Clofazimine Mesylate: A High Solubility Stable Salt

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 R 2 1 (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.

Full text

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
*
SSupporting 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.
■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 ΔpKaof the API
and the coformer. The ΔpKarule, 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
cutofffor 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
cocrystal−salt continuum.
5
These ΔpKaranges were recently
revised to < −1 for cocrystals, > 4 for salts, and −1 to 4 for
cocrystal, salt, or cocrystal−salt continuum state using crystallo-
graphic data from the Cambridge Structural Database (CSD)
and calculated pKa
’s in Marvin
6
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
Received: October 7, 2012
Revised: November 6, 2012
Published: November 8, 2012
Article
pubs.acs.org/crystal
© 2012 American Chemical Society 6250 dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−6259

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
derivatives
11
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 CFZ
14c
are reported in the literature.
14
Our main objective was
to explore cocrystals and salts of CFZ using a crystal engineering
approach
15
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.
■RESULTS AND DISCUSSION
Crystal Structures. Crystal structures of two reported
polymorphs of CFZ
14a
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···πinteraction (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···πand C−H···Cl
interactions. Crystallographic parameters and normalized hydro-
gen 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
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 motif
19
(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 liquid-
assisted grinding of equimolar CFZ and MLE-H in acetonitrile
solvent. Recrystallization was performed in the same solvent to
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.
Figure 2. (a) CFZ triclinic form having C−H···N dimers and such
dimers are further connected by C−H···πinteractions. (b) CFZ
monoclinic form has C−H···N, C−H···πand C−H···Cl interactions.
There is no dimer motif in the monoclinic structure.
Crystal Growth & Design Article
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give diffraction-quality dark red crystals (P21/nspace 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
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)
Table 1. Crystallographic Parameters of CFZ Salts
CFZ-NH+−MSA−CFZ-NH+−MLE−CFZ-NH+−INA−CFZ-NH+−NA−
empirical formula C27H23Cl2N4·CH3O3SC
27H23Cl2N4·C4H3O4C27H23Cl2N4·C6H4NO2C27H23Cl2N4·C6H4NO2
formula weight 569.50 589.46 596.50 596.50
crystal system monoclinic monoclinic triclinic triclinic
space group P21/cP21/nP1̅P1̅
T(K) 298(2) 298(2) 298(2) 298(2)
a(Å) 10.213(2) 11.2549(19) 9.8153(9) 11.6032(13)
b(Å) 18.008(6) 20.816(3) 12.1738(10) 15.361(2)
c(Å) 15.561(3) 12.519(2) 15.2122(10) 18.733(3)
α(°) 90.0 90 72.455(7) 91.280(11)
β(°) 102.79(2) 103.413(17) 77.844(7) 111.337(14)
γ(°) 90.0 90 66.479(8) 109.295(11)
V(Å3) 2790.9(12) 2853.0(8) 1580.7(2) 2896.5(8)
Dcalcd (g cm−3) 1.355 1.372 1.253 1.368
μ(mm−1) 9.9944 1.272 0.242 9.9970
θrange 2.91−28.83 2.69−26.31 2.80−24.71 2.68−26.31
Z442 4
range h−11 to +11 −14 to +13 −11 to +11 −12 to +12
range k−20 to +20 −25 to +26 −14 to +14 −16 to +17
range l−17 to +17 −15 to +15 −16 to +17 −20 to +20
reflections collected 28661 10779 9737 14036
total reflections 5549 4855 5381 8145
observed reflections 4299 1924 2582 4572
R1[I>2σ(I)] 0.0895 0.0870 0.0570 0.0586
wR2(all) 0.2307 0.1685 0.1502 0.1430
goodness of fit 0.737 0.909 0.908 0.998
diffractometer Oxford Gemini Oxford Gemini Oxford Gemini Oxford Gemini
CFZ-NH+−MLN−CFZ-NH+−SCL−CFZ-NH+−MSA−−H2O CFZ−acetone solvate
empirical formula C27H23Cl2N4·C3H2O4C27H23Cl2N4·C7H5O3C27H23Cl2N4·CH3O3S·H2OC
27H23Cl2N4·C2H6O
formula weight 576.44 611.50 587.51 531.46
crystal system triclinic triclinic triclinic triclinic
space group P1̅P1̅P1̅P1̅
T(K) 298(2) 298(2) 298(2) 100
a(Å) 9.8401(8) 10.8702(6) 9.5945(12) 10.0842(13)
b(Å) 12.4069(10) 11.2066 (6) 11.0456(12) 12.0695(16)
c(Å) 13.0576(10) 13.8272(8) 14.4628(11) 12.6613(16)
α(°) 74.129(1) 82.354(5) 110.173(9) 75.278(2)
β(°) 79.9900(1) 88.960(5) 95.859(9) 66.588(2)
γ(°) 67.520(1) 63.492(6) 94.929(9) 69.059(2)
V(Å3) 1932.2(7) 1492.31(17) 1419.1(3) 13.09.3(3)
Dcalcd (g cm−3) 1.400 1.361 1.375 1.348
μ(mm−1) 0.282 0.260 9.9943 0.279
θrange 1.68−26.02 2.97−24.66 2.79−26.31 1.77−26.10
Z22 2 2
range h−12 to +12 −12 to +12 −11 to +11 −12 to +12
range k−15 to +15 −12 to +13 −13to +13 −14 to +14
range l−16 to +16 −12 to +16 −18 to +18 −15 to +15
reflections collected 14254 9169 9694 13647
total reflections 5344 5085 5785 5152
observed reflections 3774 3423 3423 4418
R1[I>2σ(I)] 0.0567 0.0472 0.049 0.0486
wR2(all) 0.1703 0.1293 0.1069 0.1293
goodness of fit 1.043 1.027 0.892 1.030
diffractometer BRUKER Smart Oxford Gemini Oxford Gemini BRUKER Smart
Crystal Growth & Design Article
dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−62596252

Table 2. Normalized Hydrogen Bonds in Crystal Structures of CFZ and Its Salts
crystal forms interaction H···A/Å D···A/Å ∠D−H···A/°symmetry code
CFZ-NH+−MSA−N1−H1A···N2 2.39 2.767(4) 104 intramolecular
N1−H1A···O2 1.86 2.792(4) 175 1/2 −x, 1/2 + y, 1/2 −z
N2−H2A···N1 2.45 2.767(4) 102 intramolecular
N2−H2A···O2 1.93 2.777(4) 167 x,y,1+z
C1−H1···Cl2 2.73 3.582(3) 149 1/2 −x, 1/2 + y, 1/2 −z
C14−H14···O3 2.60 3.525(4) 166 1/2 −x, 1/2 + y, 1/2 −z
C20 −H20···O2 2.36 3.305(4) 171 1/2 + x, 1/2 −y, 1/2 + z
C27−H27B···O3 2.59 3.460(4) 148 a
C28−H28A···N3 2.48 3.457(4) 174 1 −x,1−y,1−z
CFZ-NH+−MLE−N1−H1A···O1 2.16 2.870(6) 141 1 −x,−y,1−z
N2−H2A···O1 1.82 2.853(6) 176 1 −x,−y,1−z
O2−H2B···O3 1.20 2.414(6) 164 intramolecular
C14−H14···O2 2.53 3.459(6) 175 1 −x,−y,1−z
C21−H21···O3 2.51 3.272(7) 139 intramolecular
CFZ-NH+−INA−N1−H1A···N2 2.37 2.761(4) 102 intramolecular
N1−H1A···O2 1.85 2.841(4) 172 1 −x,1−y,1−z
N2−H2A···N1 2.38 2.761(4) 105 intramolecular
N2−H2A···O2 1.88 2.748(4) 158 1 −x,1−y,1−z
C1−H1···Cl2 2.74 3.471(3) 133 x,y,−1+z
C3−H3···O1 2.55 3.239(6) 129 x,−1+y,z
C20−H20···O2 2.34 3.302(4) 169 1 + x,−1+y,z
CFZ-NH+−NA−N1−H1A···O3 2.06 2.914(5) 169 a
N1−H1A···N2 2.39 2.746(5) 106 intramolecular
N2−H2A···O3 2.18 2.974(5) 154 a
N2−H2A···O4 2.37 3.114(5) 146 a
N2−H2A···N1 2.36 2.746(5) 107 intramolecular
N6−H6A···N7 2.38 2.743(5) 106 intramolecular
N6−H6A···O1 1.99 2.837(5) 170 1 −x,1−y,1−z
N7−H7A···N6 2.36 2.743(5) 107 intramolecular
N7−H7A···O1 2.04 2.876(5) 163 1 −x,1−y,1−z
N7−H7A···O2 2.58 3.291(6) 141 1 −x,1−y,1−z
C1−H1···Cl2 2.76 3.428(4) 129 x,1+y,z
C4−H4···N8 2.52 3.445(5) 174 2 −x,1−y,−z
C17−H17···N10 2.56 3.461(9) 162 1 −x,1−y,−z
C28−H28···O3 2.49 2.812(6) 100 intramolecular
C34−H34···Cl4 2.82 3.464(4) 127 x,1+y,z
C37−H37···N3 2.60 3.500(5) 162 2 −x,1−y,−z
C50−H50···N5 2.54 3.412(6) 156 1 + x,y,z
C53−H53···O1 2.60 3.436(5) 151 1 + x,y,z
C60−H60A···N8 2.61 3.536(6) 161 2 −x,1−y,1−z
C61−H61···O1 2.43 2.773(7) 102 intramolecular
CFZ-NH+−MLN−N1−H1A···N2 2.37 2.746(3) 107 intramolecular
N1−H1A···O2 2.19 3.008(4) 166 1 −x,1−y,−z
N2−H2A···O1 2.25 3.033(3) 165 1 −x,1−y,−z
N2−H2A···O2 2.42 3.079(4) 140 1 −x,1−y,−z
O1−H3B···O3 1.10 2.467(3) 156 intramolecular
C4−H4···Cl1 2.75 3.506(3) 139 x,1+y,z
C21−H21···O4 2.47 3.139(4) 129 −x,1−y,1−z
C23−H23 ···O2···O2 2.57 3.472(4) 165 −x,1−y,−z
CFZ-NH+−SCL−N1−H1A···N2 2.49 2.795(3) 100 intramolecular
N1−H1A···O1 1.88 2.784(3) 176 1 −x,1−y,1−z
N2−H2A···N1 2.44 2.795(3) 108 intramolecular
N2−H2A···O1 2.16 2.938(3) 166 1 −x,1−y,1−z
O3−H3A···O2 1.64 2.532(3) 159 intramolecular
C4−H4···O3 2.52 3.408(4) 160 a
C2−H23···O1 2.57 3.478(4) 164 −x,2−y,1−z
CFZ-NH+−MSA−−H2ON1−H1A···O2 2.15 2.915(3) 174 −1+x,−1+y,z
N1−H1A···N2 2.51 2.801(3) 104 intramolecular
N2−H2A···O2 2.05 2.877(3) 168 −1+x,−1+y,z
N2−H2A···N1 2.45 2.801(3) 106 intramolecular
O4−H4A···O3 2.13 2.883(4) 145 a
Crystal Growth & Design Article
dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−62596253

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 P1̅space 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
molecules, and the 1D chains are arranged in ABAB fashion
(Figure 6b).
CFZ-NH+−MLN−(1:1) Salt. The crystal structure (P1̅) has
R22(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
Table 2. continued
crystal forms interaction H···A/Å D···A/Å ∠D−H···A/°symmetry code
O4−H4B···N4 2.09 2.899(3) 173 1 −x,1−y,1−z
C15−H15···O2 2.54 3.454(3) 164 1 −x,1−y,1−z
C17−H17···O1 2.55 3.437(3) 167 1 −x,1−y,1−z
a
Molecules/ions in the same asymmetric unit.
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 CFZ-
NH+−MLE−(1:1) salt. (b) The organic cation and anion extend
through C−H···O interactions in a 1D chain.
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 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 symmetry-
independent ions shown as ball-stick and thick-bonds models.
Crystal Growth & Design Article
dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−62596254

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 (P1̅space group). The intermo-
lecular 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 interactions
20
(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 pKaof the conjugate acid of
the base must be greater than the pKaof the acid to ensure proton
transfer. CFZ is a weak base (pKa8.51) and ΔpKa> 3 with the
acids used in this study (Table 3), suggesting proton transfer
according to the ΔpKarule.
5,7
Powder X-ray Diffraction. Powder X-ray diffraction
21
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 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. CFZ-
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.
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···OH
bonds between the ions mediated by water.
Crystal Growth & Design Article
dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−62596255

NH+−MLE−(Tm228.8 °C), CFZ-NH+−NA−(Tm230.6 °C),
and CFZ-NH+−SA−(Tm233.5 °C) melt above the melting point
of the API, whereas CFZ-NH+−INA−(Tm209.3 °C) and CFZ-
NH+−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 spectroscopy
22
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−1and 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.Itisdifficult 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−
1600 cm−1due 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. Thermody-
namic 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
Table 3. pKaValues of CFZ
a
and Organic Acids
b
Used in This
Study
pKa(water) ΔpKasalt
CFZ 8.51
MSA −1.92 10.43 1:1 salt
MLE 1.92, 6.27 6.59, 2.24 1:1 salt
INA 1.94 6.57 1:1 salt
NA 4.85 3.66 1:1 salt
MLN 2.83, 5.69 5.68, 2.82 1:1 salt
SCL 2.97 5.54 1:1 salt
a
http://www.drugbank.ca/drugs/DB00845.
b
http://www.wikipe-
dia.org.
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.
Table 4. Melting Points (°C) of CFZ Salts
crystalline salt mp (°C) of API mp (°C) of
coformer mp (°C) of salt
CFZ 219−221
CFZ−amorphous 136−146
CFZ-NH+−MSA−241−246
CFZ-NH+−MLE−135 228−233
CFZ-NH+−INA−310 209−211
CFZ-NH+−NA−237 230−233
CFZ-NH+−MLN−136 178−182
CFZ-NH+−SCL−159 233−235
Crystal Growth & Design Article
dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−62596256

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), CFZ-
NH+−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
concentration of CFZ-NH+−MSA−is 118 mg L−1compared to
19 mg L−1of the free base.
■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.
■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.
Table 5. FT-IR Stretching Frequencies (νs,cm
−
1
) of Clofazimine Salts
CFZ CNN−H (br) carboxylate (asym) carboxylate (sym) carboxylic acid (cofomer) υs(sym)
CFZ 1625.1 3446.7
CFZ-NH+−MSA−(1:1) 1615.7 3326.1
CFZ-NH+−MLE−(1:1) 1620.9 3314.9 1704.8 1391.4 1434.0
CFZ-NH+−INA−(1:1) 1617.0 3429.0 1516.3 1404.6 1411.8
CFZ-NH+−NA−(1:1) 1602.2 3424.8 1564.5 1389.5 1417.4
CFZ-NH+−MLN−(1:1) 1623.0 3435.4 1733.2 (CO), 1536.2 1397.1 1417.1
CFZ-NH+−SCL−(1:1) 1624.2 3432.6 1682.6, 1544.0 1378.1 1444.5
Table 6. Solubility and Dissolution of Clofazimine Salts
solid forms absorption coefficient
(mM−1cm−1)solubility after 24 h slurry in 60%
EtOH−water (mg L−1)solubility at 5 min in
powder dissolution aqueous solubility (g L−1)
of coformer
final residue after
24 h slurry
CFZ 19.51 183.75 18.69 CFZ
CFZ-amorphous 18.13 655.20 (x 3.6) 15.19 (x 0.8) CFZ
CFZ-NH+−
MSA−(1:1) 24.23 17142.44 (x 93.6) 117.39 (x 6.5) 1000.0 CFZ-NH+−MSA−
(1:1)
CFZ-NH+−
MLE−(1:1) 25.75 1107.59 (x 6.0) 70.47 (x 3.8) 82.7 CFZ-NH+−MLE−
(1:1)
CFZ-NH+−
INA−(1:1) 25.26 4994.24 (x 27.3) 106.09 (x 5.8) 15.0 CFZ-NH+−INA−
(1:1)
CFZ-NH+−NA−
(1:1) 26.89 3594.24 (x 19.6) 81.07 (x 4.5) 5.2 CFZ-NH+−NA−
(1:1)
CFZ-NH+−
MLN−(1:1) 26.65 2838.52 (x 15.4) 65.19 (x 3.6) 1.97 CFZ-NH+−MLN−
(1:1)
CFZ-NH+−SCL−
(1:1) 21.38 381.25 (x 2.1) 40.88 (x 2.2) 2.24 CFZ-NH+−SCL−
(1:1)
Figure 11. Powder dissolution experiments of CFZ and its salts in 60%
EtOH−water medium at 37 °C.
Crystal Growth & Design Article
dx.doi.org/10.1021/cg301463z |Cryst. Growth Des. 2012, 12, 6250−62596257

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−Kαradiation (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. 1.0
27
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
PLATON
29
did not show any missed symmetry. X-Seed
30
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 commer-
cial 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−Kαradiation (λ= 1.54056 Å);
40 kV; 30 mA; scanning interval 5−50°2θat 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−1for DSC. Samples were purged with a stream of dry
N2flowing at 80 mL min−1for 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’sfilter 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.
■ASSOCIATED CONTENT
*
SSupporting 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.
■AUTHOR INFORMATION
Corresponding Author
*E-mail: ashwini.nangia@gmail.com.
Notes
The authors declare no competing financial interest.
■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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