Cocrystals of Ethenzamide: Study of Structural and Physicochemical

Article pubs.acs.org/crystal

Cocrystals of Ethenzamide: Study of Structural and Physicochemical Properties Vijaya M. Hariprasad, Sunil Kumar Nechipadappu, and Darshak R. Trivedi* Supramolecular Chemistry Laboratory, Department of Chemistry, National Institute of Technology Karnataka (NITK)-Surathkal, Srinivasnagar 575 025, Karnataka, India S Supporting Information *

ABSTRACT: Pharmaceutical cocrystals of an analgesic drug ethenzamide (ETZ) with various coformers, namely, gallic acid (GA), 2-nitrobenzoic acid (2NB), 3-nitrobenzoic acid (3NB), 2,4-dinitrobenzoic acid (DNB), and 3-toluic acid (3TA) were synthesized by the solvent evaporation method. All the cocrystals were characterized by various analytical techniques, and the crystal structures were determined by the single-crystal X-ray diffraction method (SCXRD). SCXRD analysis revealed that all the synthesized cocrystals were formed through a robust supramolecular acid-amide heterosynthon except the ethenzamide/gallic acid cocrystal, where molecules interacted through O−H···O hydrogen bond involving −OH of gallic acid and oxygen of amide group of the ETZ molecule. The physicochemical properties such as stability, hygroscopicity, and solubility studies of the ETZ−GA cocrystal were evaluated. It was found that the ETZ−GA cocrystal has a higher solubility (2fold) than that of the pure ETZ drug molecule. Hygroscopic study of the ETZ−GA cocrystal revealed that synthesized cocrystal was non-hygroscopic at ∼75% RH conditions. The ETZ−GA cocrystal found to be stable for a time period of four months at ambient temperature.

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INTRODUCTION The field of crystal engineering has emerged as one of the most important areas of chemical research that bridges the solid state and supramolecular chemistry.1−4 Concepts of crystal engineering pave a new way for the design and synthesis of pharmaceutical cocrystals, which are defined as the molecular complexes of active pharmaceutical ingredients (APIs) with one or more cocrystal formers that are solids at ambient temperature,5−12 where complexes are formed via various noncovalent interactions including hydrogen bond, π−π stacking, halogen bond, van der Waals interactions, etc.13−18 Pharmaceutical cocrystals offer improvement in the physical properties of drug ingredients without affecting the biological activity of the API.19−21 Properties like solubility, stability, bioavailability, and dissolution rate may be improved by cocrystallization.22−25 In recent decades, pharmaceutical cocrystallization with biologically accepted generally recognized as safe (GRAS) coformers has shown significant improvement in the physicochemical properties of the drug molecule, and it offers a new possibility for generating patents and intellectual property.26,27 In the present study, pharmaceutical cocrystals of ethenzamide (2-ethoxybenzamide, ETZ) were synthesized by a crystal engineering approach with various aromatic carboxylic acid derivatives. Coformers used in the present study were gallic acid hydrate (GA), 2-nitrobenzoic acid (2NB), 3-nitrobenzoic acid (3NB), 2,4-dinitrobenzoic acid (DNB), and 3-toluic acid © 2016 American Chemical Society

(3TA) as shown in Scheme 1. Ethenzamide is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties.28−30 It is mainly used in combination with other active ingredients such as acetaminophen, aspirin, dipyrone, allyl iso-propyl acetyl urea, caffeine, and ibuprofen.31,32 The crystal structure of the ETZ molecule was reported recently in the literature.33 The main drawback of this drug is the lower solubility and bioavailability, which makes it a superior challenge to enhance its solubility behavior through cocrystallization. Attempts at enhancing the dissolution rate of ETZ drug molecule have been done with low molecular weight sugars34 and the drug release controlling carrier Carbopol.35 Amorphous crystalline material of ETZ was prepared with porous crystalline cellulose material, and it showed higher dissolution rate compared to pure drug.36 Gallic acid used in the present study is a GRAS molecule according to US FDA, which is a naturally occurring low molecular weight triphenolic compound, and it is a strong antioxidant and an efficient apoptosis inducing agent.37−39 It is known that GA exhibits diverse therapeutically beneficial activities.37−39 It displays antibacterial, antiviral, antitubercular, antifungal, anticholesterol, antimutagenic, antiulcer, antiobesity, and immunomodulatory activities.37−39 Apart from this, it is Received: April 21, 2016 Revised: June 22, 2016 Published: July 5, 2016 4473

DOI: 10.1021/acs.cgd.6b00606 Cryst. Growth Des. 2016, 16, 4473−4481

Crystal Growth & Design

Article

Scheme 1. Chemical Diagrams of ETZ and the Coformer Used in the Present Study

Table 1. Crystal Structure Details and the Refinement Parameters of ETZ Cocrystals crystal structure details

ETZ−GA

ETZ−2NB

ETZ−3NB

ETZ−DNB

ETZ−3TA

CCDC number mol. formula mol. mass crystal system space group a/Å b/Å c/Å α/° β/° γ/° volume/Å3 Z density [g/cm3] μ (MoKα) [mm−1] T/K reflns collected unique rflns parameter refined R1 (I > 2σ) wR2 (I > 2σ) GOF

1468148 C25H28N2O9 500.49 triclinic P1̅ 10.075(2) 11.906(2) 12.391(3) 74.771(10) 69.131(9) 69.777(10) 1286.5(5) 2 1.292 0.099 296(2) 5065 4236 415 0.0382 0.1081 0.689

1468153 C16H16N2O6 332.31 triclinic P1̅ 7.9899(3) 8.4054(3) 13.2022(5) 88.937(2) 74.884(2) 69.294(2) 798.00(5) 2 1.383 0.107 296 (2) 3038 2268 270 0.0619 0.1755 0.997

1468154 C16H16N2O6 332.31 monoclinic P21/c 12.7494(5) 15.1442(6) 8.3196(3) 90 92.840(2) 90 1604.37(11) 4 1.376 0.107 296(2) 3164 2485 270 0.0542 0.1668 1.199

1468159 C16H15N3O8 377.31 monoclinic P21/n 14.7377(5) 5.2226(2) 22.7947(8) 90 100.466(2) 90 1725.30(11) 4 1.453 0.119 296 (2) 3401 2933 293 0.0403 0.1329 1.034

1468161 C17H19NO4 301.33 monoclinic P21/n 8.3788(3) 10.8518(3) 17.9006(5) 90 99.358(2) 90 1605.95(9) 4 1.246 0.089 296(2) 3159 2596 264 0.0391 0.1352 1.128

synthesized cocrystals of ethenzamide with benzoic acid, 2fluorobenzoic acid, 2-iodobenzoic acid, salicylic acid, 2,4dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and 3,5-dihydroxybenzoic acid.46 Cocrystallization experiments in the present study were carried out with GRAS and non-GRAS compounds to study the structural and physicochemical properties of ethenzamide API (precisely to determine the solubility of ethenzamide drug in pharmaceutically acceptable cocrystal). Cocrystal screening experiments yielded five novel cocrystals of ethenzamide, out of which the ETZ−GA cocrystal is considered as pharmaceutically safe and acceptable.

having potential neuroprotective, cardioprotective, hepatoprotective, and nephroprotective abilities.37−39 Additionally GA derivatives are also found to exhibit biological and pharmacological activities such as radical scavenging, interfering with the cell signaling pathways, and apoptosis of cancer cells.37−39 Therefore, pharmaceutical cocrystallization of ETZ with gallic acid offers the development of new drug ingredients of the ethenzamide drug molecule. Various pharmaceutical cocrystals of ethenzamide have been reported in recent years with GRAS and non-GRAS compounds. A 1:1 cocrystal of ETZ and thiourea have been reported in the literature.40 Aitipamula et al. synthesized various polymorphic cocrystals of ethenzamide with saccharin,41 gentisic acid,42 ethyl malonic acid,43 and 3,5-dinitro benzoic acid.44 They also synthesized pharmaceutical cocrystals with salicylic acid, 2-chloro-4-nitrobenzoic acid, vanillic acid, 4aminobenzoic acid, 4-hydroxybenzoic acid, and fumaric acid with enhanced solubility and dissolution rate.45 Przybylek et al.

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RESULTS AND DISCUSSION Pharmaceutical cocrystals of ETZ were synthesized by the solvent evaporation method. Initially, FT-IR and DSC techniques were performed for the confirmation of cocrystal formation. All the cocrystals showed different melting points in 4474



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DSC analysis and a shift in the carbonyl stretching frequency in IR spectra with respect to their corresponding starting materials. Further cocrystals were characterized by SCXRD, PXRD, and 1H NMR techniques. SCXRD Analysis of Cocrystal. A 2:1 cocrystal of ETZ and GA was obtained by a solvent evaporation method in an ethanol solvent. Crystal structure and the refinement parameter details are listed in Table 1. The asymmetric unit of the cocrystal consists of two molecules of ETZ and one molecule of GA which belongs to the triclinic crystal system with P1̅ space group (Figure 1). Cocrystal has formed through the O−H···O

Figure 3. Cyclic network-like structure of ETZ−GA cocrystal.

(Figure 4). The primary synthon is a two-point supramolecular acid-amide heterosynthon between 2NB and ETZ with O−H··· O and N−H···O bond distance of 2.538 Å, ∠173.26° and 2.877 Å, ∠171.70° respectively. Two adjacent dimer units are interconnected through weak complementary C−H···O hydrogen bond (3.474 Å, ∠168.35°) involving aromatic C−H of ETZ and the O atom of the acid group of 2NB to form a cyclic tetrameric structure (Figure 5). These tetrameric units further self-assembled through complementary C−H···O hydrogen bond (3.473 Å, ∠148.53°) between the methyl group of ETZ and the O atom of the acid group of 2NB resulting in a linear 1D sheet-like structure (Figure 6). Overall crystal structure features weak interactions such as C−H···O hydrogen bond between 2NB molecules (3.191 Å), C−H···O hydrogen bond (3.436 Å) between ETZ and 2NB molecules, C−H···π interactions (3.549 Å) between ETZ and 2NB and π−π interaction between 2NB molecules (centroid to centroid distance of 3.735 Å). A 1:1 cocrystal of ETZ with 3NB resulted in acetonitrile solvent by the solvent evaporation method. Asymmetric unit in the cocrystal consists of one molecule each of ETZ and 3NB which belongs to the monoclinic crystal system with the space group P21/c (Figure 7). Cocrystal has formed through robust supramolecular acid-amide heterosynthon between 3NB and ETZ with O−H···O and N−H···O hydrogen bond distance of 2.562 Å (∠174.46°) and 2.923 Å (∠163.8°) respectively. Neighboring supramolecular units in the crystal structure are interconnected to each other through C−H···O hydrogen bond involving aromatic C−H of ETZ with the O atom of the acid group of 3NB (3.472 Å), C−H···O hydrogen bond (3.493 Å) involving aromatic C−H of ETZ with O of nitro group of 3NB and C−H···O hydrogen bond (3.325 Å) involving C−H of 3NB and O of the nitro group of 3NB resulting in a cyclic six component supramolecular unit (Figure 8). The supramolecular units further self-assembled through the C−H···O hydrogen bond to give a 2D sheet-like structure of ETZ−3NB cocrystal (Figure 9). The 2D sheet further extended to a 3D sheet through weak C−H···O (3.494 Å), C−H···π (3.589 Å), and π−π interactions (3.974 Å), which are responsible for the overall crystal structure stabilization of ETZ−3NB cocrystal. A 1:1 cocrystal of ETZ−DNB was obtained by a solvent evaporation method in an ethanol solvent. The asymmetric unit in the cocrystal consists of one molecule each of ETZ and DNB, which belongs to the monoclinic crystal system with P21/n space group (Figure 10). Primary supramolecular synthon is a two-point robust acid-amide heterosynthon between DNB and ETZ with the O−H···O and N−H···O hydrogen bond distance of 2.538 Å, ∠174.05° and 2.864 Å, ∠173.25° respectively. The two adjacent supramolecular units are interconnected to each other to form a cyclic tetrameric unit through complementary C−H···O hydrogen bond (3.233 Å, ∠147.30°) between aromatic C−H of ETZ and O atom of

Figure 1. Asymmetric unit of ETZ−GA cocrystal.

hydrogen bond involving the hydroxyl group of GA and the oxygen of the amide group of ETZ.47 All three hydroxyl groups of GA were involved in hydrogen bond formation with ETZ (O−H···O bond distance of 2.650 Å, ∠172.2°, 2.826 Å, ∠158.11° and 2.743 Å, ∠155.23°). Two molecules of ETZ interconnected with each through amide···amide homosynthon (N−H···O bond distance of 2.970 Å, ∠178.10° and 2.988 Å, ∠177.16°). In the crystal structure, GA forms a dimer through supramolecular acid−acid homosynthon with a complementary O−H···O hydrogen bond (2.621 Å, ∠175.32°), which in turn is connected to the neighboring supramolecular unit resulting in a cyclic eight component cage-like structure of ETZ−GA cocrystal (Figure 2). These cage-like supramolecular units

Figure 2. Eight component cyclic cage-like structure of ETZ−GA cocrystal.

further self-assembled through O−H···O (2.826 Å) and N− H···O (3.041 Å) hydrogen bonds between ETZ and GA resulting in a cyclic network-like structure of ETZ−GA cocrystal (Figure 3). Overall crystal structure features weak C−H···O hydrogen bond involving the −CH2 group of ETZ and the O of the acid group of GA (3.301 Å, ∠125.29°), C− H···O hydrogen bond involving aromatic C−H of ETZ, and the O atom of the acid group of GA (3.443 Å, ∠148.67°) and C− H···O hydrogen bond (3.286 Å, ∠124.85°) involving aromatic C−H of GA and O of ETZ. Geometrical parameters of hydrogen bond are documented in Table 2. A 1:1 cocrystal of ETZ and 2NB was yielded by a solvent evaporation method in an ethanol solvent. The asymmetric unit consists of one molecule each of ETZ and 2NB molecule which belongs to the triclinic crystal system with P1̅ space group 4475



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Table 2. Geometrical Parameters of the Intermolecular Interactions in the ETZ Cocrystals cocrystal ETZ−GA

ETZ−2NB

ETZ−3NB

ETZ−DNB

ETZ−3TA

a

D−H···Aa

H···A/Å

D···A/Å

∠D−H···A/°

symmetry code

O7−H9···O4 N1−H1···O2 O6−H8···O2 O6−H8···O7 N1−H2···O3 O8−H11···O9 O5−H7···O6 O5−H7···O2 C24−H3···O9 N2−H17···O1 N2−H18···O4 C16−H19···O4 C16−H14···O4 N2−H1···O1 C4−H10···O6 N2−H2···O4 O3−H3···O2 N1−H3···O1 N1−H2···O2 O3−H1···O4 C2−H13···O4 N1−H2···O7 N1−H1···O6 O5−H12···O8 N1−H1···O3 N1−H2···O1 O4−H3···O2

1.752 2.088 2.011 2.273 1.959 1.732 2.283 1.897 2.638 1.957 2.066 2.636 2.557 1.927 2.633 2.059 1.698 1.965 2.080 1.631 2.629 1.948 2.006 1.553 2.056 1.936 1.714

2.650 2.988 2.826 2.712 2.628 2.621 2.731 2.743 3.301 2.639 2.970 3.286 3.412 2.619 3.191 2.877 2.538 2.640 2.923 2.562 3.494 2.633 2.864 2.538 2.913 2.637 2.579

172.19 177.11 158.10 111.79 130.94 175.07 110.38 155.30 125.43 132.94 178.34 124.88 148.47 133.76 118.17 171.99 173.15 135.89 163.74 174.01 145.14 134.64 173.23 174.39 173.11 134.20 172.07

x−1, y, z x+1, y, z −x, −y+1, −z+1 x, y, z x, y, z −x, −y+2, −z x, y, z x+1, y, z −x, −y+2, −z+1 x, y, z x−1, y, z x−1, y, z −x, −y+1, −z+1 x, y, z −x, −y+2, −z+2 x, y, z x, y, z x, y, z x, y, z x, y, z −x+1, −y+1, −z+2 x, y, z x, y−2, z x, y+2, z x, y, z x, y, z x, y, z

D = donor, A = acceptor.

Figure 7. Asymmetric unit of ETZ−3NB cocrystal.

Figure 4. Asymmetric unit of ETZ−2NB cocrystal.

Figure 8. Six component supramolecular unit of ETZ−3NB cocrystal. Figure 5. Tetrameric unit of ETZ−2NB cocrystal.

Figure 9. 2D sheet-like structure of ETZ−3NB cocrystal. Figure 6. 1D sheet-like structure of ETZ−2NB cocrystal.

hydrogen bond involving C−H of the methyl group of ETZ and O atom of the nitro group of DNB (3.627 Å, ∠174.16°) resulting in a linear 1D tape-like structure of ETZ−DNB

the nitro group of DNB (Figure 11). Each tetrameric unit is further connected to each other through weak C−H···O 4476



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Figure 10. Asymmetric unit of ETZ−DNB cocrystal. Figure 14. Tetrameric unit of ETZ−3TA cocrystal.

and O of ETZ (3.470 Å, ∠161.81°) and C−H···O hydrogen bond between −C−H of the −C−H2 group of ETZ and O of ETZ (3.487 Å, ∠138.54°) resulting in a ladder-like structure of ETZ-3TA cocrystal. Stacking interaction was not observed in this cocrystal. Comparison of Crystal Structure Properties between ETZ−2NB, ETZ−3NB, ETZ−DNB, and ETZ−3TA Cocrystal. In ETZ−2NB and ETZ−3NB cocrystals apart from the strong acid-amide heterosynthon, various secondary interactions were involved such as C−H···O, C−H···π, and π−π interactions. ETZ−2NB was crystallized in the triclinic crystal system, whereas ETZ−3NB was crystallized in the monoclinic crystal system. The only differences observed in these cocrystals were the secondary interactions associated with the nitro group. In ETZ−2NB cocrystal, both oxygen atoms of the nitro group are involved in hydrogen bond formation, whereas in ETZ−3NB only one oxygen atom is involved in interaction with bifurcated hydrogen bond formation. As a result, only a slight change in crystal density was observed (less than ETZ−2NB cocrystal). When compared to ETZ−DNB cocrystal, both nitro groups of DNB were involved in secondary interactions. In addition to weak C−H···O hydrogen bond, strong N−H···O hydrogen bond involving the −N−H group of ETZ and O of the ortho nitro group of DNB have been noticed which lead to high crystal density of ETZ−DNB cocrystal.48 In ETZ−3TA cocrystal, where the nitro group is replaced by a methyl group, only a weak C−H···O hydrogen bond associated with the methyl group was observed. No stacking interactions were observed between these molecules which resulted in loosely bound crystal packing which further led to low crystal density of ETZ−3TA cocrystal when compared to all other cocrystals.49 Among these four cocrystals, ETZ−3TA has the lowest melting point, which is attributed to its lower density than all other cocrystals. ETZ−DNB with a greater number of secondary interactions has the highest melting compared to all other cocrystals. No correlation was observed in the melting points of ETZ−2NB and ETZ−3NB cocrystals with respect to their crystal density. PXRD Analysis. PXRD is the basic requisite for the identification and characterization of the synthesized cocrystal. Cocrystal formation was confirmed by PXRD analysis by comparing the diffraction pattern of cocrystal with their respective starting material. All the synthesized cocrystals exhibited different diffraction patterns with respect to ETZ and the coformers, which shows the formation of new crystalline phase/cocrystal. Further PXRD analysis data gives information about the bulk purity of synthesized cocrystal by equating the experimental XRD pattern with the calculated pattern obtained from SCXRD analysis. All the synthesized cocrystals were found to be pure in the bulk (PXRD plots are displayed in Supporting Information).

Figure 11. Tetrameric unit of ETZ−DNB cocrystal.

cocrystal (Figure 12). 1D tape further extends to a 3D structure through the N−H···O hydrogen bond between N−H of ETZ

Figure 12. 1D tape-like structure of ETZ−DNB cocrystal.

and O atom of the nitro group of DNB (3.055 Å, ∠106.09°) and π−π interactions between DNB−DNB and ETZ−ETZ with a centroid to centroid distance 5.223 and 5.223 Å respectively. A 1:1 cocrystal of ETZ and 3TA was obtained in methanol solvent by the solvent evaporation method. The asymmetric unit consists of one molecule each of ETZ and 3TA which belongs to the monoclinic crystal system with P21/n space group (Figure 13). A two-point supramolecular acid-amide

Figure 13. Asymmetric unit of ETZ−3TA cocrystal.

heterosynthon between ETZ and 3TA was observed in ETZ− 3TA cocrystal with O−H···O and N−H···O hydrogen bond distance of 2.579 Å, ∠172.18° and 2.913 Å, ∠173.23° respectively. Adjacent supramolecular units are interconnected through two different complementary C−H···O hydrogen bonds (C4−H5···O2, 3.568 Å, ∠165.39° and C5H4···O4, 3.417 Å, ∠136.40°) resulting in a cyclic tetrameric unit of ETZ−3TA cocrystal (Figure 14). Each tetrameric unit is further connected to each other in a 3D manner through C−H···O hydrogen bond between −C−H of the methyl group of 3TA 4477



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−OCH2, and −CH3 protons of ETZ were observed at 7.57− 7.59 ppm, 4.15−4.18 ppm, and 1.38−1.40 ppm respectively (NMR spectra are displayed in Supporting Information). DSC Analysis. DSC, a thermoanalytical technique, gives endothermic phase transitions of the crystal when it changes from solid state to liquid state. DSC analysis was conducted to find the temperature range at which melting of cocrystals occurs and to study their thermodynamic behavior. One can differentiate the monotropes from enantiotropes by considering the phase transition in DSC.50 The peak temperature for the melting of the cocrystals is shown in Table 3. ETZ−GA

Spectroscopic Analysis of Cocrystal. FT-IR Spectroscopy. The synthesized cocrystals were analyzed by FT-IR spectroscopy. The FT-IR spectra of cocrystals were compared with that of the starting materials in order to confirm the cocrystal formation. In the pure ETZ, GA, 2NB, 3NB, DNB, and 3TA carbonyl stretching frequency appeared at 1625 cm−1, 1697 cm−1, 1671 cm−1, 1685 cm−1, 1717 and 1684 cm−1 respectively. FT-IR analysis displayed a significant shift in the −CO stretching frequency of synthesized cocrystals compared to their respective starting materials. For ETZ− GA, ETZ−2NB, ETZ−3NB, ETZ−DNB, and ETZ−3TA cocrystals −CO stretching frequency was observed at 1671 cm−1, 1694 cm−1, 1691 cm−1, 1693 and 1689 cm−1 respectively. Further to the carbonyl stretching frequency, a shift in N−H stretching frequency was observed. Pure ETZ exhibited N−H stretching frequency at 3365 cm−1, whereas for ETZ−GA, ETZ−2NB, ETZ−3NB, ETZ−DNB, and ETZ−3TA cocrystals, it was observed at 3431 cm−1, 3423 cm−1, 3437 cm−1, 3426 and 3444 cm−1 respectively (FT-IR spectra with stretching frequencies are displayed in Supporting Information). 1 H NMR Spectroscopy. 1H NMR spectroscopy was performed in order to confirm the structure and molecular stoichiometry of various synthesized cocrystals. All the synthesized cocrystals except ETZ−GA cocrystal exhibited 1:1 molecular stoichiometry. In ETZ−GA cocrystal 1:0.5 molecular stoichiometry was observed, and it supports the crystal structure derived from SCXRD analysis. In ETZ−GA cocrystal singlet peak at 12.244 ppm corresponds to the carboxylic acid proton of GA. The hydroxyl proton of GA was observed at 9.20 and 8.85 ppm. Peaks at 7.81 ppm, 7.45 ppm, 7.12 ppm, and 7.01 ppm correspond to aromatic −CH proton of ETZ. −NH2, −OCH2, and −CH3 protons of ETZ were observed at 7.57−7.59 ppm, 4.15−4.17 ppm, and 1.382−1.40 ppm, respectively. Aromatic proton corresponds to GA was observed at 6.92 ppm. The acid proton of 2NB in ETZ−2NB cocrystal exhibited a singlet at 13.885 ppm. A peak corresponding to aromatic −CH of ETZ was observed at 7.45 ppm, 7.11−7.13 ppm, and 7.01−7.03 ppm. −NH2, −OCH2, and −CH3 protons of ETZ were observed at 7.57−7.59 ppm, 4.15−4.18 ppm, and 1.38−1.40 ppm, respectively. Aromatic −CH protons of 2NB were observed at 7.86−7.99 ppm and 7.78−7.81 ppm, respectively. In ETZ− 3NB cocrystal singlet at 13.740 ppm corresponds to the acid group of 3NB. The peak at 7.82 ppm, 7.45 ppm, 7.12 ppm, and 7.01−7.03 ppm corresponds to aromatic ETZ protons. −NH2, −OCH2, and −CH3 protons of ETZ displayed at 7.57−7.59 ppm, 4.16−4.17 ppm, and 1.39−1.40 ppm, respectively. A peak corresponding to aromatic 3NB was observed at 8.63 ppm, 8.48, 8.36, and 8.12 ppm, respectively. The acid proton of DNB in ETZ−DNB cocrystal was observed at 14.60 ppm as a singlet. Aromatic proton corresponding to DNB was observed at 8.79 ppm, 8.58−8.59 ppm, and 8.11−8.12 ppm, respectively. The peak at 7.81 ppm, 7.45 ppm, 7.11−7.12 ppm, and 7.01−7.02 ppm exemplifies the aromatic ETZ protons. −NH2, −OCH2, and −CH3 protons of ETZ exhibited peak at 7.57−7.59 ppm, 4.15−4.18 ppm, and 1.38−1.40 ppm, respectively. In ETZ−3TA cocrystal, a singlet peak at 12.891 ppm corresponds to acid proton of 3TA. Aromatic protons of 3TA were observed at 7.74−7.76 ppm, 7.37−7.43 ppm, respectively. The methyl group of 3TA was observed at 2.36 ppm. The peak at 7.81, 7.12, and 7.01 ppm represents aromatic ETZ protons. One aromatic proton of ETZ was merged with the 3TA peak at 7.37−7.43 ppm. −NH2,

Table 3. Melting Point of ETZ, Coformers, and Cocrystals API (mp °C)

coformer (mp °C)

cocrystal (mp °C)

(ETZ) 135.84

GA hydrate (256.53) 2NB (152.54) 3NB (144.06) DNB (187.90) 3TA (114.87)

156.39 103.01 111.04 125.48 79.17

cocrystal showed the melting point in between that of its starting materials, whereas all the other cocrystals melt at a lower temperature than that of their respective starting materials. The higher melting temperature of ETZ−GA cocrystal, when compared to other cocrystals, may be due to the strong hydroxyl interaction of GA with ETZ. The presence of a single endothermic peak in all the cocrystals indicates there is no phase conversion in the synthesized cocrystals51 (DSC plots are displayed in Supporting Information). Stability of Cocrystals. Owing to the significant pharmaceutical relevance of the synthesized ETZ−GA cocrystal, stability studies were evaluated in the present study. The hygroscopic nature of the cocrystal was studied by conventional weight loss method52 by keeping a known quantity of the sample in a desiccator which was filled with saturated sodium chloride solution that maintains the humidity of about ∼75% RH. The weight of the sample was recorded periodically for a time period of 1 week to check the absorbance of water molecules. The analysis showed that synthesized cocrystal was non-hygroscopic as there were no changes in weight during the study period. This was further confirmed from FT-IR and PXRD analysis. In FT-IR spectrum, the ETZ− GA cocrystal under hygroscopic study did not show any significant shift in carbonyl peak when compared to pure ETZ− GA cocrystal (Figure 15). In PXRD analysis, no changes in the diffraction pattern were observed before and after hygroscopic study (Figure 16). From the above analytical results, it was concluded that ETZ−GA cocrystal is non-hygroscopic at an accelerated humidity condition. Further to hygroscopic study, the stability of the crystalline phase of ETZ−GA cocrystal was carried out for a time period of four months. Phase purity of the cocrystal was analyzed by PXRD. ETZ−GA cocrystal was found to be stable even after four months at ambient temperature (Figure 17). Solubility Study. The major concern in numerous pharmaceutical products in the market is having poor solubility in the aqueous media. The ETZ drug molecule selected in the present study has a very low aqueous solubility. Solubility can be enhanced either by salt formation or by cocrystal formation. The ETZ molecule does not have any ionization site for salt formation; therefore, improvement in the solubility of ETZ drug molecule can only be done by cocrystallization. In the 4478



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Figure 15. Comparison of FT-IR spectrum of ETZ−GA cocrystal before and after hygroscopic study. ETZ-GA H represents spectra represents the hygroscopic samples.

Figure 16. Comparison of PXRD pattern of ETZ−GA cocrystal before and after hygroscopic study.

taken in a minimum quantity of 10% ethanol/water medium. The resulting mixture was then kept under continuous stirring for 24 h at room temperature (∼25 °C). Upon equilibrium (after 24 h) aliquots were taken and filtered through a 0.2 μ syringe filter. The concentration of ETZ was measured by UV− vis spectroscopy. Solubility data are given below in the Table 4. The equilibrium solubility data revealed about a 2-fold increment in the solubility of ETZ in ETZ−GA cocrystal than the parent ETZ API.

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CONCLUSION Five novel pharmaceutical cocrystals of ETZ with different coformers such as GA, 2NB, 3NB, DNB, and 3TA were prepared using a conventional solvent evaporation method. All the synthesized cocrystals were characterized by various Table 4. Solubility of ETZ and Its Cocrystal with GA in 10% Ethanol/Water Medium at Room Temperature

Figure 17. Comparison of FT-IR spectra of ETZ−GA cocrystal initial and after four months.

present study solubility measurement was conducted for ETZ− GA cocrystal in 10% ethanol/water medium at room temperature (∼25 °C). In the solubility study, an excess of finely powdered samples of ETZ and ETZ−GA cocrystal was

solid form

equilibrium solubility of ETZ (mg/L)a

ETZ ETZ−GA

1942.80 3744.0 (×1.92)

a

The values in the parentheses specifies the extent of increase in solubility of cocrystal relative to the pure ETZ. 4479



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analytical methods including SCXRD, PXRD, 1H NMR, FT-IR, and DSC. Crystal structure analysis of ETZ-GA cocrystal revealed that a robust supramolecular acid-amide heterosynthon was replaced by the O−H···O hydrogen bond, and in all other reported cocrystals ETZ−2NB, ETZ−DNB, ETZ−3TA, and ETZ−3NB a robust supramolecular acid-amide heterosynthon was observed. Physicochemical properties of ETZ−GA cocrystal suggest that the synthesized cocrystal is found to be stable at accelerated humid conditions (∼75% RH). Solubility study of ETZ−GA cocrystal revealed a 2-fold increment in the solubility of ETZ compared to pure ETZ drug molecule. ETZ− GA cocrystal was found to be stable for a time period of four months at ambient temperature. As a result, it is reasonable that ETZ−GA cocrystal is a promising candidate for improving solubility and development of new solid forms of the ETZ drug molecule.

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radiation (λ = 0.7107 Å) was used. Data collection was done at ambient temperature (296 K). All the crystal structures were solved by the direct method using SHELXL-2007/2014 software, and the refinement was carried out by full-matrix least-squares technique using SHELXL-2007/2014. For all the non-hydrogen atoms anisotropic displacement parameters were calculated. Hydrogen atoms which are attached to the nitrogen and oxygen atoms were located in a difference Fourier density map and refined isotropically. All the diagrams were prepared using mercury software of 3.5.1 version. Powder X-ray Diffraction (PXRD). PXRD analysis were carried out using a Joel (JDX-8P) powder X-ray diffractometer with Cu Kα radiation (λ = 1.54059 Å) as a radiation source. The voltage and current applied were 40 kV and 30 mA respectively. For the experimental analysis, samples were placed on the standard sample holder and then scanned continuously from 5 to 60° with the scan rate of 2° min−1. 1 H NMR Spectroscopy (NMR). 1H NMR analysis was carried out on a Bruker Biospin 400 MHz spectrometer (Bruker, Germany). 1H NMR was recorded in a DMSO-d6 solvent with tetramethyl silane (TMS) as the internal reference standard. For the analysis about 5−10 mg of the samples were dissolved in a DMSO-d6 solvent, and the spectra recorded with 16 scans. Infrared Spectroscopy (IR). IR spectra was recorded on a Bruker Alpha Fourier transform infrared spectrophotometer (FT-IR) which was equipped with silicon carbide as the IR source. An attenuated total reflectance accessory with a zinc selenide (ZnSe) crystal was used for the data acquisition. The samples under study were recorded with 16 scans with a sample resolution of 4 cm−1. Background data collection was done with ZnSe crystal prior to the analysis of API, coformers, and the cocrystals. Differential Scanning Calorimetry (DSC). DSC60 differential scanning calorimetry (SHIMADZU, Japan) was used for the DSC analysis. The instrument was calibrated for the temperature and enthalpy using tin standard material. For the analysis approximately 2−5 mg of samples was placed into an aluminum pan, and it was covered with a crimped lid. This weighed crimped aluminum pan was kept in the sample reference cell, and it was scanned from 30 to 350 °C at a heating rate of 10°/min under a continuously purged dry nitrogen atmosphere. Solubility Measurement. Solubility measurement for ETZ and ETZ−GA cocrystal was performed on Analytikjena Specord S600 UV−vis spectroscopy using standard 3.5 mL quartz cells (two optical windows) having a path length of 10 mm. Initial measurements involved the individual linearity study for both ETZ and cocrystal. For ETZ the absorption maxima was observed at 292 nm, and for GA absorption maxima was observed at 260 nm. Peak separation was observed in ETZ−GA cocrystal. Solubility measurement was performed in 10% ethanol/water medium at room temperature (∼25 °C). For the analysis, an excess quantity of each of the sample was taken in the minimum quantity of 10% ethanol/water and kept under continuous stirring for 24 h. Upon equilibrium, the sample was filtered through a 0.2 μ syringe filter, and the concentration of the sample was determined after sufficient dilution. Dilution correction was included in the calculation part for the concentration of drug content.

EXPERIMENTAL SECTION

Gallic acid hydrate, 2-nitrobenzoic acid, 3-nitrobenzoic acid, 2,4dinitrobenzoic acid, and 3-toluic acid were purchased from commercial suppliers and used as such without any further purification. Ethenzamide was purchased from Alfa Aesar. Analytical grade solvents were used for all the cocrystallization experiments. Solvent Evaporation Method for Cocrystallization. The pure cocrystals of ethenzamide with various coformers were synthesized by the solvent evaporation method. The experiment was conducted using various solvents at different thermal conditions. ETZ−GA Cocrystal. ETZ (100 mg, 0.605 mmol) and GA (113.82 mg, 0.605 mmol) were taken in a 1:1 stoichiometric ratio, dissolved in 10 mL of ethanol at 70 °C, and then allowed to crystallize by slow evaporation of the solvent at room temperature. After 4 days of the experiment block-shaped colorless crystals were obtained in 1:0.5 stoichiometry in the asymmetric unit. Cocrystal was obtained in methanol and acetonitrile solvents as well by the slow evaporation method. ETZ−2NB Cocrystal. ETZ (100 mg, 0.605 mmol) and 2NB (101.10 mg, 0.605 mmol) were taken in a 1:1 stoichiometric ratio, dissolved in 10 mL of ethanol solvent at 60 °C. It was left for slow evaporation of the solvent at room temperature. Block-shaped colorless crystals were obtained after 4 days of the experiment with 1:1 stoichiometry in the asymmetric unit. Other solvents such as methanol, acetonitrile, and ethyl acetate did not yield good quality crystal. ETZ−3NB Cocrystal. ETZ (100 mg, 0.605 mmol) and 3NBA (101.10 mg, 0.605 mmol) were taken in a 1:1 stoichiometric ratio. The resulting mixture was dissolved in 10 mL of acetonitrile solvent at 70 °C and left for slow evaporation of the solvent at room temperature. Colorless block-shaped crystals were obtained after 4 days of the experiment with 1:1 stoichiometry in the asymmetric unit. Cocrystallization experiment with methanol, ethanol, and ethyl acetate solvent did not yield good quality crystal. ETZ−DNB Cocrystal. ETZ (100 mg, 0.605 mmol) and DNB (128.33 mg, 0.605 mmol) in a 1:1 stoichiometric ratio was dissolved in 10 mL of ethanol at 70 °C, and allowed it for slow evaporation of solvent at room temperature. Needle-shaped colorless crystals were obtained after 12 days of experiment with 1:1 stoichiometry in the asymmetric unit. Good quality crystal was also obtained in methanol solvent. ETZ−3TA Cocrystal. ETZ (100 mg, 0.605 mmol) and 3TA (82.37 mg, 0.605 mmol) were taken in a 1:1 stoichiometric ratio and dissolved in 10 mL of methanol at 70 °C. It was left for slow evaporation of the solvent at room temperature. Needle-shaped colorless crystals were obtained after 10 days of the experiment with 1:1 stoichiometry in the asymmetric unit. Good quality crystal was also obtained in an ethanol solvent. Single Crystal X-ray Diffraction (SCXRD). X-ray diffraction data for various cocrystals studied in the present study was collected on a Bruker Apex II duo diffractometer with CCD detector. As a source of radiation for the experiment monochromatic molybdenum (Mo) Kα

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b00606. 3D representation of all the cocrystals (ETZ−GA, ETZ− 2NB, ETZ−3NB, ETZ−DNB, and ETZ−3TA) (Figure S1); FT-IR spectra of cocrystal and the comparison with their respective starting materials (Figure S2); NMR spectrum (Figure S3); DSC plots (Figure S4); PXRD comparison with experimental and simulated (Figure S5); UV−vis linearity plot of ETZ (Figure S6) (PDF) 4480



Article

Accession Codes

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CCDC 1468148, 1468153−1468154, 1468159, and 1468161 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc. cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91-824-2473205. E-mail: darshak_rtrivedi@yahoo. co.in. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors are thankful to the Director and HOD (Department of Chemistry), NITK Surathkal for providing the research facility. We acknowledge DST (Department of Science and Technology, Government of India, New Delhi, India) for SCXRD analysis (procured under FIST program). We are grateful to MIT Manipal for providing support for 1H NMR spectroscopy. S.K.N. thankful to NITK Surathkal for providing research fellowship.

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Cocrystals of Ethenzamide: Study of Structural and Physicochemical

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