Clofazimine Mesylate - American Chemical Society

Article pubs.acs.org/crystal

Clofazimine Mesylate: A High Solubility Stable Salt Geetha Bolla and Ashwini Nangia* School of Chemistry, University of Hyderabad, Central University PO, Prof. C. R. Rao Road, Gachibowli, Hyderabad 500 046, India S Supporting Information *

ABSTRACT: Clofazimine (CFZ), an antibacterial and anti-inflammatory drug, is also recommended by the World Health Organization for the treatment of leprosy in combination with dapsone and rifampicin. It is an iminophenazine derivative and classified as a Biopharmaceutics Classification System (BCS) class II drug because of poor aqueous solubility (10 mg L−1). Despite it being a very classical drug known for more than five decades, there is no systematic study of CFZ salts for solubility and stability enhancement. We report a solid form screen of CFZ with pharmaceutically acceptable coformers/acids. Salts of CFZ with methanesulfonic acid, maleic acid, isonicotinic acid, nicotinic acid, malonic acid, and salicylic acid in an equimolar ratio as well as an amorphous phase of CFZ are reported. All new solid phases were characterized by FT-IR, powder X-ray diffraction, and differential scanning calorimetry, and confirmed by single crystal X-ray diffraction. The acid proton is transferred to the imine nitrogen of CFZ in a R21(7) ring motif. The driving force for facile salt formation is the ionic N+−H···O− and N−H···O− bifurcated hydrogen bond synthon. Solubility and powder dissolution experiments were carried out in 60% EtOH−water to compare the higher solubility of salts compared to that of pure CFZ. CFZ-mesylate (1:1) is 99 times more soluble than the pure drug in water. All salts were stable for up to 24 h in 60% EtOH−water slurry medium. CFZ−MSA is the best pharmaceutical salt with high solubility and good stability.

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cocrystal−salt continuum.5 These ΔpKa ranges were recently revised to < −1 for cocrystals, > 4 for salts, and −1 to 4 for cocrystal, salt, or cocrystal−salt continuum state using crystallographic data from the Cambridge Structural Database (CSD) and calculated pKa’s in Marvin6 for over 6000 acid−base adducts.7 Statistical trends are now available to correlate calculated or solution pKa’s with the location of the H atom in the solid-state. Clofazimine (CFZ) (Figure 1) is a water insoluble iminophenazine derivative originally described in 1957. Primary clinical trials of CFZ to treat leprosy led to the World Health Organization recommended triple drug regimen. Apart from antileprosy, CFZ is effective against inflammatory and Mycobacterium tuberculosis diseases.8 It is generally used in combination with dapsone and rifampicin. CFZ also exhibited activity against disseminated Mycobacterium avium complex (MAC) disease in HIV-infected patients.9 CFZ is marketed by

INTRODUCTION A majority of drugs are preferably administrated in solid oral dosage form, such as tablet or capsule, because of high crystallinity, purity, patient compliance, convenience, and longer storage. 1 The physicochemical properties of an active pharmaceutical ingredient (API) can be modulated at a supramolecular level through novel solid-state forms, e.g., polymorphs, solvates, salts, and recently cocrystals.2 The major advantage with cocrystals is that physicochemical properties such as solubility, stability, and bioavailability of nonionized functional groups present in the API can be modulated and tuned.3 Salts are limited to ionizable APIs only and tend to hydrate easily.4 On the solubility advantage front, the increment is modest for cocrystals (4−20 fold) and very dramatic for salts (100−1000 fold).4b,c The formation of cocrystal or salt depends upon the ΔpKa of the API and the coformer. The ΔpKa rule, wherein ΔpKa = pKa (conjugate acid of base) − pKa (acid), is a useful guide to know beforehand if an acid−base complex will give a neutral cocrystal (ΔpKa < 3) or an ionic salt (ΔpKa > 3). A more practical cutoff for organic salts is taken as ΔpKa < 0 for cocrystals, ΔpKa > 3 for salts, and in the range 0 < ΔpKa < 3 there is possibility of a © 2012 American Chemical Society

Received: October 7, 2012 Revised: November 6, 2012 Published: November 8, 2012 6250

dx.doi.org/10.1021/cg301463z | Cryst. Growth Des. 2012, 12, 6250−6259

Crystal Growth & Design

Article

Figure 1. Chemical structure of clofazimine (CFZ). The highlighted N1 atom is the most preferred site for protonation. Atom numbering of CFZ is used in the crystal structures.

Novartis under the trade name Lamprene in 100 mg capsules. However efficacy of the drug is discussed only in regard to its antileprosy activity.10 A serious drawback of iminophenazine derivatives11 is that the highly hydrophobic molecular skeleton gives CFZ low water solubility (10 mg/L) and high permeability (log P = 7). CFZ is a Biopharmaceutics Classification System (BCS) class II drug.12 Its Dose number (Do), or the number of glasses of water (250 mL) required to dissolve the drug at its highest dosage, is high at 40. It is used as an antibiotic with a very long pharmacokinetic half-life of up to 70 days.13 Two polymorphs,14a a DMF solvate,14b and cyclodextrin complexes of CFZ14c are reported in the literature.14 Our main objective was to explore cocrystals and salts of CFZ using a crystal engineering approach15 of supramolecular synthons.16 Only generally regarded as safe (GRAS)17 molecules and coformers were considered for safety of the final cocrystal/salt.18 Pharmaceutical salts with methanesulfonic acid (MSA), maleic acid (MLA), isonicotinic acid (INA), nicotinic acid (NA), salicylic acid (SCL) and malonic acid (MLN) were obtained. Additionally, an acetone solvate (Figure S1, Supporting Information) and an amorphous phase (obtained from melt) of CFZ was obtained in our experiments. All novel solid phases were characterized by IR, PXRD, DSC and single crystal X-ray diffraction (see ORTEP diagrams in Figure S2, Supporting Information). Solubility and dissolution experiments were conducted in 60% EtOH−water medium in which both the free base and the salts have modest to good solubility to enable measurements.

Figure 2. (a) CFZ triclinic form having C−H···N dimers and such dimers are further connected by C−H···π interactions. (b) CFZ monoclinic form has C−H···N, C−H···π and C−H···Cl interactions. There is no dimer motif in the monoclinic structure.

MeOH−acetonitrile (1:1), diffraction quality single crystals of dark red color and plate morphology were harvested. The crystals structure was solved and refined in the monoclinic space group P21/c. There is one molecule of each ion in the asymmetric unit of CFZ-NH+−MSA−. Proton transfer from MSA-H to isopropyl imine N of CFZ resulted in R21(7) ring motif19 (Figure 3a). The two-point synthon of N+−H···O− and N−H···O− hydrogen bonds (N1−H1A···O2, 1.86 Å, 175°; N2−H2A···O2, 1.93 Å, 167°) to the mesylate oxygen makes the stoichiometric salt. Two more oxygen atoms of MSA are engaged in C−H···O interactions from aromatic C−H of the chlorophenyl ring to the sulfonate oxygen in the same layer (Figure 3b). The SO and S−O bond distances are 1.45 Å and 1.62 Å in methanesulfonic acid, whereas in the CFZ-NH+−MSA− salt the S−O distances are nearly close (1.44, 1.45, 1.47 Å). The intermediate S−O distances and the location of proton on N1 in difference electron density maps of the X-ray crystal structure confirm salt formation. CFZ-NH+−MLE− (1:1) Salt. The salt was prepared by liquidassisted grinding of equimolar CFZ and MLE-H in acetonitrile solvent. Recrystallization was performed in the same solvent to

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RESULTS AND DISCUSSION Crystal Structures. Crystal structures of two reported polymorphs of CFZ14a are discussed (Figure 2) to compare with the salt structures. The dihydrophenazine ring of CFZ is nearly planar and one chlorophenyl ring is perpendicular (89.5°), and the other phenyl ring is more planar (32.7°) in the triclinic polymorph of CFZ. The molecule may be considered as two planar units which form a butterfly at the N3−N4 axis, the angle being only 0.9° (planar skeleton) in the triclinic form. Two CFZ molecules form a C−H···N dimer interaction (3.465(3) Å, 134°) along with a weak C−H···π interaction (3.732(4) Å). There are no strong hydrogen bonds possible in this structure. Four types of N atoms (basic sites) are present. CFZ molecules in the monoclinic form assemble via C−H···N, C−H···π and C−H···Cl interactions. Crystallographic parameters and normalized hydrogen bonds are summarized in Tables 1 and 2. CFZ-NH+−MSA− (1:1) Salt. When an equimolar ratio of CFZ and methanesulfonic acid (MSA-H) was crystallized from 6251



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Table 1. Crystallographic Parameters of CFZ Salts CFZ-NH+−MSA−

CFZ-NH+−MLE−

CFZ-NH+−INA−

empirical formula formula weight crystal system space group T (K) a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Dcalcd (g cm−3) μ (mm−1) θ range Z range h range k range l reflections collected total reflections observed reflections R1 [I > 2σ(I)] wR2 (all) goodness of fit diffractometer

C27H23Cl2N4·CH3O3S 569.50 monoclinic P21/c 298(2) 10.213(2) 18.008(6) 15.561(3) 90.0 102.79(2) 90.0 2790.9(12) 1.355 9.9944 2.91−28.83 4 −11 to +11 −20 to +20 −17 to +17 28661 5549 4299 0.0895 0.2307 0.737 Oxford Gemini CFZ-NH+−MLN−

C27H23Cl2N4·C4H3O4 589.46 monoclinic P21/n 298(2) 11.2549(19) 20.816(3) 12.519(2) 90 103.413(17) 90 2853.0(8) 1.372 1.272 2.69−26.31 4 −14 to +13 − 25 to +26 −15 to +15 10779 4855 1924 0.0870 0.1685 0.909 Oxford Gemini CFZ-NH+−SCL−

C27H23Cl2N4·C6 H4NO2 596.50 triclinic P1̅ 298(2) 9.8153(9) 12.1738(10) 15.2122(10) 72.455(7) 77.844(7) 66.479(8) 1580.7(2) 1.253 0.242 2.80−24.71 2 −11 to +11 −14 to +14 −16 to +17 9737 5381 2582 0.0570 0.1502 0.908 Oxford Gemini CFZ-NH+−MSA−−H2O

empirical formula formula weight crystal system space group T (K) a (Å) b (Å) c (Å) α (°) β (°) γ (°) V (Å3) Dcalcd (g cm−3) μ (mm−1) θ range Z range h range k range l reflections collected total reflections observed reflections R1 [I > 2σ(I)] wR2 (all) goodness of fit diffractometer

C27H23Cl2N4·C3H2O4 576.44 triclinic P1̅ 298(2) 9.8401(8) 12.4069(10) 13.0576(10) 74.129(1) 79.9900(1) 67.520(1) 1932.2(7) 1.400 0.282 1.68−26.02 2 −12 to +12 −15 to +15 −16 to +16 14254 5344 3774 0.0567 0.1703 1.043 BRUKER Smart

C27H23Cl2N4·C7H5 O3 611.50 triclinic P1̅ 298(2) 10.8702(6) 11.2066 (6) 13.8272(8) 82.354(5) 88.960(5) 63.492(6) 1492.31(17) 1.361 0.260 2.97−24.66 2 −12 to +12 −12 to +13 −12 to +16 9169 5085 3423 0.0472 0.1293 1.027 Oxford Gemini

C27H23Cl2N4·CH3O3S·H2O 587.51 triclinic P1̅ 298(2) 9.5945(12) 11.0456(12) 14.4628(11) 110.173(9) 95.859(9) 94.929(9) 1419.1(3) 1.375 9.9943 2.79−26.31 2 −11 to +11 −13to +13 −18 to +18 9694 5785 3423 0.049 0.1069 0.892 Oxford Gemini

CFZ-NH+−NA− C27H23Cl2N4·C6H4NO2 596.50 triclinic P1̅ 298(2) 11.6032(13) 15.361(2) 18.733(3) 91.280(11) 111.337(14) 109.295(11) 2896.5(8) 1.368 9.9970 2.68−26.31 4 −12 to +12 −16 to +17 −20 to +20 14036 8145 4572 0.0586 0.1430 0.998 Oxford Gemini CFZ−acetone solvate C27H23Cl2N4·C2H6O 531.46 triclinic P1̅ 100 10.0842(13) 12.0695(16) 12.6613(16) 75.278(2) 66.588(2) 69.059(2) 13.09.3(3) 1.348 0.279 1.77−26.10 2 −12 to +12 −14 to +14 −15 to +15 13647 5152 4418 0.0486 0.1293 1.030 BRUKER Smart

interactions complete the molecular organization (Figure 4b). The formation of the salt was confirmed by resonance in the carboxylate bond distances (1.22, 1.26 Å) of maleate anion. CFZ-NH+−INA− (1:1) Salt. Single crystals of the salt were obtained from acetonitrile, and its X-ray structure was solved in triclinic space group P1̅. The familiar R21(7) ring motif (Figure 5)

give diffraction-quality dark red crystals (P21/n space group). Single proton transfer from the dicarboxylic acid (MLE-H) to the isopropyl imine N of CFZ gave the same R21(7) motif of salt (Figure 4a) along with intramolecular H bond S(7) motif in maleate anion. The N+−H···O− and N−H···O− hydrogen bonds are 2.16 Å, 141°, and 1.82 Å, 176°. Auxiliary C−H···O 6252



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Table 2. Normalized Hydrogen Bonds in Crystal Structures of CFZ and Its Salts crystal forms CFZ-NH+−MSA−

CFZ-NH+− MLE−

CFZ-NH+−INA−

CFZ-NH+−NA−

CFZ-NH+−MLN−

CFZ-NH+−SCL−

CFZ-NH+−MSA−−H2O

interaction

H···A /Å

D···A /Å

∠D−H···A /°

N1−H1A···N2 N1−H1A···O2 N2−H2A···N1 N2−H2A···O2 C1−H1···Cl2 C14−H14···O3 C20 −H20···O2 C27−H27B···O3 C28−H28A···N3 N1−H1A···O1 N2−H2A···O1 O2−H2B···O3 C14−H14···O2 C21−H21···O3 N1−H1A···N2 N1−H1A···O2 N2−H2A···N1 N2−H2A···O2 C1−H1···Cl2 C3−H3···O1 C20−H20···O2 N1−H1A···O3 N1−H1A···N2 N2−H2A···O3 N2−H2A···O4 N2−H2A···N1 N6−H6A···N7 N6−H6A···O1 N7−H7A···N6 N7−H7A···O1 N7−H7A···O2 C1−H1···Cl2 C4−H4···N8 C17−H17···N10 C28−H28···O3 C34−H34···Cl4 C37−H37···N3 C50−H50···N5 C53−H53···O1 C60−H60A···N8 C61−H61···O1 N1−H1A···N2 N1−H1A···O2 N2−H2A···O1 N2−H2A···O2 O1−H3B···O3 C4−H4···Cl1 C21−H21···O4 C23−H23 ···O2···O2 N1−H1A···N2 N1−H1A···O1 N2−H2A···N1 N2−H2A···O1 O3−H3A···O2 C4−H4···O3 C2−H23···O1 N1−H1A···O2 N1−H1A···N2 N2−H2A···O2 N2−H2A···N1 O4−H4A···O3

2.39 1.86 2.45 1.93 2.73 2.60 2.36 2.59 2.48 2.16 1.82 1.20 2.53 2.51 2.37 1.85 2.38 1.88 2.74 2.55 2.34 2.06 2.39 2.18 2.37 2.36 2.38 1.99 2.36 2.04 2.58 2.76 2.52 2.56 2.49 2.82 2.60 2.54 2.60 2.61 2.43 2.37 2.19 2.25 2.42 1.10 2.75 2.47 2.57 2.49 1.88 2.44 2.16 1.64 2.52 2.57 2.15 2.51 2.05 2.45 2.13

2.767(4) 2.792(4) 2.767(4) 2.777(4) 3.582(3) 3.525(4) 3.305(4) 3.460(4) 3.457(4) 2.870(6) 2.853(6) 2.414(6) 3.459(6) 3.272(7) 2.761(4) 2.841(4) 2.761(4) 2.748(4) 3.471(3) 3.239(6) 3.302(4) 2.914(5) 2.746(5) 2.974(5) 3.114(5) 2.746(5) 2.743(5) 2.837(5) 2.743(5) 2.876(5) 3.291(6) 3.428(4) 3.445(5) 3.461(9) 2.812(6) 3.464(4) 3.500(5) 3.412(6) 3.436(5) 3.536(6) 2.773(7) 2.746(3) 3.008(4) 3.033(3) 3.079(4) 2.467(3) 3.506(3) 3.139(4) 3.472(4) 2.795(3) 2.784(3) 2.795(3) 2.938(3) 2.532(3) 3.408(4) 3.478(4) 2.915(3) 2.801(3) 2.877(3) 2.801(3) 2.883(4)

104 175 102 167 149 166 171 148 174 141 176 164 175 139 102 172 105 158 133 129 169 169 106 154 146 107 106 170 107 163 141 129 174 162 100 127 162 156 151 161 102 107 166 165 140 156 139 129 165 100 176 108 166 159 160 164 174 104 168 106 145

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symmetry code intramolecular 1/2 − x, 1/2 + y, 1/2 intramolecular x, y, 1 + z 1/2 − x, 1/2 + y, 1/2 1/2 − x, 1/2 + y, 1/2 1/2 + x, 1/2 − y, 1/2 a 1 − x, 1 − y, 1 − z 1 − x, −y, 1 − z 1 − x, −y, 1 − z intramolecular 1 − x, −y, 1 − z intramolecular intramolecular 1 − x, 1 − y, 1 − z intramolecular 1 − x, 1 − y, 1 − z x, y, −1 + z x, −1 + y, z 1 + x, −1 + y, z a intramolecular a a intramolecular intramolecular 1 − x, 1 − y, 1 − z intramolecular 1 − x, 1 − y, 1 − z 1 − x, 1 − y, 1 − z x, 1 + y, z 2 − x, 1 − y, −z 1 − x, 1 − y, −z intramolecular x, 1 + y, z 2 − x, 1 − y, −z 1 + x, y, z 1 + x, y, z 2 − x, 1 − y, 1 − z intramolecular intramolecular 1 − x, 1 − y, −z 1 − x, 1 − y, −z 1 − x, 1 − y, −z intramolecular x, 1 + y, z −x, 1 − y, 1 − z −x, 1 − y, −z intramolecular 1 − x, 1 − y, 1 − z intramolecular 1 − x, 1 − y, 1 − z intramolecular a −x, 2 − y, 1 − z −1 + x, −1 + y, z intramolecular −1 + x, −1 + y, z intramolecular a

−z

−z −z +z



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Table 2. continued crystal forms

interaction O4−H4B···N4 C15−H15···O2 C17−H17···O1

a

H···A /Å

D···A /Å

∠D−H···A /°

2.09 2.54 2.55

2.899(3) 3.454(3) 3.437(3)

173 164 167

symmetry code 1 − x, 1 − y, 1 − z 1 − x, 1 − y, 1 − z 1 − x, 1 − y, 1 − z

Molecules/ions in the same asymmetric unit.

Figure 5. (a) The basic bimolecular unit of CFZ-NH+−INA− (1:1) salt with the R21(7) ring motif. (b) The bimolecular units form a chain through C−H···Cl interactions.

Figure 3. (a) Proton transfer from methanesulfonic acid to CFZ secondary imine N atom to give the R21(7) ring motif in CFZ−MSA (1:1) salt. (b) These salt pairs are further interlinked through C−H···O and C−H···Cl interactions.

Figure 4. (a) Proton transfer from maleic acid to CFZ forms CFZNH+−MLE− (1:1) salt. (b) The organic cation and anion extend through C−H···O interactions in a 1D chain.

of N1−H1A···O2 (1.85 Å, 172°) and N2−H2A···O2 (1.88 Å, 158°) is present. The remaining structural features are similar to the previous salts. CFZ-NH+−NA− (1:1) Salt. The salt of CFZ and nicotinic acid crystallized from acetonitrile−MeOH (1:1) in P1̅ space group. There are two symmetry-independent CFZ-NH+ (shown as ball and stick (CFZ1) and thick bonds model (CFZ2) (Figure 6) and nicotinate anions in the asymmetric unit. The two chloro phenyl rings in CFZ-NH+−NA− are conformationally different. The R21(7) ring motif (Figure 6a) is formed by N+−H···O− and N− H···O− hydrogen bonds with crystallographic independent

Figure 6. (a) The R21(7) ring motif of in the CFZ-NH+−NA− salt. (b) Weak C−H···N interactions between the 1D chains of symmetryindependent ions shown as ball-stick and thick-bonds models.

molecules, and the 1D chains are arranged in ABAB fashion (Figure 6b). CFZ-NH+−MLN− (1:1) Salt. The crystal structure (P1)̅ has 2 R2 (9) ring motif (Figure 7a) of N+−H···O− and N−H···O− hydrogen bonds (2.19 Å, 166°; 2.42 Å, 140°) because now both oxygen atoms of the carboxylate group accept H bonds. The 6254



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Figure 7. (a) The basic unit of bimolecular R22(9) ring motif present in CFZ-NH+−MLN (1:1) salt. (b) The ions extend through C−H···O and C−H···Cl interactions in 2D sheets. Figure 8. (a) The R21(7) ring motif in CFZ-NH+−SCL−. (b) Cl···Cl interactions and π-stacking in the structure.

resonance stabilized C−O distances (1.24, 1.21 Å) in the malonate confirm salt formation. Additional C−H···O and C− H···Cl interactions interactions make 2D sheets (Figure 7b). CFZ-NH+−SCL− (1:1) Salt. A salicylate salt of CFZ was crystallized from acetonitrile (P1̅ space group). The intermolecular R21(7) ring motif of N+−H···O−, N−H···O− hydrogen bonds (1.88 Å, 176°; 2.16 Å, 166°) and intramolecular S(6) O− H···O motif (1.64 Å) make the ionic salt (Figure 8a). Interhalogen Cl···Cl interactions20 (Figure 8b) connect the ions and which are π-stacked to the next layer (3.25 Å). Salt formation was confirmed by carboxylate C−O distances (1.26, 1.26 Å). CFZ-NH+−MSA−−H2O (1:1:1) Salt Hydrate. A hydrate of CFZ mesylate (1:1:1) was obtained from moist MeOH− CH3CN (1:1). The R21(7) ring motif of the salts (Figure 9a) persists in the hydrate. Water molecules are bonded to the mesylate O and phenazine N in a 1D chain wherein water molecules bridge the ions through O−H···N (2.09 Å, 173°) and O−H···O (2.13 Å, 145°) H bonds (Figure 9b). The sulfonate anion S−O distances (1.44, 1.44, 1.46 Å) confirm proton transfer. CFZ is dark red because of extended conjugation. The color changes from dark red to black upon salt formation, a visual color change that can be used to monitor progress of the reaction. The main criteria for salt formation is that pKa of the conjugate acid of the base must be greater than the pKa of the acid to ensure proton transfer. CFZ is a weak base (pKa 8.51) and ΔpKa > 3 with the acids used in this study (Table 3), suggesting proton transfer according to the ΔpKa rule.5,7 Powder X-ray Diffraction. Powder X-ray diffraction21 is a reliable technique to characterize the nature of new solid forms in grinding or milling experiments. Differences in the signature peaks for the ground product compared to the peaks for the starting materials are generally taken as evidence of a new phase, be it a salt, cocrystal, hydrate, solvate or polymorph. When single crystal X-ray diffraction is possible, an overlay of the XRD lines

Figure 9. (a) Crystal structure of CFZ-NH+−MSA−−H2O (1:1:1) to show the persistent R21(7) ring motif. (b) O−H···N and O−H···O H bonds between the ions mediated by water.

(Figure S3, Supporting Information) confirms purity and homogeneity of the bulk phase. Thermal Analysis. A sharp melting endotherm in differential scanning calorimetry (DSC) is usually indicative of a pure solid phase. Generally dissociation/decomposition and/or phase changes upon heating are indicated by endo-/exotherm in DSC thermogram. CFZ exhibits a sharp melting endotherm at 219.5 °C in DSC. The CFZ-NH+−MSA− salt has the highest melting point at 241.7 °C among CFZ salts analyzed. CFZ6255



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Table 3. pKa Values of CFZa and Organic Acidsb Used in This Study pKa (water) CFZ MSA MLE INA NA MLN SCL a

8.51 −1.92 1.92, 6.27 1.94 4.85 2.83, 5.69 2.97

ΔpKa 10.43 6.59, 2.24 6.57 3.66 5.68, 2.82 5.54

http://www.drugbank.ca/drugs/DB00845. dia.org.

b

Table 4. Melting Points (°C) of CFZ Salts

salt

crystalline salt

mp (°C) of API

1:1 salt 1:1 salt 1:1 salt 1:1 salt 1:1 salt 1:1 salt

CFZ CFZ−amorphous CFZ-NH+−MSA− CFZ-NH+−MLE− CFZ-NH+−INA− CFZ-NH+−NA− CFZ-NH+−MLN− CFZ-NH+− SCL−

219−221 136−146

http://www.wikipe-

mp (°C) of coformer

mp (°C) of salt

135 310 237 136 159

241−246 228−233 209−211 230−233 178−182 233−235

1600 cm−1 due to CN stretching of CFZ (Figure S5, Supporting Information). The N−H stretching bands are broad at 3400−3500 cm−1 (Table 5). The decomposition of CFZ-NH+−MLN− at 190 °C was monitored by IR (Figure S5, Supporting Information). Solubility and Powder Dissolution. Solubility and dissolution experiments of new solid phases (polymorphs, salts, cocrystals) are important to understand and control transformations in order to achieve the desired product specifications and bioavailability.23 Solubility is a thermodynamic property whereas dissolution is a kinetic parameter. Thermodynamic stability and solubility of CFZ salts were studied in slurry experiments. This is an essential step for drug formulation development in the pharmaceutical industry. In general, salts have higher solubility than cocrystals which are in turn more soluble than the pure API. However salts tend to have a hydration problem. Solubility is the concentration of the solute in given solvent when the dissolved and undissolved particles are in a state of equilibrium. Solubility experiments of CFZ and its molecular salts were performed in alcoholic medium because of poor aqueous solubility of the drug (10 mg L−1). The solubility of CFZ at 24 h in 60% EtOH−water slurry medium (treated as equilibrium solubility) is 183.7 mg L −1. The concentration of CFZ was measured by UV−vis spectroscopy at 454 nm to avoid

NH+−MLE− (Tm 228.8 °C), CFZ-NH+−NA− (Tm 230.6 °C), and CFZ-NH+−SA− (Tm 233.5 °C) melt above the melting point of the API, whereas CFZ-NH+−INA− (Tm 209.3 °C) and CFZNH+−MLN− (178.2 °C) have lower melting points (Figure 10). The malonate salt (1:1) showed two endotherms, the first peak for melting of the salt and the second for free base CFZ since the salt dissociates after melting, as confirmed independently by DSC and IR in a heat−cool−heat experiment (Figure S4b, Figure S5g, Supporting Information). The amorphous form of CFZ obtained from the melt recrystallized at 136.3 °C and transformed to the stable form after melting at 197.8 °C (Figure S4a). Tonset (°C) values of CFZ salts are summarized in Table 4. FT-IR Spectroscopy. IR spectroscopy22 is a reliable technique to analyze hydrogen bonding changes and from the objective of this study to differentiate between cocrystal and salt. Generally free COOH stretching frequency appears at 1720− 1700 cm−1 and COO− absorbs strongly at 1650−1550 cm−1 (asymmetric) and has a weaker band at 1400 cm−1 (symmetric). N−H bending frequency is at 1550−1620 cm−1. It is difficult to assign carbonyl stretch and NH bend frequency reliably because they appear close to each other. In all CFZ salts proton transfer occurred from the carboxylic acid/sulfonic acid to the CFZ imine-N, and the salts exhibited a bathochromic shift at 1625−

Figure 10. DSC of CFZ and CFZ-NH+−MSA−, CFZ-NH+−MLE−, CFZ-NH+−INA−, CFZ-NH+−NA−, CFZ-NH+−MLN−, CFZ-NH+−SCL− salts. 6256



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Table 5. FT-IR Stretching Frequencies (νs, cm−1) of Clofazimine Salts CFZ

CN

N−H (br)

carboxylate (asym)

carboxylate (sym)

carboxylic acid (cofomer) υs (sym)

CFZ CFZ-NH+−MSA−(1:1) CFZ-NH+−MLE− (1:1) CFZ-NH+−INA−(1:1) CFZ-NH+−NA−(1:1) CFZ-NH+−MLN− (1:1) CFZ-NH+−SCL− (1:1)

1625.1 1615.7 1620.9 1617.0 1602.2 1623.0 1624.2

3446.7 3326.1 3314.9 3429.0 3424.8 3435.4 3432.6

1704.8 1516.3 1564.5 1733.2 (CO), 1536.2 1682.6, 1544.0

1391.4 1404.6 1389.5 1397.1 1378.1

1434.0 1411.8 1417.4 1417.1 1444.5

Table 6. Solubility and Dissolution of Clofazimine Salts solid forms CFZ CFZ-amorphous CFZ-NH+− MSA− (1:1) CFZ-NH+− MLE− (1:1) CFZ-NH+− INA− (1:1) CFZ-NH+−NA− (1:1) CFZ-NH+− MLN− (1:1) CFZ-NH+−SCL− (1:1)

aqueous solubility (g L−1) of coformer

absorption coefficient (mM−1 cm−1)

solubility after 24 h slurry in 60% EtOH−water (mg L−1)

solubility at 5 min in powder dissolution

19.51 18.13 24.23

183.75 655.20 (x 3.6) 17142.44 (x 93.6)

18.69 15.19 (x 0.8) 117.39 (x 6.5)

1000.0

25.75

1107.59 (x 6.0)

70.47 (x 3.8)

82.7

25.26

4994.24 (x 27.3)

106.09 (x 5.8)

15.0

26.89

3594.24 (x 19.6)

81.07 (x 4.5)

5.2

26.65

2838.52 (x 15.4)

65.19 (x 3.6)

1.97

21.38

381.25 (x 2.1)

40.88 (x 2.2)

2.24

final residue after 24 h slurry CFZ CFZ CFZ-NH+−MSA− (1:1) CFZ-NH+−MLE− (1:1) CFZ-NH+−INA− (1:1) CFZ-NH+−NA− (1:1) CFZ-NH+−MLN− (1:1) CFZ-NH+−SCL− (1:1)

concentration of CFZ-NH+−MSA− is 118 mg L−1 compared to 19 mg L−1 of the free base.

interference from the coformers at 250−280 nm. CFZ-NH+− MSA− salt has 94 times greater solubility than CFZ. Solubility enhancements for other salts are CFZ-NH+−INA− (27 fold), CFZ-NH+−NA− (20-fold), CFZ-NH+−MLN− (15-fold), CFZNH+−MLE− (6 fold), CFZ-NH+−SCL− (2 fold) (Table 6). The equilibrium solubility of CFZ-NH+−MSA− in distilled water at 24h is 996.07 mg L−1, which is about 100 times more soluble than CFZ base (9.99 mg L−1). The salt solubility correlated with aqueous solubility of the coformer. All the salts are stable after 24 h slurry experiments as confirmed by PXRD (Figure S6, Supporting Information). Amorphous CFZ converted to the crystalline form after 24 h slurry. Powder dissolution experiments were carried out to determine the rate of dissolution (Figure 11). The salts exhibited peak concentration within 10 min and maintained saturation levels for up to 90 min. The saturation

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CONCLUSIONS CFZ is an antibacterial, antileprosy, and anti-inflammatory drug. However, poor aqueous solubility has limited its wider clinical applications. The graded increase in solubility of CFZ salts should allow repurposing of this classical drug to newer therapeutic targets.24 Salts of CFZ with pharmaceutically acceptable acids were prepared by wet granulation. The color change from dark red of the free base to black indicated salt formation. Proton transfer from the acid to the imine moiety gave crystalline salts containing the recurring R21(7) synthon. All CFZ salts were characterized by spectroscopic, thermal and diffraction techniques. Clofazimine mesylate CFZ-NH+−MSA− is the most promising salt for solid form development due to its high solubility and dissolution rate and good stability in the aqueous medium. Moreover mesylates are pharmaceutically well accepted as salt formers.25 Our preliminary results suggest CFZ mesylate as an optimum salt for solid formulation to explore the diverse therapeutic potential of the drug.

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EXPERIMENTAL SECTION

Clofazimine was purchased from Tianjin Xingwei Chemical Co. Ltd., China. The coformers were purchased from Sigma-Aldrich (Hyderabad, India). All starting materials were found to be pure by 1H NMR and used directly for experiments. All other chemicals/solvents were purchased from local suppliers and are of analytical and chromatographic grade. Melting points were measured on a Fisher−Johns melting point apparatus. Water filtered through a double deionized purification system (AquaDM Bhanu, Hyderabad, India) was used in all experiments. Single crystals were obtained by the solvent evaporation method at room temperature. The new salts were characterized by IR, powder XRD, DSC, and single crystal XRD. The bulk phases matched the single crystal material.

Figure 11. Powder dissolution experiments of CFZ and its salts in 60% EtOH−water medium at 37 °C. 6257



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CFZ-NH+−MSA− (1:1) Salt. 100 mg (0.21 mmol) of CFZ and 20.25 mg (0.21 mmol) of MSA were ground in a mortar-pestle for 20 min after adding 5 drops of acetonitrile, and then kept for crystallization in a solvent mixture of methanol and acetonitrile (5 mL) at room temperature. Plate-shaped crystals were harvested at ambient conditions after 3−4 days. In the same crystallization batch CFZ-NH+−MSA− hydrate (1:1:1) was concomitantly obtained. The ground material of CFZ and MSA matched with anhydrate salt by XRD. mp 241−246 °C. CFZ-NH+−MLE− (1:1) Salt. 100 mg (0.21 mmol) of CFZ and 24.37 mg (0.21 mmol) of MLE were ground in a mortar-pestle for 20 min after adding 5 drops of acetonitrile, and then kept for crystallization in a solvent mixture of methanol and acetonitrile (5 mL) at room temperature. Block-shaped crystals were harvested at ambient conditions after 3−4 days mp 228−233 °C. CFZ-NH+−INA− (1:1) Salt. 100 mg (0.21 mmol) of CFZ and 25.85 mg (0.21 mmol) of INA were ground in a mortar-pestle for 20 min by liquid assisted grinding with acetonitrile as a solvent, and then kept for crystallization in 10 mL of the same solvent. Block morphology crystals appeared after solvent evaporation at ambient conditions after 3−4 days. mp 209−211 °C. CFZ-NH+−NA− (1:1) Salt. 100 mg (0.21 mmol) of CFZ and 25.85 mg (0.21 mmol) of NA were ground in a mortar-pestle for 20 min after adding 5 drops of acetonitrile as solvent and crystallized from a 1:1 solvent mixture of acetonitrile−MeOH. Block-shaped crystals appeared after solvent evaporation. mp 230−233 °C. CFZ-NH+−MLN− (1:1) Salt. 100 mg (0.21 mmol) of CFZ and 27.15 mg (0.21 mmol) of INA were mixed together in a mortar-pestle for 30 min after adding 5 drops of acetonitrile as solvent, and then kept for crystallization in acetonitrile−MeOH. mp 178−182 °C. CFZ-NH+− SCL− (1:1) Salt. 100 mg (0.21 mmol) of CFZ and 28.98 mg (0.21 mmol) of SCL were ground in a mortar-pestle for 20 min after adding 5 drops of acetonitrile and then kept for crystallization in 10 mL of 1:1 mixture of MeOH−acetonitrile to give crystals of the salt. mp 233−236 °C. Single Crystal X-ray Diffraction. Single crystals were mounted on the goniometer of Oxford Gemini (Oxford Diffraction, Yarnton, Oxford, UK) or Bruker Smart (Bruker−AXS, Karlsruhe, Germany) X-ray diffractometer equipped with an Mo−Kα radiation (λ = 0.71073 Å) source, and reflections were collected at 298(2) K. Data reduction was performed using CrysAlisPro 171.33.55 software.26 Crystal structures were solved and refined using Olex2, ver. 1.027 with anisotropic displacement parameters for non-H atoms. Hydrogen atoms were experimentally located through Fourier difference electron density maps in all crystal structures. All C−H atoms were geometrically fixed using the HFIX command in SHELX-TL,28 and O−H, N−H and were located in difference electron density maps. A check of the final .cif files in PLATON29 did not show any missed symmetry. X-Seed30 was used to prepare the figures and packing diagrams. Crystallographic parameters of crystal structures are summarized in Table 1. Hydrogen bond distances in Table 2 are neutron-normalized to fix the D−H distance to its accurate neutron value in the X-ray crystal structure (O−H 0.983 Å, N−H 0.82 Å, C−H 1.083 Å). FT−IR Spectroscopy. A Thermo-Nicolet 6700 FT-IR spectrometer (Waltham, MA, USA) was used to record IR spectra. IR spectra were recorded on samples dispersed in KBr pellets. Data were analyzed using the Omnic software (Thermo Scientific, Waltham, MA). Powder X-ray Diffraction. Microcrystalline powders of commercial and ground bulk samples were analyzed by X-ray powder diffraction on a Bruker AXS D8 powder diffractometer (Bruker-AXS, Karlsruhe, Germany). Experimental conditions: Cu−Kα radiation (λ = 1.54056 Å); 40 kV; 30 mA; scanning interval 5−50° 2θ at a scan rate of 1° min−1; time per step 0.5 s. The experimental PXRD patterns and calculated PXRD patterns from single crystal structures were compared to confirm purity of the bulk phase using Powder Cell.31 Thermal Analysis. DSC was performed on a Mettler-Toledo DSC 822e module. Samples were placed in open alumina pans for TGA and in crimped but vented aluminum sample pans for DSC. Typical sample size is 3−5 mg for DSC. The temperature range was 30−300 °C at a heating rate of 2 °C min−1 for DSC. Samples were purged with a stream of dry N2 flowing at 80 mL min−1 for DSC.

Dissolution and Solubility Measurements. Powder dissolution rate (PDR) measurements were carried out on a USP-certified Electrolab TDT-08L Dissolution Tester (Electrolab, Mumbai, MH, India). A calibration curve was obtained for all the new solid phases (salts) including CFZ by plotting absorbance vs concentration of UV− vis spectra curves on a Thermo Scientific Evolution EV300 UV−vis spectrometer (Waltham, MA, USA) for known concentration solutions in 60% EtOH−water medium. The absorbance of known concentration of CFZ and salts were considered at 454 nm (λmax). Slope of the plot from the standard curve gave the molar extinction coefficient (ε) by applying the Beer−Lambert’s law. Equilibrium solubility was determined in the same medium using the shake-flask method.32 To obtain the equilibrium solubility, an excess amount of each solid material was stirred for 24 h in 5 mL of 60% EtOH−water medium to obtain a supersaturation condition of solution system. After 24 h of stirring, the aqueous solution was filtered by Whatman’s filter paper, and absorbance was measured at 454 nm with proper dilution. The concentration of CFZ in all solid phases was calculated and reported as the equilibrium solubility of that particular solid form. 100 mg of the each solid (drug, salt) was taken in 900 mL of 60% EtOH−water medium at 37 °C with the paddle rotating at 150 rpm. At regular interval of 5−10 min, 5 mL of the dissolution medium was withdrawn and replaced by an equal volume of fresh medium to maintain a constant volume. Samples were filtered through 0.2 μm nylon filter and assayed for drug content spectrophotometrically at 454 nm on a Thermo-Nicolet EV300 UV−vis spectrometer. There was no interference to the CFZ UV−visible maxima at 454 nm by the coformer λmax because the latter absorbs at 260−275 nm in the UV region. The amount of drug dissolved at each time interval was calculated using the calibration curve by UV−vis spectroscopy.

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ASSOCIATED CONTENT

S Supporting Information *

ORTEP diagrams, PXRD plots, DSC thermograms, IR spectra, and crystallographic .cif files (CCDC Nos. 909155−909162) are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the DST for JC Bose fellowship (SR/S2/JCB-06/ 2009) and CSIR for Pharmaceutical Cocrystals (01(2410)/10/ EMR−II) research funding, and DST (IRPHA) and UGC (PURSE grant) for providing instrumentation and infrastructure facilities. GB thanks the UGC for fellowship.

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REFERENCES

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Clofazimine Mesylate - American Chemical Society

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