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Title:
Cocrystals of nutraceuticals protocatechuic acid and quercetin
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Book
Language:
English
Creator:
Pujari, Twarita Anil
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
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Subjects / Keywords:
Supramolecular chemistry
Bioavailability
Hydrogen bond
Bcs classification
Solubility
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: The cocrystallization of two or more pure compounds by crystal engineering to create a new functional material is of a great academic and industrial interest. Pharmaceutical cocrystallization has allured a lot of attention by means of altering the physicochemical properties of Active Pharmaceutical Ingredient (API) such as solubility, stability and bioavailability. Crystal engineering of nutraceuticals can produce novel compounds such as pharmaceutical cocrystals. To establish the importance of nutraceutical cocrystallization and its use; polyphenols, a major class of nutraceuticals and potential disease preventing agents, are the appropriate targets. The work herein focuses on two polyphenols, protocatechuic acid and quercetin, which are strong antioxidants. The cocrystals of quercetin have been synthesized, aiming to modify its poor water solubility and bioavailability which limits its usage. On the other hand, cocrystals of water soluble protocatechuic acid are also prepared to establish its use as a cocrystal former. Seven novel cocrystals of protocatechuic acid and two novel cocrystals of quercetin are obtained and are characterized by FTIR, DSC (Differential Scanning Calorimetry), PXRD (Powder X-Ray Diffraction), single crystal x-ray diffraction and TGA (Thermo Gravimetric Analysis). The new crystal forms have also been studied via dissolution. Dissolution studies show alteration in solubility of a target molecule by its cocrystal irrespective of solubility of the cocrystal former. Overall, the study helps in understanding the role of crystal engineering and its utility.
Thesis:
Thesis (M.S.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
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System requirements: World Wide Web browser and PDF reader.
Statement of Responsibility:
by Twarita Anil Pujari.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains 88 pages.

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aleph - 002317586
oclc - 660813501
usfldc doi - E14-SFE0003065
usfldc handle - e14.3065
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ABSTRACT: The cocrystallization of two or more pure compounds by crystal engineering to create a new functional material is of a great academic and industrial interest. Pharmaceutical cocrystallization has allured a lot of attention by means of altering the physicochemical properties of Active Pharmaceutical Ingredient (API) such as solubility, stability and bioavailability. Crystal engineering of nutraceuticals can produce novel compounds such as pharmaceutical cocrystals. To establish the importance of nutraceutical cocrystallization and its use; polyphenols, a major class of nutraceuticals and potential disease preventing agents, are the appropriate targets. The work herein focuses on two polyphenols, protocatechuic acid and quercetin, which are strong antioxidants. The cocrystals of quercetin have been synthesized, aiming to modify its poor water solubility and bioavailability which limits its usage. On the other hand, cocrystals of water soluble protocatechuic acid are also prepared to establish its use as a cocrystal former. Seven novel cocrystals of protocatechuic acid and two novel cocrystals of quercetin are obtained and are characterized by FTIR, DSC (Differential Scanning Calorimetry), PXRD (Powder X-Ray Diffraction), single crystal x-ray diffraction and TGA (Thermo Gravimetric Analysis). The new crystal forms have also been studied via dissolution. Dissolution studies show alteration in solubility of a target molecule by its cocrystal irrespective of solubility of the cocrystal former. Overall, the study helps in understanding the role of crystal engineering and its utility.
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Bcs classification
Solubility
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Cocrystals of Nutraceuticals: Protocatechuic Acid and Quercetin by Twarita Anil Pujari A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemis try College of Arts and Sciences University of South Florida Major Professor: Michael J. Zaworotko Ph.D. Mohamed Eddaoudi, Phd. Abdul Malik, Ph.D Date of Approval: July 17, 2009 Keywords: s upramolecular chemistry, bioavailability, hydrogen bond, bcs classification, solubility Copyright 2009 Twarita Anil Pujari

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|| KRISHANARPANMASTU || || AVADHUT CHINTAN SHREE GURUDEV DATTA ||

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ACKN OWLEDGEMENTS I would like to express my sincere thanks to my professor Dr. Michael J. Zaworotko for giving me an opportunity to conduct research under his expert supervision. His valuable guidance will go a long way towards my passion in chemistry. I am a lso thankful to Dr. W ayne C. Guida and Dr. Abdul Malik for being my committe e members and giving me useful suggestions and encouragement Big thanks to all my colleagues for their support. I will cherish all brainstorming sessions blended with fun times. I also thank Faculty and Staff of the Department of Chemistry, University of South Florida, for their friendly accommodation. A special thanks to Dr. B. G. Ugarkar, Dr. R.C. Chikate, Dr. A.D. Natu, Dr. M.J. Pujari, late Dr. S.Y.Gadre and all other professo rs for building my chemistry foundation. in laws and all near and dear ones for their constant encouragement My heartfelt thanks to our pillar of support, my grandfather, whose principles I idealize. Aba, you ar e always with us! W ithout his love and constant support it would have not been achievable.

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i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES v ABSTRACT i x 1. INTRODUCTION 1 1 .1 Nutraceuticals 1 1.2 Crystal Engineering 2 1 .2.1 Supramolecular Chemistry 3 1.2.2 Supramolecular Synthons 3 1.2.3 Cocrystal s 4 1.2.4 Pharmaceutical Cocrystal s 6 1.2.5 Nutraceutical Cocrystal s 11 1.2.6 Synth esis of Cocrystal s 11 1.3 Solubility and Bioav a ilability 1 1 1.3.1 Noyes Whitney Equation 12 1.3.2 BCS Classification 1 2 1.4 Cocrystal Design 1 3 1.4.1 Cambri dge Structural Database (CSD) 1 3 1.4.2 CSD Statistics for Acids and Phenols 14 1.4.3 Co crystal Formers 22

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ii 1.5 R eferences 26 2. PROTOCATECHUIC ACID 30 2.1 Introduction 30 2.1.1 Protocatechuic acid in CSD 31 2.2 Cocr ystals of Protocatechuic acid 32 2.2.1 Experimental Section 3 3 2.2.2 Cocrystallization via Solution Crystallization 33 2.2.3 Cocrystal lization via G rinding and Slurry conversion 36 2.2.4 FTIR, PXRD, Diff erential Scanning Calorimetry and Thermo Gravimetric Analysis 37 2.2.5 Deter mination of Crystal Structure 45 2.2.6 Results and Discussion 51 2.3 Diss olution studies 65 2 4 R e f e r e n c e s 6 6 3. QUERCETIN 6 9 3.1 Introduction 6 9 3.1.1 Quercetin in CSD 6 9 3.2 Cocrystals of Quercetin 71 3.2.1 Experimental Section 71 3.2.2 Cocrystallization via Solution Crystallization 72 3.2.3 Cocrystallization via Grindi n g and Slurry conversion 73 3.2.4 FTIR, PXRD, Diff erential Scanning Calorimetry and Thermo G ravimertic A nalysis 74 3.2.5 Determination of Crystal Structure 7 8

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iii 3.2.6 Results and Discussion 7 9 3.3 Dissolution Studies 84 3 4 R e f e r e n c e s 8 7 4. C ONCLUSION AND FUTURE DIRECTION 8 8

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iv LIST OF TABLES Table 1.1 : Summary of the CSD statistics for phenols with other functionalities 18 Table 1.2 : Summary of the CSD statistics for acids with other functionalities 22 Table 1.3 : C ocrystal formers c ontaining amide functionality 23 Table 1.4: C ocrystal formers containing purines 24 Table 1.5: C ocrystal formers containing aromatic nitrogen base 25 Table 2.1: Summary of the cocrystals of PA synthesized 33 Table 2.2: Comparison of solvent ev aporation, grinding and slurry techniques 37 Table 2.3: Crystallographic data of cocrystals of PA 5 0 Table 2.4: C N C angles in n e utral isonicotinamide cocrystals from CSD 55 Table 2.5: C N C angles in n e utral nicotinamide cocrystals from CSD 61 Tab le 3.1: Su mmary of cocrystals of quercetin obtained 72 Table 3.2: Comparison of solvent evaporation, g rinding and slurry techniques 73 Table 3.3: Crystallographic data for cocrystals of quercetin 7 8

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v LIST OF FIGURES Figure 1.1 : Relationship between nu traceuticals and other health products 1 Figure 1.2 : Examples of supramolecula r (a) carboxylic acid homosynthon (b) carboxylic acid pyridine heterosynthon 4 Figure 1.3 : Crystal structure of the triclinic form of quinhydrone 4 Figure 1.4 : The Hoogsteen base pair 5 Figure 1 5 : Representation of pharmaceutical cocrsytal 6 Figure 1 6 : 1 :1 cocrystal of carbamazepine sa ccarine 7 Figure 1 7 : Dissolution profiles of Intraconazole and its cocrystals 8 Figure 1 8 : Dissolution profile of fluox et ine HCl and its cocrystals 8 Figure 1 9 : Hydrogen bonding interactions between the carboxylic acid of the glutaric acid molecule with amide and pyridine groups of the drug molecule 9 Figure 1 1 0 : Dog plasma concentration with time for (a) 5 mg/kg dosing and ( b ) 50 mg/kg dosing o f a drug (dark circles) and cocrystal (open circles) 9 Figure 1.1 1 : 1:1 cocrystal of melamine and cy a nuric acid 1 0 Figure 1.1 2 : The Biopharmaceutic s Classification System (BCS) as defined by the FDA 1 3 Figure 1.1 3: Identification of supramolecular synthons using CSD 1 4 Figure 1.1 4 : (a) Supramolecular OH OH homosynthon (b) Histogram of O H O hydrogen bond lengths in phenols 1 5 Figure 1.1 5 : (a) Supramolecular heterosynthon of phenolic OH N arom (b) Histogram of OH N arom hydrogen bond lengths in phenols 16 Figure 1.16: Supramolecular heterosynthons in phenol and primary amide 16

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vi Figure 1.1 7 : (a) Supramolecular heterosynthon in phenol and carbonyl moiety (aldeh yde/ketone) (b) Histogram of OH O=C hydrogen bond lengths 17 Figure 1. 18 : Histogram of OH ... CO 1 7 Figure 1. 19 : Carboxylic acid dime r s and catemers 19 Figure 1.2 0 : Histogram of O H O hydr ogen bond lengths in carboxylic acid dimers retrieved from CSD 19 Figure 1.2 1 : (a) COOH N arom supramolecular heterosynthon (b) Histograms of O H N arom hyd rogen bond lengths in carboxylic acid a r omatic nitrogen bases retrieved from CSD 20 Figure 1.2 2 : (a) COOH O=C supramolecular heterosynthon (b) Histograms of COOH O=C hydrogen bond lengths in carboxylic acid wit h carbonyl functionality 20 Figure 1.2 3 : (a) COOH CONH 2 supramolecular heterosynthon (b) Histograms of NH 2 O=C hydrogen bond lengths (c) Histograms of OH O=C hydrogen bond lengths in carb oxylic acid with amide functionality 21 Figure 2.1 : Structure of Protocatechuic acid (PA) 30 Figure 2.2: A popular Roselle variety 31 Figure 2.3: Hydrogen bonding in Protocatechuic acid monohydrate 31 Figure 2.4: Hydrogen bonding in Protocatechuic acid ac etonitrile solvate 32 Figure 2.5 : Synthesis and s ingle crystal of 1 34 Figure 2.6 : Synthesis and s ingle crystal of 2 34 Figure 2.7 : Synthesis and s ingle crystal of 3 3 5 Figure 2.8 : Synthesis and s ingle crystal of 5 35 Figure 2.9 : Synthesis of 6 3 6 Figure 2.10 : S ynthesis and single crystal of 7 36 Figure 2.11: Cocrystal 1: (a) PXRD comparison (b) DSC (C) IR 39 Figure 2.12: Cocrystal 2: (a) PXRD comparison (b) DSC (C) IR 40

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vii Figure 2.13: Cocrystal 3: (a) PXRD comparison (b) TGA (C) DSC (d) IR 4 2 Figure 2.1 4: Cocrystal 4: (a) PXRD comparison (b) DSC (C) IR 4 4 Figure 2.15: Cocrystal 5: (a) PXRD comparison (b) DSC (C) IR 4 5 Figure 2.16: Cocrystal 6: (a) PXRD comparison (b) DSC ( c ) IR 4 7 Figure 2.17: Cocrysta l 7: (a) PXRD comparison (b) TGA ( c ) IR (d) DSC before heating (e) DSC after heating 49 Figure 2.18: Each PA molecule is hydrogen bonded to three molecules caprolactam through phenol carbonyl supramolecular h eterosynthon 51 Figure 2.1 9: Formation of a tape with alternate PA and isonicotinamide molecules by acid pyridine supramolecular heterosynthon 52 Figure 2.20: One molecule of PA is hydrogen bonded to five molecules of isonicotinamide 53 Figure 2.21: PA molecules forming acid acid supramolecular homosynthon and supramolecular heterosynthons with two water molecules and one isonicotinic acid molecule. 56 Figure 2.22: Formation of isonicotinic acid chains in 3 56 Figure 2 .23: Acid dimer of PA molecules forming supramolecular heterosynthon 5 8 with theophylline dimers Figure 2.24: Formation of sheets in 5 5 9 Figure 2.25: One molecule of PA is hydrogen bonded to two molecules of PA through OH OH supramolecular homosynthon, one molecule of nicotinamide through acid amide supramolecular heterosynthon and with another nicotinamide molecule th rough OH NH 2 supramolecular heterosynthon 60 Figure 2.26 : Overall packing of 6 61 Figure 2.2 7 : Chain s of disordered PA and water molecules hydrogen bond with 61 carbamazepine dimers Figure 2.2 8 : Comparison of dissolution profiles of PA and its cocrystals 64 Figure 2.2 9 : Pi stacking in (a) PA nicotinamide (b) PA isonicotinamide cocrystals 6 5

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viii Figure 3.1 : Structure of quercetin 6 8 Figure 3.2 : OH carbonyl supramolecular heterosynthon in quercetin dihydrate 70 Figure 3.3 : Intermolecular interactions in the 1:1:1 cocrystal solvate of quercetin, 70 caffeine and methanol Figure 3.4 : Intermolecular interactions in the 1:1 Quercetin Isonicotinamide cocrystal 71 Figure 3.5 : Synthesis and single crysta l of 1 72 Figure 3.6 : Synthesis and si ngle crystal of 2 7 3 Figure 3. 7 : Cocrystal 1 : (a) PXRD comparison (b) TGA (c) DSC (d) IR 75 Figure 3. 8 : Cocrystal 2 : (a) PXRD comparison (b) TGA (c) DSC (d) IR 7 7 Figure 3. 9 : Quercetin dimers hydrogen bonding with iso nicotinic acid molecules 79 Figure 3. 10 : Formation of a chain of isonicotinic a cid molecul es with acid pyridin supramolecular heterosynthon 80 Figure 3.1 1 : DSC after heating cocrystal 1 81 Figure 3.1 2 : Quercetin dimer hydrogen bonding with one theobromine molecule through carbonyl phenol supramolecular heterosynthon 81 Figure 3.1 3 : Formation of sheet structure in 2 82 Figure 3.1 4 : (a) PXRD p attern (b) DSC (C) TGA of attempts of synthesizing anhydrous cocrystal of 2 84 Figure 3.1 5 : Dissolution profiles of dihydrate of 1:1 cocrystal of quercet in and theobromine, in 1:1 EtOH/Water (V/V%) system at 360 nm 85

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ix COCRYSTALS OF NURACEUTICALS: PROTOCATECHUIC ACID AND QUERCETIN Twarita Anil Pujari ABSTRACT The cocrystal l ization of two or more pure compounds by crystal engineering to create a new functional material is of a great academic and industrial inter est. Pharmaceutical cocrysta l lization has allured a lot of attention by means of altering the physicochemical properties of Active Pharmaceutical Ingredient (API) such as solubility, stability and bioavailability. Crystal engineering of nutraceuticals can produce novel compounds such as pharmaceutical cocrystals. To establish the importance of nutraceutical cocrystallization and its use; polyphenols, a major class of nutraceuticals and potential disease preventing agents, are the appropriate targets. The w ork herein focuses on two polyphenols, protocatechuic acid and quercetin, which are strong antioxidants. The cocrystals of quercetin have been synthesized aiming to modify its poor water solubility and bioavailability which limits its usage. On the other hand, cocrystals of water soluble protocatechuic acid are also prepared to establish its use as a cocrystal former. Seven novel cocrystals of protocatechuic acid and two novel cocrystals of quercetin are obtained and are characterized by FT IR, DSC (Differe ntial Scanning Calorimetry), PXRD (Powder X Ray Diffraction), single crystal x ray diffraction and TGA (Thermo Gravimetric Analysis). The new crystal forms have also been studied via dissolution. Dissolution studies show alteration in solubility of a targe t molecule by its cocrystal irrespective of solubility of the cocrystal former. Overall, the study helps in understanding the role of crystal engineering and its utility.

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1 1. INTRODUCTION 1.1 Nutraceutical Nutraceutical is the term used to describe a medici nal or nutritional component which is present in food, plant or any naturally occurring material and which is used for improvement of health by preventing or treating a disease. 1 Nutraceuticals overlap with the other health products such as pharmaceuticals and herbals. 1 The complex relationship is shown in Figure 1.1. Fig ure 1.1 Relationship between nu traceuticals and other health products Pharmaceuticals are usually classe d as medicines by law however n utraceuticals are available over the counter. 2 Dr. Stephen De Felice, founder and chairman of the Foundation for Innovation in Medicine (FIM, USA), first coined the term Nutraceuticals in 1976. 3 He defined it as a food, or parts of food, that provide medical or health benefits, including the prevention an d treatment of disease 1 The broad usage includes supplementation for poor diet, to improve overall health, to delay the onset of age related diseases, for stress, after illness etc. 1 However dietary supplements in the US can be

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2 marketed without FDA appro val. Thus Foundation for the Innovation in Medicine (FIM) introduced the Nutraceutical Research and Education Act in Congress i n October 1999. 1 It helped nutraceuticals to establish a new legal classification. 4 Nutraceuticals are popularly available in fo rms of tablets, capsules, softgels soft chews etc. 1 An American survey of consumer preferences for different formulations carried out in 2004 reveals that new drug deliver y systems such as chewing gums and patches are overcoming the traditional forms suc h as capsules. 5 Nutraceuticals includes wide range of products such as po lyphenols ( Epigallocatechin gallate (EGCG) from tea or resveratrol from red grapes), vitamins, oils from fish and flax, calcium fortified juices, theobromine from cacao tree, caffeine from coffee leaves etc. 1 1.2 Crystal Engineering A crystal, solid material, in which molecules are arranged in an orderly repeating pattern extending in all three spatial dimensions, is an ultimate case of molecular recognition. A series of complex mol ecular recognition with high level of precision supermolecules par excellence 6 The rational design of new crystalline materials led to the development of new field of crystal engineering. 7 In 1955, R. Pepinsky first coined the term crystal engineering who crystallized organic ions with metal containing complexes. 8 Subsequently in 1971 G.M.J. Schmidt implemented it in the photodimerization of cinnamic acid. 9 A working definition of crystal engineering was given by Dr. Desiraju in 198 9 crystal engineering is the understanding of intermolecular interactions in the context of crystal packing and in the utilization of such understanding in the design of new solids with desired physical and chemical properties 10 Crystal engineering ha s been explored vastly in different applications such as non linear optics (NLO ) 11 porous materials, 12 photographic m aterials 13 and coordination polymers. 14 Pharmaceutical one of the most significant sector has start ed utilizing the concept of crystal engineering very recently and it has been noted that crystal engineering has a great impact on Active Pharmaceutical Ingredients (APIs). APIs are among the most valuable crystalline substances and crystal engineering ha s been successfully applied in the ge neration of cocrystals of APIs with improved

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3 physicochemical properties. 15 ,31 1.2.1 Supramolecular Chemistry Supramolecular chemistry is referred as chemistry beyond the molecules Traditional chemistry focuses on covalent bond while supramolecular chem istry deals with noncovalent interactions. In 1890 Nobel laureate Hermann Emil Fischer suggested that enzyme substrate interactions take the form of a "lock and key", pre empting the concepts of molecular recognition and host guest chemistry. Gradually non covalent bonds were understood in more detail and the importance of supramolecular chemistry was well established by 1987. Nobel Prize for chemistry was awarded to Donald J. Cram, Jean Marie Lehn and Charles J. Pedersen in recognition of their work in this area. 1 7 Supramolecular chemistry has wide range of applications in different fields such as material technology, catalysis, data storage and processing and green chemistry. Apart from these ; supramolecular chemistry has been used in the development of ne w pharmaceutical therapies by understanding the interactions at a drug binding site. Moreover it has a great impact on the studies of drug delivery and protein protein i nteractions. 1.2.2 Supramolecular Synthons In 1967 E.J. Corey introduced a concept of a synthon 1 8 as structural units within molecules which can be formed and/or assembled by known or conceivable synthetic operations. On the similar lines recognizing that crystal engineering is the solid state supramolecular equivalent of organic synthes is a term supramolecular synthon was introduced by Dr. Desiraju in 1995. 1 9 Supramolecular synthons structural units within supermolecules which can be formed and/or assembled by known or conceivable intermolecular interactions Supramolecular synthon s are spat ial arrangements of intermolecular interactions between complementary functional groups and simplifies unders tanding of a crystal structure. Supramole cular synthons can be separated in two distinct categories, Supramolecular homosynthons and supr amolecular heterosynthon s. Supramolecular homosynthons are composed of identical self complementary

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4 functionalities 20 such as carboxylic acid dimers 2 1 (Figure 1.2 a) and amide dimers 22 Supramolecular heterosynthons are composed of different but complement ary functionalities such as carboxylic acid amide 2 3 and carboxylic acid pyridine 2 4 (Figure 1.2 b). (a) (b) Figure 1. 2 : Examples of supramolecular (a) carboxylic acid homosynthon (b) carboxylic ac idpyridine heterosynthon 1.2.3 Cocrystals Cocrystal s are a long known little studied class of compounds. The first entry of a cocrystal was reported in 1844 by Whler ; the cocrystal of benzoquinone and hydroquinone ( Refcode: QUIDON, Figure 1.3). 2 5 Sub sequently they were studied as organic molecular compounds 2 6 However until 1960 the structural information on cocrystals was absent and term complexes came in to the picture. Figure 1. 3: Crystal structure of the triclinic form of quinhydrone many people came up with different nucle ic base complexes 2 7 and the term cocrystal was first used in this context 2 8 In 1963, K.Hoogsteen came up with the new base pair known as which can be considered as the first prototypal cocryst al. He studied the formation and properties of free radicals in a

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5 cocrystal between 1 methylthy a mine and 9 methyladenine ( Refcode: MTHMAD F igure 1. 4 ). Later t he term cocrystal was subsequently popularized by Margaret Etter. 2 9 Figure 1. 4 : The Hoogsteen b ase pair T he definition of a cocrystal is still a topic of debate today 30 A broad definition of a cocrystal was given by Dunitz; 3 1 I n this vi ew hydrates, solvates, inclusion compounds, clathrates c an be recognized as cocrystals. In Zaworotko research group and others we have been using a definition; a cocrystal is a stoichiometric multiple component crystal in which all components are solid under ambient condi tions when in their pure form. These c omponents consist of a target molecule or ion and a molecular cocrystal former(s) and when in a cocrystal they coexist at the molecular level within a single crystal 32 ,33 This definition distinguishes cocrystals from the other broad group of multiple com ponent compounds. This emerging field of cocrystal allows modifying the composition of matter and physicochemical properties of a molecule without breaking or forming a covalent bond. Thus there are different applications for cocrystals. The most importa nt field of pharmaceutics is discussed in detail in section 1.4. Other relevant fields include solid state synthesis and chiral resolution In 1982 Etter et. al. reported a nucleophilic substitution reaction, the firs t reaction in the context of solid sta te synthesis 3 4 Recently Cheney et. al. have reported the condensation of amines and acid anhydrides synthesizing imides. 3 5

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6 In 1848 Louis Pasteur reported spontaneous resolution of sodium ammonium Tartrate 3 6 Generally racemates are known to be the resul t of 90% racemic mixtures. 3 7 Chiral sep aration being a tedious process, the chiral cocrystal formers can offer an enhanced possibility of separating enantiomers by fractional crystal lization since the resulting co crystals are diastereomeric in nature. The well known example of chiral separation is a spontaneous resolution of racemic mandelic aci d by (s) alanine and (R) cystine. 3 8 1.2.4 Pharmaceutical Cocr ystals An attractive subset of cocrystals is pharmaceutical cocrystals. Pharmaceutical cocrystals are defined as co crystals in which the target molecule or ion is an active pharmaceutical ingredie nt, API, and it bonds to the co crystal former(s) through hydrogen bonds. 39 Figure 1. 5 : Represe ntation of pharmaceutical cocrys tal Delivery of an API is gene rally developed in variety of solid forms and administered in different ways such as amorphous, salt, polymorphs, solvates. 40 The properties such as stability, solubility and bioavailability of a drug are direct ed by its solid form. Thus it gives an opportunity to a researcher to develop the best suitable form of a drug with improved physicochemical properties. First pharmaceutical cocrystal was reported in 1934 by Heyden et al. 4 1 He patented cocrystals of Bar biturates with 4 oxy 5 nitropyridine, 2 ethoxy 5 acetaminopyridine, N methyl alpha pyridone and aminopyridine Complexes of a class

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7 of lactam antibiotics with parabens and related compounds were patented by Eli Lilly in 1995. 4 2 Scientif ic literature da ta base is getting enriched with number of pharmaceutical cocrystals with improved physicochemical properties. Carbamazepine: Saccharin 1:1 cocrystal ( UNEZAO01 ) (Figure 1. 6 ), developed in 2003 by Almarsson and coworkers, was studied in vivo in dogs f or the ir oral pharmacokinetics. It showed the improved bioavailability than the marketed carbamazepine drug, T egretol. 43 Figure 1. 6 : 1:1 cocrystal of carbamazepine saccarine Sporanox capsules containing itraconazole as an active ingredient used as an anti fungal agent and is extremely water in soluble. The aqueous dissolution of 2:1 cocrystal of i traconazole and succinic acid and other two cocrystals were studied in 0.1 N HCl at 25C which showed the improved dissolution profile (Figure 1. 7 ) Pharmacokinetic study of itraconazole cocrystals also revealed that co crystal formulation of the API gives similar oral bioavailability to the Sporanox form in the animal trial using a dog model. 4 4

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8 Figure 1. 7 : Dissolution profiles of Intraconazole and its cocrystals Prozac an antidepressant drug, contains Fluoxetine hydrochloride as an API. In 2004, Childs et.al studied dissolution profile of the three cocrystals of fluoxine HCl in water (Figure 1. 8 ). 4 5 Fluox eti ne HCl: Succinic acid cocrystal showed two fold increa se in solubility as compared to fluox et ine HCl itself. It has also been observed that cocrystal may decrease the solubility a s in the case of fluox et ine HCl: b enzoic acid. Figure 1. 8 : Dissolution profile of fluox et ine HCl and its cocrystals

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9 2 [4 (4 chlo ro 2 fluorophenoxy) phenyl] pyrimidine 4 carboxamide, a sodium channel blocker (SCB) was developed for the treatment and prevention of surgical, chronic and neuropathic pain. Many formulation trials failed to improve the bioavailability of a drug. 1:1 coc r ystal of a drug (SCB) with glutaric acid (Figure 1. 9 ) which was one of the hit from cocry s tal screening showed physical and chemical stability of a drug. Co crystal increases in vitro rate of dissolution by 18 times when compared to the pure API (Figure 1. 1 0 ) Bioavailability studie s on dogs confirmed that the co crystal improved the plasma AUC values by three times at both low (5 mg/kg) and high dose (50 mg/kg). 4 6 Figure 1. 9 : Hydrogen bonding interactions between the carboxylic acid of the glutaric acid molecule with the amide and pyridine groups o f the drug molecule Figure 1.1 0 : Dog plasma concentration with time for (a) 5 mg/kg dosing and (b) 50 mg/kg dosing of a drug (dark circles) and cocrystal (open circles)

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10 The cocrystal of Sildenafil citrate (a ctive pharmaceutical ingredient in Viagra ) with aspirin was patented in 2007 which showed the improved solubility properties in acidic conditions Also it has shown the thermodynamic stability up to 165 0 C. 4 7 Amgen developed a transient receptor potentia l vanilloid 1 antagonist, AMG 517, for treatment of acute and chronic pain. The free base of AMG 517 is associated with problems such as insolubility in water and buffers at physiological pH. Ba k and coworkers 4 8 synthesized 10 cocrystals of AMG 517 and the physicochemical properties such as particle size, solubility, stability, hygroscopicity, thermal behavior and structu ral characteristics of these co crystals were studied in detail. Furth e r the solubility studies of these c ocrystals showed the increase in solubility and the ir conver sion to AMG 517 hydrate. In early 2007, pet food crisis was observed in China. More than 24,000 clinical assessments were found with acute renal failure within 3 months in Hong Kong. It was observed that melamine, protein con te nt in the pet food, forms a cocrystal with cy a nuric acid (QACSUI) another content of pet food. Even though melamine and cy a nuric acid are nontoxic individually the resulting 1:1 cocrystal (Figure 1.1 1 ) is highly insoluble in water resulting in toxic kidne y stones. It is a first example of co crystals altering clinically relevant physical properties in a negative manner 4 9 Figure 1.1 1 : 1:1 cocrystal of melamine and cy a nuric acid C ocrystals or pharmaceutical cocrystals are novel as they can afford crystal form diversity. The utility in terms of improving physicochemical properties of a drug represents an opportunity to patent and market clinically improved crystal forms of available drugs in form of new drugs. In conclusion cocrystals and pharmaceutical c ocrystals are offering new avenue s to the pharmaceutical industry.

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11 1.2.5 Nutraceutical Cocrystals As explained in 1.1 nutraceuticals are the naturally occuring nutritional products that have health related benefits and thus have an added advantage over p harmaceuticals Many nutraceuticals have a proven record in drug discovery such as aspirin and penicillin. Nutraceuticals have established safety record and are readily available under Good Manufacturing Practices (GMP). This will speed up the drug to clin ic by lowering the preclinical burden and toxicity risk. Even though nutraceuticals are the potential drug candidates many of them possess low water solubility and hence bioavailability. Improving bioavailability by synthesizing a nutraceutical cocrystal has a large intellectual property potential. On the other hand, highly water soluble, being safe, can serve as a cocrystal former for a less soluble API to improve its solubility. 1.2.6 Synthesis of Cocrystals A range of methodologies for the synthesis o f cocrystal s has been reported in the literature Single crystals are commonly developed via slow evaporation of a solution containing stoichiometric amounts of target molecule and co c rystal former. The choice of the solvent is crucial in obtaining a cocry stal since role of a solvent in nucleation of crystals is poorly understood. Solid state grinding has been established from 19 th century. T he very first cocrystal of quino ne and hydroquinone was synthesized via grinding. 2 5 The technique of adding small am ount of solvent i.e. solvent drop grinding or liquid assisted grinding was shown to promote cocrystal formation. 50 Automated grinding ballmills are also de veloped for efficient synthesis Another technique is the growth from melt in which two components ar e placed together and melt is formed. 5 1 The resulting solution is seeded by remaining solid material and a cocrystal is formed. Slurry preparation, 5 2 sublimation are the other suitable methodologies. 1.3 Solubility and Bioavailability Solubility and bi oavailability of a drug are important parameters in drug development process. Intrinsic solubility and dissolution of a drug are prerequisites for oral drug delivery. The fundamentals of the dissolution are studied by Noyes Whitney

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12 equation 5 3 and the corre lation of it with bioavailability leads to a Biopharmaceutics Classification system (BCS) 1.3.1 Noyes Whitney Equation In 1897 the first dissolution experiment was performed by Noyes and Whitney for benzoic acid and lead chloride. 5 3 The observations o f this experiment was formulated in terms of law which wa s expressed as the rate at which a solid substance dissolves in its own solution is proportional to the difference between the concentration of that solution and concentration of the saturated sol ution Its mathematical expression is as follows, Where C S represents the solubility of the substance or the concentration of its saturated solution; C the concentration at the expiration of time t and k a constant. Further with the series of the experi ment in 1900 Erich Brunner and Stanislaus von Tolloczko showed that the rate of the dissolution depends on the exposed surface, the rate of the stirring, temperature, structure of the surface and the arrangement of the apparatus. 5 4 Thus modified mathematic al expression came out to be, Where k 1 is constant and S is the surface are a It implemented that the rate of dissolution of a drug will increase if the bulk solubility will increase. 5 5 In the period of 1950 1980 series of experiments showed that the rat e of dissolution affects the bioavailability of drug. And it is now well recognized that bioavailability gets influenced critically by solubility. 1.3.2 BCS Classification While understanding the complexity of the drug absorption process Amidon and cowor kers developed a simplified macroscopic approach and correlated drug absorption and intestinal permeability. 5 6 In addition they developed a model for water insoluble drugs. From these models it was well established that drug absorption is controlled by

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13 th ree factors; absorption number, dissolution number and dose number. All these observations lead them to set a standard for drug bioavailability and Biopharmaceutics Classification S ystem (BCS) was proposed. According to BCS a substance is classified on the basis of its aqueous solubility and intestinal permeability, and four drug classes were defined i.e., high solubility/high permeability (Class I), low solubility/high permeability (Class II), high solubility/low permeability (Class III), low solubility/lo w permeability (Class IV) (Figure 1.1 2 ) According to BCS a drug substance is considered highly soluble when the highest strength is soluble in 250 m l or less of aqueous media over the pH range of 1.0 7.5; otherwise, the drug substance is considered to be poorly soluble. 5 7 BCS class II drugs present a challenge to cocrystallization field where cocrystals can make a difference by altering the bulk solubility of a drug and hence altering the rate of a dissolution and furthermore the bioavailability of an API. It has already been demonstrated with cocrystals of carbamazepine, itraconazole and fluoxetine h ydrochloride as explained in section 1.2.4. Figure 1.1 2 : The Biopharmaceutics Classification System as defined by the FDA 1.4 Cocrystal Design 1.4.1 Cambr idge Structural Database (CSD) A prerequisite in cocrystal design is Cambridge Structural Database (CSD). CSD is a database that was developed in 1965 at Cambridge University by Olga

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14 Kennard. 5 8 CSD is a d epository of X ray and neutron diffraction data of organics, organometallics and metal complexes containing up to 500 non H atoms. The database is a collection of bibliographic, information re ferencing t he c hemical connectivity in terms of two dimensional (2 D ) structural formula and three dimensional ( 3D ) molecular structure and crystallographic data such as u nit cell dimensions, s pace group, a tomic coordinate data (3D crystal structure) CSD comprises of mainly four programs. ConQuest allows searching and retrieving the information, Mercury for structure v isualization, Vista gives numerical analysis and PreQuest which is used for database creation. CSD database is rapidly increasing and August 2008 release contains structural information of 456,628 organic, metal organic and organometallic crystal structur es. Designing a cocrystal first involves a CSD survey which helps in the identification and understanding of supramolecular synthons of functional groups present in target molecule. Analysis of intermolecular contacts facilitates the selection of a cocrys tal former. Figure 1.1 3 : Identification of supramolecular synthons using CSD 1.4.2 CSD statistics for Acids and Phenols The chosen targeted molecules for the study contain phenol and acid functionalities Thus it is necessary to know about the statist ics of existing synthons of acid and phenol with other functional groups present in the CSD. All searches were carried out by considering following and structures with 3D coordinates. The raw data inclu des any datasets with competing hydrogen bonding functionality and refined data excludes the same

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15 Figure 1.1 4 : (a) Supramolecular OH OH homosynthon (b) Histogram of O H O hydrogen bond lengths in phenols The CSD a na lysis reveals 7193 entries con taining at least one phenolic OH moiety Phenols can form supramolecular homosynthon (Figure 1.1 4 a ). The raw search contains 1028 (14.29%) entries that exhibit OH OH supramolecular homosynthon The percentage of supramolecula r homo synthon increases to around 59% when competing functionalities are absent. Figure 1.1 4 b shows the histogram for OH O hydrogen bond. The range of the hydrogen bond is 2.459 3.040 with the mean value of 2.809 (2) . The results are summarized in table 1.1 There are 690 structures containing both phenolic OH and aromatic nitrogen base, out of which 3 49 (50.58%) entries exhibit OH N arom supramolecular heterosynthon (Figure 1.1 5 a) where as 87 (12.60%) entries contain OH OH supramolecular homosynthon When the competing hydrogen bonding groups are absent, there are 136 entries containing both phenolic OH and aromatic nitrogen base and out of these 38 (27.94%) shows OH OH supramolecular homosynthon and 108 (79.41%) contain OH Narom supramolecular heterosynth on which explains the reliability of OH N arom synthon. The OHN arom supramolecular heterosynthon occurs within the range 2.515 3.066 (ON) with an average ON of 2.746 () as shown (Figure 1.1 5 b). (a) (b)

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16 Figure 1 .1 5 : (a) Supramolecular heterosynthon of phenolic OH N arom (b) Histogram of OH N arom hydrogen bond lengths in phenols F igure 1.1 6 : Supramolecular heterosynthons in phenol and primary amide CSD holds 81 structures containi ng both phenol and amide functionalities. Phenols can hydrogen bond with amide s in two different fashions. Raw data reveals that supramolecular homosynthon of amide (30 37.03% ) dominates over the phenol amide supramolecular hete r osynthon (Figure 1.1 6 ). S tatistics obtained from refined data (3) is too limited to generalize the hierarchy of the se synthon s The CSD analysis on phenols and carbonyl moiety was also carried out. It shows the presence of 1668 structures. Supramolecular heterosynthon OH O=C (Fig ure 1.1 7 a) is present in 599 (35.91%) entries whereas 212 (12.70%) entries contain the supramolecular OH OH homosynthon. The % of supramolecular heterosynthon increases to 64% when competing groups are absent. The cut off range for OH O=C is 2.413 3.038 with an average of 2.760 (12) . (a) (b)

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17 Figure 1.1 7 : (a) Supramolecular heterosynthon in phenol and carb onyl moiety (aldeh yde/ketone) (b) Histogram of OH O=C hydrogen bond lengths There are 363 entries containing both pheno l and acid moieties. 90 (24.79%) of them s hows phenol carbonyl OH O=C supramolecular het e rosynthon The cut off range for OH O=C synthons is 2.613 3.039 with the average value of 2.785 ( 4 ) Figure 1. 18 : Histogram of OH O=C and (a) (b)

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18 Table 1. 1 : Summary o f the C SD statistics of phenols with other functionalities structures with 3D coordinates Functional group present No. of structures Raw data No. of structures Refined data OH (Phenol) 7193 1028 ( 14.29% ) OH OH 216 127 (58.79%) OH OH OH & N arom 690 87 (12.60%) OH OH 3 49 (50.58%) OH N arom 136 38 (27.94%) OH OH 108 (79.41%) OH N arom OH & primary amide 81 30 (37.03%) CONH2 CONH2 15 (18.52%) OH O=C(NH2) 21 (25.92%) OH NH2 3 1 (3 3.33%) CONH2 CONH2 2 (66.67%) OH O=C(NH2) 3 (100%) OH NH2 OH & O=C 1668 212 (12.70%) OH OH 599 (35.91%) OH O=C 245 53 (21.63%) OH OH 153 (62.45%) OH O=C OH & carboxylic acid 363 70 (19.28%) OH OH 106 (29.20%) COOH COOH 90 (24.79%) OH O=C (acid) 58 21 (%) OH OH 47 (%) COOH COOH 17 (29.31%) OH O=C (acid Carboxylic acid is one of the important functional group in crystal engineering. Carboxylic acid can act as both hydrogen bond donor as well as acceptor thus can form a complementary unit with themselves. Two types of association are observed, dimers and catemers (Figure 1.19) The CSD data reveals 5943 entries which contain at least one carboxylic acid functional group. The carboxylic acid dimer was observed in 1871 (31.48%) entries with O O range 2.50 3.0 0 with mean value of 2.650 (1) and catemers are observed in 176 (2.96%) entries. Whereas when other functionalities are absent, out of 683 entries 547 (80%) show acid dimers and 54 (7.9%) show acid catemers.

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19 F igure 1. 19 : Carboxylic acid dime r s and catemers Figure 1.2 0 : Histogram of O H O hydrogen bond lengths in carboxylic acid dimers retrieved from CSD

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20 Figure 1.2 1 : (a) COOH N arom supramolecular heterosynthon (b) Histograms of OH N arom hydrogen bond lengths in carboxylic acid a r omatic nitrogen bases retrieved from CSD Further analysis of CSD reveals 645 entries containing both carboxylic acid and aromatic nitrogen base. The ability of carboxylic acids and aromatic nitrogen to form reliable carboxylic acid aromatic nitrogen supramolecul ar heterosynthon has been already established. 58 It shows 94.78% occurrence of COOH N arom supramolecular heterosynthon when other competing groups are not present. The cut of f range for the synthon is 2.50 3.045 with mean value of 2.652 (2) Figu re 1.2 2 : (a) COOH O=C supramolecular heterosynthon (b) Histograms of COOH O=C hydrogen bond lengths in carboxylic acid with carbonyl functionality

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21 The analysis of carboxylic acid and carbonyl (aldehydes/ketones) reveals 621 entries with approximately simil ar occurrence of supramolecular heterosynthon and supramolecular homosynthon. The occurrence of supramolecular heterosynthon increases from 26 to 30% in case of refined data but supramolecular homosynthon dominates (46.73%). The cut of f range for COOH O is 2.451 3.030 with mean value of 2.698 (6) The analysis of carboxylic acids and amides reveals that the COOH CONH 2 supramolecular heterosynthon shows 49.44% reliability of occurrence in the presence of other competing groups whereas the percentage incre ases to 64.3% in the absence of other hydrogen bonding functionalities. The total number of entries for the refined data is limited to 28. The cut off range for NH 2 O=C is 2.809 3.061 with mean value of 2.940 (9) and for OH O=C is 2.418 2.713 mean 2.5 64 (5) . Figure 1.2 3 : (a) COOH CONH 2 supramolecular heterosynthon (b) Histograms of NH 2 O=C hydrogen bond lengths (c) Histograms of OH O=C hydrogen bond lengths in carboxylic acid with amide functionality All above results for carboxylic acid are s ummarized in Table 1.2.

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22 Table 1. 2 : Summary of the C SD statistics for acids with other functional ities Functional group present No. of structures Raw data No. of structures Refined data COOH 5943 1871( 31.48% dimers) 176 (2.96% catemer) COOH COOH 6 83 547( 80% dimers) 54 (7.9% catemer) COOH COOH COOH & N arom 645 32 (4.96%) COOH COOH 486 (75.35%) COOH N arom 134 9 (6.7%) COOH COOH 127 (94.78%) COOH N arom COOH & primary amide 178 8 (4.49%) COOH COOH 52 (29.21%) CONH 2 CONH 2 88 (49.44%) COOH CONH 2 28 1 COOH COOH 3 CONH 2 CONH 2 18 (6 4 3%) COOH CONH 2 COOH & O=C 621 158(25.44%) COOH COOH 164 (26.40%) COOH O=C 199 93 (46.73%) COOH COOH 78(39.20%) COOH O=C Upon reviewing the CSD statistics obtained for phenols and acids aromatic nitrogen bases seem to be promising cocrystal formers Also carbonyls, amides and purines would be promising. 1.4.3 Cocrystal formers A c ocrystal former hydrogen bond s with the ta r get molecule forming a cocrys tal. The data obtained from CSD stati stics helps us to choose suitabl e cocrystal formers for a target molecule. A cocrystal former must also be pharmaceutically acceptable or even approved. Even though the capacity of available cocrystal formers is not fixed they are limited to generally regarded as safe (GRAS) or a compoun d that has already been approved by food and drug administration (FDA) Other characteristic features those are taken in to an account are solubility, toxicity, pKa and cost. Tables 1.3, 1.4 and 1.5 summarize the cocrystal formers chosen for targeted molec ules.

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23 Table 1.3: Cocrystal formers containing amide functionality Structure Mol. formula, Name Mol. Wt. M.P ( 0 C) Cost pKa LD 50 mg/kg Solubility mg/ml C 6 H 11 NO Caprolactam 113.16 68 70 20.30/500 g 16.61 Acidic 0.14 basic 1210 (Rat oral) 4560 Soluble C 6 H 10 N 2 O 2 Piracetam 142.16 153 47.40/25g 15.67 Acidic 0.62 basic 12000 (Mouse subcutan eous) 84 Soluble C 15 H 12 N 2 O 2 Oxcarbazepine 252.27 219 221 65/10mg 13.73 basic 0.53 acidic 4470 (rat, oral) 0.35 Very Slightly soluble C 15 H 12 N 2 O Ca rbazepine 236.37 203 205 41.70/5g 13.94 0.20Acid ic 0.49 0.20 basic 4025 (Rat oral) 0.078 Practically Insoluble

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24 Table 1.4: Cocrystal formers containing purines Structure Mol. formula, Name Mol. Wt. M.P ( 0 C) Cost pKa LD 50 mg/kg Solubility mg/ ml C 7 H 8 N 4 O 2 Theobromine 180.16 357 358 24.80/25g 9.90 basic 0.59acidi c 300 (dog) 6.1 Slightly soluble C 8 H 10 N 4 O 2 Caffeine 194.13 238 57.70/250 g 0.73 basic 355 (Rat oral) 20 Slightly Soluble C 5 H 5 N 5 Adenine 135.13 361 63.70/25g 10.43 bas ic 745 (Rat oral) 0.976 Very Slightly soluble C 7 H 8 N 4 O 2 Theophylline 180.16 271 25.90/100 g 8.600.5 0 Acidic 1.450.7 0 basic 225 (Rat oral) 4.1 Slightly soluble

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25 Table 1.5: Cocrystal formers containing aromatic nitrogen base Structu re Mol. formula, Name Mol. Wt. M.P ( 0 C) Cost pKa LD 50 mg/kg Solubility mg/ml C 5 H 8 N 2 3,5 dimethyl pyrazole 96.13 105 108 49.20/100 g 15.2 basic 3.91acidi c 1060 (mouse, oral) 9 Slightly soluble C 8 H 14 N 2 O 2 Isonicotinamid e 122.12 155 157 39.40/100 g 1 4.96 acidic 3.39 basic 106* Soluble C 8 H 10 N 4 O 2 Nicotinamide 122.12 128 130 18.20/100 g 14.83 acidic 3.54 basic 1680 (Rat subcutan eous) 600* Slightly soluble C 6 H 4 NO 2 Isonicotinic acid 123.11 >300 (decomposes) 13.30/50g 1.94 acidic 4.88 bas ic 3123 (mouse, oral) 16 Sparingly soluble C 6 H 4 NO 2 Nicotinic acid 123.11 236 239 14.50/100 g 2.17 basic 4.82 acidic 5000 (rat, subcutan eous) 32 Sparingly soluble Practically determined values

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26 1 5 R e f e r e n c e s 1. Brian Lockwood, Nutraceuticals, Pharmaceutical Press: London, UK, 2007 2. Rapport L.; Lockwood B. Nutraceuticals London: Pharmaceutical press, 2002. 3. Mannion m, Nutraceutical revolution continues at Foundation for Innovation in Medicine Conference, Am J Nat Med 1998 5, 30 33. 4. Foundation for innovation in Medicine, 2006 www.fimdefelice.com 5. Write T. Nutraceuticals World 2005 42, 44 6. Dunitz, J. D. Pure Appl. Chem. 1991 63 177. 7. (a) Desiraju, G. R. Crystal Engineering: the Design of Organic Solids ; Elsevier: Amsterdam, 1989 (b) Braga, D. Chem. Commun. 2003 2751. (c) Moulton, B.; Zaworotko, M. J. Chem. Rev. 2001 101 1629. 8. Pepinsky, R. Phys. Rev. 1955 100 971. 9. Schmidt, G. M. Pure Appl. Chem. 1971 27 647. 10. Desiraju, G. R. Crystal Engineerin g: the Design of Organic Solids ; Elsevier:Amsterdam, 1989 11. Panunto, T. W.; Urbanczyk Lipkowska, Z.; Johnson, R.; Etter, M. C. J. Am.Chem. Soc. 1987 109 7786. 12. Russell, V. A.; Evans, C. C.; Li, W. J.; Ward, M. D. Science 1997 276 5 75. 13. Taylor, L. D.; Warner, J. C. US 5338644 A 19940816 Cont. of US 5,177,262 14. (a) Moulton, B.; Zaworotko, M. J. Curr. Opi. Solid State and Mater.Sci. 2002, 6, 117 123. (b) McManus, G. J.; Perry IV, J. J.; Perry, M.; Wagner, B. D.; Zaworotko, M. J. J. Am. Chem. Soc. 2007, 129, 9094 9101. 15. Blagden, N.; de Matas, M.; Gavan, P. T.; York, P. Advanced drug delivery Reviws, 2007 ; 59, 617 630 16. Lehn, J. M., Struct. Bonding 1973 16 1. 17. "Chemistry and Physics Nobels Hail Disc overies on Life and Superconductors; Three Share Prize for Synthesis of Vital Enzymes" Harold M. Schmeck Jr. New York Times October 15, 1987 1 8 Corey, E. J. (1967). Pure Appl. Chem. 14, 19 37 1 9 Desiraju, G. R. (1995). Angew. Chem. Int. Ed. Engl. 34, 2311 2327

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27 20 Walsh, R. D. B.; Bradner, M. W.; Fleischman, S.; Morales, L. A.; Moulton, B.; Rodrguez Hornedo, N.; Zaworotko, M. J. Chem. Commun. 2003 186. 2 1 (a) Leiserowitz, L. Acta Crystallogr. 1976 B32 775. (b) Etter, M. C. J. Am. Chem. Soc. 1982 104 1095 2 2 ( a) Leiserowitz, L.; Hagler, A. T. Proc. Royal Soc. London Series A 1983 388 133 175. (b) Weinstein, S.; Leiserowitz, L.; Gil Av, E. J. Am. Chem. Soc. 1980 102 2768 2772. (c) Leiserowitz, L.; Tuval, M. Acta Crystallogr. 1978 B34 1230 1247. 2 3 (a) Aakeroy, C. B.; Beatty, A. M.; Helfrich, B. A.; Nieuwenhuyzen, M. Cryst. Growth Des. 2003 3 159 165. (b) Aakeroy, C. B.; Beatty, A. M.; Helfrich, B. A. Angew. Chem. Int. Ed. 2001 40 32 40 3242. (c) Vishweshwar, P.; Nangia, A.; Lynch, V. M. Cryst. Growth Des. 2003 3 783 790. (d) Reddy, L. S.; Nangia, A.; Lynch, V. M. Cryst. Growth Des. 2004 4 89 94. (e) Videnovaadrabinska, V.; Etter, M. C. J. Chem. Crystallogr. 1 995 25 823 829. 2 4 a) Arora, K. K.; Pedireddi, V. R. J. Org. Chem. 2003 68 9177 9185. (b) Steiner, T. Acta Crystallogr. 2001 B57 103 106. (c) Vishweshwar, P.; Nangia, A.; Lynch, V. M. J. Org. Chem. 2002 67 556 565. (d) Etter, M. C. ; Adsmond, D. A. J. Chem. Soc.Chem. Commun. 1990 589 591. (e) Aakeroy, C. B.; Beatty, A. M.; Helfrich, B. A. J. Am. Chem. Soc. 2002 124 14425 14432. (f) Bhogala, B. R.; Vishweshwar, P.; Nangia, A. Cryst. Growth Des. 2002 2 325 3 28. 2 5. F. Whler, Justus Liebigs Ann. Chem., 51 153 (1844) 2 6 J. S. Anderson, Nature, 140 583 584 (1937) 2 7 (a)Hoogsteen, K. Acta Cryst 1963 ; 16, 907 (b). Kim, S. H.; Rich, A. J. Mol. Biol. 1969 ; 42, 87 (c) Hoogsteen, K. Acta Crys 1959 ; 12, 822. (d) O'Brien, E. J. Acta Cryst 1967 ; 23, 92. 2 8 Schmidt, J.; Snipes, W. Int. J. Radiat. Biol 1967 ; 13, 101 109. 2 9 Etter, M. C. J. Phys. Chem 1991 ; 95, 4601 4610. 30 (a) G. R. Desiraju, CrystEngComm, 2003 5 466 467 (b) J. D. Dunitz, CrystEngComm, 2003 4 506 (c) A. D. Bond, CrystEngComm, 2007 9 833 834 (d) C. B. Aakery, M. E. Fasulo and J. Desper, Mol. Pharm., 2007 4 317 322 (e) G. P. Stahly, Cryst. Growth Des., 2007, 7 1007 1026 (f) J. Zukerman Schpector and

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28 E. R. T. Tiekink, Zeit. Fur Kristallogr., 2008 223 233 234 (g) A. Parkin, C. J. Gilmore and C. C. Wilson, Zeit. Fur Kristallogr., 2008, 223 430. 31 Dunitz, J. D. Cryst Eng Comm. 2003 ; 5 506. 32 N. Shan and M. J. Zaworotko, Drug Disco Today. 13 440 446 (2008). 33 C. B. Aakery .; A. M. Beatty .; B. A. Helfrich Angew. Chem., Int. Ed., 2001 40 34 M. C. Etter, G. M. Frankenbach and J. Bernstein, Tetrahedron Lett., 1989 30 3617 3620 3 5 M. L. Cheney, G. J McManus, J. A. Perman, Z. Wang and M. J. Zaworotko, Cryst. Growth Des., 2007 7 616 617. 36 L. Pasteur, Ann. Chim. Phys., 24 442 (1848) 37 O. Wallach, Justus Liebigs Ann. Chem., 286 90 143 (1895) 38. (a) Z. Q. Hu.; D. J. Xu .; Y. Z. Xu .; J. Y. WU.; M. Y. Chiang .; Acta Crystallogr., 2002 C58 612 614 (b) Z. Q. H u.; D. J. Xu .; Y. Z. Xu, Acta Crystallogr., 2004 E60 269 271. 39 Almarsson and M. J. Zaworotko, Chem. Commun., 1889 1896 (2004) 40 Ann, M. Thayer, C & EN 2007; 17 4 1 French patent: F. von Heyden et al., 769 586, (1934) 4 2 Eli lilly patent: J. G. Amos, J. M. Indelicato, C. E. Pasini and S. M. Reutzel, Bicyclic beta lactam/paraben complexes 1995 US5412094 (A1), JP7048383 (A), FI943081 (A), BR 9402561 (A) and EP0637587 (B1) 4 3 M. B. Hickey, M. L. Peterson, L. A. Scoppettuolo, S. L. Morrisette, A. Vetter, H. Guzman, J. F. Remenar, Z. Zhang, M. D. Tawa, S. Haley, M. J. Zaworotko and . Almarsson, Eur. J. Pharma. Biopharma., 2007 67 112 119. 4 4 J. F. Remenar, S. L. Morissette, M. L. Peterson, B. Moulton, J. M. MacPhee, H. R. Guzmn and . Almarsson, J. Am. Chem. Soc ., 2003 125 4 5 S. L. Childs, L. J. Chyall, J. T. Dunlap, V. N. Smolenskaya, B. C. Stahly and G. P. Stahly, J. Am. Chem. Soc ., 2004 126 4 6 D. P. McNamara, S. L. Childs, J. Giordano, A. Iarriccio, J. Cassidy, M. S. Shet, R. Mannion, E. O'Donnell and A. Park, Pharma. Res ., 2006 23, 1888 1897

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29 4 7 Zegarac, M. et al., Pharmaceutically accepta ble cocrystalline forms of sildenafil, WO 2007/080362 A1 4 8 A. Bak, A. Gore, E. Yanez, M. Stanton, S. Tufekcic, R. Syed, A. Akrami, M. Rose, S. Surapaneni, T. Bostick, A. King, S. Neervannan, D. Ostovic and A. Koparkar, J. Pharm. S ci., 2008 97 3942 3956. 4 9 B. Puschner, R. H. Poppenga, L. J. Lowenstine, M. S. Filigenzi and P. A. Pesavento, J. Vet. Diagn. Invest., 2007 19 616 624 50 N. Shan, F. Toda and W. Jones, Chem. Commun ., 2002 2372 2373 5 1 K. Chadwick, R. J. Dave y and W. Cross, CrystEngComm 2007 9, 732 734 5 2 Childs, S. L.; Rodrguez Hornedo, N.; Reddy, L. S.; Jayasankar, A.; Maheshwari, C.; McCausland, L.; Shipplett, R.; Stahly, B. C. CrystEngComm 2008 10 856 864. 5 3 Noyes, A.A., Whitney, W.R., 1897. The rate of solution of solid substances in their own solutions. J. Am. Chem. Soc. 19, 930 934. 5 4 Bruner, L., Tolloczko, S., 1900. U¨ ber die Auflo¨sungsgeschwindigkeit Fester K¨orper. Z. Phys. Chem. 35, 283 290 5 5 A. Dokoumetzidis, P. Mac heras International Journal of Pharmaceutics 2006 321 1 11 5 6 G. L. Amidon, H. Lennernas, V. P. Shah, and J. R. Crison. A theoretical basis for a biopharmaceutics drug classification: the correlation of in vitro drug product d issolution and in vivo bioavailability. Pharm. Res. 12 :413 420 (1995). 56. Chi Yuan Wu; Leslie Z. Benet Pharmaceutical Research, 2005 22, 1 57. www.ccdc.cam.ac.uk/products/csd/ 58. Lehn, J. M., Struc t. Bonding 1973 16 1

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30 2. PROTOCATECHUIC ACID 2.1 Introduction Protocatechui c acid (PA Figure 2.1 ), (3,4 dihydroxybenzoic acid, C 7 H 6 O 4 Mol. Wt. 154.12, M.P. 20 5 0 C) is a phenolic acid 1 in the broad class of polyphenols of the nutraceuticals. It is widely avail able in oil, vegetables, fruits and tea. I t i s mainly found in Roselle ( Hibiscus sabdariffa ) (Figure 2.2) which is a species of hibiscus found in old world tropics. Figure 2.1: Structure of Protocatechuic acid (PA) PA is a strong antioxidant and in pharmaceutics it has been used as an anticarcinogenic agent. 2 3, 4 PA is known for chemoprevention as well as tumor progression. It i nduces apoptosis of human leukemia cells while on the other hand it enhances tumor promotion in mouse skin cells. 5 It has been predicted that higher dosage of PA enhances tumor. 6 The solubility of PA in water is 10mg/ml and LD 50 is 800mg/kg which makes PA a potential cocrystal former. PA (Figure 2 .1) contains two phenolic groups and a carboxylic acid group. Both can serv e as hydrogen bond donor/acceptor. Based on the CSD search, occurrence of supramolecular heterosynthons between polyphenols with amides, basic nitrogen and purines would be promising in the context of cocrystals. Thus different cocrystal formers containing such functionality were selected on the basis of their solubility, toxicity, pKa and cost as summarized in tables 1. 3 1. 4 and 1. 5 The study herein foc uses to establish the use of PA as a cocrystal former

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31 to improve the physicochemical properties of the target molecules. Figure 2.2: A popular Roselle variety 2. 1.1 Protocatechuic acid in CSD The CSD analysis on PA revealed that the crystal structure of pure PA is not known however there are t hree polymorphs ( four entries ) of PA monohydrate (Ref codes : BIJDON, BIJDON1 BIJDON2 and BIJDON3 ) and an entry of PA acetonitrile solvate (EDUWUW) Figure 2. 3 represents the crystal packing of monohydrate of PA for ref code BIJDON3. The coordinates for BIJDON, BIJDON1 and BIJDON2 are not available Figure 2. 3 : Hydrogen bonding in Protocatechuic acid monohydrate The c rystal structure of monohydrate of PA (BIJDON3) reveals that PA forms a noncentrosymmetric carboxylic acid dimer ; a supramolecular homosynthon (Figure 2.3 a) (O O: 2.717 and 2.592 ) and these dimers form a chain through a water molecule Water molecules insert themselves between PA dimer chain s forming a tetramer unit (b) (a)

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32 (F igure 2.3 b) T hree p henolic OH groups (two from the same PA molecule and a third from other PA molecule) are hydrogen bonded to water molecule and o ne water molecule is hydrogen bonded to three molecules of PA Figure 2.4: Hydrogen bonding in Protocatechuic acid acetonitrile solvate In the crystal structure of the acetonitrile solvate of PA PA forms a carboxylic acid dimer (O O: 2.606 ) One of the p henolic OH groups of PA is bonded to a phenolic OH of another pair of a PA dimer (OH O: 2.688 ) to form a zig zag chain. An acetonitrile molecule is trapped in between these chains via hydrogen bonding between nitrogen atom of acetonitrile molecule and phenol moiet ies of PA (OH N: 2.819 ) (F igure 2. 4 ). 2.2 Cocrystals of Proto catechuic acid S even novel cocrystals of PA with C aprolactam, I sonicotinamide, Isonicotinic acid (hydrated and anhydrated cocrystals) T heophylline, Ni cotinamide and C arbamazepine are obtaine d As discussed in section 2. 1 carboxylic acid moiety and phenolic OH groups of PA are likely to form hydrogen bonds. The se functional groups were u sed to form cocrystals with caprolactam, isonicotinamide, isonico tinic acid,

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33 theophylline, carbamazepine and nicotinamide. The summary of cocrystals synthesized is listed in table 2.1 2. 2 .1 Experimental Section All chemicals were obtained from commercial sources and used without further purification. Solvents were also purchased from commercial sources and distilled before use. S ingle crystals of cocrystals 1 to 3 and 5 to 7 were obtained by slow evaporation of stoichiometric amounts of starting materials in an appropriate solvent as summarized in table 2 .2 and they wer e removed from their mother liquors before complete evaporation of the solvent S amples were characterized by powder X ray diffraction, infrared spectroscopy differential scanning calorimetry thermo gravimetric analysis (for cocrystal 3) and single crysta l X ray data was collected for cocrystals 1 3 and 5 7 Table 2.1: Summary of the cocrystals of PA synthesized Cocrystal Target molecule Structure and Ratio Melting Point ( 0 C) Yield (%) 1 Caprolactam Anhydrous (1:1) 131 95.4 2 Isonicotinamide Anhydrous (1:1) 195 65.9 3 Isonicotinic acid Monohydrate (1:1:1) 224 62.5 4 Isonicotinic acid Anhydrous (1:1:1) 223 5 Theophylline Anhydrous (1:1) 254 49.5 6 Nicotinamide Anhydrous (1:1) 20 1 28.4 7 Carbamazepine Anhydrous (1:1) 89.2 2.2. 2 Cocrystallization via Solution Crystallization 1:1 cocrystal of PA and Caprolactam 1:

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34 Figure 2.5 : Synthesis and s ingle crystal of 1 PA (15.4 mg, 0.0999mmol) and caprolactam (11.3 mg, 0.0998mmol) were dissolved in 1ml of methanol and 3ml of water by heating. The resulting solution was allowed to stand at room temperature and slowly evapor ate. Colorless crystals (25. 5 mg, 0.0954 mmol, 95. 5% ) were harvested after 13 days and exhibited a melting point of 131 0 C. 1:1 cocrystal of PA and Isonicotinamide 2: Figure 2.6 : Synthesis and s ingle crystal of 2 PA (15.4 mg, 0.0999 mmol) and isonicotinamide (12.2 mg, 0.0998mmol) were dissolved in a mixture of 1ml of methanol and 3ml of water by heating. The resulting solution was allowed to slowly evaporate at room temperature. Colorless crystals of II (18.2 mg, 0.0659 mmol, 65.9 %) were harvested after 8 days and exhibited a melting point of 19 5 0 C. Monohydrate of 1:1 PA and Isonicotinic acid 3:

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35 Figure 2.7 : Synthesis and s ingle crystal of 3 PA (15.4 mg, 0.0999mmol) and isonicotinic acid (12.3 mg, 0. 0999 mmol) were d issolved in 5ml of 1:1 mixture of water and methanol b y heating. The resulting solution was placed at room temperature and allowed to slow ly evaporat e The colorless needle crystals (17.3, 0.0586 mmol, 62.5%) were harvested after 13 days and exhibited melting point of 224 0 C Cocrystal of P A and Isonicotinic a cid 4 : Cocrystal of anhydrate of p rotocatechuic acid and i sonicotinic acid was prepared by dehydrating 3 Crystals of 3 were heated in an oven at 120 o C for 48 hou rs thereby affording cocrystal 4 Unfortunately the single crystal was not harvested. The coc rystal exhibited a melting point of 223 0 C 1:1 cocrystal of PA and Theophylline 5 : Figure 2.8 : Synthesis and s ingle crystal of 5 PA (15.4 mg, 0.09mmol) and theophylline (18.0 mg, 0.09mmol) were dissolved in a 4ml of me thanol and the resulting solution was allowed to slowly evaporate at room temperature. Colorless blocks of crystals of 5 (Figure 2.8) (16.5 mg, 0.0497 mmol, 49.5%) were harvested after 3 days and exhibited a melting point of 254 0 C 1:1 cocrystal of PA and Nicotinamide 6:

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36 Figure 2.9: Synthesis of 6 PA (30.8 mg, 0. 1 9 9 mmol) and nicotinamide (24.4 mg, 0. 1 9 9 mmol) were slurried in a 1ml of methanol for 2 hrs and the resulting mixture was dissolved in 1ml of water and then allow ed to slowly evaporate at room temperature. Colorless thin crystals of 6 ( 15.7 mg, 0.0546 mmol, 28.4% ) were harvested after 7 days and exhibited a melting point of 20 1 0 C. Dihydrate of 1: 2 cocrystal of PA and Carbamazepine 7 : Figure 2.10 : Synthesis and s ingle crystal of 7 PA (15.4 mg, 0.09mmol) and carbamazepine (18.0 mg, 0.09 mmol) were dissolved in a 5ml of methanol and the resulting solution was allowed to slowly evaporate at room temperature. Colorless crystals of 7 (2 9 7 mg, 0.0264 mmol, 89.2%) were harvested after 2 days. Crystals were also harvested by dissolving the starting materials in same quantities by heating in a mixture of water ( 3 ml) and acetone ( 1 ml) after 3 days. 2.2. 3 Cocrystallization via Grinding and Slurry conversion A ttempts were made to synthesize the cocrystals using different methodologies d escribed in 1.2.6. For grinding starting materials were ground in a mortar and pestle

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37 using respective solvents and resulting powders were analyzed by X ray po wder diffraction. Also Cocrystal 5 was synthesized using an automated grinder ( Retseh MM301 ball mill ) for 90 min. The results from slurry and grinding are summarized in table 2. 2. Table 2.2: Comparison of solvent evaporation, grinding and slurry technique s Cocrystal Molar ratio used Crystallization solvent Solvent for grinding Solvent for slurry 1 1:1 MeOH/Water DMF Water 2 1:1 MeOH/Water Water Water 3 1:1 MeOH/ Water 5 1:1 MeOH Water 6 1:1 MeOH Water Water 7 1:1 MeOH and Water/Acetone MeOH 2 .2.4 FTIR, PXRD Differential Scanning Calorimetry and Thermo G ravimetric A nalysis All samples were characterized by IR spectroscopy using a Nicolet Avatar Smart Miracle 320 FT IR instrument. The bulk samples were analyzed by X ray powder dif fraction. PXRD analysis was performed using a Bruker D8 ADVANCE, /2 dif f ractometer Experimental conditions: Cu K radiation ( = 1.54056 ); 50 kV and 4 0 mA. Scanning interval: 3 40 0 ; time per step: 0. 5 s ; step size: 0.02 0 Thermal analysis was ca rried out using TA instrument DSC 2920 differential scanning calorimeter. Aluminum pans were used for the experiment for all the samples. Temperature calibrations were made using indium as the standard. An empty pan, sealed in the same way as the sample, w as used as a reference. The thermograms were run at a scanning of 10 0 C/ min from 30 0 C to the required temperature on 1 3mg of the sample Thermogravimet r ic analysis was carried out using STM6000. R esults are summarized in figures 2.11 2.17.

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38 (a) (b)

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39 Figure 2. 11 : Cocrystal 1 : (a) PXRD comparison (b) DSC (C) IR (a) (c)

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40 Figure 2.1 2 : Cocrystal 2 : (a) PXRD comparison (b) DSC (C) IR (c) (b)

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41 (b) (a )

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42 Figure 2.1 3 : Cocrystal 3 : (a) PXRD comparison (b) TGA (c) DSC (d) IR (a) (d) (c)

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43 (a) (b)

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44 Figure 2.1 4 : Cocrystal 4 : (a) PXRD comparison (b) DSC (c) IR (a) (c)

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45 Figure 2.1 5 : Cocrystal 5 : (a) PXRD comparison (b) DSC (c) IR (b) (c)

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46 (a) (b)

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47 Figure 2.1 6 : Cocrystal 6: (a) PXRD comparison (b) DSC (c) IR (c) ( a )

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48 ( b ) ( c )

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49 Figure 2.17: Cocrystal 7: (a) PXRD comparison (b) TGA (c) IR (d) DSC ( e ) DSC after heating cocrystal 7 2.2.5 Determination of Crystal Structure Single crystals of cocrystals 1 3 and 5 7 were examined under a microscope and single cry stal X ray diffraction data was collected on a Bruker AXS SMART APEX CCD diffractometer with monochromatized Mo K radiation ( = 0.71073 ) connected to a KRYO FLEX low temperature device. (d) (e)

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50 T able 2. 3 : Crystallographic data for cocrystals of PA

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51 2.2.6 Results and Discussion Figure 2. 1 8 : Each PA molecule is hydrogen bonded to three molecules of caprolactam through phenol carbonyl supramolecular heterosynthon The crystal structure of 1 reveals that PA exists as carboxylic acid dimer [ O O: 2.659 (3) ] and dimers are further connected to caprolactam molecules through hydrogen bonding between the carbonyl moiety of PA and N H moiety of caprolactam [ NH O: 2.882 (3) ] forming a heterocatemer Each PA molecule is connected to three molecules of caprolactam. Additional hydrogen bonding affords a three dimensional network These interactions help to establish the reported interactions. caprolactam is an organic molecule which has been listed on GRAS list. Two polymorphs ( Refcodes : CAPLAC and CAPLAC01 and eight cocrystals are reported in CSD for caprolactam [Ref. codes: CAPRES (with 4 chlororesorcinol ), KEWZUE ( 4,4' Biphe nol ), QQQGHM and QQQGHM01(with resorcinol ), QQQGHP (with hydroquinone ), QQQGHS and QQQGHS01(with 5 methylresorcinol ) and QQQGHV (with naphtho l)]. The coordinates are not available for five of them (QQQGHM, QQQGHP, QQQGHS, QQQGHS01, QQQGHV). In CAPRES and KEWZUE it can be caprolactam exists as lactam dimer forming a supramolecular homosynhon with CO HN distance s 2.901 3.014

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52 caprolactam in addition to the lactam dimer (CO HN: 2.950 ); supramolecular heterosynthon within the phenolic moiety of resorcinol and lactam (CO HO: 2.665 and NH O: 2.998 ) has been also observed with lactam act ing as bot h hydrogen bond donor and acceptor to a phenolic OH moiety. In the case of 1 PA breaks the lactam dimer fo rming a heterocatemer with supramolecular heterosynthon between carbonyl moiety of carboxylic acid and phenolic group of PA having bond distances of NH O: 2.882 (3) and OH O: 2.736 (3) respectively. Major peaks at 7.2, 9.1, 17.0, 21.1, 24.4, 25.5 and 28.0 degrees in experimental powder patterns matches with calculated PXRD (Figure 2.11a). The DSC shows sharp endotherm at 131.07 0 C (Figur e 2.11 b) which falls between the melting points of the two starting materials (Protocatechic acid: 20 5 0 C Caprolactam: 68 0 C) Figure 2.1 9 : Formation of a tape with alternate PA and isonicot inamide molecules by acid pyridine supramolecular heterosynthon The single crystal X ray structure of 2 reveals that i sonicotinamide molecules break the PA acid dimers and forms tapes through an acid pyridine [COOH N arom (O N : 2.669 (10) ) ] and amide phenol [N O: 3.042 (13) ] supramolecular heterosynthon s These tapes with alternate molecules of PA and isonicotinamide are further connected to each other through carbonyl amide and carbony l phenol

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53 supramolecular hetersynthons [N O: 3.021 (13) and O O: 2.762 (10) ] The C N C angle of pyridine ring in isonicotinamide was observed to be 116. 7 0 0 Figure 2. 20 : One molecule of PA is hydrogen bonded to five molecules of isonicotinamide There a re three entries for isonicotinamide with two polymorphs of it (EHOWIH EHOWIH01 and E HOWIH02 ) in CSD. Recently Seaton et. al. reported monohydrate of isonicotinamide. 19 Apart from this forty one cocrystals are reported till date in CSD. [AJAKAX (with hex 2 enoic acid ), AJAKEB (with 4 nitrobenzoic acid ), AJAKIF (with 3,5 dinitrobenzoic acid and 4 methylbenzoic acid ), ASAXOH (with 3 Nitrobenzoic acid ), ASAXUN (with 4 fluorobenzoic acid ), BUDWEC (with b enzoic acid ), BUDZUV (with 3,5 d initrobenzoic acid and 3 methylbenzoic acid ), BUFBIP (with 3,5 Dinitrobenzoic acid and 4 (N,N dimethylamino) benzoic acid ), BUFQAU (with 3,5 Dinitrobenzoic acid and 4 hydroxy 3 methoxycinnamic acid ), JAWWEK ( cis,cis 1,3,5 Cyclohexanetricarboxylic acid ), LUNMAI (with cinnamic acid ) LUNMEM ( 3 hydroxybenzoic acid ), LUNMIQ (with 3 (N,N dimethylamino) benzoic acid ), LUNMOW (with 3 ,5 bis (trifluoromethyl) benzoic acid ), LUNMUC (with 12 bromododecanoic acid ), LUNNAJ (with monochloroacetic acid ), LUNNEN (with fumaric acid monoethyl ester ), LUNNIR (with 4 ketopimelic acid ), LUNNOX (with fumaric acid ), LUNNUD and LUNNUD01 (with succinic acid ), LUNPAL ( d,l mandelic acid ), LUNPEP (with thiodiglycolic acid ), MELYEI (with p yridine 2,6 dicarboxylic acid

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54 and water), ULAWAF (with Oxalic acid ), UL AWEJ (with m alonic acid ), ULAWOT and ULAXAG (with g lutaric acid ), ULAWUZ and ULAXEK (with a dipic acid ), VAKTOR ( 4 h ydroxybenzoic acid ), VAKTUX (with r esorcinol ), VAKVEJ (with p hloroglucinol and water) VAKVIN (with h ydroquinone ), VETVUM (with 1 Ethyl 6 fl uoro 1,4 dihydro 4 oxo 7 (1 piperazinyl) 3 quinolinecarboxylic acid and chloroform ), XAQPOV (with 3,5 dinitrobenzoic acid and 3,4 dimethoxycinnamic acid ), XAQQEM (with 2 hydroxybenzoic acid ), ULAWAF01(with cis oxalic acid), ULAWAF02 (with trans oxalic acid ), YIPCEG (with iodine ) and YIPCIK (with p tetrafluorodi iodobenzene ). Among these structures 3 5 of them are with carboxylic acids The crystal structures of 34 form acid pyridine su pramolecular h eterosynthon with exception of one (VAKTOR) having carboxyli c acid amide and phenol pyridine supramol ecular hetero synthon s In accordance with these observations as predicted this dominant acid pyridine supramolecular heterosynthon was observed in case of 2 The C N C angle in aromatic nitrogen moieties are known to be sensitive to protonation. 7 Also the geometrical features of carboxylic acid and carboxylate are known to be different. 8 Thus e xamination of quantitative differences such as C N C angle of pyridine to protonation at nitrogen and distinction in C O bon d lengths between carboxylate and carboxylic acid will further provide an information whether 2 is a cocrystal or a salt. 9 If t he proton transfer from a carboxylic acid to base i.e pyridyl nitrogen in the case of 2 which lead to formation of a salt, takes place the carboxylate ion shows the same bond distance for C O. In the case of cocrystal formation bond lengths of C O and C OH differ which is observed in 2 ; the distances are 1.222 and 1.323 From table 2.4 it has been observed that C N C angle in 41 coc rystals of isonicotinamide with 35 carboxylic acids, 1 zwitterion, 3 phenols and 2 halogenated molecules is in the range of 11 6 90 to 1 19 91 0 T he C N C angle in 2 is 116.70 0 which can be correlated with the observed range supporting the neutral nat ure of isonicotinamide in the structure. The difference in pKa of aromatic nitrogen base and carboxylic acid is used as an

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55 pharmaceutical industry p K a difference gre ater that 2 or 3 are used for salt formation. 10 pKa = pKa(base) pKa(acid) for 2 is 1.06 which suggests the formation of n e utral bond. Hence in 2 ; pyridine nitrogen is not protonated giving rise to formation of neutral cocrystal and not a salt. S olution NM R is also a useful technique to conclude whether it is a salt or cocrystal. PXRD comparison shows major peaks are present in the experimental powder x ray diffraction pattern at 16.0, 18.1, 24.6, 25.7, 26.7, 29.3, 29.6 and 31.5 0 2 positions match with th e calculated powder pattern (Figure 2.1 2 a). Also powder pattern from water slurry shows the formation of cocrystal. DSC shows a sharp endotherm at 195.29 0 C (Figure 2.1 2 b) which falls in between the melting points of starting materials (PA : 205 0 C ; Isonico tinamide: 155 157 0 C ). Table 2.4: C N C angles in neutral isonicotinamide cocrystals f rom CSD Refcode C N C angle ( 0 ) Refcode C N C angle ( 0 ) Refcode C N C angle ( 0 ) AJAKAX 117.60 LUNMUC 117.16 ULAXAG 117.35 AJAKEB 118.63 LUNNAJ 119.15 ULAXEK 118.22 AJAK IF 117.68 LUNNEN 118.19 VAKTOR 116.90 ASAXOH 117.29 LUNNIR 117.69 VAKTUX 117.48 117.60 ASAXUN 117.03 LUNNOX 118.24 VAKVEJ 117.29 117.63 BUDWEK 117.68 LUNNUD 117.58 VAKVIN 117.33 BUDZUV 117.69 LUNNUD01 118.01 VETVUM 115.88 BUFBIP 117.66 LUNPAL 117.46 X AQPOV 117.39 BUFQAU 118.46 LUNPEP 117.46 118.28 XAQQEM 117.86 JAWWEK 116.86 117.35 MELYEI 117.35 ULAWAF01 119.67 LUNMAI 117.86 ULAWAF 119.47 ULAWAF02 119.91 LUNMEM 118.18 ULAWEJ 118.62 119.02 119.37 119.48 120.58 ULAWUZ 118.05 LUNMIQ 117.25 ULAWOT 117 .38 117.55 LUNMOW 117.74

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56 Figure 2. 2 1 : PA molecules forming acid acid supramolecular homosynthon and supramolecular heterosynthons with two water molecules and one isonicotinic acid molecule. The single crystal X ray analysis of 3 shows PA molecule exis t as a carboxylic acid dimer [ O H O: 2.616 (6) ] and isonicotinic acid molecules exist as zwitterions. One of the phenolic group of PA is hydrogen bonded to the isonicotin ic acid through water molecule [ H 2 O OH : 2.621 (5) ] and the remaining phenolic moiety is hydrog en bonded to isonicotinic acid [ O H O: 2.706 (6) ] PA molecules forms dimers and isonicotinic acid molecules form a chain hanging out to either side of protocatechuic acid dimers (Figure 2.2 2 ). The z witterionic nature of isonicotinic acid molecu le is supported by C N C angle of 121.50 0 in pyridine ring. Figure 2.2 2 : Formation o f isonicotinic acid chains in 3

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57 An analysis of the CSD shows no cocrystals of isonicotinic acid. But t he crystal structure of neutral isonicotinic acid is known in CSD with ref. code ISNICA. Also there are five entries involving isonicotinate ion. [ AJECAT (with 3,5 dinitrosalicylate), FETXIM ( 4 Aminopyridinium and water ), REFFIS (with R 1,1' binaphthalene 2,2' diyl phosphate), XECDUF and XECDUF01 (with chloride ) and YERX UP (with 2,5 dichloro 4 hydroxy 3,6 dioxocyclohexa 1,4 dien 1 olate and water) ] Based on these observations Zaworotko et. al recently published that the COO H N arom supramolecular heterosynthon that would be expected if a cocrystal were to form. 1 1 This ex pected supramolecular synthon is present in c ocrystal 3 Further whether 3 is a salt or a zwitterion ic cocrystal can be distinguished as explained in the case of 2 ; by the examination of C N C angle and the location of proton by C O distances in carboxylat e and carboxylic acid. The C N C angle of 3 supports the non existence of neutral isonicotinic acid molecule since the C N C angle in neutral isonicotinic acid is 118.91 0 The salt is an ionic species whereas zwitterion is a species which carries a total n et charge of zero but carries formal positive and negative charges on different atoms within the molecule. In 3 examination of bond lengths in carboxylic acid moiety (C O: 1.224 and 1.259 ) resulted the presence of carboxylate ion This carboxylate car rying formal negative charge and pyridyl nitrogen of isonicotinic acid carrying a formal positive charge because of a proton tr ansfer give s rise to overall neutral molecule Hence 3 is a zwiterion ic cocrystal T he major peaks at 13.3, 17.3, 19.1, 22.9, 23 .8, 25.2, 27.8 and 39.3 o from the experimental powder x ray diffraction pattern (Figure 2.1 3 a) matches with the calculated powder pattern. DSC shows three endotherms (Figure 2.1 3 c) The endotherm at 115.10 0 C corresponds to solvent molecule which is a wat er molecule and endotherm at 224.20 0 C correspond s to cocrystal of anhydrate The third endotherm around 300 0 C may correspond to the decomposition of isonicotinic acid. TGA (Figure 2.1 3 b) supports the loss o f water molecule (5.24%) which correlates the cal culated % loss (6.09%). Attempts to obtain single crystal of 4 were unsuccessful. But the powder X ray diffraction data after heating 3 shows the formation of new cocrystal (Figure 2.1 4 a)

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58 which is well supported by DSC. The absence of first endotherm from 3 shows the absence of water molecule in the 4 Also endotherm at 223.19 0 C correlates with the endotherm present in 3 Figure 2.2 3 : Acid dimer of PA molecule s forming supramolecular heterosynthon with theophylline dimers The single crystal X ray crysta llographic study analysis of 5 reveals that PA forms a acid dimer [ O H --O: 2.636 (19) ] and theophylline form dimers with N H O=C contacts at a distance of 2.781 (2) The two remaining phenolic groups form PA hydrogen bond s with theophylline molecules t hrough OH --O=C [ 2.703 (17) ] and OH --N contacts [ 2.774 ( 2 ) ] to establish an overall sheet structure. Each PA molecule is hydrogen bonded to two theophylline molecules through phenolic OH moieties (Figure 2.2 3 ). Theophylline is a naturally occurring dru g (present in tea) used in the treatment of respiratory diseases such as asthma. The crystal structure of anhydrous and monohydrate of theophylline as well as number of cocrystals have been reported to date in CSD [R eference codes : CSATEO (with 5 chlorosal icylic acid); DUXZAX(with urea); SULTHE (with sulfathiazole); THOPBA (with phenobarbital);TOPPNP (with p nitrophenol);WUYROX (with N (2 aminoethyl) carbamate); XEJWUF (with oxalic

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59 acid); XEJXAM (with malonic acid), XEJXEQ (with maleic acid); XEJXIU (with g lutaric acid); ZAYLOA (with 5 fluorouracil and water) and ZEXTIF (with p nitroaniline) ; KIGKAN (with citric acid and water); KIGLAO (with sorbic acid); KIGLES (with salicylic acid); KIGLIW (with 1 hydroxy 2 napthoic acid); KIGLOC (with 4 hydroxy benzoic ac id); KIGLUI (with N (4 hydroxyphenyl)acetamide ); XOBCUN (with saccharin ) ] In theophylline there are three sites of hydrogen bond acceptor and also one site of hydrogen bond donor. Out of 23 cocrystals 9 of them shows theophylline dimers in the crystal str ucture. In 5 it can be observed th at this dimer as well as phenol imidazole and phenol carbonyl supramolecular heterosynthon is present. Figure 2. 2 4 : Formation of sheet s in 5 PXRD of experimental pattern shows major peaks at 11.6, 15.8, 22.8, 25.2 and 27 .3 o which are present in the calculated powder x ray diffraction pattern (Figure 2.1 5 a). The sharp endotherm in DSC (Figure 2.15 b) at 254.28 0 C falls in between the melting points of the starting materials ( PA : 205 0 C ; Theophylline: 274 0 C ). The single crys tal X ray studies of 6 reveals that PA and nicotinamide forms an acid amide dimer [ N O: 2.875 (18) and O O: 2.639 (17) ] One unit of dimer is hydrogen bonded to o ther in helical fashion in which amide moiety is hydrogen b onded to carbonyl moiety of PA [ N O: 2.990 (2) ] Also aromatic pyridine nitrogen of nicotinamide hydro gen bonds to phenolic OH of PA [ N arom O: 2.671 (17) ] to form the helix. Dimer units in the helix create pi pi stacking (centroid to centroid distance 3.750 ). Nicotinamide is a small m olecule of vitami n B family. Nicotinamide serves as a target molecule because of its high medicinal value such as anti anxiety, 1 2 anti inflammatory 1 3 1 4 Two polymorphs of nicotinamide are

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60 known in CSD with three entries (Refcodes : NICOAM01, NICOAM02, NICOAM03) There are eleven cocrystals of nicotinamide are reported in CSD till date. FIFLAI, UCOTUC, VIGDAR, IACNCA, JEMDIP, PEQBES, UNEZES, XAQPUB, XAQQIQ, CIPHAL, JILZOU. Figure 2.2 5 : One molecule of PA is hydrogen bonded to tw o molecules of PA through OH OH supramolecular homosynthon, one molecule of nicotinamide through acid amide supramolecular heterosynthon and with another nicotinamide molecule through OH NH 2 supramolecular heterosynthon Among 11 cocrystals ; 8 of them are w ith carboxylic acid and in these 8 cocrystal 1 is phenolic acid. Except XAQQIQ in rest all 7 cocrystals carboxylic acid pyridin supramolecular heterosynthon was observed. XAQQIQ exhibits carboxylic acid primary amide and phenol pyridine supramolecula r hete rosynthon. This was expected in the case of 6 and observed the same. The C N C angle for nicotinamide cocrystals are summarized in table 2.5, which shows the range of 116.81 to 118.25. In case of 6 the observe d C N C angle is 120.39 which still falls in th e n e utral range of C N C angle. pKa of nicotinamide and protocatechuic acid is 0.91 which supports the formation of neutral bond as explained in case of 2

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61 Table 2. 5 : C N C angles in neutral nicotinamide cocrystals f rom CSD Figure 2.2 6 : Overall packing of 6 The majors peaks at 8.4, 11.5, 16.7and 19.2 o f rom the experimental PXRD data m atches with the calculated PXRD pattern (Figure 2.1 6 a). DSC shows the sharp endotherm at 200 0 C (Figure 2.16b) which falls in between the melting points of starting material (PA: 205 0 C and nicotinamide: 128 131 0 C). Figure 2.27 : Chain of d isordered PA and water molecules hydrogen bonding with c arbamazepine dimers Refcode C N C angle ( 0 ) Refcode C N C angle ( 0 ) Refcode C N C angle ( 0 ) FIFLAI 117.98 PEQBES 118.00 XAQQIQ 117.39 IACNCA 117.44 UCOTUC 116.81 JILZOU 118.25 118.42 JEMDIP 117.05 XAQPUB 117.47

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62 In s ingle crystal analysis of dihydrate of PA carbamazepine cocrystal; PA is not very well defined in the structure. The reason can be disorder or twinning in the crystal. Model of the disorder shows that PA forms chins with water molecules and hydrogen bonded to carbamazepine dimers. R factor is approximately 5% so model can be reliable. Structure can solved in lower space group but can not be refined very well. 2.3 Dissolution Studies To addr ess solubility, dissolution and o ther physicochemical properties; cocrystals have come up with a promising future as explained in section 1.2.4. Having understood the importance of it the efforts are underway to correlate the solubility and stability of a cocrystal with properties of pure components and if one can predict the trend in solubility To concentrate on this issue Hornedo et.al has recently published the study of 25 cocrystals of carbamazepine with their dissolution data and t hey have concluded t hat (1) solubility incr eases with the solubility of cocrystal components, (2) ligand transition concentrations increase with cocrystal and ligand solubilities for cocrystals of the same drug, and (3) ligand solubility about 10 fold higher than drug leads to cocrystal being more soluble than drug. 1 5 For further contribution to this regard dissolution studies of c ocrystals of PA have been carried out. In formulating and controlling the drug quality, dissolution is commonly used technique in pharmaceutical industries. 1 6 The common analytical methods used for the quantification i n dissolution studies are mainly spec trophotometric, chromatographic, mass spectrometric and potentiometric 1 7 UV/VIS spectrophotometry is the most prominent method. Cocrystals 1 2 and 5 6 are chosen for the dissolution studies. Water solutions of a ll cocrystals show absorption band in UV/V IS The dissolution studies are carried out with respect to PA and 310 nm was chosen to be the common wavelength for all cocrystals where absorption of cocrystal formers does not interfere with the absorption of PA. As kinetic dissolution is influenced by particle size distribution and experimental apparatus, all the cocrystals were sieved using ASTM standard sieve to obtain particle sizes between 53 75 m. All the experiments were carried out at room temperature and in set of three for the reliability unde r constant and same stirring speed. Calibration curves

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63 are determined in set of three with seven varying dilutions. Approximately 80 100 ml of water was used and an aliquot was drawn from the resulting slurry of cocrystal at regular time intervals using a syringe. The filtrate (use of 0.25 m nylon filter) was diluted with water and UV absorption was recorded. Results are plotted as concentration (mg/ml) versus time (min) and are summarized in figure 2.2 8 It has been observed that apart from cocrystal 1 all other cocrystals 2, 4, and 5 lo wer the solubility of PA. The solubility of cocrystal formers is in the order of caprolactam > nicotinamide > isonicotinamide > theophylline. Solubility of theophylline is less than the pure PA and all others are having higher solubility than PA. It was ob served based on Hornedo et.al. studies 1 5 as the solubility of cocrystal former increases the solubility of cocrystal increases and thus cocrystals 1,2 and 6 should increase PA solubility and 5 should decrease the solubility of PA. As predicted 1 showed an incre ase in solubility of PA about one and half fold and 5 showed about four fold decrease in solubility of PA But 2 and 6 are the outliners. As per the assumption as isonicotinamide and nicotinamide are more soluble cocrystal formers and furthermore nicotinam ide has higher solubility than isonicotinamide, 6 predicted to be higher in dissolution profile than 2 and PA. The observed data is contradictory to the assumptions and thus cocrystals afford solubility in unpredictable manner.

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64 Figure 2.2 8 : Comparison o f dissolution profiles of PA and its cocrystals To correlate dissolution profiles the detail comparison of crystal packing was carried out. As shown in figure 2.16 and 2.20 acid acid dimer of PA is intact while in case of 2 and 6 as shown in figures 2.17 and 2.22 acid dimer is interrupted and acid pyridine and acid amide supramolecular heterosynthons are observed respectively. Furthermore isonicotinamide and nicotinamide cocrystals show pi stacking (Figure 2.2 9 ) that may be increasing the lattice energy ca using lower solubility. Pi stacking is stronger in 6 than 2. Another observation is, as solubility decreases melting point of cocrystal increases. Melting temperatures of pharmaceutical crystals have been found an important indicator in solubility predict ion. In structurally similar pharmaceutical crystalline drugs, high melting drugs are generally recognized to possess lower solubility. 1 8 Even though it is generally expected that solubility and melting points are correlated it has not been observed in cas e of study of carbamazepine cocrystals. 1 5 Dissolution profiles of cocrystals also suggest the thermodynamic stability of the cocrystals In conclusion PA as a small molecule has altered the solubility of its cocrystals with caprolactam, isonicotinamide ni cotinamide and thophylline. The trend of the solubility is independent of the solubility of a cocrystal former in case of PA since

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65 direct proportionality was not observed. But it correlates with melting temperature of a cocrystal and higher temperature low ers the solubility. Figure 2.2 9 : Pi stacking in (a) PA nicotinamide (b) PA isonicotinamide cocrystal s In conclusion s even novel cocrystals of PA have been developed using the strategies of crystal engineering. The OH OH supramolecu lar homosynthon has been defeated by supramolecular heterosynthon in all cocrystals As per the CSD statistics phenol pyridine supramolecular heterosynthon is more reliable than phenol phenol supramolecular homosynthon and it has been s een in cocrystals of PA nicotinamide. But in case of cocrystals of PA with isonicotinamide and isonicotinic acid ... pyrine supramolecular heterosynthon overcomes the phenol pyridine supramolecular homosynthon as reported in case of competition of supramolecular synthons when b oth COOH and OH moieties are present with aromatic nitrogen base. 1 1 In isonicotinamide, isonicotinic acid and nicotinamide molecules aromatic nitrogen acts as a free base and accepts hydrogen from phenol or acid moiety. Theophylline has a tendency to form dimer leaving carbonyl as hydrogen accepter which has been also observed in case of PA theophylline cocrystal. Furthermore dissolution studies of cocrystals shows an a lteration in solubility of PA with the formation of cocrystal The solubility o f a cocrys tal is irrespective of the solubility of the cocrystal former. (b) (a)

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66 2 4 R e f e r e n c e s 1 Claudine Manach et. al. Am J Clin Nutr, 2004 79, 727 47 2. Ueda, J.; Saito N.; Shimazu,Y; Ozawa. T, Arch. Biochem. Biophys 1996 333, 377 84 3. Tanaka, T; Kawamori; T; Oh nishi, M; Mori; H, Cancer Res., 1993 53, 3908 3913 4. Tanaka, T; Kawamori; T; Ohnishi, M; Mori; H, Hara A, 1994 54, 2359 2365 5. Babich, H; Sedletcaia, A; Kenigsberg, B., Pharmacol. Toxicol. 2002 91 (5): 245 53 6. Nakamura, Y; Torikai, K; Ohto, Y; Muraka mi, A; Tanaka, T; Ohigashi, H. Carcinogenesis 2000 21 (10): 1899 907 7. (a) Boenigk, D.; Mootz, D. J. Am. Chem. Soc. 1988 110 2135; (b) Cowan, J. A.; Howard, J. A. K.; Intyre, G. J.; Lo, S. M. F.; Williams, I. D. Acta Crystallogr 2003 B59 794. 8. Borthwick, P. W. Acta Crystallogr. B 1980 36 628. 9 Bis, J. A.; Zaworotko, M. J. Cryst. Growth Des 2005 5 1169 1179 10. Stahl, P.H.; Wermuth, C. G. ed. Handbook of pharmaceutical salts: properties, selection, and use; Internationa l Union of Pure and Applied Chemistry, VHCA; Wiley VCH: Weinheim, New York, 2002. 1 1 Tanise, R. Shattock.; Kapildev, K. Arora.; Peddy, Vishweshwar.; and Michael, J. Zaworotko. Cryst. Growth Des. 2008 8 (12), 4533 4545 1 2 Tallman, J. F; Paul, S. M; Skolnick, P; Gallager, D. W. Science. 1980, 207 (4428), 274 81. 1 3 Niren, N. M. Cutis, 2006 77 1 4 Green, K. N; Steffan, J. S; Martinez Coria, H; Sun, X; Schreiber, S. S; Thompson, L. M; LaFerla, F. M. J Neuroscience 2008 1 5 David J. Good.; Hornedo, Cryst. Growth Des 2009 9 (5), 2252 64 1 6 Merck Research Laboratories, Pharmaceutical Research & Development, PA. 19486 1 7 Qingxi, Wang.; Decheng, Ma1.; John, P. Higgins. Disssolution Techonologie. 2006 1 8 (a) Grant, D. J. W.; Higuchi, T., Solubility Beha V ior of Organic Compounds ; Wiley: New York, 1990 ; p liii. (b) Yalkowsky, S. H. Solubility and Solubilization in Aqueous Media ; American Chemical Society: Washington, D.C., 1999 ; 61 77. 19. Colin C. Seaton. ; Andrew Parkin.; Chick C. Wilson.; Nicholas Blagden,

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67 Crystal Growth & Design 2009, 9, 1, 47 56

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68 3. Quercetin 3.1 Introduction Figure 3.1: Structure of Quercetin Fla vonoids are a group of phenolic plant compounds which are secondary metabolite s of plant 1 To date, more than 6000 flavonoids have been identified. 2 They are present in most edible fruits and vegetables, but the type of flavonoids obtained from different dietary sources varies. 3 Flavonoids and flavonoid containing products have bee n reported to possess biological activities. 4 Flavonoids are divided in to several subgroups. They consist of 2 benzene rings (A and B), which are connected by an oxygen containing pyrene ring (C). Flavonoids containing a hydroxyl group in position C 3 of t he C ring are classified as 3 hydroxyflavonoids e.g. flavono ls and those lacking it as 3 de oxyflavonoids e.g. flavanones and flavones. Quercetin (3, pentahydroxyflavanone, C 15 H 10 O 7, Mol. W t. 302.326, M.P.316 0 C) is one o f the most prevalent flavonol present in health food. Several studies have been carried out on the daily intake of querce tin in different countries and the importance of different foods in quercetin sources. Hertog et al concluded that wine was the major source of quercetin in Ital y, while onion and apples contributed most in the United States, Finland, Greece, and former Yugoslavia. 5 Recently Hakkinen et al estimated that onions, followed by tea, apples, and berries are the major sources of

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69 quercetin in Finland. Besides its anti oxidative and anticancer properties, quercetin also has been reported to benefit prostatitis and interstitial cystitis 6, 7 Thus quercetin can be view ed as a promising pharmacological agent because of the popular usage of products containing quercetin. Ac cording to BCS classification quercetin is a low soluble (0.45g/l) molecule. The bioavailability of quercetin is 5. 3 % in rats and LD 50 is 163 mg/kg. 8 This low solubility and bioavailability limits the usage of quercetin. As explained in section 1.2.4; c ocr lystallization can alter the solubility and bioavailability of the target molecule. The study herein focuses to synthesize cocrystals of quercetin to improve its solubility and thus bioavailability. Quercetin contains four phenolic OH groups and one hydro xyl group which can act acceptor/donor and one carbonyl and one ether moiety which can act as a hydrogen bond acceptor. Based on the CSD search as discussed in section 1. 4.2 for the occurrence of supramolecular synthons between polyphenols and other funct ionalities, aromatic nitrogen, carbonyls and amides would be promising. Thus different cocrystal formers were selected on the basis of their solubility, toxicity, pKa and cost as summarized in tables 1. 3, 1.4 and 1.5. 3.1.1 Quercetin in CSD To date, the crystal structure of pure quercetin is not known but CSD contains two entries for quercetin dihydrate (Ref. codes: FEFBEX, FEFBEX01). In the crystal packing of quercetin dihy d rate quercetin exists as dimer throu gh phenolic OH carbonyl sypramo lecular heter osynthon. These two functionalities form intermolecular hydrogen bonding too within the quercetin molecule. This quercetin dimers are hydrogen bonded to other dimers through water molecule. This extended hydrogen bonding network forms a sheet structure.

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70 Figure 3.2: OH carbonyl supramolecular heterosynthon in quercetin dihydrate Figure 3.3: Intermolecular interactions in the 1:1:1 cocrystal solvate of quercetin, caffeine and methanol Apart from the CSD entries, Zaworotko et.al patented three cocrysta ls of quercetin; 1:1:1 cocrystal of quercetin, caffeine and methanol, 1:1 cocrystal of quercetin and isonicotinamide and de solvated cocrystal of quercetin and caffeine. 9 The single crystal analysis for de solvated cocrystal of quercetin caffeine is not av ailable. The crystal structure of cocrystal of methanol solvate of quercetin and caffeine shows that caffeine interacts with quercetin molecule through phenolic OH N arom (2.831 A o ) and

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71 OH O=C (2.716 A o ) supramolecular heterosynthon. Another carbonyl of ca ffeine molecule hydrogen bonds to methanol molecule (OH O: 2.712 ). Quercetin molecules hydrogen bonds with each other via OH OH (2.774 2.847 ) supramolecular homosynthon. Overall it forms stacked sheets. The crystal structure of quercetin and isoni cotinamide shows that quercetin and isonicotinamide forms a four component assembly composed of two phenolic OH O=C (from isonicotinamide) (2.607 ) supramolecular heterosynthon and two NH OH (3.028 ) supramolecular heterosynthons. Quercetin molecule form s a dimer with OH OH (2.765 ) supramolecular homosynthon. Figure 3.4: Intermolecular interactions in the 1:1 Quercetin Isonicotinamide cocrystal 3.2 Cocrystals of quercetin Two novel cocrystals of quercetin with isonicotinic acid and theobromine were prepared As discussed in 3.1 phenolic OH groups and carbonyl group were utilized to form a cocrystal with the functional groups from isonicotinic acid and theobromine. The summary of cocrystals synthesized is listed in table 3.1. 3.2.1 Experimental Secti on All the chemicals were obtained from commercial sources and used without further purification. Solvents were also purchased from commercial sources and distilled before use. The single crystals of cocrystals 1 and 2 we re obtained by slow evaporation of

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72 stoichiometric amounts of starting materials in an appropriate solvent as summarized in table 3.2 and they were removed from their mother liquors before complete evaporation of the solvent had occurred. All samples were characterized by powder X ray diffr ac tion, infrared spectroscopy, differential scanning calorimetry thermogravimetric analysis and single crystal X ray diffraction. Table 3. 1 : Summary of cocrystals of quercetin obtained Cocrystal Target molecule Structure and Ratio Melting Point ( 0 C) Yield (%) 1 Isonicotinic acid Monohydrate (1:1:1) 283 95. 5 2 Theobromine Dihydrate (1:1:2) 294 65.9 3 .2.2 Cocrystallization via Solution Crystallization 1:1:1 cocrystal of quercetin, isonicotinic acid and water 1: Figu re 3 5 : Synthesis and single crystal of 1 Quercetin dihydrate (33.8 mg, 0.0999 mmol) was dissol ved in 10ml of acetone and iso nicotinic acid (12.3 mg, 0.0999 mmol) was dissolved in 2mL of water by heating on a hotplate. The resulting solutions were filtered together and placed in a refrigerator to slowly evaporate. After 8 days yellow plate crystals of 1 (26.4mg, 0.0595 mmol, 95.5%) were obtained and exhibited a melting point of 283 0 C. 1:1:2 cocrystal of quercetin, theobromine and water 2:

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73 Figure 3 6 : Synthesis and single crystal of 2 Quercetin dihydrate (33.8 mg, 0.0999 mmol) and theobromine (18.0 mg, 0. 0999mmol) were dissolved in 5 ml of ethanol and 10ml of 1:1 mixture of water and ethanol respectively by heating. Resul ting solutions were filtered together and placed in the refrigerator for slow evaporation. The crystals of 2 (18.2mg, 0.0351mmole, 65.9%) were harvested after 4 days and exhibited a melting point of 294 0 C 3 .2.3 Cocrystallization via Grinding and Slurry c onversion Using different methodologies attempts were made to synthesize the cocrystal as described in section 1.2.6. Only cocrystal 2 was successfully obtained by grinding as well as slurry conversion. Table 3.2: Comparison of solvent evaporation, grind ing and slurry techniques Cocrystal Molar ratio used Crystallization solvent Solvent for grinding Solvent for slurry 1 1:1 MeOH/Water 2 1:1 Et OH/Water Ethanol Ethanol 3 .2.4 FTIR, PXRD Differential Scanning Calorimetry and Thermo G ravim etric A nalysis All samples were characterized by IR spectroscopy using a Nicolet Avatar Smart Miracle 320 FT IR instrument. The bulk samples were analyzed by X ray powder diffraction. PXRD analysis was performed using a Bruker D8 ADVANCE, /2 difractometer. Experimental conditions: Cu K radiation ( = 1.54056 ); 50 kV and 40 mA. Scanning interval: 3 40 0 ; time per step: 0.5 s; step size: 0.02 0 Thermal analysis was carried out using a TA instruments DSC 2920 differential scanning ca lorimeter. Aluminum pans were used for the experiment for all the samples. Temperature

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74 calibrations were made using indium as the standard. An empty pan, sealed in the same way as the sample, was used as a reference. The thermograms were run at a scanning of 10 0 C/ min from 30 0 C to the required temperature on 1 3mg of the sample. Results are summarized in figures 3. 7 and 3. 8 (a) (b)

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75 Figure 3. 7 : Cocrystal 1 : (a) PXRD comparison (b) TGA (c) DSC (d) IR (d) (c)

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76 (a) (b)

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77 Figure 3. 8 : Cocrystal 2 : (a) PXRD comparison (b) TGA (c) DSC (d) IR (d) (c)

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78 3 .2.5 Determination of Crystal Structure Single crystals of cocrystals 1 and 2 were examined under a microscope and single crystal X ray diffraction data were collected on a Bruker AXS SMART APEX CCD diffractometer with monochromatized Mo K radiation ( = 0.71073 ) connected to a KRYO FLEX low temperature device. Table 3.3: Crystallographic data for cocrysta ls of quercetin Cocrystal 1 Cocrystal 2 Formula C 21 H 17 N O 10 C 22 H 22 N 4 O 11 MW 443.36 518.44 Crystal System Monoclinic Tr iclinic S pace group C2/c P 1 a () 34.7256(4) 7.0110(4) b () 7.43960(10) 11.2992(5) c () 14.3727(2) 14.9618(7) (deg) 90 106.548(3) (deg) 90.07 101.816(3) (deg) 90 94.797(3) V /( 3 ) 3713.11(8) 1099.29(9) Dc/ m gcm 3 1.586 1.566 Z 8 2 1 1 2 range 2.54 to 67.41 3.17 to 67.25 Nref./Npara. 3207 / 357 3641 / 422 T /K 100(2) 293(2) R1 [I>2sigma(I)] 0.0369 0.0 430 w R2 0.0896 0. 1039 GOF 1.047 1.028 Abs coef./mm 1 1.102 1.097

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79 3 .2.6 Results and Discussion Figure 3. 9 : Quercetin dimers hydr ogen bonding with isonicotinic acid molecules through phenol carboxylate supramolcular heterosynthon The single crystal X ray analysis of 1 reveals that quercetin molecules form dimer s through hydroxyl carbonyl (OH O=C ) supramolecular homosynthon. Phenolic OH b moiety forms a n intermolecular hydrogen bond with the carbonyl functionality. Phenolic OH e is hydrogen bonding to water molecule. And remaining two phenolic OH groups from ring A and ring B are engaged in hydrogen bonding with one molecule of isonic otinic acid which are in zwiterionic forms and one molecule of water. Thus each quercetin molecule is hydrogen bonded to three molecules of water, one molecule of quercetin and two molecules of isonicotinic acid (Figure 3. 9 ). Isonicotinic acid forms a cha in with expected acid pyridi n e supramolecular heterosynthon (Figure 3.1 0 ). As expected from the CSD statistics explained in 1.4.2 acid pyridin e supramolecular heterosynthon was observed and quercetin dimer is intact. As explained in section 2.2.6 for cocry stal 3 (cocrystal of PA with isonicotinic acid), examination of C N C ang le and location of a proton will distinguish 1 from a neutral cocrystal or a salt or a zwitreionic cocrystal. The C N C angle of 121.70 0 suggests the non existence of neutral isonicot inic acid molecule in comparison with the C N C angle is neutral isonicotinic acid molecule (118.91 0 ). The location of the proton can be

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80 confirmed by the C O distances in carboxylate and in carboxylic acid. In 1 examination of these bond lengths resulted in the presence of carboxylate ion (C O: 1.244 and 1.263 ) where as in case of neutral isonicotinic acid molecule these distances are 1.215 and 1.294 Furthermore both the formal charges are present on the same molecule resulting a overall neutral s pecies. Thus 1 is zwiterionic cocrystal and not a salt. Figure 3.1 0 : Formation of a chain of isonicotinic acid molecules with acid pyridin supramolecular heterosynthon The major peaks at 5.1, 10.3, 12.2, 13.3, 15.3, 20.5, 24.4 and 30.8 o from the experime ntal powder x ray diffraction pattern (Figure 3.7 a) matches with the calculated powder pattern. DSC sho ws three endotherms (Figure 3.7 c). The endotherm at 196.17 0 C corresponds to solvent molecule which is a water molecule and endotherm at 282.68 0 C is cor responding to cocrystal of anhydrate. The third endotherm around 320 0 C may be corresponding to the decomposition of isonicotinic acid and que rcetin as well. TGA (Figure 3.7 b) supports the loss of water molecule (4.85%) which correlates the calculated % lo ss (4.05%). Attempts to dehydrate 1 by heating in vacuum oven were carried out. DSC showed disappearance of the endotherms at 196 0 C and 283 0 C but the endotherm of decomposition temperature of quercetin and isonicotinic acid was present. Cocrystal c omponent s might be falling apart from each other and decomposing individually.

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81 Figure 3.11: DSC after heating cocrystal 1 Figure 3.1 2: Q uercetin dimer hydrogen bonding with one theobromine molecule through carbonyl phenol supramolecular heterosynthon The sing le crystal X ray crystallographic study analysis of 2 reveals that quercetin molecules exist as a d imer through hydroxyl carbonyl [ OH O: 2.616 (2) A 0 ] supramolecular homosynthon. These dimers are further hydrogen bonding to theobromine dimers [ CO HN: 1.916 A 0 ] via phenol carbonyl (OH O: 2.752 (2) A 0 ) supramolecular he terosynthon from one side and through water molecule from other side T he remaining phenolic group is hydrogen bonded to N arom of theobromine through water molecule (OH OH: 2.796 A 0 and H2O N arom : 2.822 (3) A 0 ]. Overall it forms a sheet.

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82 Adjacent sheets are linked to each other through water molecule to give an overall 3D network (Figure 3.1 3 ). Figure 3.1 3 : Formation of sheet structure in 2 Theobromine is a molecule from xanthine family and a major constituent of chocolate. Theobromine is a bitter alkaloid of cacao plant. The name theobromine is derived from theobroma meaning food of the gods in Greek. The use of theobromine in cancer has been patented 10 and currently theobromine is u sed as a vasodilator, diuretic and heart stimulant. 11 The crystal structure for pure theobromine is known in CSD (Ref. code: SEDNAQ). It reveals that theobromine molecules form dimer through amide amide supramolecular homosynthon. Apart from the pure struc ture two cocrystals [Ref. code: CSATBR (with 5 chlorosalicylic acid) and HIJYEF (malonic acid)] are known. In both the cocrystals it can be observed that theobromine forms a cid amide dimer and imidazoly l nitrogen acts as a hydrogen bond acceptor and forms supramolecular synthon with respective carboxylic acid OH moiety. In case of 2 theobromine amide dimer is observed and imidazolyl nitrogen acts as hydrogen acceptor through supramolecular heterosynthon with water molecule. Carbonyl functionality of theob romine forms supramolecular heterosynthon with phenolic OH a of quercetin which correlates with CSD statistics explained in 1.4.2. Attempts to synthesize the anhydrous cocrystal of theobromine were made. The cocrystal was heated in vacuum oven at 120 0 C for 3 hours. DSC showed dehydrated product. When PXRD pattern of 2 and dehydrated product was compared it was observed that all the peaks from the 2 were present but shifted to left in dehydrated product. After 30 min. TGA was recorded and it was observed that cocrystal contains 1 1 % of water. It can be concluded that cocrystal

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83 might not be stable and during the course of the investigation cocrystal absorbs water from air giving a hydrated product. (a) (b)

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84 Figure 3.1 4 : (a) PXRD pattern (b) DSC (C) TGA of att empts of synthesizing anhydrous cocrystal of 2 3.3 Dissolution Studies Low solubility and bioavailability limits the use of quercetin as a therapeutic target molecule. The dissolution studies of cocrystals of quercetin with isonicotinamide and caffeine me thanol solvate are already patented and it has been shown the six fold increase in solubility of quercetin in both the cases. 9 Furthermore de solvated cocrystal of quercetin caffeine shows 21 fold increase. Also in case PA molecule; as explained in section 4.3; the direct proportionality to the solubility of cocrystal former was not observed. Thus dissolution study was carried out with 2 Attempts to study cocrystal 1 were unsuccessful. The scaling up of the product was attempted which resulted in the preci pitation of isonicotinic acid with the cocrystal. As explained in section 2.3 UV/VIS was used to carry out dissolution experiment. As quercetin did not show any absorbance in water a suitable solvent in which the cocrystal is soluble and shows absorptio n was found. (c)

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85 The first choice of the solvent was 1:1 ethanol/water (V/V%) since previous studies we re carried out in the same solvent system. The standard calibration curve was plotted by preparing standard solution of a cocrystal in EtOH/water system. Th e bulk powder of a cocrystal was sieved in order to get the particle size between 53 75 m using ASTM standard sieves. Experiment was carried out in set of three using 80 ml of the solvent and 800 mg of cocrystal The resulting slurry was stirred for four hours and at regular intervals of time an aliquot was drawn, filtered through 0.25 m ny lon filter, diluted with appropriate amount of same solvent system and UV absorption was recorded. The result is plo tted as concentration (mg/ml) Verses time (min) (Figure 3.1 5 ). Figure 3.1 5 : D issolution profiles of dihy d rate of 1:1 cocrys tal of querce tin and theobromine, in 1:1 EtOH/Water (V/V%) system at 360 nm It can be viewed from the plot that the solubility of the cocrystal maximizes within first five minutes and then slowly goes down to that of quercetin dihydrate. But the solubility of the querc etin has been improved by approximately 1.5 fold. Even though it is not a significant change it can be concluded that solubility of cocrystal is independent of the solubility of cocrystal former. In conclusion two novel cocrystals of quercetin ha ve been de veloped using crystal engineering strategies. As expected from CSD statistics in case of 1 supramolecular heterosynthon has been observed. Observed quercetin dimer in quercetin 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 100 200 300 Concentration (mg/ml) Time (min) Quercetin dihydrate; hydroxyl carbonl Rel.Sol.1; mp=316 degreeC 1:1:2 theobromine, water; hydroxyl carbonyl dimer intact; Rel.Sol. 0.7; mp=294 degree C

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86 dihydrate and cocrystal of quercetin isonicotinamide is also pre sent in both cocrystals (1 and 2). The tendency of theobromine forming dimer leaving carbonyl moiety as hydrogen acceptor is also observed. A nhydrous cocrystals of 1 and 2 are found to be unstable. Dissolution study of 2 shows increase in solubility of quer cetin dihyrate and similarly as discussed in case of PA, direct proportionality between solubility of cocrystal former and solubility of cocrystal is not observed.

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87 3 4 R e f e r e n c e s 1. D.F. Birt et al. Pharmacology & Therapeutics, 2001 90, 157 177. 2. Groot, H. Rauen, U. Fundamentals of Clinical Pharmacology 1998, 12:249 55. 3. Jitka, Psotova1.; Iarka, ChlopCikova1.; Petra, Miketova1.; Jan, Hrbac.; Vilim, imanek. Phytother. Res 2004 ; 18, 516 521. 4 (a) Waladkhani, A. R., & Clemens, M. R. (1998). Effe ct of dietary phytochemicals on cancer development. Int J Med 1, 747 753 (b)Wattenberg, L. W. (1992a). (b) Inhibition of carcinogenesis by minor dietary constituents. Cancer Res 52 (suppl.), 2085s 2091s. 5. Hertog, M. G. L., Kromh out, D., Aravanis, C., Blackburn, H., Buzina, R., Fidanza, F., Giampaoli, S., Jansen, A., Menotti, A., Nedeljkovic, S., Pekkarinen, M., Simic, B. S., Toshima, H., Feskens, E. J. M., Hollman, P. C. H., & Katan, M. B. (1995). Flavonoid intake a nd long term risk of coronary heart disease and cancer in the Seven Countries Study. Arch Intern Med 155, 381 386 6 Young, J. F.; Nielsen, S. E.; Haraldsdottir, J. et al. Am J Clin Nutr 1999 ; 69(1): 87 94. 7 White, L.; Mavor, S. Herbs, Health. Love land, Colo: Interweave Press; 1998: 22, 84 8. Xiao Chen.; Ophelia Q. P.Yin.; Zhong Zuo.; Moses S. S. ChowPharmaceutical Research, 2005, 22, 6, 892. 9. Zaworotko et.al. (2008/153945) Nutraceutical cocrystal compositions 10. US patent 6693104 "Theobromine with an anti carcinogenic activity" granted 2004 02 17 11 William Marias Malisoff (1943). Dictionary of Bio Ch emistry and Related Subjects Philosophical Library. pp. 311, 530, 573. ISBN B0006AQ0NU.

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88 4. CONCLUSION AND FUTURE DIRECTIONS Development of crystal engineering has led the foundation for supramolecular chemistry. The importance of supramolecular che mistry has been established by a vast array of applications. Discovery of new forms of drugs such as hydrates, solvates, polymorphs and cocrystals are overcoming the problems associated with drugs such as stability, solubility and bioavailability. Cocrysta ls have proved their importance in this regards and attracting pharmaceutical industries vastly. Using the strategies of crystal engineering the work herein has led to develop novel cocrystals of protocatechuic acid and quercetin with pharamaceutically acc epted molecules (cocrystal formers) such as caprolactam, isonicotinamide, isonicotinic acid, theophylline, nicotinamide and theobromine. Cocrystals are synthesized and structurally characterized. Cocrystals of protocatechuic acid establish its use as cocry stal former. Furthermore the effect of these cocrystals has been studied in regards with the solubility of pure protocatechuic acid and quercetin. Dissolution studies of protocatechuic acid have been carried out to study the trends in the solubility of coc rystals with respect to a cocrystal former. The predicted direct proportionality has not been observed. But the trend in the melting point of a cocrystal and its solubility has been observed. As the melting point of a cocrystal increases the solubility dec reases. Thus crystal packing of a cocrystal influences its solubility. The study herein contributes in the limited literature available regarding to the predictability of cocrystal solubility. Attempts to improve solubility of quercetin via cocrystallizat ion were achieved. Quercetin theobromine dihydrate cocrystal results in 1.5 fold increase in solubility of quercetin. As observed in case of protocatechuic acid, direct relationship between solubility of quercetin and theobromine is not observed. Thus eval uation of physicochemical properties of pharmaceutical cocrystals and its predictability is an important area for additional research. The results reported herein could be the subject of future contributions.