Soluble Salts and Cocrystals of Clotrimazole

Soluble Salts and Cocrystals of ClotrimazoleSudhir Mittapalli,† M. K. Chaitanya Mannava,‡ U. B. Rao Khandavilli,‡ Suryanarayana Allu,†

and Ashwini Nangia*,†,‡

†School of Chemistry, ‡Technology Business Incubator, University of Hyderabad, Prof. C. R. Rao Road, Central University PO,Hyderabad 500 046, India

*S Supporting Information

ABSTRACT: Novel crystalline adducts of clotrimazole with pharmaceuticallyacceptable coformers were prepared. Five salts and two cocrystals of theantimycotic drug clotrimazole (CLT) were crystallized with carboxylic acidcoformers adipic acid (ADA), 2,5-dihydroxybenzoic acid (25DHBA),2,4,6-trihydroxybenzoic acid (246THBA), p-coumaric acid (PCA), caffeic acid(CFA), maleic acid (MA), and suberic acid (SBA). Molecular overlay diagramof clotrimazole in the four salts (CLT−25DHBA, 1:1; CLT−246THBA, 1:1;CLT−PCA, 1:1; CLT−CFA−ANI, 1:1:1) and CLT−ADA (1:0.5) cocrystalshowed conformational flexibility of the phenyl rings in the triaryl methanemolecule. The X-ray crystal structures are sustained by N+−H···O−/ N−H···Ohydrogen bonds. The solid-state forms were well characterized and analyzedby PXRD, FT-IR, and DSC and confirmed by single crystal X-ray diffraction(except for CLT−MA and CLT−SBA adducts). 15N ss-NMR indicated inter-molecular proton transfer in CLT−MA, and the chemical shifts are consistent with salt formation. The acidic coformers forCLT base were selected based on the ΔpKa rule of 3. Solubility measurements showed improved solubility by a factor of 2.9(CLT−25DHBA), 14.0 (CLT−246THBA), 1.3 (CLT−PCA), 2.8 (CLT−CFA), and 22.4 (CLT−MA) for salts and 5.0 incocrystals (CLT−ADA and CLT−SBA) compared to CLT in 65% EtOH−water.

■ INTRODUCTIONA majority of active pharmaceuticals ingredients (APIs) in themarket show poor physicochemical properties, mainly solubilityand stability, and this poses serious problems for clinicaldevelopment and can lead to late stage drug failure.1 Improvingthe solubility and bioavailability of poorly water-soluble drugs is adifficult challenge for pharmaceutical scientists. In addition tosalts and cocrystals, other techniques such as micronization,micellar solution, oil encapsulation, and amorphous phase bymeans of solid dispersions by using polymers, cyclodextrins,and additives have been widely used.2−5 However, salt formationand cocrystallization of the API are the first-choice methods toimprove the solubility and bioavailability of poorly soluble drugsusing crystal engineering principles6−9 without changing theirchemical structure. Salt formation is a preferred method toimprove physicochemical properties because it makes the drugmolecule ionized, and furthermore, other favorable factors, suchas high melting point, crystallinity, filtration, stability, etc., arealso optimized in salts.10,11 Salt formation can improve solubilitymore than 2000-fold compared to a factor of 50 for cocrystals12,13

and a mere 5 with polymorphs (these are upper limit numbers).Cocrystal formation is a recent strategy for improving thesolubility and stability of nonionizable drugs.14−17 There aremultiple techniques to prepare cocrystals such as solutioncrystallization, solid-state grinding, solvent-assisted grinding,mechanochemical synthesis, etc.18−20 Pharmaceutical cocrys-tals21,22 are a subset of the broader cocrystal class wherein amolecular or ionic API and aGRAS coformer (generally regarded

as safe by the US FDA)23 are combined in stoichiometric ratio ina crystalline lattice. The solubility of cocrystal former shouldtypically be 10 times higher than that of the pure API. Remenar et al.reported the first example of a pharmaceutical cocrystal to improvethe solubility of antifungal drug itraconazole.24

Clotrimazole (1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole) is an imidazole derivative used as an antifungal agent.The drug can also work against different strains of Plasmodiumfalciparum.25 Malaria has become a global peril due to the spreadof resistance to quinolone based antimalarial drugs such as quinine,chloroquine,26 and mefloquine. The World Health Organization(WHO) has recommended artemisinin combination therapy(ACT) as a first line treatment for uncomplicated malaria insteadof artemisinin based monotherapies.27 Pharmaceutical companiesare generally averse to registering drug products for tropicalparasitic diseases including malaria due to increased cost ofdevelopment and inadequate commercial returns.28 Efforts todevelop new drugs through “repurposing” and “piggy back”29 arenew ways to reduce cost of drugs targeting multidrug resistantplasmodium species.According to the Biopharmaceutical Classification System

(BCS), clotrimazole is a class II drug30 of poor aqueous solubility(0.49 μg/mL) and high permeability (log P 6.30). In comparisonwith other antimalarial drugs like quinine and chloroquine,

Received: February 23, 2015Revised: April 3, 2015Published: April 13, 2015

Article

pubs.acs.org/crystal

© 2015 American Chemical Society 2493 DOI: 10.1021/acs.cgd.5b00268Cryst. Growth Des. 2015, 15, 2493−2504

pubs.acs.org/crystal

http://dx.doi.org/10.1021/acs.cgd.5b00268

clotrimazole shows better activity against chloroquine resistantmalarial parasites because of its complex forming ability withfree heme.31 Because of its poor and erratic bioavailability,Cmax is reached after 6 h when administered orally. The drugconcentration for 50% inhibition of parasite was 0.2−1.1 μM,and CLT at >2 μM can cause complete inhibition of parasitereplication.32 CLT is slightly basic in nature because of thepresence of nitrogen atoms (pKa of N2 6.62; Chemaxoncalculator). Clotrimazole is stable at pH 1.2, 4.5, 6.8, and 7.5buffer solution,33 but it degrades in strongly acidic and basicmedia and at high temperature. The byproducts in stronglyacidic medium are (o-chlorophenyl)diphenyl methanol andimidazole34 (Scheme 1).

The crystal structure of clotrimazole was reported by Songet al. in 1998.35 Prabagar et al. reported β-cyclodextrin inclusioncomplexes of clotrimazole to increase oral bioavailability.Borhade et al. prepared clotrimazole nanoemulsion to improvedrug solubility and dissolution rate.36,37 There were no reportson salts and cocrystals of clotrimazole to modulate its solubilityand stability. Salts and cocrystals of clotrimazole were crystallizedusing solvent-assistant grinding, melting, and rotary evaporation,neat grinding, etc. We report salts of CLT with 25DHBA,246THBA, PCA, CFA, MA, and cocrystals with ADA, SBA(Figure 1). The products were characterized by PXRD, IR, DSC,and single crystal XRD (except for MA and SBA adducts). Thelatter two complexes were characterized by 15N ss-NMR to assignthe proton position in the structure.

■ RESULTS AND DISCUSSION

Clotrimazole (pKa 6.62) is expected to form salts with GRASorganic acids (pKa 2−4), which are not so strong as to degradeclotrimazole. The ΔpKa rule of 3

39−42 states that salt formationrequires at least three units pKa

38 difference, whereas ΔpKa of<1 means a neutral cocrystal. ΔpKa 1−3 is a gray zone ofintermediate proton states. We note that the ΔpKa rule is wellbehaved in clotrimazole (Table 1). Notably, salt formation isobserved in the case of 25DHBA, 246THBA, PCA, CFA, MA,and cocrystal with ADA and SBA coformers.

Crystal Structure Description. CLT−ADA Cocrystal(1:0.5). The ground material of CLT and ADA in stoichiometricratio (1:0.5) was crystallized from EtOH. The same cocrystalstructure in P1 ̅ space group was obtained by crystallization of thecocrystal powder from EtOH−CHCl3 (1:1). One CLT and halfdiacid molecule are present in the asymmetric unit. The protonof ADA is hydrogen bonded to imidazole nitrogen of CLTtogether with an auxiliary C−H···O interaction (O1−H1A···N2,1.84 Å, 174°; C14−H14···O2, 2.80 Å, 118°; Figure 2a). Thecarboxylic acid CO bond distances in ADA (1.19 and 1.31 Å)suggest that CLT−ADA is a cocrystal (ΔDCO > 0.08 Å).Bifurcated C−H···O interactions connect CLT to carbonyloxygen (O2) (Figure 2b) and dimeric C−H···Cl interactions.Crystallographic data and hydrogen bonds are listed inTables 2 and 3.

CLT−25DHBA Salt (1:1). This salt was prepared by takingequimolar amount of the components in CHCl3−EtOH (1:1),and the crystal structure was solved in space group P1 ̅. Thestructure contains one CLT−NH+ cation and 25DHBA− anionthrough proton transfer from 25DHBA to the imidazole N2of CLT (N2−H2A···O1, 1.8 Å, 169°; Figure 3a). The phenolichydroxy group ortho to the acid group is involved inintramolecular hydrogen bond (O3−H3A···O1, 1.85 Å, 146°)and the second hydroxy group interacts with O2 (carboxylate) ofadjacent 25DHBA− (O4−H4A···O2, 1.91 Å, 168°) in a R2

2(14)dimer ring motif43,44 (Figure 3a). Auxiliary C−H···O (2.46 Å,152°; 2.56 Å, 139°; Figure 3b) and weak Cl···Cl interactionsstabilize the structure (Figure 3c).

CLT−246THBA Salt (1:1). The title salt was prepared in bulkby liquid-assisted grinding of CLT and 246THBA in equimolarratio from acetone. Single crystals were harvested from CHCl3−EtOH (1:1), and the crystal structure (P21/n space group)confirms CLT−NH+ and 246THBA− ions in the asymmetricunit, with the proton being transferred to the basic N2 of CLT(N2−H2A···O1, 1.71 Å, 173°; C14−H14···O2, 2.78 Å, 119°)(Figure 4a). The intra molecular hydrogen bonds (O3−H3A···O2, 1.67 Å, 152°; O4−H4A···O1, 1.69 Å, 151°) and the

Scheme 1. Degradation of Clotrimazole in Acidic Medium

Figure 1.Molecular structures of the CLT and the list of coformers usedin this study.

Table 1. pKa Valuesa of API and Coformers Used in This

Study

API/coformer pKa ΔpKa cocrystal/salt

CLT 6.62ADA 3.92, 4.7 2.7, 1.92 1:0.5 cocrystal25DHBA 2.53 4.09 1:1 salt246THBA 1.83 4.79 1:1 saltPCA 4.0 2.62 1:1 saltCFA 3.64 2.98 1:1 saltMA 3.55, 5.69 3.07, 0.93 1:0.5 saltSBA 4.15, 4.90 2.47, 1.72 1:0.5 cocrystal

apKa calculations were carried out in ChemAxon calculator (ref 38).

Crystal Growth & Design Article

DOI: 10.1021/acs.cgd.5b00268Cryst. Growth Des. 2015, 15, 2493−2504

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p-hydroxyl donor makes intermolecular hydrogen bond with thecarboxylate (O5−H5A···O2, 1.81 Å, 174°) (Figure 4b) to makea wave-like arrangement (Figure 4c). Coformer acid moleculesare arranged in a corrugated sheet and CLT molecules hang onalternately through N2−H2A···O1 bond.CLT−PCA Salt (1:1). Single crystals of CLT−PCA were

obtained from CHCl3 and the structure was determined in spacegroup P212121. The crystal structure contains one molecule ofeach ion in the asymmetric unit. Similar to above salts, CLT−NH+ and PCA− ions are bonded through ionic N2−H2A···O3(1.78 Å, 161°; Figure 5a). The acid moieties extend viaO1−H1A···O2 (1.90 Å, 174°) and C27−H27···O2 (2.49 Å,130°) interactions in a 1D tape and further by C10−H10···Cl1(2.73 Å, 161° ; Figure 5b) interaction at the chlorine atomof CLT.CLT−CFA−ANI Salt (1:1:1). This salt was prepared by liquid-

assisted grinding with acetone by taking equimolar CLT andcaffeic acid, and the product was characterized by powder XRD,IR. The product was recrystallized from anisole−ethanol, and inthis process anisole was included in the crystal lattice (P21/cspace group). Anisole molecule was included in the crystal latticeCLT−CFA−ANI (1:1:1) to give CLT−NH+, CFA−, and anisole.A proton is transferred from CFA to imidazole N2 of CLT (N2−H2A···O3, 1.80 Å, 172°). The acid forms two types of tetramericunits, R4

4(18), and R44 (38) ring motif via O1−H1A···O3

(1.95 Å, 149°), O2−H2C···O4 (1.82 Å, 161°) H bonds, andCLT−NH+ ions are arranged alternately above and below theplane of acid tetramers sustained by ionic N2−H2A···O3 bonds(Figure 6a,b). The oxygen of carboxyl group forms bifurcatedO−H···O and N−H···O motif with the OH group of CFA andNH donor of CLT.

Conformations of CLT. CLT contains three freely rotatablephenyl rings and one imidazole ring connected to a single tertiarycarbon atom. CLT exhibits different conformations in its multi-component crystal structures (Figure 7).

Powder X-ray Diffraction. Overlay of the experimentalpowder XRD pattern on the calculated lines for the crystalstructure confirms purity and homogeneity of the bulk phase forCLT−ADA, CLT−25DHBA, CLT−246THBA,and CLT−PCA(Figure 8). CLT−CFA salt was crystallized from anisole−EtOHsolvent mixture, and it included anisole in the crystal lattice togive CLT−CFA−ANI (1:1:1). The powder diffraction pattern ofthe ground products and the starting materials are shown inFigure S17 (Supporting Information). Microcrystalline powderof CLT−MA and CLT−SBA was obtained from acetone bygrinding for about 20 min, but good quality single crystals did notdevelop from the solvent. The difference in powder patterncompared to starting materials (Figures S1 and S2, SupportingInformation) suggests the formation of a novel solid form, which

Figure 2. (a) Two molecules of CLT are connected to one molecule of adipic acid by O−H···N synthon. (b) Chains extend via C−H···Cl interactions,and also we can see the C−H···O interactions.



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Table2.CrystallographicDataof

CLT

Add

ucts

CLT

−ADAcocrystal(1:0.5)

CLT

−25DHBAsalt(1:1)

CLT

−246T

HBAsalt(1:1)

CLT

−PC

Asalt(1:1)

CLT

−CFA

−ANIsaltsolvate(1:1:1)

empiricalform

ula

C22H

17ClN

2·0.5(C

6H

10O

4)C22H

18ClN

2·C7H

5O4

C22H

18ClN

2·C7H

5O5

C22H

18ClN

2·C9H

7O3

C23H

17ClN

2·C9H

7O4.C7H

8Oform

ulawt

417.90

498.94

514.94

508.98

644.12

crystalsystem

triclinic

triclinic

monoclinic

orthorhombic

monoclinic

spacegroup

P1̅̅

P1̅̅P2

1/n

P212

121

P21/c

T(K

)298(2)

298(2)

298(2)

298(2)

100(2)

a(Å)

8.730(9)

10.172(10)

12.0214(14)

10.3536(13)

12.4974(17)

b(Å)

9.654(9)

10.646(8)

13.8128(16)

14.9452(14)

14.527(2)

c(Å)

13.254(13)

13.041(9)

15.9926(18)

16.4976(19)

17.789(2)

α(deg)

74.48(9)

108.469(7)

90.00

9090.00

B(deg)

84.281(8)

102.963(8)

97.934(2)

9096.028(2)

γ(deg)

85.466(8)

107.069(8)

90.00

9090.00

V(Å

3 )1069.4(18)

1200.12(17)

2630.1(5)

2552.8(5)

3211.7(7)

Dcalcd(g

cm−3 )

1.298

1.381

1.300

1.324

1.332

μ(m

m−1 )

0.203

0.199

0.187

0.186

0.168

θrange

2.73−26.37

2.94−26.37

1.96−28.3

2.69−26.37

1.64−25.00

Z/Z

12/1

2/1

4/1

4/1

4/1

rangeh

−6to

10−12

to12

−15

to16

−12

to12

−14

to14

rangek

−12

to12

−13

to13

−18

to18

−11

to18

−17

to17

rangel

−16

to16

−15

to16

−21

to21

−20

to12

−21

to21

reflectio

nscollected

7803

8431

30171

7083

30355

totalreflectio

ns4368

4889

6337

4539

5662

observed

reflectio

ns3471

3471

3515

1495

4791

R1[I>2σ(I)]

0.058

0.050

0.082

0.074

0.075

wR2(all)

0.083

0.125

0.161

0.125

0.237

goodness-of-fi

t0.776

1.031

1.097

0.829

1.087

X-ray

diffractom

eter

OxfordXcalibur

OxfordXcalibur

BrukerSm

art

OxfordXcalibur

BrukerSm

art



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is supported by DSC (Figure 9) and TGA (Figures S18−S19,Supporting Information).Infrared Spectroscopy. In CLT salts, a proton is transferred

from the coformer acid to the imidazole nitrogen (N2) of CLT.

Normally free COOH group stretching frequency occurs at1730−1700 cm−1, and the COO− group absorbs strongly at1640−1540 cm−1 (asym). The CN absorption peak of CLTappears at 1646.7 cm−1, and for salts a bathochromic shift is

Table 3. Hydrogen Bond Geometry in CLT Crystal Structures (Neutron-Normalized)

cocrystal/salt interaction D−H (Å) H···A (Å) D···A (Å) ∠D−H···A (Å) symmetry code

CLT−ADA (1:0.5) O1−H1A···N2 0.82 1.84 2.654(4) 174 −1+x,y,zC21−H21···O2 0.93 2.45 3.377(4) 172 1+x,1+y,z

CLT−25DHBA (1:1) N2−H2A···O1 0.86 1.80 2.65(3) 169 −x,1−y,−zO3−H3A ···O1 0.82 1.85 2.570(3) 146 intramolecularO4−H4A···O2 0.82 1.91 2.714(3) 168 −x,1−y,−zC14−H14···O3 0.93 2.56 3.318(3) 139 x,−1+y,zC15−H15···O2 0.93 2.46 3.309(3) 152 1+x,y,z

CLT−246THBA (1:1) N2−H2A···O1 0.93 1.71 2.651(3) 173 1+x,y,zO3−H3A···O2 0.96 1.67 2.553(3) 152 intramolecularO4−H4A···O1 0.87 1.69 2.526(4) 151 intramolecularO5−H5A···O2 0.88 1.81 2.683(3) 174 1/2−x,1/2+y,1/2−zC12−H12···O4 0.93 2.57 3.375(4) 145 1/2+x,1/2−y,−1/2+zC8−H8···O5 0.93 2.58 3.350(4) 141 3/2−x,−1/2+y,1/2−zC15−H15···O3 0.93 2.50 3.276(3) 141 1/2+x,1/2−y,1/2+z

CLT−PCA (1:1) O1−H1A···O2 0.82 1.90 2.713(8) 174 −1+x,y,zN2−H2A···O3 0.86 1.78 2.608(8) 161 2−x,−1/2+y,1/2−zC27−H27···O2 0.93 2.49 3.170(10) 130 −1+x,y,zC10−H10···Cl1 0.93 2.73 3.624(8) 161 1/2+x,1/2−y,z

CLT−CFA−ANI (1:1:1) O1−H1A···O3 0.82 1.95 2.689(3) 149 x,3/2−y,−1/2+zN2−H2A···O3 0.86 1.81 2.661(3) 172 x,3/2−y,−1/2+zO2−H2C···O4 0.82 1.82 2.611(3) 161 −x,−1/2+y,1/2−z

Figure 3. (a) Proton transferred from the acid to imidazole nitrogen of CLT and O−H···O hydrogen bond forming R22(14) motif. (b) Auxiliary

C−H···O interactions. (c) Salt pairs extend through Cl···Cl (type-I) interactions.



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observed with respective to CLT. In the case of adipic acid andsuberic acid adducts (neutral cocrystals), the CO stretchingfrequencies are observed at 1694.5 and 1700.0 cm−1 (no protontransfer and COOH group peak is similar to the pure coformer),whereas when the COOH proton is transferred to theimidazolium nitrogen of CLT, the COO− (asym stretch)frequency is red-shifted, and the N−H (stretch) frequency ispresent (Table 4 and Figures S3−S9, Supporting Information).Solid-State NMR. 15N CP−MAS is the best tool to identify

salt formation or inter-/intramolecular proton transfer.45,46

Clotrimazole consists of two nitrogen atoms (N1, N2), andtheir chemical shifts were observed at 186 and 262 ppm. Transferof the proton from the carboxylic acid to the N2 of CLT increasesshielding of the nitrogen to result in an upfield chemical shift.Salts showed significant decrease in the 15N chemical shift values(shielding) compared to pure clotrimazole (most deshieldedN2), indicating salt formation (Figure S10, SupportingInformation), whereas there is not much change observed inADA and SBA cocrystals (Table 5). The 1:0.5 stoichiometryof CLT−MA salt and CLT−SBA cocrystal was confirmed by1H NMR proton integration (Figures S11 and S12, SupportingInformation).Thermal Analysis. Clotrimazole showed a sharp endotherm

at 148 °C without any phase transformation. The groundmaterial of CLT and ADA cocrystal melts at 134.9 °C (mp adipicacid 151−153 °C). The melting points of other CLT salts with25DHBA, 246THBA, PCA, CFA, MA, and SBA cocrystal are

154.5, 150, 170.3, 141.1, 122, and 130 °C, respectively. CLT−PCA exhibited the highest melting point (170.3 °C), and CLT−MA has the lowest melting point (122 °C). DSC thermogramsare displayed in Figure 9 and of CLT−246THBA in Figure S13(Supporting Information), and melting points are listed inTable 6.

Solubility and Dissolution Studies. Poor solubility is themajor issue in the pharmaceutical industry for many drugsbecause poor pharmacokinetic and pharmacodynamic pro-perties limit bioavailability. Improvement in dissolution rateand solubility by means of supramolecular modification of anAPI is a crystal engineering strategy. Solubility experiments ofCLT (a BCS class II drug) and its cocrystals/salts (CLT−ADA,CLT−25DHBA, CLT−246THBA, CLT−PCA, CLT−CFA,CLT−MA, and CLT−SBA)were performed in 65% EtOH−H2Omedium due to poor aqueous solubility of CLT (0.49 μg/mL). Thesolubility of CLT in 65% EtOH−H2O after 24 h is 4.4 mg/L.Equilibrium solubility experiments for salts were performed for24 h (the salts were stable for more than 24 h as confirmed byPXRD, Figure S14−16, Supporting Information). Normallysolubility and dissolution is measured by plotting a calibrationcurve for the chromophore in the drug molecule using UV−visspectroscopy. For CLT−25DHBA, CLT−246THBA, CLT−PCA,and CLT−CFA, the coformer absorption interferes with that ofCLT (at 262 nm) due to the aromatic ring in the coformer acid.Hence the solubility of these salts was determined by analyticalHPLC (Table 7) using acetonitrile and 1% acetic acid as themobile

Figure 4. (a) Proton transfer from coformer acid to CLT. (b) Salt pairs extend through O−H···O bonds. (c) The chains extend in wave with CLThanging above and below the plane.



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phase (1:1 v/v). The solubility of CLT−MA salt is 22.45 timeshigher than that of the pure drug, while ADA and SBA cocrystalswere higher by 5 times. Intrinsic dissolution was measured overa 4 h period to give values of 0.972, 0.370, 1.740, and 1.658mg/mLfor CLT, CLT−ADA, CLT−MA, and CLT−SBA (Figure 10).The dissolution rate of CLT−MA and CLT−SBA adducts were1.7 times higher than CLT, while CLT−ADA cocrystal decreasedby 2.6 times. The area under the curve AUC0−4h for CLT−MA ishigh at 1234 mg h/L due to high equilibrium solubility (measuredat 24h) and high solution concentration of the drug (measured at240 min). Dissolution and AUC measurements on CLT salts witharomatic carboxylic acids are pending because it is a tedious task torecord the 12 or so readings over 240 min using HPLC (and alsomaking calibration curves). The overlapping peaks from thechromophore of the drug and the coformer make difficult a rapidUV−vis analysis of CLT concentration.

■ CONCLUSIONSWe have prepared seven novel solid forms of CLT by usingthe ΔpKa rule of 3, CLT−ADA, CLT−SBA cocrystals, andCLT−25DHBA, CLT−246THBA, CLT−PCA, CLT−CFA,and CLT−MAsalts. In salts structures, the carboxylic acid protonis transferred to the imidazole nitrogen (N2) of the CLT throughionic N+−H···O− hydrogen bond except in CLT−ADAcocrystal, which is sustained by neutral COOH···N hydrogenbond. Solubility experiments were performed in 65% EtOH−water for CLT−25DHBA, CLT−246THBA, CLT−PCA, CLT−CFA, and CLT−MAsalts and CLT−ADA and CLT−SBAcocrystals. The solubility of CLT−MA salt is 22 times higher

compared to CLT and net drug dissolved in 4 h (the earlydissolution phase for any drug) is 1.79 times. These preliminarystudies encourage us to explore a soluble salt of clotrimazole forantimalarial therapy47 in the future.

■ EXPERIMENTAL SECTIONClotrimazole was purchased from Yarrow chemicals, Mumbai, India.Solvents (purity >99%) and other coformers were purchased fromHychem Laboratories (Hyderabad, India) and Sigma−Aldrich(Hyderabad, India). Water filtered through a double deionizer purifica-tion system (Aqua DM, Bhanu, Hyderabad, India) was used for allexperiments.

CLT−ADA Cocrystal (1:0.5). Clotrimazole and adipic acid salt wasobtained by grinding (1:0.5) stoichiometric ratio of CLT (344.83 mg,1 mol) and ADA (73.07 mg, 0.5 mol) in a mortar and pestle for 15 minusing acetone as solvent. The formed salt was characterized by PXRD,IR, and DSC. Colorless single crystals suitable for single-crystal XRDwere developed by upon dissolving the material in ethanol−CHCl3(1:1) solvent mixture left for solvent evaporation at ambient conditionsfor 3−4 days; mp 134.9 °C.

CLT−25DHBA Salt (1:1). Clotrimazole and 2,5-dihydroxy benzoicacid were gently ground in equimolar (1:1) stoichiometry of CLT(344.83 mg, 1 mol) and 25DHBA (154.12 mg, 1 mol) in a mortar andpestle for 15 min using acetone as solvent. The formed salt wascharacterized by PXRD, IR, and DSC. Colorless single crystals suitablefor single crystal XRDwere developed by upon dissolving the material inethanol−CHCl3 (1:1) solvent mixture and left for solvent evaporation;mp 154.5 °C.

CLT−246THBA Salt (1:1). Clotrimazole and 2,4,6-tri hydroxyben-zoic acid were ground in 1:1 stoichiometric ratio of CLT (344.83 mg,1 mol) and 246THBA (170.12 mg, 1 mol) in a mortar and pestle for

Figure 5. (a) Proton transfer from acid to CLT. (b) Salt pairs extend via C−H···Cl interactions.



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15 min using acetone as solvent. The formed salt was characterized byPXRD, IR, and DSC. Colorless single crystals suitable for single-crystalXRD were developed by upon dissolving the material in ethanol−anisole (1:1) solvent mixture left for solvent evaporation. mp 150 °C.

CLT−PCA Salt (1:1). Clotrimazole and p-coumaric acid salt wereground in 1:1 stoichiometry of CLT (344.83 mg, 1 mol) and PCA(164.16 mg, 1 mol) in a mortar and pestle for 15 min using acetone assolvent. The formed salt was characterized by PXRD, IR, and DSC.Colorless single crystals suitable for single-crystal XRD were developedby upon dissolving the material in ethanol−anisole (1:1) solventmixture left for solvent evaporation; mp 170 °C.

CLT−CFA (1:1). Clotrimazole and caffeic acid salt was obtained bygrinding (1:1) stoichiometric ratio of CLT (344.83 mg, 1 mol) and CFA(180.16 mg, 1 mol) in a mortar and pestle for 15 min using acetone assolvent. The formed salt was characterized by PXRD, IR, and DSC.Colorless single crystals suitable for single-crystal XRD were developedby upon dissolving the material in ethanol−anisole (1:1) solventmixture left for solvent evaporation; mp 141 °C.

CLT−MA Salt (1:0.5). Clotrimazole and maleic acid salt wasobtained by grinding (1:0.5) stoichiometric ratio of CLT (344.83 mg,1 mol) and MA (58.03 mg, 0.5 mol) in a mortar and pestle for 15 min

Figure 6. (a) CFA forms a tetramer R44(18) ring motif via O−H···O hydrogen bonds. (b) CFA forms a tetramer R4

4 (38) ring motif viaO−H···O hydrogen bonds. (c) The two tetrameric units are arranged alternately. The included solvent molecules of anisole are not shown forclarity.

Figure 7.Overlay of CLT in salts and cocrystals to show the changes inmolecular conformations of phenyl groups. Black, CLT; red, CLT−ADA; green, CLT−25DHBA; magenta, CLT−246THBA; purple,CLT−PCA; blue, CLT−CFA−ANI.



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using acetone as solvent. The formed salt was characterized by PXRD,IR, and DSC; mp 122 °C.CLT−SBA Cocrystal (1:0.5). Clotrimazole and suberic acid salt was

obtained by grinding (1:0.5) stoichiometric ratio of CLT (344.83 mg,1 mol) and SBA (87.1 mg, 0.5 mol) in a mortar and pestle for 15 min

using acetone as solvent. The formed salt was characterized by PXRD,IR, and DSC; mp 130 °C.

The coformers used to crystallize binary systems but did notresult in any phase change are displayed in Table S1, SupportingInformation.

Figure 8. Overlay of experimental PXRD patterns of novel crystal forms of CLT on the calculated lines from the X-ray crystal structure.

Figure 9. DSC thermograms of CLT salts/cocrystal.



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X-ray Crystallography. X-ray reflections for the CLT−ADA,CLT−2,5DHBA, and CLT−PCA were collected on OxfordXcalibur Gemini Eos CCD diffractometer at 298 K using Mo Kαradiation (λ = 0.7107 Å). Data reduction was performed usingCrysAlisPro (version 1.171.33.55),48 and OLEX249 was to solve andrefine the structures. X-ray reflections for CLT−246 THBA andCLT−CFA were collected on Bruker SMART-APEX CCD

diffractometer equipped with a graphite monochromator and Mo Kαfine-focus sealed tube (λ = 0.71073 Å). Data reduction was performedusing Bruker SAINTSoftware.50 Intensities were corrected forabsorption using SADABS,51 and the structure was solved andrefined using SHELX-97.52,53 All non-hydrogen atoms were refinedanisotropically. Hydrogen atoms on hetero atoms were locatedfrom difference electron density maps, and all C−H hydrogens werefixed geometrically. Hydrogen bond geometries were determined inPlaton.54 X-Seed55 was used to prepare packing diagrams. Crystalstructures are deposited as part of the Supporting Information andmay be accessed at www.ccdc.cam.ac.uk/data_request/cif (CCDC nos.1050767−1050771).

Table 4. Stretching and Bending Frequencies of CLT Adducts

compdCN(cm−1)

N−H(cm−1)

carboxylate/carboxylic acid (asym)(cm−1)

carboxylate (sym)(cm−1)

carboxylic acid (coformer) CO stretch(cm−1)

CLT 1646.7CLT−ADA 1567.2 1694.5 1693.1CLT−25DHBA 1618.3 3426.2 1567.9 1381.3 1669.6CLT−246THBA 1634.7 3449.3 1603.0 1420.9 1663.3CLT−PCA 1604.3 3453.4 1587.1 1374.2 1672.3CLT−CFA 1640.4 3437.6 1585.7 1379.1 1646.0CLT−MA 1584.4 3483.1 1701.8, 1622.0 1443.2 1706.8CLT−SBA 1640 1700.0 1702.8

Table 5. 15N ss-NMR Chemical Shift Values (δ, ppm)a

compd chemical shift of N1, N2 (ppm)

CLT 186, 262CLT−ADA (1:0.5) 184, 242CLT−SBA (1:0.5) 183, 239CLT−25DHBA (1:1) 189.2, 191CLT−246THBA (1:1) 175, 190CLT−MA (1:0.5) 184, 193

aN2 is hydrogen bonded to the COOH group and N1 is the internalimidazole nitrogen.

Table 6. Melting Points of CLT Cocrystals and Salts fromDSC

S. no. compdmelting point of

API/coformer (°C)melting point ofcocrystal/salt (°C)

1 CLT 1482 CLT−ADA (1:0.5) 152 134.93 CLT−25DHBA

(1:1)203 154.5

4 CLT−246THBA(1:1)

210 150.0

5 CLT−PCA (1:1) 211 170.36 CLT−CFA (1:1) 223 141.17 CLT−MA (1:0.5) 135 122.08 CLT−SBA (1:0.5) 142 130.0

Table 7. Intrinsic Dissolution Rate and Solubilitya of CLT Cocrystals/Salts

compdaqueous solubility ofAPI/coformer mg/mL

molar extinctioncoefficient (ε/mM cm)

equilibriumsolubility mg/L

solution concentration inmg/mL (240 min)

area under the curve, AUC 0−4 h(mg h)/L

CLT 0.00049 1.609 4.40 0.972 427CLT−ADA 23 0.750 22.06 (× 5.0) 0.370 (× 0.38) 234.58CLT−MA 780 1.000 98.79 (× 22.43) 1.740 (× 1.79) 1233.82CLT−SBA 11.9 0.563 22.01 (× 4.99) 1.658 (× 1.70) 867.23CLT−25DHBA 5 13.11 (× 2.97)CLT−246THBA 18.6b 61.87 (× 14.04)CLT−PCA 18.3b 5.84 (× 1.32)CLT−CFA 54b 12.48 (× 2.83)

aEquilibrium solubility was measured for all samples using HPLC method. Dissolution curve requires multiple measurements, and this value wasdetermined for the aliphatic carboxylic acid coformers only where there is no interference for the UV−vis maximum peak of CLT with those forthe coformers, i.e., the aromatic carboxylic acids were excluded from dissolution measurements. bAqueous solubility of compounds is taken fromhttp://www.chemspider.com/.

Figure 10. Intrinsic dissolution rate of CLT and its cocrystals and salts in65% EtOH−H2O.



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Powder X-ray Diffraction. Powder X-ray diffraction was recordedon Bruker D8 Advance diffractometer (Bruker-AXS, Karlsruhe,Germany) using Cu Kα X-radiation (λ = 1.5406 Å) at 40 kV and 30 mApower. X-ray diffraction patterns were collected over the 2θ range 5−50° ata scan rate of 1°/min.Vibrational Spectroscopy. Nicolet 6700 FT−IR spectrometer

with a NXR FT−Raman module was used to record IR spectra.IR spectra were recorded on samples dispersed in KBr pellets.Solid−State NMR Spectroscopy. Solid-state 15N−NMR spectra

were recorded on Bruker Avance 400 MHz spectrometer (Bruker−Biospin, Karlsruhe, Germany). ss-NMR measurements were carried outon Bruker 4 mm double resonance N15CP−MAS probe in zirconiarotors with a Kel-F cap at 5.0 kHz spinning rate with a cross-polarizationcontact time of 4ms and a delay of 8 s. 15N−NMR spectra were recordedat 40 MHz and referenced to the glycine N, and then chemical shifts arerecalibrated to nitromethane (δglycine = −347.6 ppm).Thermal Analysis. DSC was performed on a Mettler Toledo DSC

822e module. Samples were placed in crimped but vented aluminumsample pans. The typical sample size is 4−6 mg, temperature range was30−250 °C at 5 °C/min. Samples were purged by a stream of nitrogenflowing at 150 mL/min.Dissolution and Solubility Measurements. The solubility curves

of CLT salts and cocrystal weremeasured using theHiguchi and Connormethod in 65% ethanol−water medium at 37 °C. First, the absorbanceof a known concentration of the salt was measured at the given λmax(CLT at 262 nm) in 65% ethanol−water medium on Thermo ScientificEvolution 300 UV−vis spectrometer (Thermo Scientific, Waltham,MA). These absorbance values were plotted against several knownconcentrations to prepare the concentration vs intensity calibrationcurve. From the slope of the calibration curves, molar extinctioncoefficients for CLT salts were calculated and the respective molarextinction coefficients 1.609, 0.75, 1.0, and 0.563 were used to determinethe intrinsic dissolution. An excess amount of the sample was addedto 6 mL of 65% ethanol−water medium. The supersaturated solutionwas stirred at 500 rpm using a magnetic stirrer at 30 °C. After 24 h,the suspension was filtered through Whatman’s 0.45 μm syringe filter.The filtered aliquots were diluted sufficiently, and the absorbance wasmeasured at the given λmax. The intrinsic dissolution studies of CLT saltswas done using CLT 100 mg, CLT−MA 100 mg, CLT−SBA 100 mg,and CLT−ADA 100 mg (0.289, 0.248, 0.231, and 0.239 mol of eachcompound, respectively). This was directly poured into 500 mL of 65%ethanol−water medium. The paddle rotation was fixed at 150 rpm, anddissolution experiments were continued up to 240 min at 37 °C.At regular intervals, 5 mL of the dissolution medium was withdrawn andreplaced by an equal volume of fresh medium to maintain a constantvolume. The AUC was calculated using the linear trapezoidal rule ofdrug bioavailability. The nature of the solid samples after diskcompression and solubility/dissolution measurements was verified byPXRD to know if there is any phase transition.

■ ASSOCIATED CONTENT

*S Supporting InformationCrystallographic information files: CCDC nos. 1050767−1050771; IR comparison of new solid phases with their startingmaterials; solid-state 15N NMR chemical shift (ppm) values ofCLT salts and cocrystals; 1H NMR of CLT−MA salt shows 1:0.5stoichiometric ratios of the components; 1H NMR of CLT−SBAcocrystal shows 1:0.5 stoichiometric ratios of the components;DSC thermogram of CLT−246THBA salt; comparison ofpowder XRD pattern of salts and cocrystals with the calculatedline pattern from the X-ray crystal structure shows stability ofthese adducts in the solubility medium (65% EtOH−water) for 1day; PXRD comparison for the CLT−CFA salt with its startingmaterials; TGA analysis for CLT−MA salt; TGA analysis forCLT−SBA cocrystal; pKa values for coformers used in this study.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Phone: 91 40 23134854. Fax: 91 40 23010567. E-mail:[email protected].

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This research was funded by J.C. Bose fellowship SR/S2/JCB-06/2009 and SERB scheme SR/S1/OC-37/2011. DST−IRPHAand UGC−PURSE are thanked for providing instrumentationand infrastructure facilities. S.M. and S.A. thank UGC andUniversity of Hyderabad for fellowship. M.K.C.M. and U.B.R.K.thank Crystalin Research, Hyderabad, for support.

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