Salts of Hydrates of Imiquimod, an Immune Response Modifier

DOI: 10.1021/cg900647y

Salts of Hydrates of Imiquimod, an Immune Response Modifier T. Lakshmi Kumar,† Peddy Vishweshwar,*,† J. Moses Babu,† and K. Vyas‡

2009, Vol. 9 4822–4829

†

Department of Analytical Research, Discovery Research, Dr. Reddy’s Laboratories Ltd., Miyapur, Hyderabad 500 049, India, and ‡Analytical Research Department, Integrated Product Development, Dr. Reddy’s Laboratories Ltd., Bachupalli, Hyderabad 500 072, India Received June 10, 2009; Revised Manuscript Received September 9, 2009

ABSTRACT: Imiquimod free base 1, an immune response modifier, its hydrochloride salt 2, hydrochloride salt monohydrate 3, salts with maleic acid 4, fumaric acid 5, succinic acid 6, adipic acid 7, and 4-hydroxybenzoic acid 8 have been prepared and characterized by single crystal X-ray diffraction. The carboxylic acid salts 4-8 were obtained by solution crystallization experiments of imiquimod hydrochloride with carboxylic acid group containing molecules. Imiquimod contains a 2-aminopyridine moiety which is known to form salts when crystallized with acidic carboxylic acid moiety containing molecules. During the crystallization, hydrochloride was liberated from the imiquimod and the basic pyridine moiety was protonated by the carboxylic acids. Dicarboxylic acid salts 5-7 crystallized in 2:1 stoichiometry; however, 2, 4, and 8 crystallized in a 1:1 ratio. Imiquimod maleate salt 4 crystallized as sesquihydrate, whereas imiquimod fumarate (5) crystallized as hexahydrate. Two aliphatic-R,ω-dicarboxylic acid salts of imiquimod (6 and 7) crystallized as tetrahydrates. Notably, all the dicarboxylic acid salts of imiquimod are crystallized as hydrates. Imiquimod hydrochloride salt was isolated as both anhydrous and hydrous forms (2 and 3). Molecular arrangements in these salt crystal structures are described with supramolecular homo- and heterosynthons. Imiquimod free base 1 forms a molecular tape through 2-aminopyridine-aminoimidazole heterosynthon III. All the carboxylic acid salts of imiquimod 4-8 form robust and recurring charge-assisted 2-aminopyridinium-carboxylate supramolecular heterosynthon V and aminoimidazole homosynthon II. All the salt formers in this study are US-FDA Generally Recognized As Safe (GRAS) substances. The present examples suggest that co-crystallization experiments are useful not only to make cocrystals but also to discover unexpected novel forms of active pharmaceutical ingredients.

1. Introduction Solid active pharmaceutical ingredients (APIs) can be either crystalline (a regular arrangement of molecules) or amorphous (no defined long-range order). Owing to the greater stability and other reasons, crystalline forms are preferred.1 Generally, crystalline active ingredients may have solid-state problems such as stability, solubility/dissolution, etc. that hampers the development of a drug substance. However, such properties can be modulated by adding a suitable nontoxic compound to the drug substance to prepare multicomponent forms such as salts2 and co-crystals.3 In general, salts of an acidic or basic API have been prepared to modulate the physiochemical properties. An estimated half of all drug molecules used in medicinal therapy are in the salt form. However, recent developments in the solid-state research of APIs suggest that co-crystals3 are new crystalline forms of a drug substance to modulate the solid-state properties. In particular, co-crystals are advantageous if an API is not acidic/basic enough to form salts or if the salt screening does not produce viable solid phase. Further, the co-crystals are useful for the life-cycle management of the APIs. Solvates/ hydrates are realistic possibilities for single and multicomponent forms.4 In general, solvates/hydrates are serendipitous results discovered during crystallization of a solid compound from solvent(s), and such forms are selected for the development if the anhydrous form does not yield a viable solid phase. There are many ways molecules can assemble in a crystal; hence, each arrangement is considered as a separate form (polymorphs), and each form exhibits different physicochemical *Corresponding author. E-mail: [email protected]. pubs.acs.org/crystal

Published on Web 10/16/2009

properties.5 Hence, screening polymorphs of the selected free API or salt or co-crystal or solvate/hydrate is also very important. Crystal engineering6 is a fast growing field in solid-state research with a wide range of applications in material science and pharmaceuticals. Crystal engineering deals with analysis of intermolecular interactions between the molecules in crystals and utilization of such knowledge for designing new solids with a desired arrangement of molecules to obtain desired physicochemical properties. In crystals, molecular recognition events between the molecules occurs through noncovalent interactions (such as hydrogen bonds, halogen bonds, π-π stacking, etc.) which are described as supramolecular synthons.7 Supramolecular synthon formation between selfcomplementary functional groups are described as “homosynthons” and those comprised of different but complementary functional groups as “heterosynthons”.8 In general, supramolecular heterosynthon formation is favored if a crystal (single or multicomponent) contains different but complementary functional groups. Hence, supramolecular heterosynthons are widely used as a design tool for the selection of suitable co-crystal formers to prepare co-crystals of a given API. Imiquimod (1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine) (1) (Scheme 1) is marketed by 3M pharmaceuticals under the trade name Aldara.9 It exhibits antiviral activity and is used to treat certain diseases of the skin, such as skin cancer and genital and anal warts. It causes interferon production which triggers the patient’s immune response against the tumors. The molecular structure of imiquimod shows that it contains a 2-aminopyridine and imidazole moieties, available for hydrogen bonding or for salt formation. The 2-aminopyridine r 2009 American Chemical Society

Article

Crystal Growth & Design, Vol. 9, No. 11, 2009 Scheme 1

Table 1. The Carboxylate C-O/CdO Bond Distances and C1-N1-C8 Angles of Imiquimod in Crystal Structures 1-8 crystal structure 1 2 3 4

6 7 8 a

2. Experimental Section Imiquimod free base and imiquimod hydrochloride salt were received from Dr. Reddy’s Holdings Ltd., (DRH). All the salt formers (maleic acid, fumaric acid, succinic acid, adipic acid, and 4-hydroxybenzoic acid) were purchased from Aldrich and used as such for crystallization experiments. In 4-8 experiments, imiquimod hydrochloride and mono- or dicarboxylic acids were dissolved in organic solvent(s) and kept for slow evaporation crystallization to obtain co-crystals. However, the crystallization resulted in single crystals of imiquimod carboxylate salts. The crystallization experiments are described below. Preparations. Imiquimod, 1: Single crystals of 1 have been obtained by slow evaporation of imiquimod aqueous solution. Powder X-ray diffraction (PXRD: 2θ, °): 10.66, 11.24, 12.24, 14.16, 15.24, 18.14, 19.04, 21.42, 21.96, 24.12, 24.36, 29.22, and 31.70.

C-O/CdO (A˚)a

1.268(4) 1.275(4) 1.233(6) 1.271(6) 1.234(4) 1.269(4) 1.227(4) 1.306(4) 1.253(3) 1.261(3) 1.237(5) 1.247(5) 1.244(7) 1.263(7) 1.263(4) 1.270(4)

5

is a basic moiety, hence known to form salts with acidic carboxylic acid group containing molecules.10 The WO 2006/070408 A2 patent claims the imiquimod maleate salt, and three polymorphs for both the oxalate and fumarate salts. The patent describes that the above salts were prepared as a method for the preparation of substantially pure imiquimod.11 For any new crystalline form, solid-state structural studies are important for unambiguous characterization. Interestingly, for imiquimod API, only the free base crystal structure is known to date (Cambridge Structural Database (CSD) V5.30, May 2009 update).12 In this contribution, we describe the synthesis and structural features of various salts of imiquimod, obtained during the co-crystallization experiments of imiquimod hydrochloride with carboxylic acid group containing molecules. All the carboxylate salts of imiquimod are sustained through a robust 2-aminopyridinium-carboxylate supramolecular heterosynthon [graph set: R22(8)]. Further, interestingly, the majority of the imiquimod salts are crystallized as hydrates.

4823

C1-N1-C8 (°) 118.9(2) 124.51(19) 124.40(16) 124.4(2) 124.8(2)

123.9(2) 124.2(2) 125.2(3) 124.2(2)

Carboxylic acid distances are in italics.

Salt 2: Imiquimod 3 HCl (70 mg) was dissolved in 2 mL of methanol and kept for slow evaporation at ambient conditions. Single crystals have been obtained after few days. PXRD (2θ, °): 8.64, 10.18, 17.34, 21.98, 25.80, and 29.54. Salt 3: Imiquimod 3 HCl (70 mg) was dissolved in 2 mL of water and kept for slow evaporation at ambient conditions. Single crystals of 3 have been obtained after a few days. PXRD (2θ, °): 8.52, 10.78, 11.36, 13.82, 16.22, 17.12, 17.36, 22.82, 23.22, 24.38, 25.26, 26.50, 27.44, 30.68, and 33.06. Salt 4: 55.5 mg (0.2 mmol) of imiquimod 3 HCl and 23.3 mg (0.2 mmol) of maleic acid were dissolved in 5 mL of hot methanol and kept for slow evaporation at ambient conditions. Single crystals were formed after few days. Salt 5: Imiquimod 3 HCl (55.5 mg, 0.2 mmol) and fumaric acid (11.6 mg, 0.1 mmol) were dissolved in 5 mL of hot methanol and kept for slow evaporation at ambient conditions. Single crystals have been isolated after a few days. Salt 6: 55.5 mg (0.2 mmol) of imiquimod 3 HCl and 11.8 mg (0.1 mmol) of succinic acid were dissolved in 5 mL of hot water and kept for slow evaporation at ambient conditions. Single crystals were formed after a few days. Salt 7: Imiquimod 3 HCl (55.5 mg, 0.2 mmol) and adipic acid (14.6 mg, 0.1 mmol) were dissolved in 5 mL of hot water and kept for slow evaporation at ambient conditions. Single crystals have been obtained after a few days. Salt 8: Imiquimod 3 HCl (55.5 mg, 0.2 mmol) and 4-hydroxybenzoic acid (27.6 mg, 0.2 mmol) were dissolved in 5 mL of hot methanol and kept for slow evaporation at ambient conditions. Single crystals were formed after a few days. Powder X-ray Diffraction (PXRD). X-ray powder diffractograms have been obtained on Rigaku D/MAX-2200 model diffractmeter equipped with horizontal goniometer in θ/2θ geometry. The Cu KR radiation was used and the samples were scanned between 2θ: 3-45°. Single Crystal X-ray Diffraction (SXRD). X-ray data for the single crystals 1-8 were collected at room temperature on Rigaku AFC-7S diffractometer equipped with a mercury CCD detector using graphite monochromated Mo KR (λ = 0.7107 A˚) radiation. The structures were solved with direct methods (SIR92 or SIR2004)13 and refined using least-squares procedure (CRYSTALS or SHELXL-97)14 using the crystal structure 3.8.1 software. The non-hydrogen atoms were refined anisotropically, and the hydrogen atoms bonded to N and O were located in the difference Fourier map and refined isotropically. The hydrogen atoms bonded to carbons were positioned geometrically and refined in the riding model approximation with C-H = 0.95 A˚, and with U(H) set to 1.2Ueq(C). Crystallographic information of crystal structures 1-8 are summarized in Table 2 and hydrogen bond geometries are listed in Table 3. CCDC 715284-715291 contains the supplementary

(C14H17N4)þ 3 (C7H5O3)378.43 298 monoclinic P21/n 6.1024(17) 29.750(8) 10.996(3) 90 105.439(3) 90 1924.2(9) 4 1.306 0.090 22096 4255 1626 0.060 0.116

Crystal Growth & Design, Vol. 9, No. 11, 2009

8

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Lakshmi Kumar et al. crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: þ44 1223 336033).

(C14H17N4) 3 (C6H8O4)-0.5 3 (H2O)2 349.41 298 triclinic P1 6.973(5) 9.380(7) 15.239(12) 101.300(5) 98.408(8) 103.615(10) 930.3(12) 2 1.247 0.090 10729 3774 869 0.065 0.133 (C14H17N4) 3 (C4H4O4)-0.5 3 (H2O)2 335.38 298 monoclinic P21/c 13.442(5) 6.462(2) 20.599(7) 90 95.270(4) 90 1781.8(10) 4 1.250 0.091 19959 3680 1153 0.060 0.132

þ

(C14H17N4) 3 (C4H2O4)-0.5 3 (H2O)3 352.39 298 triclinic P1 8.627(6) 10.687(8) 10.934(8) 94.462(2) 111.655(7) 102.912(10) 898.8(11) 2 1.302 0.098 10214 3625 2367 0.069 0.086

6

þ þ

5

(C14H17N4) 3 (Cl)- 3 (H2O) 294.78 298 monoclinic P21/c 8.0251(16) 20.698(4) 9.1806(19) 90 103.9550(5) 90 1479.9(5) 4 1.323 0.260 17484 2891 2534 0.051 0.170

766.81 298 triclinic P1 11.154(3) 12.411(4) 15.563(5) 73.737(10) 70.195(10) 77.643(12) 1928.9(10) 2 1.320 0.099 22236 8421 4153 0.067 0.127

4

(C14H17N4) 3 (Cl)276.77 298 triclinic P1 7.668(4) 9.621(5) 11.036(6) 106.412(3) 99.630(5) 105.995(4) 723.7(7) 2 1.270 0.256 7150 2622 2018 0.052 0.159

þ

C14H16N4

240.31 298 orthorhombic P212121 8.145(2) 9.764(3) 15.771(4) 90 90 90 1254.3(6) 4 1.272 0.080 14924 1588 1331 0.052 0.148

empirical formula

formula weight T/K crystal system space group a/A˚ b/A˚ c/A˚ R/° β/° γ/° V/A˚3 Z Fcalc/g cm-3 μ/mm-1 reflns collected independent reflns observed reflns R1 wR2

þ

3 2 1

Table 2. Crystallographic Parameters of 1-8

(C14H17N4)þ2 3 (C4H3O4)-2 3 (H2O)3

7

3. Results and Discussion Co-crystallization of APIs with two or more components to obtain binary or ternary co-crystals is a current focus of solidstate researchers to modulate the physicochemical properties of APIs. Co-crystallization of two or more solid components may yield either co-crystal or other forms. For example, binary co-crystallization of solid components may result in the formation of (i) co-crystal, (ii) salt, (iii) solid solution, (iv) polymorphs of either or both components, (v) physical mixture, and (vi) unexpected forms. All the i-iv categories can be anhydrous or may contain solvent/water molecules. The above-mentioned categories are not limited; other forms are also possible. Herein we discuss the formation of unexpected multicomponent crystalline forms of imiquimod, obtained during the co-crystallization of imiquimod hydrochloride salt with organic substances consisting of at least one carboxylic acid group. Childs et al. have shown that co-crystals of Fluoxetine hydrochloride salt can be prepared by co-crystallizing the API salt with carboxylic acid moiety containing molecules. The results reveal that one of the driving forces for the formation of co-crystals is the O-H 3 3 3 Cl- hydrogen bond between the components.3m Following these lines, in order to obtain co-crystals, imiquimod 3 HCl was co-crystallized with carboxylic acid group containing substances such as maleic acid, fumaric acid, succinic acid, adipic acid, and 4-hydroxybenzoic acid (listed in the US-FDA Generally Recognized As Safe (GRAS) substances).15 However, co-crystallization attempt of imiquimod 3 HCl with maleic acid and 4-hydroxybenzoic acid (1:1) interestingly resulted in the formation of 1:1 imiquimod carboxylate salts (4 and 8). Similarly, imiquimod 3 HCl was co-crystallized with fumaric acid, succinic acid, and adipic acid in a 2:1 molar ratio; however, these crystallization experiments also resulted in imiquimod carboxylate salts (2:1) (5-7). In all these salts, 4-8 the basic pyridine moiety was protonated by the carboxylic acid moieties. The loss of HCl during the solution co-crystallization experiments could be due to heating the solution to dissolve the components or lower solubility of the imiquimod carboxylate salts. The free imiquimod base is not soluble in most of the organic solvents. Hence the above carboxylate salts could not be prepared by the slow evaporation crystallization method. Salts and co-crystals are multicomponents forms. Characterizing a multicomponent form as a salt or co-crystal is important for both scientific and legal perspectives. There are various spectroscopic techniques for characterization, but single crystal X-ray/neutron diffraction is noteworthy. In crystal structures, carboxylic acid C-O/CdO bond distances and pyridine C-N-C angles are sensitive to the proton transfer from the carboxylic acid to the pyridine moiety.10 Hence, the neutral or ionic nature of the carboxylic acid and pyridine moieties can be established by measuring the carboxylic acid C-O/CdO bond distances and C-N-C angle of pyridine group. In general, a neutral system consists of approximately C-O: ∼1.30 A˚, CdO: ∼1.20 A˚, C-N-C < 120° (∼116-118°) and ionic compounds with ca. C-O/CdO: ∼1.25 A˚, C-N-C > 120° (∼122-125°). The C-O/CdO bond distances, C-N-C angles, and location of hydrogen atoms covalently bonded to nitrogen and oxygen atoms in the

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Crystal Growth & Design, Vol. 9, No. 11, 2009

Table 3. Hydrogen Bond Geometries in Crystal Structures, 1-8 structure D-H 3 3 3 Aa 1 N2-H...N3 2 3

4

5

6

8

2.17 3.107(4)

D-H 3 3 3 A/° 178

2.21 2.21 2.63 2.17 1.89 2.33 2.34 2.48 2.56 2.66 1.81 1.92 1.45 1.43 2.37 1.89 2.00 1.75 2.10 2.16 2.15 2.29 2.40 2.53 2.59 1.89 1.87 2.07 1.99 1.81 1.82 1.84 2.15 2.43 2.54 1.93 1.93

O4-H...O2-

1.98 2.884(5)

161

O3-H O4

2.20 2.894(5)

164

N1þ-H...O1N2-H...O2N2-H...N3 C10-H...O3 O3-H...O1O3-H...O2O4-H...O3 N1þ-H...O1N2-H...O2N2-H...N3 O3-H...O1-

1.77 1.87 2.05 2.37 1.82 1.79 1.85 1.68 1.89 2.10 1.62

2.699(4) 2.829(4) 2.981(4) 3.267(5) 2.788(6) 2.760(6) 2.817(9) 2.679(5) 2.759(5) 2.957(6) 2.612(3)

176 179 172 158 169 164 166 173 152 149 175

N1þ-H...O2-

1.62 2.586(3)

172

1.795 2.786(3)

170

2.08 2.971(3) 2.36 3.297(4)

158.4 168

N2-H

...

O1-

N2-H...N3 C10-H...O3 a

D3 3 3 A/A˚

N2-H...N1 N1þ-H...Cl1N2-H...Cl1N2-H...N3 N1þ-H...O1 O1-H...Cl1O1-H...Cl1N2-H...Cl1N2-H...Cl1C11-H...Cl1O9-H...O4 O9-H...O8 O7-H...O5O3-H...O2O11-H...O6N1þ-H...O5N2-H...O6N5þ-H...O9 N6-H...O1N2-H...N3 N6-H...N7 C24-H...O2C10-H...O11 C19-H...O7 C35-H...O8 O3-H...O2O4-H...O1O3-H...O5 O4-H...O3 O5-H...O4 N1þ-H...O1N2-H...O2N2-H...N3 C10-H...O3 C14-H...O2O3-H...O2O4-H...O1-

...

7

H3 3 3 A/A˚

3.129(4) 3.055(3) 3.299(3) 3.021(3) 2.745(3) 3.080(2) 3.177(2) 3.325(2) 3.336(2) 3.627(2) 2.722(4) 2.844(4) 2.436(4) 2.421(5) 2.887(5) 2.743(3) 2.844(4) 2.714(4) 2.975(4) 2.995(4) 2.985(4) 3.233(4) 3.312(5) 3.465(4) 3.526(4) 2.821(4) 2.845(3) 2.926(5) 2.909(4) 2.732(5) 2.720(3) 2.758(4) 3.027(4) 3.362(5) 3.423(5) 2.821(5) 2.820(5)

156 170 140 157 176 166 171 166 150 176 159 157 173 137 115 175 169 165 152 152 162 174 160 168 170 176 176 159 168 167 172.7 174 147 166 154 168 161

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Scheme 2. Supramolecular Homo- and Heterosynthons Studied in the Paper

symmetry -1/2 þ x, 1/2 - y, 1-z -x, 1/2 - y, 1 - z x, y, z -x, 2 - y, -z -1 - x, 1 - y, -z x, 1/2 - y, 1/2 þ z x, y, z x, 1/2 - y, 1/2 þ z x, 1/2 - y, 1/2 þ z x, y, z 2 - x, 1 - y, 1 - z x, y, 1 þ z 1 - x, 1 - y, 1 - z x, y, z x, y, z 1 - x, 1 - y, 1 - z 1 - x, 1 - y, 1 - z 1 - x, 1 - y, 1 - z -x, 1 - y, 1 - z x, y, z 1 - x, -y, 1 - z -x, 1 - y, 1 - z -x, 1 - y, 1 - z 1 - x, -y, 1 - z 1 - x, -y, 1 - z 1 - x, 1 - y, -z -1 þ x, y, z x, y, z x, y, 1 þ z 1 - x, -y, 1 - z x, y, z x, y, z x, y, z 2 - x, 1 - y, 2 - z 1 - x, 1 - y, 2 - z -x, 1 - y, -z 1 - x, 1 - y, 2 - z 1 - x, -1/2 þ y, 3/2 þ z 1 - x, 1/2 þ y, 3/2 þ z -x, 1/2 þ y, 3/2 - z x, y, z x, y, z 1 - x, -y, 2 - z x, -1 þ y, z 1 þ x, y, z x, y, z x, 1 þ y, z x, y, z x, y, z 1 - x, -y, 1 - z -1/2 þ x, 1/2 -y, -1/2 þ z 1/2 þ x, 1/2 -y, -1/2 þ z 1/2 þ x, 1/2 - y, -1/2 þ z 3 - x, 1 - y, 1 - z 2 - x, 1 - y, 1 - z

D = donor, A = acceptor.

difference Fourier map indicates that the multicomponent forms 4-8 are salts not cocrystals (Table 1). All the multicomponent forms 2-8 are salts; however, 4-7 additionally consist of water molecules, hence they can be well described as “salt hydrates”. If a crystal structure (single or multicomponent) contains different but complementary functional groups, the probability

of formation of a heterosynthon is greater than a homosynthon.8 A recent analysis of the CSD reveals that 77% of compounds that contain at least one 2-aminopyridine and carboxylic acid groups result in the formation of 2-aminopyridine-carboxylic acid (neutral) or 2-aminopyridiniumcarboxylate (ionic) supramolecular heterosynthons. The occurrence of heterosynthon formation increases to 97% in the absence of other competing donors and acceptors.10 All the carboxylic acid salts of imiquimod 4-8 form robust ionic 2-aminopyridinium-carboxylate supramolecular heterosynthon V and aminoimidazole homosynthon II. The structural features of 1-8 are described below. Imiquimod 1 has crystallized in the orthorhombic crystal system with one molecule in the asymmetric unit (space group P212121, Table 2). The molecule contains a 2-aminopyridine and aminoimidazole moieties, available for hydrogen bonding. Imiquimod can pack in at least two molecular arrangements with 2-point synthon utilizing its strong hydrogen bond donors and acceptors: (i) A molecular tape formation through alternating 2-aminopyridine homodimer (I, Scheme 2) and aminoimidazole homodimers (II); or (ii) a molecular tape generation via 2-aminopyridine-aminoimidazole heterodimer (III). The crystal packing reveals that imiquimod attains the latter pattern with crinkled tape along [100], parallel to the (0 3 1) plane (Figure 1, Table 3). The simulated PXRD of the crystal structure matches with (i) the experimental PXRD of polycrystalline imiquimod, (ii) the experimental PXRD of the imiquimod patented form (see Figure 1 in WO 2006/070408 A2 patent),11 and (iii) simulated PXRD of the reported imiquimod crystal structure (CSD Refcode: GAQTAU).12 It suggests that the single crystal used for the X-ray crystal structure determination, polycrystalline material, patented form, and the reported crystal structure have the same solid phase. Imiquimod hydrochloride 2 crystals have been obtained from methanol solvent. The crystal structure shows that it is crystallized in the triclinic crystal system (space group P1, Table 2). Protonation has taken place on the basic pyridine moiety (N1 nitrogen) of imiquimod. All the hydrogen atoms attached to N are located in the difference Fourier map and refined isotropically. N1-H hydrogen atom location and the C1-N1-C8 angle (124.51°) confirm the protonation on the pyridine ring (Table 1). The 2-aminopyridinium moiety of imiquimod forms a strong centrosymmetric N1þ-H 3 3 3 Cl1- hydrogen bonded dimer with Cl1- acceptors (synthon IV, Scheme 2) and the aminoimidazole moiety forms centrosymmetric

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Lakshmi Kumar et al.

Figure 1. Molecular tape of imiquimod free base, 1, sustained through supramolecular heterosynthon III.

Figure 3. Crystal structure of imiquimod HCl salt hydrate, 3. Protonated imiquimod, water, and chloride ions form molecular tapes along [001].

Figure 2. Molecular tape of imiquimod hydrochloride salt 2. Notice that tape is formed by alternating imidazole homosynthon II and 2-aminopyridinium-chloride synthon IV.

aminoimidazole homodimer II resulting in a linear tape parallel to the (3 3 7) plane (Figure 2). Such tapes stack each other with weak C-H 3 3 3 Cl- interactions (Table 3). The simulated PXRD of the crystal structure matches with the experimental PXRD of polycrystalline imiquimod 3 HCl salt. Thus, single crystal represents the bulk polycrystalline solid phase. Imiquimod hydrochloride has crystallized as monohydrate from water. The salt hydrate, 3, crystallized in the monoclinic crystal system (P21/c space group) (Table 2). Location of N1-H hydrogen atom and the C1-N1-C8 angle 124.40(16)° suggests the pyridinium cation formation (Table 1). The crystal structure shows that pyridinium cation forms a N1þ-H 3 3 3 O1 hydrogen bond with water oxygen and water hydrogen bonded to chloride ions through O1-H 3 3 3 Cl1hydrogen bonds. Imiquimod amine N-H donors are hydrogen bonded to the chloride ions via N2-H 3 3 3 Cl1- H-bonds (Table 3). Such hydrogen bonding results in molecular tapes along [001], parallel to the (1 0 0) plane (Figure 3). Imidazole nitrogen (N3) is not involved in conventional hydrogen bonding. The simulated PXRD of the crystal structure matches with the experimental PXRD of polycrystalline salt hydrate, 3. Thus, single crystal represents the bulk polycrystalline solid phase. Maleic acid salt of imiquimod 4 was obtained by mixing equimolar amounts of imiquimod 3 HCl and maleic acid in methanol. The crystal structure shows that the salt has adopted a triclinic crystal system with two independent molecules of imiquimod, two maleate and three water molecules

in the asymmetric unit (space group P1; Table 2). Hence, salt 4 is imiquimod maleate (1:1) sesquihydrate. Two hydrogen atoms on O10 water could not be located in the difference Fourier map. The imiquimod pyridine moieties were protonated by the maleic acids (Table 1). Both the maleates form very short intramolecular charge-assisted O-H 3 3 3 O- [D/A˚: 2.436(4) and 2.421(5)] hydrogen bonds. The crystal packing shows that one independent imiquimod is forming centrosymmetric aminoimidazole homodimer II and connected to one independent maleate through heterosynthons V. Two independent water molecules formed a water tetramer16 and connected to maleate carboxylate oxygens via Owater-H 3 3 3 O-carboxylate H-bonds resulting in linear chains along [001] (Figure 4a). Another independent imiquimod also formed a centrosymmetric aminoimidazole dimer (synthon II), hydrogen bonded to a second independent maleate and third water molecule, resulting in linear chains as shown in Figure 4b. Such different molecular chains mentioned above are stacked on each other in 3 3 3 ABABAB 3 3 3 fashion, connected by O9water-H 3 3 3 O8carboxylate and O10water-H 3 3 3 O4carboxylate hydrogen bonds along [100] (Table 3). As mentioned in the introduction, WO 2006/070408 A2 patent discloses a crystalline form of imiquimod maleate salt.11 The simulated PXRD of the salt 4 crystal structure is different from the patented maleate form. Hence, salt 4 is a new imiquimod maleate form. Crystal structure of salt 5 shows that the asymmetric unit consists of one molecule of protonated imiquimod, half fumarate and three water molecules (triclinic system, P1 space group; Tables 1 and 2). Hence, salt 5 is imiquimod fumarate (2:1) hexahydrate. Fumarate resided on an inversion center special position. Crystal packing shows that one fumarate is connected to two protonated imiquimods through heterosynthons V and protonated imiquimods also forms homosynthon II. Such assembly results in zigzag molecular chains along [011]. Water molecules form a six-membered ring through O-H 3 3 3 O hydrogen bonds.16 Such hexamers link the

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Figure 6. Crystal structure of imiquimod succinate salt hydrate, 6. Notice the molecular chain formation by succinate and protonated imiquimod molecules through heterosynthons V and homosynthons II. Such chains are connected by water molecules through Owater-H 3 3 3 O-carboxylate hydrogen bonds. Few atoms on imiquimod were deleted for clarity.

Figure 4. Molecular chains in imiquimod maleate salt hydrate, 4. (a) Linear chains are formed by one independent imiquimod, maleate and two water molecules through synthons II, V, and water tetramer along [001]. (b) Another chain is formed by second independent imiquimod, maleate and third water molecules along [001]. Such different molecular chains stack each other in 3 3 3 ABABAB 3 3 3 fashion, connected by O9water-H 3 3 3 O8carboxylate and O10water-H 3 3 3 O4carboxylate hydrogen bonds along [100].

Figure 5. Imiquimod fumarate (2:1) hexahydrate crystal structure, 5. Notice zigzag molecular chains formed by fumarate and protonated imiquimod molecules through heterosynthon V and homosynthon II. Such chains are connected by water hexamers through Owater-H 3 3 3 O-carboxylate hydrogen bonds. A few atoms on imiquimod were deleted for clarity.

molecular chains through Owater-H 3 3 3 O-carboxylate hydrogen bonds (Figure 5, Table 3). Here we describe some inaccuracies in the WO 2006/070408 A2 patent and the importance of characterization of crystalline forms of a compound by single crystal X-ray diffraction. The patent claims three polymorphs of imiquimod fumarate salt (forms I, II, and III). The simulated PXRD of the salt 5 crystal structure matches with the patented fumarate form III. Hence, form III is

Figure 7. Crystal structure of imiquimod adipate salt hydrate, 7. Molecular chains are formed by adipate and protonated imiquimod molecules through heterosynthons V and homosynthons II. Such chains are connected by water tetramers through Owater-H 3 3 3 O-carboxylate hydrogen bonds. A few atoms on imiquimod were deleted for clarity.

most likely 2:1 imiquimod fumarate hexahydrate. However, the patent does not describe the stoichiometry and hydrate form of the salt. In the patent, the protonation site was shown at the amine (as -NH3þ).11 However, the salt 5 crystal structure and other structures (salts 2-4, 6-8) unambiguously prove that the pyridine nitrogen was protonated in all the salts. The asymmetric unit of salt 6 crystal structure consists of one molecule of protonated imiquimod, half succinate and two water molecules (monoclinic system, P21/c space group; Tables 1 and 2). Hence, salt 6 is 2:1 imiquimod succinate tetrahydrate. Succinate occupied the inversion center special position. Crystal packing shows that one succinate is connected to two protonated imiquimods through heterosynthons V and protonated imiquimods also forms homosynthon II. Such an arrangement resulted in the formation of a zigzag molecular chain along [110]. Four water molecules connect the molecular chains through Owater-H 3 3 3 O-carboxylate hydrogen bonds (Table 3) as shown in Figure 6. Another aliphatic dicarboxylic acid, adipic acid, also crystallized in 1:2 stoichiometry with imiquimod. The crystal structure of salt 7 shows that the asymmetric unit consists of one molecule of protonated imiquimod, half adipate and two water molecules (triclinic, P1 space group; Tables 1 and 2). Hence, salt 7 is 2:1 imiquimod adipate tetrahydrate. Adipate resided on an inversion center special position, similar to fumarate and succinate in salts 5 and 6, respectively. Crystal packing shows that one adipate is connected to two protonated imiquimods through heterosynthons V and protonated imiquimods also forms homosynthon II. Such arrangement of molecules results in the formation of zigzag molecular chains

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carboxylate and protonated imiquimod molecules through heterosynthons V and homosynthons II. Such chains are connected by water molecules through Owater-H 3 3 3 O-carboxylate hydrogen bonds. 4. Conclusions

Figure 8. Crystal structure of imiquimod 4-hydroxybenzoate salt, 8. 4-Hydroxybenzoate molecules are connected in a head-to-tail fashion through O-H 3 3 3 O- hydrogen bonds. Protonated imiquimod homodimers are connected to 4-hydroxybenzoic acid chains through heterosynthons V. A few atoms on imiquimod were deleted for clarity.

along [101]. Four water molecules form a water tetramer through O-H 3 3 3 O hydrogen bonds and connect the molecular chains through Owater-H 3 3 3 O-carboxylate hydrogen bonds (Table 3) as shown in Figure 7. The only monocarboxylic acid in this study, 4-hydroxybenzoic acid, crystallized in 1:1 stoichiometry with imiquimod. The asymmetric unit consists of 4-hydroxybenzoate and protonated imiquimod (monoclinic, P21/n; Tables 1 and 2). In the salt 8, 4-hydroxybenzoate molecules are connected in a head-to-tail fashion through charge-assisted O-H 3 3 3 Ohydrogen bonds along [101]. Protonated imiquimod homodimers (synthon II) are connected to 4-hydroxybenzoate molecular chains through heterosynthons V (Figure 8). Interestingly, all the imiquimod salts (except 8) are crystallized as hydrates. Imiquimod maleate salt (4) crystallized as sesquihydrate, whereas the trans-isomer, fumarate salt (5), crystallized as hexahydrate. Two aliphatic-R,ω-dicarboxylic acid salts of imiquimod (6 and 7) crystallized as tetrahydrates. Interestingly, all the dicarboxylic acid salts of imiquimod are in the hydrate form. The imiquimod hydrochloride salt exists in both the anhydrous and monohydrate forms (2 and 3). Only the salt 8 is in anhydrous form. The CSD and present 4-8 crystal structures suggest that 2-aminopyridinium cation and carboxylate anion forms robust 2-aminopyridiniumcarboxylate supramolecular heterosynthon V through charge-assisted Nþ-H 3 3 3 O- and N-H 3 3 3 O- hydrogen bonds. Since water consists of strong oxygen acceptor and two hydrogen bond donors, it has the ability to disrupt even robust synthons. Interestingly, even though water is present in 4-7 carboxylate salts, the heterosynthon V and homosynthon II are persistent, thus substantiating the robustness. In the three 2:1 salts 5-7, molecular chains are formed by

We have obtained and structurally characterized imiquimod and its seven salts with GRAS substances such as HCl, maleic acid, fumaric acid, succinic acid, adipic acid, and 4-hydroxybenzoic acid. Imiquimod formed salts with fumaric acid, succinic acid, and adipic acid in 2:1 molar ratio; however, it has crystallized in 1:1 stoichiometry with maleic acid and 4-hydroxybenzoic acids. In all the salts 2-8, the basic pyridine moiety was protonated by the salt formers. Except salt 8, all the imiquimod salts are crystallized as hydrates. Imiquimod maleate salt 4 crystallized as sesquihydrate, whereas fumarate salt 5 crystallized as hexahydrate. Two aliphaticR,ω-dicarboxylic acid salts of imiquimod (6 and 7) are crystallized as tetrahydrates. Interestingly, all the dicarboxylic acid salts of imiquimod are in the hydrate form. Two forms (anhydrous and hydrous) of imiquimod hydrochloride salt have been isolated (salt 2 and 3) and characterized. Imiquimod free base 1 forms a molecular tape through 2-aminopyridineaminoimidazole heterosynthon III. All the carboxylic acid salts of imiquimod 4-8 form robust 2-aminopyridinium-carboxylate supramolecular heterosynthon V and aminoimidazole homosynthon II. The present examples suggest that co-crystallization experiments are useful not only to make co-crystals but also to discover unexpected novel forms of APIs. The loss of HCl during the crystallization will be studied in the future. Acknowledgment. We are grateful to the management of Dr. Reddy’s Discovery Research for their encouragement. We thank Dr. Reddy’s Holding Ltd. for the gift of imiquimod free base and its HCl salt.

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