Clofazimine mesylate: A high solubility stable salt

Article abstract of DOI:10.1021/cg301463z

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 Biopharmac

Full text of DOI:10.1021/cg301463z

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 R2 (7) ring motif. The driving force for facile salt formation is the ionic N+−H···O− and N−H···O−
1
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.
cocrystal−salt continuum.5 These ΔpKa ranges were recently
INTRODUCTION
■
revised to < −1 for cocrystals, > 4 for salts, and −1 to 4 for
A majority of drugs are preferably administrated in solid oral
dosage form, such as tablet or capsule, because of high
cocrystal, salt, or cocrystal−salt continuum state using crystallo-
graphic data from the Cambridge Structural Database (CSD)
crystallinity, purity, patient compliance, convenience, and longer
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.
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
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
© 2012 American Chemical Society
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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
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.
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.
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
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 hydro-
gen bonds are summarized in Tables 1 and 2.
imine N of CFZ resulted in R2 (7) ring motif19 (Figure 3a). The
1
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
CFZ-NH+−MSA− (1:1) Salt. When an equimolar ratio of
CFZ and methanesulfonic acid (MSA-H) was crystallized from
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Crystal Growth & Design
Article
Table 1. Crystallographic Parameters of CFZ Salts
CFZ-NH+−MSA−
CFZ-NH+−MLE−
CFZ-NH+−INA−
CFZ-NH+−NA−
empirical formula
formula weight
crystal system
space group
T (K)
C27H23Cl2N4·CH3O3S
569.50
C27H23Cl2N4·C4H3O4
589.46
C27H23Cl2N4·C6 H4NO2
596.50
C27H23Cl2N4·C6H4NO2
596.50
monoclinic
P21/c
monoclinic
P21/n
triclinic
triclinic
P1
̅
298(2)
P1
̅
298(2)
298(2)
298(2)
a (Å)
10.213(2)
18.008(6)
15.561(3)
90.0
11.2549(19)
20.816(3)
12.519(2)
90
9.8153(9)
12.1738(10)
15.2122(10)
72.455(7)
77.844(7)
66.479(8)
1580.7(2)
1.253
11.6032(13)
15.361(2)
18.733(3)
91.280(11)
111.337(14)
109.295(11)
2896.5(8)
1.368
b (Å)
c (Å)
α (°)
β (°)
γ (°)
V (Å3)
102.79(2)
90.0
103.413(17)
90
2790.9(12)
1.355
2853.0(8)
1.372
D
calcd (g cm−3
)
μ (mm−1
θ range
Z
)
9.9944
1.272
0.242
9.9970
2.91−28.83
4
2.69−26.31
4
2.80−24.71
2
2.68−26.31
4
range h
range k
range l
−11 to +11
−20 to +20
−17 to +17
28661
−14 to +13
− 25 to +26
−15 to +15
10779
−11 to +11
−14 to +14
−16 to +17
9737
−12 to +12
−16 to +17
−20 to +20
14036
reflections collected
total reflections
observed reflections
R1 [I > 2σ(I)]
wR2 (all)
5549
4855
5381
8145
4299
1924
2582
4572
0.0895
0.0870
0.0570
0.0586
0.2307
0.1685
0.1502
0.1430
goodness of fit
diffractometer
0.737
0.909
0.908
0.998
Oxford Gemini
CFZ-NH+−MLN−
Oxford Gemini
CFZ-NH+−SCL−
Oxford Gemini
CFZ-NH+−MSA−−H2O
Oxford Gemini
CFZ−acetone solvate
empirical formula
formula weight
crystal system
space group
T (K)
C27H23Cl2N4·C3H2O4
576.44
C27H23Cl2N4·C7H5 O3
611.50
C27H23Cl2N4·CH3O3S·H2O
587.51
C27H23Cl2N4·C2H6O
531.46
triclinic
triclinic
triclinic
triclinic
P1
̅
298(2)
P1
̅
298(2)
P1
̅
298(2)
P1
̅
100
a (Å)
9.8401(8)
12.4069(10)
13.0576(10)
74.129(1)
79.9900(1)
67.520(1)
1932.2(7)
1.400
10.8702(6)
11.2066 (6)
13.8272(8)
82.354(5)
88.960(5)
63.492(6)
1492.31(17)
1.361
9.5945(12)
11.0456(12)
14.4628(11)
110.173(9)
95.859(9)
94.929(9)
1419.1(3)
1.375
10.0842(13)
12.0695(16)
12.6613(16)
75.278(2)
66.588(2)
69.059(2)
13.09.3(3)
1.348
b (Å)
c (Å)
α (°)
β (°)
γ (°)
V (Å3)
D
calcd (g cm−3
)
μ (mm−1
θ range
Z
)
0.282
0.260
9.9943
0.279
1.68−26.02
2
2.97−24.66
2
2.79−26.31
2
1.77−26.10
2
range h
range k
range l
−12 to +12
−15 to +15
−16 to +16
14254
−12 to +12
−12 to +13
−12 to +16
9169
−11 to +11
−13to +13
−18 to +18
9694
−12 to +12
−14 to +14
−15 to +15
13647
reflections collected
total reflections
observed reflections
R1 [I > 2σ(I)]
wR2 (all)
5344
5085
5785
5152
3774
3423
3423
4418
0.0567
0.0472
0.049
0.0486
0.1703
0.1293
0.1069
0.1293
goodness of fit
diffractometer
1.043
1.027
0.892
1.030
BRUKER Smart
Oxford Gemini
Oxford Gemini
BRUKER Smart
give diffraction-quality dark red crystals (P21/n space group).
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
Single proton transfer from the dicarboxylic acid (MLE-H) to the
1
isopropyl imine N of CFZ gave the same R2 (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
obtained from acetonitrile, and its X-ray structure was solved in
1
triclinic space group P1. The familiar R (7) ring motif (Figure 5)
̅
2
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Crystal Growth & Design
Article
Table 2. Normalized Hydrogen Bonds in Crystal Structures of CFZ and Its Salts
crystal forms
CFZ-NH+−MSA−
interaction
H···A /Å
D···A /Å
∠D−H···A /°
symmetry code
intramolecular
N1−H1A···N2
N1−H1A···O2
N2−H2A···N1
N2−H2A···O2
C1−H1···Cl2
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
1/2 − x, 1/2 + y, 1/2 − z
intramolecular
x, y, 1 + z
1/2 − x, 1/2 + y, 1/2 − z
1/2 − x, 1/2 + y, 1/2 − z
1/2 + x, 1/2 − y, 1/2 + z
a
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
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
CFZ-NH+− MLE−
CFZ-NH+−INA−
C3−H3···O1
x, −1 + y, z
1 + x, −1 + y, z
a
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
CFZ-NH+−NA−
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
C4−H4···N8
2 − x, 1 − y, −z
1 − x, 1 − y, −z
intramolecular
x, 1 + y, z
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
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
CFZ-NH+−MLN−
−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
CFZ-NH+−SCL−
C2−H23···O1
N1−H1A···O2
N1−H1A···N2
N2−H2A···O2
N2−H2A···N1
O4−H4A···O3
−x, 2 − y, 1 − z
−1 + x, −1 + y, z
intramolecular
−1 + x, −1 + y, z
intramolecular
a
CFZ-NH+−MSA−−H2O
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Crystal Growth & Design
Table 2. continued
crystal forms
Article
interaction
H···A /Å
D···A /Å
∠D−H···A /°
symmetry code
O4−H4B···N4
C15−H15···O2
C17−H17···O1
2.09
2.54
2.55
2.899(3)
3.454(3)
3.437(3)
173
164
167
1 − x, 1 − y, 1 − z
1 − x, 1 − y, 1 − z
1 − x, 1 − y, 1 − z
a
Molecules/ions in the same asymmetric unit.
Figure 5. (a) The basic bimolecular unit of CFZ-NH+−INA− (1:1) salt
Figure 3. (a) Proton transfer from methanesulfonic acid to CFZ
1
1
secondary imine N atom to give the R2 (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.
with the R2 (7) ring motif. (b) The bimolecular units form a chain
through 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.
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.
1
Figure 6. (a) The R2 (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.
CFZ-NH+−NA− (1:1) Salt. The salt of CFZ and nicotinic acid
crystallized from acetonitrile−MeOH (1:1) in P1 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
molecules, and the 1D chains are arranged in ABAB fashion
(Figure 6b).
CFZ-NH+−MLN− (1:1) Salt. The crystal structure (P1) has
̅
R2 (9) ring motif (Figure 7a) of N+−H···O− and N−H···O−
2
1
R2 (7) ring motif (Figure 6a) is formed by N+−H···O− and N−
hydrogen bonds (2.19 Å, 166°; 2.42 Å, 140°) because now both
H···O− hydrogen bonds with crystallographic independent
oxygen atoms of the carboxylate group accept H bonds. The
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Crystal Growth & Design
Article
2
Figure 7. (a) The basic unit of bimolecular R2 (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.
1
Figure 8. (a) The R2 (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 (P1 space group). The intermo-
̅
lecular R2 (7) ring motif of N+−H···O−, N−H···O− hydrogen
1
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−
1
CH3CN (1:1). The R2 (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
Figure 9. (a) Crystal structure of CFZ-NH+−MSA−−H2O (1:1:1) to
1
show the persistent R2 (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.
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
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-
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Crystal Growth & Design
Article
a
b
Table 3. pKa Values of CFZ and Organic Acids Used in This
Study
Table 4. Melting Points (°C) of CFZ Salts
mp (°C) of
coformer
crystalline salt
CFZ
mp (°C) of API
mp (°C) of salt
pKa (water)
ΔpKa
10.43
salt
219−221
136−146
CFZ
MSA
MLE
INA
NA
8.51
CFZ−amorphous
CFZ-NH+−MSA−
CFZ-NH+−MLE−
CFZ-NH+−INA−
CFZ-NH+−NA−
CFZ-NH+−MLN−
CFZ-NH+− SCL−
−1.92
1.92, 6.27
1.94
1:1 salt
1:1 salt
1:1 salt
1:1 salt
1:1 salt
1:1 salt
241−246
228−233
209−211
230−233
178−182
233−235
6.59, 2.24
6.57
135
310
237
136
159
4.85
3.66
MLN
SCL
2.83, 5.69
2.97
5.68, 2.82
5.54
a
b
http://www.drugbank.ca/drugs/DB00845. http://www.wikipe-
dia.org.
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).
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 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 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−
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
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.
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Crystal Growth & Design
Table 5. FT-IR Stretching Frequencies (νs, cm− ) of Clofazimine Salts
Article
1
CFZ
CN
N−H (br)
carboxylate (asym)
carboxylate (sym)
carboxylic acid (cofomer) υs (sym)
CFZ
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
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)
1704.8
1391.4
1404.6
1389.5
1397.1
1378.1
1434.0
1411.8
1417.4
1417.1
1444.5
1516.3
1564.5
1733.2 (CO), 1536.2
1682.6, 1544.0
Table 6. Solubility and Dissolution of Clofazimine Salts
absorption coefficient
solubility after 24 h slurry in 60%
solubility at 5 min in
aqueous solubility (g L−1
)
final residue after
solid forms
CFZ
(mM−1 cm−1
)
EtOH−water (mg L−1
)
powder dissolution
of coformer
24 h slurry
19.51
18.13
24.23
183.75
18.69
CFZ
CFZ
CFZ-amorphous
655.20 (x 3.6)
17142.44 (x 93.6)
15.19 (x 0.8)
117.39 (x 6.5)
CFZ-NH+−
1000.0
82.7
15.0
5.2
CFZ-NH+−MSA−
MSA− (1:1)
(1:1)
CFZ-NH+−
25.75
25.26
26.89
26.65
21.38
1107.59 (x 6.0)
4994.24 (x 27.3)
3594.24 (x 19.6)
2838.52 (x 15.4)
381.25 (x 2.1)
70.47 (x 3.8)
106.09 (x 5.8)
81.07 (x 4.5)
65.19 (x 3.6)
40.88 (x 2.2)
CFZ-NH+−MLE−
MLE− (1:1)
(1:1)
CFZ-NH+−
CFZ-NH+−INA−
INA− (1:1)
(1:1)
CFZ-NH+−NA−
CFZ-NH+−NA−
(1:1)
(1:1)
CFZ-NH+−
1.97
2.24
CFZ-NH+−MLN−
MLN− (1:1)
(1:1)
CFZ-NH+−SCL−
CFZ-NH+−SCL−
(1:1)
(1:1)
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−1 compared to
19 mg L−1 of 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
1
crystalline salts containing the recurring R2 (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.
Figure 11. Powder dissolution experiments of CFZ and its salts in 60%
EtOH−water medium at 37 °C.
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Crystal Growth & Design
Article
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.
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.
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.
ASSOCIATED CONTENT
* Supporting Information
■
S
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.
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 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−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
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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