Anion-/cation-directed reaction routes to polymorphic forms of a pyrazole-type ligand and its coordination compounds with zinc. Key structural differences between polymorphs’

Article abstract of DOI:10.1007/s11224-015-0734-1

Synthetic paths toward the two polymorphs of a monohydrate, one anhydrous polymorph of 1-carboxamidino-5-hydroxy-3-methylpyrazole (hcmp) and two polymorphs of zinc complexes containing hcmp ligand are presented. By choosing ions which are not part of the final product, it is possible to direct the synthesis toward the particular polymorph. In all three modifications of hcmp, the same hydrogen bonding motif appears, leading to formation of similar molecular chains. Differences arise due to different modes of chain aggregation and the presence of solvent water. Analysis of the crystal packing and the energetic features of hcmp polymorphs is made using the PIXEL model. The thermal decomposition processes are examined using differential scanning calorimetry and thermogravimetry. Analysis of crystal packing in the two polymorphs of zinc complex suggests the key role of the hydrogen bonding capacity of the aqua ligand for the appearance of the two polymorphic forms. In both polymorphs of zinc complex, stacking interactions have an important role. However, the enhanced hydrogen bonding capacity of the aqua ligand influences the formation of multistacking arrangement.

Full text of DOI:10.1007/s11224-015-0734-1

Struct Chem
DOI 10.1007/s11224-015-0734-1
ORIGINAL RESEARCH
Anion-/cation-directed reaction routes to polymorphic forms
of a pyrazole-type ligand and its coordination compounds
with zinc. Key structural differences between polymorphs’
1
Berta Barta Hollo Katalin Meszaros Szecsenyi Maria Deli Lorand Kiss
1
2
2
•
•
•
•
´
Alfred Kallay-Menyhard
´
´
´
´
´
´ ´
3,4
5
6
ˇ
´ ´ ´
Vukosava Zivkovic-Radovanovic Zoran D. Tomic
•
•
´
´
´
Received: 11 September 2015 / Accepted: 23 December 2015
Ó Springer Science+Business Media New York 2016
Abstract Synthetic paths toward the two polymorphs of a
monohydrate, one anhydrous polymorph of 1-carboxam-
idino-5-hydroxy-3-methylpyrazole (hcmp) and two poly-
morphs of zinc complexes containing hcmp ligand are
presented. By choosing ions which are not part of the final
product, it is possible to direct the synthesis toward the
particular polymorph. In all three modifications of hcmp,
the same hydrogen bonding motif appears, leading to for-
mation of similar molecular chains. Differences arise due
to different modes of chain aggregation and the presence of
solvent water. Analysis of the crystal packing and the
energetic features of hcmp polymorphs is made using the
PIXEL model. The thermal decomposition processes are
examined using differential scanning calorimetry and
thermogravimetry. Analysis of crystal packing in the two
polymorphs of zinc complex suggests the key role of the
hydrogen bonding capacity of the aqua ligand for the
appearance of the two polymorphic forms. In both poly-
morphs of zinc complex, stacking interactions have an
important role. However, the enhanced hydrogen bonding
capacity of the aqua ligand influences the formation of
multistacking arrangement.
Keywords Polymorphism Á PIXEL calculations Á
Intermolecular interactions Á Pyrazole-based ligand and Zn
complex
Introduction
A large group of pyrazole derivatives and/or their coordi-
nation compounds show biological activity [1–4] or anti-
inflammatory [5], analgesic [6] or anti-diabetic [7] effect. As
the number of pathogens resistant to widely used antibiotics
is constantly growing, finding new compounds with a wide
spectrum of antimicrobial or antiviral activity but low tox-
icity toward normal cells is a challenging task. Coordination
of biologically active ligands with metal ions often alters the
activity that may be enhanced [8, 9] or decreased [10] by
complex formation. As our interest in pyrazole-type com-
pounds dates back more than a decade [11–13], the goal of
this study was to prepare a potentially bioactive ligand,
1-carboxamidino-5-hydroxy-3-methylpyrazole (hcmp), and
its complexes in the form of single crystals. The reactions of
the ligand were carried out with zinc(II) and magnesium(II)
salts in aqueous and ethanolic solutions in order to see how
the changes in reaction conditions affect the composition/
structure/morphology of the formed compounds. Namely,
the bioavailability of the active pharmaceutical ingredients
(API) depends not only on its structure, but also on the
´
& Zoran D. Tomic
zorant@vin.bg.ac.rs
1
Faculty of Sciences, University of Novi Sad, Trg Dositeja
Obradovica 3, Novi Sad 21000, Serbia
2
Biological Research Centre HAS, Institute of Biophysics,
Szeged, Hungary
3
Laboratory of Plastics and Rubber Technology, Department
of Physical Chemistry and Materials Science, Budapest
University of Technology and Economics, M}uegyetem
rakpart 3. H/1, Budapest 1111, Hungary
4
Research Center for Natural Sciences, Institute of Materials
Science and Environmental Chemistry, Hungarian Academy
´
´
of Sciences, Magyar tudosok korutja 2, Budapest 1117,
Hungary
¨
5
6
Faculty of Chemistry, University of Belgrade,
P.O. Box 51, Belgrade 11158, Serbia
Laboratory of Theoretical Physics and Condensed Matter
ˇ
Physics, VINCA’ Institute of Nuclear Sciences, University of
Belgrade, P.O. Box 522, Belgrade 11001, Serbia
123

Struct Chem
physical form. The physical form of API is often determined
by the conditions of crystallization, and very often, one
compound exists in more than one crystal form [14]. By
controlling the nucleation and growth process, nanocrystals
with desired composition and morphology may be obtained
[15]. Here, depending on the reaction conditions, we
obtained two solvatomorphs and the anhydrous form of
hcmp and two polymorphic complexes of zinc in well-re-
peatable chemical processes. The crystal structures of the
ligand and the zinc complexes were determined. The poly-
morphism of the ligand in solid state is discussed on the
basis of its tautomerism in solution which is supported by
spectral data. The desolvation temperatures of the two hcmp
polymorphs and the thermal decomposition temperatures of
the anhydrous forms of hcmp have been discussed in view of
the energetic analysis of the crystal packing. Analysis of
crystal packing in Zn-complex polymorphs has been done
with the aim to determine the most significant structural
features, which differentiate the two polymorphs. Addi-
tionally, the cytotoxicity of the compounds was tested on
human intestinal epithelial (Caco-2) cells using viability
MTT (with tetrazolium salts) and lactate dehydrogenase
(LDH) assays.
aims was to prepare a complex of hcmp with MgII, the
complex formation reaction was carried out using MgCO3.
However, instead of the corresponding complex, hcmp
crystallized in the form of polymorph 2 that was prepared
by the following procedure. 1 mmol (0.16 g) hcmp was
dissolved in water at 90 °C. To the warm solution of hcmp
(1 mmol), solid MgCO3 (1 mmol) was added. The mixture
was refluxed for 10 min at 90 °C, and then glacial acetic
acid was added dropwise until the dissolution of MgCO3.
The pH of the reaction mixture was set to 10 by ammonia
solution drops and left at room temperature. After
2–3 days, yellowish single crystals were obtained and fil-
tered off. In this way, hcmp crystallizes with one water
molecule. Solvent change from water to ethanol in the
same synthetic procedure resulted in formation of anhy-
drous hcmp (3). The crystallization at room temperature
takes about 3 weeks. Yields: 84 % (1), 32 % (2) and 10 %
(3). Mr (C5H10N4O2) = 158.16 g mol-1. All crystalline
ligand types are well soluble in DMF and DMSO at room
temperature and partly soluble by heating in EtOH and
water. KM(DMF) = 1.0 S cm2 mol-1
.
[Zn(hcmp–H)2H2O]
To prepare complex of ZnII with hcmp, the ligand-to-metal
molar ratio was varied in 3:2, 2:1 and 1:1 proportions. The
optimal ligand-to-metal molar ratio for the complexation
with ZnII was 2:1 in an ethanolic solution. The reaction was
carried out by the mixing of zinc(II) salt (0.5 mmol) dis-
solved in 5 cm3 EtOH with a solution of hcmp (1 mmol) in
15 cm3 EtOH by heating and intensive stirring. After
standing for 24 h at room temperature, to the reaction
mixture 3 cm3 of ammonia solution was added. The addition
of ammonia solution promoted the deprotonation of the
ligand, namely the precipitation of the neutral complex. At
the same time, as a result of zinc ammine formation, the
concentration of ZnII is lowered, assisting thus the single-
crystal formation. In 2–3 days, single crystals were formed
from the solution containing ZnCl2 (uncolored), ZnBr2(light
orange) and Zn(NO3)2 (light yellow). With ZnBr2 and
ZnCl2, the ligand coordinates to form polymorph I. Differ-
ently, with Zn(NO3)2Á6H2O the polymorph II was obtained.
The precipitates were filtered off and washed with EtOH.
Yields: [85 % for I and [81 % for II. Both polymorphs,
Mr(ZnC10H16N8O3) = 361.68 g mol-1, are well soluble in
DMSO and DMF at mild heating, but in common solvents as
EtOH, MeOH, water or acetone, the complexes are hardly
soluble. KM(DMF) = 1.9 S cm2/mol.
Experimental
Materials and measurements
All chemicals were commercial products of analytical
reagent grade. The thermogravimetric and differential
scanning calorimetric data were recorded on SDT Q600
thermal analyzer, TA Instruments, at a heating rate of
10 °C min-1 in nitrogen atmosphere (100 cm3 min-1
)
with sample masses of *3 mg using alumina crucibles.
The non-isothermal phase change curves were recorded
using a PerkinElmer DSC 7 apparatus. Sample masses
were between 3 and 5 mg and measured at 10 °C min-1
heating and cooling rates under continuous nitrogen flow
(20 cm3 min-1) in hermetically sealed aluminum pans.
The molar conductivity of the freshly prepared DMF
solutions of hcmp and the complexes was measured on a
digital conductivity meter Jenway 4010.
Synthesis of the compounds
1-Carboxamidino-5-hydroxy-3-methylpyrazole (hcmp)
The polymorph hcmp (1) was obtained by the condensation
reaction of aminoguanidine hydrochloride and ethyl ace-
toacetate in an aqueous solution, in the presence of sodium
acetate as described by Erkin and Krutikov [16], and
crystallized in the form of monohydrate. As one of our
Crystal structure determination and refinement
Single-crystal X-ray diffraction data of compounds 1–3 and
I–II were collected on an Oxford Diffraction Gemini S
123

Struct Chem
Materials and methods for biological activity studies
four-circle diffractometer equipped with a Sapphire CCD
˚
detector, using CuKa radiation (k = 1.54184 A) at room
All reagents were purchased from Sigma-Aldrich Kft.,
Hungary, unless otherwise indicated. Phosphate buffer
solution (PBS, pH 7.4) and Dulbecco’s modified Eagle’s
medium (DMEM) were purchased from Gibco, Invitrogen.
Fetal bovine serum from Lonza, Switzerland. Cytotoxicity
detection kit measuring lactate dehydrogenase (LDH)
release from Roche, Switzerland. Ninety-six-well cell
culture plates form Orange, UK. Solubility measurements,
cell culture and the viability assays are described in detail
in the Supporting Material.
temperature. The data reduction was done using the Oxford
Diffraction program CrysAlisPro [17]. Empirical absorp-
tion correction using spherical harmonics implemented in
the SCALE3 ABSPACK [18] scaling algorithm were
applied for compounds 1, 3, I and II. Numerical absorption
correction using a multifaceted crystal model, as imple-
mented in CrysAlisPro [17] program system, was applied
for compound 2. The structures were solved by direct
methods using SIR92 program [19] as implemented in
WinGX program system [20]. Compound 3 crystallizes in
space group P212121, but it is a weak anomalous scatterer,
and the absolute structure cannot be determined reliably
from the Flack parameter. All non-hydrogen atoms were
refined anisotropically using SHELXL-97 [21] by applying
a full-matrix least-squares method based on F2, including
all reflections. The hydrogen atoms belonging to the methyl
group in 1 were located from difference electron-density
map, for 2–3 and I–II methyl hydrogens are generated at
ideal positions. The hydrogen atoms belonging to the
nitrogen atoms, and pyrazole ring carbon atom, were found
in the difference electron-density maps. All hydrogen
atoms were refined using riding model. The molecular
diagrams were generated using ORTEP-3 for Windows
[22]. For packing diagrams, the programs CAMERON [23]
and Mercury [24] were employed. Geometrical calcula-
tions were carried out using the program PLATON [25].
The crystal data and refinement parameters are summarized
in Table 1. In the amidine fragment, C1–N1/C1–N2 bond
lengths are 1.310(2)/1.313(2), 1.312(2)/1.309(2) and
Discussion
The synthesis and the structure, determined by spectro-
scopic methods, of hcmp are published earlier [16]. The
crystal structures of the two polymorphs of a monohydrate
and the solvent-free form obtained by different, well-re-
peatable synthetic procedures are described for the first
time here. It is known that in ionic salts, anion participates
in molecular self-assembly in coordination compounds
[29]. Our results refer to the significant role of the anion in
the crystallization process affecting the symmetry of the
crystal even when it is not a part of the molecule. The
molar conductivity values in DMF solution of the ligands
1–3 (1.0 S cm2 mol-1) and the complexes I and II
(1.9 S cm2 mol-1), in accordance with the structure, refer
to their non-electrolytic character [30].
˚
1.318(5)/1.301(5) A in molecules of 1, 2 and 3, respec-
Crystal and molecular structures of the ligand
tively. Close similarity of two C–N distances indicates that
amidine fragment is protonated and exists in two resonant
forms (Scheme 1). Consequently, hcmp in all three modi-
fications exists in the form of zwitterion with negative
charge located on the pyrazolone-O and positive charge
located at the protonated amidine fragment.
A perspective view and atom numbering scheme of 1 is
shown in Fig. 1. The same labeling scheme is applied for
the polymorph 2 and the solvent-free form 3.
Geometry of the selected nonbonding contacts is given
in Table 2.
1
According to its H NMR and IR spectrum [16], 5-hy-
droxy-1-carboxamidino-3-methylpyrazole (hcmp) in solu-
tion exists predominantly in the lactim form (Scheme 1a)
with strong interaction between hydroxyl and imino
groups. However, in the crystal structure of all three
polymorphic modifications, the amidine moiety is proto-
nated and hcmp exists as zwitterion with two resonance
forms (Scheme 1b, c).
Calculation of intermolecular energies
Molecule–molecule pairwise energies in 1–3 were calcu-
lated by the CLP program package [26] (version 12.5.2014).
Crystal coordinates were used for non-hydrogen atoms,
while the H atoms were normalized using the RETCIF
module of the CLP software [26]. Molecular electron-den-
sity calculations of 1–3 were performed in Gaussian 09 [27]
at the MP2/6-31G (d,p) level. These electron densities were
used to calculate intermolecular interaction energies in 1–3
by the PIXEL [28] method using a distance cutoff from the
Bond lengths of hcmp in all three polymorphic modifi-
cations are similar. All interatomic distances can be con-
sidered normal, and the pyrazole rings are planar within
experimental error. The only significant variation in the
molecular geometry is the rotation of the carboxamidine
group relative to pyrazole ring. Dihedral angle between the
mean planes of the pyrazole ring and carboxamidine group
˚
central molecule of 30 A. The positions of atoms for the
purpose of this calculation were obtained using RETCIF and
RETCOR modules of the CLP software.
123

Struct Chem
Table 1 Crystal data and structure refinement for compounds 1–3, I and II
1
2
3
I
II
Empirical formula
Formula weight
C5H8N4OÁH2O
158.17
C5H8N4OÁH2O
158.17
1.54184
Orthorhombic
Pbca
C5H8N4O
140.15
1.54184
Orthorhombic
P212121
7.767(5)
8.102(5)
10.966(5)
90
C10H16N8O3Zn
361.70
C10H16N8O3Zn
361.70
˚
Wavelength (A)
1.54184
Monoclinic
P21/n
1.54184
Monoclinic
P21/c
1.54184
Monoclinic
P21/n
Crystal system
Space group
˚
a (A)
7.8258(3)
11.5640(5)
8.7744(3)
90
12.4996(4)
9.8078(4)
12.8102(5)
90
10.5350(2)
13.0020(4)
11.0961(2)
90
8.8637(3)
7.3260(2)
23.3707(7)
90
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
102.929(4)
90
90
90
96.168(2)
90
98.550(3)
90
90
90
3
˚
V (A )
773.93(5)
4
1570.5(1)
8
690.1(7)
4
1511.10(6)
4
1500.72(8)
4
Z
Dcalc (g/cm3)
1.357
1.338
1.349
1.590
1.601
l (CuKa) (mm-1
)
0.908
0.895
0.841
2.514
2.531
F(000)
336
672
296
744
744
h range (°)
Ref. collected
6.4–72.3
2602
6.7–72.4
3774
6.8–72.1
1477
4.2–72.5
5525
3.8–72.5
5287
Unique ref. (Rint
Data/parameters
GOF on F2
)
1485 (0.023)
1485/101
1.07
1528 (0.023)
1528/102
1.05
1134(0.013)
1134/92
1.08
2920(0.020)
2920/201
1.05
2891(0.028)
2891/201
1.04
wR (all data)
R (all data)
3
Residual density (e/A )
0.1182
0.1212
0.0486
-0.22/0.24
1002040
66.2
0.1246
0.0536
-0.15/0.13
1002192
66.0
0.0933
0.1142
0.0393
0.0399
0.0507
˚
-0.17/0.20
1002039
67.3
-0.25/0.26
1002041
68.2
-0.36/0.41
1002042
68.7
CCDC No.
Pack. coefficient
Scheme 1 Tautomeric and resonant forms of hcmp
is 0.53°, 5.76° and 12.31° in 1, 2 and 3, respectively.
Intramolecular energies, as calculated in Gaussian 09 [27]
at the MP2/6-31G(d,p) level, indicate that intramolecular
energy of 1 is higher for 4.63 and 15.18 kJ/mol relative to 2
and 3, respectively. Inspection of nonbonding interactions
involving the carboxamidine group in 1–3 indicates that
this difference is associated with different environment of
carboxamidine group (Table 2). In the solvatomorphic
forms 1 and 2, the protonated carboxamidine group is
hydrogen-bonded to the two molecules of water and one
hcmp molecule. In anhydrous form 3, the carboxamidine
group interacts with two molecules of hcmp. It can be
Fig. 1 View of asymmetric unit of 1. Same labeling scheme is
applied for the polymorph 2 and the solvent-free form 3
therefore assumed that the steric effects, dependent on the
type of surrounding molecules (water or hcmp), influence
the orientation of the carboxamidine group. It should be
123

Struct Chem
Table 2 Geometry of the
selected nonbonding contacts in
1–3
˚
˚
D–HÁÁÁA
HÁÁÁA(A)
D–HÁÁÁA(°)
DÁÁÁA(A)
Symmetry code
1
N2–H4ÁÁÁO1
O2–H10ÁÁÁN4
O2–H9ÁÁÁO1
N2–H3ÁÁÁO2
N1–H2ÁÁÁO2
N1–H1ÁÁÁO1
Stacking interactions
1.92
2.09
1.89
2.11
1.99
1.97
172
159
165
151
173
131
2.814(2)
2.893(2)
2.759(2)
2.917(2)
2.880(2)
2.673(2)
-1/2?x, 1/2-y, 1/2?z
3/2-x, 1/2?y, 1/2-z
1/2?x, 1/2-y, 1/2?z
-1/2?x, 1/2-y, 1/2?z
-1?x, y, z
˚
Cg1ÁÁÁCg1(A)
3.6922(9)
1-x, -y, -z
2
N2–H4ÁÁÁO1
O2–H10ÁÁÁN4
N1–H2ÁÁÁO2
N2–H3ÁÁÁO2
O2–H9ÁÁÁO1
N1–H1ÁÁÁO1
3
1.96
2.04
2.00
2.04
1.87
1.97
165
164
171
157
162
135
2.835(2)
2.897(2)
2.880(2)
2.930(2)
2.717(2)
2.678(2)
x, 1/2-y, 1/2?z
-1/2?x, 1/2-y, 1-z
1-x, -y, 1-z
1/2?x, y, 1/2-z
N1–H2ÁÁÁO1
N2–H3ÁÁÁO1
N2–H4ÁÁÁN4
N1–H1ÁÁÁO1
1.88
1.92
2.32
1.98
173
155
151
133
2.766(4)
2.798(4)
3.064(5)
2.684(4)
1-x, -1/2?y, 1/2-z
1/2-x, -y, -1/2?z
1/2?x, -1/2-y, -z
Cg1 refer to the centroid of the ring formed by C2–C3–C4–N3–N4
noted that conformational flexibility of the –C(NH2)2 group
is partly reduced by the intramolecular hydrogen bond N1–
H1…O1 (Table 2) which is present in all three forms of
hcmp. Aggregation of molecules in polymorphs 1 and 2 is
achieved through the same hydrogen bonding motifs
(Table 2), and the same 1D supramolecular motif, molec-
ular chain, is formed (Fig. 2a). The molecules of hcmp are
directly connected through the N2–H4ÁÁÁO1 H-bonds and
also via the hydrogen bonds involving solvent water
molecule (N1–H2•••O2 and O2–H9•••O1) forming chains
of molecules related by a glide plane. The similarity of the
molecular chains formed in 1 and 2 is reflected in the
mutual orientation of the consecutive pyrazole moieties.
Thus, dihedral angle between the mean planes of the
neighboring pyrazole rings within the same chain is 75.1°
and 75.8° in compounds 1 and 2, respectively. This simi-
larity is illustrated in Fig. 2, where the parts of the crystal
structures of 1 (black) and 2 (gray) are overlaid. Connec-
tion between the neighboring chains in both 1 and 2 is
achieved through the interactions of hcmp with water
(Table 2; Fig. 2b, c). While the intrachain geometry and
associated intermolecular contacts show close similarity in
polymorphs 1 and 2, aggregation of these chains into the
crystal lattice differentiate the two structures. In 1
(Fig. 2b), hcmp molecules from the neighboring chains are
oriented in an antiparallel fashion, giving rise to stacking
interaction with a centroid-to-centroid separation of
˚
3.69 A. Dissimilar to 1, the hcmp molecules from the
neighboring chains in 2 (Fig. 2c) are mutually rotated
forming the torsion angle O1–Cg–Cgi–O1i of 131.73°,
while the closest centroid-to-centroid separation increases
˚
to 6.27 A (Fig. 2c; i = 1/2 ? x, y, 1/2–z; Cg1 designate
centroid of the ring C2–C3–C4–N3–N4). The closer
approach of the hcmp rings in the structure 1 is probably
facilitated by the hydrogen bonding involving two water
molecules (Fig. 2b) and O1 and N4 acceptors of stacked
rings. This is not the case with 2 where only one water
molecule is situated between the rings.
In polymorph 3, basic supramolecular motif could be
also described as molecular chain, formed by the propa-
gation of the same type of hydrogen bonds as in 1 and 2,
the N2–H3ÁÁÁO1 (Fig. 3b). Molecules in the chain are
related by a screw axis and arranged in the crystallographic
c direction. Dihedral angle between the mean planes of the
neighboring pyrazole rings within the same chain is 73.59°,
not significantly different from 1 and 2. Neighboring chains
are linked through the N–HÁÁÁO and N–HÁÁÁN H–bonds
(Fig. 3a; Table 2).
In summary, it can be said that in polymorphs 1, 2 and 3,
the basic supramolecular motif, common to all three
structures, is a molecular chain formed by the N–HÁÁÁO
hydrogen bonds. What makes the difference between the
123

Struct Chem
Fig. 2 Overlay diagram of
molecular chains (a) in 1 (black)
and 2 (gray) depicting the
similarity of the intrachain
geometry. Part of the crystal
structure illustrating different
orientation of neighboring
chains in 1 (b) and 2 (c).
Dashed line indicates closest
ring–ring separation
(d) between the neighboring
chains
Fig. 3 Crystal packing of 3. View down the c axis (a) shows arrangement of molecules into chains and interchain links. View down the a axis
(b) depicts details of intrachain and interchain connections
three modifications is the way these chains connect into
crystal structure. The above observations pose two ques-
tions: Does these structural differences in the aggregation
of molecular chains lead to significant difference in the
stability of the crystal, and what is the relative importance
of various nonbonding contacts for the formation of par-
ticular molecular arrangement in the crystal? In order to
investigate the significance of nonbonding contacts for the
aggregation of molecules, common approach is to compare
observed geometrical properties with a vast quantity of
empirical data related to the geometry of intermolecular
contacts [31]. However, ranking the contacts observed in a
particular structure in terms of their importance while
relaying only on geometrical criteria is complicated by the
presence of a number of close nonbonding atom–atom
contacts. These contacts often have similar geometrical
features, and there are no clear criteria for connecting the
particular structural features with its significance in the
process of molecular aggregation. One of the methods to
overcome this problem is quantitative evaluation of crystal
lattice energy and partition of energy into pairwise mole-
cule–molecule contributions [32]. To gain more insight
into the relative importance of various nonbonding con-
tacts, pairwise molecule–molecule energies of 1–3 were
calculated using PIXEL method implemented in CLP
program [26, 34]. To identify the most significant inter-
molecular contacts, we looked at the molecule–molecule
energies. Interaction energies of the selected molecular
123

Struct Chem
pairs, partitioned into electrostatic (Coulombic, EC),
polarization (EP), dispersion (Ed) and repulsive (Er) con-
tributions, together with associated intermolecular con-
tacts, are given in Table 3. In polymorphs 1–3, the largest
interaction energy corresponds to the hydrogen bond
formed between the pyrazolone oxygen and amino hydro-
gen. In 1 and 2, the molecular chains are formed through
the hcmpÁÁÁhcmp and hcmpÁÁÁH2O interactions. Connection
between the molecular chains is achieved through the
interaction of hcmp with water. In 3, hcmp molecules are
linked directly and pack in a herringbone fashion. The
packing coefficients [33] are not significantly different in 1,
2 and 3 (Table 1). Due to the zwitterionic nature of the
molecule (Scheme 1), dominant contribution to the stron-
gest intermolecular interaction comes from the coulombic
term (Table 3). Partition of the total energy of pairwise
interactions indicates that electrostatic forces are dominant
in other intermolecular contacts, too. The only exception to
this is stacking interaction of hcmp molecules in 1
(Tables 2, 3). In this case, dominant contribution to the
intermolecular energy comes from dispersion forces, while
the coulombic energy has a positive sign and destabilizes
the stacking arrangement. The largest energy of inter-
molecular interaction in 3 is somewhat lower than the
largest energy observed in 1 and 2. However, the second
largest energy of interaction in 3 is larger than the second
largest energy found in 1 and 2.
It should be noted that the presence of stacking inter-
actions supported by two pairs of hydrogen bonds involv-
ing water did not lead to larger temperature of dehydration
in 1, in comparison to 2 where the stacking interactions are
absent.
Crystal and molecular structures of the complexes I
and II
Coordination of hcmp to Zn in the presence of chloride or
bromide ions leads to the formation of [Zn(hcmp–H)2H2O]
in the polymorphic form I. If nitrate ion is used instead, the
polymorph II is formed. In complexes I and II, the Zn
atom is five-coordinated with two hcmp ligands bonded
through the N4 atom of pyrazole ring and amidinium
nitrogen N2. The fifth position is occupied by a water
molecule (Fig. 4a).
The Addison distortion index s [35] (s = 0 for a square
pyramid and s = 1 for a trigonal bipyramid) is 0.84 and
0.72 for complex molecules I and II, respectively. Hence,
for both I and II, the coordination geometry is best
described as distorted trigonal bipyramid. Similarity of the
molecular geometries in polymorphs I and II is evident
from the comparison of relevant bonds and angles. Overlay
of the molecules (Fig. 4b) of I (red) and II (green) illustrate
the similarity of the molecular shape. Hence, both com-
plexes have similar capacity for the formation of
Table 3 PIXEL [32] interaction energies (kJ/mol) in the crystals 1–3, between molecular pairs related by a symmetry code, and the associated
intermolecular contacts
Molecular pair
Ecoul
Epol
Edisp
Erep
Etot
Associated intermolecular contacts*
Symmetry code
1
hcmpÁÁÁhcmp
hcmpÁÁÁH2O
hcmpÁÁÁH2O
hcmpÁÁÁH2O
hcmpÁÁÁH2O
hcmpÁÁÁhcmp
2
-73.2
-51.5
-50.0
-23.9
-30.3
4.2
-23.3
-23.4
-20.3
-9.3
-15.6
-17.9
-10.3
-7.8
47.9
58.3
54.9
19.0
35.6
10.3
-64.2
-34.4
-25.7
-22.0
-21.7
-17.7
N2–H4ÁÁÁO1
O2–H10ÁÁÁN4
O2–H9ÁÁÁO1
N2–H3ÁÁÁO2
N1–H2ÁÁÁO2
Cg1ÁÁÁ.Cg1
-1/2?x, 1/2-y, 1/2?z
3/2-x, 1/2?y, 1/2-z
1/2?x, 1/2-y, 1/2?z
-1/2?x, 1/2-y,1/2?z
-1?x, y, z
-16.1
-5.6
-10.9
-26.6
1-x, -y, -z
hcmpÁÁÁhcmp
hcmpÁÁÁH2O
hcmpÁÁÁH2O
hcmpÁÁÁH2O
hcmpÁÁÁH2O
3
-72.6
-48.7
-34.3
-25.5
-47.4
-22.3
-22.4
-15.9
-12.0
-23.0
-15.1
-16.0
-10.2
-9.0
42.8
53.7
34.0
23.4
62.2
-67.2
-33.4
-26.3
-23.2
-19.2
N2–H4ÁÁÁO1
O2–H10ÁÁÁN4
N1–H2ÁÁÁO2
N2–H3ÁÁÁO2
O2–H9ÁÁÁO1
x, 1/2-y,1/2?z
-1/2?x, 1/2-y, 1-z
1-x, -y, 1-z
-11.0
1/2?x, y, 1/2-z
hcmpÁÁÁhcmp
hcmpÁÁÁhcmp
hcmpÁÁÁhcmp
-74.2
-57.9
-26.4
-28.5
-23.1
-15.7
-17.0
-20.8
-17.4
58.1
43.7
24.5
-61.6
-58.1
-35.0
N1–H2ÁÁÁO1
N2–H3ÁÁÁO1
N2–H4ÁÁÁN4
1-x, -1/2?y, 1/2-z
1/2-x, -y, -1/2?z
1/2?x, -1/2-y, -z
Cg1 refers to the centroid of the ring formed by C2–C3–C4–N3–N4
123

Struct Chem
Fig. 4 a ORTEP view of I. The atom labeling schemes in I and II are identical. Methyl hydrogens have been removed for clarity. b Overlay of
molecular structures in I (black) and II (gray)
intermolecular interactions. The most notable difference in
the molecular geometry of I and II is the orientation of the
coordinated water molecule relative to the Zn–O2 bond
(Fig. 5). In complex I, the water molecule is oriented so
that angle between the mean plane of the water atoms and
Zn–O2 bond is 0.4(2)°. In II, water molecule is tilted rel-
ative to Zn–O2 bond and the associated angle is 40.4(2)°.
This difference influences the capacity of coordinated
water for intermolecular bonding since, due to the steric
factors, oxygen is more easily accessible for neighboring
molecules in II than in I. This is reflected in the type and
geometry of intermolecular contacts. Geometry of the
selected nonbonding contacts in I and II is given in
Tables 4 and 5. In I, water is hydrogen-bonded to two
neighboring molecules through the O2–H2ÁÁÁO1 and O2–
H2aÁÁÁO1a H-bonds. In II, water is involved in same
hydrogen bonding motif with two neighboring molecules,
but additionally accepts hydrogen bond from the third
molecule N1–H2…O2. Difference in the position of
coordinated water and the associated hydrogen bonds are
depicted in Fig. 5.
In I, water is hydrogen-bonded to pyrazolone oxygen of
the two neighboring molecules. Both pyrazole rings of a
complex molecule are involved in stacking interactions
˚
with ring–ring distances of 3.478(1) and 3.464(2) A
(Fig. 6). Propagation of this interaction leads to the
aggregation of molecules into chains running along the
crystallographic a direction. Neighboring molecular chains
are connected through the N–HÁÁÁO H-bonds (Tab 5,
Fig. 7).
In polymorph II, coordinated water serves as a donor of
hydrogen bonds toward the two neighboring molecules as
in polymorph I. But dissimilar to I, it also serves as an
acceptor of hydrogen bond from the third molecule (N1–
H…O2) (Fig. 8, colored in black). In the resulting
arrangement, only one of the pyrazole rings forms stack-
ing contact similar to I (Fig. 8, p2), while hydrogen-do-
nating molecule (Fig. 8, colored in black) is involved in
the additional stacking arrangement (Fig. 8, p1). These
stacking interactions (p1, p2, Fig. 8) propagate in the
crystallographic b direction (Fig. 9). Hence, interplay of
N2–H3•••O1a, O2–H8•••O1a and stacking interactions
lead to formation of multistacked chain of molecules.
Neighboring chains are connected through the N1–
H2•••O2, N1a–H2a•••O1 and O2–H9•••O1 H-bonds
(Fig. 9).
Thermal data of the compounds
The bioavailability of pharmaceuticals strongly depends on
its solubility that, in turn, depends on the corresponding
crystalline form. DSC studies of polymorphs are especially
useful to detect and quantify polymorphism [36] or to
determine the transitions between the different polymor-
phic forms [37]. Even the solubility of a solid may be
measured as a function of temperature by differential
Fig. 5 Details of crystal structures of I (a) and II (b) showing
different orientation of the coordinated water relative to Zn–O bond.
Relevant hydrogen bonding is depicted as dashed lines
123

Struct Chem
Table 4 Geometry of selected
intermolecular contacts in I
˚
˚
D–HÁÁÁA
HÁÁÁA(A)
D–HÁÁÁA(°)
DÁÁÁA(A)
Symmetry code
N1–H2ÁÁÁO1i
2.26
2.19
1.89
1.81
159
164
176
177
3.124(3)
3.109(3)
2.699(3)
2.670(3)
i = x, 1/2-y, -1/2?z
i = x, 1/2-y, -1/2?z
ii = 1-x, -y, -z
N1a–H2aÁÁÁO1ai
O2–H8ÁÁÁO1aii
O2–H9ÁÁÁO1iii
iii = 2-x, -y, 1-z
Stacking interactions
p1 [Cg1ÁÁÁCg1iv = 3.478(1) A]
iv = 2-x, -y, 1-z
v = 1-x, -y, -z
˚
v
˚
p2 [Cg2ÁÁÁCg2 = 3.464(2) A]
Cg1 and Cg2 are the centroids of pyrazole rings N3–C4 and N3a–C4a, respectively
Table 5 Geometry of selected
intermolecular contacts in II
˚
˚
D–HÁÁÁA
HÁÁÁA(A)
D–HÁÁÁA(°)
DÁÁÁA(A)
Symmetry code
N1–H2ÁÁÁO2i
2.19
2.08
2.19
2.49
1.80
1.82
158
162
165
145
164
169
3.058(3)
2.950(3)
3.108(3)
3.318(3)
2.619(3)
2.668(3)
i = x, -1?y,z
N1a–H2AÁÁÁO1ii
ii = -1/2-x, 1/2?y, 1/2-z
iii = -x, -y, 1-z
N2–H3ÁÁÁO1aiii
N2A–H3aÁÁÁO1ii
ii = -1/2-x, 1/2?y, 1/2-z
iv = -x, 1-y, 1-z
O2–H8ÁÁÁO1aiv
O2–H9ÁÁÁO1v
v = 1/2-x, 1/2?y, 1/2-z
Stacking interactions
p2 [Cg3ÁÁÁCg3vi = 3.620(2)]
p2 [Cg3ÁÁÁCg3vii = 3.742(2)]
vi = -x, -y, 1-z
vii = -x, 1-y, 1-z
Cg3 is the centroid of pyrazole ring N3a–C4a
Fig. 6 View of the crystal packing of I showing the formation of two molecular chains (colored in black and gray). Hydrogen bonds and
stacking interactions (p1, p2) are depicted in dotted and waved lines, respectively
scanning calorimetry of the saturated solution. Here, we
used simultaneous thermogravimetric/differential scanning
calorimetric (TG/DSC) measurements to compare the
thermal decomposition temperatures and the possible phase
transitions between polymorphic forms of compounds 1–3
and I–II. For the sake of comparison, DTG/DSC curves
were used for the corresponding compounds. In Fig. 10,
the thermal decomposition of different forms of the ligand
is depicted, while Fig. 11 shows the comparison of the
thermal behavior of the complexes.
123

Struct Chem
Monohydrates 1 and 2 loose the crystal water below
100 °C. The amount of water corresponds to the stoichio-
metric quantity (calcd: 11.39 %): 10.5 and 11.1 % in 1 and
2, respectively. The DTG and DSC maximum for dehy-
dration in compound 2 appear at a little higher temperature
(ca. 5 °C, see Fig. 10). After water evaporation, the
dehydrated ligands are stable and decompose at DTG onset
temperatures of 249 and 243 °C in 1 and 2, respectively.
The anhydrous ligand 3 is stable to 226 °C. Above 350 °C,
the decomposition of 1–3 in nitrogen is practically finished
with about 25 % tar residue.
In principle, some of the polymorphic transitions may be
reversible. However, such transition between 1 and 2 in the
measured temperature range is not detected. Neither is
visible the melting of the compounds. The steep start in
DSC curve above 200 °C may refer to melting, but
Fig. 7 View of crystal packing in I down axis c showing the
arrangement of molecular chains
Fig. 8 View of the crystal
structure of II, showing the
significant hydrogen bonds and
formation of multistacked chain
Fig. 9 Formation of molecular chains in II and connections between them. Regions involved in multistacked interactions are labeled with p
123

Struct Chem
Fig. 10 DTG and DSC curves for the ligand forms 1–3
Fig. 11 DTG and DSC curves for complexes I and II
according to the corresponding DTG curves it is accom-
panied by decomposition.
difference in decomposition scheme is in the rate of the
first decomposition step that is somewhat higher in II. The
decomposition is not completed up to 700 °C. There is no
sign of phase transition between the polymorphs. The
comparison of the decomposition mechanism of the ligands
and the complexes can be found in ESI.
In complexes I and II, the trigonal bipyramidal envi-
ronment around zinc is established by NN-coordination of
two mono-deprotonated hcmp ligand molecules and an
aqua ligand. However, the high decomposition temperature
(Fig. 11) suggests that water molecules are not present.
TG/MS measurements (see ESI) support this observation.
Namely, despite the coordination of H2O, it evaporates at
room temperature during the storage. Water can be detec-
ted in the freshly prepared compound only. The anhydrous
complexes have practically the same decomposition tem-
perature (DTG onset 157–158 °C). The only significant
In order to trace the phase transitions in solvatomorphs,
DSC measurements were taken in the temperature range
from 50 to -50 °C for all the forms of the ligand as well as
for the complexes. On heating, 2 shows an endothermic
solid–solid phase transition at -1.6 °C peak temperature
(see Fig. 12). On cooling 2 far below the equilibrium
temperature (temperature range -25 and -45 °C), several
123

Struct Chem
hcmp (3) is crystallized. The reaction of the hcmp with
MgCO3 instead of coordination resulted in a formation a
new polymorph of hcmpÁH2O (2). Depending on the
reaction conditions, two stable polymorphs of zinc com-
plex were isolated. The polymorphism in both cases
depends on the reaction conditions, namely on the ions,
which do not take part in complex formation.
Comparison of intramolecular energies of 1–3 shows
that intramolecular energy decreases in the order
1 [ 2 [ 3. Examination of the strength of pairwise mole-
cule–molecule interactions indicates that in 1 and 2, the
aggregation of molecules into chains gives the main con-
tribution to the stability of crystal lattice. Water molecules
located between the chains interact with these chains with
much lower energy. In 3, molecules are packed in her-
ringbone fashion with dominant contribution of electro-
static forces to the stabilization of crystal lattice. Main
structural difference between 1 and 2 is the different mode
of aggregation of the otherwise similar molecular chains.
This is caused by different interchain nonbonding interac-
tions and particularly by the presence of stacking interac-
tions in 1, between the two pyrazole rings supported by
four nonbonding O–HÁÁÁN H-bonds. In 2, there are no
stacking interactions and the link between the chains is
achieved by the pair of O–HÁÁÁN H-bonds. Interestingly, the
presence of stacking interactions supported by hydrogen
bonds did not lead to larger temperature of dehydration of
the compound 1, relative to 2. It is significant to note the
same mutual orientation of the pyrazole rings involved in
stacking interactions in 1, I and II. In all three cases,
pyrazole rings are oriented in antiparallel fashion.
Fig. 12 Phase transitions of the ligand 1 and 2 by DSC
small exothermic peaks appear due to the time necessary
for nucleation. On the second heating, the endothermic
peak at -2.1 °C refers to a reversible phase transition.
However, under dynamic conditions the transformation
between phases is not entirely complete. In complexes
I and II, no phase transition was detected.
Biological activity
Toxicity of I and II on Caco-2 cells
Analysis of intermolecular contacts in I and II and
visual inspection of the resulting molecular aggregation
suggest the key role of the coordinated water in the for-
mation of two polymorphic forms, I and II. Enhanced
hydrogen bonding capacity of coordinated water in II rel-
ative to I is associated with the formation of multistacked
chain of molecules in II.
MTT viability and LDH cell toxicity tests were used to
determine the non-toxic concentrations on Caco-2 human
intestinal epithelial cells. From the hcmp ligand series for
3, no cell damaging effect was measured by MTT assay for
24-h treatment in the concentration B1000 lM. Complex
formation notably enhanced the biological activity of the
ligand, and the toxic concentration was more than 30 times
higher on Caco-2 cells for both I and II (C30 lM) mea-
sured by a metabolic assay. With LDH assay, a significant
release of LDH was observed at 1 lM and higher con-
centrations of II in Caco-2 cells. On the contrary, despite
the minor structural differences between I and II, the
release of LDH in the solution of complex I started only by
increasing its concentration to above 30 lM.
Despite the similar structures, the dehydration appears at
somewhat higher temperatures in 2 than in 1, while the
stability of the dehydrated 1 is somewhat higher (252 and
248 °C onsets for 1 and 2). The decomposition temperature
of the anhydrate form 3 starts at 230 °C. This indicates that
the stability of the compounds decreases in the order
3 [ 2 [ 1. Both the monohydrate complexes I and II loose
the crystal water at room temperature. The only significant
difference in decomposition scheme is in the rate of the
first decomposition step that is higher in II.
Conclusions
No significant biological activity was found in hcmp
ligands. The toxicity of the zinc(II) complexes I and II is
considerably higher. In spite of the high similarity in their
structures, polymorph II shows a markedly higher release
of LDH compared to that of I.
In aqueous solution, in the presence of sodium acetate, the
monohydrate of hcmpÁH2O is obtained in the form of
polymorph 1, while in ethanolic solution the anhydrous
123

Struct Chem
Acknowledgments This work was supported by the Ministry of
Education and Science of the Republic of Serbia (continuation of the
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¯
helpful discussions.
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