation and oxidative degradation. Moreover,
the oxidative processes predominate in this step having in
view both the exothermic effects, as well as the increased
quantities of CO2, NO and NO2 over that of nitrogen
containing fragments. As result of these reactions, several
exothermic processes, one of them being very strong, can
be noticed on DTA curve. Also a part of cobalt(II) is
oxidised at Co(III) having in view that the final product is
Co3O4 (found/calcd. overall mass loss: 78.6/78.8 %). The
nature of the final product was confirmed by powder X-ray
diffraction data (ASTM 78-1970).
The complex (3) loses the water molecules (Fig. 6) in a
range that proves their coordination nature [50, 51]. Fur-
thermore, in the MS spectra in the temperature range
50–170 °C, only fragment with m/z 18 can be noticed. The
fragments identified in the evolved gases (Cl, NO, CH3Cl)
indicate that chloride elimination and ligand bonds cleav-
age together with some moieties oxidation occur in the
second step. On the other hand, the small endothermic
event observed on DTA curve can be the result of several
123

L. Calu et al.
123

Thermal behaviour of some novel biologically active complexes with a triazolopyrimidine…
40
0.3
80
60
40
20
0
TG
0.4
TG
0
20
40
60
80
0
20
40
60
80
0.2
30
20
10
0
0.2
0.1
DTG
DTG
0.0
0.0
–0.2
–0.4
–0.6
–0.8
–1.0
–0.1
–0.2
–0.3
–0.4
–0.5
DTA
DTA
–10
–20
1000
200
600
Temperature/°C
0
800
400
200
600
Temperature/°C
1000
0
800
400
Fig. 7 TG, DTG and DTA curves of complex (4)
Fig. 4 TG, DTG and DTA curves of complex (1)
100
80
0.4
Compound (4) is anhydrous and as result its decompo-
sition starts at 252 °C with methyl group elimination
(Fig. 7). In the next step, the partial chloride anion elimi-
nation (75 %) proceeds according with the identified
products in the gaseous mixture. This behaviour could be
the result of chloride anion involving in different interac-
tions in the compound network, besides coordinative ones.
Fragments such as Cl, CO2, HCO2, H2O, NO, NO2 detected
in the mass spectra of eliminated products indicate that in
the third step several processes such as chloride elimina-
tion, bond cleavage, rearrangements and oxidative degra-
dation occurs. As consequence, in the 544–787 °C range,
two small exothermic events can be observed on the DTA
curve profile. The residual mass at 787 °C corresponds to
zinc (II) oxide (ASTM 036-1451) generations (found/-
calcd. overall mass loss: 76.1/76.5 %).
TG
0
0.2
20
40
60
80
DTG
60
40
20
0
0.0
–0.2
–0.4
–0.6
DTA
–20
1000
200
600
Temperature/°C
0
800
400
Fig. 5 TG, DTG and DTA curves of complex (2)
3
TG
0
100
2
Taking into account all above data, complexes can be
formulated as [Co2(pmtp)2Cl4(OH2)4] (1), [Ni2(pmtp)2
Cl4(OH2)4]Á2H2O (2), [Cu2(pmtp)2Cl4(OH2)2] and [Zn2
(pmtp)2Cl4], respectively (Scheme 1). For Zn(II) the
tetrahedral stereochemistry was proposed having in view
its anhydrous nature, the small number of ligands and its
preference for this kind of surroundings in complexes with
both large and negatively charged ligands such as chloride
anion.
80
60
40
20
0
1
20
DTG
0
40
–1
–2
–3
–4
–5
60
DTA
80
–20
200
600
Temperature/°C
1000
0
800
400
Antimicrobial activity against planktonic cells
Fig. 6 TG, DTG and DTA curves of complex (3)
The compound (1) exhibited good microbicidal activity
against E. coli, K. pneumoniae reference strains, and
MRSA (MIC of 125 lg mL-1) and moderate activity
against B. subtilis (MIC of 250 lg mL-1). The compound
(2) exhibited good antimicrobial activity against B. subtilis
(MIC of 125 lg mL-1) and moderate activity against K.
pneumoniae 806 clinical strain and P. aeruginosa reference
strain. Compound (3) exhibited very good activity against
B. subtilis strain (MIC of 62.50 lg mL-1) and moderate
activity against a wide range of Gram-positive and Gram-
exothermic and endothermic overlapped processes. A CN
fragment is eliminated from the organic matrix in the next
step according with the mass loss and products as CO2,
H2O, NO and the decomposition proceeds with oxidative
degradation of the organic matrix. According with powder
X-ray diffraction profile, the final product formed at
850 °C is CuO (ASTM 5-661) (found/calcd. overall mass
loss: 78.6/78.1 %).
123

L. Calu et al.
Table 4 The MIC values (lg mL-1) for complexes (1)–(4)
Table 5 The complexes influence on biofilm formation (MBEC
value, lg mL-1
)
Strain
(1)
(2)
(3)
(4)
Strain
(1)
(2)
(3)
(4)
E. coli ATCC 25922
E. coli 832
125
500 250
62.50
1000
62.50
250
E. coli ATCC 25922
E. coli 832
62.50
62.50
62.50
–
250
–
125
62.50
–
250 [1000 500
125 [1000 250
250
K. pneumoniae ATCC 134202
K. pneumoniae 806
K. pneumoniae ATCC 134202
K. pneumoniae 806
–
62.50
125
31.25
125
[1000
[1000
[1000
250 250
250 250
500 500
62.50
62.50
125
–
P. aeruginosa ATCC 27853
P. aeruginosa 392
[1000
500
P. aeruginosa ATCC 27853
P. aeruginosa 392
–
62.50
125
–
–
250
S. aureus MRSA 1263
S. aureus ATCC 25923
B. subtilis ATCC 6633
125 [1000 500
125
S. aureus MRSA 1263
S. aureus ATCC 25923
B. subtilis ATCC 6633
62.50
–
250
62.50
31.25
31.25
[1000 [1000 250
62.50
–
62.50
31.25
250
125
62.50 62.50
125
62.50
negative strains including clinical resistant ones. Com-
pound (4) proved to be most active, exhibiting low MIC
values against E. coli, K. pneumoniae, S. aureus and B.
subtilis reference strains, good activity against MRSA
strains and moderate activity against K. pneumoniae clin-
ical strain (Table 4).
It is worth to mention that a MIC value [500 lg mL-1
was considered as corresponding to a low, B500 to
C250 lg mL-1 to moderate, \250 to C125 lg mL-1 to
good and \125 lg mL-1 to a very good antimicrobial
activity [53].
Conclusions
A new derivative 5-phenyl-7-methyl-1,2,4-triazolo[1,5-
a]pyrimidine (pmtp) was synthesised and fully charac-
terised by single crystal X-ray diffraction. Complexes of
type M(pmtp)Cl2ÁnH2O (M: Co, Ni, Cu, Zn) with this
ligand were synthesised and characterised through a wide
range of physico-chemical methods.
The IR spectra of complexes exhibit the characteristic
bands for triazolopyrimidine ring. Electronic spectra of
Co(II) and Ni(II) complexes are characteristic for octahe-
dral stereochemistry, while that of Cu(II) complex display
the pattern of square pyramidal surrounding. These data
were furthermore confirmed by magnetic behaviour at
room temperature and EPR spectrum.
Antimicrobial activity against biofilm embedded
cells
The tested compound exhibited a very good anti-biofilm
activity at concentrations lower than the MIC value,
demonstrating their potential to act as anti-pathogenic
agents, which inhibit the expression of different virulence
features (in the present case, the ability of bacterial strains
to form biofilms on the inert substratum), without inter-
fering with microbial growth. The advantages of anti-
pathogenic versus microbicidal agents are the low proba-
bility of selecting resistance and the low risk of side effects
caused by the high doses required for the occurrence of
therapeutic action and by the induced dysbiosis of the
normal microbiota [54].
The thermal analyses evidenced processes as water and
chloride anion elimination as well as fragmentation and
oxidative degradation of the organic ligand. The formed
gaseous products during decomposition were monitored by
MS measurements. The final product of decomposition was
metal (II) oxide, except for cobalt (II) complex where
Co3O4 is formed. The results are in good agreement with
the complexes composition.
Complexes exhibited moderate to very good antimi-
crobial activity against a wide spectrum of Gram-positive
and Gram-negative bacterial strains, both reference and
clinical resistant ones. The most effective compound
proved to be (4) and (3), both against planktonic and bio-
film cells. The minimal biofilm eradication values were
two to four times lower than the minimal inhibitory con-
centrations demonstrating the potential of the obtained
complexes to act as anti-pathogenic agents.
For the compounds (1) and (2), the anti-biofilm micro-
bial spectrum was similar to that of microbicidal one,
including also the clinical E. coli and P. aeruginosa strains,
but the MBEC values were two to four times lower than the
MIC ones. The compound (3) exhibited moderate to very
good anti-biofilm activity against all tested strains, the
MBEC values being generally two times lower than the
MIC values. For compound (4) the anti-biofilm spectrum
was identical with the microbicidal one, by the MBEC
values were in most cased two times lower than the MIC
values (Table 5).
Acknowledgements The authors thank for EPR data to researcher
˘
Gabriela Ioni¸ta and for magnetic measurements at room temperature
to researcher Nicolae Stanica, both from ‘‘Ilie Murgulescu’’ Physical
˘
Chemistry Institute of Romanian Academy.
123

Thermal behaviour of some novel biologically active complexes with a triazolopyrimidine…
´
´
´
16. Caballero AB, Rodrıguez-Dieguez A, Salas JM, Sanchez-Moreno M,
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