Solid Forms of Ciprofloxacin Salicylate: Polymorphism, Formation

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Solid Forms of Ciprofloxacin Salicylate: Polymorphism, Formation Pathways and Thermodynamic Stability Artem O. Surov, Nikita A. Vasilev, Andrei V. Churakov, Julia Stroh, Franziska Emmerling, and German L. Perlovich Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b00185 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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Crystal Growth & Design

Solid Forms of Ciprofloxacin Salicylate: Polymorphism, Formation Pathways and Thermodynamic Stability Artem O. Surova, Nikita A. Vasileva, Andrei V. Churakovb, Julia Strohc, Franziska Emmerlingc, German L. Perlovicha,* aInstitution

of Russian Academy of Sciences, G.A. Krestov Institute of Solution Chemistry RAS, 153045, Ivanovo, Russia. E-mail: [email protected]

bInstitute

of General and Inorganic Chemistry RAS, Leninskii Prosp. 31,119991, Moscow, Russia.

cFederal

Institute for Materials Research and Testing (BAM), Richard-Willstaetter-Str. 11,

12489 Berlin, Germany *To whom correspondence should be addressed: Telephone: +7-4932-533784; Fax: +7-4932336237; E-mail [email protected]

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Abstract

The crystallization of ciprofloxacin – an antibacterial fluoroquinolone compound - with salicylic acid resulted in the isolation of five distinct solid forms of the drug, namely an anhydrous salt, two polymorphic forms of the salt monohydrate, methanol and acetonitrile solvates and the saltcocrystal hydrate. The salicylate salts were investigated by different analytical techniques ranging from powder and single crystal X-ray diffractometry (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), variable temperature powder X-ray diffraction (VT-PXRD), dynamic vapor sorption analysis (DVS), dissolution and solubility investigations. Real-time in situ Raman spectroscopy was used to investigate the mechanochemical formation pathways of the different solid polymorphs of ciprofloxacin salicylate. The mechanism of the phase transformation between the crystalline forms was evaluated under mechanochemical conditions. It was found that the formation pathway and kinetics of the grinding process depends on the form of the starting material and reaction conditions. The analysis of the solid-state thermal evolution of the hydrated salts revealed the two-step mechanism of dehydration process which proceeds through a formation of the distinct intermediate crystalline products.

1. Introduction Fluoroquinolones represent an important class of the broad-spectrum antibiotics that are widely used for the treatment of several types of bacterial infections and inflammations.1 Ciprofloxacin (CIP) is an important fluoroquinolone, which belongs to the second generation of drugs and show strong activity against Gram-negative and Gram-positive bacteria.2 Despite the powerful biological activity in vivo, fluoroquinolones suffer from the low solubility at neutral pH3 and insufficient permeability across biological membranes.4 This is by far the most challenging case for the drug development as well as their formulation design. It has been recently suggested that anhydrous ciprofloxacin exhibits limited solid-state solubility due to the ACS Paragon Plus Environment

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strong intermolecular interaction between zwitterionic species in a crystal lattice.5,

6

In the

neutral pH range ciprofloxacin is also zwitterionic and has neutral overall charge, making it practically insoluble in aqueous media. One of the common procedures to improve the aqueous solubility of such drugs is the salt formation using a suitable counterion. Removal of the dipole in zwitterions such as CIP via salt formation will often result in improved solubility, due to a decrease in the crystal lattice energy and an increase of hydration energy. Therefore, efforts have been made to extend the range of CIP solid forms and to modify its poor solubility performance through the salt preparation with different organic acids, including aliphatic and aromatic carboxylic acids,7-11 artificial sugars12, 13 and other drug compounds.14, 15 However, only several of the reported solid forms contain GRAS (“Generally Recognized as Safe”) counterions (such as citrate, maleate, fumarate, succinate etc.) and thus may be considered pharmaceutically relevant. Despite a considerable diversity of the reported ciprofloxacin salts, only few of them are known to be polymorphic.16,

17

Polymorphism is of great importance in the pharmaceutical

industry as different polymorphs of the drug substance can show different stability, solubility, dissolution rate, pharmacological activity, bioavailability.18-20 Polymorphic forms of a compound are thermodynamic phases, which are stable in a particular range of conditions (temperature, pressure, humidity, etc.). A thermodynamically less stable polymorph can spontaneously transform into the stable one during processing or storing. This, in turn, may result in a dramatic change of the pharmacokinetic parameters and therapeutic activity of a drug. Moreover, the metastable phase may even “disappear” completely despite recreating crystallization conditions where it was easily obtained previously.21, 22 Therefore, systematic screening for polymorphs has become an essential step in the drug development. The aims of this step are to select a solid polymorphic form with optimal properties and to provide reproducible conditions for the crystallization process. However, the mechanisms that lead to the crystallization of different



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polymorphs remain poorly understood making the polymorphism phenomenon largely unpredictable. In recent years, mechanochemistry has been increasingly exploited for the screening of polymorphic forms of single- and multi-component crystals.

23-25

In the case of fluoroquinolone

salts, it has been shown that mechanochemical synthesis of pharmaceutical salt of ciprofloxacin with maleic acid resulted in the formation of three distinct polymorphic modifications of the ciprofloxacin maleate.17 In addition, the third polymorph of the ciprofloxacin saccharinate has been recently discovered entirely due to the application of the mechanochemical approach.26 These examples indicate that mechanochemistry is the suitable tool to explore the polymorphic potential of the fluoroquinolone multi-component crystals. Here, we report a novel pharmaceutical salt composed of ciprofloxacin and salicylic acid (Figure 1) which is found to exist in five distinct solid forms, namely an anhydrous salt, two polymorphic forms of the salt monohydrate, methanol and acetonitrile solvates and salt-cocrystal hydrate. Salicylic acid is well known anti-inflammatory agent and an important active metabolite of aspirin. Salts of salicylic acid with choline27 and benzidamine28 have been used to treat mildmoderate pain, to reduce fever and inflammation. Salicylic acid also has bacteriostatic, fungicidal, and keratolytic actions. It has been reported that salicylic acid may be an appropriate cotreatment antimicrobial agent along with fluoroquinolone antibiotics for treatment of Pseudomonas keratitis.29 In addition, antibiotics can be prescribed along with anti-inflammatory drugs to treat stomach ulcer, chronic bacterial infection and an unremitting inflammatory response.30 Recently, a number of drug–drug salt forms of ciprofloxacin14 and norfloxacin31 with non-steroidal anti-inflammatory drugs have been reported. A broad range of analytical techniques was applied to characterize the salicylate salts, including powder and single crystal X-ray diffractometry (XRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), variable temperature powder X-ray diffraction (VT-PXRD), dynamic vapor sorption analysis (DVS), dissolution and solubility


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investigations. In addition, in situ monitoring by the Raman spectroscopy32,

33

was used to

investigate the mechanochemical formation pathways of different solid polymorphs of the ciprofloxacin salicylate under variable reaction conditions. As a result, direct insights into the mechanisms of the phase transformations between the crystalline forms under variable humidity and mechanochemical conditions could be provided.

Figure 1. Molecular structures of ciprofloxacin and salicylic acid

2. Materials and Methods 2.1. Compounds and solvents Ciprofloxacin (C17H18FN3O3, anhydrous, 98%) and salicylic acid (C7H6O3, 99%) were purchased from Acros Organics. All the solvents were available commercially and used as received without further purification. 2.2. Mechanochemical experiments The mechanochemical synthesis were performed using a Fritsch planetary micro mill, model Pulverisette 7, in 12 ml agate grinding jars with ten 5 mm agate balls at a rate of 500 min-1 for 50 min. In a typical experiment, 100 mg of the equimolar ciprofloxacin/salicylic acid mixture was placed into a grinding jar, and 60 μl of a solvent was added with a micropipette. 2.3. Solution crystallization Ciprofloxacin salicylate monohydrate form I ([CIP+SA+H2O] (1:1:1) form I). Equimolar amounts of ciprofloxacin and salicylic acid were dissolved in water/ethanol mixture (1:1 v:v) and



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stirred at 60-70 °C until a clear solution was obtained. The solution was slowly cooled and kept in a fume hood at the room temperature until a crystalline material was formed. This procedure was reliable only in the preliminary stage of the crystallization experiments but lacked reproducibility after appearance of the form II of ciprofloxacin salicylate monohydrate. Ciprofloxacin salicylate monohydrate form II ([CIP+SA+H2O] (1:1:1) form II). Equimolar amounts of ciprofloxacin and salicylic acid were dissolved in various water/organic solvent mixtures (1:1 v:v) and stirred at 60-70 °C until a clear solution was obtained. For the preparation of the water/organic solvent mixtures, the following solvents were used: methanol, ethanol, isopropanol, acetonitrile. The resulting solution was seeded by the ground powder of [CIP+SA+H2O] (1:1:1) form II, slowly cooled and kept in a fume hood at room temperature. Ciprofloxacin salicylate methanol solvate ([CIP+SA+MeOH] (1:1:1)). Equimolar amounts of ciprofloxacin and salicylic acid were dissolved in hot methanol (50-55 °C) under stirring. The clear solution was slowly cooled and kept in a refrigerator at ca. 3 °C until a crystalline material was formed. Ciprofloxacin salicylate acetonitrile solvate ([CIP+SA+ACN] (1:1:1)). Ciprofloxacin and salicylic acid in a 1:2 molar ratio were dissolved in hot acetonitrile at 50-60 °C. The single crystals of [CIP+SA+ACN] (1:1:1) were obtained by placing the resulting solution in a refrigerator at 3 °C. Ciprofloxacin 3.67 hydrate (CIP·3.67H2O) was prepared by slurring anhydrous ciprofloxacin in water for 2 days. The resulting material was air-dried and identified by comparing the experimental and calculated powder XRD patterns.

2.4. Single Crystal XRD Single-crystal XRD data were collected on the diffractometer SMART APEX II (Bruker AXS, Germany) using graphite-monochromated MoKα radiation (λ = 0.71073 Å). Absorption corrections based on measurements of equivalent reflections were applied.34 The structures were


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solved by direct methods and refined by full matrix least-squares on F2 with anisotropic thermal parameters for all non-hydrogen atoms.35 In the structure [CIP+SA+H2O] (1:2.5:1) one of the two solvent salicylic acid molecules is rotationally disordered over two sites with equal occupancies. In [CIP+SA+H2O] (1:1:1) form I the solvent water molecule is disordered over two sites with occupancies ratio 0.89/0.11. In the structures [CIP+SA+H2O] (1:1:1) form II, [CIP+SA+I] (1:1:1), and [CIP+SA+MeOH] (1:1:1) all hydrogen atoms were found from difference Fourier map and refined isotropically. In [CIP+SA+H2O] (1:2.5:1) all hydrogen atoms were placed in calculated positions and refined using a riding model. As for [CIP+SA+H2O] (1:1:1) form I all carbon hydrogen atoms and H atoms of the minor component of disordered water molecule were placed in calculated positions and refined using a riding model; all others (hydroxy, amino and water) were located from difference Fourier map and refined isotropically. The crystallographic data for [CIP+SA+H2O] (1:1:1) form I, [CIP+SA+H2O] (1:1:1) form II, [CIP+SA+ACN] (1:1:1), [CIP+SA+MeOH] (1:1:1), and [CIP+SA+H2O] (1:2.5:1) have been deposited by the Cambridge Crystallographic Data Centre as supplementary publications under the CCDC numbers 1895171, 1895172, 1895175, 1895174, and 1895173, respectively. This information can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. 2.5 Powder XRD (PXRD) PXRD data of the bulk materials were recorded under ambient conditions on the BraggBrentano diffractometer D8 Advance (Bruker AXS, Germany) with the copper X-ray source (λCuKα1 = 1.5406 Å) and the high-resolution position-sensitive detector LYNXEYE XE-T. The samples were prepared into the plate sample holders and rotated with 15 min-1 during the data acquisition. The diffractograms were acquired in the 4–40 ° 2θ range with step size of 0.015 °. VT-PXRD experiments were performed on D8 Advance diffractometer equipped with the Modular Temperature Chamber (MTC-FURNACE). The measured sample was prepared into the corundum sample holder and heated under a nitrogen purge gas from 30 °C up to 200 °C with



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the heating rate of 60 °C·min-1 while rotating with 15 min-1. A VT-PXRD patterns were acquired under isothermal conditions at every 10 °C step in the range of 4-30 ° 2θ for 10 min/acquisition. 2.6 Raman spectroscopy The in situ Raman spectroscopy experiments were performed in the vibration ball mill Pulverisette 23 (Fritsch, Germany) in a Perspex jar for 60 min at 30 Hz. Raman measurements were performed using a Raman RXN1™ Analyser (Kaiser Optical Systems, France). The spectra were collected using a laser radiation with a wavelength of λ = 785 nm and a contactless probe head (working distance 1.5 cm, spot size 1.0 mm). Raman spectra were recorded every 30 s with an acquisition time of 5 s and 5 accumulations. NIR excitation radiation at λ = 785 nm and an irradiation of 6.6 W·cm-2 were performed. 2.7. DSC Thermal analysis was carried out using a differential scanning calorimeter with a refrigerated cooling system (Perkin Elmer DSC 4000, USA). The sample was heated in sealed aluminum sample holder at the rate of 10 °C·min-1 in a nitrogen atmosphere. The unit was calibrated with indium and zinc standards. The accuracy of the weighing procedure was ± 0.01 mg. 2.8. TGA TGA was performed on a TG 209 F1 Iris thermomicrobalance (Netzsch, Germany). Approximately 10 mg of the sample was added into a platinum crucible. The samples were heated at a constant heating rate of 10 °C·min-1 and purged throughout the experiment with a dry argon stream at 30 ml·min-1. 2.9. DVS DVS measurements were performed on a DVS Resolution instrument (Surface Measurement Systems Ltd., UK) equipped with the Raman spectrometer (i-Raman® Plus, B&W Tek, USA). The temperature was maintained at a constant level of 25 ± 0.1 ºC. The sample of [CIP+SA] (1:1) was initially dried for several hours under a stream of nitrogen at 50 ºC to obtain equilibrium dry mass. The reference mass was recorded, and the sorption-desorption analysis


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was then carried out in the RH range of 0-90 % with the step size of 5 %. The sample mass was equilibrated according to dm/dt ≤ 0.002 mg·min-1 for at least 10 min before the RH was changed. Raman spectra of the samples were recorded at each RH step. An isotherm was calculated from the complete sorption and desorption profile. 2.10 Water activity measurements The influence of water activity on stability of hydrated/dehydrated forms of salts was studied using a series of the isopropanol/water (IPA/H2O) binary mixtures with a varying water content. The water activity of the binary mixtures was calculated from the following polynomial equation proposed by Zhu et al.:36 aw = 0.0215 + 2.828·xw – 1.837·xw2 - 2.380·xw3 + 2.379·xw4,

(1)

where aw is the water activity, xw is the mole fraction of water in a solution. In a typical experiment, 30 mg of [CIP+SA] (1:1) was suspended in 1.6 ml of IPA/H2O mixture in a sealed vial and left to shake at 25 °C for at least 5 days. After equilibration, the suspension was centrifuged, and the precipitate was collected, carefully dried at room temperature and analyzed by PXRD. 2.11. Aqueous solubility experiments Dissolution and solubility measurements were carried out by the shake-flask method in hydrochloric buffers with pH 1.2, pH 2.0 and a phosphate buffer with pH 6.8 at 25.0 ± 0.1 °C. For solubility and dissolution experiments, the “Standard Ambient Temperature and Pressure” (SATP), i.e. 25.0 °C (298.15 K) and 101.325 kPa, conditions were used, as they are the most frequently chosen reference parameters for solubility measurements of drug compounds, including fluoroquinolones. In the dissolution experiments, the excess amount of each sample was suspended in 10 ml of the buffer solution preheated to 25.0 °C in Pyrex glass tubes. Aliquots of the suspension were withdrawn at the specific time intervals and filtered through the 0.22 μm Rotilabo® PTFE syringe filter (Carl Roth, Germany). The drug content in the solution phase was determined after suitable dilution on the Cary 50 UV-VIS spectrophotometer (Varian, Australia)



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at the reference wavelength (λ = 277 nm). For the solubility measurements, the excess amount of the solid form was suspended in 2 ml of the solvent and allowed to equilibrate under shaking at 25.0 °C for 48 hours. The results are stated as the average of three replicated experiments. The stability of the salts in the dissolution and solubility experiments was monitored by analyzing the samples collected from the bottom phase using PXRD. 2.12. Computational Methods The geometry of the crystal structures of the [CIP+SA+H2O] (1:1:1) polymorphs was optimised using the Crystal14 software suite37 with B3LYP functional with the Grimme dispersion correction (D2)38 and 6-31G(d,p) basis set. The space groups and the unit cell parameters of the crystals obtained in the single crystal XRD studies were fixed and structural relaxations were limited to the positional parameters of atoms. As the starting point in the solidstate density functional theory (DFT) computations, the coordinates of heavy atoms were used directly from the single crystal experiment with hydrogen atomic positions normalized to the standard X‒H distances from neutron diffraction data. The default CRYSTAL options were used to provide the sufficient accuracy in evaluating of the Coulomb and Hartree-Fock exchange series and a grid was used in evaluating the DFT exchange-correlation contribution. Tolerance on energy controlling of the self-consistent field convergence for the geometry optimizations and frequency computations was set to 10‒10 hartree. The mixing coefficient of Hartree-Fock/KohnSham matrices was set to 25 %. The number of points in the numerical first derivative calculation of the analytic nuclear gradients was equal 2. The shrinking factor of the reciprocal space net was set to 3. Relative stability of the polymorphs was established by the comparing of the computed total cell energies (referred to the number of molecules in the unit cell, Z). The intermolecular interactions of the [CIP+SA+H2O] (1:1:1) polymorphic forms were quantified using the Hirschfeld surface analysis performed in the CrystalExplorer17 software39 on the basis of the experimental crystal structures.


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Void Map Calculations were carried out via Mercury 3.1040 software after removing the solvent molecules from the unit cell. The conditions were set as follows: probe radius 0.8 Å, approx. grid spacing 0.7 Å, calculate using the contact surface.

3. Results and Discussion 3.1. Preparation and identification of the solid forms of ciprofloxacin salicylate The propensity of ciprofloxacin to form various crystalline products with salicylic acid was observed by analyzing the results of different synthesis techniques. Ciprofloxacin salicylate was synthesised via mechanochemical treatment, slurry and solution crystallization of the components in presence of water, different pure organic solvents and their aqueous mixtures. Identification of solid forms obtained by different syntheses and estimation of their phase purity were carried out by the single crystal and powder XRD, DSC and Raman spectroscopy. The water content and composition of the hydrated salts were derived from TGA. The full set of solid forms obtained and identified by different screening methods is summarized in Table S1. It was established in the literature that the reaction of an acid with a base is expected to form a salt if the difference in ΔpKa = pKa(base) – pKa(acid) is greater than 4.41 For ciprofloxacin (pKa = 8.74 for the piperazine fragment3) and salicylic acid (pKa = 2.9842), the ΔpKa value is equal to 5.76, which strongly suggests proton transfer and salt formation to occur. The pKa of the COOH group in the fluoroquinolone drug (6.09) is at least three orders less acidic than the organic acid used, and hence zwitterionic form of the drug is not expected. Combination of ciprofloxacin and salicylic acid resulted in the formation of five new solid forms of ciprofloxacin salicylate: anhydrous salt [CIP+SA] (1:1), two polymorphic forms of the salt monohydrate [CIP+SA+H2O] (1:1:1) (form I and form II), methanol solvate [CIP+SA+MeOH] (1:1:1) and acetonitrile solvate [CIP+SA+ACN] (1:1:1). Preliminary mechanochemical and solution crystallization experiments of ciprofloxacin and salicylic acid in the water/ethanol mixture led to the formation of the hydrated product with low ACS Paragon Plus Environment


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dehydration temperature (≈ 30 °C) (Figure 2a). The results of TGA revealed the sample mass loss of ca. 3.60 % over the temperature range of 25-100 C, which corresponds to one water molecule per one molecule of the salt (Figure S1a). According to TG analysis, the sample starts to decompose at 180 C (Figure 2a). The second endothermic event in the DSC curve (Tfus(onset) ≈ 230 °C) corresponds to the melting process, which is accompanied by rapid decomposition of the salt (Figure 2a). The water content and the total composition of this phase, later referred to as [CIP+SA+H2O] (1:1:1) form I, were also confirmed by the single crystal XRD (Table S2). Interestingly, subsequent grinding, crystallization and slurry experiments using various water/organic solvent mixtures or pure water produced a new crystalline form of ciprofloxacin salicylate which differed from the known form I in terms of powder patterns (Figure 2b) and dehydration temperature (Tdehyd(onset) ≈ 89 °C). According to TGA, this form showed however the same drug/acid/water molar ratio as in [CIP+SA+H2O] (1:1:1) (Figure S1b). It can be thus concluded, that the obtained material is a new polymorph of ciprofloxacin salicylate monohydrate, which has been designated as form II. Moreover, repeated syntheses of the form I, by using the same recipe and the conditions, resulted exclusively in a new phase II, while form I of [CIP+SA+H2O] (1:1:1) displayed “disappearing polymorph” behavior. This term was first introduced by Bernstein21,

22

and is widely used nowadays to

characterize the metastable phases with transient existence.43-46

(a) (b) Figure 2. Experimental (a) DSC curves and (b) PXRD patterns of [CIP+SA] (1:1) and


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polymorphs of [CIP+SA+H2O] (1:1:1). Mechanochemical treatment of the equimolar amounts of ciprofloxacin and salicylic acid in the presence of neat IPA, ACN and EtOAc resulted in the formation of the third solid form of the salt, which had distinct diffraction patterns (Figure 2b) and showed no evidence of a mass loss under heating up to the melting temperature (Figure S1c). Therefore, this phase was considered to be an anhydrous salt of ciprofloxacin salicylate with 1:1 molar ratio, later referred to as [CIP+SA] (1:1). The pure samples of the anhydrous form could be obtained only via mechanochemical route in the presence of selected solvents, while slurry and crystallization techniques always provided the mixture of products (Table S1). Crystallization experiments from the methanol and acetonitrile solutions at lower temperature (3 °C) yielded two new solvates of ciprofloxacin salicylate. Each phase was obtained solely as few individual single crystals, which were readily isolated from the mother liquor and analyzed by single-crystal XRD (Table S2). However, it was impossible to obtain these solvates as bulk samples by conventional methods (mechanochemical reaction, slurry and solution crystallization at room temperature) due to their low thermodynamic stability in contrast to the anhydrous and monohydrate forms of the salt. Similar behavior has been previously observed for solvates of arbidol47 and ciprofloxacin maleate monohydrate.17

3.2 Raman spectroscopy The formation of new solid forms of ciprofloxacin salicylate was also characterized by the Raman spectroscopy. The spectrum of pure ciprofloxacin contains the strong bands at 1590 and 1375 cm-1 corresponding to antisymmetric and symmetric vibrations of the carboxylic group (Figure 3).5 This fact confirms that ciprofloxacin exists in its zwitterionic form, which is in good agreement with the powder XRD data (Figure S2). Another strong absorption band at 1624 cm-1 corresponds to the stretching vibrations of the carbonyl group ν(C=O) of the pyridone moiety.48 Formation of the salt between ciprofloxacin and salicylic acid causes new weak vibration band at ACS Paragon Plus Environment


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1720 cm-1, indicating the presence of the protonated carboxylic group in the molecular structure of ciprofloxacin.49 The strong bands at 1540 and 1390 cm-1 can be attributed to antisymmetric and symmetric ν(O–C–O) vibrations, respectively. A comparison of the spectra of both monohydrate polymorphs reveals that there are several band shifts and peak-shape changes occurring between the forms I and II (Figure 3). The most prominent changes can be observed in the peak positions responsible for vibrations of the carboxylic group as well as in the region of 1370-1310 cm-1. In contrast, the Raman spectra of [CIP+SA] (1:1) and form I of the monohydrate appear similar, indicating that the supramolecular surroundings of the molecules in these solid forms are closely comparable. These solid forms can be differentiated based on the Raman signal of [CIP+SA+H2O] (1:1:1) form I at 1379 cm-1, which is absent in [CIP+SA] (1:1) (Figure 3).

Figure 3. Experimental Raman spectra of CIP, [CIP+SA] (1:1) and polymorphs of [CIP+SA+H2O] (1:1:1). The appearance of the band at at 1720 cm-1 attributed to the protonated carboxylic group in the molecular structure of ciprofloxacin is indicated by a dashed red frame.

3.3 Crystal structures of the ciprofloxacin salicylate monohydrate polymorphs. The relevant crystallographic data of the ciprofloxacin salicylate monohydrate polymorphs (form I and II) and its solvates with methanol and acetonitrile are given in Table S2. The forms I ACS Paragon Plus Environment

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and II crystallize in the triclinic P and monoclinic P21/n space groups, respectively, with one ciprofloxacin cation, one salicylate anion, and water molecule in the asymmetric unit. In both forms, the protonated ciprofloxacin cations are connected to the salicylate anions and water via charge-assisted N+–H···O‒ and N+–H···O hydrogen bonds. In turn, water molecules act as bridges between the components forming C(6) (form I) or C(8) (form II) hydrogen bonded chains (green dash lines in Figure 4),50,

51

which propagate along the a-axis. In form I, water

molecules are also responsible for the formation of the ring R44 (16) motives (blue dash lines in Figure 4a), assembling the neighboring chains of salicylate moieties into a distinct layer. In the form II, water molecules not only accept H-bonds from the piperazine ring of ciprofloxacin, but also serve as donors of hydrogen bonding for the carboxylic groups of the drug (blue dash lines in Figure 4b), thus stabilizing the adjacent ciprofloxacin ions into a layer. The packing arrangement of the polymorphs is found to be typical for fluoroquinolone multi-component crystals. It consists of conventional layers of columnar π-stacks of ciprofloxacin separated by domains containing the counterions and water molecules. In both polymorphs, the ciprofloxacin moieties are arranged into similar infinite columnar stacks along the a-axis with interplanar distances of 3.297 Å in form I and 3.282 Å in form II. As a result, the salicylate anions and water molecules can be packed in different ways inside the interlayer space (Figure S3).

(a) (b) Figure 4. Illustration of hydrogen bond patterns in the crystals of [CIP+SA+H2O] (1:1:1) (a)



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form I and (b) form II The calculated volume of solvent accessible voids using probe radius of 0.8 Å after removing the solvent molecules resulted in the value of 5.9 % of the cell volume for form I and 2.7 % for form II. The comparison of these values indicate that the dehydration of the form I should proceed more readily than that of the form II, which is consistent with the results of the thermal analysis. In both forms, these voids are discontinuous, what means they are not connected as a channel (so-called isolated site hydrates19). The acetonitrile and methanol solvates contain void channels with volume of 13.0 % and 9.1 % of the cell volume, respectively, suggesting an easy ‘escape route’ for the solvent molecules (Figure S4). These packing features are in good agreement with the low stability of these solvates and explain the difficulties in their preparation. To better understand the differences in crystalline environments of the ciprofloxacin ions in the polymorphs, Hirshfeld surface analyses were performed. The relative contributions of the selected intermolecular interactions in the crystals of the both [CIP+SA+H2O] (1:1:1) polymorphs are shown in Figure 5. It is evident that the relative contributions of the dominant interactions in the form I and form II are comparable, comprising 38.4 % and 34.9 % for H…H, 30.1 % and 31.7 % for O…H contacts, respectively. Significant differences between two forms are found for C…H and F…H interactions, which comprise 6.9 % and 11.6 % for the form I, and 7.7 % and 4.9 % for the form II, respectively. These differences can be attributed to the rearrangement of the C–H··· and F…H contacts formed by ciprofloxacin towards the layers containing the salicylate anions.


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Figure 5. Relative contributions of the main intermolecular contacts to the Hirshfeld surface area for the form I (bottom) and form II (top) of [CIP+SA+H2O] (1:1:1) Solid state DFT calculations revealed that the total energy of the unit cell (referred to the number of molecules in the unit cell, Z) obtained for the triclinic form I is 19 kJ∙mol-1 higher than that for the monoclinic form II (Table S3), suggesting that form II is more favorable in terms of the energy of the intermolecular interactions than form I. This result agrees with all the experimental evidences described above.

3.4 Moisture-dependent stability of solid forms The relative stability of anhydrous and hydrated forms of ciprofloxacin salicylate was investigated simultaneously by DVS and in situ Raman spectroscopy. In addition, slurry experiments in water/isopropanol mixtures of various compositions (water activity) with the subsequent PXRD analysis were performed. The DVS studies of the [CIP+SA] (1:1) salt indicate full hydration at 90 % RH resulting in the formation of the [CIP+SA+H2O] (1:1:1) form I, which was confirmed by the Raman spectra of the materials (Figure 6a, Figure S5). The dehydration of form I occurs only at 10 % RH, leading to a large hysteresis between the sorption and desorption isotherms and indicating that the process is kinetically controlled. In contrast, [CIP+SA+H2O] (1:1:1) form II was found to be stable within the range of 0 to 90 % RH at 25 °C, absorbing 0.4 % of water at 90 % RH (Figure ACS Paragon Plus Environment


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6b, Figure S6). The simultaneous analysis of the form II by DVS and Raman spectroscopy indicated no evidence of phase transformation. The reversible phase transition between [CIP+SA] (1:1) and [CIP+SA+H2O] (1:1:1) form I (Figure S5) suggests low energy barrier for the hydration process and indicates that the crystal structures of these two solid salt forms are very similar in terms of packing arrangements. The observed behavior of [CIP+SA+H2O] (1:1:1) form II shows its high kinetic stability with respect to the water vapor pressure. It also leads to the conclusion that water molecules are tightly bound within the host structure, resulting in a high dehydration temperature of the form II (Figure 2a).

(a)

(b)


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Figure 6. Water sorption (full points) and desorption (empty points) curves of (a) [CIP+SA] (1:1) and (b) [CIP+SA+H2O] (1:1:1) form II at 25 °C; inset: Raman spectra of the sample during DVS analysis. The water activity experiments revealed that the [CIP+SA] (1:1) form was stable in the aqueous solution up to aw ≤ 0.20. At aw ≥ 0.21, [CIP+SA] (1:1) transformed into [CIP+SA+H2O] (1:1:1) form I. The latter existed only in a narrow water activity range of 0.21 ≥ aw ≥ 0.26. Further increase in the water activity leads to spontaneous and irreversible transition to the form II, which is thermodynamically preferred solid form of the salt (Figure S7). Based on the experimental results, it is possible to rationalize the transformation pathways between different forms in terms of their energy barriers. As shown above, the activation barrier for the [CIP+SA] (1:1) → [CIP+SA+H2O] (1:1:1) form I solid-solid conversion is low enough to be overcome by moisture content which induces the nucleation of the new phase. In the DVS experiment, the form I → form II transformation is not observed as the process seems to be associated with the high activation energy and requires other conditions to occur. The results of DVS studies show that disappearing polymorph [CIP+SA+H2O] (1:1:1) form I can be caught in a “kinetic trap” and isolated as a pure phase without any contaminations. Under slurry conditions, the solute–solvent interactions diminish the nucleation barrier, accelerate the nucleation and growth rate of the stable phase, leading to a spontaneous form I → form II transition at aw > 0.26. A similar phenomenon has been recently described by Tieger et al.52 for different polymorphic forms of sitagliptin L-tartrate, the stability order of which altered depending on the aw value of the water/organic solvent mixture.

3.5 In-situ investigations of the mechanochemical reactions by Raman spectroscopy The mechanochemical formation pathway of different solid forms of ciprofloxacin salicylate were evaluated by in situ Raman spectroscopy. The outcomes of the following reactions were investigated: (1) grinding of the stoichiometric physical mixture of ciprofloxacin and salicylic



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acid in the presence of water, isopropanol or water/organic solvent mixtures; (2) neat grinding of a stoichiometric physical mixture of ciprofloxacin 3.67 hydrate and salicylic acid; (3) mechanochemical reaction of anhydrous ciprofloxacin salicylate in the presence of water. The analysis of the Raman spectra reveals that the mechanochemical formation of ciprofloxacin salicylate monohydrate from the pure components is a two-step process. Under grinding conditions starting materials readily convert into the metastable [CIP+SA+H2O] (1:1:1) form I within first 4 min, followed by the rapid formation into the thermodynamically stable form II after 9.5 min of grinding (Figure 7a). It was observed that the amount of solvent (water) participating in the reaction mainly affects the duration of the first stage of the process (i. e. induction time), accelerating the conversion of reactants to form I at higher water/ciprofloxacin molar ratios. The use of water/organic solvent mixtures also accelerates the reaction and considerably reduces the time window for the existence of the metastable form I.

(a)


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(b) Figure 7. Time resolved in situ Raman spectra for mechanochemical reaction of (a) anhydrous ciprofloxacin and salicylic acid in the presence of water and (b) ciprofloxacin 3.67 hydrate and salicylic acid under neat grinding condition. The rectangular boxes highlight the development of the characteristic bands of the form I (blue) and form II (red). In the case of ciprofloxacin 3.67 hydrate and salicylic acid, the formation of the [CIP+SA+H2O] (1:1:1) form II was completed after 17 min of the neat grinding experiment. No evidence of any intermediate products was detected by the Raman spectroscopy, suggesting a continuous formation pathway of the [CIP+SA+H2O] (1:1:1) form II from the parent compounds (Figure 7b). Similar continuous process is observed for the formation of the anhydrous ciprofloxacin salicylate from ciprofloxacin and salicylic acid in the presence of isopropanol (Figure S8). In contrast, ciprofloxacin and salicylic acid do not reacted under neat grinding condition (Figure S9), indicating high activation energy for this reaction which may result from the strength of the ciprofloxacin crystal lattice.6 Apparently, crystallization water in ciprofloxacin 3.67 hydrate induces a mechanochemical process enabling the salt formation. Similar reaction mechanism has been previously observed for the mechanochemical synthesis of cocrystals53, 54 and coordination polymers.55



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Milling of the anhydrous ciprofloxacin salicylate ([CIP+SA] (1:1)) in the presence of water leads to the rapid formation of the [CIP+SA+H2O] (1:1:1) form I. This metastable polymorph persists in the reaction for ca. 20 min and can be isolated as a pure phase (Figure 8). The experimental results suggest that the pathway of the mechanochemical formation of form II depends on the initial crystalline form of ciprofloxacin (anhydrate or 3.67 hydrate) used for the synthesis. The differences in the reaction mechanisms and kinetics can be attributed to the particular features of the nucleation process of the salt monohydrate. It seems likely that the mechanochemical crystallization of [CIP+SA+H2O] (1:1:1) from the initial components might result in the competitive nucleation of both form I and form II, so that first formed form I is contaminated by the small amount of seeds of the stable form II which promote and accelerate transformation from I to II. If the [CIP+SA] (1:1) is used as a reactant, a low kinetic barrier to transform [CIP+SA+H2O] (1:1:1) into the form I led to selective formation of this metastable polymorph. The nucleation of form II is thus hindered, providing longer stability of the form I under the mechanochemical treatment.

Figure 8. Time resolved in situ Raman spectra for the mechanochemical treatment of the anhydrous ciprofloxacin salicylate in the presence of water. The rectangular boxes highlight the development of the characteristic bands of the form I (blue) and form II (red).


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3.6 Thermal analysis As mentioned above, the DSC and TGA data indicate considerable difference in the dehydration temperatures of the [CIP+SA+H2O] (1:1:1) polymorphs. Form I starts to release the solvent at 30 °C, while form II is found to be thermally stable up to 89 °C. However, the products of the polymorph dehydration have similar melting temperatures, which coincide with the melting point of the anhydrous [CIP+SA] (1:1) form (Figure 2a). To further investigate the solid-state thermal evolution of the salts and analyze their dehydration mechanisms, VT-PXRD experiments were performed under the nitrogen atmosphere. The dehydration temperatures for the [CIP+SA+H2O] (1:1:1) polymorphs obtained by VT-PXRD agree with the DSC and TGA results, indicating consistency between different thermal methods. The in situ monitoring of the heating process for the polymorphs of [CIP+SA+H2O] (1:1:1) revealed occurrence of the intermediate phases which are formed immediately after the solvent release (Figure 9). It can be assumed that direct transition of the hydrate structure to the known anhydrous form is not possible due to high energy barrier associated with significant reorganization of the salt molecular packing.

(a) (b) Figure 9. Variable temperature powder XRD patterns for (a) [CIP+SA+H2O] (1:1:1) form I and (b) form II. The characteristic peaks of the initial monohydrate form (blue) and of the anhydrous product (red) are highlighted by rectangular boxes. The observed unhydrated products are found to be stable in a wide temperature window (ca. 80 °C). The further rising of the temperature promotes transformation of the intermediate phases ACS Paragon Plus Environment


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to the known [CIP+SA] (1:1) form, which is followed by the melting of the compound. The shift of the peak positions to lower 2θ angles in the temperature range of 180–200 °C may be caused by the decomposition of the sample. It is noteworthy that dehydration of form I and form II gave intermediate products which differ from each other and from the anhydrous form in the powder patterns (Figure S10). These facts indicate that the transformation pathway of the polymorphic hydrates into anhydrate strongly depends on the crystal structure of the parent form. According to the classification scheme proposed by Petit and Coquerel,56 the observed behavior of the hydrates corresponds to the two-step mechanism of dehydration defined as a class I cooperative crystallization (or class I-C.C.). It implies that release of water molecule leads to deformation of the parent structure to form a highly defective anhydrous material which preserves the longrange order. This is followed by the nucleation and growth process of the stable phase due to the reconstruction of a crystal lattice from an intermediate product. Additional isothermal experiments at 140 °C showed apparent stability of the intermediate product of dehydration of form II during 3 hours of exposure (Figure S11). This result indicates that the transformation of the intermediate phase to the [CIP+SA] (1:1) is kinetically controlled process. In the case of the anhydrous form, no evidence of any phase transformation was observed during the VT-PXRD experiment (Figure S12).

3.7 Solution stability and dissolution of ciprofloxacin salicylates The aim of the next part of this work was to explore the influence of crystal structure and hydration level of the ciprofloxacin salicylate solid forms on their stability and solubility in aqueous solutions with pharmaceutically relevant pHs at 25 °C. The solid form stability at pH 1.2, pH 2.0, water and pH 6.8 was monitored by analyzing samples of the bottom phase after 12 hours of shaking, using PXRD. Analysis of the solid phases recovered after the preliminary stability experiments at pH 1.2 showed that regardless of the initial salt form ([CIP+SA] (1:1), [CIP+SA+H2O] (1:1:1) form I


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or form II), the final bottom phase contained the previously unseen solid material which powder patterns differ from those of the initial compounds and of the known solid ciprofloxacin salicylate forms. This fact indicates that all the known forms of ciprofloxacin salicylate were unstable upon dissolution at pH 1.2 and underwent a solution-mediated phase transformation to a thermodynamically more stable phase. Single crystals of the new phase of the diffraction quality were obtained by slow evaporation of the saturated solution of the components at pH 1.2 (Table S2). According to the single crystal XRD, the asymmetric unit of the new solid salt form contains one ciprofloxacin cation, one salicylate anion, one and a half molecule of salicylic acid and one water molecule (Figure S13). Hence, this compound, belongs to a special class of the multi-component crystals called salt-cocrystal hydrates.57 In the buffer solutions with pH ≥ 2.0, the [CIP+SA+H2O] (1:1:1) form II was found to be the only solid phase at equilibrium. Recently, we observed similar pH-depended stability behavior in our experiments with salts of norfloxacin and fumaric acid.11 It has been shown that the pH-depended stability issue can rationalized in terms of alteration of the solubility ratio of individual components at different pH values, leading to significant changes in distribution of stability regions on a ternary phase diagram for a system. This phenomenon is commonly observed in cocrystals of different drug/coformer ratios.58 The dissolution profiles for the unstable [CIP+SA] (1:1) and [CIP+SA+H2O] (1:1:1) form I salts at pH 2.0 demonstrate a so-called “spring and parachute” behavior,59 generating supersaturation of the drug with respect to the solubility level of the stable form60-62 (Figure 10a, Table S4). The metastable forms show a small apparent solubility improvement against the parent drug, while the equilibrium solubility of [CIP+SA+H2O] (1:1:1) form II is found to be slightly below the concentration level, corresponding to the solubility of pure CIP at pH 2.0. Similar solubility ratio at acidic conditions has been observed previously for the ciprofloxacin salts with fumaric acid.11



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In the pH 6.8 medium, however, the solubility advantage (Ssalt/SCIP) of the stable form of the salt reaches ≈ 8.2 times of the parent drug level (Figure 10b, Table S4). Both of the metastable [CIP+SA] (1:1) and [CIP+SA+H2O] (1:1:1) form I salts transform to form II of the monohydrate within 2 hours. Similar to the pH 2.0 medium, the [CIP+SA] (1:1) phase is superior to form I in its apparent solubility, which indicates a lower thermodynamic stability and a greater driving force for the conversion of the anhydrate to form II of monohydrate under the considered conditions.

(a) (b) Figure 10. Dissolution profiles and transformation pathways for [CIP+SA] (1:1) and [CIP+SA+H2O] (1:1:1) form I in the (a) pH 2.0 and (b) pH 6.8 solutions at 25 °C.

4. Conclusions In this work, a series of crystalline products formed by ciprofloxacin and salicylic acid, namely anhydrous salt, two polymorphic forms of salt monohydrate, methanol and acetonitrile solvates and hydrated salt-cocrystal, have been obtained and characterized. Despite a considerable diversity of the reported fluoroquinolone salts, ciprofloxacin salicylate monohydrate described in this work is the only known example of a three-component polymorphic system. However, the form I of the [CIP+SA+H2O] (1:1:1) salt turned out to be an example of a “disappearing polymorph” as it has been observed only in the preliminary experiments, further attempts to obtain this polymorph by conventional methods were unsuccessful. Alternative ways to isolate the elusive form I were found by analyzing results of the in situ monitoring of moisture ACS Paragon Plus Environment

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sorption/desorption and mechanochemical synthesis processes. According to DVS studies, [CIP+SA] (1:1) and [CIP+SA+H2O] (1:1:1) form I undergo a moisture induced reversible phase transition which allows to obtain the disappearing polymorph as a pure phase. In situ Raman spectroscopy data revealed that the monohydrate form I could be also isolated via mechanochemical treatment of either anhydrous initial components or the [CIP+SA] (1:1) salt in the presence of water. The VT-PXRD experiments showed that the dehydration of form I and form II of [CIP+SA+H2O] (1:1:1) resulted in the distinct intermediate crystalline products which further transform into the known [CIP+SA] (1:1) form at elevated temperature. Thus, the observed behavior of the hydrates corresponds to the two-step mechanism of dehydration and could be defined as a class I cooperative crystallization.56 The dissolution studies show that transformation pathways of the solid forms are pH-dependent, resulting in the formation of the complex salt-cocrystal hydrate phase at pH 1.2. In the solutions with pH ≥ 2.0, the [CIP+SA+H2O] (1:1:1) form II was found to be the only equilibrium solid phase. The stability relationships of the [CIP+SA+H2O] (1:1:1) polymorphs has been validated by DFT calculations, confirming that form II is the thermodynamically most stable one.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Results of DSC and TGA analyses, packing arrangement of [CIP+SA+H2O] (1:1:1) form I and form II, Void maps in the crystal structures of the salt solvates, in situ Raman spectra collected during the DVS experiments, time resolved in situ Raman spectra for mechanochemical reactions, results of the VT-PXRD experiments, the full set of solid forms obtained and identified by different screening methods, crystallographic data for the solid forms, results of the solid state DFT calculations, solubility values of the solid forms.



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Acknowledgements Artem O. Surov gratefully acknowledges a research scholarship (funding program ID 57378441, personal ref. No. 91692531) from Deutsche Akademische Austauschdienst (DAAD). We thank “the Upper Volga Region Centre of Physicochemical Research” for technical assistance with TGA experiments. X-ray diffraction studies were performed at the Centre of Shared Equipment of IGIC RAS.

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For Table of Contents Use Only

Solid Forms of Ciprofloxacin Salicylate: Polymorphism, Formation Pathways and Thermodynamic Stability

Artem O. Surov, Nikita A. Vasilev, Andrei V. Churakov, Julia Stroh, Franziska Emmerling, German L. Perlovich

The crystallization of ciprofloxacin – an antibacterial fluoroquinolone compound - with salicylic acid resulted in the isolation of five distinct solid forms of the drug, namely an anhydrous salt, two polymorphic forms of the salt monohydrate, methanol and acetonitrile solvates and the saltcocrystal hydrate. Real-time in situ Raman spectroscopy was used to investigate the mechanochemical formation pathways of the different solid polymorphs of ciprofloxacin salicylate.


Solid Forms of Ciprofloxacin Salicylate: Polymorphism, Formation

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