Pharmaceutical salts of biologically active hydrazone compound salinazid: Crystallographic, solubility, and thermodynamic aspects

Article abstract of DOI:10.1021/acs.cgd.5b01681

The crystal structures of salts of the active pharmaceutical ingredient (API) called salinazid with dicarboxylic acids and acesulfame were determined by single-crystal X-ray diffraction method. The crystals contain hydrogen bond motifs of different structure and complexity, the energies of which were estimated by using the quantum theory of atoms in molecules and crystals (QTAIMC) methodology. It was found that the driving force for facile the oxalate and malate salts formation is the bifurcated N⁺-H···O^- and N⁺-H···O hydrogen bond synthon, while salinazid malonate is mainly stabilized via a "classic" pyridinium-carboxylate heterosynthon. The oxalate and acesulfame salts of salinazid were found to be stable during aqueous dissolution experiments, providing a substantial solubility improvement compared to pure API (33 and 18 times, respectively). However, the malonate and malate salts dissolved incongruently and rapidly underwent a solution-mediated transformation to form pure salinazid. Based on the solubility data of the stable salts and of the pure components, the Gibbs free energy of the salts formation were calculated to be -21.2 kJ·mol^-1 for salinazid oxalate and -22.6 kJ·mol^-1 for salinazid acesulfame.

Full text of DOI:10.1021/acs.cgd.5b01681

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Article
Pharmaceutical salts of biologically active hydrazone compound
salinazid: crystallographic, solubility and thermodynamic aspects
Artem O. Surov, Alexander P. Voronin, Anna A. Simagina, Andrei V. Churakov, and German L. Perlovich
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b01681 • Publication Date (Web): 30 Mar 2016
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Pharmaceutical salts of biologically active
hydrazone compound salinazid: crystallographic,
solubility and thermodynamic aspects
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Artem O. Surova, Alexander P. Voronina, Anna A. Simaginaa, Andrei V. Churakovb, German L.
Perlovicha,*
aInstitution of Russian Academy of Sciences, G.A. Krestov Institute of Solution Chemistry RAS,
153045, Ivanovo, Russia. Eꢀmail: glp@iscꢀras.ru
bInstitute of General and Inorganic Chemistry RAS, Leninskii Prosp. 31,119991, Moscow,
Russia.
*To whom correspondence should be addressed: Telephone: +7ꢀ4932ꢀ533784; Fax: +7ꢀ4932ꢀ
336237; Eꢀmail glp@iscꢀras.ru
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Abstract
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The crystal structures of salts of the active pharmaceutical ingredient (API) called salinazid with
dicarboxylic acids and acesulfame were determined by singleꢀcrystal Xꢀray diffraction method.
The crystals contain hydrogen bond motifs of different structure and complexity, the energies in
which were estimated by using the quantum theory of atoms in molecules and crystals
(QTAIMC) methodology. It was found that the driving force for facile the oxalate and malate
salts formation is the bifurcated N+–H···Oꢀ and N+–H···O hydrogen bond synthon, while
salinazid malonate is mainly stabilized via a “classic” pyridiniumꢀcarboxylate heterosynthon.
The oxalate and acesulfame salts of salinazid were found to be stable during aqueous dissolution
experiments, providing a substantial solubility improvement compared to pure API (33 and 18
times, respectively). However, the malonate and malate salts dissolved incongruently and rapidly
underwent a solutionꢀmediated transformation to form pure salinazid. Based on the solubility
data of the stable salts and of the pure components, the Gibbs free energy of the salts formation
were calculated to be ꢀ21.2 kJ·mol–1 for salinazid oxalate and ꢀ22.6 kJ·mol–1 for salinazid
acesulfame.
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Keywords: hydrazone derivatives; pharmaceutical salts; Xꢀray diffraction; solidꢀstate DFT
calculations; noncovalent interactions; solubility; formation thermodynamics.
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1. Introduction
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Selection of an appropriate dosage form for a drug compound is a crucial step in the process of
drug development.1 It has been reported that approximately 80% of all medications are delivered
on the market as solidꢀstate formulations (e.g., tablets, capsules, etc.) due to their evident
advantages in terms of stability, longer storage, production and transportation economy, and
convenience of the intake.2ꢀ5 Pharmaceutical solids are classified as enteral drug forms. This
implies the oral route of administration of the active pharmaceutical ingredient (API) and
delivery through the gastrointestinal tract. However, pharmaceutically relevant physicochemical
properties of an API, such as solubility, chemical and thermodynamic stability, bioavailability,
etc., usually have to be modulated and tuned to provide the optimal API solid form for practical
use. One of the straightforward ways to considerably improve the physicochemical and
biological properties of an API without modifying its pharmacophore structure is to develop
novel solid forms such as salts or coꢀcrystals. It has been reported that salt formation is the most
frequently chosen method in the pharmaceutical industry and today more than 50% of APIs are
marketed as salts.6 In the realm of salts, the main challenge to consider is selection of a suitable
counterꢀion, which would provide desired properties of a new solid form. For weakly basic APIs,
salt formation with inorganic acids (such as hydrochloric acid) has often been the most obvious
and convenient way to improve bioavailability of a drug. However, there are some potential
drawbacks of using, for example, hydrochloride salts, namely, their high acidity in parenteral
formulations, the risk of corrosion of industrial equipment, a decreased solubility in the stomach
due to a common ion effect, etc.7 These problems can be partly avoided by using
pharmaceutically acceptable carboxylic acids and other relatively strong organic acids that are
able to form stable coꢀcrystal/salt forms with weakly basic APIs.8
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This work is a continuation of our previous studies9 concerning new crystalline forms of the
biologically active hydrazone compound salinazid (Slz) ((2ꢀhydroxybenzaldehyde)isonicotinoyl
hydrazone) (Figure 1). Hydrazones represent a special group of Schiff base compounds, that are
of a great interest to the pharmaceutical and medicinal chemistry because of broad spectrum of
biological activities.10,11 Hydrazone derivatives have been recognized as potent agents for
treatment of drugꢀresistant forms of tuberculosis12ꢀ14 and reported to have lower toxicity than
isoniazid (an important firstꢀline antituberculosis drug) due to the blockage of –NH2 group.15
Moreover, the methoxyꢀ and hydroxyꢀ substituted hydrazones have been demonstrated to possess
antioxidant16 and antitumoral activities.17
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In this work, we discuss the crystal structures and physicochemical properties of the salinazid
salts with wellꢀknown pharmaceutically acceptable dicarboxylic acids, namely oxalic acid,
malonic acid and malic acid (Figure 1). In addition, well known sugar substitute called
acesulfame was used as a salt former. Acesulfame is an aliphatic calorieꢀfree sweetener, and its
potassium salt is widely used in food products and pharmaceutical formulations. It has been
reported that organoleptic properties of a formulation can be improved by adding acesulfame,
which suppress the bitter taste of a drug.18 However, examples of using acesulfame as a guest
molecule to prepare novel solid forms are limited. Multicomponent crystals containing
acesulfame are currently described for the following drugs: griseofulvin,19 haloperidol,20 5ꢀ
fluorocytosine,21 theophylline,22 ciprofloxacin and norfloxacin.23
All the novel solid phases were characterized by Xꢀray diffraction and thermal techniques.
Energies of the strongest intermolecular interactions in the crystals were estimated using the
solid state DFT calculations with the subsequent quantum theory of atoms in molecules and
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crystals (QTAIMC) analysis of the periodic electron density. Water solubility, stability and
formation thermodynamics of the salinazid salts were also investigated and analyzed.
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Figure 1. Molecular structures of salinazid, dicarboxylic acids and acesulfame used in this work.
Flexible torsion angles in the salinazid molecule are numbered and indicated by τ1, τ2 and τ3.
2. Material and Methods
2.1. Compounds and solvents
Salinazid ((2ꢀhydroxybenzaldehyde)isonicotinoyl hydrazone, C13H11N3O2, 99%) was obtained
from Interbioscreen Ltd. Oxalic acid dihydrate (C2H2O4·2H2O, 99.5%) and acesulfame
potassium (C4H4KNO4S, 99%) were purchased from SigmaꢀAldrich. Malonic (C3H4O4, 99%)
and DLꢀmalic (C4H6O5, 99%) acids were purchased from Acros Organics. All the solvents were
of analytical grade and used as received without further purification. Acesulfame potassium was
neutralized with HCl to obtain free acesulfame acid according to the procedure described by
Velaga et al.24 Acesulfame acid was identified as form I by Xꢀray powder diffraction (XRPD)
and differential scanning calorimetry (DSC) (see Figure S1 of the supporting information).
2.2. Crystallization procedure
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Due to considerable difference in solubilities of salt components in common organic solvents,
a significant excess of acids (except for acesulfame acid) was needed to keep the conditions
when only the salt is a thermodynamically stable solid phase. For the crystallization experiments
of salinazid with malonic and malic acids, salinazid (50 mg, 0.2mM) and acids were dissolved in
10 ml of methanol in a 1:10 molar ratio and stirred at 50ꢀ60°C until a clear solution was
obtained. The solution was slowly cooled and kept at room temperature. Diffraction quality
yellow crystals of the malonate and malate salts appeared inside the solution over a period of 2ꢀ3
days.
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An attempt to crystallize salinazid with oxalic acid using the same experimental condition as
described above resulted in etherification of the acid molecule to form a hydrated salt of
salinazid with monomethyl oxalate. The relevant crystallographic details and the asymmetric unit
with displacement ellipsoids for this complex are given in the supporting information (Figure S2
and Table S1). Crystals of salinazid oxalate suitable for Xꢀray analysis were obtained after 2ꢀ3
days by slow evaporation of acetonitrile solution with the API to acid molar ratio equal to 1:2.
For the crystallization experiment of salinazid with acesulfame acid, equimolar amounts of
components (0.2mM) were dissolved in 6 ml of methanol in a 1:1 molar ratio and stirred at 50ꢀ
60°C until a clear solution was obtained. The resulting solution was allowed to crystallize at
room temperature. Yellow crystals of acesulfame salt suitable for a Xꢀray experiment were
collected in several days.
The bulk samples of the salts were obtained by slurring equimolar amounts of salinazid and
corresponding acid in methanol (acetonitrile for salinazid oxalate) for several hours at room
temperature.
2.3. X-ray diffraction experiments
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Singleꢀcrystal Xꢀray diffraction data were collected on a Bruker SMART APEX II
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diffractometer using graphiteꢀmonochromated MoKα radiation (λ = 0.71073 Å) using
ωꢀscan
mode . Absorption corrections based on measurements of equivalent reflections were applied.25
The structures were solved by direct methods and refined by full matrix leastꢀsquares on F2 with
anisotropic thermal parameters for all nonꢀhydrogen atoms.26 All hydrogen atoms were found
from the difference Fourier map and refined isotropically. Data were collected at 183K for the
crystals of [Slz+Oxalic] (1:1), [Slz+Acesulfam] (1:1) and at 150K for the [Slz+Malonic] (1:1),
[Slz+Malic] (1:1) salts.
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The crystallographic data have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publications under the CCDC numbers 1437855ꢀ1437859. This
information may be obtained free of charge from the Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
Xꢀray powder diffraction (PXRD) data were recorded under ambient conditions in Braggꢀ
Brentano geometry by a Bruker D8 Advance diffractometer with CuKα1 radiation (λ = 1.5406
Å).
2.4. DSC experiments
Thermal analysis was carried out using a Perkin Elmer DSC 4000 differential scanning
calorimeter with a refrigerated cooling system (USA). Approximately 1ꢀ2 mg of the solid sample
was heated in sealed aluminum sample holders in the temperature range of 25−280 °C at the rate
of 10°C·minꢀ1 in a nitrogen atmosphere. The unit was calibrated with indium and zinc standards.
The accuracy of the weighing procedure was ±0.01 mg.
2.5 Aqueous dissolution.
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The solubilities of the solids were determined by the shakeꢀflask method at 25.0 ± 0.1°C. An
excess of each sample was added into pyrex glass tubes with 10 ml of degassed water. After 24
hours the solid phase was filtered through 0.2 ꢀm PTFE syringe filter, and the concentration of a
compound in a solution was determined by UV−vis spectroscopy (Varian Cary 50) at the
reference wavelength (330 nm). Residual solid materials at the end of the solubility experiments
were collected and analyzed by using PXRD.
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2.6 Phase solubility studies
Solubility studies were performed as described by Connors.27 Excess amounts of salinazid
were added to 2 ml of the aqueous solutions containing different concentrations of the
corresponding acids. The samples were shaken at 25.0 ± 0.1°C to attain equilibrium (24 hours),
an aliquot was filtered using a 0.2 ꢀm filter (Rotilabo® syringe filter, PTFE). The drug assay
was measured by UV−vis spectroscopy (Varian Cary 50) at the reference wavelength (330 nm).
2.7 Computational procedure
The CRYSTAL14 software28 was used to perform the DFT calculations with periodic
boundary conditions (solidꢀstate DFT) at the B3LYPꢀD2/6ꢀ31G** level of theory. In the present
study we explored a simplified approach. It is widely used for the description of the particular
types of intermolecular interactions (conventional Hꢀbonds and/or CꢀH…O contacts).29ꢀ31 In this
approach the crystalline wavefunction is computed using the experimental space group, lattice
parameters and atom coordinates. The Bader analysis was performed with TOPOND1432,33 and
the energies of the particular noncovalent interactions, Eint, were evaluated according to Mata et
al.34 as
Eint = 0.429·Gb (in atomic units)
(1)
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It has been reported that equation (1) yields reasonable values of Eint for intermolecular
interactions with different strength from strong chargeꢀassisted hydrogen bonds to weak vanꢀderꢀ
Waals contacts.35ꢀ39
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3. Results and Discussion
It is widely accepted in the literature that whether an API and a guest molecule form a salt or
coꢀcrystal can be predicted in terms of the ∆pKa rule (ꢁpKa = pKa(base) ‒ pKa(acid)).40ꢀ42 When the
ꢁpKa is greater than 3, the components tend to form a salt. If ꢁpKa ≤ 0, a coꢀcrystal is likely to
be formed. In the ꢁpKa range between 0 and 3, however, the ionization state of molecules in a
crystal remains hardly predictable. The pKa value based on pyridine nitrogen for salinazid is
found to be 3.6,43 which is quite close to that of its structural precursor isoniazid (3.5).
Considering the first ionization constants of the acid molecules used in this work, the ꢁpKa
values are equal to 2.3 for oxalic acid (pKa,1 = 1.3), 0.8 for malonic acid (pKa,1 = 2.8), 0.2 for
malic acid (pKa,1 = 3.4), and 1.6 for acesulfame acid (pKa,1 = 2.0). It is evident that for all the
considered drug/coformer pairs the ꢁpKa should be considered in the salt−coꢀcrystal continuum.
Therefore, the resulting ionization state of the components in the multicomponent crystal cannot
be reliably predicted.
3.1 Crystal structures of the salts and pattern of intermolecular interactions
Relevant crystallographic details for all the studied crystalline complexes are given in Table 1.
Protonation of the pyridine ring of Slz by acid groups of the salt formers was confirmed by the
single crystal Xꢀray diffraction data. This was evidenced by the proton location, analysis of the
CꢀO bonds length in the acid molecules and the CꢀNꢀC angle value in the pyridine ring.40 For
example, CꢀO, C=O bond distances of 1.30, 1.20 Å and angles of 117ꢀ118° indicate a neutral
synthon, whereas an intermediate distance of 1.25 Å and a of 120ꢀ122° mean an ionized
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pyridinium state.44ꢀ47 This fact suggests that proton transfer in the studied crystals may be
facilitated by the crystalline environment and/or crystallization solvent.48,49
Table 1. Crystallographic data for salinazid salts
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[Slz+Oxalic] (1:1)
[Slz+Malonic] (1:1) [Slz+Malic] (1:1)
[Slz+Acesulfam] (1:1)
Crystal data
Chemical formula C13H12N3O2·C2HO4 C13H12N3O2·C3H3O4 C13H12N3O2·C4H5O5 C13H12N3O2·C4H4NO4S
Crystal size (mm) 0.22 × 0.20 × 0.03
0.20 × 0.20 × 0.15
345.31
0.25 × 0.20 × 0.10
375.34
0.42 × 0.17 × 0.01
404.40
Monoclinic, P21/c
Fw
Crystal
space group
Temperature (K)
a (Å)
b (Å)
c (Å)
α (°)
β (°)
331.28
system, Monoclinic, P21/n
Monoclinic, P21/c
Triclinic, P¯1
183
150
150
183
19.295(4)
5.5361(11)
27.666(5)
90
95.408(3)
90
2942.0(10)
8
0.12
13.2240(8)
9.3797(6)
12.4908(8)
90
101.213(1)
90
7.7100(15)
10.443(2)
11.671(2)
106.805(3)
97.485(3)
109.110(3)
823.3(3)
2
15.7874(18)
7.9201(9)
14.7285(17)
90
107.129(2)
90
1759.9(3)
4
0.23
γ (°)
V (Å3)
Z
1519.75(17)
4
ꢂ (mmꢀ1)
0.12
0.12
Data collection
1.23 – 27.00
26648
6399, 0.035
5054
2.68 – 27.99
13970
1.88 – 26.00
7009
2.70 – 26.99
15579
3826, 0.029
3042
θ
range (°)
total refl.
unique refl., Rint
refl. with I > 2σ(I)
3668, 0.021
3177
3207, 0.017
2721
Refinement
No. of parameters 537
286
306
317
0.038
0.092
1.03
0.29, ꢀ0.19
0.038
0.112
1.05
0.037
0.101
1.04
0.034
0.091
1.03
R1[I > 2σ(I)]
wR2 (all data)
Goof
ꢁρmax, ꢁρmin (e Åꢀ3)
0.39, ꢀ0.22
0.79, ꢀ0.23
0.36, ꢀ0.39
The salinazid molecule has two donor and two acceptor sites suitable for hydrogen bonding
(Figure 1). The pyridine nitrogen atom is capable of forming neutral or chargeꢀassisted hydrogen
bonds, while the site containing an amino group (NꢀH) and a number of nearby CꢀH groups are
also able to interact with the highly electronegative atoms of neighbour molecules. The main
acceptors of hydrogen bonding in the Slz molecule are the oxygen atom of the hydrazide group
and hydroxyl oxygen attached to the phenyl ring. Additionally, the aromatic fragments of Slz can
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participate in phenyl CꢀH···O contacts and other weak intermolecular interactions, which
stabilize the crystal lattice. However, simple examination of the molecular packing arrangement
based only on geometrical criteria cannot provide reliable information about the strength of
intermolecular contacts, since the existence of a short contact between atoms does not imply by
default presence of an intermolecular (noncovalent) interaction.50 Beside that, even for strong
hydrogen bonds the geometric criteria cannot provide the exact value of Eint but only its variation
range. Therefore, the pattern of intermolecular interactions in the crystals has been additionally
investigated in terms of the quantum theory of atoms in molecules and crystals (QTAIMC).51,52 It
has been reported that this method is one of the most applicable approaches to estimate the
strength of interactions of different nature in a uniform manner that would be in good agreement
with the experiment.35ꢀ39 In the present paper, the QTAIMC analysis was utilized to evaluate the
energies of Hꢀbonds and CꢀH···O contacts and to compare the relative strength of different
structural motifs of the intermolecular interactions in the crystals of the salts.
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3.1.1 [Slz+Oxalic] (1:1)
Salinazid oxalate crystallizes in the monoclinic P21/n space group with two conformationally
distinct oxalate anions and two planar API cations in the asymmetric unit. The conformations of
salinazid molecules are nearly identical, whereas the main differences are observed between the
oxalate ions. In the first of the symmetry unequivalent molecules (mol. F ꢀ flat), the carboxyl
groups lie approximately in the same plane, so the torsion angle ∠O2ꢀC1ꢀC2ꢀO3 equals ꢀ164.6°
(Figure 2b). In the second molecule (mol. T ꢀ twisted), the deprotonated carboxyl group is
twisted in relation to the protonated one through the angle of ∠O6ꢀC3ꢀC4ꢀO8 = ꢀ102.3° (Figure
2a). The CSD analysis (version 5.36, may 2015 update)53 has shown that the “twisted”
conformation of the oxalate ion is rare in crystal structures (Figure S3). Therefore, the
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stabilization of this higherꢀenergy conformation in a crystal requires a considerable amount of
nonꢀcovalent interaction energy. From XꢀRay data one can observe that oxygen atoms of the
twisted carboxylic group are located in proximity to the hydrazide NꢀH and several CꢀH groups
of both symmetry independent Slz molecules (Figure 2a). The QTAIMC analysis confirms that
the total energy of hydrogen bonds and CꢀH...O contacts accepted by atoms O7 and O8 of the Tꢀ
oxalic ion are 44 and 41 kJ·mol–1, respectively (Table S2).
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In the crystal, the oxalate ions are linked with each other by OꢀH···Oꢀ hydrogen bonds to form
infinite chains along the bꢀaxis with C(5) graph set notation. Each chain contains oxalate
conformers of one type only (“flat” or “twisted”). It is notable that the energy of the O5ꢀ
H5···O7ꢀ bond (d(O5···O7ꢀ) = 2.589 Å) in the chain of Tꢀoxalic ions is ~42 kJ·mol–1 (Figure 2a),
while for the Fꢀoxalic ions the O1ꢀH1···O4ꢀ bond reaches ca. 76 kJ·mol–1 (Figure 2b) (Table
S2). The remarkably high electron density in the bond critical point for the latter interaction (ρb =
0.090 a.u.) and short O1···O4ꢀ distance (2.463 Å) indicate its partially covalent nature.54 The
stronger Hꢀbonds between the flat oxalate anions compared to the twisted ones are possibly
caused by the better charge delocalization in the Fꢀoxalic ions.
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Figure 2. Intermolecular hydrogen bonds (blue) and C–H···O contacts (green) along the chain of
(a) Tꢀanions of oxalic acid, (b) Fꢀanions of oxalic acid, (c) packing arrangement of the
[Slz+Oxalic] salt along bꢀaxis. The interaction energies are given in kJ·molꢀ1.
The API and oxalic acid molecules do not form the expected acidꢀpyridine synthon, instead the
Fꢀmolecules of oxalic acid accept the bifurcated N23+ꢀH33···O3(O1) Hꢀbonds from the
protonated pyridine group of molecule B. In addition, N13+ꢀH13···O4ꢀ bond links Fꢀoxalate ion
and molecule A (Figure 2b). The energy of the N23+ꢀH33···O3 hydrogen bond (d(N23+···O3) =
2.762 Å) is roughly equal to that of N13+ꢀH13···O4ꢀ (d(N13+···O4) = 2.758 Å) (26 versus 29
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kJ·mol–1) due to the charge delocalization in the deprotonated carboxylic group of the acid, while
the N23+ꢀH33···O1 bond (d(N23+···O1) = 2.862 Å) has only ca. 16 kJ·mol–1 (Table S2). It is
interesting to note that QTAIMC analysis did not confirm the existence of the (3;ꢀ1) critical point
corresponding to N13+ꢀH13···O2 contact, despite the fact that geometrical criteria (d(N13+ꢀO2)
= 3.029 Å, ∠( N13+ꢀH13···O2) = 120.8°) suggest formation of a weak hydrogen bond.
Considering the energy of several CꢀH···O contacts (Figure 2b), the strength of the oxalateꢀ
pyridinium synthons is estimated to be 52 kJ·mol–1 for the BꢀF pair and 37 kJ·mol–1 for AꢀF
pair.
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A number of the (3;ꢀ1) bond critical points corresponding to weak C–H···O contacts between
the symmetry unequivalent salinazid molecules have been located by the QTAIMC analysis. The
A molecules are arranged in centrosymmetric dimers interacting via the C12ꢀH12...O11
(d(C12···O11) = 3.218 Å) and C11ꢀH11...O12 (d(C11···O12) = 3.366 Å) contacts with a total
Eint value of ca. 18 kJ·mol–1 (Figure S4a). In the case of B molecules, only weak C32ꢀH32...O21
(d(C32···O21) = 3.530 Å) contacts (Eint ≈ 6 kJ·mol–1) are detected (Figure S4b).
In the [Slz+Oxalic] (1:1) crystal, different motifs of Hꢀbonds are united into a complex chain
consisting of salinazid and oxalate ions to form a distinct layer. The neighboring layers,
however, are not hydrogen bonded to each other and interact only via C–H···O contacts and van
der Waals forces (Figure 2c).
3.1.2 [Slz+Malic] (1:1)
In the crystal of the [Slz+Malic] (1:1) salt, the malate anions are assembled into C(7) chains by
O21ꢀH21···O24ꢀ hydrogen bonds (d(O21···O24ꢀ) = 2.634 Å), and each chain consists of one
enantiomer only (Figure3a). The Hꢀbonds between malate ions in chains are the strongest nonꢀ
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covalent interactions in the crystal with the energy about 36 kJ·mol–1 (Table S3). In addition,
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R22 (10)
ring motifs through the O25–H25···O23 hydrogen
malate ions of different chirality form
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bonds (d(O25···O23) = 2.841 Å) between the hydroxyl and carboxylic groups (ca. 25 kJ·mol–1),
connecting the neighboring chains into a single layer (Figure 3a). The pyridine nitrogen of Slz is
involved in the relatively weak bifurcated N3+–H3···O24ꢀ (O25) bonds with the deprotonated
carboxyl group and hydroxyl group of the malate ion (d(N3+···O24ꢀ) = 2.739 Å, d(N3+···O25) =
3.002 Å) with ca. 27 and ca. 11 kJ·mol–1, respectively (Figure 3b). This value is nearly equal to
the energy of the AꢀF bifurcated synthon in the [Slz+Oxalic] (1:1) structure with similar
topology (see Tables S2 and S3).
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The O23 atom of the acid carboxylic group accepts the N1ꢀH6···O23 (d(N1···O23) = 2.856 Å)
bond from the hydrazide group of API, which brings additional 19 kJ·mol–1 to the APIꢀacid
interaction energy (Figure 3b). It has to be pointed out that, like in [Slz+Oxalic], the energy of
acidꢀacid interactions in the crystal (ca. 61 kJ·molꢀ1) is larger than acidꢀpyridine synthon energy
(ca. 38 kJ·molꢀ1), suggesting the structureꢀforming role of the acid chains in the salt.
The packing arrangement of the salt can be described as alternating layers of malate and
salinazide ions in the (001) planes (Figure 3c). The layers are segregated so that there are clear
regions with hydrogen bonding between Slz and malate ions and regions where only Slz
molecules interact via van der Waals forces.
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Figure 3. (a) Hydrogen bonded chain and ring motives between malate ions, (b) hydrogen bonds
(blue) and C–H···O contacts (green) in the crystal of [Slz+Malic], (c) packing arrangement of
the [Slz+Malic] salt along aꢀaxis. The interaction energies are given in kJ·molꢀ1.
3.1.3 [Slz+Malonic] (1:1)
The asymmetric unit of the [Slz+Malonic] (1:1) salt contains one Slz cation and one malonate
anion linked by the charge assisted N3+–H3···O21ꢀ hydrogen bond (d(N3+···O21ꢀ) = 2.516 Å;
Eint = 72 kJ·mol–1), which is the strongest Hꢀbond among all other hydrogen bonds present in the
crystal structure (Table S4). This level of energy is in good agreement with the reported value for
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3ꢀhydroxypyridinium benzoate crystal, where a similar proton transfer forms an N+–H···O
bond.38 The N3+–H3···O21ꢀ Hꢀbond, along with C5ꢀH5···O22 contact, completes a nearly planar
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robust pyridiniumꢀcarboxylate heterosynthon (R2 (7) in graph set notation) with the estimated
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energy of 78 kJ·mol–1 (Figure 4a).
Malonate ions are assembled into C(6) chains along the cꢀaxis via O23ꢀH23···O21ꢀ hydrogen
bonds (d(O23···O21ꢀ) = 2.624 Å) with moderate strength (ca. 36 kJ·mol–1) (Figure 4a). Unlike
the above discussed structures, the energy of acidꢀacid interactions in the crystal is significantly
lower than that in the acidꢀpyridine synthon (36 against 78 kJ·mol–1) (Table S4). It would be
interesting to note that the strong affinity of Slz to malonic acid and the high vapor pressure of
the latter may be the reason for spontaneous salt formation when two solids are simply brought
in contact with one another55 (see Figure S5). This effect was not observed (at least by the naked
eye) for the systems with oxalic and malonic acids.
Another N1–H6···O22 hydrogen bond (d(N1···O22) = 2.926 Å) with energy ca. 19 kJ·mol–1
connects two neighboring [Slz+Malonic] units to form a C(11) hydrogen bonded chain along the
bꢀaxis (Table S4). The O22 atom also accepts two C4ꢀH4···O22 and C7ꢀH7···O22 contacts
(d(C4···O22) = 3.260 Å; d(C7···O22) = 3.549 Å), which brings additional ca. 15 kJ·mol–1
(Figure 4a). In a chain, nearly planar [Slz+Malonic] units are packed in a perpendicular manner
to form an angle of ca 88° to each other (Figure 4b). The neighboring units are additionally
stabilized by the weak C5ꢀH5···O1 contacts (≈ 9 kJ·mol–1) between the Slz molecules (Figure
S6a).
Combinations of different chain motifs result in formation of complex Hꢀbonded rings
including 30 or more atoms (not shown). In contrast to the [Slz+Oxalic] (1:1) and [Slz+Malic]
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that the components are linked to each other through various Hꢀbond motifs.
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Figure 4. (a) Hydrogen bonds (blue) and main C–H···O contacts (green) in the crystal of
[Slz+Malonic], (b) packing arrangement of the [Slz+Malonic] salt along cꢀaxis. The interaction
energies are given in kJ·molꢀ1.
3.1.4 [Slz+Acesulfame]
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The crystal structure of [Slz+Acesulfame] (1:1) is similar to that of the salinazid saccharinate
salt reported by us previously.9 In the asymmetric unit, Slz and acesulfame molecules are
connected by a strong N3+–H3···O6 hydrogen bond (d(N3+···O6) = 2.587 Å; Eint = 51 kJ·mol–1)
involving the pyridinium nitrogen of the API and the carbonyl oxygen atom of the oxathiazin
ring of the salt former (Table S5). The relatively flat API and hydrogenꢀbonded acesulfame
molecules lie approximately in the same plane to form a distinct unit (Figure 5a). The N1–
H6···N4 hydrogen bond (d(N1···N4) = 2.587 Å) with Eint ≈ 17 kJ·mol–1 connects two
neighboring [Slz+Acesulfame] units constructing a hydrogen bonded ribbon consisting of API
and acesulfame ions, which is expended along the bꢀaxis (Figure 5b). In the previously studied
[Slz+Saccharin] (1:1) crystal, N1ꢀH6 group and adjacent CꢀH groups of the API form nonꢀ
covalent interactions with oxygen atoms of the SO2 group of saccharin.9 In the case of
[Slz+Acesulfame] (1:1), this site of the Slz molecule interacts with N4, O4 and O6 atoms of
acesulfame through the N1ꢀH6···N4 bond as well as two CꢀH···O contacts of different strength
(Figure 5a). These interactions taken all together contribute ca. 34 kJ·mol–1 to the lattice energy
(Table S5).
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Similar to the salinazid saccharinate crystal, [Slz+Acesulfame] units in a chain form an angle
of ca 69° with each other. The chains are packed into layers in the crystallographic (100) planes,
with the acesulfame ions pointing towards the layer surfaces (Figure 5b). Similar to the
[Slz+Malonic] (1:1) crystal, the Slz molecules interact with each other via relatively strong C5ꢀ
H5···O1 (d(C5···O1) = 3.040 Å) contacts (16 kJ·mol–1) to form C(6) motifs (see Figure S6b).
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Figure 5. (a) Hydrogen bonds (blue) and main C–H···O contacts (green) in the crystal of
[Slz+Acesulfame], (b) packing arrangement of the [Slz+Acesulfame] salt along cꢀaxis. The
interaction energies are given in kJ·molꢀ1.
3.1.5 Concluding remarks on the salinazid salts with dicarboxylic acids.
The structure analysis has revealed that in the [Slz+Oxalic] (1:1), [Slz+Malic] (1:1) and
[Slz+Malonic] (1:1) crystals, acid molecules tend to interact with each other via recurring chain
motives of Hꢀbonds. The main reason for that is most likely the 1:1 molar ratio between the API
and acid molecules in crystals leaving one carboxylic group of the acid free to form hydrogen
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bonds with surroundings. The QTAIMC analysis has confirmed that there is an energy
preference behind the chain structure of Hꢀbonds. In the [Slz+Oxalic] (1:1) crystal, the energy of
acidꢀacid interactions is ca. 1.5 times stronger than the total energy of the bifurcated N+–H···O
hydrogen bonds between the API and acid ions. It is not surprising that a similar structure of the
hydrogen bond network is also found in salts of oxalic acid with different weak pyridine bases,
such as 4ꢀ(hydroxyiminomethyl)pyridinium hydrogen oxalate (COSDUK), pyridinium oxalate
(DEFCUM), nicotinamidium oxalate (LICLEP), 4ꢀaminopyridinium oxalate (MOSNAK), 2ꢀ
aminoꢀ5ꢀnitropyridinium oxalate (QOCCUI).
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Combination of chain and bifurcated motifs is also typical for [Slz+Malic] (1:1) crystal. Malic
acid has a hydrogen bonding site, the spatial geometry of which is essentially close to that in
oxalic acid (Figure 6).
Figure 6. Hydrogen bonding sites in the oxalate and malate ions
The O24carboxyl···O25hydroxyl distance in malic acid (2.728 Å) is comparable with the
O3carboxyl···O1 carboxyl distance in oxalic acid (2.649 Å), which probably explains the formation of
similar bifurcated Hꢀbonded synthons between the API and respective acid molecules.
In [Slz+Malonic] (1:1) the bifurcated Hꢀbonded synthon cannot be formed between the API
molecules and the acid due to sterical hindrances in malonic acid. Instead, the API and malonic
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acid are connected by a stable pyridiniumꢀcarboxylate heterosynthon, which is the strongest nonꢀ
covalent interaction in the crystal.
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Therefore, in terms of energy hierarchy of the Hꢀbonds, the [Slz+Oxalic] and [Slz+Malic]
crystals may be considered as chains of acids to which the API molecules are attached. In the
[Slz+Malonic] salt, however, the acidꢀacid Hꢀbonded chains act as linkers of the APIꢀacid
pyridiniumꢀcarboxylate heterosynthons. This competition between different motifs of Hꢀbonds is
not trivial and hard to predict though, so additional studies of an extended set of salts are needed
in order to clarify the structureꢀenergy relationships.
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3.2 Conformational analysis
The conformational state of the salinazid molecule can be described by using at least three
torsion angles, namely τ1 (∠C2ꢀC3ꢀC6ꢀN1), τ2, (∠C6ꢀN1ꢀN2ꢀC7) and τ3, (∠N2ꢀC7ꢀC8ꢀC9) (see
Figure 1). The torsion angles τ1 and τ3 are responsible for the rotation of the pyridine and phenyl
rings, respectively. The torsion angle τ2 defines the conformation of the central spacer unit
between these two rings. The angle between the leastꢀsquares planes of the aromatic rings, β, was
also taken in consideration.
Table 2. Selected torsion angles and dihedral angles between planes of aromatic rings, β, of
salinazid molecule in different crystal forms.
τ1 (C2ꢀC3ꢀC6ꢀN1),º τ2 (C6ꢀN1ꢀN2ꢀC7),º τ3 (N2ꢀC7ꢀC8ꢀC9),º β,º
[Slz+Oxalic] (1:1)
178.5
(C12ꢀC13ꢀC16ꢀN11) (C16ꢀN11ꢀN12ꢀC17) (N12ꢀC17ꢀC18ꢀC19)
176.5 ꢀ174.6 ꢀ2.6
(C32ꢀC33ꢀC36ꢀN21) (C36ꢀN21ꢀN22ꢀC37) (N22ꢀC37ꢀC38ꢀC39)
ꢀ177.2
ꢀ2.6
2.8
mol A
[Slz+Oxalic] (1:1)
1.7
mol B
[Slz+Malonic] (1:1)
[Slz+Malic] (1:1)
178.0
144.0
176.0
ꢀ4.0
4.9
10.9
30.2
4.0
ꢀ177.2
168.9
[Slz+Acesulfame] (1:1) ꢀ168.9
[Slz+Saccharin] (1:1) 169.2
ꢀ0.3
7.90
ꢀ175.35
3.4
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Slz ion after geometry
157.1
174.6
0.6
30.2
optimizationa
aGeometric optimization for the salinazid ion was performed using the GAUSSIAN09 program at the B3LYP/6ꢀ
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Table 2 indicates that conformational differences between the API molecules in the salts are
small. The τ2 torsion angle values change by no more than ±7º from 180º. Most of the τ3 values
are found to be close to 0º. The pyridine ring (τ1) is mainly coplanar to the rest of the molecule.
For [Slz+Malic] (1:1), however, the pyridine orientation is relatively further twisted from
planarity, increasing the β values up to ca 30º.
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In order to investigate conformational preferences of the API molecules, a search of the CSD
(version 5.36, may 2015 update)53 for crystal structures of Nꢀisonicotinoyl
arylaldehydehydrazones derivatives was performed. The search constraints were: 3ꢀD
coordinates were determined, only organics, no powder structures, not disordered, R < 0.1. As a
result, 145 hits, including hydrates, solvates, salts and coꢀcrystals, were retrieved and analyzed.
The distribution of the τ1, τ2 and τ3 torsion angles from the retrieved CSD set is described in the
supporting information (Figures S7). Herein, we mainly focus on analysis of the dihedral angle
between the planes of aromatic rings (β), which is an integral conformational characteristic and
the most informative parameter to consider. Figure 7 shows that the distribution profile of βꢀ
parameter has a maximum at ca 9º, whereupon the occurrence frequency gradually decreases as
β increases.
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Figure 7. Distribution of dihedral angle between planes of aromatic rings, β, in Nꢀisonicotinoyl
arylaldehydehydrazone derivatives from retrieved CSD set (145 hits).
Most structures are located between 0 º and 30º, indicating that hydrazon derivatives tend to be
flat in a crystal. The crystal structures of the salts described here are generally consistent with the
populations obtained from the CSD survey. In the [Slz+Malic] (1:1) crystal, the API
conformation is found to be close to the geometry of an isolated Slz ion (Table 2). This
minimumꢀenergy conformation, however, is rare in crystals, which suggests that conformation of
hydrazon molecules is under the influence of supramolecular surroundings. The packing energy
gained in the case of nearly planar geometry must be greater than the conformational energy
penalty caused by the deviation of the molecule from its optimal geometry.
3.3 Thermal analysis.
Thermal stability of the salinazid salts was evaluated by the DSC method. The DSC results for
the salts and pure salinazid are shown in Figure 8, and the thermal data are tabulated in Table 3.
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Figure 8. DSC curves of salinazid and corresponding salts recorded at 10 °C⋅min–1 heating rate.
Table 4. Thermophysical data for salinazid salts.
ꢁH Tfus , kJ·mol–1a
Tfus (salt), °C (onset)
Tfus (acid), °C
[Slz+Oxalic] (1:1)
[Slz+Acesulfame] (1:1)
[Slz+Malic] (1:1)
[Slz+Malonic] (1:1)
[Slz+Saccharin] (1:1)b
Salinazidb
221.2± 0.5
202.2 ± 1.0
169.7 ± 0.8
162.6 ± 0.5
195.8 ± 0.3
249.0 ± 0.4
72.5 ± 1.6
≈ 31
189.0
121.3
130.6
135.2
227.0
54.1 ± 2.0
59.9 ± 1.5
64.9 ± 1.0
42.5 ± 1.5
aFor the salts, the values correspond to a mole of molecules in the asymmetric unit.
bData taken from ref. 9.
As Table 4 indicates, the melting temperatures for most of the salts are located in between the
melting temperatures of respective pure components. An exception is seen only for salinazid
saccharinate.9 The [Slz+Oxalic] (1:1) salt demonstrates the largest thermal stability (221.2 °C),
while the least Tfus values are observed in salinazid malate (169.7 °C) and salinazid malonate
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(162.6 °C). Interestingly, the melting temperatures of these salts qualitatively correlate with the
energy of OꢀH···O interactions between the respective acid ions in the salt crystals (see 3.1).
This fact indicates that the acidꢀacid interactions in the crystals provide a significant impact to
the thermal stability of the salts, altering the melting behavior of the API over a significant
temperature range (≈60 °C).
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The DSC curve of [Slz+Acesulfame] (1:1) demonstrates a sharp endothermic peak at 203°C,
which is immediately followed by an intense exothermic peak indicating decomposition process
of acesulfame. A similar thermal behavior has been observed for the acesulfame salts with 5ꢀ
fluorocytosine21 and coꢀcrystal with griseofulvin.19 Analysis of the salts thermal stability against
melting points of the corresponding pure acids did not reveal any clear correlation. This,
however, is not unexpected since the acids used are not structurally consistent.
3.4 Aqueous solubility and salts stability
Solubility and stability in aqueous media are the most important physicochemical properties to
consider when preparing new solid forms of an API. The solubility values of salinazid and its
salts in water at 25°C are shown in Figure 9 and tabulated in Table 5.
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Figure 9. Solubility of the salinazid salts in water at 25°C. Stable salts are colored in blue.
Unstable salts are colored in red. Solubility of pure salinazid is shown in brown color.
The largest concentration gain of API is observed during the dissolution of salinazid oxalate,
which is found to be ca. 33 times more soluble than the parent drug. The [Slz+Acesulfame] (1:1)
salt shows a relatively moderate 18ꢀfold solubility enhancement in relation to pure API. This
value is comparable to that for the salinazid saccharinate (20 times).9 In general, the
concentration level of salinazid decreases in the following order, [Slz+Oxalic] >
[Slz+Saccharine] > [Slz+Acesulfame] > [Slz+Malonic] > [Slz+Malic] > Slz.
However, XRPD analysis of the residual material recovered after the experiment has revealed
that only two salts, namely [Slz+Oxalic] (1:1) and [Slz+Acesulfame] (1:1), remain stable during
the dissolution experiment (Figure S8 a,b). Whereas, the [Slz+Malonic] (1:1) and [Slz+Malic]
(1:1) salts dissolve incongruently and undergo a solutionꢀmediated transformation to form pure
salinazid in the bottom phase (Figure S8 c,d).
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For the stable salts, the free energy of formation in a particular solvent can be estimated be the
following relation:57
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pKa,B − pKa,A
p
p


10
SA ⋅SB
ꢁG0 = −RT ⋅ln
(2)




f
app
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K
sp


where R is the gas constant, T is the temperature, Ksappp is the apparent solubility product of the
salt, SAp and SBp are the solubility of pure A and B in a solvent, pKa,A and pKa,B are dissociation
constants of components. In order to ensure proximity of the intrinsic pKa value of each
compound and to attain consistency with the reported solubility data for the pure acids, all the
solubility measurements were performed in an unbuffered water solution. It should be noted that
app
sp
K
is expressed in terms of concentration, with the approximation that the activity coefficient
for all the species is 1.0, and the impact of the ionic strength on the activity coefficient of the
solution is neglected.
Equation (2) also suggests that for a salt to be thermodynamically stable, the following
condition has to be fulfilled:58
a,B − pKa,A
K
<10pK
SAp ⋅SBp
(3)
app
sp
The numerical values for the rightꢀhand and leftꢀhand sides of equation (3) and the Gibbs
energies of formation for the stable salinazid salts are shown in Table 5.
Table 5. Solubility of salts (SAPI) and corresponding pure acids (Sacid), Ksp values and Gibbs
energies of the salts formation in water at 25°C.
10pK
SAp ⋅SBp
a,B − pKa,A
app
ꢁG0f
SAPI·104
mol·Lꢀ1
Sacid
Stability
K
sp
mol·Lꢀ1
kJ·mol–1
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[Slz+Oxalic] (1:1)a
[Slz+Acesulfame] (1:1)a
[Slz+Saccharin] (1:1)a
[Slz+Malic] (1:1)
[Slz+Malonic] (1:1)
Salinazid
19.0 ± 1.5
10.1± 1.0
1.6b
4.2c
3.6·10ꢀ6
1.1·10ꢀ6
1.8·10ꢀ2
9.5·10ꢀ3
2.4·10ꢀ5
4.0·10ꢀ4
2.0·10ꢀ3
‒
ꢀ21.2
ꢀ22.6
ꢀ7.3
‒
Stable
Stable
Stable
Unstable
Unstable
‒
1.7·10ꢀ2d 1.3·10ꢀ6
11.3
±
0.2
0.3
0.3
0.02d
4.4b
5.7b
‒
‒
‒
‒
2.5
4.8
±
‒
±
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‒
0.57
±
aFor the stable salts, the numbers represent concentration of each of API and acid in a stoichiometric
solution in equilibrium with the salt.
bData taken from the ref. 59.
cThis work.
dData taken from the ref. 9.
app
sp
The
K
values for the stable [Slz+Oxalic] (1:1) and [Slz+Acesulfame] (1:1) salts were
calculated from the solubility experiments and found to be considerably smaller than the product
a,B − pKa,A
of 10pK
SAp ⋅SBp . In addition, the Gibbs energies of formation of the salts derived from
equation (2) were found to be more negative than that for [Slz
1).9 It suggests a greater thermodynamic stability of the [Slz
+
Saccharine] (1:1) (ꢀ7.3 kJ·mol–
+
Oxalic] (1:1) and
[
Slz+Acesulfame] (1:1) salts in relation to salinazid saccharinate. It has to be pointed out that the
free energy of formation for the acesulfame salt is ca. 3 times larger than that of saccharinate
salt, despite the similarity in the packing arrangement of the crystals (see 3.1.4). This issue seems
likely to be related to a considerable difference in the lattice free energy for pure acesulfame and
saccharine. The difference can be qualitatively estimated by considering melting temperatures of
the compounds: 121.3°C for acesulfame acid and 227.0°C for saccharine.
The dissociation of the [Slz+Malonic] (1:1) and [Slz+Malic] (1:1) salts in water occurs almost
immediately. It was observed by changing color of the powders: from yellow for the salts to
white for pure salinazid. Low stability of the respective salts in water indicates that they do not
obey the condition (3).
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