Structural Landscape of Pure Enrofloxacin and Its Novel Salts

Article pubs.acs.org/crystal

Structural Landscape of Pure Enrofloxacin and Its Novel Salts: Enhanced Solubility for Better Pharmaceutical Applicability Maheswararao Karanam and Angshuman Roy Choudhury* Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Sector 81, Knowledge City, S. A. S. Nagar, Manauli PO, Punjab, India 140306 S Supporting Information *

ABSTRACT: The crystal structures of anhydrous enrofloxacin (EFC) (1), enrofloxacin hexahydrate (EFC·6H2O) (2), enrofloxacin maleate (EFC-M) (3), enrofloxacin hemifumarate (EFCF) (4), enrofloxacin hemisuccinate (EFC-S) (5), enrofloxacin hemioxalate (EFC-O) (6), enrofloxacin acetate (EFC-A) (7) and ammonium salt of enrofloxacin (Am-EFC) (8) have been determined by using single crystal X-ray diffraction (SCXRD). The crystals of 1 were grown from a dilute solution of EFC in dilute ammonium hydroxide. The crystals of 2 were grown from a solution of EFC with nicotinic acid. Solvent drop grinding experiments on enrofloxacin with various dicarboxylic acids resulted in four new salts (3−6). When a solution of EFC in glacial acetic acid evaporated to dryness over a period of time, crystals of 7 were grown. Crystals of 8 were found to grow when EFC was dissolved in liquid ammonia and solution was slowly evaporated at room temperature (RT). All these products (1−8) were characterized by 1H and 13C NMR spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). Single crystal X-ray diffraction study has indicated that the products 3−6 had a cation of enrofloxacin and an anion of the acid in common. Four of these eight products have been found to contain water of crystallization. Three of these salts were found to have one neutral acid molecule. The proton transfer and hydrate stoichiometry were confirmed from single crystal X-ray diffraction. Solubility of 1 and 3−6 was determined in water (pH = 6.8) using UV−vis spectroscopy at RT. The alteration between the neutral, zwitterionic, anionic and cationic forms EFC has led to very interesting structures of these salt/cocrystals and also the pure forms (1 and 2).

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INTRODUCTION In generating modified drug products, the use of supramolecular chemistry1 and the concept of crystal engineering2 have gained enormous interest in recent years. This is due to the possibility for enhanced solubility3 and bioavailability3 of the newly modified drug products (polymorphs, salt and cocrystals). These new materials are mostly synthesized using noncovalent interactions, which are weak enough to modulate and obtain different possible geometries among the interacting molecules (rings, chains, sheets etc.) and strong enough to pack the molecules together to form a supramolecular assembly.4 The active pharmaceutical ingredients (API) in their pure forms have a range of solubility in our biological systems. A number of drug molecules available in the market suffer from formulation difficulties due to their poor solubility in water, which in turn results in the inconstant bioavailability.5 Hence efforts are being made to modify these drugs to form salts or cocrystals with suitable organic molecules to form new composites. The selection of the salt/cocrystal formers for the formation of the salt/cocrystal with various APIs based on the crystal engineering concepts is well documented in the literature.6 © 2013 American Chemical Society

In recent years, we have focused our studies on a number of different classes of drug molecules which are poorly soluble in water. We have been exploring the possibilities of these drug molecules to form salts or cocrystals with pharmaceutically acceptable small organic molecules (acids and bases). Our recent efforts to cocrystallize fluconazole using a number of cocrystal formers have resulted in the development of five new polymorphs of fluconazole.7 This observation indicates that the presence of a cocrystal former in stoichiometric amount during the crystallization of an API may lead to the formation of new polymorphs of the API instead of forming a new salt/cocrystal. We have been involved in studying the cocrystallization of a number of drugs belonging to the fluoroquinolone class of antibiotics known for their use in the treatment of bacterial infections.8 Most of these drug molecules suffer from their poor solubility in water at pH ≈ 7 due to their existence as zwitterions.9 The importance of salt formation of ciprofloxacin and norfloxacin, both belonging to the fluoroquninolone class Received: December 14, 2012 Revised: February 18, 2013 Published: February 20, 2013 1626

dx.doi.org/10.1021/cg301831s | Cryst. Growth Des. 2013, 13, 1626−1637

Crystal Growth & Design

Article

Scheme 1. The Proposed Scheme To Obtain Different Salts of Enrofloxacin

Scheme 2. Structures of the Cocrystal Formers Used in This Study

of antibiotics, has been demonstrated recently.10 Enrofloxacin (EFC, Scheme 1) belonging to this class of compounds has been found to show good responses toward veterinary use and was approved for the treatment of individual pets and domestic animals.11 Its antibacterial activity was found to be concentration dependent.12 In the current manuscript, we intend to describe the anhydrous form of enrofloxacin (1), which is found to exist in the neutral form and the hexahydrate form (2), which exists as the zwitterionic form in the solid state (Scheme 1). The bulk enrofloxacin is available in the market in anhydrous form and has poor water solubility (0.45 mg/mL).13 The solubility of some of the APIs has improved by the formation of a salt with suitable organic counterionic compounds.14 To choose a number of pharmaceutically acceptable counterions/cocrystal formers (Scheme 2) to form

salts/cocrystals with EFC, we have utilized crystal engineering concepts (Table 1). The crystal structures of enrofloxacin have been reported in the literature as metal complexes (CSD Refcodes MIRBIZ; PETRIQ; PETROW; QUJQUI)15 and three organic salts, namely 2-hydroxyethanaminium enrofloxacinate (CSD Refcode = PAGZED),16a enrofloxacinium picrate (CSD Refcode = IKAGIL)16b and as citrate monohydrate.16c No effort has been made to study the solubility of these metal complexes and salts. In the current manuscript, we report the results of our studies on enrofloxacin using slow evaporation, cocrystallization and solvent assisted grinding experiments. The crystal structure of anhydrous (1) and the hexahydrate (2) of enrofloxacin along with the salts of enrofloxacin with maleic acid (3), fumaric acid (4), succinic acid (5), oxalic acid (6), acetic acid (7) and ammonium hydroxide (8) have been 1627



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studied by using powder and single crystal X-ray diffraction techniques. The acids yielding the salts have pKa less than 5, and hence they are considered to be highly ionizable. When these acids were crystallized together with EFC molecule, having basic nitrogen, the proton transfer has been found to be highly suitable. All the products (1−8) were also characterized by 1H and 13C NMR spectroscopy, Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) studies. The 1H and 13C NMR and FTIR spectra on 1− 8 confirmed that the backbone of the EFC molecule remained unaffected except for the ionization due to salt formation. The solubility of 1 and 3−6 has been determined in water (pH = 6.8) using UV−vis spectroscopy at RT. The enhanced solubility of the new salts (3−7) indicates that these salts can be used for better formulations.

Table 1. List of Cocrystallization Experiments Performed ratio expt no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

cocrystal former D(−)

tartaric acid tartaric acid fumaric acid maleic acid succinic acid glutaric acid nicotinamide nicotinic acid oxalicacid dihydrate 4-aminobenzoic acid salicylic acid methyl 4hydroxybenzoate propyl 4-hydroxybenzoate γ-aminobutyric acid citric acid acetic acid 25% ammonia solution L(+)

EFC

conformer

result

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1

new phasea new phasea EFC-F EFC-M EFC-S new phasea no new phase EFC·6H2Ob EFC-O no new phase new phasea no new phase

1 1 1 solvent solvent

1 1 1 EFC-A Am-EFC

new phasea no new phase new phasea

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EXPERIMENTAL SECTION

Materials. Enrofloxacin, purchased from Sigma-Aldrich, was used without further purification. The cocrystal formers were obtained from various commercial suppliers such as Sigma Aldrich, Merck and Spectrochem Pvt. Ltd., India, etc. Analytical grade solvents were obtained from Sisco Research Laboratories Pvt. Ltd., India. PXRD patterns of all the starting materials were recorded (Figures S1−S17 in the Supporting Information) on a Rigaku Ultima IV powder diffractometer using parallel beam geometry equipped with a Cu Kα source, 2.5° primary and secondary soller slits, 0.5° divergence slit with 10 mm height limit slit, sample rotation stage (120 rpm) attachment and DTex Ulta detector. The X-ray generator operated at

a

Single crystals were not obtained. bWhen the product of solid state grinding was crystallized in methanol, we obtained crystals of EFC·6H2O.

Table 2. Crystallographic Data for Enrofloxacin Salts

CCDC no. empirical formula formula wt temp/K cryst syst space group a/Å b/Å c/Å α/deg β/deg γ/deg vol/Å3 Z ρcalc mg/mm3 μ/mm−1 F(000) cryst size/mm3 2θ range for data collection/deg index ranges

reflns collected indep reflns data/restraints/ params goodness-of-fit on F2 final R indexes [I ≥ 2σ(I)] largest diff peak/ hole/e Å−3

EFC (1)

EFC·6H2O (2)

EFC-M (3)

EFC-F (4)

EFC-S (5)

915550 C19H22N3O3F 359.40 100.0 monoclinic P21/c 13.9345(4) 6.8516(2) 25.1563(5) 90.00 133.7980(10) 90.00 1733.55(8) 4 1.377 0.102 760.0 0.4 × 0.3 × 0.2 3.38 to 52.74

915549 C19H34FN3O9 467.49 298.0 triclinic P1̅ 8.037(3) 11.030(8) 13.186(8) 79.170(5) 78.873(10) 88.102(10) 1126.5(11) 2 1.378 0.114 500.0 0.2 × 0.1 × 0.1 4.46 to 50.04




909005 909007 909006 C20H29FN3O8 C23H32FN3O8 C19H31FN4O6 458.46 497.52 430.48 100.0 298.0 100.0 triclinic triclinic triclinic P1̅ P1̅ P1̅ 7.0198(10) 7.2779(4) 8.7779(3) 9.3767(19) 12.2704(8) 9.8015(4) 17.249(4) 14.5883(9) 12.8782(4) 85.137(3) 95.722(4) 106.472(2) 83.594(3) 99.508(4) 90.408(2) 73.497(4) 102.931(4) 91.071(2) 1080.1(4) 1239.57(13) 1062.25(7) 2 2 2 1.410 1.333 1.346 0.115 0.106 0.106 486.0 528.0 460.0 0.2 × 0.1 × 0.1 0.2 × 0.2 × 0.1 0.4 × 0.2 × 0.2 4.54 to 49.42 2.86 to 51.34 3.3 to 49.42

−17 ≤ h ≤ 17, −8 ≤ k ≤ 8, −31 ≤ l ≤ 31 11883 3462 3462/0/323

−9 ≤ h ≤ 9, −12 ≤ k ≤ 13, −15 ≤ l ≤ 15 8206 3946 3946/7/322

−8 ≤ h ≤ 8, −14 ≤ k ≤ 14, −15 ≤ l ≤ 14 8156 3664 3664/0/400

−8 ≤ h ≤ 8, −16 ≤ k ≤ 16, −16 ≤ l ≤ 16 11458 4233 4233/0/320

−8 ≤ h ≤ 8, −15 ≤ k ≤ 15, −16 ≤ l ≤ 16 10667 3787 3787/0/317

−3 ≤ h ≤ 7, −10 ≤ k ≤ 11, −19 ≤ l ≤ 20 4527 3409 3409/0/307

−6 ≤ h ≤ 8, −14 ≤ k ≤ 10, −17 ≤ l ≤ 16 11882 4581 4581/0/423

−10 ≤ h ≤ 10, −11 ≤ k ≤ 10, −14 ≤ l ≤ 15 9241 3615 3615/0/395

1.064

1.044

0.935

1.030

0.948

1.028

0.995

1.088

R1 = 0.0324, wR2 = 0.0935 0.26/−0.23

R1 = 0.0777, wR2 = 0.2523 0.58/−0.66

R1 = 0.0680, R1 = 0.0389, wR2 = 0.1360 wR2 = 0.0935 0.24/−0.21 0.26/−0.22

R1 = 0.0776, wR2 = 0.1819 0.44/−0.38

R1 = 0.0472, R1 = 0.0581, wR2 = 0.1095 wR2 = 0.1506 0.35/−0.39 0.58/−0.28

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EFC-O (6)

EFC-A (7)

Am-EFC (8)

R1 = 0.0379, wR2 = 0.0949 0.24/−0.28



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Table 3. Hydrogen Bond Geometry Parameters in Enrofloxacin and Its Salts D−H···A EFC O1−H1···O3 C17−H17B···F1 C12−H12B···N3 C13−H13A···O3 C11−H11···O3 C13−H13B···O1 C14−H14A···O1 C17−H17A···O1 EFC·6H2O C14−H14A···F1 O5−H5B···O6 O9C−H9A···O10 O8D−H8B···O10 C3−H3···O7 O9−H9B···O7 C3−H3···O5 O8D−H8A···O7 N3−H3B···O4 N3−H3A···O4 O7−H7A···O4 O6−H6A···O1 O6−H6A···O3 O6−H6B···O6 O10−H10B···O2 N3−H3B···O9C N3−H3A···O9C O5−H5A···O3 O7−H7B···O8D C18B−H18D···O8D O4−H4A···O8D O4−H4A···O9C O10−H10A···O2 EFC-M O1−H1···O3 N3−H3A···O4 O5−H5···O6 C6−H6···O7 C9−H9···O4 EFC-F O1−H1···O3 O4−H4···O6 N3−H3A···O6 C6−H6···O5 C21−H21···O1 EFC-M O1−H1···O3 O4−H4···O6 N3−H3A···O6 C6−H6···O5 C3−H3···O7 C21−H21···O1 EFC-S O1−H1···O3 O5−H5···O6 N3−H3A···O6 C21−H21A···O1 EFC-O O7−H7A···O8 O1−H1···O3 O6−H6A···O7

(D···H)/Å

D (D···A)/Å

d (H···A) /Å

∠D−H···A/deg

symmetry

0.93(3) 0.98(1) 0.95(2) 0.95(1) 0.93(1) 0.97(1) 0.98(2) 1.00(1)

2.547(2) 2.893(1) 3.546(1) 3.275(1) 3.210(1) 3.455(1) 3.558(1) 3.611(2)

1.66(3) 2.26(1) 2.67(1) 2.34(1) 2.43(1) 2.55(1) 2.75(1) 2.84(2)

158(2) 121(1) 154(1) 168(1) 141(1) 156(1) 140(1) 134(1)

x, y, z x, y, z −x + 1, −y + 2, −z + 1 −x, +y − 1/2, −z + 1/2 −x, +y + 1/2, − z + 1/2 x + 1, −y + 1/2 + 1, +z + 1/2 −x, +y − 1/2, − z + 1/2 −x, +y + 1/2, −z + 1/2

0.97(1) 0.85(4) 0.85(9) 0.85(8) 0.93(4) 0.85(9) 0.93(4) 0.85(8) 0.91(5) 0.91(5) 0.85(4) 0.85(5) 0.85(5) 0.85(5) 0.85(4) 0.91(5) 0.91(5) 0.85(4) 0.85(4) 0.97(15) 0.85(4) 0.85(4) 0.85(4)

2.888(7) 2.775(7) 2.756(11) 2.736(9) 3.675(6) 2.976(11) 3.650(6) 2.843(10) 2.942(7) 2.942(7) 2.789(6) 2.818(6) 3.005(6) 2.787(7) 2.826(5) 3.255(12) 3.255(12) 2.796(5) 2.843(10) 3.386(18) 2.713(9) 2.608(9) 2.762(6)

2.23(1) 1.96(1) 2.09(1) 1.89(1) 2.99(1) 2.23(1) 2.75(1) 2.12(1) 2.06(1) 2.06(1) 1.97(1) 2.02(1) 2.43(1) 2.16(1) 1.99(1) 2.60(1) 2.60(1) 1.96(1) 2.15(1) 2.56(1) 1.95(1) 1.78(1) 1.94(1)

124(1) 158(1) 135(1) 173(1) 132(1) 146(1) 162(1) 143(1) 164(1) 163(1) 162(1) 156(1) 125(1) 130(1) 166(1) 129(1) 129(1) 166(1) 139(1) 143(1) 148(1) 164(1) 163(1)

x, y, z x, y, z x, y, z x, y, z x, +y + 1, +z x, +y + 1, +z x, +y + 1, +z x, +y + 1, +z −x, −y + 2, −z −x, −y + 2, −z −x, −y + 2, −z −x, −y + 2, −z −x, −y + 2, −z −x, −y + 1, −z −x + 1, −y + 2, −z −x + 1, −y + 2, −z − 1 −x + 1, −y + 2, −z − 1 x, +y − 1, +z x, +y − 1, +z x, +y − 1, +z x − 1, +y, +z + 1 x − 1, +y, +z + 1 x + 1, +y, +z − 1

0.82(5) 0.96(5) 1.15(9) 0.89(4) 0.98(4)

2.544(5) 2.775(6) 2.426(8) 3.614(9) 3.563(7)

1.81(5) 1.83(5) 1.30(9) 2.80(5) 2.68(5)

150(5) 168(5) 165(8) 154(3) 150(3)

x, y, z x, y, z x, y, z −x + 1, −y + 1, −z + 1 x, +y + 1, +z

0.92(3) 1.08(3) 0.94(2) 0.95(1) 0.95(1)

2.513(2) 2.557(2) 2.667(2) 3.528(2) 3.276(2)

1.63(3) 1.50(3) 1.73(2) 2.67(1) 2.59(1)

159(2) 163(2) 174(2) 151(1) 129(1)

x, y, z x, y, z −x + 1, −y + 2, −z + 1 x + 1, +y, +z + 1 −x + 1, −y + 1, −z + 1

0.92(3) 1.08(3) 0.94(2) 0.95(1) 0.95(1) 0.95(1)

2.513(2) 2.557(2) 2.667(2) 3.528(2) 3.617(2) 3.276(2)

1.63(3) 1.50(3) 1.73(2) 2.67(1) 2.82(1) 2.59(1)

159(2) 163(2) 174(2) 151(1) 141(1) 129(1)

x, y, z x, y, z −x + 1, −y + 2, −z + 1 x + 1, +y, +z + 1 x, +y −1, +z −x + 1, −y + 1, −z + 1

0.96(5) 0.84(1) 0.71(6) 0.99(1)

2.510(4) 2.569(5) 2.705(7) 3.402(8)

1.60(5) 1.74(1) 2.01(6) 2.48(1)

157(6) 171(1) 169(5) 155(1)

x, y, z −x + 1, −y, −z + 1 −x, −y, −z + 1 x + 1, +y, +z − 1

0.87(1) 0.94(4) 0.87(1)

2.901(4) 2.563(3) 2.787(4)

2.14(1) 1.66(4) 1.94(1)

146(1) 158(3) 162(1)

x, y, z x, y, z x, y, z

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Table 3. continued D−H···A EFC-O N3−H3···O4 O7−H7B···O5 O8−H8B···O4 C9−H9···F1 O6−H6B···O8 N3−H3···O5 EFC-A N3−H3A···O5 N3−H3A···O4 O1−H1···O3 O6−H6A···O5 O8−H8A···O4 O8−H8B···O2 Am-EFC N4−H4B···N3 O5−H5B···O4 O6−H6B···O5 O4−H4E···O2 N4−H4A···O3 O6−H6A···O2 N4−H4C···O6 O5−H5A···O1 N4−H4D···O1 O4−H4F···O3

(D···H)/Å

D (D···A)/Å

d (H···A) /Å

∠D−H···A/deg

0.92(3) 0.87(1) 0.87(1) 0.95(1) 0.87(1) 0.92(3)

2.754(3) 2.651(3) 2.796(3) 3.374(3) 2.858(3) 2.849(3)

1.89(3) 1.80(1) 1.93(1) 2.52(1) 2.00(1) 2.25(3)

157(2) 169(1) 177(1) 150(1) 167(1) 122(2)

x, y, z x, +y − 1, +z x, +y − 1, +z x, +y − 1, +z x + 1, +y, +z −x, −y + 1, −z + 1

0.98(3) 0.98(3) 0.86(3) 0.97(4) 1.03(6) 0.82(4)

3.079(3) 2.729(3) 2.532(3) 2.566(4) 2.887(4) 2.973(4)

2.42(2) 1.76(3) 1.76(4) 1.61(4) 1.88(6) 2.17(4)

124(2) 173(3) 147(4) 171(4) 165(4) 164(4)

x, y, z x, y, z x, y, z x, y, z x, y, z −x + 1, −y, −z + 2

0.96(2) 0.84(3) 0.93(4) 0.89(3) 0.92(3) 0.88(3) 1.00(3) 0.87(3) 1.01(3) 0.88(3)

2.918(2) 2.879(3) 2.835(3) 2.751(2) 2.869(3) 2.830(2) 2.914(2) 2.830(2) 2.889(3) 2.847(2)

1.96(2) 2.04(3) 1.91(4) 1.88(3) 1.98(3) 1.95(3) 1.98(3) 1.96(3) 1.88(3) 2.06(3)

175(2) 173(3) 176(3) 164(2) 162(2) 175(3) 154(2) 177(3) 172(2) 149(2)

x, y, z x, y, z x, y, z −x + 2, −y + x, +y, +z + 1 x, +y, +z + 1 x−1, +y, +z −x + 1, −y + −x + 1, −y + −x + 1, −y +

40 kV and 40 mA power settings. The data were collected over an angular range 3 to 60° with a scanning speed of 5° per minute with 0.02° step. Data processing and Kα2 striping were performed with PDXL17 package. The powder patterns were indexed using the program DICVOL,18a which is available in DASH 3.2.18b Crystallization of the Salts. a. Solvent Evaporation Method. Pure enrofloxacin was dissolved in dilute (5%) ammonia solution, and the evaporation of this solution at 45 °C resulted in the formation of crystals of anhydrous enrofloxacin (1). Pure enrofloxacin was dissolved in liquor ammonia, and the solution was allowed to evaporate at RT to obtain single crystals of salt 8. Crystals of salt 7 were grown by slow evaporation of a solution of enrofloxacin in glacial acetic acid at RT. The crystals of salt 7 were formed after about a week. The crystals were collected by decanting acetic acid and were dried in air for 1 day. Some of the single crystals were used to record PXRD for salt 7 and salt 8 (Figures S16 and S17 in the Supporting Information). b. Solvent Drop Grinding Method. An equimolar mixture of EFC and the cocrystal formers was made by accurate weighing, and the mixtures were ground in an agate mortar and pestle for approximately 10 min. PXRD data (scan 1) were recorded for this ground sample. The samples were recovered and were mixed with approximately 200 μL of methanol to make a paste. This paste was then ground for 10− 15 min till the mixture was free from the solvent and became freeflowing. Another PXRD (scan 2) was recorded on this sample. It was observed that the PXRD in scan 2 was different from that of scan 1. Further, approximately 200 μL of methanol was added to the recovered sample after scan 2 and the same procedure was followed to prepare the desired salt. Several successive grindings with methanol were carried out and PXRD were recorded (scan 3, scan 4, etc.) till two consecutive scans were found to be identical. The resulting powder samples were transferred to 5 mL beakers, dissolved in different solvents and the solvent mixtures. The beakers were then covered with paraffin film, and 2−3 holes were made in the paraffin film to allow the solvent to evaporate slowly at 4 °C in a refrigerator. On complete evaporation of the solvent, single crystals suitable for single crystal X-ray diffraction were obtained. This procedure was followed for 17 different cocrystal formers. Table 1 lists all the observations in these experiments. Although we could identify 12 new

symmetry

1, −z + 1

1, −z + 1 1, −z + 1 1, −z + 1

(distinctly different from the respective starting materials) crystalline phases of enrofloxacin, only four of them resulted in the formation of single crystals suitable for SCXRD study. The PXRD patterns obtained for all these experiments are reported in Figures S1−S15 in the Supporting Information. Single Crystal X-ray Diffraction. The single crystals obtained in these experiments were analyzed by single crystal X-ray diffraction. Single crystal X-ray diffraction experiments were carried out on a Bruker AXS KAPPA APEX-II CCD diffractometer (monochromatic Mo Kα radiation) equipped with Oxford cryosystem 700Plus. The data sets were collected at 100 K. Data collection and unit cell refinement for the data sets were done using the Bruker APEX-II19 suite, data reduction and integration were performed by SAINT V7.685A19 (Bruker AXS, 2009) and absorption corrections and scaling were done using SADABS V2008/119 (Bruker AXS). The crystal structures were solved and refined by using SHELXS9720 and SHELXL9720 respectively, available within Olex221 or WinGx22 packages. Single crystal data and refinement results are listed in Table 2. The anhydrous form was found to crystallize in monoclinic P21/c space group while all other structures reported in this manuscript were found to crystallize in triclinic P1̅ space group. All the figures, including the packing and interaction diagrams, were generated using Mercury.23 Geometric calculations, including the leastsquares plane angles, were done using PARST24 and PLATON.25 Thermal Analysis. The melting points of the salts 1−8 were determined by DSC and the melting points along with the melting enthalpies are listed in Table 4. The DSC traces are reported in Figures S25−S33 in the Supporting Information. The DSC data of 1 indicate a single melting transition at 226.5 °C while that of 2 indicate the loss of water molecules at 69.6 °C. 2 was found to melt at 226.4 °C. The maleate salt (3) was found to melt at 225.5 °C, the fumarate (4) at 267.4 °C, the succinate (5) at 212.8 °C and oxalate (6) at 244.6 °C with the loss of water at 221.2 °C. The acetate salt (7) lost the water molecule at 92.4 °C and melted at 226.9 °C. The ammonium salt (8) released ammonia at 67.2 °C, and the residue melted at 227.7 °C. FTIR Measurements. FTIR spectra of 1−8 were recorded on a Perkin-Elmer Spectrum 2 FTIR spectrometer in the range of 400− 1630



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Table 4. Saturation Solubility Data for 1 and 3−6 in Water (pH = 6.8) and Melting Point Data for 1−8 compound EFC EFC·6H2O EFC-M EFC-F EFC-S EFC-O EFC-A Am-EFC

λmax (in water)

ελmax (in water)

276

21179

0.615

277 275 274 275 275 273

20754 14561 21595 57727 28874

4.70 (8) 9.09 (15) 20.65 (34) 16.21 (26) highly soluble not determined

saturation solubility in water (mg/mL)

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RESULTS

1. Enrofloxacin Anhydrous: EFC. Anhydrous form of enrofloxacin (EFC) was found to crystallize in the P21/c space group with Z = 4. The asymmetric unit was found to contain one neutral enrofloxacin molecule (Figure 1). The piperazine ring in EFC and all other structures described in this manuscript has been found to adopt a stable chair conformation. The intramolecular O−H···O hydrogen bond (Table 3) has been observed in this structure and in the structures of EFC-M, EFC-F, EFC-S, EFC-O and EFC-A salts where EFC exists as a cation. This intramolecular O−H···O hydrogen bond is not observed in EFC·6H2O, where EFC exists as a zwitterion, and in Am-EFC, which is found to have the anionic form of EFC. A short intramolecular C−H···F hydrogen bond (Table 3) has been observed in this molecule. The same C−H···F hydrogen bond is observed in all the crystal structures reported in this manuscript. The conformation of the piperazine ring in EFC and in all other structures reported in this manuscript has been found to be in the chair form irrespective of the charge on the N3 atom. The crystal structure is stabilized by a number of different weak C−H···O and C− H···N hydrogen bonds (Table 3) in EFC. 2. Enrofloxacin Hexahydrate: EFC·6H2O. The data were collected at room temperature as it was observed that the crystals lost their single crystal nature and developed several cracks while being cooled below 250 K. The asymmetric unit of the EFC·6H2O was found to contain one molecule of enrofloxacin in the zwitterionic form and six water molecules (Figure 2). One of these water molecules and the ethyl group, connected to the piperazine ring, have been found to be disordered. The crystal packing is mainly through a 3D network of strong O−H···O hydrogen bonds (Table 3) formed by water molecules present around the enrofloxacin molecule. 3. Enrofloxacinium Monomaleate (1:1): EFC-M. The data for EFC-M were also collected at room temperature because the crystals lost their single crystal nature and developed several cracks while being cooled below 250 K. The asymmetric unit of the EFC-M was found to contain one molecule of enrofloxacin cation and one molecule of monomaleate anion. The acidic hydrogen of the maleic acid

melting point (°C) from DSC 226.55 226.42 225.45 267.38 212.77 244.62 226.89 227.69

4000 cm−1 (Figures S18−S24 in the Supporting Information). Samples were prepared using dry KBr in the form of a pellet, and data were processed by using Spectrum software. Solubility Studies. The solubility of the salts (3 to 6) and of the anhydrous form (1) was determined by using UV−vis spectroscopy as reported by Reddy et al.,10 based on the method proposed by Higuchi and Connor in 1965.26 A measured quantity (approximately 5−25 mg) of each of the salts was completely dissolved in a large excess (100 mL) of deionized water (pH = 6.8). These stock solutions were suitably diluted to prepare 3−4 primary standard solutions for generating the calibration curves. The λmax values for all the salts were then determined using a double beam UV−vis spectrophotometer (Lab India UV3000+). The absorbance values of the primary standard solutions were determined at the respective λmax values. The absorbance values were plotted in y-axis, and the concentrations were plotted in x-axis, and the points were fitted with a straight line (calibration curve). Simultaneously saturated solutions of each of the salts were prepared by stirring an excess amount of the salt in 2 mL of deionized water in a 5 mL sealed vial at 25 °C and at 1500 rpm for 24 h. These solutions were then centrifuged at 10000 rpm for 10 min. The supernatant solution was then diluted 1000 times (first 1 mL diluted to 100 mL and then 1 mL of this diluted to 10 mL) using ELIX water. The absorbance of the clear 1000 times diluted solution was determined at λmax of the salt, and the concentration of the salt was determined using the calibration curve, which was generated earlier. The solubility was calculated by multiplying the concentration of the dilute solution by 1000. Table 4 lists the solubility of 1 and 3−7.

Figure 1. ORTEP diagram of the enrofloxacin (ellipsoids drawn at 50% thermal probability). 1631



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Figure 2. ORTEP diagram of the EFC·6H2O (ellipsoids drawn at 30% thermal probability; disordered part has been removed for clarity).

Figure 3. ORTEP diagram of the EFC-M (ellipsoids drawn at 30% thermal probability).

Figure 4. ORTEP diagram of the EFC-F (ellipsoids drawn at 50% thermal probability). 1632



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Figure 5. ORTEP diagram of the EFC-S (ellipsoids drawn at 50% thermal probability level).

Figure 6. ORTEP diagram of the EFC-O (ellipsoids drawn at 50% thermal probability).

are a couple of very weak C−H···O hydrogen bonds observed in the packing. 4. Enrofloxacinium Difumarate Fumaric Acid (2:1:1): EFC-F. The unit cell of EFC-F was found to contain two molecules of enrofloxacin cation, one difumarate anion and one neutral fumaric acid molecule. The acidic hydrogen atom (H3A) of one of the −COOH groups of fumaric acid has been found to be transferred to the piperazine nitrogen (N3) of EFC (Figure 4). The enrofloxacin cation and the difumarate anion

was found to be transferred to the piperazine nitrogen of EFC (Figure 3). One charge assisted intramolecular O−H···O− hydrogen bond has been observed in maleic acid monoanion along with the intramolecular O−H···O hydrogen bond in enrofloxacin cation as mentioned earlier. One of the two acidic protons (H3A) of the maleic acid has been transferred to piperazine ring nitrogen (N3), and this proton is found to form a charge assisted N+−H···O− hydrogen bond (Table 3). There 1633



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Figure 7. ORTEP diagram of the EFC-A (ellipsoids drawn at 30% thermal probability).

Figure 8. ORTEP diagram of the Am-EFC (ellipsoids drawn at 30% thermal probability).

are connected to each other by a strong charge assisted N+− H···O− hydrogen bond (Table 3) as observed in EFC-M. The neutral fumaric acid and the fumarate anion are found to form molecular chains by charge assisted O−H···O− hydrogen bonds (Table 3). 5. Enrofloxacinium Disuccinate Succinic Acid (2:1:1): EFC-S. The unit cell of EFC-S was found to contain two enrofloxacinium cations, one disuccinate anion and one neutral succinic acid molecule, similar to EFC-F. The acidic hydrogen atom (H3A) of one of the −COOH groups of succinic acid has been found to be transferred to the piperazine nitrogen (N3)

(Figure 5). All the strong and weak hydrogen bonds observed in EFC-F are also present in this structure (Table 3). It is noteworthy that the crystal structures of EFC-M and EFC-S are isostructural. 6. Enrofloxacinium Dioxalate Hexahydrate (2:1:6): EFC-O. The unit cell of the EFC-O was found to contain two molecules of enrofloxacinium cations, one dioxalate anion and six water molecules (Figure 6). Both the acidic hydrogen atoms (H3A) of the oxalic acid have been transferred to the piperazine nitrogen (N3) of two enrofloxacin molecules. A 1634



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number of strong O−H···O− and N−H···O− hydrogen bonds are responsible for the formation of the crystal lattice (Table 3). 7. Enrofloxacinium Acetate Acetic Acid Monohydrate (1:1:1:1): EFC-A. The data set on the crystals of EFC-A was also collected at room temperature because the crystals lost their single crystal nature and developed several cracks while being cooled below 250 K. The asymmetric unit of EFC-A was found to contain one enrofloxacinium cation, one acetate anion and one neutral acetic acid along with one water molecule (Figure 7). The acidic hydrogen of the acetic acid was found to be transferred to the piperazine nitrogen. As observed in EFCO, a number of strong O−H···O− and N−H···O− hydrogen bonds are responsible for the formation of the crystal lattice (Table 3). 8. Ammonium Enrofloxacinate Trihydrate (1:1:3): AmEFC. The asymmetric unit of Am-EFC was found to contain one ammonium cation and one enrofloxacinate anion along with three water molecules (Figure 8). The acidic hydrogen of the carboxylic acid of EFC was found to be transferred to nitrogen of the ammonia. Each ammonium cation (N4) was found to be connected to three different enrofloxacinate anions through (a) N+−H···N with N3, (b) N+−H···O− with O1 and O2, and (c) N+−H···O with O3, and one water molecule (O6) by strong N+−H···O hydrogen bonds (Table 3). It was found that this salt completely released ammonia on grinding the single crystals for recording PXRD. As a result Am-EFC was found to get converted to the same anhydrous form of EFC as reported in this manuscript (Figure S17 in the Supporting Information).

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energy calculations on EFC molecule by rotating the piperazine ring with the ethyl substitution such that the H···F distance became longer and longer in small steps up to 3.64 Å from 2.26 Å. We observed that the energy of the EFC molecule started increasing with the increase in the H···F distance (Table S1 and Figure S34 in the Supporting Information). This observation further reinforces that the intramolecular C−H···F hydrogen bond also provides additional stability to the EFC molecule, both in the gas phase and in the solid state. The importance of this intramolecular C−H···F hydrogen bond can only be ascertained by high resolution charge density analysis on the EFC crystal. The experimental charge density analysis on EFC is presently being carried out by us. The results of this study will be highlighted in our future publication. The existence of the anhydrous and hexahydrate forms of EFC was reported earlier by spectroscopic and PXRD techniques in a patent.27 The simulated PXRD patterns from our SCXRD data for EFC and EFC·6H 2 O were found to be identical to the corresponding forms reported in the said patent. The nature (neutral, zwitterionic, cationic and anionic) of EFC molecule, in the anhydrous or hydrated form, in the solid state was not discussed in the same patent and also was not shown experimentally prior to our investigations. On careful observation of the crystal structure of EFC·6H2O, it indicates that the EFC molecules are surrounded by a large number of water molecules. But, these water molecules are not strongly hydrogen bonded to the EFC molecule. Therefore, it may be stated that EFC molecule is hydrophobic in nature. When the single crystals of EFC·6H2O were ground and the PXRD data were recorded on the powdered sample, it was found that the powdered sample lost all the water molecules of crystallization and got converted to the anhydrous form (1) reported in this manuscript. The same phenomenon was observed when the crystals of EFC·6H2O were exposed to ambient conditions for a long time or heated gently for a few minutes. When anhydrous (1) or the hexahydrate (2) form of EFC was stirred in water, a suspension of EFC was formed instead of a clear solution. This observed poor solubility of EFC in water may be attributed to the hydrophobic nature of EFC molecule in either neutral (1) or zwitterionic form (2). The salts of EFC with different organic acids were found to contain EFC in its cationic form in the solid state. EFC in its cationic form in these salts has been found to be strongly hydrogen bonded to the carboxylate anions. When these salts were added to water, clear solutions were formed. This may be attributed to the fact that EFC cations are strongly hydrogen bonded to the carboxylate anions, which in turn are known to form strong hydrogen bonds with water molecules when dissolved in water. Hence we observed 30- to 110-fold increase in the solubility of these salts in water, when compared to the anhydrous form. Am-EFC is also expected to be highly soluble in water as the ammonium cation is strongly hydrogen bonded to three EFC molecules and one water molecule in the solid state. This assembly, when added to water, is expected to result in a clear solution instead of a suspension of EFC in water. Enrofloxacinium citrate monohydrate has recently been reported in the literature.16c It is interesting to note that the PXRD pattern of enrofloxacinium citrate monohydrate reported by Golovnev et al. is markedly different from the product obtained by us using solvent drop grinding of EFC with citric acid (Figure S15 in the Supporting Information).

RESULTS FROM SOLUBILITY STUDIES

The results of the solubility determinations are listed in the Table 4. Commercially available enrofloxacin used in all our experiments was found to have a solubility of 0.61 mg/mL, which is similar to the earlier report.13 The salts EFC-M, EFCF, EFC-S and EFC-O were found to have 30 to 110 times higher solubility (in water of pH 6.8) than the commercially available enrofloxacin. EFC-A was found to be highly soluble in water of pH 6.8. The solubility of Am-EFC could not be determined as this salt could not be produced in large quantity (100−200 mg).

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DISCUSSION It is evident from the above results that enrofloxacin is highly capable of forming salts with a number of different organic acids listed in Table 1. The existence of EFC in neutral, zwitterionic, cationic and anionic forms in the solid state has been elucidated in this manuscript. The EFC molecule has been found to display weak intramolecular C−H···F hydrogen bond in all the structures reported herein. The H···F distances have been found to be close to 2.2 Å and the ∠C−H···F ranging between 117° and 128°. We have used the coordinates of EFC molecule from the CIF of EFC as an input file for Gaussian0928 for geometry optimization of this molecule using the second order Møller−Plesset (MP2) method29 and 6-31+G* basis set. When we compared the optimized geometry obtained from this MP2 calculation with that of the input geometry of EFC, we found that the two geometries are nearly similar. In fact, we observed that the H···F distance in the optimized geometry remained 2.266 Å, and the ∠C−H···F became 120.25°, which is also the same as compared to 121(1)o found in the structure of EFC (Table 1). We then carried out a number of single point 1635



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Pharm. Biopharm. 2007, 67, 112−119. (f) Jung, M.-S.; Kim, J.-S.; Kim, M.-S.; Alhalaweh, A.; Cho, W.; Hwang, S.-J.; Velaga, S. P. J. Pharm. Pharmacol. 2010, 62, 1560−1568. (g) Blagden, N.; de Matas, M.; Gavan, P. T.; York, P. Adv. Drug Delivery Rev. 2007, 59, 617−630. (4) Espinosa-Lara, J. C.; Guzman-Villanueva, D.; Arenas-García, J. I.; Herrera-Ruiz, D.; Rivera-Islas, J.; Román-Bravo, P.; Morales-Rojas, H.; Höpfl, H. Cryst. Growth Des. 2013, 13 (1), 169−185, DOI: 10.1021/ cg301314w. (5) (a) Datta, S.; Grant, D. J. W. Nat. Rev. Drug Discovery 2004, 3, 42−57. and references therein. (b) Meyer, M. C. Bioavailability of drugs and bioequivalence. In Encyclopedia of Pharmaceutical Technology; Marcel Dekker Inc.: New York, 1998; Vol. 2, pp 33−58. (6) (a) Almarsson, Ö .; Zaworotko, M. J. Chem. Commun. 2004, 1889−1896. (b) Childs, S. L.; Chyall, L. J.; Dunlap, J. T.; Smolenskaya, V. N.; Stahly, B. C.; Stahly, G. P. J. Am. Chem. Soc. 2004, 126, 13335− 13342. (c) Vishweshwar, P.; McMahon, J. A.; Bis, J. A.; Zaworotko, M. J. J. Pharm. Sci. 2006, 95, 499−516. (d) Jones, W.; Motherwell, W. D. S.; Trask, A. V. MRS Bull. 2006, 31, 875−879. (e) RodríguezHornedo, N. Mol. Pharmaceutics 2007, 4, 299−300. (f) Shan, N.; Zaworotko, M. J. Drug Discovery Today 2008, 13, 440−446. (g) Schultheiss, N.; Newman, A. Cryst. Growth Des. 2009, 9, 2950− 2967. (h) Aitipamula, S.; Chow, P. S.; Tan, R. B. H. CrystEngComm 2009, 11, 889−895. (i) Friscic, T.; Jones, W. J. Pharm. Pharmacol. 2010, 62, 1547−1559. (j) Babu, N. J.; Nangia, A. Cryst. Growth Des. 2011, 11, 2662−2679. (k) Upadhyay, N.; Shukla, T. P.; Mathur, A.; Manmohana; Jha, S. K. Int. J. Pharm. Sci. Rev. Res 2011, 8, 144−148. (l) Desiraju, G. R. In Pharmaceutical Salts and Co-crystals; Wouter, J., Queré, L., Eds.; RSC Publishing: Cambridge, 2011. (7) Karanam, M.; Dev, S.; Choudhury, A. R. Cryst. Growth Des. 2012, 12, 240−252. (8) (a) Andersson, M. I.; MacGowan, A. P. J. Antimicrob. Chemother. 2003, 51, 1−11. (b) Lohray, B. B.; Baskaran, S.; Rao, B. S.; Mallesham, B.; Bharath, K. S.; Reddy, B. Y.; Venkateswarlu, S.; Sadhukhan, A. K.; Kumar, M. S.; Sarnaik, H. M. Bioorg. Med. Chem. Lett. 1998, 8, 525− 528. (c) Takahashi, H.; Hayakawa, I.; Akimoto, T. Yakushigaku Zasshi 2003, 38, 161−179. (d) Wiles, J. A.; Bradbury, B. J.; Pucci, M. J. Expert. Opin. Ther. Pat. 2010, 20, 1295−1319. (e) Abbanat, D.; Morrow, B.; Bush, K. Curr. Opin. Pharmacol. 2008, 8, 582−592. (f) Rothlin, R. P. Medicina 1999, 59, 3−7. (g) Elmas, M.; Trans, B.; Kaya, S.; Bas, A. L.; Yazar, E.; Yarsan, E. Can. J. Vet. Res. 2001, 65, 64− 67. (9) (a) Sorgel, F.; Kinzig, M. Am. J. Med. 1993, 94, 44S−55S. (b) Riley, C. M.; Kindberg, C. G.; Stella, V. J. International Telesymposium; Prous Science Publishers: Barcelona, Spain, 1989; pp 21−36. (c) Rosen, T.; Chu, C. D. W.; Lico, I. M.; Fernandes, P. B.; Marsh, K.; Shen, L.; Cepa, V. G.; Pernet, A. G. J. Med. Chem. 1988, 31, 1598−1611. (d) Lizondo, M.; Pons, M.; Gallardo, J. E. J. Pharm. Biomed. Anal. 1997, 15, 1845−1849. (10) Reddy, J. S.; Ganesh, S. V.; Nagalapalli, R.; Dandela, R.; Solomon, K. A.; Kumar, K. A.; Goud, N. R.; Nangia, A. J. Pharm. Sci. 2011, 100, 3160−3176. (11) Prescott, J. F.; Baggot, J. D. In Antimicrobial Therapy in Veterinary Medicine 2nd ed.; Iowa State University Press: 1993. (12) TerHune, T. N.; Skogerboe, T. L.; Shostrom, V. K.; Daniel, M. S.; Weigel, J. Am. J. Vet. Res. 2005, 66, 342−349. (13) (a) Seedher, N.; Agarwal, P. Indian J. Pharm. Sci. 2009, 1, 82− 87. (b) Lizondo, M.; Pons, M.; Gallardo, J. E. J. Pharm. Biomed. Anal. 1997, 15, 1845−1849. (14) Brittain, H. G. Cocrystal Systems of Pharmaceutical Interest: 2010. Cryst. Growth Des. 2012, 12, 1046−1054. (15) (a) Tarushi, A.; Psomas, G.; Raptopoulou, C. P.; Psycharis, V.; Kessissoglou, D. P. Polyhedron 2009, 28, 3272. (b) Efthimiadou, E. K.; Katsarou, M.; Sanakis, Y.; Raptopoulou, C. P.; Karaliota, A.; Katsaros, N.; Psomas, G. J. Inorg. Biochem. 2006, 100, 1378. (c) Recillas-Mota, J.; Flores-Alamo, M.; Moreno-Esparza, R.; Gracia-Mora, J. Acta Crystallogr., Sect. E 2007, 63, m3030. (d) Tarushia, A.; Raptopoulou, C. P.; Psycharis, V.; Terzis, A.; Psomas, G.; Kessissoglou, D. P. Bioorg. Med. Chem. 2010, 18, 2678−2685.

CONCLUSION The crystal structures of the anhydrous and the hexahydrate forms of enrofloxacin with five new salts with weak organic acids and one salt with ammonium cation have been reported for the first time in this manuscript. These crystal structures allow us to understand the nature (hydrophobic or hydrophilic) of EFC molecule and also describe the existence of EFC in four different molecular forms (neutral, zwitterionic, cationic and anionic) in the solid state. The enrofloxacin molecule is found to exist as a neutral molecule in the solid state, and water induced proton transfer led to the formation of the zwitterionic form of the enrofloxacin in the hexahydrate. The generation of the new salts by solvent assisted grinding in the case of EFC is found to be highly rewarding. Experiments are in progress to determine solubilities and to grow single crystals of the new phases reported in the Table 1. In the current manuscript, the structural landscape of EFC along with the enhanced solubility of the materials studied has been described in detail.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

* Supporting Information S

CIF files and PXRD patterns for EFC as discussed in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.K. thanks Department of Science and Technology (DST), India, for research fellowship, and A.R.C. thanks DST for the funding of the fast track project entitled “Co-crystallization of Active Pharmaceutical Ingredients Pathway for Enhanced Properties”, FT-SR/2008-09. We thank Dr. Adrene Freeda D’cruz for suggesting suitable grammatical corrections in the manuscript. Dr. Sagarika Dev and Ms. Gurpreet Kaur are acknowledged for their contribution in the computational studies, and IISER Mohali is acknowledged for providing X-ray diffraction, NMR spectrometer, FTIR and UV−vis spectrophotometers, other research facilities and infrastructural support.

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REFERENCES

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Structural Landscape of Pure Enrofloxacin and Its Novel Salts

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