Hepatoprotective Cocrystals and Salts of Riluzole: Prediction

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Hepatoprotective Cocrystals and Salts of Riluzole: Prediction, Synthesis, Solid State Characterization and Evaluation Balvant Yadav, Sridhar Balasubramanian, Rahul B Chavan, Rajesh Thipparaboina, V GM Naidu, and Nalini R. Shastri Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01514 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Crystal Growth & Design

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Hepatoprotective Cocrystals and Salts of Riluzole: Prediction, Synthesis, Solid State Characterization and Evaluation

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Balvant Yadava, Sridhar Balasubramanianb, Rahul B Chavana, Rajesh Thipparaboinaa,

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V.G.M. Naiduc, Nalini R Shastria*

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a

Solid State Pharmaceutical Research Group (SSPRG), Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, 500037, India. b

X-ray Crystallography Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana, 500007, India c

Department of Pharmacology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, 500037, India

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* Corresponding author. Nalini R Shastri

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Tel. +91-040-23423749

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Fax. +91-040-23073751

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E-mail: [email protected], [email protected]

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Address: Department of Pharmaceutics, National Institute of Pharmaceutical Education & Research (NIPER), Balanagar, Hyderabad, India, Pin code – 500037

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ACS Paragon Plus Environment

Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Riluzole is a drug, used to slow the course of amyotrophic lateral sclerosis. Due to its unique

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structure and functionalities, it is able to form both salts and cocrystals. This is a BCS class II

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drug with poor solubility and causes hepatotoxicity which limits its application. The present

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study aims towards development of novel solid forms of riluzole to address the said

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limitations. Apart from this, an attempt has been made to develop a prediction model using

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software tools to identify the appropriate synthons for formation of cocrystals. It was

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observed that out of 33 coformers selected, prediction results were in agreement with the

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experimental outcome for 25 coformers, which demonstrated the potential of the model

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developed. Seven new solid forms of riluzole, five cocrystals with ferulic acid, syringic acid,

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vanillic acid, cinnamic acid and proline; two salts with 2,4 dihydroxybenzoic acid and

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fumaric acid were successfully developed. All the solid forms were characterized by DSC,

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powder XRD, FTIR and single crystal XRD. Single crystal X-ray analysis of the all solid

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form shows R22(8) motif between riluzole and coformers through N-H···O and O-H···N bond

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except riluzole-proline zwitterionic cocrystal. In riluzole-fumaric acid, partial proton transfer

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of O to N due to acidic H atom disorder has been observed. Dissolution profiles of all the

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solid forms were comparable to that of plain riluzole and complete drug release was observed

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within 60 min for all systems. In vivo hepatotoxicity study with riluzole-ferulic acid and

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riluzole-syringic acid in mice model revealed its potential hepatoprotective effect to

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counterattack the hepatotoxic adverse effects of riluzole.

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Keywords: Crystal engineering, prediction model, salts, hepatotoxicity, nutraceuticals

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1

Introduction

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In recent times, solid state manipulation of active pharmaceutical ingredients (APIs) have

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gained popularity to fortify the pharmaceutical and biopharmaceutical attributes such as

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solubility, dissolution, bioavailability and stability. These solid state manipulation strategies

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usually involve generation of new polymorphs,1 salts,2 amorphous systems,3 co amorphous

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systems,4 and cocrystals.5 Among these, salts can effectively improve the pharmaceutical

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attributes of ionisable API where as cocrystals are reported for alteration of physicochemical

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properties such as solubility,6 compressibility,7 bioavailability,8 melting point,9 and

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hygroscopicity10 of poorly ionisable or non-ionisable APIs. Designing a new cocrystal

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requires in-depth understanding of chemistry of molecules, which can be solved by crystal

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engineering approach. 'Crystal engineering’ as defined by Desiraju mainly deals with the

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understanding of intermolecular interactions and utilization of such understanding in

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designing new solids with desired physical and chemical properties.11 Synthon approach is

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commonly used for generation of cocrystals which decides the direction and strength of bond.

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Apart from synthon approach, various prediction models are reported in the literature to aid

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in the selection of appropriate coformers for cocrystallization. Most of the prediction model

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are based on the calculation of difference in lattice energies of cocrystal and its component,12

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and calculation of gas phase molecular electrostatic potential surfaces of the individual

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components.13 These prediction models possess some limitations. Hence, there exists a scope

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to develop a robust and more accurate prediction tool which will speed up the process of

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cocrystal generation by reducing number of the screening experiments.

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Riluzole (2-amino-6-trifluoromethoxybenzothiazol) is used to slow the course of

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amyotrophic lateral sclerosis (ALS).14 Riluzole exhibits strong neuroprotection,15

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anticonvulsant,16 antidepressant effects17 and sedative properties.18 The molecular weight of

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the compound is 234.20 g/mol and the empirical formula is C8H5F3N2OS. It is almost



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insoluble in water at pH 7.4. Mondal P et al., had reported various cocrystals of this drug

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with succinic acid, glutaric acid, adipic acid, sorbic acid, pimelic acid, suberic acid, azelic

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acid, sebacic acid, 4- dimethylaminopyridine and nicotinic acid and a malonate salt for

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increasing the solubility and dissolution.19

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Severe hepatotoxicity was reported in clinical data for riluzole, which limits its use in

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patients.20, 21 This necessitates careful consideration in the selection process of coformers. In

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addition to cocrystal formation ability, the selection of coformers should entail evaluation of

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the toxicity profile of the coformers, the potential therapeutic benefits provided and their

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ability to tackle the hepatotoxicity problem of riluzole. Apart from the routine list of

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pharmaceutically acceptable salt/ cocrystal formers, there is need to consider an additional

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group of molecules as possible coformers i.e. naturally occurring compounds, nutraceuticals.

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These naturally occurring coformers besides aiding cocrystal formation may additionally

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provide some physiological benefits to the patients such as antioxidant, anti-inflammatory,

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anti-aging and hepatoprotective activity. Hence, the present study was designed with the

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objective of developing the novel solid forms i.e. cocrystals or salts using hepatoprotective

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coformers, mainly nutraceuticals like ferulic acid, vanillic acid and syringic acid. These

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nutraceuticals selected had already proven their hepatoprotective22-25 potential in addition to

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their antioxidant,26 and antiepileptic27 properties. Additionally, an attempt was made to

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develop a simple prediction model for screening of the coformers. The model was designed

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to predict the possible synthon groups in drug and coformers which would participate in

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hydrogen bond formation during cocrystal generation. Total 33 coformers were employed in

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this study; structures of these coformers are represented in figure S1, supporting information.


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2

Experimental section

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2.1

Material

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Riluzole was kindly gifted by Suven Life Sciences, Hyderabad, India. Ferulic acid, syringic

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acid, vanillic acid, cinnamic acid, 2,4 dihydroxybenzoic acid and fumaric acid were

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purchased from Alfa Aesar, India. Proline was purchased from Avra Synthesis Private Ltd.,

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India. All other solvents and chemicals were of analytical grade. HPLC grade acetonitrile was

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purchased from Merck, India. Double distilled water was generated in house.

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2.2

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Hydrogen bond formation between riluzole and coformers were predicted using Materials

108

Studio 7.0 (Accelrys Inc., USA) software tools. Amorphous cells were constructed to identify

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the interaction sites and possible synthons. Briefly, MOL files were downloaded from

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Chemical Entities of Biological Interest (CHEBI). Amorphous cell was constructed, after

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structure clean up and adjustment of hydrogen atoms with the following parameters for

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temperature, cell type and configurations at 298 K, periodic and 1-100 respectively. Post

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processing involved calculation of hydrogen bonds and assignment of bond length to identify

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the possible synthons.

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2.3

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2.3.1 Liquid assisted grinding

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Preliminary screening for novel forms was carried out by grinding drug and coformers in 1:1,

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1:2 and 2:1 molar ratio in an agate mortar in the presence of 120 µL of methanol for 30 min.

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Riluzole-ferulic acid, riluzole-syringic acid, riluzole-vanillic acid, riluzole-cinnamic acid,

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riluzole-proline, riluzole-2,4-dihydroxybenzoic acid and riluzole-fumaric acid were

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successfully synthesized by liquid assisted grinding.

Prediction and Modelling

Solid form screening



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2.3.2 Recrystallization experiments

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Recrystallization experiments were carried out by dissolving the drug and coformer (1:1

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molar ratio) in methanol or ethanol in a beaker at 55-65 °C. The beaker was covered with

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pin-holed aluminium foil and left at room temperature for slow evaporation. Single crystals

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obtained after 2-3 days were filtered, air-dried, and stored in a glass vial until further analysis.

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Information regarding the crystallization experiments is provided in supporting information

128

table S1. Single crystals were obtained from all recrystallization systems except riluzole-

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syringic acid, wherein single crystals suitable for structural analysis were not obtained.

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2.4

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2.4.1 DSC

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Thermal analysis of plain riluzole and novel solid forms were carried out using Mettler

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Toledo DSC system operating with Stare software to determine the melting point and

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enthalpy. DSC instrument was calibrated for temperature and heat flow using high purity

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indium standard. Accurately weighed samples (5−10 mg) were scanned at a heating rate of 10

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°C /min from 20 to 250 °C in aluminium crimped pans with pinhole. The measurements were

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conducted in nitrogen gas environment with a purging rate of 60 mL/min.

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2.4.2 Powder X-ray Diffraction (P-XRD)

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P-XRD patterns of samples were recorded at room temperature using PANalytical X’Pert

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PRO X-ray Powder Diffractometer (Eindhoven, Netherlands), using Ni-filtered Cu Kα

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radiation (λ = 1.5406 Å). The data were recorded over a scanning 2θ range of 2° to 50° at

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step time of 0.045 steps/0.5 s.

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2.4.3 FTIR

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IR discs were prepared by using 100 mg potassium bromide and 2 mg of samples with

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blending. The blend was subjected to compression under vacuum at a pressure of 12 ρsi for 3

Characterization


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min. FTIR was performed using a PerkinElmer IR spectrophotometer with a suitable holder

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for the disc. FTIR spectra were obtained in the range of 4000 to 400 cm−1.

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2.4.4 Thermogravimetric Analysis (TGA)

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TGA of riluzole-2,4 dihydroxy benzoic acid methanol solvate was carried out using ExStar

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TGA/DTA 7200 instrument operating with Muse software. Sample was loaded in alumina

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crucibles and heated at a rate of 20 °C/min over a temperature range of 30 to 300 °C under a

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nitrogen purge of 60 mL/min.

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2.4.5 Single Crystal Analysis

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The intensity data for riluzole-ferulic acid was collected at room temperature using a Bruker

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Smart Apex CCD diffractometer with graphite monochromated MoKα radiation

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(λ=0.71073Å) by the ω-scan method. Preliminary lattice parameters and orientation matrices

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were obtained from four sets of frames. Integration and scaling of intensity data were

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accomplished using the program SAINT.28 The structures were solved by direct methods

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using SHELXS and refinement was carried out by full-matrix least-squares technique using

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SHELXL.29 Anisotropic displacement parameters were calculated for all non-hydrogen

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atoms. X-ray data of riluzole-vanillic acid, riluzole-cinnamic acid, riluzole-proline, riluzole-

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2,4 dihydroxy benzoic acid methanolate and riluzole-fumaric acid were collected at room

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temperature on a Bruker D8 QUEST instrument with an IµS Mo micro source (λ = 0.7107 A)

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and a PHOTON-100 detector. The raw data frames were reduced and corrected for absorption

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effects using the Bruker Apex 3 software suite programs. The structure was solved using

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intrinsic phasing method and further refined with the SHELXL program and expanded using

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Fourier techniques. The H atoms bound to the N and O atoms were located in difference

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Fourier maps, and their positions and isotropic displacement parameters were refined. C-

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bound H atoms were located in a difference density map but were positioned geometrically

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and included as riding atoms, with C—H distance = 0.93 -0.96 Å and with Uiso(H) values of



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1.2Ueq(C). Atoms O1 and F1 of riluzole-2,4 dihydroxy benzoic acid methanolate were

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disordered and the site-occupancy factors of the disordered atoms O1/O1D/F1/F1D were

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refined to 0.688(7) and 0.312(8). The anisotropic displacement parameters for the disordered

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atoms were restrained to be similar. The C1-O1 distance was constrained to 1.32(1) Å. The H

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atom bound to the O atom of the fumaric acid of riluzole-fumaric acid during the initial

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refinement shows high Uiso(H) value. Contoured difference Fourier maps show significant

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electron density at the location of the potential H-atom site and the electron density is

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smeared along the O···N axis in riluzole-fumaric acid. However, the electron density has a

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maximum at atom O. Hence the H atoms were treated as disorder over two sites (H1O and

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H1N) and refined with their site-occupancy factors to 0.67 (3) and 0.33 (3). The disordered H

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atoms were located in difference Fourier and refined as riding, with Uiso(H) =

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1.2Ueq(N)/1.5Ueq(O), using constrained distance of 0.82 and 0.86 Å for O—H and N—H,

183

respectively.

184

2.4.6 In vitro Dissolution Study

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Dissolution study was performed for the plain riluzole and novel solid forms of riluzole. USP

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apparatus II (paddle type) was selected for the studies. The dissolution studies were

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performed in 1000 ml of media maintained at 37± 0.5 °C, at 50 rpm. Phosphate buffer (pH

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6.8) was used as a dissolution media.20 Plain drug and all novel solid forms equivalent to 20

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mg of drug were added to the dissolution media. All samples were passed through BSS

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#sieve no. 44 and retained on BSS #sieve no. 60 before addition of dissolution media.

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Aliquots of 5 ml were withdrawn at predetermined time points (5, 10, 15, 30, 45, 60, 120,

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180, and 240 min) substituting the same with equal quantity of fresh dissolution media.

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Samples were filtered using 0.22 µ filters and analyzed by a validated HPLC method after

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suitable dilution with the mobile phase. Dissolution profiles of plain drug and novel solid


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forms were evaluated for the amount of drug released at 15 min (Q15), % dissolution

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efficiency (DE) at 15 min and similarity factor using DDsolver.30

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2.4.7 HPLC Method

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Drug was quantified using a HPLC system (e2695 Waters) consisting of a HPLC pump, an

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automated injector equipped with a UV detector (2998 PDA) and an auto sampler. Formic

200

acid (0.005 % v/v) in water and acetonitrile were used as mobile phase at a flow rate of 1

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ml/min in an isocratic mode on an Inertsustain C18 (5µ, 4.6 x 100 mm) column. After

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suitable dilution, 20 µL of sample was injected, and the absorbance of elute was recorded at

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222 nm. Details of the HPLC method used are given in supporting information table S2.

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2.4.8 In vivo hepatotoxicity study

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In vivo hepatotoxicity study for plain drug and novel solid forms containing hepatoprotective

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coformers were carried out in 6-7 weeks old, male BALB/c mice weighing 20-25 gm.

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Animals were obtained from the central animal facility, National Institute of Pharmaceutical

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Education and Research (NIPER), Hyderabad, India. Animals were housed at 22 ± 2 °C, 50-

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60 % relative humidity (RH), under 12 hr natural light/dark cycle. Standard pellet diet and

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water was given ad libitum. The study protocol was duly approved by the Institutional

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Animal Ethics Committee (IAEC), NIPER, Hyderabad, India (NIP/12/2016/PE/220). The

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animals were divided into six groups (control group, riluzole treated group, ferulic acid

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treated group, syringic acid treated group, riluzole-ferulic acid cocrystal treated group and

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riluzole-syringic acid cocrystal treated group). All treatment doses were prepared in 0.5 %

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sodium carboxy methyl cellulose to form a suspension. Plain drug riluzole and cocrystals

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(riluzole-ferulic acid and riluzole-syringic acid were administered orally to animals in a dose

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equivalent to 25 mg/kg of body weight. While, ferulic acid and syringic acid were given at an

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equivalent dose of ferulic acid and syringic acid present in riluzole-ferulic acid and riluzole-

219

syringic acid cocrystal respectively. The blood samples were collected at 24 hr after dosing



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via retro orbital route in vials prefilled with heparin solution. The blood samples were

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centrifuged at 3000 rpm for 10 min, plasma was collected and analysed for enzyme levels.

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The enzyme levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline

223

phosphatase (ALP) in plasma were estimated using commercial kits (Accurex, India) as per

224

manufacturer’s protocol.

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2.4.9 Statistical analysis

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The data obtained in animal study were expressed as the mean of replicate determinations

227

(n=5) ± standard error of mean (S.E.M). Statistical comparisons were made using one-way

228

analysis of variance (ANOVA). The intergroup variations were analyzed by “Tukey’s

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multiple comparison test” using the Graph Pad Prism software, Version 5.0.

230

3

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With its unique fluorine containing chemical structure and easily accessible functional

232

groups, riluzole is found to have propensities to form salt or cocrystals through hydrogen or

233

ionic interactions. Hence, to screen the appropriate coformers for generation of novel

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cocrystal or salt of riluzole, prediction studies were carried out using Material Studio

235

software. Outcome of prediction study are depicted in supporting information table S3, along

236

with the predicted bond length and rank orders. From the

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concluded that some of the coformers such as ferulic acid, fumaric acid, malonic acid,

238

succinic acid, ascorbic acid, azelic acid, cinnamic acid and 2,4 dihydroxybenzoic acid form

239

intermolecular (amine- carboxylic acid) hydrogen bond with riluzole (figure 1 and table S3

240

supporting information). Other coformers namely glutaric acid and sebacic acid showed the

241

presence of intermolecular interactions between carboxylic acid-halogen. In contrast,

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phenolic –OH of adipic acid showed molecular interactions with the sulphur of riluzole.

243

Experimental outcome of the screening experiments of this current study with 24 coformers

244

and 9 coformers (adipic acid, azelaic acid, 4-dimethylaminopyridine, glutaric acid, maleic

Result and discussion


prediction study, it can be

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acid, nicotinic acid, sorbic acid, suberic acid and succinic acid) already reported for this drug

246

by Mondal P et al,19 was compared with the prediction outcome (table 1). It was observed

247

that out of 33 coformers reported in table 1, prediction outcome showed similarity with the

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outcome of crystallization experiments of riluzole with the 25 coformers. Coformers in the

249

same analogue series have not shown similar pattern for cocrystal formation in the prediction

250

study. Ferulic and cinnamic acids have given cocrystals but not related caffeic and p-

251

coumaric acids. This could be attributed to the fact that this prediction model was based on

252

molecular dynamics, wherein the orientation of the drug and coformers during amorphous

253

cell construction may vary from co-former to co-former despite their analogous similarity.

254

However, it is interesting to note that the prediction behaviour matched with the experimental

255

behaviour in these molecules demonstrating the potential utility of this model. Additionally,

256

single crystal analysis of few novel solid forms of riluzole with (ferulic acid, vanillic acid,

257

fumaric acid, cinnamic acid and 2,4-dihyroxybenzoic acid) depicted similar type of

258

intermolecular interactions between drug and coformers to that of predicted results for

259

molecular interactions.

260

Furthermore, the difference in pKa of acid and base (∆pKa) was used to predict the formation

261

of cocrystal or salt. ∆pKa < 0 often gives a cocrystal, ∆pKa > 3 may form a salt and ∆pKa in

262

the range of 0 to 3 may result in salt-cocrystal hybrids31-34. ∆pKa value for the screened drug-

263

coformer systems were calculated and depicted in table 1, and S4 supporting information,

264

wherein novel solid systems like riluzole-ferulic acid, riluzole-syringic acid, riluzole-vanillic

265

acid, riluzole-cinnamic acid and riluzole-proline showed negative value of ∆pKa which

266

correspond to the cocrystal formation, while ∆pKa values for riluzole-2,4-dihydroxybenzoic

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acid methanolate and riluzole-fumaric acid were found to be 0.69 and 0.77, respectively,

268

which led to the assumption that riluzole-2,4-dihydroxybenzoic acid methanolate and

269

riluzole-fumaric acid may form cocrystal salt continuum zone.



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Table 1 Comparative analysis of prediction and experimental outcome (# cocrystals and salts reported by Mondal P et al.)19 Sr. No.

System

1 2 3 4

Riluzole-adipic acid# Riluzole- ascorbic acid Riluzole-azelaic acid# Riluzole-benzoic acid

0.63,-1.6 -0.37 -0.75 -0.40

5 6 7 8 9

-0.82 -3.0 -0.66 1.01 0.69

11 12

Riluzole-caffeic acid Riluzole-chrysin Riluzole-cinnamic acid Riluzole-citric acid Riluzole2,4dihydroxybenzoic acid Riluzole- 4 dimethylaminopyridine# Riluzole-ferulic acid Riluzole-fumaric acid

13 14 15 16 17 18

Riluzole-gallic acid Riluzole-glutaric acid# Riluzole-hesperetin Riluzole-maleic acid Riluzole-malic acid Riluzole-malonic acid#

19

Riluzole-nicotinic acid#

20 21

Riluzole-p-coumaric acid Riluzole-proline

22

Riluzole-quercetin

23 24 25 26 27 28

Riluzole-salicylamide Riluzole-salicylic acid Riluzole-sebacic acid Riluzole-silibinine Riluzole-sorbic acid# Riluzole-suberic acid#

29

Riluzole-succinic acid#

30 31

Riluzole-syringic acid Riluzole-tartaric acid

32 33

Riluzole-vanillic acid Riluzole-vanillin

10

∆pKa

-5.9 -0.78 0.77, 0.64 -0.64 -0.54 -4.12 1.97,-2.4 0.4,-1.3 0.97,1.89 -0.95 -0.2 1.86,0.74 -2.07,4.68 -4.57 0.83 -0.92 -3.95 -0.96 -0.72 -0.46,1.92 -0.13 0.82,0.54 -0.71 -3.58

Prediction model intermolecular interactions Intermolecular Intermolecular Intermolecular Absence of interaction Intramolecular Intramolecular Intermolecular Intramolecular Intermolecular

Cocrystal or salt formation

Melting point (°C)

Predicted outcome

Experimental outcome

Yes Yes Yes No

Yes No Yes No

115 97 110 101

No No Yes No Yes

No No Yes No Yes

102 110 145 72 157

Absence of interaction Intermolecular Intermolecular

No

Yes

96

Yes Yes

Yes Yes

159 163

Intramolecular Intermolecular Intramolecular Intramolecular Intramolecular Intermolecular

No Yes No No No Yes

No Yes No Yes No Yes

86 120 105 153 78 139

Absence of interaction Intramolecular

No

Yes

118

No

No

92

Absence of interaction Intramolecular

No

Yes

206

No

No

101

Intramolecular Intramolecular Intermolecular Intramolecular Intramolecular Absence of interaction Intermolecular

No No Yes No No No

No No Yes No Yes Yes

130 110 125 109 91 136

Yes

Yes

133

Intermolecular Intermolecular

Yes Yes

Yes Yes

142 158

Intramolecular Intramolecular

No No

Yes No

139 78

272


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273 274 275 276 277 278


Figure 1 Prediction study outcome: (a) riluzole-adipic acid, (b) riluzole-ascorbic acid, (c) riluzoleazelic acid, (d) riluzole-benzoic acid, (e) riluzole-caffeic acid, (f) riluzole-chrysin, (g) riluzolecinnamic acid, (h) riluzole-citric acid, (i) riluzole-p-coumaric acid, (j) riluzole-2,4-dihydroxybenzoic acid, (k) riluzole-2,4 -dimethylaminopyridine, (l) riluzole-ferulic acid, (m) riluzole-fumaric acid, (n) riluzole-gallic acid, (o) riluzole-glutaric acid, (p) riluzole-hesperetin, (q) riluzole-maleic acid, (r)



279 280 281 282

riluzole-malic acid, (s) riluzole-malonic acid, (t) riluzole-nicotinic acid, (u) riluzole-proline, (v) riluzole-quercetin, (w) riluzole-salicylic acid, (x) riluzole-salicylamide, (y) riluzole-sebacic acid, (z) riluzole-silibinin, (aa) riluzole-sorbic acid, (ab) riluzole-suberic acid, (ac) riluzole-succinic acid, (ad) riluzole-syringic acid, (ae) riluzole- tartaric acid, (af) riluzole-vanillic acid and (ag) riluzole-vanillin

283

3.1

284

Single endotherm at 119.2 °C corresponding to its melting appeared in the DSC curve of

285

riluzole (figure 2). The melting endotherm for coformers such as ferulic acid, syringic acid,

286

fumaric acid, cinnamic acid, 2,4 dihydroxybenzoic acid, proline, and vanillic acid were

287

observed at 172.8, 208.4, 207.1, 133.8, 194.9, 219.4 and 211.5 °C respectively. The

288

generated novel solid forms such as riluzole-ferulic acid, riluzole-syringic acid, riluzole-

289

fumaric acid, riluzole-cinnamic acid, riluzole-2,4-dihydroxybenzoic acid methanolate,

290

riluzole-proline and riluzole-vanillic acid gave single melting endotherms at 159.4, 142.4,

291

163.1, 145.5, 157.3, 206.9 and 139.7 °C respectively (figure 2). Riluzole-2,4-

292

dihydroxybenzoic acid methanolate gave a small endotherm at 86 °C indicating a presence

293

of solvate form, which complemented the single crystal structural analysis. The onset of

294

melting and ∆H values for all novel forms along with coformers, and respective thermograms

295

along with controls are given in supporting information (table S5). Changes in melting point

296

and enthalpy indicated the formation of new solid forms, which were additionally confirmed

297

by P-XRD studies.

DSC


Page 14 of 33

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298 299 300 301 302

Figure 2 Overlay of DSC thermogram of R- riluzole, RFRA-riluzole-ferulic acid, RSYRA-riluzolesyringic acid, RVNLA– riluzole-vanillic acid, RCA-riluzole-cinnamic acid, RPRO-riluzole-proline, RDHBA methanolate riluzole-2, 4-dihydroxybenzoic acid methanolate and RFUM-riluzole-fumaric acid

303

3.2

304

FTIR analysis of plain riluzole and novel solid forms were performed to detect the presence

305

of hydrogen bond between drug and coformers. Plain riluzole showed characteristic N-H

306

stretching band at 3371.5, 3279.4 cm−1 which confirms the presence of primary amine group

307

in riluzole. Apart from this, presence of peak at 1606.6 and 1327.1 cm−1 in the spectra

308

correspond to N-H bending and C-N stretching, respectively. O-H and -C=O stretching

309

corresponding to the carboxylic acids appear in the region of 3400-2400 cm−1 and 1720-1680

310

cm−1, respectively (figure S4, supporting information). It is expected that due to hydrogen

311

bond formation in novel solid forms, there will be shift in these stretching peaks. Specifically,

312

-N-H stretching and bending peaks at 3279.0 cm−1 and 1606.5 cm−1 showed significant shift

313

in IR spectra of novel solid forms. Significant shift in the characteristic peaks of riluzole

314

confirms the formation of novel solid forms. To differentiate formation of cocrystal and salt

FTIR



315

by FTIR, presence or absence of carboxylic acid O-H stretching peak (2500-3200 cm−1) in

316

FTIR spectra of solid forms is considered as an indicator.19 As highlighted in the figure S4

317

supporting information, generated cocrystals showed carboxylic acid -O-H stretching peak

318

that confirmed the presence of carboxylic acid group, while this peak was absent in fumaric

319

acid and DHBA based solid forms confirming formation of salt for these two coformers.

320

3.3

321

As shown in figure 3, characteristic peaks of riluzole-ferulic acid, riluzole-syringic acid,

322

riluzole-vanillic acid, riluzole-cinnamic acid, riluzole-proline, riluzole-2, 4-dihydroxybenzoic

323

acid methanolate and riluzole-fumaric acid in PXRD patterns differ from riluzole and its

324

corresponding coformer. Riluzole has shown characteristic peaks at 2θ values 9.0, 13.5, 18.1

325

and 26.4 whereas new characteristic peaks appeared in riluzole-ferulic acid (13.8, 14.9, 27.3

326

and 27.9); riluzole-syringic acid (13.2, 14.9, 18.4 and 26.2), riluzole-vanillic acid (14.3, 15.2,

327

20.3, 26.5 and 26.9), riluzole-cinnamic acid (11.7, 18.8, 23.9 and 27.3), riluzole-proline

328

(11.1, 16.7, 20.9 and 22.5), riluzole-2,4 dihydroxybenzoic acid (8.1, 24.8, 26.9, and 27.5) and

329

riluzole-fumaric acid (12.2, 22.6, 25.0 and 26.9). The appearance of new peaks in PXRD

330

pattern when compared with R and respective coformers, collaborate the generation of novel

331

solid forms. Bulk purity of all generated system was confirmed by superimposable

332

experimental and simulated diffraction patterns overlay (supporting information figure S5).

333

Overall, outcome of DSC, PXRD, FTIR and single crystal XRD confirmed the occurrence of

334

the new solid forms.

335

Plain salicylamide and riluzole-salicylamide combination from liquid assisted grinding

336

showed a melting point at 141.0 and 138.7 ⁰C, respectively in DSC (data not shown).

337

Although the riluzole-salicylamide combination gave a melting point which was intermediate

338

to the starting components, the PXRD result of the combination showed overlapping peaks as

PXRD


Page 16 of 33

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339

that of plain drug and salicylamide indicating no cocrystal formation (figure S6 supporting

340

information).

341 342 343 344 345 346 347

Figure 3 Overlay of diffractograms of R-riluzole, FRA- ferulic acid, RFRA-riluzole-ferulic acid, VNLA-vanillic acid, RVNLA-riluzole-vanillic acid, SYRA-syringic acid and RSYRA-riluzolesyringic acid, CA- cinnamic acid, RCA- riluzole-cinnamic acid, PRO-proline, RPRO-riluzole-proline, DHBA-2,4-dihydroxybenzoic acid, RDHBA methanolate- riluzole-2,4-dihydroxybenzoic acid methanolate, FUM-fumaric acid and RFUM-riluzole-fumaric acid. (* indicates new peaks in solid forms).

348

3.4 TGA



349

Riluzole-2, 4-dihydroxybenzoic acid methanolate showed a weight loss of 3.248 % (0.0819

350

mg) in the region of 86-100 °C in TGA thermogram (figure S2, supporting information). This

351

finding is in line with the DSC outcome and the single crystal structural analysis. The

352

outcome hence confirms the presence of solvate in novel solid form, riluzole-2,4-

353

dihydroxybenzoic acid methanolate. However, the discrepancy in weight loss during TGA

354

analysis from the theoretical mass loss of one molecule of methanol might have occurred due

355

to slipping of methanol from the crystal lattice at lower temperature.35 This was confirmed by

356

integration of results from 25 ⁰C up to 100 and 154 ⁰C, which showed enhancement in

357

percentage weight loss (figure S2 and S3, supporting information).

358

3.5

359

All six novel solid forms (riluzole-ferulic acid, riluzole-vanillic acid, riluzole-cinnamic acid,

360

riluzole-proline, riluzole-2,4-dihydroxybenzoic acid methanolate and riluzole-fumaric acid)

361

crystallized in monoclinic system. The state of the carboxylic acid group (whether neutral or

362

anionic) can be determined from the C-O and C = O bond lengths. For a neutral carboxylic

363

acid group, the C-O and C=O distances are around 1.2 and 1.3A˚, respectively. However,

364

when deprotonation occurs, both C-O bond lengths will be similar, around 1.25 A˚.36 Based

365

on the bond distances listed in table 3, riluzole-ferulic acid, riluzole-vanillic acid and riluzole-

366

cinnamic acid exist as cocrystals. In case of riluzole-proline, internal proton transfer takes

367

place from carboxylic acid to the N atom of the pyrrolidine ring and exists as zwitter ion. As

368

a result, the C-O distances of the carboxylic group of proline are similar. However, riluzole-

369

proline as a whole exists as zwitterionic cocrystal.37 The near equality of C-O distances of

370

carboxylic group of riluzole-2,4-dihydroxybenzoic acid methanolate, show deprotonation

371

from O to N and confirm the presence of salt. However, in riluzole-fumaric acid, the C-O

372

bond distances are intermediate between the neutral and ionic because of partial proton

373

transfer between O to N. The geometry of bond angles of carboxylic acid group also show

Single crystal structure analysis


Page 18 of 33

Page 19 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


374

similar trend for neutral, ionic and intermediate state. The crystal structures of riluzole-ferulic

375

acid,

376

dihydroxybenzoic acid methanolate and riluzole-fumaric acid are mainly determined by

377

N−H···O and O−H···O hydrogen bonds. Hydrogen bond geometry of crystal had been

378

explained in table 4 for all systems.

riluzole-vanillic

acid,

riluzole-cinnamic

acid,

riluzole-proline,

riluzole-2,4-

Table 2 Crystallographic parameter

379 Identification code

Riluzoleferulic acid

Riluzolevanillic acid

Riluzole cinnamic acid

Riluzole-proline

Riluzolefumaric acid

1557240

Riluzole-2,4dihydroxybenzo ic acid methanolate 1557241

CCDC deposition no. Empirical formula Formula weight

1538080

1557243

1557244

C8H5F3N2OS, C10H10O4 428.38

C8H5F3N2OS,C8 H8O4 402.34

C8H5F3N2OS+, C9H8O2382.35

C8H5F3N2OS C5H9NO2 349.33

C8H6F3N2OS+ C7H5O4-,CH4O 420.36

C8H5.33F3N2OS,0 .5(C4H3.34O4) 292.24

Temperature K Wavelength Å

294(2) 0.71073

294(2) 0.71073

294(2) 0.71073

294(2) 0.71073

294(2) 0.71073

294(2) 0.71073

Crystal system Space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) Volume Å3

Monoclinic P21/c 6.366 (8) 7.5981(9) 38.608 (5) 90 93.901(2) 90 1863.1(4)

Monoclinic P21/n 6.655 (6) 35.02 (3) 7.437 (8) 90 95.61(2) 90 1725 (3)

Monoclinic P21/c 7.602 (4) 6.397 (3) 35.158 (18) 90 90.946 (14) 90 1709.5 (14)

Monoclinic P21 8.457 (14) 5.425 (9) 16.02 (3) 90 97.54 (2) 90 729 (2)

Monoclinic P21/n 13.874 (13) 8.694 (8) 15.219 (12) 90 97.102 (13) 90 1822 (3)

Monoclinic P21/C 8.140 (2) 15.658 (3) 9.508 (2) 90 94.46 (3) 90 1208.1 (5)

1557242

Z

4

4

4

2

4

4

Density (calculated) Mg/m3

1.527

1.549

1.486

1.592

1.533

1.607

Absorption

0.237

0.250

0.240

0.275

0.245

0.312

coefficient mm-1 F(000)

880

824

784

360

864

592

Crystal size mm

0.480x0.320x

0.420x0.380x0.2 80

0.380x0.360x0.2 80

0.400x0.360x0.2 20

0.420x0.180x0.1 60

0.360x0.220x0.1 80

0.270 θ range

1.05-28.364

2.36-28.345

2.317-28.356

2.429-28.38

2.697-28.470

2.510-28.382

Range h Range k Range l Reflections collected Observed reflections Data / restraints / parameters Goodness-of-fit on F2

-8 to 8 -10 to 10 -51 to 49 20608

-8 to 8 -46 to 46 -9 to 9 43639

-9 to 10 -8 to 8 -46 to 46 17852

-10 to 11 -7 to 7 -21 to 21 17399

-18 to 18 -11 to 11 -20 to 19 56736

-10 to 10 -20 to 20 -12 to 11 28755

4514

4280

4256

3644

4596

3022

4514/0/279

4280/0/262

4256/0/248

3644/1/224

4596/81/297

3022/0/183

1.087

1.150

1.075

1.076

1.051

1.029

Final R indices [I>2σ(I)] R1, wR2 R indices (all data) R1, wR2

0.0547, 0.1395

0.0623, 0.1661

0.0511, 0.1389

0.0394, 0.0900

0.0705, 0.2034

0.0381, 0.1040

0.0600, 0.1436

0.0722, 0.1723

0.0562, 0.1427

0.0497, 0.0942

0.0845, 0.2197

0.0420, 0.1088



Page 20 of 33

380 Table 3 C-O bond distances and angles value of novel solid forms

381

Compounds Riluzole-ferulic acid Riluzole-vanillic acid Riluzole-cinnamic acid Riluzole-proline Riluzole-2,4-dihydroxybenzoic acid methanolate Riluzole-fumaric acid

Bond distances (Ǻ) 1.218 (2) 1.308 (2) 1.219 (2) 1.311 (2) 1.219 (2) 1.305 (2) 1.238 (4) 1.243 (4) 1.255 (3) 1.259 (3)

Bond angles (°) 122.90 (18) 114.03 (17) 122.50 (2) 114.40 (2) 122.91 (17) 113.89 (17) 118.3 (3) 116.2 (2) 119.3 (2) 117.9 (2)

1.2228 (16)

120.43 (12)

1.2911 (16)

114.88 (11)

Table 4 Hydrogen bond geometry in crystal structures

382 Compounds Riluzoleferulic acid

Riluzolevanillic acid

Riluzolecinnamic acid Riluzoleproline

Riluzole2,4dihydroxy benzoic acid methanolate

Interaction N(2)-H(1N)···O(1)#1 N(2)-H(2N)···O(1) O(3)-H(3O)···O(4) O(2)-H(2O)···N(1) N(2)-H(1N)···O(3) N(2)-H(2N)···O(3)#1 O(2)-H(1O)···N(1) O(4)-H(2O)···F(2)#2 O(4)-H(2O)···O(5) N(2)-H(1N)···O(3) N(2)-H(2N)···O(3)#1 O(2)-H(1O)···N(1) N(3)-H(1N)···N(2)#1

d(D-H) 0.88(3) 0.90(3) 0.82(3) 0.96(4) 0.85(4) 0.88(4) 0.88(4) 0.74(4) 0.74(4) 0.93(3) 0.85(3) 0.92(3) 0.98(5)

d(H···A) 2.11(3) 1.95(3) 2.09(3) 1.74(3) 2.07(4) 2.18(4) 1.80(4) 2.50(4) 2.13(4) 1.92(3) 2.16(3) 1.75(3) 2.59(4)

d(D···A) 2.925(3) 2.840(3) 2.611(3) 2.690(2) 2.908(4) 2.988(3) 2.671(3) 3.126(4) 2.602(3) 2.846(3) 2.921(3) 2.661(2) 3.163(6)

Hepatoprotective Cocrystals and Salts of Riluzole: Prediction

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